Article

Annexin 1 regulates cell proliferation by disruption of cell morphology and inhibition of cyclin D1 expression through sustained activation of the ERK1/2 MAPK signal

November 2003
Experimental Cell Research 290(1):93-107

November 2003
290(1):93-107

DOI:10.1016/S0014-4827(03)00310-0

Source
PubMed

Authors:

Louise Alldridge

The Peninsula College of Medicine and Dentistry

Clare Bryant

University of Cambridge

Cellular proliferation is controlled by the integration and coordination of extracellular signals. This study explores the role of the protein annexin 1 (ANXA1) in the regulation of such events. We show that ANXA1 has a cell-type independent, anti-proliferative function through sustained activation of the ERK signaling cascade. Moreover, ANXA1 reduces proliferation by ERK-mediated disruption of the actin cytoskeleton and ablation of cyclin D1 protein expression and not by ERK-mediated induction of the cyclin-dependent kinase, CDK2, inhibitor p21(cip/waf). Finally, ANXA1 regulates the ERK pathway at a proximal location, by SH2 domain-independent association with the adapter protein Grb-2. In summary, overexpression of ANXA1 mediates the disruption of normal cell morphology and inhibits cyclin D1 expression, therefore reducing cell proliferation through proximal modulation of the ERK signal transduction pathway.

Novel Pathogenic, Biomarker and Therapeutic Potentials of Foxm1 in Glioma

Article

May 2019

Seidu A. Richard

Gliomas embody entirely prime brain tumors of glial cell or neuroepithelial derivation. Glioma is still one of the lethal human cancers notwithstanding contemporary innovations in both diagnostic techniques and therapeutic schemes. Also, glioma carries the lowest survival rate as compare to other cancers 5 years after definitive diagnosis. FoxM1 was initially identified as HFH-11, WIN, MPP2, as well as Trident. It’s an evolutionary well-maintained, with common winged helix DNA-binding domain. FoxM1 actively participates in gliomagenesis via several pathways like, FoxM1/MELK/EZH2 signaling, FoxM1/BMI-1/Ink4a/Arf/Ink4b signaling, FoxM1/IPO7/Hh signaling as well as FoxM1/PLAGL2/Wnt/β-catenin signaling. FoxM1 also augments’ the stimulation of Akt as well as secretion of survivin, cyclin E, and cyclin D1. Furthermore, FoxG1 contributes to glioma invasiveness via MMPs especially MMP-4 and MMP-9. Nevertheless, FoxM1 contributes to glioma angiogenesis via VEGF and transcription stimulators like HIF-1 and STAT3 have been implicated as VEGF facilitators. FoxM1 has also proven to a promising diagnostic and prognostic biomarker in glioma. Moreover, FoxM1 has therapeutic potential in glioma either alone or in combination with other agents. This review therefore focuses on the novel pathogenic, biomarker and therapeutic potentials of FoxM1 in Glioma.

Novel Pathogenic, Biomarker and Therapeutic Potentials of Foxm1 in Glioma

Article

Full-text available

Jul 2019

Seidu A. Richard

Gliomas embody entirely prime brain tumors of glial cell or neuroepithelial derivation. Glioma is still one of the lethal human cancers notwithstanding contemporary innovations in both diagnostic techniques and therapeutic schemes. Also, glioma carries the lowest survival rate as compare to other cancers 5 years after definitive diagnosis. FoxM1 was initially identified as HFH-11, WIN, MPP2, as well as Trident. It's an evolutionary well-maintained, with common winged helix DNA-binding domain. FoxM1 actively participates in gliomagenesis via several pathways like, FoxM1/ MELK/EZH2 signaling, FoxM1/BMI-1/Ink4a/Arf/Ink4b signaling, FoxM1/IPO7/Hh signaling as well as FoxM1/ PLAGL2/Wnt/β-catenin signaling. FoxM1 also augments' the stimulation of Akt as well as secretion of survivin, cyclin E, and cyclin D1. Furthermore, FoxG1 contributes to glioma invasiveness via MMPs especially MMP-4 and MMP-9. Nevertheless, FoxM1 contributes to glioma angiogenesis via VEGF and transcription stimulators like HIF-1 and STAT3 have been implicated as VEGF facilitators. FoxM1 has also proven to a promising diagnostic and prognostic biomarker in glioma. Moreover, FoxM1 has therapeutic potential in glioma either alone or in combination with other agents. This review therefore focuses on the novel pathogenic, biomarker and therapeutic potentials of FoxM1 in Glioma.

Evaluation of Cellular Toxicity in Hep G2 Cell and Endothelial Cells on Different Medical Biopolymer Supports

Article

Full-text available

Jul 2018

Charles Fokunang

ANXA1 affects murine hair follicle growth through EGF signaling pathway

Article

Dec 2020
GENE

Annexin A1 (ANXA1), a calcium-dependent phospholipid binding protein expressed in animals, plants and microorganisms, participates in various cellular physiological activities. Previous proteomics analysis indicates that the level of ANXA1 in mice dorsal skin changes during hair growth cycle, we speculate that ANXA1 may play an important role in hair follicle (HF) development. Thus, Anxa1 knock-out (KO) and over-expression (OE) mice were constructed to test its function. Our results showed that in addition to the diameter of HF and hair shaft, ANXA1 could participate in hair growth by affecting the density of HF, and the proliferation of hair follicle stem cells (HFSCs). Meanwhile, molecular analysis showed that EGF signaling pathway is involved in the function of ANXA1. The expression of Anxa1 is negatively correlated with the levels of Egf, Notch1, Mkk7, and phosphorylated AKT1 and ERK/2 proteins. The levels of Egf, Notch1, Mkk7 and phosphorylation of AKT1 and ERK/2 increased in Anxa1 KO mice but decreased in Anxa1 OE mice. Taken together, our results suggested that ANXA1 could affect the hair growth by regulating the HFSCs proliferation through EGF signaling pathway.

Core Matrisome Protein Signature During Periodontal Ligament Maturation From Pre-occlusal Eruption to Occlusal Function

Article

Full-text available

Mar 2020

The pre-occlusal eruption brings the molars into functional occlusion and initiates tensional strains during mastication. We hypothesized that upon establishment of occlusal contact, the periodontal ligament (PDL) undergoes cell and extracellular matrix maturation to adapt to this mechanical function. The PDL of 12 Wistar male rats were laser microdissected to observe the proteomic changes between stages of pre-occlusal eruption, initial occlusal contact and 1-week after occlusion. The proteome was screened by mass spectrometry and confirmed by immunofluorescence. The PDL underwent maturation upon establishment of occlusion. Downregulation of alpha-fetoprotein stem cell marker and protein synthesis markers indicate cell differentiation. Upregulated proteins were components of the extracellular matrix (ECM) and were characterized with the matrisome project database. In particular, periostin, a major protein of the PDL, was induced following occlusal contact and localized around collagen α-1 (III) bundles. This co-localization coincided with organization of collagen fibers in direction of the occlusal forces. Establishment of occlusion coincides with cellular differentiation and the maturation of the PDL. Co-localization of periostin and collagen with subsequent fiber organization may help counteract tensional forces and reinforce the ECM structure. This may be a key mechanism of the PDL to adapt to occlusal forces and maintain structural integrity.

Evaluation of cellular toxicity in Hep G2 cell and endothelial cells on different medical biopolymer supports

Article

Jun 2018

Charles Fokunang

Annexin A1, Annexin A2, and Dyrk 1B are upregulated during GAS1-induced cell cycle arrest: GAS1-induced cell arrest and 2DE profiles

Article

Full-text available

Oct 2017

GAS1 is a pleiotropic protein that has been investigated because of its ability to induce cell proliferation, cell arrest, and apoptosis, depending on the cellular or the physiological context in which it is expressed. At this point, we have information about the molecular mechanisms by which GAS1 induces proliferation and apoptosis; but very few studies have been focused on elucidating the mechanisms by which GAS1 induces cell arrest. With the aim of expanding our knowledge on this subject, we first focused our research on finding proteins that were preferentially expressed in cells arrested by serum deprivation. By using a proteomics approach and mass spectrometry analysis, we identified 17 proteins in the 2-DE protein profile of serum deprived NIH3T3 cells. Among them, Annexin A1 (Anxa1), Annexin A2 (Anxa2), dual specificity tyrosine-phosphorylation-regulated kinase 1B (Dyrk1B) and Eukaryotic translation initiation factor 3, F (eIf3f) were upregulated at transcriptional level regarding proliferative NIH3T3 cells. Moreover, we demonstrated that Anxa1, Anxa2, and Dyrk1b are upregulated at both the transcriptional and translational levels by the overexpression of GAS1. Thus, our results suggest that the upregulation of Anxa1, Anxa2, and Dyrk1b could be related to the ability of GAS1 to induce cell arrest and maintain cell viability. Finally, we provided further evidence showing that GAS1 through Dyrk 1B leads not only to the arrest of NIH3T3 cells but also maintains cell viability. This article is protected by copyright. All rights reserved

Proteomic Analysis of Stage-II Breast Cancer from Formalin-Fixed Paraffin-Embedded Tissues

Article

Full-text available

Jan 2016
BMRI

Breast cancer is the most frequently occurring disease among women worldwide. The early stage of breast cancer identification is the key challenge in cancer control and prevention procedures. Although gene expression profiling helps to understand the molecular mechanism of diseases or disorder in the living system, gene expression pattern alone is not sufficient to predict the exact mechanisms. Current proteomics tools hold great application for analysis of cancerous conditions. Hence, the generation of differential protein expression profiles has been optimized for breast cancer and normal tissue samples in our organization. Normal and tumor tissues were collected from 20 people from a local hospital. Proteins from the diseased and normal tissues have been investigated by 2D gel electrophoresis and MALDI-TOF-MS. The peptide mass fingerprint data were fed into various public domains like Mascot, MS-Fit, and Pept-ident against Swiss-Prot protein database and the proteins of interest were identified. Some of the differentially expressed proteins identified were human annexin, glutathione S-transferase, vimentin, enolase-1, dihydrolipoamide dehydrogenase, glutamate dehydrogenase, Cyclin A1, hormone sensitive lipase, beta catenin, and so forth. Many types of proteins were identified as fundamental steps for developing molecular markers for diagnosis of human breast cancer as well as making a new proteomic database for future research.

The Role of Annexin A1 in DNA Damage Response in Placental Cells: Impact on Gestational Diabetes Mellitus

Preprint

Full-text available

May 2023

The functions of annexin A1 (ANXA1) that is expressed on membranes and in cytoplasmic granules have been fully described. Nonetheless, the role of this protein in protecting against DNA damage in the nucleus is still emerging and requires further investigation. Here, we investigated the involvement of ANXA1 in DNA damage response in placental cells. Placenta was collected from ANXA1 knockout mice (AnxA1-/-) and pregnant women with gestational diabetes mellitus (GDM). The placental morphology and ANXA1 expression, which are related to the modulation of cellular response markers in the presence of DNA damage, were analyzed. Total area of AnxA1-/- placenta was smaller because of reduced labyrinth zone, enhanced DNA damage, and impaired base excision repair (BER) enzymes, which resulted in the induction of apoptosis in the labyrinthine and junctional layers. The placentas from pregnant women with GDM showed reduced expression of AnxA1 in the villous compartment, increased DNA damage, apoptosis, and reduction of enzymes involved in the BER pathway. Our translational data provide valuable insights into the possible involvement of ANXA1 in the response of placental cells to oxidative DNA damage and represent advancement in investigations on the mechanisms involved in placental biology.

The Role of Annexin A1 in DNA Damage Response in Placental Cells: Impact on Gestational Diabetes Mellitus

Article

Full-text available

Jun 2023
INT J MOL SCI

The functions of annexin A1 (ANXA1), which is expressed on membranes and in cytoplasmic granules, have been fully described. Nonetheless, the role of this protein in protecting against DNA damage in the nucleus is still emerging and requires further investigation. Here, we investigated the involvement of ANXA1 in the DNA damage response in placental cells. Placenta was collected from ANXA1 knockout mice (AnxA1−/−) and pregnant women with gestational diabetes mellitus (GDM). The placental morphology and ANXA1 expression, which are related to the modulation of cellular response markers in the presence of DNA damage, were analyzed. The total area of AnxA1−/− placenta was smaller due to a reduced labyrinth zone, enhanced DNA damage, and impaired base excision repair (BER) enzymes, which resulted in the induction of apoptosis in the labyrinthine and junctional layers. The placentas of pregnant women with GDM showed reduced expression of AnxA1 in the villous compartment, increased DNA damage, apoptosis, and a reduction of enzymes involved in the BER pathway. Our translational data provide valuable insights into the possible involvement of ANXA1 in the response of placental cells to oxidative DNA damage and represent an advancement in investigations into the mechanisms involved in placental biology.

Annexin A1 affects tumor metastasis through epithelial- mesenchymal transition: a narrative review

Article

Full-text available

Jan 2021

Background and objective: Annexin A1 (annexin I, ANXA1), the first discovered member of the annexin superfamily, plays important roles in tumor development, invasion, metastasis, apoptosis and drug resistance based on tumor type-specific patterns of expression. The acquisition of the epithelial-mesenchymal transition (EMT) characteristics is an essential mechanism of metastasis because they increase the mobility and invasiveness of cancer cells. Cancer invasion and metastasis remain major health problems worldwide. Elucidating the role and mechanism of ANXA1 in the occurrence of EMT will help advance the development of novel therapeutic strategies. Hence, this review aims to attract everyone's attention to the important role of ANXA1 in tumors and provide new ideas for clinical tumor treatment. Methods: The PubMed database was mainly used to search for various English research papers and reviews related to the role of ANXA1 in tumors and EMT published from November 1994 to April 2022. The search terms used mainly include ANXA1, EMT, tumor, cancer, carcinoma, and mechanism. Key content and findings: This article mainly provides a summary of the roles of ANXA1 and EMT in tumor metastasis as well as the various mechanisms via which ANXA1 facilitates the occurrence of EMT, thereby affecting tumor metastasis. In addition, the expression of ANXA1 in different metastatic tumor cell lines and its roles in tumorigenesis and development are also elaborated. This article has found many tumorous therapeutic targets related to ANXA1 and EMT, further confirming that ANXA1 has a huge potential for the diagnosis, treatment and prognosis of certain cancers. Conclusions: Both the abnormal expression of ANXA1 and the occurrence of EMT are closely related to the invasion and metastasis of tumors, and more interestingly, ANXA1 can impact EMT directly or indirectly by mediating signaling pathways and adhesion among cells. We need more studies to elucidate the effects of ANXA1 on tumor invasion, migration and metastasis through EMT in vitro and in vivo clearly, and ultimately in patients to identify more therapeutic targets.

Pathobiological functions and clinical implications of annexin dysregulation in human cancers

Article

Full-text available

Sep 2022

Annexins are an extensive superfamily of structurally related calcium- and phospholipid-binding proteins, largely conserved and widely distributed among species. Twelve human annexins have been identified, referred to as Annexin A1-13 (A12 remains as of yet unassigned), whose genes are spread throughout the genome on eight different chromosomes. According to their distinct tissue distribution and subcellular localization, annexins have been functionally implicated in a variety of biological processes relevant to both physiological and pathological conditions. Dysregulation of annexin expression patterns and functions has been revealed as a common feature in multiple cancers, thereby emerging as potential biomarkers and molecular targets for clinical application. Nevertheless, translation of this knowledge to the clinic requires in-depth functional and mechanistic characterization of dysregulated annexins for each individual cancer type, since each protein exhibits varying expression levels and phenotypic specificity depending on the tumor types. This review specifically and thoroughly examines the current knowledge on annexin dysfunctions in carcinogenesis. Hence, available data on expression levels, mechanism of action and pathophysiological effects of Annexin A1-13 among different cancers will be dissected, also further discussing future perspectives for potential applications as biomarkers for early diagnosis, prognosis and molecular-targeted therapies. Special attention is devoted to head and neck cancers (HNC), a complex and heterogeneous group of aggressive malignancies, often lately diagnosed, with high mortality, and scarce therapeutic options.

Identification of Annexin A1 as a novel regulator of oriented cell divisions in mammary epithelial cell

Thesis

Nov 2021

Maria Fankhaenel

Oriented cell divisions (OCDs) represent a fundamental mechanism for tissue morphogenesis, repair and differentiation where the mitotic spindle is oriented along a specific polarity axis. Early research identified the evolutionarily conserved Gαi/LGN/NuMA ternary complex that mediates orientation of the mitotic spindle by being restricted to specific cortical regions. The mechanisms that control the recruitment of these proteins to the cortex remain unfolding, particularly in epithelial systems such as the mammary gland. The mammary gland represents a unique organ that develops predominantly after birth where postnatal morphogenesis of the mammary gland drives dramatic tissue turnover and remodelling. Thus, differentiation and proliferation are constantly balanced to allow normal mammary gland development and homeostasis. How mammary epithelial cells regulate mitotic spindle orientation, hence OCDs, to accompany the rapid and constant tissue turnover is not well understood. This study aimed to identify novel factors that regulate the LGN-mediated spindle orientation machinery and determine how their dysregulation affects OCDs in mammary epithelial cells. By combining co-immunoprecipitation with mass spectrometry, the LGN interactome at the cell cortex of mitotic mammary epithelial cells was characterised and the membrane-associated protein Annexin A1 (ANXA1) was identified as a novel partner of LGN. Confocal and time-lapse microscopy demonstrated a critical role of ANXA1 in regulating the position and planar orientation of the mitotic spindle by instructing the accumulation and restriction of the LGN complex at the lateral cortex. Moreover, loss of ANXA1 leads to mitotic spindle misassembly and chromosome segregation defects, affecting the dynamics and progression of mitosis. Collectively the present study identified ANXA1 as a novel intrinsic cue of OCDs in mammary epithelial cells. Given increasing evidence of a link between OCD and tumorigenesis, this work is not only important for advancing our understanding of normal epithelial biology but also elucidating how imbalance of OCDs can contribute to the abnormal cell behaviour observed in cancer.

Effects of Formyl Peptide Receptor Agonists Ac9-12 and WKYMV in In Vivo and In Vitro Acute Inflammatory Experimental Models

Article

Full-text available

Jan 2022

Formyl peptide receptors (Fprs) are a G-protein-coupled receptor family mainly expressed on leukocytes. The activation of Fpr1 and Fpr2 triggers a cascade of signaling events, leading to leukocyte migration, cytokine release, and increased phagocytosis. In this study, we evaluate the effects of the Fpr1 and Fpr2 agonists Ac9-12 and WKYMV, respectively, in carrageenan-induced acute peritonitis and LPS-stimulated macrophages. Peritonitis was induced in male C57BL/6 mice through the intraperitoneal injection of 1 mL of 3% carrageenan solution or saline (control). Pre-treatments with Ac9-12 and WKYMV reduced leukocyte influx to the peritoneal cavity, particularly neutrophils and monocytes, and the release of IL-1β. The addition of the Fpr2 antagonist WRW4 reversed only the anti-inflammatory actions of WKYMV. In vitro, the administration of Boc2 and WRW4 reversed the effects of Ac9-12 and WKYMV, respectively, in the production of IL-6 by LPS-stimulated macrophages. These biological effects of peptides were differently regulated by ERK and p38 signaling pathways. Lipidomic analysis evidenced that Ac9-12 and WKYMV altered the intracellular lipid profile of LPS-stimulated macrophages, revealing an increased concentration of several glycerophospholipids, suggesting regulation of inflammatory pathways triggered by LPS. Overall, our data indicate the therapeutic potential of Ac9-12 and WKYMV via Fpr1 or Fpr2-activation in the inflammatory response and macrophage activation.

Knockdown of Annexin-A1 Inhibits Growth, Migration and Invasion of Glioma Cells by Suppressing the PI3K/Akt Signaling Pathway

Article

Full-text available

Mar 2021

ANXA1, which can bind phospholipid in a calcium dependent manner, is reported to play a pivotal role in tumor progression. However, the role and mechanism of ANXA1 involved in the occurrence and development of malignant glioma are still not well studied. Therefore, we explored the effects of ANXA1 on normal astrocytes and glioma cell proliferation, apoptosis, migration and invasion and the underlying mechanisms. We found that ANXA1 was markedly up-regulated in glioma cell lines and glioma tissues. Down-regulation of ANXA1 inhibited normal astrocytes and glioma cell proliferation and induced the cell apoptosis, which suggested that the consequences of loss of Annexin 1 are not specific to the tumor cells. Furthermore, the siRNA-ANXA1 treatment significantly reduced tumor growth rate and tumor weight. Moreover, decreasing ANXA1 expression caused G2/M phase arrest by repressing expression levels of cdc25C, cdc2 and cyclin B1. Interestingly, ANXA1 did not affect the expressions of β-catenin, GSK-3β and NF-κB, the key signaling molecules associated with cancer progression. However, siRNA-ANXA1 was found to negatively regulate phosphorylation of AKT and the expression and activity of MMP2/-9. Finally, the decrease of cell proliferation and invasiveness induced by ANXA1 down-regulation was partially reversed by combined treatment with AKT agonist insulin-like growth factor-1 (IGF-1). Meanwhile, the inhibition of glioma cell proliferation and invasiveness induced by ANXA1 down-regulation was further enhanced by combined treatment with AKT inhibitor LY294002. In summary, these findings demonstrate that ANXA1 regulates proliferation, migration and invasion of glioma cells via PI3K/AKT signaling pathway.

Annexin A1 (ANXA1): A Systematic Review of Its Role in Inflammation

Article

Jan 2021

Inflammation is a body response towards any injury or tissue damage. It involves the accumulation of neutrophils and release of inflammatory mediators. ANXA1, a 37 kDa glucocorticoid inducible protein plays an important role in resolving inflammation. The unique N-terminal and its mimetic peptide exert strong anti-inflammatory actions. This study was conducted to review the roles of ANXA1 in inflammation and identify any other reported roles. Electronic search was done whereby a total of 3797 articles were located from three databases, namely Ovid MEDLINE, Science Direct, and PubMed. Articles on ANXA1 and inflammation were selected based on inclusive criteria and review papers were excluded. Bias analysis was performed based on bias risk tool and 27 articles were included in the study. It was found that ANXA1 was able to resolve inflammation in many inflammatory diseases. Upon treatment with glucocorticoid, ANXA1 is induced and its significant expressions in tissues are important in resolving inflammation. However, this effect can be reversed by administering an anti-annexin antibody. This protein also acts on members of all formyl peptide receptors (FPR) and activates them to initiate reaction. It acts mainly by causing the death of neutrophils through apoptosis. In addition, ANXA1 is identified as a marker in cancer cells which determines the survival rates. In conclusion, ANXA1 is a key modulator in resolving inflammation in many diseases and is actively being induced upon glucocorticoid treatment.

Overexpression of LVRN impedes the invasion of trophoblasts by inhibiting epithelial–mesenchymal transition

Article

Full-text available

Dec 2020

Laeverin (LVRN) was first detected on the outer layer of the chorion laeve and migrating extravillous trophoblasts (EVTs). It is an enzyme that plays an important role in the placentation and pathophysiology of preeclampsia (PE). Previous studies have indicated that LVRN may be required for the invasion of human trophoblast cells. Paradoxically, LVRN was found to be highly expressed in the trophoblasts of PE patients with impaired invasive capacities. In this study, we detected the expression of LVRN in the placentas of PE patients (n=5) and normal term pregnancy women (n=5) as a control group by immunohistochemistry. LVRN was elevated in decidua (P=0.0083) and villi (P=0.0079) of PE patients. Next, LVRN was overexpressed via adeno-associated virus-mediated gene transfer in trophoblastic cell lines HTR8, Swan71, and JAR. Matrigel transwell assay and wound healing assay showed that overexpression of LVRN impeded the invasion of these three cell lines. Western blot analysis showed that LVRN overexpression caused downregulation of N-cadherin and vimentin and upregulation of E-cadherin, suggesting the inhibitory role of LVRN in epithelial–mesenchymal transition (EMT). Moreover, our data indicated that long noncoding RNA NONSTAT103348 (lnc10-7) was elevated in PE patients. Silencing lnc10-7 led to decreased LVRN expression. Taken together, although the basal level of LVRN may be crucial for cell invasion, overexpression of LVRN may abrogate the cell invasiveness, suggesting a multifaceted role of LVRN in the pathogenesis of PE.

Upregulation of annexin A1 protein expression in the intratumoral vasculature of human non–small-cell lung carcinoma and rodent tumor models

Article

Full-text available

Jun 2020
PLOS ONE

Annexin A1 (anxA1) is an immunomodulatory protein that has been proposed as a tumor vascular target for antitumor biologic agents, yet to date the vascular expression of anxA1 in specific tumor indications has not been systematically assessed. Attempts to evaluate vascular anxA1 expression by immunohistochemistry are complicated by a lack of available antibodies that are both specific for anxA1 and bind the N-terminal–truncated form of anxA1 that has previously been identified in tumor vasculature. To study the vascular expression pattern of anxA1 in non–small-cell lung carcinoma (NSCLC), we isolated an antibody capable of binding N-terminal–truncated anxA127-346 and employed it in immunohistochemical studies of human lung specimens. Lung tumor specimens evaluated with this antibody revealed vascular (endothelial) anxA1 expression in five of eight tumor samples studied, but no vascular anxA1 expression was observed in normal lung tissue. Tumor microarray analysis further demonstrated positive vascular staining for anxA1 in 30 of 80 NSCLC samples, and positive staining of neoplastic cells was observed in 54 of 80 samples. No correlation was observed between vascular and parenchymal anxA1 expression. Two rodent tumor models, B16-F10 and Py230, were determined to have upregulated anxA1 expression in the intratumoral vasculature. These data validate anxA1 as a potential vascular anti-tumor target in a subset of human lung tumors and identify rodent models which demonstrate anxA1 expression in tumor vasculature.

Phosphorylation of the multi functional signal transducer B-cell adaptor protein (BCAP) promotes recruitment of multiple SH2/SH3 proteins including GRB2

Article

Full-text available

Sep 2019

B-cell adaptor protein (BCAP) is a multimodular, multifunctional signal transducer that regulates signal transduction pathways in leukocytes, including macrophages, B cells, and T cells. In particular, BCAP suppresses inflammatory signaling by Toll-like receptors (TLRs). However, how BCAP itself is regulated and what its interaction partners are is unclear. Here, using human immune cell lines, including THP-1 cells, we characterized the complex phosphorylation patterns of BCAP and used a novel protein complex trapping strategy, called virotrap, to identify its interaction partners. This analysis identified known interactions of BCAP with phosphoinositide-3-kinase (PI3K) p85 subunit and NCK adaptor protein (NCK), together with previously unknown interactions of BCAP with SH2 and SH3 domain-containing adaptor proteins, notably growth factor receptor-bound protein 2 (GRB2) and CRK-like proto-oncogene, adaptor protein (CRKL). We show that the SH3 domain of GRB2 can bind to BCAP independently of BCAP phosphorylation status, suggesting that the SH2 domains mediate interactions with activated receptor tyrosine kinase complexes including the CD19 subunit of the B-cell receptor. Our results also suggested that the PI3K p85 subunit binds to BCAP via SH3 domains forming an inactive complex that is then activated by sequential binding with the SH2 domains. Taken together, our results indicate that BCAP is a complex hub that processes signals from multiple pathways in diverse cell types of the immune system.

Identification of gene networks in childhood epilepsy: evidence from focal cortical dysplasia

Conference Paper

Aug 2016

Fatma Scerif

Introduction: Focal cortical dysplasia (FCD) is a malformation of cortical development that is a frequent cause of multidrug resistant paediatric epilepsy. FCD type IIb (FCDIIb) is characterised by a population of unique abnormally enlarged cells known as balloon cells (BCs). The understanding of the molecular abnormalities underlying FCDIIb is poor. It is unclear if BCs are the key pathological cell or if there are other types of cells that are important in the pathogenesis of the disease. Interactions between such cell types are also unknown. Methods: Bioinformatic analyses of gene expression data were used to identify networks of genes that could characterise FCD and to identify putative diagnostic biomarkers. These markers were validated at the protein level by immunohistochemistry. Furthermore, various techniques allowed for the investigation of possible signalling between different cell types in FCDIIb. Results: Several of the gene networks and biomarkers were differentially expressed between FCDIIb and control. Analysis of one particular network showed that some components were expressed in BCs but others were expressed in a potentially novel cell population that was only present in FCDIIb. The importance of cellular heterogeneity of FCDIIb was further supported by the finding that BCs may have a unique senescence-associated secretome that could be involved in cell-to-cell communications. Conclusion: A genomic characterisation of FCD has provided a better understanding of the cellular abnormalities in FCDIIb from a molecular perspective. In addition to the identification of several differentially expressed gene networks, the results also provide an interesting insight into cell-to-cell communications between different cell types in FCDIIb, which could be important in driving the pathogenesis of the disease.

Advancements of Annexin A1 in inflammation and tumorigenesis

Article

Full-text available

Apr 2019

Annexin A1 is a Ca²⁺-dependent phospholipid binding protein involved in a variety of pathophysiological processes. Accumulated evidence has indicated that Annexin A1 has important functions in cell proliferation, apoptosis, differentiation, metastasis, and inflammatory response. Moreover, the abnormal expression of Annexin A1 is closely related to the occurrence and development of tumors. In this review article, we focus on the structure and function of Annexin A1 protein, especially the recent evidence of Annexin A1 in the pathophysiological role of inflammatory and cancer. This summary will be very important for further investigation of the pathophysiological role of Annexin A1 and for the development of novel therapeutics of inflammatory and cancer based on targeting Annexin A1 protein.

Annexin-A1 – A Blessing or a Curse in Cancer?

Article

Mar 2019
TRENDS MOL MED

Annexin-A1 (ANXA1), a potent endogenous immunomodulatory protein has been implicated in multiple functions essential in cancer, including cell proliferation, apoptosis, chemosensitivity, metastasis, and invasion. ANXA1 expression is varied depending on tumor type, and there are contradictory reports on its role in the regulation of proliferation and tumor growth. Here, we summarize the differing reports on cell proliferation and metastasis and attempt to discuss the reasons behind these different effects. ANXA1 plays a role as a homeostatic protein that regulates essential transcription factors and miRNAs. A more coherent understanding of ANXA1 in cancer could present a more biologically meaningful and clinically relevant strategy.

Annexins in Influenza Virus Replication and Pathogenesis

Article

Full-text available

Nov 2018

Influenza A viruses (IAVs) are important human respiratory pathogens which cause seasonal or periodic endemic infections. IAV can result in severe or fatal clinical complications including pneumonia and respiratory distress syndrome. Treatment of IAV infections is complicated because the virus can evade host immunity through antigenic drifts and antigenic shifts, to establish infections making new treatment options desirable. Annexins (ANXs) are a family of calcium and phospholipid binding proteins with immunomodulatory roles in viral infections, lung injury, and inflammation. A current understanding of the role of ANXs in modulating IAV infection and host responses will enable the future development of more effective antiviral therapies. This review presents a comprehensive understanding of the advances made in the field of ANXs, in particular, ANXA1 and IAV research and highlights the importance of ANXs as a suitable target for IAV therapy.

Annexin A1 in inflammation and breast cancer: a new axis in the tumor microenvironment

Article

Full-text available

Aug 2018

Targeting inflammation in cancer has shown promise to improve and complement current therapies. The tumor microenvironment plays an important role in cancer growth and metastasis and -tumor associated macrophages possess pro-tumoral and pro-metastatic properties. Annexin A1 (ANXA1) is an immune-modulating protein with diverse functions in the immune system and in cancer. In breast cancer, high ANXA1 expression leads to poor prognosis and increased metastasis. Here, we will review ANXA1 as a modulator of inflammation, and discuss its importance in breast cancer and highlight its new role in alternative macrophage activation in the tumor microenvironment. This review may provide an updated understanding into the various roles of ANXA1 which may enable future therapeutic developments for the treatment of breast cancer.

Connections of Annexin A1 and Translocator Protein-18 KDa on Toll Like Receptor Stimulated BV-2 Cells

Article

Full-text available

Apr 2018

Background: Annexin A1 (ANXA1) and Translocator Protein-18KDa (TSPO) down-regulate neuroinflammation. We investigated the role of recombinant ANXA1 (rANXA) on TSPO functions on Toll Like Receptor (TLR) activated microglia. Methods: BV-2 cells (murine microglia), were stimulated by E. coli Lipopolysaccharide (LPS) and treated with rANXA1 in order to measure TSPO expression and inflammatory parameters. Anti-sense ANXA1 and TLR4 and TSPO shRNA, as well as pharmacological treatments, were employed to assess the mechanisms involved. Results: LPS-stimulated BV-2 cells caused overexpression of TSPO, which was inhibited by: pharmacological blockade of TLR4 or TLR4 mRNA silencing; inhibition of myeloid differentiation primary response gene 88 (MyD88) dimerization; or blocking of nuclear factor κB (NF-κB) activation. rANXA1 treatment impaired LPS-induced TSPO upregulation by down-modulating MyD88 and NF-κB signaling; the effect was abolished by WRW4, an antagonist of formyl peptide receptor 2 (FPR2). rANXA1 treatment also downregulated interleukin 1β (IL-1β) and tumor necrosis factor-α (TNFα) secretion in LPS-stimulated BV-2 cells. TSPO knockdown in BV-2 cells augmented LPS-induced TNFα secretion and abolished the inhibitory effect of rANXA1 on TNFα secretion evoked by LPS. Conclusions: exogenous ANXA1 down-modulates LPS-induced TSPO via MyD-88/NF-κB pathways, and constitutive TSPO is pivotal for the control of ANXA1 on TNFα secretion. TSPO actions may be involved with the mechanisms of ANXA1 on inflammatory brain diseases.

Comparative Assay of 2D and 3D Cell Culture Models: Proliferation, Gene Expression and Anticancer Drug Response

Article

Full-text available

Apr 2018
CURR PHARM DESIGN

Background: In vitro tests allow establishing experimental variables. However, in vitro results cannot be extrapolated to in vivo tests. Considering that three-dimensional (3D) culture has been one of the best ways to portray the in vivo system of most cell types, it is possible to carry out assays with a great clinical relevance for the analysis of the screening, action and resistance of antitumor drugs. Objective: Thus, the objective of the present study was to compare between 2D and 3D cell culture forms to conclude which is the most suitable model for preclinical in vitro drug testing. Method: We evaluated the proliferation, genetic expression and chemoresistance of prostate tumor cell lines, PC- 3, LNCaP and DU145. Prostate tumor cell lines PC-3, LNCaP and DU145 were treated with the antineoplastic drugs paclitaxel and docetaxel and evaluated with cytotoxicity, cell proliferation and gene expression assays in 2D and magnetic 3D bioprinting cultures. Results: Lower cell proliferation rate, more resistance to paclitaxel and docetaxel and altered gene expression profile was shown in 3D cell culture comparing with its 2D counterpart. Conclusion: 3D cell culture exhibited a more similar behavior to in vivo systems, being a promising and more reliable tool for the development of new drugs.

Expression of genes and proteins of multidrug resistance in gastric cancer cells treated with resveratrol

Article

Full-text available

Feb 2018

Multidrug resistance (MDR) is a notable problem in the use of chemotherapy. Therefore, studies aimed at identifying substances capable of overcoming resistance of cancer cells are required. Examples of these compounds are polyphenols, including resveratrol, that exert a range of various biological activities. The aim of the present study was to demonstrate the effect of 3,5,4'-trihydroxy-trans-stilbene (resveratrol) on the expression of ATP binding cassette subfamily B member 1, Annexin A1 (ANXA1) and thioredoxin (TXN) genes, and the proteins encoded by these genes, which are associated with MDR. The experiments were performed in human gastric cancer cell lines EPG85-257RDB (RDB) and EPG85-257RNOV (RNOV), which are resistant to daunorubicin and mitoxantrone, respectively, in addition to EPG85-257P (control), which is sensitive to cytostatic drugs. Cells were treated with 30 or 50 µM resveratrol for 72 h and changes in the expression levels of the genes were analysed with the use of a reverse transcription-quantitative polymerase chain reaction. The cellular levels of P-glycoprotein (P-gp), ANXA1 and TXN were evaluated using immunofluorescence and western blot analysis. Resveratrol in both concentrations has been shown to have a statistically significant influence on expression of the mentioned genes, compared with untreated cells. In RDB cells, resveratrol reduced the expression level of all analyzed genes, compared with untreated cells. Similar results at the protein level were obtained for P-gp and TXN. In turn, in the RNOV cell line, resveratrol reduced TXN expression at mRNA and protein levels, compared with untreated cells. The results of the present study indicate that resveratrol may reduce the resistance of cancer cells by affecting the expression of a number of the genes and proteins associated with MDR.

Inhibiting proliferation and migration of lung cancer using small interfering RNA targeting on Aldo-keto reductase family 1 member B10

Article

Full-text available

Nov 2017

Lung cancer is the leading cause of global cancer‑associated mortality. Genomic alterations in lung cancers have not been widely characterized, however, the molecular mechanism of tumor initiation and progression remain unknown, and no molecularly targeted have been specifically developed for its treatment and diagnosis. The present study observed the upregulation of Aldo‑keto reductase family 1 member Bio10 (AKR1B10) lung cancer tissues by analyzing two public lung cancer gene expression datasets. Further experiments in silencing AKR1B10 demonstrated that the expression of AKR1B10 was associated with cell proliferation, cell cycle, adhesion and invasion, as well as extracellular‑signal‑regulated kinase/mitogen activated protein kinase signal pathway. The overexpression of AKR1B10 in lung cancer indicates the important role of AKR1B10 in tumorigenesis. These findings suggest that AKR1B10 could be a potential diagnosis and treatment mark of lung cancer.

PPARγ Ligand-induced Annexin A1 Expression Determines Chemotherapy Response via Deubiquitination of Death Domain Kinase RIP in Triple Negative Breast Cancers

Article

Aug 2017

Metastatic breast cancer is still remain incurable so far, new specifically targeted and more effective therapies for triple negative breast cancer (TNBC) are required in the clinic. In this study, our clinical data has established that basal and claudin-low subtypes of breast cancer (TNBC types) express significantly higher levels of Annexin A1 (ANXA1) with poor survival outcomes. Using human cancer cell lines which model the TNBC subtype, we observed a strong positive correlation between expression of ANXA1 and Peroxisome Proliferator-Activated Receptor gamma (PPARγ). A similar correlation between these two markers was also established in our clinical breast cancer patients' specimens. To establish a link between these two markers in TNBC, we show de novo expression of ANXA1 is induced by activation of PPARγ both in vitro and invivo and it has a predictive value in determining chemo-sensitivity to PPARγ ligands. Mechanistically, we show for the first time PPARγ-induced ANXA1 protein directly interacts with Receptor Interacting Protein-1 (RIP1), promoting its deubiquitination and thereby activating the caspase 8-dependent death pathway. We further identified this underlying mechanism also involved a PPARγ-induced ANXA1-dependent autoubiquitination of cIAP1, the direct E3 ligase of RIP1, shifting cIAP1 towards proteosomal degradation. Collectively, our study provides first insight for the suitability of using drug-induced expression of ANXA1 as a new player in RIP1-induced death machinery in TNBCs. Hitherto, presenting itself both as an inclusion criterion for patient selection and surrogate marker for drug response in future PPARγ chemotherapy trials.

Regulation of retinal pigment epithelial cell phenotype by Annexin A8

Article

Full-text available

Dec 2017

The retinoic acid derivative fenretinide (FR) is capable of transdifferentiating cultured retinal pigment epithelial (RPE) cells towards a neuronal-like phenotype, but the underlying mechanisms are not understood. To identify genes involved in this process we performed a microarray analysis of RPE cells pre- and post-FR treatment, and observed a marked down-regulation of AnnexinA8 (AnxA8) in transdifferentiated cells. To determine whether AnxA8 plays a role in maintaining RPE cell phenotype we directly manipulated AnxA8 expression in cultured and primary RPE cells using siRNA-mediated gene suppression, and over-expression of AnxA8-GFP in conjunction with exposure to FR. Treatment of RPE cells with AnxA8 siRNA recapitulated exposure to FR, with cell cycle arrest, neuronal transdifferentiation, and concomitant up-regulation of the neuronal markers calretinin and calbindin, as assessed by real-time PCR and immunofluorescence. In contrast, AnxA8 transient over-expression in ARPE-19 cells prevented FR-induced differentiation. Ectopic expression of AnxA8 in AnxA8-depleted cells led to decreased neuronal marker staining, and normal cell growth as judged by phosphohistone H3 staining, cell counting and cleaved caspase-3 levels. These data show that down-regulation of AnxA8 is both necessary and sufficient for neuronal transdifferentiation of RPE cells and reveal an essential role for AnxA8 as a key regulator of RPE phenotype.

Ac 2-26 peptide and serine protease of Bothrops atrox similarly induces angiogenesis without triggering local and systemic inflammation in a murine model of dorsal skinfold chamber

Article

Jul 2017

Identification and dynamic changes of RNAs isolated from RALY-containing ribonucleoprotein complexes

Article

Full-text available

Apr 2017
NUCLEIC ACIDS RES

RALY is a member of the heterogeneous nuclear ribonucleoprotein family (hnRNP), a large family of RNA-binding proteins involved in many aspects of RNA metabolism. Although RALY interactome has been recently characterized, a comprehensive global analysis of RALY-associated RNAs is lacking and the biological function of RALY remains elusive. Here, we performed RIP-seq analysis to identify RALY interacting RNAs and assessed the role of RALY in gene expression. We demonstrate that RALY binds specific coding and non-coding RNAs and associates with translating mRNAs of mammalian cells. Among the identified transcripts, we focused on ANXA1 and H1FX mRNAs, encoding for Annexin A1 and for the linker variant of the histone H1X, respectively. Both proteins are differentially expressed by proliferating cells and are considered as markers for tumorigenesis. We demonstrate that cells lacking RALY expression exhibit changes in the levels of H1FX and ANXA1 mRNAs and proteins in an opposite manner. We also provide evidence for a direct binding of RALY to the U-rich elements present within the 3΄UTR of both transcripts. Thus, our results identify RALY as a poly-U binding protein and as a regulator of H1FX and ANXA1 in mammalian cells.

Comparative proteome analysis of human esophageal cancer and adjacent normal tissues

Article

Full-text available

Mar 2017

Objective(s): Ranking as the sixth commonest cancer, esophageal squamous cell carcinoma (ESCC) represents one of the leading causes of cancer death worldwide. One of the main reasons for the low survival of patients with esophageal cancer is its late diagnosis. Materials and Methods: We used proteomics approach to analyze ESCC tissues with the aim of a better understanding of the malignant mechanism and searching candidate protein biomarkers for early diagnosis of esophageal cancer. The differential protein expression between cancerous and normal esophageal tissues was investigated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Then proteins were identified by matrix-assisted laser desorption/ ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and MASCOT web based search engine. Results: We reported 4 differentially expressed proteins involved in the pathological process of esophageal cancer, such as annexinA1 (ANXA1), peroxiredoxin-2 (PRDX2), transgelin (TAGLN) andactin-aortic smooth muscle (ACTA2). Conclusion: In this report we have introduced new potential biomarker (ACTA2). Moreover, our data confirmed some already known markers for EC in our region.

Effect of Annexin A1 gene on the proliferation and invasion of esophageal squamous cell carcinoma cells and its regulatory mechanisms

Article

Full-text available

Dec 2016

The aim of this study was to examine the effect of Annexin A1 (ANXA1) on the proliferation, migration and invasion of esophageal squamous cell carcinoma (ESCC) cells and its possible mechanisms of action. After constructing the ANXA1 overexpression plasmid, we transfected this plasmid and/or microRNA (miRNA)‑196a mimic into ESCC cells (Eca109 cell line). Methyl thiazolyl tetrazolium (MTT) assay and Transwell chamber assay were performed to determine cell proliferation, migration and invasion, respectively. Western blot analysis was used to examine the protein expression levels of ANXA1, Snail and E-cadherin. RT-PCR was used to detect the expression of miRNA-196a. Our results revealed that ANXA1 expression was upregulated in the cells transfected with the ANXA1 overexpression plasmid, and cell proliferation, migration and invasion were significantly increased (p=0.004, p<0.001 and p=0.011, respectively). In the cells transfected with the miRNA‑196a mimic, miRNA‑196a expression was significantly upregulated (p<0.001). However, miRNA-196a expression was downregulated in the cells transfected with the ANXA1 overexpression plasmid. In addition, in the cells transfected with the miRNA‑196a mimic, cell proliferation, migration and invasion were significantly decreased (p=0.027, p=0.009 and p=0.021, respectively). In the cells transfected with the ANXA1 overexpression plasmid, the expression of Snail was upregulated and that of E-cadherin was downregulated. However, the opposite was observed in the cells transfected with the miRNA‑196a mimic. Our findings thus demonstrate that ANXA1 promotes the proliferation of Eca109 cells, and increases the expression of Snail, whereas it inhibits that of E-cadherin, thus enhancing the migration and invasion of ESCC cells. miRNA-196a negatively regulates the expression of ANXA1, thereby inhibiting the proliferation, invasion and metastasis of ESCC cells.

Annexin A1 contributes to pancreatic cancer cell phenotype, behaviour and metastatic potential independently of Formyl Peptide Receptor pathway

Article

Full-text available

Jul 2016

Annexin A1 (ANXA1) is a Ca2+-binding protein over-expressed in pancreatic cancer (PC). We recently reported that extracellular ANXA1 mediates PC cell motility acting on Formyl Peptide Receptors (FPRs). Here, we describe other mechanisms by which intracellular ANXA1 could mediate PC progression. We obtained ANXA1 Knock-Out (KO) MIA PaCa-2 cells using the CRISPR/Cas9 genome editing technology. LC-MS/MS analysis showed altered expression of several proteins involved in cytoskeletal organization. As a result, ANXA1 KO MIA PaCa-2 partially lost their migratory and invasive capabilities with a mechanism that appeared independent of FPRs. The acquisition of a less aggressive phenotype has been further investigated in vivo. Wild type (WT), PGS (scrambled) and ANXA1 KO MIA PaCa-2 cells were engrafted orthotopically in SCID mice. No differences were found about PC primary mass, conversely liver metastatization appeared particularly reduced in ANXA1 KO MIA PaCa-2 engrafted mice. In summary, we show that intracellular ANXA1 is able to preserve the cytoskeleton integrity and to maintain a malignant phenotype in vitro. The protein has a relevant role in the metastatization process in vivo, as such it appears attractive and suitable as prognostic and therapeutic marker in PC progression.

Additional file 1

Data

Full-text available

Oct 2009

Results for an example analysis with ArrayMining.net. The document provided contains the results and a discussion for an example analysis of microarray data with ArrayMining.net.

Study on reproductive endocrine disturbance and DNA damage mechanism of female Ruditapes philippinarum under Benzo[a]pyrene stress

Article

Oct 2023

Unraveling Potential Candidate Targets Associated with Expression of p16INK4a or p16 Truncated Fragment by Comparative Proteomics Analysis

Article

Jul 2021

Background P16 is a tumor suppressor protein that is significantly involved in cycle regulation through the reduction of cell progression from G1 phase to S phase via CDK-cyclin D/p16 INK4a /pRb/E2F cascade. The minimum functional domain of p16 has been uncovered that may function comparable to wild type p16. Objective To expand the knowledge on molecules and mechanisms by which p16 or p16 ⁶⁶⁻¹⁵⁶ fragment suppresses human fibrosarcoma cell line growth, differential proteome profiles of fibrosarcoma cells following p16 full length or the functional domain overexpression were analyzed. Methods Following transfecting HT-1080 fibrosarcoma cells with p16 full length, p16 ⁶⁶⁻¹⁵⁶ truncated form, and pcDNA3.1 empty vector, protein extract of each sample was harvested and clarified by centrifugation, and then the protein content was determined via Bradford assay. All protein extract of each sample was analyzed by two-dimensional gel electrophoresis. Immunoblot analysis was performed as further validation of the expression status of identified proteins. Results Expression of p16 or p16 ⁶⁶⁻¹⁵⁶ fragment could induce mostly common alterations (up/down-regulation) of proteome profile of HT-1080 cells. Mass spectrometry identification of the differentially expressed protein spots revealed several proteins that were grouped in functional clusters, including cell cycle regulation and proliferation, cell migration and structure, oxidative stress, protein metabolism, epigenetic regulation, and signal transduction. Conclusion The minimum functional domain of p16 could act in the same way as p16 full length. Also, these new ﬁndings can significantly enrich the understanding of p16 growth-suppressive function at the molecular level by the introduction of potential candidate targets for new treatment strategies. Furthermore, the present study provides strong evidence on the functional efficacy of the identified fragment of p16 for further attempts toward peptidomimetic drug design or gene transfer to block cancer cell proliferation.

Decreased Annexin A1 expression enhances sensitivity to docetaxel, cisplatin and 5‐fluorouracil combination induction chemotherapy in oral squamous cell carcinoma

Article

Full-text available

Jun 2021

Background Annexin A1, a member of the Annexin superfamily, has been shown to play a vital role in a broad range of molecular and cellular processes. This study aims to explore the relationship between the Annexin A1 expression and the clinical response to cisplatin, docetaxel, and 5-fluorouracil (TPF) as induction chemotherapy in patients with oral squamous cell carcinoma (OSCC). Methods This study recruited two hundred thirty-two patients from a III/IVA OSCC trial. Immunohistochemistry was used to assess the level of Annexin A1 expression. Overexpression and knockdown methods in HB96, HN4 and CAL27 cell lines were used to assess the role of Annexin A1 in the neoplastic cellular response to chemotherapy. Results We found that reduced expression of Annexin A1 conferred a prognostic benefit from induction chemotherapy based on the TPF drug combination in patients with moderately/poorly differentiated disease. Using an in vitro model, we found that low Annexin A1 enhanced cellular proliferation by activating the EGFR/AKT signalling pathway and inhibiting p27 expression. Furthermore, low Annexin A1 initiated a significant decrease in cell viability after treatment with TPF agents. In addition, downregulation of Annexin A1 promoted apoptosis induced by docetaxel, cisplatin and 5-fluorouracil, and upregulation of Annexin A1 inhibited apoptosis. Conclusion Annexin A1 may be of prognostic value in patients with locally advanced OSCC who are managed with TPF chemotherapy, as low Annexin A1 promotes chemosensitivity to TPF chemotherapy in oral cancer cells via enhanced caspase-dependent apoptosis.

Effect of Cryoablation Treatment on the Protein Expression Profile of Low-Grade Central Chondrosarcoma Identified by LC-ESI-MS/MS

Article

Full-text available

May 2021

The use of cryoablation/cryosurgery in treating solid tumors has been proven as a unique technique that uses lethal temperatures to destroy the tumors and impart better functions for the affected organs. This novel technique recently demonstrated the best clinical results in chondrosarcoma (CSA) with faster recovery, less recurrence, and metastasis. Due to the resistant nature of CSA to chemo and radiation therapy, cryoablation comes to light as the best alternative approach. Therefore, for the first time, we aimed to compare CSA-untreated with cryoablation treated samples to discover some potential markers that may provide various clues in terms of diagnosis and pathophysiology and may facilitate the development of novel methods to treat sarcoma efficiently. To find the altered proteins among both groups, a mass-based label-free approach was employed and identified a total of 160 significantly altered proteins. Among these, 138 proteins were dysregulated with <1- to -0.1-fold, 18 proteins were up-regulated with >3 folds, and four proteins were similarly expressed in the untreated group compared to the treated. Interestingly, the differential expressions of proteins from the untreated group showed contrast expressions in the treated group. Furthermore, the functional enrichment analysis revealed that most of the identified proteins from this study were associated with various significant pathways such as glycolysis, MAPK activation, PI3K-Akt signaling, extracellular matrix degradation, etc. In addition, two protein expressions, such as fibronectin and annexin-1, were validated by immunoblot analysis. Therefore, this study signifies the most comprehensive discovery of altered protein expressions to date and the first large-scale detection of protein profiles from CSA-cryoablation treated compared to untreated. This work may serve as the basis for future research to open novel treatment options for chondrosarcoma.

Identification of ANXA1 as a potential prognostic biomarker and correlating with immune infiltrates in colorectal cancer

Article

Feb 2021

Background ANXA1 is a calcium-dependent phospholipid-binding protein and is frequently associated with inflammation, cell proliferation and apoptosis. However, the relationship between ANXA1 and the prognosis of multiple tumours and tumour infiltrating immune cells remains unclear. Methods Multivariate Cox proportional regression analysis was used for signature genes exploration in the basic of colon adenocarcinoma (COAD) RNA-sequence dataset obtained from TCGA, following the identification of 267 common differentially expressed genes, including ANXA1, among three expression profile datasets (GSE41328, GSE110224, and GSE113513). The differential expression of ANXA1 in different tumours and their corresponding normal tissues were evaluated through the Tumour Immune Estimation Resource (TIMER) and Oncomine database. Subsequently, we investigated the correlation between the expression level of ANXA1 and diverse panel of infiltrating immune cells and their related gene markers in colorectal cancer using correlation analysis in TIMER and GEPIA database. Results The high expression of ANXA1 was demonstrated to be closely correlated with poor survival in patients with colorectal cancer. More importantly, we found that changes in ANXA1 expression showed a moderate to strong, and statistically significant, correlation with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells. By contrast, there are only weak correlations between ANXA1 expression and immune cell infiltration in ESCA and STAD. ANXA1 expression was considerably associated with various immune markers involving immune cell recruitment, polarization of tumour-associated macrophages, and T cell exhaustion. Conclusion ANXA1 is not only an independent risk factor in the prediction of the prognosis of colorectal cancer, but also a crucial regulator in immune cell infiltration. This study may shed light on the clinical value of ANXA1, especially in the areas of early diagnosis of colorectal cancer and therapeutic target discovery.

Biological and physical approaches on the role of piplartine (piperlongumine) in cancer

Article

Full-text available

Dec 2020

Chronic inflammation provides a favorable microenvironment for tumorigenesis, which opens opportunities for targeting cancer development and progression. Piplartine (PL) is a biologically active alkaloid from long peppers that exhibits anti-inflammatory and antitumor activity. In the present study, we investigated the physical and chemical interactions of PL with anti-inflammatory compounds and their effects on cell proliferation and migration and on the gene expression of inflammatory mediators. Molecular docking data and physicochemical analysis suggested that PL shows potential interactions with a peptide of annexin A1 (ANXA1), an endogenous anti-inflammatory mediator with therapeutic potential in cancer. Treatment of neoplastic cells with PL alone or with annexin A1 mimic peptide reduced cell proliferation and viability and modulated the expression of MCP-1 chemokine, IL-8 cytokine and genes involved in inflammatory processes. The results also suggested an inhibitory effect of PL on tubulin expression. In addition, PL apparently had no influence on cell migration and invasion at the concentration tested. Considering the role of inflammation in the context of promoting tumor initiation, the present study shows the potential of piplartine as a therapeutic immunomodulator for cancer prevention and progression.

Annexin A1: A Double-edged Sword as Novel Cancer Biomarker

Article

Jan 2020
CLIN CHIM ACTA

The occurrence, development, infiltration and metastasis of tumors are very complex processes involving the participation of many factors, some of which play a key role. Annexin A1 (ANXA1) is known as an anti-inflammatory protein. However, it has now been recognized to have a broader role beyond the inflammation, including roles in cell proliferation, differentiation, apoptosis, invasion, angiogenesis and metastasis. This review is intended to outline the research surrounding the pathophysiological effects of ANXA1 in tumors and its potential as a therapeutic and diagnostic agent. These studies comprehensively explore the expression changes of ANXA1 in cancer and further explore its mechanism of action in tumors, which is of great clinical significance for the early diagnosis, treatment and prognostic evaluation of tumors.

Proteomic characterization of mesenchymal stem cell-like populations derived from various tissue types

Chapter

Jan 2012

The evolution of proteomics has led to its application in identifying biomarkers of biological processes and pathways including signal transduction and cell development. Proteomic technologies are increasingly utilized to define the molecular mechanisms controlling mesenchymal stem/stromal cell (MSC) self-renewal, multipotency and fate. Bone marrow-derived MSCs are highly promising candidates in the field of regenerative medicine based on their high proliferative capacity, multilineage differentiation potential and immunomodulatory properties. Recently, equivalent MSC-like populations have also been isolated from adipose, dental and feto-maternal tissues. This chapter discusses the current technologies available for proteomic analysis and the studies performed on tissue-specific MSC-like populations to date.

Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b

Article

Full-text available

Dec 2017

Annexin A1 (ANXA1) down-regulation is an early and frequent event in the development of head and neck squamous cell carcinomas (HNSCC). In an attempt to identify the underlying mechanisms of reduced ANXA1 protein expression, this study investigated ANXA1 mRNA expression in HNSCC specimens by both in situ hybridization and RT-qPCR. Results showed a perfect concordance between the pattern of ANXA1 mRNA and protein detected by immunofluorescence in tumors, precancerous lesions and normal epithelia, reflecting that ANXA1 down-regulation occurs at transcriptional level. We also found that both miR-196a and miR-196b levels inversely correlated with ANXA1 mRNA levels in paired HNSCC tissue samples and patient-matched normal mucosa. In addition, endogenous levels of ANXA1 mRNA and protein were consistently and significantly down-regulated upon miR-196a and miR-196b over-expression in various HNSCC-derived cell lines. The direct interaction of both mature miR-196a and miR-196b was further confirmed by transfection with Anxa1 3′UTR constructs. Combined bioinformatics and functional analysis of ANXA1 promoter activity contributed to identify key regions and potential mediators of ANXA1 transcriptional control. This study unveils that, in addition to miR-196a, miR-196b also directly targets ANXA1 in HNSCC.

Mechanism and Functions of Protein Decarbonylation

Chapter

May 2017

Yuichiro J. Suzuki

Protein oxidation seems to play important roles in both oxidative stress and redox signaling. The authors use the term “primary protein carbonylation” when the direct metal-catalyzed oxidation of amino acid side chains is involved, and this plays a major role in oxidative stress. To understand the mechanism and nature of protein decarbonylation, they searched for decarbonylated proteins. By using the protein decarbonylation of peroxiredoxin-2 and peroxiredoxin-6 as an endpoint, they tested the role of thioredoxin and glutaredoxin-1 in catalyzing the thiol-dependent reduction of protein carbonyls. We propose two possible mechanisms through which protein decarbonylation may function: (i) fine-tuning the cell signaling mechanism that is analogous to the role of peroxiredoxin-1 phosphorylation and the hyperoxidation of other peroxiredoxins as proposed by Soo Goo Rhee and coworkers [30] and (ii) promoting site-directed mutagenesis-like reactions that may convert one amino acid into another.

Hypoxia regulates ANXA1 expression to support prostate cancer cell invasion and aggressiveness

Article

Full-text available

Nov 2016

Annexin A1 (ANXA1) is a Ca²⁺-binding protein over-expressed in the invasive stages of prostate cancer (PCa) development; however, its role in this tumor metastatization is largely unknown. Moreover, hypoxic conditions in solid tumors have been related to poor prognosis in PCa patients. We have previously demonstrated that ANXA1 is implicated in the acquisition of chemo-resistant features in DU145 PCa cells conferring them a mesenchymal/metastatic phenotype. In this study, we have investigated the mechanisms by which ANXA1 regulates metastatic behavior in LNCaP, DU145 and PC3 cells exposed to hypoxia. ANXA1 was differentially expressed by PCa cell lines in normoxia whereas hypoxic stimuli resulted in a significant increase of protein expression. Additionally, in low oxygen conditions ANXA1 was extensively secreted out-side the cells where its binding to formyl peptide receptors (FPRs) induced cell invasion. Loss and gain of function experiments performed by using the RNA interfering siANXA1 and an ANXA1 over-expressing plasmid (MF-ANXA1), also confirmed the leading role of the protein in modulating LNCaP, DU145 and PC3 cell invasiveness. Finally, ANXA1 played a crucial role in the regulation of cytoskeletal dynamics underlying metastatization process, such as the loss of adhesion molecules and the occurrence of the epithelial to mesenchymal transition (EMT). ANXA1 expression increased inversely to epithelial markers such as E-cadherin and cytokeratins 8 and 18 (CKs) and proportionally to mesenchymal ones such as vimentin, ezrin and moesin. Our results indicated that ANXA1 may be a key mediator of hypoxia-related metastasis-associated processes in PCa.

Annexin A2, up-regulated by IL-6, promotes the ossification of ligament fibroblasts from ankylosing spondylitis patients

Article

Dec 2016

Background: Annexin A2, a calcium-dependent phospholipid binding protein, is involved in osteogenesis. The objective of the present study was to explore the expression of Annexin A2 in spinal ligament tissues (LT) of ankylosing spondylitis (AS) patients and determine its pathological functions. Methods: mRNA and protein expression of Annexin A2 was detected by real-time PCR and Western blotting, respectively. Interleukin-6 (IL-6) concentration in serum was assessed by enzyme linked immunosorbent assay. Alkaline phosphatase (ALP) activity was measured with ALP activity kit on a microplate reader. Results: mRNA and protein expression of Annexin A2 in LT, and IL-6 concentration in serum were significantly increased in AS patients. Moreover, exogenous IL-6 treatment significantly up-regulated Annexin A2 expression and ALP activity. Silencing of Annexin A2 expression significantly ameliorated IL-6-induced ossification of fibroblasts from AS patients, as indicated by ALP activity, expression of proteins associated with osteogenic differentiation, including bone morphogenetic protein-2, osteocalcin and osterix, and the ratio of osteoprotegerin to receptor activator of NF-κB ligand. Further MEK inhibitor experiments suggested that Annexin A2 may exert its function through extracellular signal-related kinase pathway. Conclusions: Annexin A2, up-regulated by IL-6, may promote ligament ossification of AS patients.

Proteomic analysis of apoptosis induction by lariciresinol in human HepG2 cells

Article

Jul 2016

Lariciresinol (LA) is a traditional Chinese medicine possessing anticancer activity, but its mechanism of action remains unclear. The present study explored the effects of LA on human HepG2 cells and the underlying mechanism. Our data indicated that LA inhibited cell proliferation and induced cell cycle arrest in S phase, subsequently resulting in apoptosis in HepG2 cells. Using a proteomics approach, eight differentially expressed proteins were identified. Among them, three proteins, glyceraldehyde-3-phosphate, UDP-glucose 4-epimerase, and annexin A1, were upregulated, while the other five proteins, heat shock protein 27, haptoglobin, tropomodulin-2, tubulin alpha-1A chain, and brain acid soluble protein 1, were downregulated; all of these proteins are involved in cell proliferation, metabolism, cytoskeletal organization, and movement. Network analysis of these proteins suggested that the ubiquitin-conjugating enzyme (UBC) plays an important role in the mechanism of LA. Western blotting confirmed downregulation of heat shock protein 27 and upregulation of ubiquitin and UBC expression levels in LA-treated cells, consistent with the results of two-dimensional electrophoresis and a STRING software-based analysis. Overall, LA is a multi-target compound with anti-cancer effects potentially related to the ubiquitin-proteasome pathway. This study will increase our understanding of the anticancer mechanisms of LA.

Proteomic Characterization of Mesenchymal Stem Cell-Like Populations Derived from Various Tissue Types

Chapter

Dec 2012

The evolution of proteomics has led to its application in identifying biomarkers of biological processes and pathways including signal transduction and cell development. Proteomic technologies are increasingly utilized to define the molecular mechanisms controlling mesenchymal stem/stromal cell (MSC) self-renewal, multipotency and fate. Bone marrow-derived MSCs are highly promising candidates in regenerative medicine based on their high proliferative capacity, multi-lineage differentiation potential and immunomodulatory properties. Recently, equivalent MSC-like populations have also been isolated from adipose, dental and various feto-maternal tissues. This chapter discusses the current technologies available for proteomic analysis and the studies performed on tissue-specific MSC-like populations to date.

ATP and GTP Hydrolytic Function of N-terminally Deleted Annexin I

Article

Full-text available

Jul 2000

Annexin I is a 37 kDa member of the annexin family of calcium-dependent phospholipid binding proteins. Annexin I plays regulatory roles in various cellular processes including cell proliferation and differentiation. Recently we found that annexin I is a heat shock protein (HSP) and displays a chaperone-like function. In this paper we investigated the function of annexin I as an ATPase using 1 to 32 amino acids deleted annexin I ( I). I hydrolyzed ATP as determined by thin layer chromatography. The ability of ATP hydrolysis was inhibited by ADP, GTP and GDP, but not by the AMP, GMP and cAMP. In view of the ATP hydrolyzing function of HSP, the results support the function of annexin I as a HSP.

Increased apoptosis in U937 cells over-expressing lipocortin I (annexin I)

Article

Full-text available

Apr 2000
LIFE SCI

The potential involvement of endogenous lipocortin 1 in the process of cellular apoptosis, particularly in cells of the myelo-monocytic lineage, has been investigated. U937 cells were transfected either with an antisense or a sense DNA for lipocortin 1 and the stable clones 36.4AS clone (20–40% lower lipocortin 1 levels) and 15S (30% higher lipocortin 1 levels) were obtained. Cell apoptosis was induced by incubation with tumor necrosis factor-α: optimal responses were observed within a 24 h incubation period at a 5 concentration. Apoptosis was assessed both morphologically, by annexin V binding and cell cycle analysis with propidium iodide. Whilst no consistent difference was seen between wild type cells and clone 36.4AS, a higher incidence of apoptosis (ranging from +30% to + 60%) was observed in the 15S clone. Release of arachidonic acid from loaded cells was promoted by 24 h incubation with the cytokine, and a higher degree of release was measured in the 15S clone. These data indicate that endogenous intracellular lipocortin 1 is involved in the promotion of apoptosis in cells of the myelo-monocytic derivation.

The annexin protein lipocortin 1 regulates the MAPK/ERK pathway

Article

Full-text available

Dec 1999

Lipocortin 1 (annexin 1) is a calcium- and phospholipid-binding protein that modulates anti-inflammatory responses including those induced by lipopolysaccharide. To investigate the precise role of lipocortin 1 in regulating the lipopolysaccharide-induced signal transduction pathways, we generated stable RAW 264.7 macrophage cell lines expressing decreased and increased lipocortin 1 protein. Several RAW 264.7 clones with increased lipocortin 1 protein levels showed constitutive activation of the mitogen-activated protein kinase extracellular signal-regulated kinase, which was down-regulated following lipopolysaccharide treatment. Conversely, clones with decreased lipocortin 1 protein expression showed prolonged extracellular signal-regulated kinase activity, following lipopolysaccharide activation. Lipocortin 1 specifically regulates the components of the extracellular signal-regulated kinase pathway, since changes in lipocortin 1 protein expression had no affect on the related mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Lipocortin 1 modulated upstream components of the extracellular signal-regulated kinase pathway and associated with the adaptor protein growth factor binding protein. The downstream consequences of altered extracellular signal-regulated kinase activity were independent of the proinflammatory transcription factor nuclear factor kappa B. These data indicate that lipocortin 1 specifically regulates proximal signaling components of the extracellular signal-regulated kinase signal transduction pathway, resulting in the modulation of biochemical functions in RAW macrophages.

Annexin-1 Localization in Human Skin: Possible Association with Cytoskeletal Elements in Keratinocytes of the Stratum Spinosum

Article

Full-text available

Dec 1993

Annexin-1 (also called lipocortin-1 or p35), a putative substrate of the epidermal growth factor/receptor kinase, protein kinase C, and transglutaminase, was immunolocalized in embryonic, neonatal, adult, and diseased human epidermis. In embryonic skin intense annexin-1 immunoreactivity was found in the periderm at 54 d estimated gestational age (EGA). Later (EGA = 91-143 d), annexin-1 immunoreactivity was restricted to basal keratinocytes. In neonatal skin, basal cells were often more heavily stained than were suprabasal keratinocytes, which were also stained. Only basal keratinocytes stained in adult plantar skin, but in thin skin annexin-1 was present in the basal, suprabasal, and sometimes even in the granular layers of the epidermis. Often, annexin-1 appeared concentrated around the perimeter of cells, especially tonofilament/desmosome-rich keratinocytes of the spinous-cell layer. At high magnification, annexin-1 appeared associated with distinct structures and was very granular in appearance in the intensely stained ductal keratinocytes of eccrine sweat glands, cells that are very highly enriched in keratin tonofilaments. This striking distribution in certain keratinocytes enriched in tonofilaments suggests a role for annexin-1 in cytoskeletal functions.

The cyclin D1 gene is a target of the -catenin/LEF-1 pathway

Article

Full-text available

May 1999

β-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell–cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated β-catenin levels in colorectal cancer caused by mutations in β-catenin or by the adenomatous polyposis coli molecule, which regulates β-catenin degradation, result in the binding of β-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the β-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of β-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by β-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased β-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle.

Mitogen-activated protein kinase activation resulting from selective oncogene expression in NIH 3T3 and Rat 1a cells

Article

Full-text available

Sep 1992

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that are rapidly activated in response to a variety of growth factors in many cell types. MAPKs are activated by phosphorylation of both tyrosine and threonine residues. They are proposed to be key integrators of growth factor receptor transduction systems involving conversion of tyrosine kinase signals to serine/threonine kinase activation. We have studied the influence of specific oncogenes on the regulation of MAPK activity in NIH 3T3 and Rat 1a fibroblasts. In NIH 3T3 cells, ras or raf oncogene expression, but not gip2 oncogene expression, induces a significant constitutive MAPK activation. In contrast, in Rat 1a cells, gip2, but not ras or raf oncogene expression, induces a strong constitutive MAPK activation. The findings indicate that, in a cell type-selective manner, different oncoproteins are capable of causing the constitutive activation of MAPK. However, the magnitude of oncogene-induced MAPK activation is not directly correlated with cellular transformation in either cell type. It appears that expression of only a subset of transforming oncogenes in a specific cell type is able to alter the regulation of the MAPK activation pathway. Thus, the network of cytoplasmic serine/threonine kinases will be differentially regulated when the same oncogene is expressed in different cell types.

Abrogation of glucocorticoid-mediated inhibition of T cell proliferation by the synergistic action of IL-1, IL-6, and IFN-gamma

Article

Full-text available

Jun 1991

Glucocorticoids (GCS) inhibit the transcription of multiple activation-associated cytokine genes. By Northern blot analysis now we demonstrate that antiproliferative concentrations of dexamethasone and 6 alpha-methylprednisolone block mitogen-induced IL-2 gene expression in human peripheral blood mononuclear leukocytes in a concentration-dependent fashion. In addition, using a mitogen-induced proliferation assay of human peripheral blood mononuclear leukocytes, we show that GCS-mediated anti-proliferative effects are not blocked by rIL-1, rIL-2, rIL-3, rIL-4, rIL-5, rIL-6, rTNF-alpha, rTNF-beta, and rIFN-gamma, individually at 1 to 1000 U/ml, but are totally abrogated, in a concentration-dependent fashion, by the combination of rIL-1, rIL-6, and rIFN-gamma (25 to 50 U/ml for each cytokine). Thus, blockade of cytokine expression is the primary mechanism by which GCS inhibit mitogen-driven and alloantigen-induced T cell proliferation. The immunosuppressive effects of GCS are almost certainly exacted at the level of cytokine gene transcription.

Expression of Annexins as a function of cellular growth rate

Article

Full-text available

Aug 1990

Annexins are a structurally related family of Ca2+ binding proteins of undertermined biological function. Annexin I (also called lipocortin 1) is a substrate for the EGF-stimulated tyrosine kinase and is postulated to be involved in mitogenic signal transduction. To investigate further the involvement of lipocortin 1 in cell proliferation, we measured lipocortin 1 levels in normal diploid human foreskin fibroblasts (HFF) to determine whether its expression changed as a function of growth status. For comparison, the expression of annexin V (also called endonexin II) was measured in HFF cells. Endonexin II is a protein with similar Ca2+ and phospholipid binding properties as lipocortin 1, but it is not a substrate for tyrosine kinases. Quiescent HFF cell cultures were induced to proliferate by either subculture to lower cell density, EGF stimulation, or serum stimulation. In all three protocols, proliferating HFF cells contained three- to fourfold higher levels of lipocortin 1 and three- to fourfold lower levels of endonexin II than quiescent HFF cells. In contrast, the expression of annexin II (also called calpactin I) and annexin IV (also called endonexin I) remained relatively unchanged in growing and quiescent HFF cells. Lipocortin 1 synthesis rate was eightfold higher and its turnover rate was 1.5-fold slower in proliferating compared to quiescent HFF cells. Endonexin II synthesis rate remained constant but its turnover rate was 2.2-fold faster in proliferating compared to quiescent HFF cells. In a separate set of experiments, annexin expression levels were measured in cultures of rat PC-12 cells, a pheochromocytoma that ceases proliferation and undergoes reversible differentiation into nondividing neuronlike cells in response to nerve growth factor (NGF). After NGF treatment, PC-12 cells expressed fivefold higher levels of endonexin II and 32-fold higher levels of calpactin 1. Lipocortin 1 and endonexin I were not expressed in PC-12 cells. In summary, lipocortin 1 expression exhibited a positive correlation with cell proliferation in HFF cells. The increased expression of endonexin II in quiescent HFF cells and differentiating PC-12 cells implies that this protein may play a more prominent role in nondividing cells.

Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-Dalton substrate in intact A-431 cells

Article

Full-text available

Aug 1985

We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, is an excellent in vitro substrate for the epidermal growth factor (EGF) receptor/kinase present in membrane preparations (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). In this communication we demonstrate that the phosphorylation of the 35-kDa protein is markedly enhanced in intact, 32P-labeled, A-431 cells following exposure of the cells to EGF. The 35-kDa protein immunoprecipitated from cells treated with EGF is phosphorylated to a 20-120-fold greater extent than comparable preparations from control cells. Both phosphotyrosine and phosphoserine residues are detected in the protein after treatment of the cells with EGF. EGF-dependent phosphorylation of the 35-kDa protein is barely detected unless the intact cells are exposed to EGF for periods greater than 5 min. We suggest that endosomes containing internalized EGF X receptor/kinase complexes are primarily responsible for the observed phosphorylation of the 35-kDa protein in intact cells.

Lipocortins 1 and 2 as substrates for the insulin receptor kinase in rat liver

Article

Full-text available

Sep 1988

Lipocortins 1 and 2 are major substrates for the epidermal growth factor receptor and the pp60v-src tyrosine kinases in transformed cells. In the present study, we have characterized the phosphorylation of lipocortins 1 and 2 by the insulin receptor tyrosine kinase in vitro and in vivo. In vitro, the solubilized insulin receptor, partially purified from rat liver, catalyzed phosphorylation of human recombinant lipocortin 1 and purified bovine lipocortin 2. Phosphorylation of lipocortin 1 was increased 15-fold upon stimulation with 10(-7) M insulin. The apparent Km of the reaction was 3.3 microM and was not affected by insulin stimulation. Insulin stimulated phosphate incorporation into lipocortin 2 by 20-fold (apparent Km greater than 20 microM). Both lipocortins were phosphorylated exclusively on tyrosine residues as judged by phosphoamino acid analysis. Based upon peptide mapping, lipocortin 1 was phosphorylated on Tyr-21, a site phosphorylated by other tyrosine kinases. Polyclonal anti-phosphotyrosine antibodies recognized the tyrosine-phosphorylated lipocortin 2, but not lipocortin 1 in its phosphorylated form. In hepatocytes from normal and dexamethasone-treated rats, lipocortin 1 content was less than 50 ng/10(6) cells. Insulin-induced phosphorylation of lipocortin 1 was detected in intact hepatocytes from corticosteroid-treated animals but not in cells from normal rats. No phosphorylation of lipocortin 2 was found, although its content was approximately 100 ng/10(6) cells from normal animals and increased to approximately 1 microgram/10(6) cells following treatment of rats with dexamethasone for 4 days. Thus, although lipocortins 1 and 2 are in vitro substrates of the insulin receptor kinase, only lipocortin 1 is phosphorylated in an insulin-dependent manner in intact hepatocytes, and this is only observed after dexamethasone treatment of the rats.

Mapping Replicational Sites in the Eucaryotic cell Nucleus

Article

Full-text available

Feb 1989

We have used fluorescent microscopy to map DNA replication sites in the interphase cell nucleus after incorporation of biotinylated dUTP into permeabilized PtK-1 kangaroo kidney or 3T3 mouse fibroblast cells. Discrete replication granules were found distributed throughout the nuclear interior and along the periphery. Three distinct patterns of replication sites in relationship to chromatin domains in the cell nucleus and the period of S phase were detected and termed type I (early to mid S), type II (mid to late S) and type III (late S). Similar patterns were seen with in vivo replicated DNA using antibodies to 5-bromodeoxyuridine. Extraction of the permeabilized cells with DNase I and 0.2 M ammonium sulfate revealed a striking maintenance of these replication granules and their distinct intranuclear arrangements with the remaining nuclear matrix structures despite the removal of greater than 90% of the total nuclear DNA. The in situ prepared nuclear matrix structures also incorporated biotinylated dUTP into replication granules that were indistinguishable from those detected within the intact nucleus.

Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat

Article

Full-text available

May 1995

Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS-treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock.

Sustained effect of angiotensin II on tyrosine phosphorylation of annexin I in glomerular mesangial cells

Article

Full-text available

Jul 1993

By means of selective extraction in a Ca(2+)-chelating medium and immunoblotting, four annexins (I, II, V, and VI) were identified in both isolated rat renal glomeruli and rat glomerular mesangial cells. Upon 32P labeling of these cells in culture, annexin I was immunoprecipitated using a specific polyclonal antibody and was found to incorporate radioactivity in a constitutive manner. However, as with epidermal growth factor (200 ng/ml), addition of angiotensin II (10(-7) M), arginine-vasopressin (10(-7) M), or endothelin I (10(-7) M) resulted in a 2-3-fold stimulation of annexin I phosphorylation. The basal phosphorylation as well as the stimulating effect of angiotensin II were also detected by immunoblotting annexin extracts using an antiphosphotyrosine antibody. In addition, among various phosphotyrosyl proteins isolated from EGTA extracts by adsorption onto an anti-phosphotyrosine antibody, annexin I was specifically recognized by Western blotting using a monoclonal anti-annexin I antibody, and displayed the same increase upon cell stimulation with angiotensin II. Moreover, thin layer chromatographic analysis of phosphoamino acids present in immunoprecipitated [32P]annexin I showed an exclusive labeling of phosphotyrosine residue(s). Finally, the effect of angiotensin II was detectable after 10 min, maximal at 6 h, and present until 12 h of incubation. Using 12-h stimulation, tyrosine phosphorylation of annexin I displayed a maximum at 10(-7) to 10(-6) M angiotensin II. These data report for the first time the stimulation of annexin I tyrosine phosphorylation by biologically active peptides acting via receptors belonging to the superfamily of seven hydrophobic domain, G-protein-linked receptors, which lack an intrinsic protein tyrosine kinase. This suggests a possible role of annexin I in the mitogenic effect of angiotensin II, arginine-vasopressin, and endothelin I, which was previously observed on rat glomerular mesangial cells as well as on other cells.

Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42.

Article

Full-text available

Jun 1995

The mechanisms by which cells rapidly polarize in the direction of external signals are not understood. Helper T cells, when contacted by an antigen-presenting cell, polarize their cytoskeletons toward the antigen-presenting cell within minutes. Here we show that, in T cells, the mammalian Ras-related GTPase CDC42 (the homologue of yeast CDC42, a protein involved in budding polarity) can regulate the polarization of both actin and microtubules toward antigen-presenting cells but is not involved in other T-cell signaling processes such as those which culminate in interleukin 2 production. Although T-cell polarization appears dispensable for signaling leading to interleukin 2 production, polarization may direct lymphokine secretion towards the correct antigen-presenting cell in a crowded cellular environment. Inhibitor experiments suggest that phosphatidylinositol 3-kinase is required for cytoskeletal polarization but that calcineurin activity, known to be important for other aspects of signaling, is not. Apparent conservation of CDC42 function between yeast and T cells suggests that this GTPase is a general regulator of cytoskeletal polarity in many cell types.

Ras-Dependent and -Independent Pathways Target the Mitogen-Activated Protein Kinase Network in Macrophages

Article

Full-text available

Feb 1995

Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation.

Differential Raf requirement for activation of mitogen-activated protein kinase by growth factors, phorbol esters, and calcium

Article

Full-text available

Apr 1994

Although a pathway that requires sequential activation of Ras, Raf, and MAP kinase kinase has been proposed as the major mechanism for stimulation of mitogen-activated protein kinase (MAP kinase), alternative pathways also exist. A wide variety of extracellular stimuli have been shown to activate MAP kinase; however, the precise mechanisms by which these stimuli mediate the signaling events have not been elucidated. Using a Balb/c-derived cell line expressing a dominant-negative mutant of Raf, we determined whether Raf is required for the activation of MAP kinase by growth factors, phorbol esters, and calcium. Insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and phorbol 12,13-dibutyrate activated Ras in both mutant and control cells. However, stimulation of MAP kinase by IGF-I was nearly abolished in the dominant-negative Raf mutant. Stimulation of MAP kinase by the Ca2+ mobilizer thapsigargin was also inhibited in the presence of the Raf mutant. In contrast, EGF and phorbol 12,13-dibutyrate remained potent stimulators of MAP kinase in the dominant-negative Raf cells. The activation of MAP kinase by these stimuli can be further distinguished by differential requirements for Ca2+ and protein kinase C. These results suggest that Raf is required for the activation of MAP kinase by IGF-I and calcium, whereas EGF and possibly phorbol esters may employ alternative Raf-independent pathways for MAP kinase activation.

The mitogen-activated protein kinase signal transduction pathway

Article

Full-text available

Aug 1993

R.J. Davis

The cytoplasmic raf oncogene induces a neuronal phenotype in PC12 cells: A potential role for cellular raf kinases in neuronal growth factor signal transduction

Article

Full-text available

Jul 1993

The neuron-like differentiation of PC12 cells is induced by nerve growth factor (NGF) through stimulation of a membrane-bound protooncoprotein signaling pathway containing the NGF receptor Trk, the tyrosine kinase Src, and the GTP-binding protein Ras. The Raf-1 and B-raf protooncogenes encode cytoplasmic serine/threonine kinases that are stimulated by NGF in a Ras-dependent manner. To investigate the possible roles of cytoplasmic Raf kinases in eliciting neuronal differentiation, we have expressed the activated Raf-1 oncogene in PC12 cells. Expression of the raf oncogene results in the elaboration of a neuron-like phenotype, including neurite growth and the induction of the NGF-responsive genes NGFI-A and transin. The actions of activated Raf-1 and NGF are not additive. Furthermore, activated Raf-1 oncoprotein can prime cells for transcription-independent neurite growth by NGF and can elicit rapid neurite growth from NGF-primed cells. Our data indicate that the pathways utilized by NGF and activated raf to effect PC12 differentiation overlap and lead to the suggestion that cellular raf kinase activities play significant roles in transducing the differentiating signals of neuronal growth factors.

The SH2 and SH3 domains of the mammalian Grb-2 couple the EGF receptor to the Ras activator mSOS1

Article

Full-text available

Jun 1993

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

Regulation of p21WAF1/CIP1 Expression through Mitogen-activated Protein Kinase Signaling Pathway

Article

Full-text available

Feb 1996

p21WAF1/CIP1 is a cyclin-dependent kinase inhibitor whose expression in mammalian tissues is highly induced in response to stress as well as during normal development and differentiation. Induction of p21WAF1/CIP1 in response to DNA damage occurs through a transcriptional mechanism that is dependent on the activation of the tumor suppressor protein p53. Recent evidence indicates that p21WAF1/CIP1 can also be induced independently of p53, but the signal transduction mechanisms involved in regulating p21WAF1/CIP1 expression in these situations have not been elucidated. In this study, we have addressed the role of the mitogen-activated protein kinase signaling pathway in the induction of p21WAF1/CIP1 in response to growth factor treatment. Using an experimental approach involving cotransfection of a p21WAF1/CIP1 promoter-luciferase construct with a variety of plasmids expressing dominant positive or dominant negative mutant proteins involved in this signaling pathway, we provide evidence to support a role for mitogen-activated protein kinase in the transcriptional activation of p21WAF1/CIP1 by growth factor stimulation.

Evidence for specific annexin I-binding proteins on human monocytes

Article

Full-text available

Jul 1996

Recombinant human annexin I and a monoclonal antibody specific for this protein (mAb 1B) were used to investigate surface binding of this member of the annexin family of proteins to peripheral blood monocytes. Flow cytometric analysis demonstrated trypsin-sensitive, saturable binding of annexin I to human peripheral blood monocytes but not to admixed lymphocytes. A monoclonal antibody that blocks the anti-phospholipase activity of annexin I also blocked its binding to monocytes. These findings suggest the presence of specific binding sites on monocytes. Furthermore, surface iodination, immunoprecipitation and SDS/PAGE analysis were used to identify two annexin I-binding proteins on the surface of monocytes with molecular masses of 15 kDa and 18 kDa respectively. The identification and characterization of these annexin I-binding molecules should help us to better understand the specific interactions of annexin I with monocytes that lead to down-regulation of pro-inflammatory cell functions.

The pool of MAP kinase associated with microtubules is small but constitutively active

Article

Full-text available

Jul 1996

Mitogen-activated protein kinase (MAPK) is activated by many kinds of stimuli and plays an important role in integrating signal transduction cascades. MAPK is present abundantly in brain, where we have studied its association with microtubules. Immunofluorescence of primary hippocampal neurons revealed that MAPK staining co-localized with microtubules and biochemical analyses showed that MAPK co-purified with microtubules. Approximately 4% of MAPK in cytosolic extracts was associated with microtubules, where it was associated with both tubulin and microtubule-associated proteins (MAPs) fractions. Further fractionation of MAPs suggested that a portion of MAPK is associated with MAP2. An association with MAP2 was also demonstrated by co-immunoprecipitation and in vitro binding experiments. A similar association was shown for the juvenile MAP2 isoform, MAP2C. The pool of MAPK associated with microtubules had a higher activity relative to the nonassociated pool in both brain and proliferating PC12 cells. Although MAPK was activated by nerve growth factor in PC12 cells, the activity of microtubule-associated MAPK did not further increase. These results raise the possibility that microtubule-associated MAPK operates through constitutive phosphorylation activity to regulate microtubule function in neurons.

The Hepatocyte Growth Factor Receptor Kinase-mediated Phosphorylation of Lipocortin-1 Transduces the Proliferating Signal of the Hepatocyte Growth Factor

Article

Full-text available

Dec 1996

Hepatocyte growth factor (HGF), which is identical to scatter factor (SF) through coupling to its receptor the product of c-met oncogene, was found to induce proliferation of A549 lung carcinoma cell line, accompanied by release of prostaglandin E2 (PGE2). This activity was sensitive to 0.1-100 microM indomethacin and to 5-50 nM of verapamil. Lipocortin-1, a dexamethasone-inducible inhibitor of phospholipase A2, was shown to be phosphorylated on tyrosine 10 min upon addition of HGF and to translocate to the membrane fraction for up to 6 h upon ligand stimulation. Lipocortin-1 was found to associate in vivo with the HGF receptor species, and this association was independent of the phosphorylation state of the beta-subunit of the HGF receptor (p145betaMET. Immobilized HGF receptor kinase species associated and phosphorylated in vitro lipocortin-1, thus providing evidence that lipocortin-1 is directly phosphorylated by the p145betaMET. Incubation of A549 cells with antisense 21-mer lipocortin-1 oligonucleotides reduced the synthesis and the HGF-stimulated phosphorylation of lipocortin-1 as well as the HGF-stimulated cell proliferation. In processes where the HGF receptor tyrosine kinase is activated, phosphorylation of lipocortin-1 may function as a "signal amplifier" promoting the release of intercellular messengers (PGE2) with pluripotent roles in cell proliferation, chemotaxis, and vascular remodeling.

Partial Mediation of Glucocorticoid Antiproliferate Effects by Lipocortins

Article

Full-text available

Jan 1997

The glucocorticoids (GCs) dexamethasone (DEX) and prednisolone (PRED), in a concentration-dependent fashion, profoundly inhibit mitogen-induced proliferation of human peripheral blood mononuclear lymphocytes (PBML). This inhibition was specific for GCs, as non-GC steroids were devoid of any antiproliferative capacity. GCs enhanced the mRNA (Northern blot) and protein (Western blot) expression of the calcium and phospholipid binding proteins lipocortin I, II, and V. As a consequence of mitogenic stimulation, PBML secrete PGE2 and leukotriene B4 (LTB4). Antiproliferative concentrations of both DEX and PRED as well as recombinant lipocortin I abolished PGE2 and LTB4 production, suggesting an involvement of lipocortins in GC-mediated antiproliferative effects, possibly by inhibiting eicosanoid production and, consequently, mitogen-induced cellular proliferation. Whereas 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid mimicked DEX and PRED in inhibiting PGE2 and LTB4 production, neither 5,8,11,14-eicosatetraynoic acid nor nordihydroguaiaretic acid had any effect on mitogen-induced PBML proliferation, indicating that the GC-mediated antiproliferative effect is separate from their effects on eicosanoid release. Furthermore, neutralizing anti-lipocortin I and anti-lipocortin II mAb, while reversing the inhibitory activity of DEX and PRED on PGE2 and LTB4 production, only partially reversed DEX- and PRED-mediated antiproliferative effects. This indicates that the GC-mediated antiproliferative effect is not dependent on inhibition of eicosanoid release by lipocortins and suggests the existence of lipocortin-dependent and lipocortin-independent pathways by which GCs mediate their antiproliferative effects.

Pumiglia KM, Decker SJCell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 94:448-452

Article

Full-text available

Feb 1997

The mitogen-activated protein kinase (MAPK) cascade plays a crucial role in the transduction of extracellular signals into responses governing growth and differentiation. The effects of a specific inhibitor of the MAPK kinase (MEK)/MAPK pathway (PD98059) on nerve growth factor (NGF)-induced growth arrest and inhibition of cell cycle-dependent kinases (CDKs) have been examined. Treatment of NIH 3T3 cells expressing TRKA with PD98059 dramatically reversed the complete inhibition of growth of these cells caused by NGF. PD98059 also blocked the ability of NGF to inhibit the activities of CDK4 and CDK2, while partially preventing NGF induction of p21Cip1/WAF1. To independently evaluate the involvement of the MEK/MAPK pathway in growth arrest, an inducible activated form of the Raf-1 protooncogene (delta RAF-1:ER) was expressed in these cells. Activation of delta RAF-1:ER resulted in a prolonged increase in MAPK activity and growth arrest of these cells, with concomitant induction of p21Cip1/WAF1 and inhibition of CDK2 activity. These effects of delta RAF-1:ER activation were all reversed by treatment of cells with PD98059. These data indicate that in addition to functioning as a positive effector of growth, stimulation of the MEK/MAPK pathway can result in an inhibition of CDK activity and cell cycle arrest.

Cell cycle and post-transcriptional regulation of annexin expression in IMR-90 human fibroblasts

Article

Full-text available

Apr 1997

Based on the finding that the expression of some annexins varies dramatically as a function of cellular proliferation state [Schlaepfer and Haigler (1990) J. Cell Biol. 111, 229-238], it has been proposed that the cellular level of the annexins might be critical for the regulation of cell growth. To further test this hypothesis, we have studied the expression of various annexins in normal human IMR-90 fibroblasts synchronized by serum deprivation. Using immunoblotting, the cellular content of annexins (Anxs) II, V and VI was found to vary by less than 10% during the cell cycle. However, Anx IV expression increased by 50% during S-phase and the levels of Anxs I and VII were reduced by 40% in early G2/M. However, using RNase protection assays, the mRNAs of Anxs I and VII were found to be uniformly expressed throughout the cell cycle, suggesting that down-regulation of both proteins in G2/M occurred through a post-transcriptional process. In addition, cells transfected with Anx VII cDNA were shown to contain an amount of Anx VII similar to wild-type cells, despite the elevation of Anx VII mRNA content in transfected cells by approx. 2 orders of magnitude. Vector misconstruction or possible secretion of the overexpressed protein were ruled out using appropriate controls. Therefore, as with cell-cycle regulation, Anx VII expression in transfected cells is also controlled by post-transcriptional mechanisms. Furthermore, using pulse-chase analysis, we have determined that annexin VII, and other Anxs, have a slow turnover rate, consistent with the limited changes of expression throughout the cell cycle. Taken together, these results question the hypothesis that cellular expression of Anxs plays a general role in cell growth and support the concept that post-transcriptional mechanisms may control levels of Anxs I and VII.

Raf-Induced Proliferation or Cell Cycle Arrest Is Determined by the Level of Raf Activity with Arrest Mediated by p21 Cip1

Article

Full-text available

Oct 1997

The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.

High-Intensity Raf Signal Causes Cell Cycle Arrest Mediated by p21 Cip1

Article

Full-text available

Oct 1997

Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G1-specific cell cycle arrest through induction of p21Cip1. This in turn leads to inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracycline-dependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.

Regulation of polymorphonuclear leukocyte phagocytosis by myosin light chain kinase after activation of mitogen-activated protein kinase

Article

Apr 2000

Polymorphonuclear leukocyte (PMNL) phagocytosis mediated by FcγRII proceeds in concert with activation of the mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase ERK2. We hypothesized that myosin light chain kinase (MLCK) could be phosphorylated and activated by ERK, thereby linking the MAP kinase pathway to the activation of cytoskeletal components required for pseudopod formation. To explore this potential linkage, PMNLs were challenged with antibody-coated erythrocytes (EIgG). Peak MLCK activity, 3-fold increased over controls, occurred at 4 to 6 minutes, corresponding with the peak rate of target ingestion and ERK2 activity. The MLCK inhibitor ML-7 (10 μmol/L) inhibited both phagocytosis and MLCK activity to basal values, thereby providing further support for the linkage between the functional response and the requirement for MLCK activation. The MAPK kinase (MEK) inhibitor PD098059 inhibited phagocytosis, MLCK activity, and ERK2 activity by 80% to 90%. To directly link ERK activation to MLCK activation, ERK2 was immunoprecipitated from PMNLs after EIgG ingestion. The isolated ERK2 was incubated with PMNL cytosol as a source of unactivated MLCK and with MLCK substrate; under these conditions ERK2 activated MLCK, resulting in phosphorylation of the MLCK substrate or of the myosin light chain itself. Because MLCK activates myosin, we evaluated the effect of directly inhibiting myosin adenosine triphosphatase using 2,3-butanedione monoxime (BDM) and found that phagocytosis was inhibited by more than 90% but MLCK activity remained unaffected. These results are consistent with the interpretation that MEK activates ERK, ERK2 then activates MLCK, and MLCK activates myosin. MLCK activation is a critical step in the cytoskeletal changes resulting in pseudopod formation.

Growth hormone induces tyrosine phosphorylation of annexin I in rat osteosarcoma cells.

Article

Oct 1996

GH induces phosphorylation of a number of cellular proteins, of which several have now been identified, such as mitogen-activated protein kinase, insulin receptor substrate-1, and members of the JAK kinase and STAT families of proteins. However, other phosphorylated proteins remain unidentified. Growth factors and cytokines, including epidermal growth factor, insulin, pp60v-scr, and angiotensin II, induce a rapid phosphorylation of annexin I, a 35-kDa member of the annexin family of Ca2+ and phospholipid-binding proteins. The osteoblast-like rat osteosarcoma cell-line UMR-106.01, in which GH acts as a mitogen via a high affinity GH receptor, was used as a model for GH-induced protein phosphorylation. It is demonstrated by immunoblotting and immunoprecipitation techniques that GH induces the phosphorylation of annexin I on tyrosine residues. This phosphorylation is dose and time dependent. Induction of annexin I phosphorylation is delayed compared with that of JAK2. These results identify annexin I as a protein that becomes tyrosine phosphorylated under the influence of GH and show that phosphorylation of annexin I is a general phenomenon that follows activation of a cell by hormones or cytokines.

Control of the Cell Morphology and the S Phase Entry by Mitogen-activated Protein Kinase Kinase

Article

Apr 1999

The mitogen-activated protein kinase kinase (MAPKK)/MAP kinase (MAPK) cascade plays an important role in the growth control of mammalian cells. We have found that expression of constitutively active MAPKK induces rapid morphological changes of fibroblastic cells, which are accompanied by disruption of stress fibers and disappearance of focal adhesions. These changes took place under the conditions that inhibited cellular Ras function, suggesting a linkage between the MAPK cascade and the control of cell morphology. We further show that constitutively active MAPKK can induce expression of endogenous Fos protein, an immediately early gene product, and cause the S phase entry of G0-arrested cells. Finally, expression of the N-terminal fragment of MAPKK which encompasses the nuclear export signal sequence and the MAPK-binding site blocked both the serum-induced S phase entry of quiescent cells and the oncogenic Ras-induced morphological changes. All these results demonstrate that MAPKK is one of key molecules involved in the control of both cell morphology and cell proliferation and suggest an important role for the N-terminal region of MAPKK in the regulation of the MAPK signaling.

Suppression by Dexamethasone of Inducible Nitric Oxide Synthase Protein Expression In Vivo: A Possible Role for Lipocortin 1

Article

Mar 1998
BIOCHEM PHARMACOL

Western blot and densitometric analysis of organ homogenates from lipopolysaccharide (LPS)-treated rats (1–10 mg kg−1, i.p.) exhibited a strong induction of inducible nitric oxide synthase (iNOS) expression seen at all the doses tested (1, 3, and 10 mg kg−1, n = 3). In particular, 3 hr after challenge of rats with LPS, iNOS was detectable in the liver, kidney, aorta, spleen and lung. Dexamethasone (DEX) (0.1–1 mg kg−1; −1 hr) dose-dependently reduced iNOS expression in lung homogenates after exposure to LPS (1 mg kg−1; P < 0.05). A partial reversal of DEX-induced suppression of iNOS expression in lung homogenates 3 hr after challenge with LPS was observed in rats which received a specific anti-lipocortin 1 sheep serum (LCS3; 1 mL kg−1 24 hr prior to the steroid), with an inhibition of 35 ± 8%, as compared to animals passively immunised with normal sheep serum where dexamethasone exhibited an inhibition of 60 ± 7% (n = 4). Peritoneal macrophages collected from rats treated with LPS (1 mg kg−1; 3 hr) and cultured for 16 hr, released significant amounts of nitrite (51 ± 1 μM) into the cell supernatants; this was reduced (−70 ± 6%) after pre-treatment with dexamethasone (0.3 mg kg−1) and this effect was neutralised if animals were passively immunised with LCS3 (P < 0.01; n = 4). Thus lipocortin 1 mediates, at least in part, the inhibitory action exerted by dexamethasone on both iNOS protein expression in lung and iNOS activity (as measured by nitrite release) in primary peritoneal cells of rats.

Inhibitory effect of peptides derived from the N-terminus of lipocortin 1 on arachidonic acid release and proliferation in the A549 cell line: Identification of E-Q-E-Y-V as a crucial component

Article

Mar 1998

The ability of the glucocorticoid‐induced protein lipocortin 1 (LC1) to inhibit arachidonic acid release and cell proliferation in A549 cells may be mimicked by a sequence taken from the N‐terminal, LC1 13–25 (FIENEEQEYVQTV). We have now synthesized and tested for biological activity a library of 25 smaller peptides derived from this sequence. Peptides were tested in two assays: A549 cells were prelabelled with tritiated arachidonic acid and thapsigargin (50 n M ) and EGF (10 n M ) used to stimulate the release of this fatty acid. Cell proliferation was determined by counting cell numbers following 3 day incubation with these peptides, or controls. Many of the peptides were highly insoluble but could be more readily dissolved in aqueous solution in the presence of commercial liposomes or phosphatidyl serine (5 μ M ). Since neither of these agents alone had any effect on arachidonic acid release or cell proliferation, all peptides were tested in the presence of 5 μ M phosphatidyl serine. Under these conditions LC1 13–25 was active in both assay systems with an IC 40 of 40.7 and 57.0 μ M respectively. Deletion of amino acids from the C‐terminus of the peptide progressively diminished (2–3 fold) the molar potency of LC1 13–25 in both assays: after the removal of Val ²² biological activity was virtually undetectable or very weak (<30% of LC1 13–25 ). Removal of amino acids from the N‐terminus also lead to a progressive reduction (3–5 fold) in the molar potency of the peptides and biological activity became undetectable, or very weak, after the removal of Glu ¹⁸ . All active peptides contained the core sequence EQEYV(Glu‐Gln‐Glu‐Tyr‐Val) which seems to represent a crucial component of the pharmacophore, although this sequence on its own was inactive and the shortest peptide with significant activity was LC1 18–25 (EQEYVQTV). Methoxylation of Tyr ²¹ abolished the ability of LC1 18–25 to inhibit cell proliferation and arachidonic acid release. A cyclized version of LC1 18–25 was also tested and found to be inactive. LC1 18–25 (178 μ M ) inhibits cPLA 2 activation in A549 cells as judged by a band‐shift assay, whereas equimolar concentrations of an inactive peptide LC1 19–25 were without effect in this assay system. Several possible mechanisms whereby these peptides act are discussed in the light of LC1 biology and of the effect of glucocorticoids on cell function. British Journal of Pharmacology (1998) 123 , 975–983; doi: 10.1038/sj.bjp.0701679

Lipocortin 1 Mediates Dexamethasone-Induced Growth Arrest of the A549 Lung Adenocarcinoma Cell Line

Article

May 1992

The synthetic glucocorticoid dexamethasone (1 microM to 1 pM) strongly (maximum greater than 80%) inhibits proliferation of the A549 human lung adenocarcinoma line (EC50 greater than 1 nM) and leads to the appearance, or a further increase (approximately 3-fold) in the expression on the cell surface, of the calcium and phospholipid binding protein lipocortin (annexin) 1. Both these effects, which are shared by hydrocortisone (1 microM) but not by progesterone or aldosterone (1 microM), are inhibited by the antiglucocorticoids RU38486 and RU43044 (1 microM). The nonsteroidal antiinflammatory drugs indomethacin (1 microM) and naproxen (10 microM) and human recombinant lipocortin 1 (0.05-5.0 micrograms/ml) also produce growth arrest in this cell line. During proliferation A549 cells spontaneously release prostaglandin E2 [10-20 ng (28-57 pmol) per ml per 5-day period] into the growth medium. In concentrations that cause growth-arrest, dexamethasone, indomethacin, and lipocortin 1 abolish the generation of this eicosanoid by A549 cells. Prostaglandin E2 itself (0.01-1 pM) stimulates cell growth and partially reverses (approximately 50%) the inhibition of growth caused by dexamethasone and indomethacin. Addition of the neutralizing anti-lipocortin 1 monoclonal antibody 1A (5 micrograms/ml), but not the nonneutralizing anti-lipocortin monoclonal antibody 1B, substantially reversed (greater than 80%) the inhibitory activity of dexamethasone on both growth and prostaglandin E2 synthesis. The generation of prostaglandin E2 by A549 cells seems to be an important regulator of cell proliferation in vitro and the dexamethasone-induced suppression of proliferation in this model is attributable to eicosanoid inhibition caused by lipocortin 1.

Diversity in the Lipocort??n/Calpactin Family

Article

Nov 1988
CELL

The lipocortin family is now known to comprise at least five different proteins purified from mammalian cells, all of which bind to cellular membranes and phospholipids in a Ca2+-dependent manner. The related amino acid sequences are reflected in several shared properties, although distinct N-terminal regions may confer functional individuality, and phosphorylation of two of these proteins on tyrosine residues results in altered Ca2+/phospholipid binding. However, the elucidation of the true physiological functions of these proteins remains the greatest challenge. Whether they have roles in phospholipase regulation, membrane trafficking, or cytoskeletal organization, or whether their importance lies elsewhere, is not yet known, nor is the relevance of the phosphorylation of calpactins I and II in the context of cell growth and transformation understood. The rapid accumulation of structural and biochemical data, some of which has been reviewed here, should accelerate progress toward defining the true activities of the lipocortin-like proteins.

Inhibition of arachidonic acid metabolism is not involved in dexamethasone-induced growth inhibition in embryonic palatal development

Article

Feb 1985
Prostag Leukotr Med

Previous studies have shown that glucocorticoids induce cleft palate in susceptible strains of mice and inhibit proliferation of palatal mesenchyme cells in vivo and in culture. The present study shows that the synthetic glucocorticoid, dexamethasone (DEX), inhibits serum-stimulated arachidonic acid release in cultured mouse palatal mesenchyme cells. Arachidonic acid could neither prevent the DEX effect on cell proliferation when added in culture nor prevent glucocorticoid-induced cleft palate when administered in vivo. Furthermore, the time course for DEX-induced inhibition of arachidonic acid release (maximal by 5h) is markedly different from the time courses for both inhibition of cell proliferation in culture and cleft palate induction in vivo (3 to 4 days). These results suggest that both DEX-induced cleft palate formation and inhibition of palatal cell proliferation arise from some mechanism other than a DEX-induced inhibition of arachidonic acid metabolism.

Dexamethasone-induced inhibition of prostaglandin production does not result from a direct action on phospholipase A2 activities but is mediated through a steroid-inducible factor

Article

Aug 1982
Biochim Biophys Acta

Investigations were carried out to define the mechanisms of steroid-induced inhibition of prostaglandin secretion by rat renomedullary cells in tissue culture. Although it was strongly proposed that glucocorticoids may inhibit phospholipase A2 activity, we present several pieces of evidence against a direct action of dexamethasone on phospholipase activities. First, dexamethasone, which significantly decreases the release of labeled material from cells prelabeled with [3H]arachidonate, does not significantly alter the pattern of distribution of the radioactivity among the various classes of cell lipids. In addition, direct measurement of phospholipase A3 activity in dexamethasone-treated cells failed to show any significant decrease in the deacylation capacity. On the other hand, several indications suggest that dexamethasone may induce the secretion of a non-dialysable, transferable factor able to inhibit prostaglandin production, the mechanism of which remains to be investigated.

Ion channels. Annexins taken to task

Article

Dec 1995

Stephen E Moss

Specificity of Receptor Tyrosine Kinase Signaling – Transient Versus Sustained Extracellular Signal-Regulated Kinase Activation

Article

Feb 1995
CELL

C.J. Marshall

A number of different intracellular signaling pathways have been shown to be activated by receptor tyrosine kinases. These activation events include the phosphoinositide 3-kinase, 70 kDa S6 kinase, mitogen-activated protein kinase (MAPK), phospholipase C-gamma, and the Jak/STAT pathways. The precise role of each of these pathways in cell signaling remains to be resolved, but studies on the differentiation of mammalian PC12 cells in tissue culture and the genetics of cell fate determination in Drosophila and Caenorhabditis suggest that the extracellular signal-regulated kinase (ERK-regulated) MAPK pathway may be sufficient for these cellular responses. Experiments with PC12 cells also suggest that the duration of ERK activation is critical for cell signaling decisions.

Transformation of Mammalian Cells by Constitutively Active MAP Kinase Kinase

Article

Sep 1994

Mitogen-activated protein (MAP) kinase kinase (MAPKK) activates MAP kinase in a signal transduction pathway that mediates cellular responses to growth and differentiation factors. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells by prolonging the activated state of MAPKK and of components downstream in the signaling pathway. To test this hypothesis, constitutively active MAPKK mutants were designed that had basal activities up to 400 times greater than that of the unphosphorylated wild-type kinase. Expression of these mutants in mammalian cells activated AP-1-regulated transcription. The cells formed transformed foci, grew efficiently in soft agar, and were highly tumorigenic in nude mice. These findings indicate that constitutive activation of MAPKK is sufficient to promote cell transformation.

Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins

Article

May 1994
Biochim Biophys Acta

Lipocortin-1: cellular mechanisms and clinical relevance

Article

Apr 1994
TRENDS PHARMACOL SCI

Lipocortin-1, a 37 kDa member of the annexin superfamily of proteins, originally evoked interest as one of the 'second messengers' of the anti-inflammatory actions of the glucocorticoids. Subsequent research has shown that the protein plays a major regulatory role in systems as diverse as cell-growth regulation and differentiation, neutrophil migration, CNS responses to cytokines, neuroendocrine secretion and neurodegeneration. The role of lipocortin-1 in mediating glucocorticoid-induced effects in these systems has been demonstrated using immunoneutralization strategies and by mimicking steroid actions with highly purified or recombinant lipocortin-1 or its biologically active peptide fragments. Originally the mode of action of lipocortin-1 seemed to be largely through inhibition of prostaglandin formation, but it is now clear that it can modify other aspects of cell function, perhaps pointing to a more fundamental mechanism than was originally envisaged. In this article Rod Flower and Nancy Rothwell review the nature, possible mechanisms and clinical relevance of these diverse actions of lipocortin-1.

v-raf confers CSF-1 independent growth to a macrophage cell line and leads to immediate early gene expression without MAP-Kinase activation

Article

Jan 1994
ONCOGENE

The BAC-1.2F5 macrophage cell line depends on CSF-1 for proliferation and survival. Phosphorylation and activation of the RAF-1 kinase are among the early events in CSF-1 signal transduction. To characterize the role of RAF-1 in CSF-1-induced proliferation, we overexpressed oncogenically activated RAF-1, cellular RAF-1 and RAF-1 kinase-defective mutant proteins in BAC-1.2F5 cells. We were unable to establish stable cell lines expressing either kinase-negative or full length RAF-1 proteins, implying that expression of these molecules is not tolerated in BAC-1.2F5 cells. Oncogenically activated RAF-1 induces CSF-1-independent growth in the absence of autocrine growth factor production. Autonomous growth is not associated with dedifferentiation, since v-raf-expressing macrophages perform the same immunological functions as control cells. Intriguingly, autonomous growth correlates with the suppression of CSF-1-mediated MAP-Kinase activation and with the low constitutive expression of a number of CSF-1-inducible genes, including fos, jun, ets2, and myc, but also the genes for the inflammatory cytokines TNF alpha and IL-1 beta. Many of these genes have AP-1 binding sites in their promoters, and the v-raf-expressing cells contain constitutive AP-1 binding activity. These data indicate that RAF-1, but not MAP-Kinase, is a key component in CSF-1 mitogenic signal transduction, and are consistent with a working hypothesis in which RAF-1 mediates transcriptional activation of genes via AP-1.

A Divergence in the MAP Kinase Regulatory Network Defined by MEK Kinase and Raf

Article

May 1993

Mitogen-activated protein kinases (MAPKs) are rapidly phosphorylated and activated in response to various extracellular stimuli in many different cell types. Such regulation of MAPK results from sequential activation of a series of protein kinases. The kinases that phosphorylate MAPKs, the MAP kinase kinases (MEKs) are also activated by phosphorylation. MEKs are related in sequence to the yeast protein kinases Byr1 (from Schizosaccharomyces pombe) and Ste7 (from Saccharomyces cerevisiae), which function in the pheromone-induced signaling pathway that results in mating. Byr1 and Ste7 are in turn regulated by the protein kinases Byr2 and Ste11. The amino acid sequence of the mouse homolog of Byr2 and Ste11, denoted MEKK (MEK kinase), was elucidated from a complementary DNA sequence encoding a protein of 672 amino acid residues (73 kilodaltons). MEKK was expressed in all mouse tissues tested, and it phosphorylated and activated MEK. Phosphorylation and activation of MEK by MEKK was independent of Raf, a growth factor-regulated protein kinase that also phosphorylates MEK. Thus, MEKK and Raf converge at MEK in the protein kinase network mediating the activation of MAPKs by hormones, growth factors, and neurotransmitters.

Modulation of IL-1 -Induced Neutrophil Migration by Dexamethasone and Lipocortin 1

Article

Mar 1993

IL-1 is a pro-inflammatory cytokine which controls many features of the immune and inflammatory response. When injected into a mouse 6-day-old air-pouch, human rIL-1 (1 to 100 ng) induced in a dose-dependent fashion a migration of PMN that could be reliably assessed 4 h after injection. Both IL-1 alpha and IL-1 beta were active in this model. The effect of the cytokine was inhibited by local administration of actinomycin D (1 to 10 micrograms), alpha-melanocyte-stimulating hormone (200 micrograms), and a mAb recognizing IL-1R type I (10 micrograms). Indomethacin (1 mg/kg), an inhibitor of cyclo-oxygenase, and BW4AC (2 mg/kg), a selective lipoxygenase inhibitor, were without effect but moderate inhibition was seen with the platelet-activating factor antagonist WEB2086 (1 to 10 mg/kg). The glucocorticoid dexamethasone (0.015 to 1.5 mg/kg) potently inhibited the elicitation of neutrophils induced by IL-1 when given systemically 2 h before the cytokine. The steroid-induced anti-inflammatory protein lipocortin 1 (LC1) also produced a dose-dependent inhibition of PMN migration into the pouch with an ED50 of approximately 0.15 to 0.21 mg/kg. The denatured protein was without effect. Passive immunization of mice with a polyclonal sheep antiserum or a mAb raised against LC1 abolished the inhibitory action of dexamethasone whereas preimmune serum or control IgG were without significant effect. These findings provide further evidence that LC1 is involved in the anti-inflammatory action of glucocorticosteroids and suggest that this protein may act as an endogenous regulator of IL-1 action.

Developmental regulation of various annexins in the embryonic palate of the mouse: Dexamethasone affects expression of annexin-1

Article

Oct 1995
J Craniofac Genet Dev Biol

The annexins are a group of structurally related proteins implicated in a number of cellular processes, including growth, membrane fusion, and the effects of glucocorticoids on cellular physiology, signal transduction, and regulation of activities of phospholipase A2. Though their exact role in cellular physiology is not clear, their properties make them candidate proteins for signal transduction pathways by which growth factors and glucocorticoids modulate development of the palate. We have determined the exact cellular location and development expression of various annexins in the embryonic murine palate as a first step in assessing their developmental function. Western blot analysis revealed an increased accumulation of selected annexins in elevated palates compared to vertical (unelevated) ones. This was particularly striking for lipocortin I1 (annexin I), whose mRNA accumulated as well. Lipocortin I was expressed primarily in the apical portion of the palatal epithelium at early stages of development, but throughout the epithelium at later stages. Also, there was increased immunoreactivity for lipocortin I in the mesenchyme as development proceeded. Immunoreactivity for the endonexins (annexins IV and V) was found in the palatal epithelium and mesenchyme, whereas immunoreactivity for the 67-kDa calelectrin (annexin VI) was found only in the mesenchyme. Treatment of pregnant A/J strain mice with a cleft-palate inducing regimen of dexamethasone stimulated accumulation of lipocortin I protein and mRNA, but not lipocortin II (annexin II) protein. In contrast, the same regimen of dexamethasone did not affect levels of lipocortin I protein in palates of the glucocorticoid-less sensitive C57BL/6J strain mouse embryo. These data permit the suggestion that lipocortin I plays some critical, but as yet undefined, role in modulating ontogeny of the murine palate.

Growth hormone induces tyrosine phosphorylation of annexin I in rat osteosarcoma cells

Article

Nov 1996

Localization of five annexins in J774 macrophages and on isolated phagosomes

Article

Jun 1997

Annexins are a family of structurally related proteins which bind phospholipids in a calcium-dependent manner. Although the precise functions of annexins are unknown, there is an accumulating set of data arguing for a role for some of them in vesicular transport and, specifically, in membrane-membrane or membrane-cytoskeletal interactions during these processes. Here we describe our qualitative and quantitative analysis of the localization of annexins I-V in J774 macrophages that had internalized latex beads, both with and without IgG opsonization. Our results show that whereas all these annexins are present on both the plasma membrane and on phagosomes, the localization on other organelles differs. Annexins I, II, III and V were detected on early endosomes, while only annexin V was seen on late endocytic organelles and mitochondria. Annexins I and II distributed along the plasma membrane non-uniformly and co-localized with F-actin at the sites of membrane protrusions. We also investigated by western blot analysis the association of annexins with purified phagosomes isolated at different time-points after latex bead internalization. While the amounts of annexins I, II, III and V associated with phagosomes were similar at all times after their formation, the level of annexin IV was significantly higher on older phagosomes. Whereas annexins I, II, IV and V could be removed from phagosome membranes with a Ca2+ chelator they remained membrane bound under low calcium conditions. In contrast, annexin III was removed under these conditions and needed a relatively high Ca2+ concentration to remain phagosome bound. Because of their purity and ease of preparation we suggest that phagosomes are a powerful system to study the potential role of annexins in membrane traffic.

Participation of annexins in protein phosphorylation

Article

Jul 1997

B Rothhut

Simultaneous discovery of members of the annexin family of calcium and phospholipid binding proteins by several groups is intimately linked to the possibility that these proteins may be controlled by phosphorylation. Indeed, annexin I and annexin II have been identified as major substrates for the tyrosine kinase activity associated with epidermal growth factor receptor (EGF-R) and for the retrovirus encoded protein tyrosine kinase pp60v-arc. Both annexins are also in vitro and/or in situ substrates for platelet derived growth factor (PDGF), insulin and hepatocyte growth factor/scatter factor (HGF/SF) receptor tyrosine kinases. In addition, to serve as substrates for tyrosine protein kinases some annexins are cellular targets for serine threonine protein kinases such as protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA). Although the role of annexin phosphorylation has not been studied in detail, it is thought to influence their vesicle aggregation and phospholipid binding properties. Some annexins are also potent inhibitors of various serine/threonine and tyrosine kinases. The physiological functions of the annexins have still not been clearly defined. Therefore the identification of the ability of these proteins to undergo phosphorylation may be helpful in assigning them a precise biological role.

Novel Recruitment of Shc, Grb2, and Sos by Fibroblast Growth Factor Receptor-1 in v-Src-Transformed Cells

Article

Mar 1998

In response to fibroblast growth factor (FGF), FGF receptor-1 (FGFR-1) (flg) becomes tyrosine phosphorylated and associates with phospholipase C gamma (PLC gamma) and a 90 kDa protein. We report here that in cells transformed by v-Src, FGFR-1 becomes phosphorylated on tyrosine; however, neither PLC gamma nor p90 was found to be associated with tyrosine-phosphorylated FGFR-1. Instead, there was a strong constitutive association of FGFR-1 with the adaptor proteins Shc and Grb2 and the Ras guanine nucleotide exchange factor Sos. Association with Shc and Grb2 and Sos was not observed in response to FGF. Suramin did not prevent either tyrosine phosphorylation or Shc/Grb2/Sos association, indicating a non-autocrine mechanism. Thus, in cells transformed by v-Src, tyrosine phosphorylation of FGFR-1 results not in the expected association with PLC gamma and p90, but rather in the recruitment of the Ras activating Shc/Grb2/Sos complex. These data suggest a mechanism for Ras activation by v-Src involving phosphorylation of novel tyrosine(s) on FGFR-1.

Annexin 1 regulates cell proliferation by disruption of cell morphology and inhibition of cyclin D1 expression through sustained activation of the ERK1/2 MAPK signal

Abstract

No full-text available

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