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The role of CCN5/WISP2 protein in pancreatic islets
Journal of Diabetes
Abstract
In search of direct targets of IGF-I action, we discovered CCN5/WISP2 as a novel protein expressed in pancreatic β-cells. As a member of Cyr61 (connective tissue growth factor in human), CTGF (cysteine rich 61, in mouse), and Nov (nephroblastoma overexpressed, in chicken) family, IGF-I together with Wnt signaling stimulate its expression. When overexpressed in insulinoma cells, CCN5 promotes cell proliferation and cell survival against streptozotocin-induced cell death. The cell proliferation effect seems to be caused by AKT phosphorylation and increased cyclin D1 level. These properties resemble that of CCN2/CTGF, another isoform of the CCN family, although CCN5 is the only one within the family of six proteins that lacks the C-terminal repeat. Treatment of primary mouse islets with recombinant CCN5 protein produced similar effects, indicating either being produced as a matricellular protein or a secreted growth factor, CCN5 stimulate β-cell proliferation and regeneration in a paracrine fashion. This review also discusses its regulation by estrogen and involvement in angiogenesis and tumorigenesis.
CCN/WISP (cellular communication network factors, or Wnt-inducted secreted proteins) family of proteins consists of six extracellular matrix (ECM)-associated proteins that regulate development, cell adhesion and proliferation, ECM remodeling, inflammation, and tumorigenesis. In the last two decades, metabolic regulation by these matricellular proteins has been studied extensively, several excellent reviews have covered the roles of CCN1, -2 and − 5. In this brief review, we will focus on those lesser-known members and more recent discoveries, together with other recent articles presenting a more complete picture of the current state of knowledge. We have found that CCN2, -4, and − 5 promote pancreatic islet function, while CCN3 plays a unique and negative role. CCN3 and − 4 are pro-adiposity leading to insulin resistance, but CCN5 and − 6 are anti-adiposity. While CCN2 and − 4 promote tissue fibrosis and inflammation, all other four members are clearly anti-fibrotic. As for cellular signaling, they are known to interact with integrins, other cell membrane proteins and ECM thereby regulate Akt/protein kinase B, myocardin-related transcription factor (MRTF), and focal adhesion kinase. Yet, a cohesive mechanism of action to comprehensively explain those major functions is still lacking.
CCN5/WISP2 is a matricellular protein, the expression of which is under the regulation of Wnt signaling and IGF-1. Our initial characterization supports the notion that CCN5 might promote the proliferation and survival of pancreatic β-cells and thus improve the metabolic profile of the animals. More recently, the roles of endogenous expression of CCN5 and its ectopic, transgenic overexpression on metabolic regulation have been revealed through two reports. Here, we attempt to compare the experimental findings from those studies, side-by-side, in order to further establish its roles in metabolic regulation. Prominent among the discoveries was that a systemic deficiency of CCN5 gene expression caused adipocyte hypertrophy, increased adipogenesis, and lipid accumulation, resulting in insulin resistance and glucose intolerance, which were further exacerbated upon high-fat diet feeding. On the other hand, the adipocyte-specific and systemic overexpression of CCN5 caused an increase in lean body mass, improved insulin sensitivity, hyperplasia of cardiomyocytes, and increased heart mass, but decreased fasting glucose levels. CCN5 is clearly a regulator of adipocyte proliferation and maturation, affecting lean/fat mass ratio and insulin sensitivity. Not all results from these models are consistent; moreover, several important aspects of CCN5 physiology are yet to be explored.
We have generated the transgenic mouse line LTCFDN in which dominant negative TCF7L2 (TCF7L2DN) is specifically expressed in the liver during adulthood. Male but not female LTCFDN mice showed elevated hepatic and plasma TG levels, indicating the existence of estrogen-β-cat/TCF signaling cascade that regulates hepatic lipid homeostasis. We show here that hepatic FGF21 expression was reduced in male but not in female LTCFDN mice. The reduction was not associated with altered hepatic expression of peroxisome proliferator-activated receptor α (PPARα). In mouse primary hepatocytes (MPH), Wnt-3a treatment increased FGF21 expression in the presence of PPARα inhibitor. Results from our luciferase-reporter assay and chromatin immunoprecipitation suggest that evolutionarily conserved TCF binding motifs (TCFBs) on Fgf21 promoter mediate Wnt-3a induced Fgf21 transactivation. Female mice showed reduced hepatic FGF21 production and circulating FGF21 level following ovariectomy (OVX), associated with reduced hepatic TCF expression and β-catenin S675 phosphorylation. Finally, in MPH, estradiol (E2) treatment enhanced FGF21 expression, as well as binding of TCF7L2 and RNA polymerase II to the Fgf21 promoter; and the enhancement can be attenuated by the G-protein-coupled estrogen receptor 1 (GPER) antagonist G15. Our observations hence indicate that hepatic FGF21 is among the effectors of the newly recognized E2-β-cat/TCF signaling cascade.
The Cellular Communication Network (CCN) family of matricellular proteins comprises six proteins that share conserved structural features and play numerous biological roles. These proteins can interact with several receptors or soluble proteins, regulating cell signaling pathways in various tissues under physiological and pathological conditions. In the skeletal muscle of mammals, most of the six CCN family members are expressed during embryonic development or in adulthood. Their roles during the adult stage are related to the regulation of muscle mass and regeneration, maintaining vascularization, and the modulation of skeletal muscle fibrosis. This work reviews the CCNs proteins’ role in skeletal muscle physiology and disease, focusing on skeletal muscle fibrosis and its regulation by Connective Tissue Growth factor (CCN2/CTGF). Furthermore, we review evidence on the modulation of fibrosis and CCN2/CTGF by the renin-angiotensin system and the kallikrein-kinin system of vasoactive peptides.
Background
To analyze and identify the circular RNAs (circRNAs) involved in promoting the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) on titanium by surface mechanical attrition treatment (SMAT).
Methods
The experimental material was SMAT titanium and the control material was annealed titanium. Cell Counting Kits-8 (CCK-8) was used to detect the proliferation of hBMSCs, and alkaline phosphatase (ALP) activity and alizarin red staining were used to detect the osteogenic differentiation of hBMSCs on the sample surfaces. The bioinformatics prediction software miwalk3.0 was used to construct competing endogenous RNA (ceRNA) networks by predicting circRNAs with osteogenesis-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The circRNAs located at the key positions in the networks were selected and analyzed by quantitative real-time PCR (QRT-PCR).
Results
Compared with annealed titanium, SMAT titanium could promote the proliferation and osteogenic differentiation of hBMSCs. The total number of predicted circRNAs was 51. Among these, 30 circRNAs and 8 miRNAs constituted 6 ceRNA networks. Circ-LTBP2 was selected for verification. QRT-PCR results showed that the expression levels of hsa_circ_0032599, hsa_circ_0032600 and hsa_circ_0032601 were upregulated in the experimental group compared with those in the control group; the differential expression of hsa_circ_0032600 was the most obvious and statistically significant, with a fold change (FC) = 4.25 ± 1.60, p -values ( p ) < 0.05.
Across the years the CCNs have been increasingly implicated in the development of obesity, diabetes and its complications. Evidence for this is currently derived from their dysregulation in key metabolic pathological states in humans, animal and in vitro models, and also pre-clinical effects of their bioactivities. CCN2 is the best studied in this disease process and the other CCNs are yet to be better defined. Key steps where CCNs may play a pathogenic metabolic role include: (i) obesity and insulin resistance, where CCN2 inhibits fat cell differentiation in vitro and CCN3 may induce obesity and insulin resistance; (ii) elevated blood glucose levels to diabetes mellitus onset, where CCN2 may contribute to pancreatic beta cell and islet function; and (iii) in diabetes complications, such as nephropathy, retinopathy, liver disease (NAFLD/NASH), CVD and diabetes with heart failure. In contrast, CCN1, CCN2 and possibly CCN3, may have a reparative role in wound healing in diabetes, and CCN2 in islet cell development. In terms of CCN2 regulation by a diabetes metabolic environment and related mechanisms, the author’s laboratory and others have progressively shown that advanced glycation-end products, protein kinase C isoforms, saturated fatty acids, reactive oxygen species and haemodynamic factors upregulate CCN2 in relevant cell and animal systems. Recent data has suggested that CCN2, CCN3 and CCN6 may affect energy homeostasis including in regulating glycolysis and mitochondrial function. This paper will address the current data implicating CCNs in diabetes and its complications, focusing on recent aspects with translational clinical relevance and future directions.
Background:
Cardiac fibrosis (CF) is associated with increased ventricular stiffness and diastolic dysfunction and is an independent predictor of long-term clinical outcomes of patients with heart failure (HF). We previously showed that the matricellular CCN5 protein is cardioprotective via its ability to inhibit CF and preserve cardiac contractility.
Objectives:
This study examined the role of CCN5 in human heart failure and tested whether CCN5 can reverse established CF in an experimental model of HF induced by pressure overload.
Methods:
Human hearts were obtained from patients with end-stage heart failure. Extensive CF was induced by applying transverse aortic constriction for 8 weeks, which was followed by adeno-associated virus-mediated transfer of CCN5 to the heart. Eight weeks following gene transfer, cellular and molecular effects were examined.
Results:
Expression of CCN5 was significantly decreased in failing hearts from patients with end-stage heart failure compared to nonfailing hearts. Trichrome staining and myofibroblast content measurements revealed that the established CF had been reversed by CCN5 gene transfer. Anti-CF effects of CCN5 were associated with inhibition of the transforming growth factor beta signaling pathway. CCN5 significantly inhibited endothelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation, which are 2 critical processes for CF progression, both in vivo and in vitro. In addition, CCN5 induced apoptosis in myofibroblasts, but not in cardiomyocytes or fibroblasts, both in vivo and in vitro. CCN5 provoked the intrinsic apoptotic pathway specifically in myofibroblasts, which may have been due the ability of CCN5 to inhibit the activity of NFκB, an antiapoptotic molecule.
Conclusions:
CCN5 can reverse established CF by inhibiting the generation of and enhancing apoptosis of myofibroblasts in the myocardium. CCN5 may provide a novel platform for the development of targeted anti-CF therapies.
Loss of insulin-producing pancreatic islet β-cells is a hallmark of type 1 diabetes. Several experimental paradigms demonstrate that these cells can, in principle, be regenerated from multiple endogenous sources using signaling pathways that are also used during pancreas development. A thorough understanding of these pathways will provide improved opportunities for therapeutic intervention. It is now appreciated that signaling pathways should not be seen as "on" or "off" but that the degree of activity may result in wildly different cellular outcomes. In addition to the degree of operation of a signaling pathway, noncanonical branches also play important roles. Thus, a pathway, once considered as "off" or "low" may actually be highly operational but may be using noncanonical branches. Such branches are only now revealing themselves as new tools to assay them are being generated. A formidable source of noncanonical signal transduction concepts is neural stem cells because these cells appear to have acquired unusual signaling interpretations to allow them to maintain their unique dual properties (self-renewal and multipotency). We discuss how such findings from the neural field can provide a blueprint for the identification of new molecular mechanisms regulating pancreatic biology, with a focus on Notch, Hes/Hey, and hedgehog pathways.
The CCN family of proteins comprises the members CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6. They share four evolutionarily conserved functional domains, and usually interact with various cytokines to elicit different biological functions including cell proliferation, adhesion, invasion, migration, embryonic development, angiogenesis, wound healing, fibrosis and inflammation through a variety of signalling pathways. In the past two decades, emerging functions for the CCN proteins (CCNs) have been identified in various types of cancer. Perturbed expression of CCNs has been observed in a variety of malignancies. The aberrant expression of certain CCNs is associated with disease progression and poor prognosis. Insight into the detailed mechanisms involved in CCN-mediated regulation may be useful in understanding their roles and functions in tumorigenesis and cancer metastasis. In this review, we briefly introduced the functions of CCNs, especially in cancer.
This is the third in a series of Perspectives on intracellular signaling pathways coupled to proliferation in pancreatic β-cells. We contrast the large knowledge base in rodent β-cells with the more limited human database. With the increasing incidence of type 1 diabetes and the recognition that type 2 diabetes is also due in part to a deficiency of functioning β-cells, there is great urgency to identify therapeutic approaches to expand human β-cell numbers. Therapeutic approaches might include stem cell differentiation, transdifferentiation, or expansion of cadaver islets or residual endogenous β-cells. In these Perspectives, we focus on β-cell proliferation. Past Perspectives reviewed fundamental cell cycle regulation and its upstream regulation by insulin/IGF signaling via phosphatidylinositol-3 kinase/mammalian target of rapamycin signaling, glucose, glycogen synthase kinase-3 and liver kinase B1, protein kinase Cζ, calcium-calcineurin-nuclear factor of activated T cells, epidermal growth factor/platelet-derived growth factor family members, Wnt/β-catenin, leptin, and estrogen and progesterone. Here, we emphasize Janus kinase/signal transducers and activators of transcription, Ras/Raf/extracellular signal-related kinase, cadherins and integrins, G-protein-coupled receptors, and transforming growth factor β signaling. We hope these three Perspectives will serve to introduce these pathways to new researchers and will encourage additional investigators to focus on understanding how to harness key intracellular signaling pathways for therapeutic human β-cell regeneration for diabetes.
© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
The technologies for pancreatic β cell-specific gene overexpression or knockout are fundamental for investigations of functional genes in vivo. Here, we generated the Ins1-Cre-Dsred and Ins1-rtTA mouse models, which expressed the Cre recombinase or reverse tetracycline regulatable transactivator (rtTA) without hGH minigene under the control of mouse Ins1 promoter. Our data showed that the Cre-mediated recombination and rtTA-mediated activation could be efficiently detected at E13.5 when these models were crossed with the reporter mice (ROSA(mT/mG) or tetO-HIST1H2BJ/GFP). The Cre and rtTA expression was restricted to β-cells without leakage in the brain and other tissues. Moreover, both the transgenic lines showed normal glucose tolerance and insulin secretion. These results suggested that the Ins1-Cre-Dsred and Ins1-rtTA mice could be utilized to knockout or overexpress target genes in embryos and adults to facilitate β-cell researches.
Cell–cell and cell–matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that αvβ3 and αvβ5, two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of αvβ3 and αvβ5 integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins αvβ3 and αvβ5 and their ligands to morphogenetic events in the human endocrine pancreas.
The matricellular protein CCN5/WISP-2 represents a promising target in triple-negative breast cancer (TNBC) because treatment or induced activation of CCN5 in TNBC cells promotes cell growth arrest at the G0/G1 phase, reduces cell proliferation and delays tumor growth in the xenograft model. Our studies found that the p27(Kip1) tumor suppressor protein is upregulated and relocalized to the nucleus from cytoplasm by CCN5 in these cells and that these two events (upregulation and relocalization of p27(Kip1)) are critical for CCN5-induced growth inhibition of TNBC cells. In the absence of CCN5, p27(Kip1) resides mostly in the cytoplasm, which is associated with the aggressive nature of cancer cells. Mechanistically, CCN5 inhibits Skp2 expression, which seems to stabilize the p27(Kip1) protein in these cells. On the other hand, CCN5 also recruits FOXO3a to mediate the transcriptional regulation of p27(Kip1). The recruitment of FOXO3a is achieved by the induction of its expression and activity through shifting from cytoplasm to the nucleus. Our data indicate that CCN5 blocks PI3K/AKT signaling to dephosphorylate at S318, S253 and Thr32 in FOXO3a for nuclear relocalization and activation of FOXO3a. Moreover, inhibition of α6β1 receptors diminishes CCN5 action on p27(Kip1) in TNBC cells. Collectively, these data suggest that CCN5 effectively inhibits TNBC growth through the accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation, and thus, activation of CCN5 may have the therapeutic potential to kill TNBC.Oncogene advance online publication, 18 August 2014; doi:10.1038/onc.2014.250.
Insulin-like growth factor (IGF)-I is normally produced from hepatocytes and other sources, stimulates protein synthesis, cell survival and proliferation through receptor-mediated activation of PI3K and MAPK and targets specific molecules within the pancreatic islet cells. The current study was designed to identify novel targets that may mediate its pro-islet actions. Whole-genome cDNA microarray analysis in IGF-I overexpressing islets identified 82 genes specifically up- or down-regulated. Prominent among them was CCN5/WISP2 whose expression was increased 3- and 2-fold at the mRNA and protein levels. Dual-labeled immunofluorescence revealed that CCN5 expression was low in the β-cells of wild-type islets but was significantly induced in response to IGF-I overexpression. In vitro treatment of mouse islets with IGF-I increased both CCN5 mRNA and protein levels significantly. To define the role of CCN5 in islet cell biology, we stably overexpressed its cDNA in insulinoma MIN6 cells and detected a 2-fold increase in the proliferation of MIN6-CCN5 compared to control cells that correlated with significant elevations in the levels of cyclin D1 and the phosphorylation of Akt and Erk2. Moreover, MIN6-CCN5 cells were found to be resistant to streptozotocin-induced cell death. Using confocal microscopy and subcellular fractionation overexpressed CCN5 exhibited cytoplasmic accumulation upon stimulation by high glucose. Our results indicate that CCN5, which is minimally expressed in islet β-cells, is strongly and directly induced by IGF-I. CCN5 overexpression stimulates the proliferation of insulinoma cells, activates Akt kinase, and inhibits streptozotocin-induced apoptosis, suggesting that increased CCN5 expression contributes to IGF-I-stimulated islet cell growth and/or survival.
Chronic kidney disease is reaching epidemic proportions worldwide and there is no effective treatment. Connective tissue growth factor (CCN2) has been suggested as a risk biomarker and a potential therapeutic target for renal diseases, but its specific receptor has not been identified. Epidermal growth factor receptor (EGFR) participates in kidney damage, but whether CCN2 activates the EGFR pathway is unknown. Here, we show that CCN2 is a novel EGFR ligand. CCN2 binding to EGFR extracellular domain was demonstrated by surface plasmon resonance. CCN2 contains four distinct structural modules. The carboxyl-terminal module (CCN2(IV)) showed a clear interaction with soluble EGFR, suggesting that EGFR binding site is located in this module. Injection of CCN2(IV) in mice increased EGFR phosphorylation in the kidney, mainly in tubular epithelial cells. EGFR kinase inhibition decreased CCN2(IV)-induced renal changes (ERK activation and inflammation). Studies in cultured tubular epithelial cells showed that CCN2(IV) binds to EGFR leading to ERK activation and proinflammatory factors overexpression. CCN2 interacts with the neurotrophin receptor TrkA, and EGFR/TrkA receptor crosstalk was found in response to CCN2(IV) stimulation. Moreover, endogenous CCN2 blockade inhibited TGF-β-induced EGFR activation. These findings indicate that CCN2 is a novel EGFR ligand that contributes to renal damage through EGFR signalling.
Type 2 diabetes is characterized by both insulin resistance and progressive deterioration of β-cell function. The forkhead transcription factor FoxO1 is a prominent mediator of insulin signaling in β-cells. We reasoned that identification of FoxO1 target genes in β-cells could reveal mechanisms linking β-cell dysfunction to insulin resistance. In this study, we report the characterization of Nov/Ccn3 as a novel transcriptional target of FoxO1 in pancreatic β-cells. FoxO1 binds to an evolutionarily conserved response element in the Ccn3 promoter to regulate its expression. Accordingly, CCN3 levels are elevated in pancreatic islets of mice with overexpression of a constitutively active form of FoxO1 or insulin resistance. Our functional studies reveal that CCN3 impairs β-cell proliferation concomitantly with a reduction in cAMP levels. Moreover, CCN3 decreases glucose oxidation, which translates into inhibition of glucose-stimulated Ca(2+) entry and insulin secretion. Our results identify CCN3, a novel transcriptional target of FoxO1 in pancreatic β-cells, as a potential target for therapeutic intervention in the treatment of diabetes.
Wnt family members are critical to many developmental processes, and components of the Wnt signaling pathway have been linked
to tumorigenesis in familial and sporadic colon carcinomas. Here we report the identification of two genes, WISP-1 and WISP-2, that are up-regulated in the mouse mammary epithelial cell line C57MG transformed by Wnt-1, but not by Wnt-4. Together with
a third related gene, WISP-3, these proteins define a subfamily of the connective tissue growth factor family. Two distinct systems demonstrated WISP induction to be associated with the expression of Wnt-1. These included (i) C57MG cells infected with a Wnt-1 retroviral vector or expressing Wnt-1 under the control of a tetracyline repressible promoter,
and (ii) Wnt-1 transgenic mice. The WISP-1 gene was localized to human chromosome 8q24.1–8q24.3. WISP-1 genomic DNA was amplified in colon cancer cell lines and in human colon tumors and its RNA overexpressed (2- to >30-fold)
in 84% of the tumors examined compared with patient-matched normal mucosa. WISP-3 mapped to chromosome 6q22–6q23 and also was overexpressed (4- to >40-fold) in 63% of the colon tumors analyzed. In contrast,
WISP-2 mapped to human chromosome 20q12–20q13 and its DNA was amplified, but RNA expression was reduced (2- to >30-fold) in 79%
of the tumors. These results suggest that the WISP genes may be downstream of Wnt-1 signaling and that aberrant levels of WISP expression in colon cancer may play a role in colon tumorigenesis.
Inability to recruit new adipose cells following weight gain leads to inappropriate enlargement of existing cells (hypertrophic obesity) associated with inflammation and a dysfunctional adipose tissue. We found increased expression of WNT1 inducible signaling pathway protein 2 (WISP2) and other markers of WNT activation in human abdominal s.c. adipose tissue characterized by hypertrophic obesity combined with increased visceral fat accumulation and insulin resistance. WISP2 activation in the s.c. adipose tissue, but not in visceral fat, identified the metabolic syndrome in equally obese individuals. WISP2 is a novel adipokine, highly expressed and secreted by adipose precursor cells. Knocking down WISP2 induced spontaneous differentiation of 3T3-L1 and human preadipocytes and allowed NIH 3T3 fibroblasts to become committed to the adipose lineage by bone morphogenetic protein 4 (BMP4). WISP2 forms a cytosolic complex with the peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activator zinc finger protein 423 (Zfp423), and this complex is dissociated by BMP4 in a SMAD-dependent manner, thereby allowing Zfp423 to enter the nucleus, activate PPARγ, and commit the cells to the adipose lineage. The importance of intracellular Wisp2 protein for BMP4-induced adipogenic commitment and PPARγ activation was verified by expressing a mutant Wisp2 protein lacking the endoplasmic reticulum signal and secretion sequence. Secreted Wnt/Wisp2 also inhibits differentiation and PPARγ activation, albeit not through Zfp423 nuclear translocation. Thus adipogenic commitment and differentiation is regulated by the cross-talk between BMP4 and canonical WNT signaling and where WISP2 plays a key role. Furthermore, they link WISP2 with hypertrophic obesity and the metabolic syndrome.
The Wnt signaling pathway was initially discovered for its role in tumorigenesis and the development of Drosophila and other eukaryotic organisms. The key effector of this pathway, the bipartite transcription factor β-cat/TCF, is formed by free β-catenin (β-cat) and a TCF protein, including TCF7L2. Extensive recent investigations have highlighted the role of the Wnt signaling pathway in metabolic homeostasis and its implication in diabetes and other metabolic diseases. Genome-wide association studies have shown that several key components of the Wnt signaling pathway are implicated in metabolic homeostasis and the development of type 2 diabetes (T2D). Despite controversial observations regarding the role of Wnt signaling in the development and function of pancreatic islets, the discovery of the association between certain single nucleotide polymorphisms of TCF7L2 and T2D susceptibility has fueled great efforts to explore the role of Wnt signaling in the function of pancreatic β-cells and glucose homeostasis. Here we have introduced our basic understanding of the canonical Wnt signaling pathway, summarized our current knowledge on its implication in metabolic homeostasis and T2D, discussed the work on TCF7L2 as a T2D susceptibility gene, and presented the controversial role of Wnt signaling and TCF7L2 in pancreatic islets as well as their potential metabolic function in other organs. We then expanded our view into the crosstalk among Wnt, insulin and FOXO signaling cascades, which further illustrates the complexity of the Wnt signaling pathway in metabolic homeostasis. Finally, we have presented our perspectives.
A major goal in diabetes research is to find ways to enhance the mass and function of insulin secreting β-cells in the endocrine pancreas to prevent and/or delay the onset or even reverse overt diabetes. In this Perspectives in Diabetes article, we highlight the contrast between the relatively large body of information that is available in regard to signaling pathways, proteins, and mechanisms that together provide a road map for efforts to regenerate β-cells in rodents versus the scant information in human β-cells. To reverse the state of ignorance regarding human β-cell signaling, we suggest a series of questions for consideration by the scientific community to construct a human β-cell proliferation road map. The hope is that the knowledge from the new studies will allow the community to move faster towards developing therapeutic approaches to enhance human β-cell mass in the long-term goal of preventing and/or curing type 1 and type 2 diabetes.
Abnormalities in the cAMP/PKA signaling pathway have been linked to the formation of benign adrenal tumors, as well as a possible predisposition to adrenocortical cancer. Mutations in the G-protein coupled receptor are associated with McCune-Albright syndrome and ACTH-independent macronodular adrenal hyperplasia, while defects in cAMP-dependent protein kinase A can lead to the development of Carney's complex, as well as primary pigmented nodular adrenocortical disease (PPNAD), and micronodular adrenocortical hyperplasia (MAH). Defects in phosphodiesterases, which regulate cAMP levels, have also been demonstrated in PPNAD and MAH. The Wnt signaling pathway, which is involved in oncogenesis in a variety of tumors, has also been implicated in adrenocortical tumorigenesis. MicroRNA profiling has added to our understanding of the signaling pathways involved in tumor formation in the adrenal cortex. Will this all lead to the development of specific targets for pharmacologic therapies? In this article, we review the molecular genetics of adrenocortical tumors and refer to potential targets for pharmacologic therapy.
The gain of plasticity by a subset of cancer cells is a unique but common sequence of cancer progression from epithelial phenotype to mesenchymal phenotype (EMT) that is followed by migration, invasion and metastasis to a distant organ, and drug resistance. Despite multiple studies, it is still unclear how cancer cells regulate plasticity. Recent studies from our laboratory and others' proposed that CCN5/WISP-2, which is found intracellularly (in the nucleus and cytoplasm) and extracellularly, plays a negative regulator of plasticity. It prevents the EMT process in breast cancer cells as well as pancreatic cancer cells. Multiple genetic insults, including the gain of p53 mutations that accumulate over the time, may perturb CCN5 expression in non-invasive breast cancer cells, which ultimately helps cells to gain invasive phenotypes. Moreover, emerging evidence indicates that several oncogenic lesions such as miR-10b upregulation and activation of TGF-β-signaling can accumulate during CCN5 crisis in breast cancer cells. Collectively, these studies indicate that loss of CCN5 activity may promote breast cancer progression; application of CCN5 protein may represent a novel therapeutic intervention in breast cancer and possibly pancreatic cancer.
Members of the CCN family of matricellular proteins are crucial for embryonic development and have important roles in inflammation, wound healing and injury repair in adulthood. Deregulation of CCN protein expression or activities contributes to the pathobiology of various diseases - many of which may arise when inflammation or tissue injury becomes chronic - including fibrosis, atherosclerosis, arthritis and cancer, as well as diabetic nephropathy and retinopathy. Emerging studies indicate that targeting CCN protein expression or signalling pathways holds promise in the development of diagnostics and therapeutics for such diseases. This Review summarizes the biology of CCN proteins, their roles in various pathologies and their potential as therapeutic targets.
Type 1 and type 2 diabetes result from an absolute or relative reduction in functional β-cell mass. One approach to replacing lost β-cell mass is transplantation of cadaveric islets; however, this approach is limited by lack of adequate donor tissue. Therefore, there is much interest in identifying factors that enhance β-cell differentiation and proliferation in vivo or in vitro. Connective tissue growth factor (CTGF) is a secreted molecule expressed in endothelial cells, pancreatic ducts, and embryonic β cells that we previously showed is required for β-cell proliferation, differentiation, and islet morphogenesis during development. The current study investigated the tissue interactions by which CTGF promotes normal pancreatic islet development. We found that loss of CTGF from either endothelial cells or β cells results in decreased embryonic β-cell proliferation, making CTGF unique as an identified β cell-derived factor that regulates embryonic β-cell proliferation. Endothelial CTGF inactivation was associated with decreased islet vascularity, highlighting the proposed role of endothelial cells in β-cell proliferation. Furthermore, CTGF overexpression in β cells during embryogenesis using an inducible transgenic system increased islet mass at birth by promoting proliferation of immature β cells, in the absence of changes in islet vascularity. Together, these findings demonstrate that CTGF acts in an autocrine manner during pancreas development and suggest that CTGF has the potential to enhance expansion of immature β cells in directed differentiation or regeneration protocols.
CTNNB1/β-catenin mutations and activation of Wnt/β-catenin pathway are frequent in adult adrenocortical tumors (ACT), but data on childhood ACT are lacking.
The aim of the study was to investigate the presence of Wnt/β-catenin pathway abnormalities in childhood ACT.
Clinicopathological findings and outcome of 62 childhood ACT patients were analyzed regarding CTNNB1 mutations and the expression of Wnt-related genes (CTNNB1; WNT4, a Wnt ligand; SFRP1, DKK3, and AXIN1, Wnt inhibitors; TCF7, a transcription factor; and MYC and WISP2, target genes) by quantitative PCR and immunohistochemistry.
CTNNB1-activating mutations were found in only four of 62 ACT (6%), all of them harboring TP53 mutation. There was association between the presence of CTNNB1 mutations and death (P = 0.02). Diffuse β-catenin accumulation was found in 71% of ACT, even in ACT without CTNNB1 mutations. Compared to normal adrenals, ACT presented increased expression of CTNNB1 (P = 0.008) and underexpression of Wnt inhibitor genes: DKK3 (P < 0.0001), SFRP1 (P = 0.05), and AXIN1 (P = 0.04). With regard to Wnt/β-catenin target genes, ACT presented increased expression of WISP2 but lower expression of MYC. Higher overall survival was associated with underexpression of SFRP1 (P = 0.01), WNT4 (P = 0.004), and TCF7 (P < 0.01).
CTNNB1 mutations are not common in childhood ACT but appear to associate with poor prognosis. Nevertheless, most ACT exhibit increased expression of β-catenin and WISP2 and reduced expression of Wnt inhibitor genes (DKK3, SFRP1, and AXIN1). Thus, in addition to CTNNB1 mutations, other genetic events affecting the Wnt/β-catenin pathway may be involved in childhood adrenocortical tumorigenesis.
There is widespread interest in defining factors and mechanisms that stimulate proliferation of pancreatic islet cells. Wnt signaling is an important regulator of organ growth and cell fates, and genes encoding Wnt-signaling factors are expressed in the pancreas. However, it is unclear whether Wnt signaling regulates pancreatic islet proliferation and differentiation. Here we provide evidence that Wnt signaling stimulates islet β cell proliferation. The addition of purified Wnt3a protein to cultured β cells or islets promoted expression of Pitx2, a direct target of Wnt signaling, and Cyclin D2, an essential regulator of β cell cycle progression, and led to increased β cell proliferation in vitro. Conditional pancreatic β cell expression of activated β-catenin, a crucial Wnt signal transduction protein, produced similar phenotypes in vivo, leading to β cell expansion, increased insulin production and serum levels, and enhanced glucose handling. Conditional β cell expression of Axin, a potent negative regulator of Wnt signaling, led to reduced Pitx2 and Cyclin D2 expression by β cells, resulting in reduced neonatal β cell expansion and mass and impaired glucose tolerance. Thus, Wnt signaling is both necessary and sufficient for islet β cell proliferation, and our study provides previously unrecognized evidence of a mechanism governing endocrine pancreas growth and function.
• Cyclin D2
• diabetes mellitus
• islets of Langerhans
• pancreas
• Pitx2
The CCN (cyr61, ctgf, nov) proteins (CCN1–6) are an important family of matricellular regulatory factors involved in internal
and external cell signaling. They are central to essential biological processes such as adhesion, proliferation, angiogenesis,
tumorigenesis, wound healing, and modulation of the extracellular matrix. They possess a highly conserved modular structure
with four distinct modules that interact with a wide range of regulatory proteins and ligands. However, at the structural
level, little is known although their biological function(s) seems to require cooperation between individual modules. Here
we present for the first time structural determinants of two of the CCN family members, CCN3 and CCN5 (expressed in Escherichia coli), using small angle x-ray scattering. The results provide a description of the overall molecular shape and possible general
three-dimensional modular arrangement for CCN proteins. These data unequivocally provide insight of the nature of CCN protein(s)
in solution and thus important insight into their structure-function relationships.
CCN proteins are important modulators of development and function of adult organs. In this study, we examined the localization and expression of the six CCN family members in normal adult human skin and during wound healing in vivo. Transcript and protein expression were studied by laser-capture microdissection-coupled real-time PCR and immunohistochemistry, respectively. Our results demonstrate that CCN1, CCN4, and CCN6 are expressed at relatively low levels in normal human skin. CCN2, CCN3, and CCN5 are the most highly expressed transcripts in the epidermis. CCN3 and CCN5 proteins are prominent in epidermal keratinocytes, whereas CCN2 is primarily expressed in melanocytes. Differential expression within epidermal layers suggests that CCN3 and CCN5 are linked with keratinocyte differentiation. CCN2, CCN3 and CCN5, are the three most highly expressed transcripts in the dermis. Their respective proteins are produced to various extents by dermal fibroblasts, blood vessels, eccrine sweat glands and hair follicles. We find that most CCN family members are temporally and specifically regulated during different phases (inflammation, proliferation, and remodeling) of partial thickness wound repair. By highlighting spatial-temporal regulations of CCN family member expression in relation to cell proliferation and differentiation, our results suggest a diverse range of functions for CCN proteins in both epidermal and dermal cells, and provides a solid reference for interpretation of future studies aimed at understanding the role of CCN proteins in human skin physiology and diseases.
Connective tissue growth factor (CTGF/CCN2) belongs to the CCN family of matricellular proteins, comprising Cyr61, CTGF, NovH and WISP1-3. The CCN proteins contain an N-terminal signal peptide followed by four conserved domains sharing sequence similarities with the insulin-like growth factor binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a C-terminal growth factor cysteine knot domain. To investigate the role of CCN2 in breast cancer, we transfected MCF-7 cells with full-length CCN2, and with four mutant constructs in which one of the domains had been deleted. MCF-7 cells stably expressing full-length CCN2 demonstrated reduced cell proliferation, increased migration in Boyden chamber assays and promoted angiogenesis in chorioallantoic membrane assays compared to control cells. Deletion of the C-terminal cysteine knot domain, but not of any other domain-deleted mutants, abolished activities mediated by full-length CCN2. We have dissected the role of CCN2 in breast tumorigenesis on a structural basis.
CCN5 is a member of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family and was identified as an estrogen-inducible gene in estrogen receptor-positive cell lines. However, the role of CCN5 in breast carcinogenesis remains unclear. We report here that the CCN5 protein is localized mostly in the cytoplasm and in part in the nucleus of human tumor breast tissue. Using a heterologous transcription assay, we demonstrate that CCN5 can act as a transcriptional repressor presumably through association with histone deacetylase 1 (HDAC1). Microarray gene expression analysis showed that CCN5 represses expression of genes associated with epithelial-mesenchymal transition (EMT) as well as expression of key components of the transforming growth factor β (TGF-β) signaling pathway, prominent among them TGF-βRII receptor. We show that CCN5 is recruited to the TGF-βRII promoter, thereby providing a mechanism by which CCN5 restricts transcription of the TGF-βRII gene. Consistent with this finding, CCN5, we found, functions to suppress TGF-β-induced transcriptional responses and invasion that is concomitant with EMT. Thus, our data uncovered CCN5 as a novel transcriptional repressor that plays an important role in regulating tumor progression functioning, at least in part, by inhibiting the expression of genes involved in the TGF-β signaling cascade that is known to promote EMT.
CCN2/connective tissue growth factor (CTGF), a CCN family matricellular protein repressed in healthy hearts after birth, is induced in heart failure of various etiologies. Multiple cellular and biological functions have been assigned to CCN2/CTGF depending on cellular context. However, the functions and mechanisms of action of CCN2/CTGF in the heart as well as its roles in cardiac physiology and pathophysiology remain unknown. Transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) were generated and compared with nontransgenic littermate control (NLC) mice. Tg-CTGF mice displayed slightly lower cardiac mass and inconspicuous increase of myocardial collagen compared with NLC mice but no evidence of contractile dysfunction. Analysis of the myocardial transcriptome by DNA microarray revealed activation of several distinct gene programs in Tg-CTGF hearts involved in cardioprotection and growth inhibition. Indeed, Tg-CTGF mice subjected to ischemia-reperfusion injury by in situ transient occlusion of the left anterior descending coronary artery in vivo displayed reduced vulnerability with markedly diminished infarct size. These findings were recapitulated in isolated hearts perfused with recombinant human (h)CTGF before the ischemia-reperfusion procedure. Consistently, Tg-CTGF hearts, as well as isolated adult cardiac myocytes exposed to recombinant hCTGF, displayed enhanced phosphorylation and activity of the Akt/p70S6 kinase/GSK-3β salvage kinase pathway and induction of several genes with reported cardioprotective functions. Inhibition of Akt activities also prevented the cardioprotective phenotype of hearts from Tg-CTGF mice. This report provides novel evidence that CTGF confers cardioprotection by salvage phosphokinase signaling leading to inhibition of GSK-3β activities, activation of phospho-SMAD2, and reprogramming of gene expression.
CCN5 is one of six proteins in the CCN family. This family of proteins has been shown to play important roles in many processes, including proliferation, migration, adhesion, extracellular matrix regulation, angiogenesis, tumorigenesis, fibrosis, and implantation. In this review, we focus on the biological and putative pathophysiological roles of CCN5. This intriguing protein is structurally unique among the CCN family members, and has a unique biological activity profile as well.
Mammary oncogenesis is profoundly influenced by signaling pathways controlled by estrogen receptor alpha (ERalpha). Although it is known that ERalpha exerts its oncogenic effect by stimulating the proliferation of many human breast cancers through the activation of target genes, our knowledge of the underlying transcriptional mechanisms remains limited. Our published work has shown that the in vivo activity of LIM homeodomain transcription factors (LIM-HD) is critically regulated by cofactors of LIM-HD proteins (CLIM) and the ubiquitin ligase RING finger LIM domain-interacting protein (RLIM). Here, we identify CLIM and RLIM as novel ERalpha cofactors that colocalize and interact with ERalpha in primary human breast tumors. We show that both cofactors associate with estrogen-responsive promoters and regulate the expression of endogenous ERalpha target genes in breast cancer cells. Surprisingly, our results indicate opposing functions of LIM cofactors for ERalpha and LIM-HDs: whereas CLIM enhances transcriptional activity of LIM-HDs, it inhibits transcriptional activation mediated by ERalpha on most target genes in vivo. In turn, the ubiquitin ligase RLIM inhibits transcriptional activity of LIM-HDs but enhances transcriptional activation of endogenous ERalpha target genes. Results from a human breast cancer tissue microarray of 1,335 patients revealed a highly significant correlation of elevated CLIM levels to ER/progesterone receptor positivity and poor differentiation of tumors. Combined, these results indicate that LIM cofactors CLIM and RLIM regulate the biological activity of ERalpha during the development of human breast cancer.
Although previous in vitro studies predicted that CCN5/WISP-2 may act as an anti-invasive gene in breast cancer, the distribution pattern of CCN5 in breast cancer samples is conflicting. Thus, we systematically investigated the CCN5 expression profile in noninvasive and invasive breast tumor samples and its functional relevance in breast cancer progression. The studies showed that CCN5 expression is biphasic, such that in normal samples CCN5 expression is undetectable, whereas its expression is markedly increased in noninvasive breast lesions, including atypical ductal hyperplasia and ductal carcinoma in situ. Further, CCN5 mRNA and protein levels are significantly reduced as the cancer progresses from a noninvasive to invasive type. Additionally, we showed that CCN5 mRNA and protein level was almost undetectable in poorly differentiated cancers compared with the moderately or well-differentiated samples and its expression inversely correlated with lymph node positivity. The result was further supported by evaluating the RNA expression profile in microdissected sections using real-time PCR analysis. Therefore, our data suggest a protective function of CCN5 in noninvasive breast tumor cells. This hypothesis was further supported by our in vitro studies illuminating that CCN5 is a negative regulator of migration and invasion of breast cancer cells, and these events could be regulated by CCN5 through the modulation of the expression of genes essential for an invasive front. These include Snail-E-cadherin signaling and matrix metalloproteinase (MMP)-9 and MMP-2. Collectively, these studies suggest that the protective effect of CCN5 in breast cancer progression may have important therapeutic implications.
Chicken embryo fibroblasts (CEF) infected with a temperature-sensitive Rous sarcoma virus (RSV) mutant, tsNY72-4, express a set of pp60v-src-induced RNAs soon after shift to the permissive temperature. By subtractive and differential screening, we have cloned 12 of these sequences, 2 of which were c-fos and krox-24. Serum induced all the v-src-inducible genes tested, suggesting that these genes serve roles in normal cell division and are not specific to transformation per se. Significantly, however, v-src produced prolonged, and in some cases kinetically complex, patterns of induction compared to serum. For most of the clones, phorbol 12-tetradecanoate 13-acetate (TPA) induced mRNAs with kinetics similar to that of serum. However, one clone (CEF-4) was expressed in a biphasic manner. Another (CEF-10) was repressed by TPA at 1 hr, after which this mRNA was permanently induced. The pattern of repression-induction of CEF-10 mRNA is the inverse of protein kinase C (PKC) activity in the cell, suggesting that PKC actively represses this gene. In vivo expression of CEF-10 mRNA is restricted predominantly to the lung. A full-length CEF-10 cDNA encodes a 41-kDa protein that has an amino-terminal signal peptide for secretion, contains a markedly high number of cysteine residues, and shows no sequence similarity to known proteins.
The serine/threonine protein kinase PKB (also known as Akt) is thought to be a key mediator of signal transduction processes. The identification of PKB substrates and the role PKB phosphorylation plays in regulating these molecules have been a major focus of research in recent years. A recently developed motif-profile scoring algorithm that can be used to scan the genome for potential PKB substrates is therefore a useful tool, although additional considerations, such as the evolutionary conservation of the phosphorylation site, must also be taken into account. Recent evidence indicates that PKB plays a key role in cancer progression by stimulating cell proliferation and inhibiting apoptosis and is also probably a key mediator of insulin signalling. These findings indicate that PKB is likely to be a hot drug target for the treatment of cancer, diabetes and stroke. There are, however, a number of pitfalls of methodologies currently employed to study PKB function, and therefore caution should be used in interpretation of such experiments.
Stimulation of endogenous β-cell expansion could facilitate regeneration in patients with diabetes. In mice, connective tissue growth factor (CTGF) is expressed in embryonic β-cells and in adult β-cells during periods of expansion. We discovered that in embryos, CTGF is necessary for β-cell proliferation, and increased CTGF in β-cells promotes proliferation of immature (MafA-) insulin+ cells. CTGF over-expression, under non-stimulatory conditions, does not increase adult β-cell proliferation. Here, we tested the ability of CTGF to promote β-cell proliferation and regeneration after partial β-cell destruction. β-cell mass reaches 50% recovery after 4 weeks of CTGF treatment, primarily via increased β-cell proliferation, which is enhanced as early as 2 days of treatment. CTGF treatment increases the number of immature β-cells, but promotes proliferation of both mature and immature β-cells. A shortened β-cell replication refractory period is also observed. CTGF treatment up-regulates positive cell cycle regulators and factors involved in β-cell proliferation, including HGF, serotonin synthesis, and integrin β1. Ex vivo treatment of whole islets with recombinant human CTGF induces β-cell replication and gene expression changes consistent with those observed in vivo, demonstrating that CTGF acts directly on islets to promote β-cell replication. Thus, CTGF can induce replication of adult mouse β-cells given a permissive micro-environment.
Wingless-type MMTV integration site family member (WNT) signaling and WNT-inhibitors have been implicated in regulation of adipogenesis, insulin resistance, pancreatic function, and inflammation. Our goal was to determine serum proteins involved in WNT signaling (WNT5 and WISP2) and WNT inhibition (SFRP4 and SFRP5) as they relate to obesity, serum adipokines, insulin resistance, insulin secretion, and inflammation in humans. Study population comprised 57 insulin resistant women with polycystic ovary syndrome (PCOS) and 27 reference women. In a cross-sectional study, blood samples were obtained at fasting, during oral, and frequently sampled intravenous glucose tolerance tests. Serum WNT5, WISP2, and SFRP4 concentrations did not differ between PCOS vs. reference women. Serum WNT5 correlated inversely with weight both in PCOS and reference women, and correlated directly with insulin response during oral glucose tolerance test in PCOS women. Serum WISP2 correlated directly with fatty acid binding protein 4. Serum SFRP5 did not differ between obese (n=32) vs. nonobese (n=25) PCOS women, but reference women had lower SFRP5 (p<5×10(-6) as compared to both PCOS groups). Serum SFRP5 correlated inversely with IL-1β, TNF-α, cholesterol, and apoprotein B. These findings demonstrated that WNT5 correlated inversely with adiposity and directly with insulin response, and the WNT-inhibitor SFRP5 may be anti-inflammatory. Better understanding of the role of WNT signaling in obesity, insulin resistance, insulin secretion, lipoprotein metabolism, and inflammation is important for prevention and treatment of metabolic syndrome, diabetes and cardiovascular disease.
WNT inducible secreted protein 2 (WISP2) has been linked with a variety of human cancer types and may contribute to cancer metastasis. The current study investigated the importance of WISP2 in colorectal cancer cells, examining the impact of targeting WISP2 on Caco-2 cell invasion and motility together with potential mechanisms of action.
WISP2 expression was targeted in Caco-2 cells using a ribozyme transgene system and successful knockdown was verified using reverse transcription-polymerase chain reaction (RT-PCR). The impact of WISP2 knockout (Caco-2(WISP2 KO)) on cell growth, adhesion, motility and invasion was examined using a number of in vitro functional assays. In vitro invasion assays were repeated in the presence of wingless-type MMTV integration site family (WNT) inhibitors (FH535 and IWP-2) to investigate the role of the WNT-signalling pathway in the regulation of cell invasion by WISP2. Quantitative-PCR was conducted to measure matrix metalloproteinase (MMP) expression in control [wild-type (Caco-2(WT)) and cells containing the empty pEF6 plasmid (Caco-2(pEF6))] and Caco-2(WISP2 KO) cells.
WISP2 knockout resulted in a significant increase in Caco-2 cell invasion and motility (p<0.05 in comparison to wild-type and plasmid control Caco-2 cells). WISP2 knockout had no significant effect on Caco-2 cell growth rate in 3- and 5-day incubation and no significant impact on Caco-2 cell-matrix adhesion rates (p>0.05). Expression analysis of a number of MMPs indicated an insignificant up-regulation of MMP2, MMP9 (p>0.05) but significant up-regulation of MMP7 (p=0.025) in Caco-2(WISP2 KO) cells compared to controls. Inhibition of WNT signalling using FH535 and IWP-2 brought about a significant or borderline significant decrease in Caco-2(WISP2 KO) cell invasion (FH535 p=0.065) and (IWP-2 p=0.002) and negated the pro-invasive effect of targeting WISP2 in Caco-2 cells.
WISP2 knockout significantly increased Caco-2 cell invasion and motility. Up-regulation of MMP2, -7 and -9 may indicate that WISP2 regulates invasion and motility through MMPs. Regulation of invasion by WISP2 may involve the WNT signalling pathway.
The CCN family of proteins includes six members presently known as CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6. These proteins were originally designated CYR61, CTGF, NOV, and WISP-1, WISP-2, WISP-3. Although these proteins share a significant amount of structural features and a partial identity with other large families of regulatory proteins, they exhibit different biological functions. A critical examination of the progress made over the past two decades, since the first CCN proteins were discovered brings me to the conclusion that most of our present knowledge regarding the functions of these proteins was predicted very early after their discovery. In an effort to point out some of the gaps that prevent us to reach a comprehensive view of the functional interactions between CCN proteins, it is necessary to reconsider carefully data that was already published and put aside, either because the scientific community was not ready to accept them, or because they were not fitting with the « consensus » when they were published. This review article points to avenues that were not attracting the attention that they deserved. However, it is quite obvious that the six members of this unique family of tetra-modular proteins must act in concert, either simultaneously or sequentially, on the same sites or at different times in the life of living organisms. A better understanding of the spatio-temporal regulation of CCN proteins expression requires considering the family as such, not as a set of single proteins related only by their name. As proposed in this review, there is enough convincing pieces of evidence, at the present time, in favor of these proteins playing a role in the coordination of multiple signaling pathways, and constituting a Centralized Communication Network. Deciphering the hierarchy of regulatory circuits involved in this complex system is an important challenge for the near future. In this article, I would like to briefly review the concept of a CCN family of proteins and critically examine the progress made over the past 10 years in the understanding of their biological functions and involvement in both normal and pathological processes.
Nutrients and certain growth factors stimulate pancreatic β-cell mitogenesis, however, the appropriate mitogenic signal transduction
pathways have not been defined. In the glucose-sensitive pancreatic β-cell line, INS-1, it was found that glucose (6–18 mm) independently increased INS-1 cell proliferation (>20-fold at 15 mm glucose). Insulin-like growth factor I (IGF-I)-induced INS-1 cell proliferation was glucose-dependent only in the physiologically
relevant concentration range (6–18 mm glucose). The combination of IGF-I and glucose was synergistic, increasing INS-1 cell proliferation >50-fold at 15 mm glucose + 10 nm IGF-I. Glucose metabolism and phosphatidylinositol 3′-kinase (PI 3′-kinase) activation were necessary for both glucose and
IGF-I-stimulated INS-1 cell proliferation. IGF-I and 15 mm glucose increased tyrosine phosphorylation mediated recruitment of Grb2/mSOS and PI 3′-kinase to IRS-2 and pp60. Glucose
and IGF-I also induced Shc association with Grb2/mSOS. Glucose (3–18 mm) and IGF-I, independently of glucose, activated mitogen-activated protein kinase but this did not correlate with IGF-I-induced
β-cell proliferation. In contrast, p70S6K was activated with increasing glucose concentration (between 6 and 18 mm), and potentiated by IGF-I in the same glucose concentration range which correlated with INS-1 cell proliferation rate. Thus,
glucose and IGF-I-induced β-cell proliferation were mediated via a signaling mechanism that was facilitated by mitogen-activated
protein kinase but dependent on IRS-mediated induction of PI 3′-kinase activity and downstream activation of p70S6K. The glucose dependence of IGF-I mediated INS-1 cell proliferation emphasizes β-cell signaling mechanisms are rather unique
in being tightly linked to glycolytic metabolic flux.
Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich matricellular secreted protein that regulates diverse cell functions including adhesion, migration, proliferation, differentiation, survival, senescence and apoptosis. In the pancreas, CTGF/CCN2 regulates critical functions including β cell replication during embryogenesis, stimulation of fibrogenic pathways in pancreatic stellate cells during pancreatitis, and regulation of the epithelial and stromal components in pancreatic ductal adenocarcinoma. This article reviews the evidence establishing CTGF/CCN2 as an important player in pancreatic physiology and pathology, highlighting the specific cell types that are involved in each process and the importance of CTGF/CCN2 as a component of autocrine or paracrine signaling within or between these various cells. Translational applications, including the potential for CTGF/CCN2-based therapies in diabetes, fibrosis, or cancer, are discussed.
Current endeavors in the type 2 diabetes (T2D) field include gaining a better understanding of extracellular signaling pathways that regulate pancreatic islet function. Recent data suggest that both Bmp and Wnt pathways are operative in pancreatic islets and play a positive role in insulin secretion and glucose homeostasis. Our laboratory found the dual Bmp and Wnt antagonist Sostdc1 to be upregulated in a mouse model of islet dysmorphogenesis and nonimmune-mediated lean diabetes. Because Bmp signaling has been proposed to enhance β-cell function, we evaluated the role of Sostdc1 in adult islet function using animals in which Sostdc1 was globally deleted. While Sostdc1-null animals exhibited no pancreas development phenotype, a subset of mutants exhibited enhanced insulin secretion and improved glucose homeostasis compared with control animals after 12-wk exposure to high-fat diet. Loss of Sostdc1 in the setting of metabolic stress results in altered expression of Bmp-responsive genes in islets but did not affect expression of Wnt target genes, suggesting that Sostdc1 primarily regulates the Bmp pathway in the murine pancreas. Furthermore, our data indicate that removal of Sostdc1 enhances the downregulation of the closely related Bmp inhibitors Ctgf and Gremlin in islets after 8-wk exposure to high-fat diet. These data imply that Sostdc1 regulates expression of these inhibitors and provide a means by which Sostdc1-null animals show enhanced insulin secretion and glucose homeostasis. Our studies provide insights into Bmp pathway regulation in the endocrine pancreas and reveal new avenues for improving β-cell function under metabolic stress.
There is increasing awareness that estrogens may affect cell functions through the integration with a network of signaling pathways. The IGF system is a phylogenetically highly conserved axis that includes the insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) pathways, which are of crucial importance in the regulation of metabolism and cell growth in relationship to nutrient availability. Numerous studies nowadays document that estrogens cooperate with IGF system at multiple levels both in physiology and in disease. Several studies have focused on this bidirectional cross-talk in central nervous system, in mammary gland development and in cancer. Notably, cancer cells show frequent deregulation of the IGF system with overexpression of IR and/or IGF-IR and their ligands as well as frequent upregulation of the classical estrogen receptor (ER)α and the novel ER named GPER. Recent studies have, therefore, unraveled further mechanisms of cross-talk involving membrane initiated estrogen actions and the IGF system in cancer, that converge in the stimulation of pro-tumoral effects. These studies offer hope for new strategies aimed at the treatment of estrogen related cancers in order to prevent an estrogen-independent and more aggressive tumor progression.
WISP-2 is a Wnt-1-induced signaling protein identified as a member of CCN growth factor family. A role for this molecule during tumorigenesis is suspected but remains unproven. Here we show that WISP-2 expression was undetectable, or minimally detectable, in nontransformed human mammary epithelial cells, but was overexpressed in MCF-7 cells. Expression of WISP-2 in MCF-7 cells was modulated by serum and correlated with the serum-induced MCF-7 tumor cell proliferation, suggesting that WISP-2 is serum responsive and may be a positive regulator of tumor cell proliferation.
Members of the CCN (CYR61/CTGF/NOV) family have emerged as dynamically expressed, extracellular matrix-associated proteins that play critical roles in cardiovascular and skeletal development, injury repair, fibrotic diseases and cancer. The synthesis of CCN proteins is highly inducible by serum growth factors, cytokines, and environmental stresses such as hypoxia, UV exposure, and mechanical stretch. Consisting of six secreted proteins in vertebrate species, CCNs are typically comprised of four conserved cysteine-rich modular domains. They function primarily through direct binding to specific integrin receptors and heparan sulfate proteoglycans, thereby triggering signal transduction events that culminate in the regulation of cell adhesion, migration, proliferation, gene expression, differentiation, and survival. CCN proteins can also modulate the activities of several growth factors and cytokines, including TGF-β, TNFα, VEGF, BMPs, and Wnt proteins, and may thereby regulate a broad array of biological processes. Recent studies have uncovered novel CCN activities unexpected for matricellular proteins, including their ability to induce apoptosis as cell adhesion substrates, to dictate the cytotoxicity of inflammatory cytokines such as TNFα, and to promote hematopoietic stem cell self-renewal. As potent regulators of angiogenesis and chondrogenesis, CCNs are essential for successful cardiovascular and skeletal development during embryogenesis. In the adult, the expression of CCN proteins is associated with injury repair and inflammation, and has been proposed as diagnostic or prognostic markers for diabetic nephropathy, hepatic fibrosis, systemic sclerosis, and several types of cancer. Targeting CCN signaling pathways may hold promise as a strategy of rational therapeutic design.
Programmed cell death 4 (Pdcd4), a novel tumor suppressor, inhibits neoplastic transformation and tumor invasion. In this study, the authors found that knockdown of Pdcd4 promoted cell proliferation and up-regulated cyclin D1 expression. Previously, the authors demonstrated that Pdcd4 knockdown activated NF-κB-dependent transcription. Mutations of NF-κB binding sites on the cyclin D1 promoter attenuated the cyclin D1 promoter activity induced by Pdcd4 knockdown. In addition, knockdown of NF-κB/IκB kinase (IKK) α or IKKβ, the kinase regulating NF-κB activation, inhibited cyclin D1 promoter activity and cyclin D1 expression, indicating that up-regulation of cyclin D1 by Pdcd4 knockdown is contributed, at least in part, by NF-κB activation. To investigate the mechanism of how Pdcd4 knockdown activates NF-κB, the authors found that the levels of AKT phosphorylation and AKT kinase activity were increased in the Pdcd4 knockdown cells. Conversely, ectopic expression of Pdcd4 inhibited AKT phosphorylation and cyclin D1 expression, suggesting that Pdcd4 regulates AKT activity and cyclin D1 expression. Furthermore, knockdown of AKT in the Pdcd4 knockdown cells inhibited IKK phosphorylation, NF-κB activation, cyclin D1 promoter activity, and cyclin D1 expression as well as cell proliferation. Taken together, these findings suggest that activation of NF-κB by Pdcd4 knockdown through AKT contributes to the elevated expression of cyclin D1, thus providing new insights into how loss of Pdcd4 expression promotes tumor development.
Wisp2/CCN5 belongs to CCN family proteins which are involved in cell proliferation, angiogenesis, tumorigenesis and wound healing. Although a number of studies on the roles of Wisp2/CCN5 in cancers have been reported, no study on the expression and function of Wisp2/CCN5 in the central nervous system has been reported. In this study, we focused on Wisp2/CCN5 that was up-regulated in nervous tissues in GM3-only mice. Over-expression of Wisp2/CCN5 enhanced neurite outgrowth potently after serum withdrawal with increased phosphorylation levels of Akt and ERKs. When cells were cultured with recombinant Wisp2/CCN5 proteins, more and longer neurites were formed than in the controls. Thus, we demonstrated for the first time that Wisp2/CCN5 facilitates neurite formation in a mouse neuroblastoma cell line, Neuro2a. Akt phosphorylation induced by recombinant Wisp2/CCN5 was suppressed after knockdown of integrin β1. Moreover, Wisp2/CCN5-over-expressing cells were resistant to apoptosis induced by H(2)O(2). These results suggested that secreted Wisp2/CCN5 induces Akt and ERK phosphorylation via integrins, and consequently facilitates neurite formation and conferred resistance to apoptosis. Up-regulation of Wisp2/CCN5 in GM3-only mice should be, therefore, a reaction to protect nervous tissues from neurodegeneration caused by ganglioside deficiency.
The Ras/Raf/extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in almost all cell functions and therefore requires exquisite control of its spatiotemporal activity. Depending on the cell type and stimulus, ERK activity will mediate different antiproliferative events, such as apoptosis, autophagy and senescence in vitro and in vivo. ERK activity can promote either intrinsic or extrinsic apoptotic pathways by induction of mitochondrial cytochrome c release or caspase-8 activation, permanent cell cycle arrest or autophagic vacuolization. These unusual effects require sustained ERK activity in specific subcellular compartments and could depend on the presence of reactive oxygen species. We will summarize the mechanisms involved in Ras/Raf/ERK antiproliferative functions.
The CCN family of proteins is involved in diverse biological functions such as cell growth, adhesion, migration, angiogenesis, and regulation of extracellular matrix. We have investigated expression of CCN family genes and alternations induced by solar-simulated ultraviolet irradiation in human skin in vivo. Transcripts of all six CCN genes were expressed in human skin in vivo. CCN5 was most abundantly expressed followed by CCN2>CCN3>CCN1>CCN4>CCN6. Solar-simulated ultraviolet irradiation increased mRNA expression of CCN1 and CCN2. In contrast, mRNA levels of CCN3, CCN4, CCN5, and CCN6, were reduced. Knowledge gained from this study provides the foundation to explore the functional roles of CCN gene products in cutaneous biology and responses to solar ultraviolet irradiation.
The factors necessary for normal pancreatic islet morphogenesis have not been well characterized. Here we report that connective tissue growth factor (CTGF) is involved in the establishment of normal islet endocrine cell ratio and architecture. CTGF is a secreted protein known to modulate several growth factor-signaling pathways including TGF-beta, BMP, and Wnt. Although its role in pancreatic diseases such as pancreatitis and pancreatic cancer are well documented, a role for CTGF in normal pancreas development and function has heretofore not been examined. Using a lacZ-tagged CTGF allele, we describe for the first time the expression pattern of CTGF in the developing pancreas and the requirement of CTGF for normal islet morphogenesis and embryonic beta-cell proliferation. CTGF is highly expressed in pancreatic ductal epithelium and vascular endothelium, as well as at lower levels in developing insulin(+) cells, but becomes down-regulated in beta-cells soon after birth. Pancreata from CTGF null embryos have an increase in glucagon(+) cells with a concomitant decrease in insulin(+) cells, and show defects in islet morphogenesis. Loss of CTGF also results in a dramatic decrease in beta-cell proliferation at late gestation. Unlike CTGF null embryos, CTGF heterozygotes survive past birth and exhibit a range of islet phenotypes, including an intermingling of islet cell types, increased number of glucagon(+) cells, and beta-cell hypertrophy.
Gene regulation by steroid hormones is mediated through an interaction of the hormone receptors with DNA regulatory sequences called hormone regulatory or responsive elements (HRE). An analysis of the HRE's in the DNA of mouse mammary tumour provirus, human metallothionein IIA gene, chicken lysozyme gene, chicken and Xenopus vitellogenin genes, growth hormones genes, Moloney murine sarcoma provirus, rabbit uteroglobin gene, rat tyrosine aminotransferase gene, rat tryptophan oxygenase gene and rat acidic glycoprotein gene, yields the following consensus for positively modulated glucocorticoid responsive elements (GRE): 5'-GGTACAnnnTGTTCT-3'. This element can also mediate induction by progesterone and probably by androgens, but not by estrogens. Detailed analysis of the DNA protection pattern suggests that a dimer of the hormone receptor interacts with this palindromic 15-mer. In genes that are negatively regulated by glucocorticoids an imperfect copy of the GRE is found, and repression is probably due to competition between hormone receptor and other transcription factors or enhancer binding proteins for binding to overlapping DNA sequences. The receptors without bound hormone are able to interact specifically with DNA in vitro, but binding of hormone is needed for transcriptional activation in vivo. This could be due, at least in part, to changes in the rate parameters of the receptor-DNA interaction induced by binding of the hormone to the receptor. The possible role of precise chromatin organization in glucocorticoid induction is discussed on the basis of the nucleosome phasing found in the LTR region of mouse mammary tumour virus.
To evaluate the effect of IGF-1 on the autoimmune process of beta cell destruction, permissive non-obese diabetic (NOD) recipients were adoptively transferred with 7 x 10(6) autoreactive T cells from diabetic NOD mice and were administered subcutaneously 10 micrograms rhIGF-1, twice daily for 3 weeks. Administration of rhIGF-1 reduced the final incidence of successful transfers of diabetes observed in only 6/24 mice (25%) versus 12/21 (57%) in control mice. A marked reduction of insulitis during histological analysis of pancreatic glands was also observed. Mice treated with rhIGF-1 had a higher percentage of intact islets (48.6 +/- 12% versus 1.6 +/- 1.1%, P = 0.001) and a lower percentage of infiltrated islets. Islets from rhIGF-1-treated mice had a more intense insulin staining reflecting a higher beta cell mass, but no difference was observed in the amount of insulin content of pancreatic extracts and in the amounts of mRNA transcripts for proinsulin. No difference was also observed in the titres of three islet cell antibody (ICA)-positive sera and in the pattern of A2B5 staining. Some mice developed diabetes and severe islet cell infiltration despite rhIGF-1, thus indicating that some committed T cells were still able to invade the islets and cause beta cell destruction. The percentages of CD4+ and CD8+ T cells in the spleen of experimental mice were similar. To evaluate the effects of rhIGF-1 on cell trafficking in recipient mice, T cells from diabetic NOD Thy-1,2 mice injected into congenic NOD-N Thy-1,1 mice were monitored 3 weeks after adoptive cell transfer. The percentage of Thy-1,2+ T cells was significantly reduced in the spleen (10.8 +/- 1.3% versus 17.2 +/- 3.9%, P = 0.004) of rhIGF-1 treated mice in contrast to the thymus (68.4 +/- 7.9% versus 72.87 +/- 6.2%, P = 0.306), suggesting that rhIGF-1 could influence T cell trafficking to the lymphoid organs. The findings that rhIGF-1 has protective effects in autoimmune diabetes opens new perspectives for future experiments as well as for preventive strategies in human type I diabetes.
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The cell cycle-associated differences in the susceptibility to apoptosis were examined in HL-60 cells before and after differentiation with phorbol 12, 13-dibutyrate (PDBu). HL-60 cells in various phases of the cell cycle were separated by the counterflow centrifugal elutriation and the susceptibility to apoptosis was measured by the morphological examination and by DNA fragmentation assay. Undifferentiated HL-60 cells in S phase showed a significantly higher susceptibility to apoptosis than those in G0/G1 or G2/M phase either in the absence or presence of apoptosis-inducing reagents such as A23187, actinomycin D (Act D), and cycloheximide (CHX). In contrast, PDBu-treated HL-60 cells preferentially underwent apoptosis in G0/G1 phase. When untreated HL-60 cells enriched for G0/G1 phase were recultured in a complete medium, the percentage of apoptotic cells increased after 6-12 h in correlation with the increase in S-phase cells. When the same experiment was performed with PBDu-treated cells, spontaneous increase of apoptotic cells was observed while almost all cells remained in G0/G1 phase. Northern blot analysis revealed that undifferentiated cells expressed the same amounts of bcl-2 mRNA in each cell cycle phase, whereas G0/G1-predominant reduction of bcl-2 mRNA was noted in PDBu-treated cells. There was no difference in the amounts of CD11b mRNA between G0/G1 fraction and S+G2/M fraction of differentiated HL-60 cells. BCL-2 overexpression could almost completely abrogate the G0/G1-predominant induction of apoptosis in differentiated HL-60 cells. These results suggest that G0/G1 cell cycle arrest and down-regulation of bcl-2 mRNA in G0/G1 phase might be associated with apoptosis in differentiated HL-60 cells whereas the weakness of chromatin structure in S phase might be related to apoptosis in undifferentiated HL-60 cells.