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HB-EGF is targeted to the INM. HB-EGF-V5-C was transiently transfected into the cells. (A and B) Cells were treated with either TPA, bFGF, or EGF, then stained with an anti-V5 mAb. (C) Cells were treated with TPA and extracted with Triton X-100, then fixed and stained with either anti-V5 mAb or #H6. (D) Cells were permeabilized with digitonin and fixed. The cells were incubated with (bottom) or without (top) Triton X-100 and labeled with mAbD and anti-lamin B antibodies. Bar, 10 μm. (E) Ultra-thin sections were stained with anti-V5 and 15-nm gold–conjugated second antibodies. PM; plasma membrane. Bar, 200 nm.
Source publication
Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of...
Citations
... Increased cAMP production mediates phosphorylation of c-Src which in turn induces the production of a matrix metalloproteinase (MMP) [27] that mediates epidermal growth factor receptor (EGFR) transactivation [28]. The implication of a membrane located heparin-binding EGF-like growth factor (proHB-EGF) that is cleaved to yield a soluble EGFR ligand and a transmembrane-cytoplasmic fragment is crucial in this transactivation [29,30]. Proximal kinases like PI3K and distal kinases like Akt and ERK become activated and initiate a downstream signaling cascade that leads to the phosphorylation of transcription factors bound to EREs that increase the expression of apoptotic genes [31]. ...
... Transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) or GeneJuice (Merck Millipore, Rahway, NJ, USA) in accordance with the manufacturers' instructions. Unless stated otherwise, 17-20 hours after transfection, cells were fixed with 4% paraformaldehyde and immunofluorescence was performed as described previously [54] using appropriate primary and secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and coverslips were mounted using Prolong Gold Antifade Reagent with 4 0 ,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Carlsbad, CA, USA). Cells were viewed using an Olympus IX81 or Olympus BX53 epifluorescence microscope. ...
Cell motility is related to the higher-order structure of chromatin. Stimuli that induce cell migration change chromatin organization; such stimuli include elevated histone H3 lysine 9 trimethylation (H3K9me3). We previously showed that depletion of histone H3 lysine 9 methyltransferase, SUV39H1, suppresses directional cell migration. However, the molecular mechanism underlying this association between chromatin and cell migration remains elusive. The Golgi apparatus is a cell organelle essential for cell motility. In this study, we show that loss of H3K9 methyltransferase SUV39H1 but not SETDB1 or SETDB2 causes dispersion of the Golgi apparatus throughout the cytoplasm. The Golgi dispersion triggered by SUV39H1 depletion is independent of transcription, centrosomes, and microtubule organization, but is suppressed by depletion of any of the following three proteins: LINC complex components SUN2, nesprin-2, or microtubule plus-end-directed kinesin-like protein KIF20A. In addition, SUN2 is closely localized to H3K9me3, and SUV39H1 affects the mobility of SUN2 in the nuclear envelope. Further, inhibition of cell motility caused by SUV39H1 depletion is restored by suppression of SUN2, nesprin-2, or KIF20A. In summary, these results show the functional association between chromatin organization and cell motility via the Golgi organization regulated by the LINC complex.
... For instance, injecting HBEGF into the paw of mice caused painful mechanical hypersensitivity and severe pain and blocking the ErbB receptor could alleviate RA pain and joint inflammation [39,40]. In vivo, there are two different structural forms of HBEGF including proHBEGF (transmembrane protein) and sHBEGF (soluble protein) [41,42]. proHBEGF is a precursor for sHBEGF and can be cleaved at the plasma membrane to yield sHBEGF. proHBEGF takes part in juxtacrine activity and sHBEGF engages in paracrine activity. ...
Background
Fibroblasts are important structural cells in synovium and play key roles in maintaining the synovial homeostasis. By single-cell RNA sequencing (scRNA-seq), subpopulation of synovium-resident cells has been reported to protect intra-articular structures from chronic inflammation and promote tissue repair. However, a significant number of researchers have concentrated on the role of fibroblasts in the progress of rheumatoid arthritis (RA) while few reports had described the contribution of distinct fibroblast subsets in the RA remission. It is helpful to understand the role of fibroblast subpopulations in the RA process to provide predictive biomarkers and address RA remission mechanisms. Here, we found HBEGF+ fibroblasts that contributed to RA remission by integrating scRNA-seq datasets and bulk RNA sequencing (bulk RNA-seq) datasets.
Method
Three single-cell RNA datasets of cells harvested from RA patients were processed and integrated by Seurat and Harmony R packages. After identifying cell types by classic marker genes, the integrated dataset was used to run CellChat for analysis of cell-cell communication. Specially, EGF signaling pathway was found and HBEGF+ fibroblasts were identified based on HBEGF expression. Differential expressed genes of HBEGF+ were shown in heatmap and volcano plot and used to run gene ontology (GO) enrichment analysis. Next, bulk RNA-seq datasets of synovium under different conditions (health, osteoarthritis (OA), rheumatoid arthritis, before and after classical treatment) were compared to show expression change of HBEGF and gene markers that are mainly expressed by HBEGF+ fibroblasts such as CLIC5, PDGFD, BDH2, and ENPP1. Finally, two single-cell RNA sequencing datasets of synovial cells from mice were integrated to identify Hbegf+ fibroblasts and calculate the population of Hbegf+ fibroblasts under different joint conditions (health, K/BxN serum transfer arthritis (STA), and remission of STA).
Result
After integrating three single-cell RNA sequencing datasets, we identified 11 clusters of synovial cells, such as fibroblasts, mural cells, endothelial cells, CD4+ T cells, CD8+ T cells, natural killer cells, synovium macrophage, peripheral blood macrophages, plasma cells, B cells, and STMN1+ cells. We found fibroblasts had an extensive communication network with other clusters and interacted with synovial macrophages through EGF signaling pathway via analysis of cell-cell communication between synovial cells. HBEGF, ligand to EGF signaling pathway, was highly expressed by a subset of fibroblasts and macrophages, and EGFR, receptor to EGF signaling pathway, was highly expressed by fibroblasts and meniscus cells. Moreover, HBEGF was downregulated under RA state and it had an increase after classical treatment. We then defined fibroblasts with high expression of HBEGF as HBEGF+ fibroblasts. In addition, we also found that HBEGF+ fibroblasts highly expressed CRTAC1, ITGB8, SCARA5, THBS4, and ITGBL1, genes relative to encoding extracellular matrix proteins and engaged in positive regulation of cell migration and motility, cellular component movement, and cell growth by GO enrichment analysis. We eventually identified HBEGF+ fibroblasts specially expressed CLIC5, PDGFD, BDH2, and ENPP1, which positively correlated with the expression of HBEGF. Moreover, the expression of CLIC5, PDGFD, BDH2, and ENPP1 was downregulated under RA state and elevated by classical therapy. On the contrary, the HBEGF+ macrophages specially expressed SLAMF8, GK, L1RN, and JAK2, which negatively correlated with the expression of HBEGF. The expression was upregulated in SLAMF8, GK, L1RN, and JAK2 under the RA state, whereas it was decreased after classical treatment. In mice, the number of Hbegf+ fibroblasts was reduced in the RA synovium but increased in the RA remitting synovium.
Conclusions
HBEGF+ fibroblasts play a role in the remission of rheumatoid arthritis, and HBEGF has potential to become a novel biomarker for prediction of RA progress.
... The cells were fixed with 4% paraformaldehyde and immunostaining was mostly performed as described previously using appropriate primary and secondary antibodies (Hieda et al., 2008) unless stated otherwise. For HUTS4 mAb staining, cells were fixed and stained without Triton X-100 permeabilization. ...
The linker of nucleoskeleton and cytoskeleton (LINC) complex is composed of the inner nuclear membrane-spanning SUN proteins and the outer nuclear membrane-spanning nesprin proteins. The LINC complex physically connects the nucleus and plasma membrane via the actin cytoskeleton to perform diverse functions including mechanotransduction from the extracellular environment to the nucleus. Mammalian somatic cells express two principal SUN proteins, namely SUN1 and SUN2. We have previously reported that SUN1, but not SUN2, is essential for directional cell migration; however, the underlying mechanism remains elusive. Because the balance between adhesive force and traction force is critical for cell migration, in the present study, we focused on focal adhesions (FAs) and the actin cytoskeleton. We observed that siRNA-mediated SUN1 depletion did not affect the recruitment of integrin β1, one of the ubiquitously expressed focal adhesion molecules, to the plasma membrane. Consistently, SUN1-depleted cells normally adhered to extracellular matrix proteins, including collagen, fibronectin, laminin, and vitronectin. In contrast, SUN1 depletion reduced the activation of integrin β1. Strikingly, the depletion of SUN1 interfered with the incorporation of vinculin into the focal adhesions, whereas no significant differences in the expression of vinculin were observed between wild-type and SUN1-depleted cells. In addition, SUN1 depletion suppressed the recruitment of zyxin to nascent focal adhesions. These data indicate that SUN1 is involved in the maturation of focal adhesions. Moreover, disruption of the SUN1-containing LINC complex abrogates the actin cytoskeleton and generation of intracellular traction force, despite the presence of SUN2. Thus, a physical link between the nucleus and cytoskeleton through SUN1 is required for the proper organization of actin, thereby suppressing the incorporation of vinculin and zyxin into focal adhesions and the activation of integrin β1, both of which are dependent on traction force. This study provides insights into a previously unappreciated signaling pathway from the nucleus to the cytoskeleton, which is in the opposite direction to the well-known mechanotransduction pathways from the extracellular matrix to the nucleus.
... Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) or Gene Juice (Merck Millipore, Darmstadt, Germany) in accordance with the manufacturer's instructions. Immunofluorescence was performed as described previously49 using appropriate primary and secondary antibodies (Jackson ImmunoResearch Laboratories, Soham, UK), and coverslips were mounted using Prolong Gold Antifade Reagent with 4′, 6-diamidino-2-phenylindole (DAPI) (Life Technologies, Waltham, MA, USA) or stained with Hoechst 33,342 and mounted using Fluorescent Mounting Medium (S3023; Dako, Tokyo, Japan). Cells were viewed using an Olympus IX81 with Plan Apo 60 × /NA1.4 or Olympus BX53 with UPlanS Apo 40 × /NA 0.95 objective lens. ...
The morphology of the Golgi complex is influenced by the cellular context, which strictly correlates with nuclear functions; however, the mechanism underlying this association remains elusive. The inner nuclear membrane SUN proteins, SUN1 and SUN2, have diverse functions together with the outer nuclear membrane nesprin proteins, which comprise the LINC complex. We found that depletion of SUN1 leads to Golgi complex dispersion with maintenance of ministacks and retained function for vesicle transport through the Golgi complex. In addition, SUN2 associates with microtubule plus-end-directed motor KIF20A, possibly via nesprin-2. KIF20A plays a role in the Golgi dispersion in conjunction with the SUN2-nesprin-2 LINC complex in SUN1-depleted cells, suggesting that SUN1 suppresses the function of the SUN2-nesprin-2 LINC complex under a steady-state condition. Further, SUN1-knockout mice, which show impaired cerebellar development and cerebellar ataxia, presented altered Golgi morphology in Purkinje cells. These findings revealed a regulation of the Golgi organization by the LINC complex.
... In some cases proteins may first travel out to the Golgi before retrograde return to take advantage of this pathway. Joining this route are some proteins that are internalized at the plasma membrane and pass through the endosomal system [5][6][7] . Recently, it has also been shown that some proteins endocytosed from the plasma membrane to the endosomal system are delivered to the nucleoplasm via fusion of nuclear-envelope associated endosomes directly with the ONM and passage through the Sec61 translocon in the INM 8,9 . ...
Nuclear-cytoplasmic communication is not limited to nuclear pores, with both proteins and RNA using alternative routes between these compartments. We previously characterized cytoplasmic capes (large invaginations of the nuclear envelope in Drosophila ), which are enriched for the membrane-bound EGF receptor ligand mSpitz, endosome-related organelles and ubiquitylated proteins. Closely associated with capes are groups of perinuclear vesicles resembling those seen at sites of RNP export via a budding mechanism. Here, we demonstrate that mSpitz delivery to capes requires passage through the endosomal system. We also show that capes are closely associated with sites of non-canonical RNP export as well as the dFrizzled2 receptor C terminal fragment, a core component of this export pathway. Video microscopy of glands in intact larvae indicates that cytoplasmic capes are stable structures that persist for at least 90 minutes without conspicuous growth. We further show that capes appear with the growth of the salivary gland rather than its developmental stage. Finally, we show that the large F-actin binding protein β H -spectrin, which modulates endosomal trafficking, as well as its partner α-spectrin are required for cape formation. Cytoplasmic capes therefore represent a subspecialization of the nuclear envelope where endosomal trafficking and RNP export are closely associated.
Synopsis
We further characterize large invaginations of the nuclear envelope called cytoplasmic capes in Drosophila . The EGF receptor ligand mSpitz is concentrated in capes and we show that it traffics to this compartment via endosomes. The presence of RNP and the dFrizzled2 receptor C-terminal fragment also indicates that non-canonical RNA export is concentrated at capes. In vivo imaging shows that capes persist for at least 90 minutes. Finally, the large F-actin crosslinker α/β ◻ -spectrin is shown to be required for cape formation.
Abstract Figure
... ADAM-12 can, therefore, contribute to the cleavage of HB-EGF and thereby enhance HB-EGF immunoexpression. Due to its transmembrane location, Hieda et al. [42] believe that the cytoplasmic portion of HB-EGF translocates to the nucleus when cleaved. ProHB-EGF is mostly expressed on the cell surface; however, in the presence of the appropriate stimuli, it is cleaved and probably undergoes retrograde transport to the Golgi apparatus through endosome recycling. ...
... ProHB-EGF is mostly expressed on the cell surface; however, in the presence of the appropriate stimuli, it is cleaved and probably undergoes retrograde transport to the Golgi apparatus through endosome recycling. It is subsequently directed to the nucleus, where it diffuses or is actively transported before binding the nuclear membrane [42]. After pro-HB-EGF cleavage, the domain that translocates to the nucleus inhibits proteins that regulate and control the cell cycle, such as PLZF and Bc16. ...
Background:
Among cancers affecting the oral cavity, adenoid cystic carcinoma (ACC) is a relatively common malignant neoplasm. It has high rates of metastasis and recurrence and is associated with significant morbidity. During the progression of ACC, the oxygen concentration is reduced in specific areas of the tumour microenvironment, leading to intratumoural hypoxia. The expression of NOTCH1, a disintegrin and metalloproteinase 12 (ADAM-12), hypoxia-inducible factor 1 alpha (HIF-1α), and heparin-binding epidermal growth factor (HB-EGF) under hypoxic conditions has been implicated in invadopodia formation, tumour invasiveness, and metastasis. The aim of this study was to analyse the expression of these proteins to elucidate the mechanisms underlying ACC invasiveness.
Methods:
Fifteen ACC samples and 10 normal-looking salivary gland (SG) samples were used to investigate the expression of these proteins by immunohistochemistry. Primary antibodies against NOTCH1, ADAM-12, HIF-1α, and HB-EGF were used.
Results:
The immunoexpression of all proteins was higher in ACC samples than in SG samples (p < 0.05).
Conclusions:
There was increased expression of proteins associated with hypoxia and tumour invasiveness in ACC samples, which indicates a possible role of these proteins in the biological behaviour of this tumour.
... Unexpectedly, HBEGF was also present in the nuclei of the tammar diapause blastocyst with ERBB4 present in the cytoplasm and possibly the membrane in both the tammar and mink embryo. Both the transmembrane and mature form of HBEGF can translocate to the nucleus, but whilst mature HBEGF is implicated in cell proliferation, nuclear localization of the transmembrane form acts as a reservoir, which can be rapidly released and cleaved to the mature form upon oxidative stress [133][134][135]. The antibody used to detect HBEGF in the tammar could recognize both forms; hence, it is unknown what specific function HBEGF is having at this time. ...
Embryonic diapause is period of developmental arrest which requires coordination of a molecular cross-talk between the endometrium and blastocyst to ensure a successful reactivation, but the exact mechanisms are undefined. The objectives of this study were to screen the tammar blastocyst for potential diapause control factors and to investigate the potential for members of the epidermal growth factor (EGF) family to coordinate reactivation. A select number of factors were also examined in the mink to determine whether their expression patterns were conserved across diapause species. The full-length sequences of the tammar genes of interest were first cloned to establish their level of sequence conservation with other mammals. The uterine expression of EGF family members EGF and heparin-binding EGF, HBEGF and their receptors (EGFR and ERBB4) was determined by quantitative RT-PCR and immunohistochemistry. Both HBEGF and EGF were significantly upregulated at reactivation compared to diapause. In the blastocyst, the expression of the potential diapause factors Forkhead box class O family members (FOXO1, FOXO3 and FOXO4), tumor protein 53 (TP53), cyclin-dependent kinase inhibitor 1A (CDKN1A) and the EGF family were examined by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence. Nuclear (and hence active) FOXO expression was confirmed for the first time in a mammalian diapause blastocyst in both the tammar and the mink CDKN1A was also expressed, but TP53 is not involved and EGFR was not detected in the blastocyst. These results indicate that the EGF family, FOXOs and CDKN1A are promising candidates for the molecular control of embryonic diapause in mammals.
... In a series of reports, we have characterized EGFR ligands, emphasizing juxtacrine and paracrine signaling, ectodomain shedding, intracellular trafficking and mRNA stability [17][18][19][20][21][22] . Here, we used EGF and AREG as the representatives of cognate growth factors, and set to understand their mechanism of action using immortalized mammary epithelial cell lines, MCF10A and HMT-3522 S1. ...
Epithelial cell plasticity is controlled by extracellular cues, but the underlying mechanisms remain to be fully understood. Epidermal growth factor (EGF) and amphiregulin (AREG) are high- and low-affinity ligands for EGF receptor (EGFR), respectively. EGFR signaling is known to promote epithelial-mesenchymal transition (EMT) by the activation of ERK and the induction of an EMT transcription factor, ZEB1. Here, we demonstrate that ligand-switching between EGF and AREG at equivalent molarity reversibly interconverts epithelial and mesenchymal-like states of EGFR signal-dependent mammary epithelial cells. The EGF- and AREG-cultured cells also differ in their epithelial characteristics, including the expression of cell surface markers, the mode of migration and the ability for acinus-formation. The ligand-switching between EGF and AREG temporally alters strength of the shared EGFR-ERK signaling. This alteration inverts relative expression levels of ZEB1 and its antagonizing microRNAs, miR-205 and miR-200c, those are critical determinants of the epithelial phenotype. Further, AREG-induced EGFR accumulation on the plasma membrane compensates for the weak association between AREG and EGFR. The EGFR dynamics enables AREG to support proliferation as efficiently as EGF at equivalent molarity and to maintain epithelial characteristics. Our findings reveal a role of EGFR ligands-generated signal strength in the regulation of mammary epithelial cell plasticity.
... Considering the above challenges in using decoy antibodies to block localized HB-EGF signaling, we next turned to specific protease inhibition as an alterative therapeutic strategy. In addition to blocking generation of soluble HB-EGF ectodomain in the cell supernatant, protease inhibition may also prevent the generation of c-terminal HB-EGF fragments that traffic to the nuclear membrane and influence transcriptional activity 22,23 . Indeed, using immunofluorescence we found that α -HBEGF decoy antibodies had no significant impact on nuclear accumulation of HB-EGF c-terminus, while metalloproteinase inhibition did (Fig. S6). ...
Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, "decoy" antibodies have been developed to sequester ligands including heparin-binding epedermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor if ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometrotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.
