C3G overexpression in glomerular epithelial cells during anti-GBM-induced glomerulonephritis
2008; Elsevier BV; Volume: 75; Issue: 1 Linguagem: Inglês
10.1038/ki.2008.448
ISSN1523-1755
AutoresVictoriya A. Rufanova, Elias A. Lianos, Anna Alexanian, Елена А. Сорокина, Mukut Sharma, Ann McGinty, Andrey Sorokin,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe guanine nucleotide exchange factor C3G, along with the CrkII adaptor protein, mediates GTP activation of the small GTPase proteins Rap1 and R-Ras, facilitating their activation of downstream signaling pathways, which had been found to be important in the pathogenesis of glomerulonephritis. We found that expression of C3G protein was upregulated in glomerular epithelial cells in an experimental model of accelerated anti-GBM antibody-induced glomerulonephritis expression. To determine the consequence of its increased expression, we transfected C3G (using adenoviral constructs) into cultured glomerular epithelial cells and measured the activated forms (i.e., GTP-bound) forms of Rap1 and R-Ras. Activation of Rap1 was not affected by C3G; however, the basal level of GTP-bound R-Ras was decreased. Further, C3G over-expression enhanced the activation of R-Ras in response to endothelin. Overexpression of C3G also led to a significant reduction in glomerular epithelial cell spreading and decreased the cells' E-cadherin expression and augmented their migration. We found that C3G was overexpressed in accelerated anti-GBM antibody-induced glomerulonephritis and suggest that this modulates glomerular epithelial cell morphology and behavior. The guanine nucleotide exchange factor C3G, along with the CrkII adaptor protein, mediates GTP activation of the small GTPase proteins Rap1 and R-Ras, facilitating their activation of downstream signaling pathways, which had been found to be important in the pathogenesis of glomerulonephritis. We found that expression of C3G protein was upregulated in glomerular epithelial cells in an experimental model of accelerated anti-GBM antibody-induced glomerulonephritis expression. To determine the consequence of its increased expression, we transfected C3G (using adenoviral constructs) into cultured glomerular epithelial cells and measured the activated forms (i.e., GTP-bound) forms of Rap1 and R-Ras. Activation of Rap1 was not affected by C3G; however, the basal level of GTP-bound R-Ras was decreased. Further, C3G over-expression enhanced the activation of R-Ras in response to endothelin. Overexpression of C3G also led to a significant reduction in glomerular epithelial cell spreading and decreased the cells' E-cadherin expression and augmented their migration. We found that C3G was overexpressed in accelerated anti-GBM antibody-induced glomerulonephritis and suggest that this modulates glomerular epithelial cell morphology and behavior. In experimental models of proliferative (crescentic) glomerulonephritis (GN), induced by antibody against the glomerular membrane (GBM), and in human crescentic GN, injury to the glomerular visceral epithelial cells (GECs), also known as podocytes, appears to be of pivotal importance.1.Griffin S.V. Petermann A.T. Durvasula R.V. et al.Podocyte proliferation and differentiation in glomerular disease: role of cell-cycle regulatory proteins.Nephrol Dial Transplant. 2003; 18: vi8-vi13PubMed Google Scholar, 2.Bariety J. Bruneval P. Meyrier A. et al.Podocyte involvement in human immune crescentic glomerulonephritis.Kidney Int. 2005; 68: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 3.Jefferson J.A. Johnson R.J. Experimental mesangial proliferative glomerulonephritis (the anti-Thy-1.1 model).J Nephrol. 1999; 12: 297-307PubMed Google Scholar To support the glomerular filtration rate, the renal GEC develop microtubule-based thick processes with sophisticated branching morphology and have thin actin-based projections (podocyte foot processes), which spread and form cell–cell contacts in mature cells. Actin filaments are thought to be important in orchestrating the function of various molecules during the formation of podocyte foot processes. The molecular mechanism of foot process formation seems to include small guanosine triphosphate (GTP)-binding proteins. One common mechanism utilized by small GTPases to regulate cellular function is to cycle between the inactive guanosine diphosphate-bound state and the active GTP-bound state. Guanine nucleotide exchange factors (GEFs) facilitate guanosine diphosphate dissociation and allow the more abundant GTP to rebind, whereas GTPase-activating proteins accelerate GTP hydrolysis to complete the cycle. In a number of pathological processes, small GTPases are upregulated due to overexpression, mutation, intensification of particular signaling pathways, or abnormal GEFs activity that are critical for the onset or progression of the disease.4.Zeghouf M. Guibert B. Zeeh J.C. et al.Arf, Sec7 and Brefeldin A: a model towards the therapeutic inhibition of guanine nucleotide-exchange factors.Biochem Soc Trans. 2005; 33: 1265-1268Crossref PubMed Google Scholar This suggests that GEFs can be promising targets for the inhibition of small GTPase-dependent pathways that are amplified or diminished in diseases. The GEF, C3G, predominantly catalyzes the guanine nucleotide exchange reaction for the Ras family of GTPases. Absence of C3G causes embryonic lethality in mice via the primary defect in vascular-supporting cells of mesenchymal origin, and cells derived from such embryos demonstrate impaired cell adhesion, delayed spreading, and enhanced cell migration.5.Voss A.K. Gruss P. Thomas T. The guanine nucleotide exchange factor C3G is necessary for the formation of focal adhesions and vascular maturation.Development. 2003; 130: 355-367Crossref PubMed Scopus (59) Google Scholar However, the mechanisms by which C3G regulates these actin-dependent cellular functions are poorly understood. C3G-mediated Rap1 activation is required for cell adhesion and cell spreading, but suppresses cell migration.6.Ohba Y. Ikuta K. Ogura A. et al.Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis.EMBO J. 2001; 20: 3333-3341Crossref PubMed Scopus (180) Google Scholar Recently, a functional link was uncovered between C3G/Rap1 and the FGF2/Akt/Gsk3β/β-catenin pathway, where C3G is involved as a self-limiting mechanism for proliferation signals.7.Voss A.K. Krebs D.L. Thomas T. C3G regulates the size of the cerebral cortex neural precursor population.EMBO J. 2006; 25: 3652-3663Crossref PubMed Scopus (37) Google Scholar Furthermore, York et al. proposed that the activation of Rap1 by C3G represents a common mechanism to induce sustained activation of the mitogen-activated protein (MAP) kinase cascade in cells that express B-Raf.8.York R.D. Yao H. Dillon T. et al.Rap1 mediates sustained MAP kinase activation induced by nerve growth factor.Nature. 1998; 392: 622-626Crossref PubMed Scopus (745) Google Scholar Our previous studies in rat models of proliferative GN have demonstrated that both extracellular signal-regulated kinase (ERK) and stress-activated protein kinase exhibit disease-associated activation, albeit with differing kinetics, and that ERK activation in glomeruli is accompanied by increased expression of its specific upstream activator, MEK1.9.Bokemeyer D. Guglielmi K.E. McGinty A. et al.Activation of extracellular signal-regulated kinase in proliferative glomerulonephritis in rats.J Clin Invest. 1997; 100: 582-588Crossref PubMed Scopus (73) Google Scholar Significant evidence has emerged that members of Ras family of small GTPases modulate ERK activation in a cell-context-dependent manner.8.York R.D. Yao H. Dillon T. et al.Rap1 mediates sustained MAP kinase activation induced by nerve growth factor.Nature. 1998; 392: 622-626Crossref PubMed Scopus (745) Google Scholar, 10.Kooistra M.R. Dube N. Bos J.L. Rap1: a key regulator in cell–cell junction formation.J Cell Sci. 2007; 120: 17-22Crossref PubMed Scopus (233) Google Scholar, 11.Hattori M. Minato N. Rap1 GTPase: functions, regulation, and malignancy.J Biochem. 2003; 134: 479-484Crossref PubMed Scopus (149) Google Scholar, 12.Wang Y. Sheibani N. PECAM-1 isoform-specific activation of MAPK/ERKs and small GTPases: implications in inflammation and angiogenesis.J Cell Biochem. 2006; 98: 451-468Crossref PubMed Scopus (35) Google Scholar Signaling through the small GTPases Rap1 and R-Ras contributes to the regulation of a wide variety of integrin-mediated processes including cell–cell and cell–matrix adhesion, actin polymerization, cell migration, and spreading.13.Bos J.L. de Bruyn K. Enserink J. et al.The role of Rap1 in integrin-mediated cell adhesion.Biochem Soc Trans. 2003; 31: 83-86Crossref PubMed Google Scholar, 14.Ada-Nguema A.S. Xenias H. Sheetz M.P. et al.The small GTPase R-Ras regulates organization of actin and drives membrane protrusions through the activity of PLCepsilon.J Cell Sci. 2006; 119: 1307-1319Crossref PubMed Scopus (62) Google Scholar Interestingly, enhanced Rap1 activity was found to be involved in p38 MAP kinase regulation, ERK suppression, increased cell adhesion, and was observed in samples from heart with right ventricular outflow tract obstruction and renal tumors.15.Konstantinov I.E. Coles J.G. Boscarino C. et al.Gene expression profiles in children undergoing cardiac surgery for right heart obstructive lesions.J Thorac Cardiovasc Surg. 2004; 127: 746-754Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 16.Habib S.L. Phan M.N. Patel S.K. et al.Reduced constitutive 8-oxoguanine-DNA glycosylase expression and impaired induction following oxidative DNA damage in the tuberin deficient Eker rat.Carcinogenesis. 2003; 24: 573-582Crossref PubMed Scopus (32) Google Scholar By contrast, decreased R-Ras activity was linked to such biological effects as augmented cell motility and migration.14.Ada-Nguema A.S. Xenias H. Sheetz M.P. et al.The small GTPase R-Ras regulates organization of actin and drives membrane protrusions through the activity of PLCepsilon.J Cell Sci. 2006; 119: 1307-1319Crossref PubMed Scopus (62) Google Scholar Taken together, these data allow us to hypothesize that the Ras family of GTPases GEF, C3G, can be important for controlling the signaling cascades in proliferative GN. We have carried out a study of C3G expression in inflammatory kidney disease followed by analysis of C3G biological significance in cultured podocytes. Western blot analysis of protein lysates obtained from isolated glomeruli revealed a significant increase in levels of GEF C3G 1, 3, and 7 days after induction the accelerated (crescentic) form of anti-GBM GN (Figure 1). Levels of the adaptor protein Grb2 were not different between glomeruli of control and experimental animals (Figure 1, lower panel). On day 14 following administration of anti-GBM antibody, we observed significantly increased proteinuria (Figure 2), crescent formation (Figure 3a and b), glomerular sclerosis (Figure 3c), and periglomerular inflammatory cell infiltrates (Figure 3, compare a to d). C3G expression (assessed by immunostaining) was prominent in GEC (black arrows in Figure 3c), within cellular crescents (Figure 3b) and, occasionally, in tubular epithelium. Very little or no C3G staining was observed in glomerular cells of control animals. To confirm the localization of C3G staining, we carried out double immunostaining for C3G and nestin as a mature podocytes marker, which is upregulated in response to injury17.Thorner P.S. Ho M. Eremina V. et al.Podocytes contribute to the formation of glomerular crescents.J Am Soc Nephrol. 2008; 19: 495-502Crossref PubMed Scopus (101) Google Scholar (Figure 4). Glomerulus from control animal was virtually free of brown C3G staining and was characterized by prominent blue signal from anti-nestin antibody (Figure 4a). After GN induction, C3G staining was enhanced and colocalized with nestin forming gray to black color (Figure 4b).Figure 3Kidney morphology and C3G immunohistochemical detection in glomerulus after induction of glomerulonephritis. Representative morphological (Masson's Trichrome, a, d) and immunohistochemical staining for C3G (b, c, e) in kidney cortical sections obtained on day 14 following onset of anti-GBM glomerulonephritis (a–c, f) and in control animals (d, e). (b) and (c) represent two different animals with GN. In (a), a crescentic glomerulus with periglomerular infiltrate is shown. In (c), arrows indicate immunolocalization of C3G in glomerular epithelial cells. Also shown is a negative control (stained with an isotype-matched IgGs) (f), corresponding to the glomerulus shown in (c). Cell nuclei in (b), (c), (e), and (f) are conterstained with hematoxylin, original magnifications × 200.View Large Image Figure ViewerDownload (PPT)Figure 4C3G and nestin immunohistochemical detection in glomerulus after induction of glomerulonephritis. Double immunostainig for C3G (brown cyptoplasm) and for nestin (blue cytoplasm) showing changes after glomerulonephritis induction. (a) Normal glomerulus from control animal demonstrates very little if any C3G signal and many cells are nestin-positive. (b) Crescent glomerulus characterized by increased C3G staining. Majority of nestin-positive cells express C3G (gray to black color, marked with arrows). Cell nuclei are conterstained with Nuclear Fast Red, original magnifications × 400.View Large Image Figure ViewerDownload (PPT) To investigate the possibility that C3G protein in glomeruli originates from infiltrating macrophages, levels of the macrophage marker ED-118.Floege J. Eng E. Young B.A. et al.Factors involved in the regulation of mesangial cell proliferation in vitro and in vivo.Kidney Int Suppl. 1993; 39: S47-S54PubMed Google Scholar were assessed in glomeruli isolated from control animals and animals with anti-GBM antibody-induced GN. At 12 h following injection of anti-GBM antibody, C3G levels in glomeruli isolated from two experimental animals were indistinguishable from levels in glomeruli from a control animal (Figure 5a, upper panel). In contrast, ED-1 levels were greatly increased only in glomerular lysates of the two experimental animals (Figure 5a, lower panel). At later time points (24 and 48 h and on days 3 and 7 following induction of GN), the differences in glomerular C3G expression between control and experimental animals were clearly manifested (Figures 1 and 5b). In contrast to the striking upregulation of C3G expression, Grb2 did not exhibit a disease-associated increase in expression levels (Figures 1 and 5b). Similarly, the level of expression of glomerular CrkII did not significantly change in animals with GN (Figure 5b). Immunohistochemistry data also did not reveal significant C3G staining in inflammatory infiltrates. As detected by Rap1 affinity binding assay, Rap1 activation in response to endothelin-1 (ET-1) was not significantly affected in GEC infected with adenovirus encoding C3G (Figure 6a, upper panel). The amount of GTP-bound Rap1 from three independent experiments was further quantified using densitometry analysis (Figure 6b). We observed a marked effect of C3G overexpression on ET-1-induced changes in R-Ras GTP loading (Figure 7a and b). First, the basal level of R-Ras GTP-loading was decreased in cells overexpressing C3G (Figure 7a, upper panel, lane 6 versus lane 1 or 3). These differences were quantified by densitometry (Figure 7b). Statistically significant alterations between the two groups (AdGFP and AdC3G) were found at basal level (time 0 min, P<0.01) and following a 20 min exposure to ET-1 (P<0.05). Within the AdGFP group, R-Ras GTP loading following 5 min of ET-1 stimulation was significantly lower than that at 0 min (P<0.01). The same effect was observed in uninfected GEC control (Figure 7a, U). Within the AdC3G group, R-Ras GTP loading at 20 min was significantly higher than that at 0 min (P<0.001) or that at 5 min (P<0.02). In summary, GEC overexpressing C3G demonstrated a dramatic decrease in the basal level of GTP-bound R-Ras and a significant increase in the level of GTP-bound R-Ras 20 min following ET-1 stimulation. In our experiments, C3G overexpression had no effect on cultured GEC attachment to fibronectin with or without ET-1 stimulation (data not shown). In accordance with R-Ras activation data, C3G upregulation significantly reduced cell size during spreading (Figure 8a). In addition, GEC spreading under ET-1 treatment was significantly diminished in control cells, reflecting normal actin dynamic response to contractile stimulus, but remained unaffected in C3G-overexpressing population (Figure 8a). To further explore changes in intracellular actin balance with or without C3G overexpression and ET-1 stimulation, we labeled filamentous actin in cultured GEC with phalloidine, conjugated with fluorochrom (Figure 8b). After C3G overexpression and ET-1 stimulation, amount of polymerized actin fibers was increased compared with unstimulated GFP-expressing control. After C3G upregulation, cell migration was agumented compared with GFP control as measured by wound area closing (Figure 9a and b) and by number of migrating cells into the opened area (Figure 9c) in scratch assay. Next, we analyzed proteins, involved into adherens and tight-junction formation and/or into migration signaling. In line with observed changes in cell morphology and behavior, we detected significant decrease in E-cadherin and podocin expression after C3G upregulation (Figure 9d). ET-1 treatment for 24 h led to similar reduction of E-cadherin protein level in GFP-expressing group compared with C3G counterpart (Figure 9d), correlating with spreading data (Figure 8a). We observed no change in vinculin and p130Cas expression. However, substrate-binding domain activation of p130Cas (measured as Tyrosin 165 phosphorylation) was enhanced after C3G overexpression without any significant changes in response to 24 h ET-1 stimulation. We demonstrated for the first time that GEFs C3G is upregulated in the model of anti-GBM GN. Little is known about regulation of C3G expression. There are few studies reporting downregulation19.Okino K. Nagai H. Nakayama H. et al.Inactivation of Crk SH3 domain-binding guanine nucleotide-releasing factor (C3G) in cervical squamous cell carcinoma.Int J Gynecol Cancer. 2006; 16: 763-771Crossref PubMed Scopus (23) Google Scholar as well as overexpression of C3G isoforms20.Gutierrez-Berzal J. Castellano E. Martin-Encabo S. et al.Characterization of p87C3G, a novel, truncated C3G isoform that is overexpressed in chronic myeloid leukemia and interacts with Bcr-Abl.Exp Cell Res. 2006; 312: 938-948Crossref PubMed Scopus (29) Google Scholar, 21.Hirata T. Nagai H. Koizumi K. et al.Amplification, up-regulation and over-expression of C3G (CRK SH3 domain-binding guanine nucleotide-releasing factor) in non-small cell lung cancers.J Hum Genet. 2004; 49: 290-295Crossref PubMed Scopus (33) Google Scholar during development of human cancers. However, mechanism of differentiated expression of C3G currently remains unclear. Intense glomerular macrophage infiltration is known to occur during the early stages of the accelerated variant of anti-GBM antibody-mediated crescentic GN employed in the present studies. Isolated glomeruli western blotting results (Figures 1 and 5) indicate that glomerular C3G overexpression accompanies the course of anti-GBM antibody-induced GN and, although it could be triggered by macrophage infiltration in glomeruli, it did not result from infiltrating macrophages overexpressing C3G. Two weeks after disease induction when macrophage number inside the glomerulus mostly reverts to normal, based on immunolocalization of C3G (Figures 3c and 4b), we propose that GEC are the likely site where C3G overexpression takes place in the anti-GBM model of immune glomerular injury. We demonstrate that C3G overexpression in cultured GEC results in modulation of ET-1-induced signaling via the small GTPase R-Ras. Our choice of ET-1 stimulation was based on knowledge that ET-1 is important in both the normal and the diseased kidney.22.Kohan D.E. Endothelins in the normal and diseased kidney.Am J Kidney Dis. 1997; 29: 2-26Abstract Full Text PDF PubMed Scopus (337) Google Scholar, 23.Simonson M.S. Dunn M.J. Endothelin peptides and the kidney.Annu Rev Physiol. 1993; 55: 249-265Crossref PubMed Scopus (61) Google Scholar ET-1, acting in concert with other constrictor peptides, cytokines, and growth factors, contributes to proliferative renal diseases.22.Kohan D.E. Endothelins in the normal and diseased kidney.Am J Kidney Dis. 1997; 29: 2-26Abstract Full Text PDF PubMed Scopus (337) Google Scholar In immune complex GN there is increased glomerular ET-1 expression24.Wu H. Wang H. Wang Y. et al.The role for endothelin in mesangial proliferative glomerulonephritis.J Tongji Med Univ. 1998; 18: 122-125Crossref PubMed Google Scholar and G-protein-coupled ET-A and ET-B receptor blockade reduces glomerular cell proliferation and proteinuria.25.Gomez-Garre D. Largo R. Liu X.H. et al.An orally active ETA/ETB receptor antagonist ameliorates proteinuria and glomerular lesions in rats with proliferative nephritis.Kidney Int. 1996; 50: 962-972Abstract Full Text PDF PubMed Scopus (66) Google Scholar We observed diminished GTP loading of R-Ras in cells overexpressing C3G, but increased level of GTP-bound R-Ras at later time-points after ET-1 stimulation (Figure 7). Recently, in wide range of cell lines, it was demonstrated that R-Ras regulates exocytosis.26.Takaya A. Kamio T. Masuda M. et al.R-Ras regulates exocytosis by Rgl2/Rlf-mediated activation of RalA on endosomes.Mol Biol Cell. 2007; 18: 1850-1860Crossref PubMed Scopus (42) Google Scholar Several reports describe that podocytes are capable of secretory exocytosis.27.Rastaldi M.P. Armelloni S. Berra S. et al.Glomerular podocytes contain neuron-like functional synaptic vesicles.FASEB J. 2006; 20: 976-978Crossref PubMed Scopus (83) Google Scholar, 28.Roselli S. Moutkine I. Gribouval O. et al.Plasma membrane targeting of podocin through the classical exocytic pathway: effect of NPHS2 mutations.Traffic. 2004; 5: 37-44Crossref PubMed Scopus (73) Google Scholar Endosomes, before recycling to membrane, contain R-Ras in GTP-bound form. When endosomes are fused with the cell membrane, R-Ras is converted to guanosine diphosphate-bound form by membrane-located GTPase-activating proteins. ET-1 could stimulate endosome recycling29.Bremnes T. Paasche J.D. Mehlum A. et al.Regulation and intracellular trafficking pathways of the endothelin receptors.J Biol Chem. 2000; 275: 17596-17604Crossref PubMed Scopus (206) Google Scholar to plasma membrane compartments containing GTPase-activating proteins and hence decrease R-Ras GTP binding. We speculate, that C3G overexpression in GEC promotes fusion of endosomes with the cell membrane, an event that may not require involvement of GEF activity of C3G, but could be controlled through C3G-mediated protein–protein interactions.30.Guerrero C. Martin-Encabo S. Fernandez-Medarde A. et al.C3G-mediated suppression of oncogene-induced focus formation in fibroblasts involves inhibition of ERK activation, cyclin A expression and alterations of anchorage-independent growth.Oncogene. 2004; 23: 4885-4893Crossref PubMed Scopus (28) Google Scholar This could explain why C3G overexpression reduced the basal R-Ras GTP level in unstimulated GEC (Figure 5, lane 6 compared with lane 1). To the extent that ET-1 stimulates GEF activity of C3G in plasma membrane compartments, C3G would facilitate GTP binding of R-Ras in ET-1-stimulated GEC. Both increased and decreased GTP loading can be important in GTPase signaling.31.Riedl J.A. Brandt D.T. Batlle E. et al.Down-regulation of Rap1 activity is involved in ephrinB1-induced cell contraction.Biochem J. 2005; 389: 465-469Crossref PubMed Scopus (16) Google Scholar Reduced R-Ras GTP level usually correlates with reduction in cell–cell and cell–matrix interactions presumably through the inactivation of integrins.32.Elowe S. Holland S.J. Kulkarni S. et al.Downregulation of the Ras-mitogen-activated protein kinase pathway by the EphB2 receptor tyrosine kinase is required for ephrin-induced neurite retraction.Mol Cell Biol. 2001; 21: 7429-7441Crossref PubMed Scopus (153) Google Scholar H-Ras and its downstream effector kinase Raf-1 suppress integrin activation in a manner that is independent of protein synthesis and mRNA transcription and correlates with activation of the ERK/MAP kinase pathway.33.Hughes P.E. Renshaw M.W. 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Bridge formation with denuded GBM was proposed to be a precursor to crescent development.39.Kriz W. LeHir M. Pathways to nephron loss starting from glomerular diseases-insights from animal models.Kidney Int. 2005; 67: 404-419Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar Together with observation of C3G-positive cells in glomerular crescents (Figures 3b and 4b) formed after induction of anti-GBM GN, our findings allow us to speculate that possible C3G-R-Ras function is to enable cells to migrate in response to kidney injury. In support of this hypothesis, we found that C3G overexpression in cultured GEC resulted in the reduced spreading associated with enhanced filamentous actin formation and in the increased migration. Since endogenous C3G directly interacts with E-cadherin at initial cell–cell contacts,10.Kooistra M.R. Dube N. Bos J.L. Rap1: a key regulator in cell–cell junction formation.J Cell Sci. 2007; 120: 17-22Crossref PubMed Scopus (233) Google Scholar colocalizes with filametous actin, and is involved into cell adhesions formation and maturation, C3G overexpression indeed can modulate signaling involved in attachment to substrate and to neighboring cells. Adhesion of GEC within crescents is a regulated process as several adhesion molecules are expressed within forming crescents and their expression is modified during evolution of crescents.40.Patey N. Lesavre P. Halbwachs-Mecarelli L. et al.Adhesion molecules in human crescentic glomerulonephritis.J Pathol. 1996; 179: 414-420Crossref PubMed Scopus (33) Google Scholar Of the various adhesion molecules identified, platelet/endothelial cell adhesion molecule-1 was shown to recruit Crk–C3G complexes12.Wang Y. Sheibani N. PECAM-1 isoform-specific activation of MAPK/ERKs and small GTPases: implications in inflammation and angiogenesis.J Cell Biochem. 2006; 98: 451-468Crossref PubMed Scopus (35) Google Scholar, whereas crosslinking of ICAM-1 induces tyrosine phosphorylation of the cytoskeleton-associated protein p130Cas, which associates with Crk and C3G and is involved into filamentous actin assembling.41.Etienne S. Adamson P. Greenwood J. et al.ICAM-1 signaling pathways associated with Rho activation in microvascular brain endothelial cells.J Immunol. 1998; 161: 5755-5761PubMed Google Scholar Formation of cadherin-based cell–cell junctions (adherens junctions) among GEC present within crescents also occurs.42.Ng Y.Y. Fan J.M. Mu W. et al.Glomerular epithelial-myofibroblast transdifferentiation in the evolution of glomerular crescent formation.Nephrol Dial Transplant. 1999; 14: 2860-2872Crossref PubMed Scopus (89) Google Scholar, 43.Usui J. Kanemoto K. 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