Epithelial protein lost in neoplasm modulates platelet-derived growth factor–mediated adhesion and motility of mesangial cells
2014; Elsevier BV; Volume: 86; Issue: 3 Linguagem: Inglês
10.1038/ki.2014.85
ISSN1523-1755
AutoresHaruko Tsurumi, Yutaka Harita, Hidetake Kurihara, Hidetaka Kosako, Kenji Hayashi, Atsuko Matsunaga, Yuko Kajiho, Shoichiro Kanda, Kenichiro Miura, Takashi Sekine, Akira Oka, Kiyonobu Ishizuka, Shigeru Horita, Motoshi Hattori, Seisuke Hattori, Takashi Igarashi,
Tópico(s)Cellular Mechanics and Interactions
ResumoMesangial cell migration, regulated by several growth factors, is crucial after glomerulopathy and during glomerular development. Directional migration requires the establishment of a polarized cytoskeletal arrangement, a process regulated by coordinated actin dynamics and focal adhesion turnover at the peripheral ruffles in migrating cells. Here we found high expression of the actin cross-linking protein EPLIN (epithelial protein lost in neoplasm) in mesangial cells. EPLIN was localized in mesangial angles, which consist of actin-containing microfilaments extending underneath the capillary endothelium, where they attach to the glomerular basement membrane. In cultured mesangial cells, EPLIN was localized in peripheral actin bundles at focal adhesions and formed a protein complex with paxillin. The MEK–ERK (extracellular signal-regulated kinase) cascade regulated EPLIN–paxillin interaction and induced translocalization of EPLIN from focal adhesion sites to peripheral ruffles. Knockdown of EPLIN in mesangial cells enhanced platelet-derived growth factor–induced focal adhesion disassembly and cell migration. Furthermore, EPLIN expression was decreased in mesangial proliferative nephritis in rodents and humans in vivo. These results shed light on the coordinated actin remodeling in mesangial cells during restorative remodeling. Thus, changes in expression and localization of cytoskeletal regulators underlie phenotypic changes in mesangial cells in glomerulonephritis. Mesangial cell migration, regulated by several growth factors, is crucial after glomerulopathy and during glomerular development. Directional migration requires the establishment of a polarized cytoskeletal arrangement, a process regulated by coordinated actin dynamics and focal adhesion turnover at the peripheral ruffles in migrating cells. Here we found high expression of the actin cross-linking protein EPLIN (epithelial protein lost in neoplasm) in mesangial cells. EPLIN was localized in mesangial angles, which consist of actin-containing microfilaments extending underneath the capillary endothelium, where they attach to the glomerular basement membrane. In cultured mesangial cells, EPLIN was localized in peripheral actin bundles at focal adhesions and formed a protein complex with paxillin. The MEK–ERK (extracellular signal-regulated kinase) cascade regulated EPLIN–paxillin interaction and induced translocalization of EPLIN from focal adhesion sites to peripheral ruffles. Knockdown of EPLIN in mesangial cells enhanced platelet-derived growth factor–induced focal adhesion disassembly and cell migration. Furthermore, EPLIN expression was decreased in mesangial proliferative nephritis in rodents and humans in vivo. These results shed light on the coordinated actin remodeling in mesangial cells during restorative remodeling. Thus, changes in expression and localization of cytoskeletal regulators underlie phenotypic changes in mesangial cells in glomerulonephritis. Mesangial cell migration is essential in renal recovery after mesangial injury. In the Thy1 model, an experimental animal model of mesangial proliferative nephritis, mesangial injury causes mesangiolysis, as manifested by attenuation or dissolution of the mesangial matrix and degeneration of mesangial cells.1.Morita T. Yamamoto T. Churg J. Mesangiolysis: an update.Am J Kidney Dis. 1998; 31: 559-573Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar This process is followed by reconstitution of the mesangium, which is accomplished by coordinated migration and proliferation of mesangial cells2.Haseley L.A. Hugo C. Reidy M.A. et al.Dissociation of mesangial cell migration and proliferation in experimental glomerulonephritis.Kidney Int. 1999; 56: 964-972Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar originating in the hilar and extraglomerular mesangium. Glomerular reconstitution after mesangial injury is controlled by several growth factors.3.Kohno M. Yasunari K. Minami M. et al.Regulation of rat mesangial cell migration by platelet-derived growth factor, angiotensin II, and adrenomedullin.J Am Soc Nephrol. 1999; 10: 2495-2502PubMed Google Scholar Experiments involving infusion or systemic overexpression have characterized platelet-derived growth factor (PDGF) as the most potent stimulus of mesangial cell proliferation.4.Floege J. Eitner F. Alpers C.E. A new look at platelet-derived growth factor in renal disease.J Am Soc Nephrol. 2008; 19: 12-23Crossref PubMed Scopus (241) Google Scholar PDGF-mediated mesangial cell migration is also an essential process in glomerular development. Deletion of PDGF-B or PDGF receptor β, expressed in endothelial cells or mesangial cells in the glomeruli, respectively, causes defective migration of mesangial cells into the capillary tuft, resulting in a single ballooning capillary loop.5.Lindahl P. Hellstrom M. Kalen M. et al.Paracrine PDGF-B/PDGF-Rbeta signaling controls mesangial cell development in kidney glomeruli.Development. 1998; 125: 3313-3322Crossref PubMed Google Scholar, 6.Lindahl P. Johansson B.R. Leveen P. et al.Pericyte loss and microaneurysm formation in PDGF-B-deficient mice.Science. 1997; 277: 242-245Crossref PubMed Scopus (1740) Google Scholar, 7.Bjarnegard M. Enge M. Norlin J. et al.Endothelium-specific ablation of PDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities.Development. 2004; 131: 1847-1857Crossref PubMed Scopus (274) Google Scholar However, the mechanisms that regulate the morphology and migratory phenotype of mesangial cells remain unclear. Epithelial protein lost in neoplasm (EPLIN) was originally identified as a genetic product that is downregulated or lost in a number of human epithelial tumor cells.8.Maul R.S. Chang D.D. EPLIN, epithelial protein lost in neoplasm.Oncogene. 1999; 18: 7838-7841Crossref PubMed Scopus (69) Google Scholar Two known isoforms of EPLIN, α and β, are generated from alternative promoter usage from a single gene and differ only at the 5′ ends.9.Chen S. Maul R.S. Kim H.R. et al.Characterization of the human EPLIN (epithelial protein lost in neoplasm) gene reveals distinct promoters for the two EPLIN isoforms.Gene. 2000; 248: 69-76Crossref PubMed Scopus (31) Google Scholar Both isoforms contain a centrally located LIN11-ISL1-MEC3 domain that may allow homodimerization,10.Maul R.S. Song Y. Amann K.J. et al.EPLIN regulates actin dynamics by cross-linking and stabilizing filaments.J Cell Biol. 2003; 160: 399-407Crossref PubMed Scopus (120) Google Scholar as well as N- and C-terminal actin-binding sites that flank the LIN11-ISL1-MEC3 domain. Through these sites, EPLIN cross-links and bundles actin filaments to form stable filament structures such as stress fibers.10.Maul R.S. Song Y. Amann K.J. et al.EPLIN regulates actin dynamics by cross-linking and stabilizing filaments.J Cell Biol. 2003; 160: 399-407Crossref PubMed Scopus (120) Google Scholar Extracellular signal-regulated kinase (ERK)–mediated phosphorylation of EPLIN contributes to actin filament reorganization and enhances NIH 3T3 cell motility.11.Han M.Y. Kosako H. Watanabe T. et al.Extracellular signal-regulated kinase/mitogen-activated protein kinase regulates actin organization and cell motility by phosphorylating the actin cross-linking protein EPLIN.Mol Cell Biol. 2007; 27: 8190-8204Crossref PubMed Scopus (58) Google Scholar Decreased EPLIN expression in some cancer cells results in enhanced motility and transformation due to instability of the actin filament structures and rapid filament turnover. Although the relevance of EPLIN in cancer cells has been documented,12.Jiang W.G. Martin T.A. Lewis-Russell J.M. et al.Eplin-alpha expression in human breast cancer, the impact on cellular migration and clinical outcome.Mol Cancer. 2008; 7: 71Crossref PubMed Scopus (95) Google Scholar,13.Zhang S. Wang X. Iqbal S. et al.Epidermal growth factor promotes protein degradation of epithelial protein lost in neoplasm (EPLIN), a putative metastasis suppressor, during epithelial-mesenchymal transition.J Biol Chem. 2013; 288: 1469-1479Crossref PubMed Scopus (29) Google Scholar few studies have examined the roles of EPLIN in normal tissues or in the pathophysiology of noncancerous diseases. EPLIN transcript levels are particularly high in the kidney.8.Maul R.S. Chang D.D. EPLIN, epithelial protein lost in neoplasm.Oncogene. 1999; 18: 7838-7841Crossref PubMed Scopus (69) Google Scholar In this study, we found a strong expression of the EPLIN protein in glomerular mesangial cells, particularly in the mesangial processes and mesangial angles, and a markedly decreased expression in mesangial proliferative nephritis. In addition, EPLIN is associated with stability of focal adhesion and regulates PDGF-induced mesangial cell migration. As a previous study using northern blot analysis demonstrated a strong expression of the EPLIN transcript in the kidney,8.Maul R.S. Chang D.D. EPLIN, epithelial protein lost in neoplasm.Oncogene. 1999; 18: 7838-7841Crossref PubMed Scopus (69) Google Scholar we examined whether either or both EPLIN isoforms was expressed. Using reverse transcription PCR amplification with isoform-specific primers, both isoforms of EPLIN mRNA were found to be expressed throughout the kidney in rats, including renal cortex, medulla, and glomeruli (Figure 1a). Immunohistochemical staining of adult rat kidney tissues revealed that EPLIN protein was expressed in glomeruli, tubular epithelial cells, and extraglomerular vascular endothelial cells (Figure 1b). Similar staining patterns were observed in human kidney sections (Figure 1c). To analyze the localization of EPLIN in glomeruli, cryosections of adult rat kidney were double-labeled with several glomerular cell markers. EPLIN colocalized with Thy1, a mesangial cell marker, but not with intercellular adhesion molecule-2, an endothelial cell marker, or zonula occludens-1, a podocyte marker; this indicates that EPLIN is highly expressed in mesangial cells in the glomeruli (Figure 2a). EPLIN was not completely colocalized with Thy1. Punctiform signals of EPLIN, distinct from Thy1 signals, were observed near the capillary endothelium and mesangial angles (Figure 2aJ, K and L). EPLIN signals corresponding to the mesangial angles were also detected in human glomerulus (Figure 2b). A more detailed localization of EPLIN was examined using immunoelectron microscopy (Figure 2c). Immunogold particles for EPLIN were mainly detected in the periphery of the cytoplasmic processes of mesangial cells at the mesangial angles (Figure 2cA and B) and in the mesangial cell–cell adhesion sites (Figure 2cC).Figure 2Localization of epithelial protein lost in neoplasm (EPLIN) in mesangial cells. (a) Localization of EPLIN in adult rat glomeruli. Sections were costained for the cell markers intercellular adhesion molecule-2 (ICAM-2), zonula occludens (ZO)-1, or Thy1 (E30) and EPLIN. Merged images are shown in G, H, and I. Higher-magnification images corresponding to squares in G, H, and I are shown in J, K, and L, respectively. Scale bar=50μm. (b) Localization of EPLIN in adult human glomeruli. Sections were costained for zonula occludens-1 (ZO-1) (A) and EPLIN (B). A merged image is shown in C. A higher-magnification image corresponding to the square in C is shown in D. Scale bar=50μm. (c) Immunogold staining for EPLIN detected using immunoelectron microscopy. Immunogold particles for EPLIN were detected in the periphery of the cytoplasmic processes of mesangial cells at the mesangial angles (A, B, arrow). Higher-magnification image corresponding to the square in A is shown in B. The particles were also detected in mesangial cell–cell adhesion sites (C, arrows). En, endothelial cell; GBM, glomerular basement membrane; M, mesangial cell. Scale bars=100nm. (d) EPLIN expression in neonatal rat glomerulus. Section was costained for podocalyxin (A) and EPLIN (B). A merged image is shown in C. Scale bar=20μm.View Large Image Figure ViewerDownload (PPT) During glomerular development, mesangial cells populate the core of the glomerular tuft and connect the capillary loops via deposition of the mesangial matrix. In the late S-shaped body stage to early capillary loop stage, when the mesangial cells derived from the metanephric mesenchyme are recruited to the developing nephron, EPLIN was observed in the central mesangial areas, which is distinct from the outer layers formed by column-shaped podocytes (Figure 2d). As EPLIN is implicated in growth control or motility of invasive tumors, we hypothesized that glomerulonephritis may affect EPLIN expression in vivo; therefore, we investigated EPLIN expression in the rat anti-Thy1.1 model of glomerulonephritis in which the intraglomerular mesangium is destroyed by an antibody-mediated reaction followed by tissue repair mediated by migrating and proliferating mesangial cells originating from the extraglomerular or hilar mesangium.14.Daniel C. Albrecht H. Ludke A. et al.Nestin expression in repopulating mesangial cells promotes their proliferation.Lab Invest. 2008; 88: 387-397Crossref PubMed Scopus (51) Google Scholar Before anti-Thy1.1 injection (day 0), EPLIN protein was expressed in the mesangial area, whereas α-smooth muscle actin (α-SMA), a marker for activated mesangial cells, was hardly detected (Figure 3a). Three or five days after antibody injection, when α-SMA staining in the mesangial area was apparent, the immunofluorescence signals for EPLIN markedly decreased. Western blotting of glomerular lysates confirmed the decreased EPLIN expression during acute phase of the models (Figure 3b). On day 15, Thy1 expression recovered to normal levels (Supplementary Figure S1 online), whereas EPLIN expression was still significantly lower than that at day 0. These results indicated that de novo expression of α-SMA, a molecular marker for phenotypical changes in mesangial cells, inversely correlated with EPLIN expression. Furthermore, changes in EPLIN expression were not fully coincident with those in Thy1 expression. Download .jpg (.12 MB) Help with files Supplementary Figure S1 To evaluate EPLIN expression in human mesangial proliferative nephritis, we performed immunohistochemical analysis using kidney biopsy samples from two patients with membranoproliferative glomerulonephritis (MPGN) and two patients with IgA nephropathy (Figure 3c and d). Both MPGN patients had presented with moderate hematuria and proteinuria at the time of renal biopsy. EPLIN expression at mesangial angles or in the mesangial cytoplasm contiguous with endothelial cells was segmentally decreased in both patients, especially in the area of mesangial proliferation in glomeruli (Figure 3c). In the glomeruli of two IgA nephropathy patients, EPLIN expression in the glomerular tufts with proliferating mesangial cells was segmentally decreased (Figure 3d). These results indicate that EPLIN expression is dynamically regulated during the mesangial proliferative process in rodents and humans in vivo. In cultured human mesangial cells, intense signals for EPLIN were observed at the sites of focal adhesion, partially colocalizing with paxillin (Figure 4a), suggesting that a subfraction of the two proteins may form a complex in mesangial cells. EPLIN signals were also partially localized to peripheral actin bundles. As a result of their colocalization at focal adhesions, we performed co-immunoprecipitation experiment to investigate whether EPLIN interacts with paxillin. As shown in Figure 4b, we confirmed endogenous EPLIN–paxillin interaction in cultured human mesangial cells. To further verify this interaction, we performed in situ proximity ligation assay (PLA) in cultured human mesangial cells. The in situ PLA technology generates localized, discrete signals where two proteins of interest (EPLIN and paxillin, in this case) are in close proximity. As shown in Figure 4c, complex formation of EPLIN and paxillin was detected as red dots especially in the cell periphery. By using overexpressed HEK293T cells, both EPLIN isoforms interacted with paxillin (data not shown), indicating that the N-terminus of EPLIN β is not required for the interaction. In human glomeruli, signals for paxillin, which is expressed in both podocytes and mesangial cells,15.Matsuura S. Kondo S. Suga K. et al.Expression of focal adhesion proteins in the developing rat kidney.J Histochem Cytochem. 2011; 59: 864-874Crossref PubMed Scopus (12) Google Scholar partially overlapped with those of EPLIN in mesangial cells (Figure 4d). As PDGF has a key role in the pathogenesis of mesangial proliferative glomerulonephritis,16.Iida H. Seifert R. Alpers C.E. et al.Platelet-derived growth factor (PDGF) and PDGF receptor are induced in mesangial proliferative nephritis in the rat.Proc Natl Acad Sci USA. 1991; 88: 6560-6564Crossref PubMed Scopus (288) Google Scholar,17.Gesualdo L. Pinzani M. Floriano J.J. et al.Platelet-derived growth factor expression in mesangial proliferative glomerulonephritis.Lab Invest. 1991; 65: 160-167PubMed Google Scholar we assessed morphological and molecular changes of human mesangial cells in response to PDGF (Figure 5a, Supplementary Figure S2 online). When serum-starved human mesangial cells were stimulated with PDGF, ruffles formed within 30min; EPLIN staining on focal adhesions and stress fibers significantly decreased, and EPLIN appeared as bright lines along the peripheral ruffles. This indicates that subcellular localization of EPLIN is dynamically modified by signals downstream of PDGF. Download .jpg (.24 MB) Help with files Supplementary Figure S2 We previously reported that EPLIN is directly phosphorylated by ERK in PDGF-treated fibroblasts.11.Han M.Y. Kosako H. Watanabe T. et al.Extracellular signal-regulated kinase/mitogen-activated protein kinase regulates actin organization and cell motility by phosphorylating the actin cross-linking protein EPLIN.Mol Cell Biol. 2007; 27: 8190-8204Crossref PubMed Scopus (58) Google Scholar As this phosphorylation regulates its association with F-actin,11.Han M.Y. Kosako H. Watanabe T. et al.Extracellular signal-regulated kinase/mitogen-activated protein kinase regulates actin organization and cell motility by phosphorylating the actin cross-linking protein EPLIN.Mol Cell Biol. 2007; 27: 8190-8204Crossref PubMed Scopus (58) Google Scholar we hypothesized that EPLIN localization to focal adhesions is also regulated by the ERK pathway. PDGF-induced ruffle formation and dissociation of EPLIN from focal adhesions were abrogated by pretreatment with the mitogen-activated protein kinase (MEK) inhibitor U0126 (Figure 5b, Supplementary Figure S3 online), indicating that these changes were ERK dependent. Effects of ERK on the interaction between EPLIN and paxillin were also investigated. Co-immunoprecipitation between EPLIN and paxillin was abrogated by the expression of constitutively active MEK (Figure 5c). These results indicated that activation of the MEK–ERK pathway decreases EPLIN localization at focal adhesions. Download .jpg (.15 MB) Help with files Supplementary Figure S3 To clarify the role of EPLIN at peripheral ruffles and focal adhesions, we analyzed the effects of small interfering RNA (siRNA)–mediated knockdown of EPLIN in mesangial cell morphology. Treatment with two different siRNAs effectively decreased EPLIN at the protein level (Figure 6a and b). To analyze morphological changes caused by PDGF, cultured mesangial cells transfected with control siRNA or EPLIN siRNA were stimulated with PDGF for 30min. Ruffling and disassembly of peripheral focal adhesions were categorized into four classes based on the staining patterns of F-actin and paxillin (Figure 6c, Supplementary Materials and Methods online). As shown in Figure 6d, knockdown of EPLIN alone did not significantly affect the cytoskeletal actin arrangement or the number and localization of focal adhesion sites compared with control siRNA-treated cells (Figure 6d). On PDGF treatment, peripheral ruffles were formed in both control and EPLIN knockdown cells, and no difference in the degrees of ruffling between them was observed (Figure 6d, upper panel). Further, the effects of EPLIN on focal adhesion formation were determined by measuring paxillin-positive sites at the cell periphery. Without PDGF treatment, both control and EPLIN knockdown cells were classified as type I or II, indicating that EPLIN knockdown itself did not affect focal adhesion stability. In contrast, after PDGF treatment, EPLIN depletion significantly enhanced the decrease in focal adhesions at the cell periphery (Figure 6d, lower panel). These results suggested that EPLIN localizes to focal adhesions and inhibits PDGF-induced focal adhesion disassembly. Download .doc (.04 MB) Help with doc files Supplementary Information We examined the roles of EPLIN in mesangial cell proliferation and directed migration. A cell count proliferation assay revealed that EPLIN depletion had no effect on the proliferation rate (data not shown). However, in a modified Boyden chamber assay, treatment with two distinct siRNAs targeting EPLIN significantly promoted cell migration in response to PDGF (Figure 7). Thus, our results reveal that EPLIN suppresses PDGF-induced mesangial cell migration. The mesangial cell body contains a maze of short tortuous F-actin fibers organized in bundles.18.Cortes P. Mendez M. Riser B.L. et al.F-actin fiber distribution in glomerular cells: structural and functional implications.Kidney Int. 2000; 58: 2452-2461Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Mesangial cells extend tongue-like protrusions underneath the capillary endothelium toward the mesangial angles. The F-actin fibers are connected to the extracellular matrix through focal adhesions, pulling the glomerular basement membrane inward by interconnecting with the opposing mesangial angles, thus generating inward-directed forces that balance the expansile forces caused by pressure gradients across the glomerular basement membrane.19.Kriz W. Elger M. Mundel P. et al.Structure-stabilizing forces in the glomerular tuft.J Am Soc Nephrol. 1995; 5: 1731-1739PubMed Google Scholar Mesangial cells express several focal adhesion proteins whose expression levels are altered in mesangial proliferative glomerulosclerosis.15.Matsuura S. Kondo S. Suga K. et al.Expression of focal adhesion proteins in the developing rat kidney.J Histochem Cytochem. 2011; 59: 864-874Crossref PubMed Scopus (12) Google Scholar,20.Kagami S. Shimizu M. Kondo S. et al.Up-regulation of integrin-linked kinase activity in rat mesangioproliferative glomerulonephritis.Life Sci. 2006; 78: 1794-1800Crossref PubMed Scopus (13) Google Scholar In this study, we demonstrated that EPLIN is concentrated at the periphery of the cytoplasmic processes of mesangial cells. EPLIN interacts with focal adhesion, and this association is regulated by PDGF signaling. Decreased EPLIN expression observed in mesangial proliferative glomerulonephritis may result in augmented PDGF-induced mesangial migration. These findings unravel the novel association of EPLIN with focal adhesion complex, and the mechanism of the coordinated actin remodeling in mesangial cells during glomerular damage and restoration. During repair after glomerular injury, mesangial cells undergo phenotypic changes, which can be recognized by markers of mesangial cell activation. Differentiated mesangial cells express nonmuscle isoforms of actin, namely β- and γ-cytoplasmic actin.21.Johnson R.J. Iida H. Alpers C.E. et al.Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation.J Clin Invest. 1991; 87: 847-858Crossref PubMed Scopus (432) Google Scholar In human and experimental rat glomerulonephritis, on the other hand, mesangial cells acquire myofibroblast-like characteristics, expressing smooth muscle cell–type actin or α-SMA.21.Johnson R.J. Iida H. Alpers C.E. et al.Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation.J Clin Invest. 1991; 87: 847-858Crossref PubMed Scopus (432) Google Scholar Regulation of the actin cytoskeleton in mesangial cells is also mediated by various actin-binding proteins, for example, profilin, which promotes the addition of actin monomers to the barbed ends, is expressed in mesangial cells, and shows increased expression in Thy1 nephritis22.Tamura M. Tanaka H. Yashiro A. et al.Expression of profilin, an actin-binding protein, in rat experimental glomerulonephritis and its upregulation by basic fibroblast growth factor in cultured rat mesangial cells.J Am Soc Nephrol. 2000; 11: 423-433PubMed Google Scholar and experimental diabetic nephropathy.23.Clarkson M.R. Murphy M. Gupta S. et al.High glucose-altered gene expression in mesangial cells. Actin-regulatory protein gene expression is triggered by oxidative stress and cytoskeletal disassembly.J Biol Chem. 2002; 277: 9707-9712Crossref PubMed Scopus (89) Google Scholar Drebrin, which binds to F-actin and profilin, is also upregulated in experimental nephritis. It is localized in the mesangial processes, which extend in various directions throughout the process of tissue repair.24.Peitsch W.K. Hofmann I. Endlich N. et al.Cell biological and biochemical characterization of drebrin complexes in mesangial cells and podocytes of renal glomeruli.J Am Soc Nephrol. 2003; 14: 1452-1463Crossref PubMed Scopus (39) Google Scholar,25.Ichimura K. Kurihara H. Sakai T. Involvement of mesangial cells expressing alpha-smooth muscle actin during restorative glomerular remodeling in Thy-1.1 nephritis.J Histochem Cytochem. 2006; 54: 1291-1301Crossref PubMed Scopus (21) Google Scholar Drebrin competes with F-actin–binding proteins, such as α-actinin, tropomyosin, and fascin,26.Grintsevich E.E. Galkin V.E. Orlova A. et al.Mapping of drebrin binding site on F-actin.J Mol Biol. 2010; 398: 542-554Crossref PubMed Scopus (40) Google Scholar to stabilize filopodia and direct cell migration in response to environmental signals. In contrast, EPLIN, which cross-links and bundles actin filaments, is downregulated in Thy1 nephritis and some forms of human mesangial proliferative glomerulonephritis, suggesting that EPLIN is a marker of mesangial cell differentiation. Thus, the phenotypic changes in mesangial cells during glomerular injury occur concurrently with the induction or suppression of actin cytoskeleton regulatory proteins. Cell migration is a complex, highly regulated process that involves the continuous formation and disassembly of adhesion (adhesion turnover), which is spatiotemporally regulated by complex intermolecular interactions.27.Margadant C. Kreft M. Zambruno G. et al.Kindlin-1 regulates integrin dynamics and adhesion turnover.PLoS One. 2013; 8: e65341Crossref PubMed Scopus (23) Google Scholar,28.Webb D.J. Donais K. Whitmore L.A. et al.FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly.Nat Cell Biol. 2004; 6: 154-161Crossref PubMed Scopus (1072) Google Scholar During cell migration, adhesion formation takes place at the leading edge of protrusions, whereas disassembly occurs both at the cell rear and at the base of protrusions. This study elucidates the mechanism of PDGF-induced mesangial migration through the regulation of focal adhesions by EPLIN. First, PDGF-induced MEK/ERK activation alters subcellular localization of EPLIN from focal adhesions to peripheral ruffles. This translocation may be regulated by ERK-dependent interaction of EPLIN with paxillin. The role of EPLIN in focal adhesion complex formation is also indicated by the fact that depletion of EPLIN significantly decreased peripheral focal adhesion sites after PDGF treatment. Therefore, EPLIN may stabilize focal adhesions, presumably by interaction with their component paxillin. To date, several paxillin-binding proteins have been reported to regulate focal adhesion turnover downstream of the kinase cascade, for example, the endosomal adaptor protein APPL1 inhibits the turnover of leading-edge adhesion by impairing Src-mediated tyrosine phosphorylation of Akt.29.Broussard J.A. Lin W.H. Majumdar D. et al.The endosomal adaptor protein APPL1 impairs the turnover of leading edge adhesions to regulate cell migration.Mol Biol Cell. 2012; 23: 1486-1499Crossref PubMed Scopus (26) Google Scholar Paxillin kinase linker regulates the development of front–rear cell polarity and cell migration, a process regulated by its phosphorylation by PDGF and interaction with paxillin.30.Yu J.A. Deakin N.O. Turner C.E. Paxillin-kinase-linker tyrosine phosphorylation regulates directional cell migration.Mol Biol Cell. 2009; 20: 4706-4719Crossref PubMed Scopus (46) Google Scholar EPLIN may form a regulatory complex at the focal adhesion and link adhesion complex to F-actin, regulating signal intensity downstream of humoral effectors. Thus, the loss of EPLIN in glomerulonephritis may result in destabilization of actin filament networks and focal adhesions, leading to enhanced cell motility. Although the mechanism regulating EPLIN expression in mesangial proliferative glomerulonephritis remains unknown, expression and proper localization of cytoskeletal regulators can modulate mesangial cell morphology and migration during glomerulonephritis. Further studies using gene knockout are required to fully elucidate the molecular mechanism by which mesangial migration is regulated during mesangial repair in vivo. The following antibodies were obtained from commercial sources: rabbit polyclonal anti-EPLIN antibody A330-103A (Bethyl, Montgomery, TX) for immunostaining of human tissue samples; rabbit polyclonal anti-EPLIN antibody A300-225A (Bethyl) for immunoblotting of human tissue samples; mouse monoclonal anti-zonula occludens-1 antibody (Zymed, San Francisco, CA); mouse monoclonal α-SMA antibody (Progen, Heidelberg, Germany); rabbit monoclonal anti-β-actin antibody (Cell Signaling, Danvers, MA); mouse anti-CD34 antibody M7165 (Dako, Copenhagen, Denmark); mouse antipaxillin antibody (BD, Franklin Lakes, NJ); mouse anti-GLEPP1 antibody (BioGenex, San Ramon, CA); BODIPY FL Phallacidin B607 (Life Technologies, Grand Island, NY); mouse monoclonal anti-c-Myc antibody 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA); PDGF-BB (Sigma, St Louis, MO); U0126 (Promega, Madison, WI); and agarose beads conjugated with the 9E10 anti-Myc antibody (Santa Cruz Biotechnology). The rabbit anti-EPLIN antibody11.Han M.Y. Kosako H. Watanabe T. et al.Extracellular signal-regulated kinase/mitogen-activated protein kinase regulates actin organization and cell motility by phosphorylating the actin cross-linking protein EPLIN.Mol Cell Biol. 2007; 27: 8190-8204Crossref PubMed Scopus (58) Google Scholar was used for studies of rat samples, and mouse anti-intercellular adhesion molecule-2 antibody D12,31.Notoya M. Shinosaki T. Kobayashi T. et al.Intussusceptive capillary growth is required for glomerular repair in rat Thy-1.1 nephritis.Kidney Int. 2003; 63: 1365-1373Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar mouse anti-Thy1.1 antibody E30,25.Ichimura K. Kurihara H. Sakai T. Involvement of mesangial cells expressing alpha-smooth muscle actin during restorative glomerular remodeling in Thy-1.1 nephritis.J Histochem Cytochem. 2006; 54: 1291-1301Crossref PubMed Scopus (21) Google Scholar and mouse anti-rat podocalyxin antibody clone N3 (ref. 32.Kurihara H. Harita Y. Ichimura K. et al.SIRP-alpha-CD47 system functions as an intercellular signal in the renal glomerulus.Am J Physiol Renal Physiol. 2010; 299: F517-F527Crossref PubMed Scopus (22) Google Scholar) were used as previously described. Human embryonic kidney HEK293T cells (ATCC, Manassas, VA) were maintained as previously described.33.Harita Y. Kurihara H. Kosako H. et al.Phosphorylation of nephrin triggers Ca2+ signaling by recruitment and activation of phospholipase C-gamma 1.J Biol Chem. 2009; 284: 8951-8962Crossref PubMed Scopus (58) Google Scholar Primary human mesangial cells (Lonza, Walkersville, MD) were routinely cultured and maintained as previously described.34.Sugenoya Y. Yoshimura A. Yamamura H. et al.Smooth-muscle calponin in mesangial cells: regulation of expression and a role in suppressing glomerulonephritis.J Am Soc Nephrol. 2002; 13: 322-331PubMed Google Scholar Cells (passages 7–9) were proliferated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum until 80% confluency was reached. Before stimulation, cells were rendered quiescent by maintaining them in serum-free medium for 24h. Transfections were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) as per the manufacturer's instructions. The sequences of siRNAs used were as follows: nonsilencing control (QIAGEN Cat#1027281, Venlo, The Netherlands), EPLIN siRNA#1 (5′-GGUGAACCAACUCAAACUATT-3′), and EPLIN siRNA#2 (5′-GUGGAAGGAAGAUCUCUGATT-3′). The siRNA duplexes were transfected into human mesangial cells using Lipofectamine RNAiMAX (Invitrogen) as per the manufacturer's instructions. The DNA fragments encoding mouse EPLIN-α (161–753) and EPLIN-β (1-753) were amplified using PCR and cloned into pCMV-Tag3-Myc (Stratagene, LaJolla, CA). Constitutively active HA-tagged MEK was previously described.11.Han M.Y. Kosako H. Watanabe T. et al.Extracellular signal-regulated kinase/mitogen-activated protein kinase regulates actin organization and cell motility by phosphorylating the actin cross-linking protein EPLIN.Mol Cell Biol. 2007; 27: 8190-8204Crossref PubMed Scopus (58) Google Scholar Mouse paxillin α was generated using PCR and cloned into pcDNA3.1 (Life Technologies). Green fluorescent protein was inserted immediately downstream of the paxillin α gene. After rats were anesthetized, kidneys were removed for RNA extraction. Glomeruli were isolated using differential sieving methods. Total RNA from the whole kidney, cortex, medulla, and glomeruli was extracted using TRIZOL (Invitrogen). Then, 0.5μg of total RNA was reverse-transcribed using a One-Step RNA PCR Kit (Takara, Tokyo, Japan). Single-strand complementary DNA was amplified using primers specific for rat EPLIN-α (forward 5′-GTCCGTAGACAAGATGGAATC-3′ and reverse 5′-CCACGCCTCTCTGAGACCTC-3′; 724bp) and rat EPLIN-β (forward 5′-CAGCTTGGAGTTTTAACTTTC-3′ and reverse 5′-CCACGCCTCTCTGAGACCTC-3′; 799bp). The amplified products were electrophoresed on 1% agarose gel, stained with ethidium bromide, and photographed under a ultraviolet lamp. Kidney biopsy samples were obtained from patients admitted to the Kidney Center, Tokyo Women's Medical University. MPGN patient 1 presented with hematuria and proteinuria, and was diagnosed with MPGN on kidney biopsy at 7 years of age. MPGN patient 2 was diagnosed with complement-associated glomerulonephritis, formerly called MPGN. Hematuria and proteinuria were detected on school urinalysis at 12 years of age. Renal biopsy was performed at 14 years of age. Patient 1 of IgA nephropathy had moderate hematuria and proteinuria from 6 years of age. Patient 2 of IgA nephropathy presented hematuria and proteinuria from 8 years of age. She was diagnosed with IgA nephropathy on kidney biopsy at 10 years of age. Normal tissue samples (adult donor kidneys) were used as control. Samples were collected after receipt of informed consent from the patients. This study was approved by the ethics committee of the Tokyo Women's Medical University. Cultured mesangial cells transfected with control or EPLIN siRNA were stimulated with or without 50ng/ml PDGF for 30min. Cells were stained with phalloidin (green) and anti-paxillin (red). The degree of ruffling was categorized into four classes, as exemplified in Figure 6c. Cells that had almost no ruffling formation were categorized as I. Cells with ruffling in less or more than 50% of entire periphery were categorized as II or III. Cells that had ruffling in almost the entire periphery were categorized as IV. The degree of peripheral focal adhesions was also categorized into four classes, as exemplified in Figure 6c. Cells having paxillin-positive dots at the tip of the F-actin in almost the entire periphery were categorized as I. Cells that had focal adhesions > or <50% of the entire periphery were categorized as II or III. Cells that had almost no focal adhesions in entire periphery were categorized as IV. The images of randomly selected 30 cells were analyzed by three blinded observers. Data are means±s.d. of three independent experiments. Statistical analysis was carried out using Student's t-test. PLA was performed with the Duolink in situ PLA kit (Olink Bioscience, Uppsala, Sweden). Human mesangial cells were cultured on coverslips coated with collagen type 1 and fixed and permeabilized. After being washed with phosphate-buffered saline, the cells were blocked in blocking solution at 37°C for 30min. After being washed, the samples were incubated with diluted primary antibodies, followed by the corresponding secondary antibodies conjugated with the PLA oligonucleotide probes (Duolink PLA Rabbit PLUS and PLA Mouse MINUS proximity probes) for 2h at 37°C. Hybridization and ligation of the connector oligonucleotides, a rolling-circle amplification, and detection of the amplified DNA products using detection reagent Red were performed using the Duolink detection reagent kit according to the manufacturer's protocol. The samples were costained with phalloidin (green) and 4,6-diamidino-2-phenylindole (blue) before mounting. We express our thanks to Professor S Ohno, Professor A Suzuki, and Dr T Hirose, Department of Molecular Biology, Yokohama City University School of Medicine, for their valuable suggestions on cell morphological analysis; and Professor M Mizuguchi, School of International Health, The University of Tokyo, for his valuable comments. This work was supported in part by a Grant-in-Aid for Scientific Research (B) (22390204 to YH, Se.H, and TI) and Grant-in-Aid for Scientific Research (C) (25461617 to HT, YH, Hi.K, and MH) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Figure S1. Recovery of epithelial protein lost in neoplasm (EPLIN) expression after induction of anti-Thy1 glomerulonephritis was lagged behind that of Thy-1 expression. Figure S2. Localization of epithelial protein lost in neoplasm (EPLIN) and paxillin in platelet-derived growth factor (PDGF)–treated mesangial cells. Figure S3. Localization of epithelial protein lost in neoplasm (EPLIN) at focal adhesions is regulated by the platelet-derived growth factor (PDGF)–extracellular signal-regulated kinase (ERK) axis. Supplementary Methods Supplementary material is linked to the online version of the paper at http://www.nature.com/ki
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