Non-redundant role of Shc in Erk activation by cytoskeletal reorganization

We have shown previously that cytoskeletal reorganization (CSR) induced by pharmacological reagents such as colchicine or cytochalasins can up-regulate the urokinase-type plasminogen activator (uPA) gene via the Ras/Erk signaling pathway. In this present study using the small interfering RNA technique, we have found that ShcA adapter proteins play a rather active role in CSR-induced Erk activation, contrary to their mostly redundant role in other signaling pathways, e.g. growth factor-induced Erk activation, where Grb2 can bind directly to the receptor tyrosine kinase and activate Erk in the absence of ShcA. ShcA knockdown abolished CSR-induced activation of both Erk and the uPA promoter. Expression of small interfering RNA-escaping silent mutants of p52 or p46 but not p66 ShcA isoform efficiently rescued CSR-induced Erk activation. Moreover, we have shown that phosphorylation of either Tyr-239/Tyr-240 or Tyr-313 in p52(ShcA) can mediate CSR-induced Erk activation equally well. In a quest for molecules upstream of ShcA in this signaling, we found that CSR-induced ShcA tyrosine phosphorylation, its association with Grb2, Erk activation, and uPA gene expression were all dependent on Rho kinase, p38 mitogen-activated protein kinase, and Src. In summary, we have found a novel, non-redundant role for ShcA in contrast to its redundant role in many other signaling pathways.     

ShcA Protects against Epithelial–Mesenchymal Transition through Compartmentalized Inhibition of TGF-β-Induced Smad Activation

Epithelial–mesenchymal transition (EMT) is a normal cell differentiation event during development and contributes pathologically to carcinoma and fibrosis progression. EMT often associates with increased transforming growth factor-β (TGF-β) signaling, and TGF-β drives EMT, in part through Smad-mediated reprogramming of gene expression. TGF-β also activates the Erk MAPK pathway through recruitment and Tyr phosphorylation of the adaptor protein ShcA by the activated TGF-β type I receptor. We found that ShcA protects the epithelial integrity of nontransformed cells against EMT by repressing TGF-β-induced, Smad-mediated gene expression. p52ShcA competed with Smad3 for TGF-β receptor binding, and down-regulation of ShcA expression enhanced autocrine TGF-β/Smad signaling and target gene expression, whereas increased p52ShcA expression resulted in decreased Smad3 binding to the TGF-β receptor, decreased Smad3 activation, and increased Erk MAPK and Akt signaling. Furthermore, p52ShcA sequestered TGF-β receptor complexes to caveolin-associated membrane compartments, and reducing ShcA expression enhanced the receptor localization in clathrin-associated membrane compartments that enable Smad activation. Consequently, silencing ShcA expression induced EMT, with increased cell migration, invasion, and dissemination, and increased stem cell generation and mammosphere formation, dependent upon autocrine TGF-β signaling. These findings position ShcA as a determinant of the epithelial phenotype by repressing TGF-β-induced Smad activation through differential partitioning of receptor complexes at the cell surface.     

Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway.

Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal PTB domain. We have isolated cDNAs encoding for a third Shc isoform, p66shc. The predicted amino acid sequence of p66shc overlaps that of p52shc and contains a unique N-terminal region which is also rich in glycines and prolines (CH2). p52shc/p46shc is found in every cell type with invariant reciprocal relationship, whereas p66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine-phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms stable complexes with Grb2. However, unlike p52shc/p46shc it does not increase EGF activation of MAP kinases, but inhibits fos promoter activation. The isolated CH2 domain retains the inhibitory effect of p66shc on the fos promoter. p52shc/p46shc and p66shc, therefore, appear to exert different effects on the EGFR-MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors.     

ERK phosphorylates p66shcA on Ser36 and subsequently regulates p27kip1 expression via the Akt-FOXO3a pathway: implication of p27kip1 in cell response to oxidative stress

Mice deficient for p66shcA represent an animal model to link oxidative stress and aging. p66shcA is implicated in oxidative stress response and mitogenic signaling. Phosphorylation of p66shcA on Ser36 is critical for its function in oxidative stress response. Here we report the identification of ERK as the kinase phosphorylating p66shcA on Ser36. Activation of ERKs was necessary and sufficient for Ser36 phosphorylation. p66shcA interacted with ERK and was demonstrated to be a substrate for ERK, with Ser36 being the major phosphorylation site. Furthermore, in response to H2O2, inhibition of ERK activation repressed p66shcA-dependent phosphorylation of FOXO3a and the down-regulation of its target gene p27kip1. Down-regulation of p27 might promote cell survival, as p27 played a proapoptotic role in oxidative stress response. As a feedback regulation, Ser36 phosphorylated p66shcA attenuated H2O2-induced ERK activation, whereas p52/46shcA facilitated ERK activation, which required tyrosine phosphorylation of CH1 domain. p66shcA formed a complex with p52/46ShcA, which may provide a platform for efficient signal propagation. Taken together, the data suggest there exists an interplay between ERK and ShcA proteins, which modulates the expression of p27 and cell response to oxidative stress.     

Regulation of interleukin-1alpha expression by integrins and epidermal growth factor receptor in keratinocytes from a mouse model of inflammatory skin disease

Transgenic mice expressing beta1 integrins in the suprabasal epidermal layers have sporadic skin hyperproliferation and inflammation correlated with activation of extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase and increased interleukin (IL)-1alpha production. We investigated the link between aberrant integrin expression, Erk activation, and expression of IL-1alpha. Transgenic keratinocytes had higher basal Erk activity and IL-1alpha levels than nontransgenic controls and were more sensitive to stimulation of Erk activity and IL-1alpha production by IL-1alpha, 12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), and serum. Inhibition of Erk in transgenic keratinocytes reduced basal IL-1alpha levels and the stimulation of IL-1alpha production by serum or phorbol ester, demonstrating that Erk could regulate IL-1alpha expression. TPA or IL-1alpha treatment resulted in rapid down-regulation of the EGF receptor in transgenic cells, indicative of transactivation. Inhibition of transactivation blocked basal and TPA or IL-1alpha induced Erk activation, but not IkappaBalpha degradation, and abolished increased IL-1alpha production in transgenic cells. In transgene-negative cells, constitutive activation of IL-1-dependent signaling by wild type or kinase-dead IRAK1 stimulated IL-1alpha production independent of Erk. We conclude that suprabasal integrin expression leads to Erk activation and increased IL-1alpha expression by potentiating activation of the EGF receptor. These results provide a mechanism by which aberrant integrin expression triggers epidermal hyperproliferation and inflammation.     

Specific beta1 integrin site selectively regulates Akt/protein kinase B signaling via local activation of protein phosphatase 2A

Integrin transmembrane receptors generate multiple signals, but how they mediate specific signaling is not clear. Here we test the hypothesis that particular sequences along the beta(1) integrin cytoplasmic domain may exist that are intimately related to specific integrin-mediated signaling pathways. Using systematic alanine mutagenesis of amino acids conserved between different beta integrin cytoplasmic domains, we identified the tryptophan residue at position 775 of human beta(1) integrin as specific and necessary for integrin-mediated protein kinase B/Akt survival signaling. Stable expression of a beta(1) integrin mutated at this amino acid in GD25 beta(1)-null cells resulted in reduction of Akt phosphorylation at both Ser(473) and Thr(308) activation sites. As a consequence, the cells were substantially more sensitive to serum starvation-induced apoptosis when compared with cells expressing wild type beta(1) integrin. This inactivation of Akt resulted from increased dephosphorylation by a localized active population of protein phosphatase 2A. Both Akt and protein phosphatase 2A were present in beta(1) integrin-organized cytoplasmic complexes, but the activity of this phosphatase was 2.5 times higher in the complexes organized by the mutant integrin. The mutation of Trp(775) specifically affected Akt signaling, without effects on other integrin-activated pathways including phosphoinositide 3-kinase, MAPK, JNK, and p38 nor did it influence activation of the integrin-responsive kinases focal adhesion kinase and Src. The identification of Trp(775) as a specific site for integrin-mediated Akt signaling supports the concept of specificity of signaling along the integrin cytoplasmic domain.     

Activation of the GP IIb-IIIa complex induced by platelet adhesion to collagen is mediated by both alpha2beta1 integrin and GP VI

alpha2beta1 integrin, CD36, and GP VI have all been implicated in platelet-collagen adhesive interactions. We have investigated the role of these glycoproteins on activation of the GP IIb-IIIa complex induced by platelet adhesion to type I fibrillar and monomeric collagen under static conditions. In the presence of Mg2+, platelet adhesion to fibrillar collagen induced activation of the GP IIb-IIIa complex and complete spreading. Anti-alpha2beta1 integrin and anti-GP VI antibodies inhibited the activation of the GP IIb-IIIa complex by about 40 and 50%, respectively, at 60 min although minimal inhibitory effects on adhesion were seen. Platelet spreading was markedly reduced by anti-alpha2beta1 integrin antibody. The combination of anti-alpha2beta1 integrin with anti-GP VI antibody completely inhibited both platelet adhesion and activation of the GP IIb-IIIa complex. Anti-CD36 antibody had no significant effects on platelet adhesion, spreading, and the activation of the GP IIb-IIIa complex at 60 min. Aspirin and the thromboxane A2 receptor antagonist SQ29548 inhibited activation of the GP IIb-IIIa complex about 30% but had minimal inhibitory effect on adhesion. In the absence of Mg2+, there was significant activation of the GP IIb-IIIa complex but minimal spreading was observed. Anti-GP VI antibody completely inhibited adhesion whereas no effect was observed with anti-alpha2beta1 integrin antibody. Anti-CD36 antibody partially inhibited both adhesion and the activation of the GP IIb-IIIa complex. Platelet adhesion to monomeric collagen, which requires Mg2+ and is exclusively mediated by alpha2beta1 integrin, resulted in partial activation of the GPIIb-IIIa complex and spreading. No significant effects were observed by anti-CD36 and anti-GP VI antibodies. These results suggest that both alpha2beta1 integrin and GP VI are involved in inside-out signaling leading to activation of the GP IIb-IIIa complex after platelet adhesion to collagen and generation of thromboxane A2 may further enhance expression of activated GP IIb-IIIa complexes.     

A streptococcal collagen-like protein interacts with the alpha2beta1 integrin and induces intracellular signaling

The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa)(n) that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an alpha2beta1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-alpha2beta1 and anti-beta1 integrins monoclonal antibodies. Accordingly, C2C12 cells stably expressing alpha2beta1 integrin as the only collagen-binding integrin show productive cell adhesion activities on p176 that can be blocked by an anti-alpha2beta1 integrin antibody. In addition, p176 promotes tyrosine phosphorylation of p125(FAK) of C2C12 cells expressing alpha2beta1 integrin, whereas parental cells do not. Furthermore, C2C12 adhesion of human lung fibroblast cells to p176 induces phosphorylation of p125FAK, p130CAS, and p68Paxillin proteins. In a domain swapping experiment, we show that integrin binds to the collagenous domain of the Scl protein. Moreover, the recombinant inserted domain of the alpha2 integrin interacts with p176 with a relatively high affinity (K(D) = 17 nm). Attempts to identify the integrin sites in p176 suggest that more than one site may be involved. These studies, for the first time, suggest that the collagen-like proteins of prokaryotes retained not only structural but also functional characteristics of their eukaryotic counterparts.     

Induction of uPA gene expression by the blockage of E-cadherin via Src- and Shc-dependent Erk signaling

Loss of E-cadherin-mediated cell-cell adhesion and expression of proteolytic enzymes characterize the transition from benign lesions to invasive, metastatic tumor, a rate-limiting step in the progression from adenoma to carcinoma in vivo. A soluble E-cadherin fragment found recently in the serum and urine of cancer patients has been shown to disrupt cell-cell adhesion and to drive cell invasion in a dominant-interfering manner. Physical disruption of cell-cell adhesion can be mimicked by the function-blocking antibody Decma. We have shown previously in MCF7 and T47D cells that urokinase-type plasminogen activator (uPA) activity is up-regulated upon disruption of E-cadherin-dependent cell-cell adhesion. We explored the underlying molecular mechanisms and found that blockage of E-cadherin by Decma elicits a signaling pathway downstream of E-cadherin that leads to Src-dependent Shc and extracellular regulated kinase (Erk) activation and results in uPAgene activation. siRNA-mediated knockdown of endogenous Src-homology collagen protein (Shc) and subsequent expression of single Shc isoforms revealed that p46(Shc) and p52(Shc) but not p66(Shc) were able to mediate Erk activation. A parallel pathway involving PI3K contributed partially to Decma-induced Erk activation. This report describes that disruption of E-cadherin-dependent cell-cell adhesion induces intracellular signaling with the potential to enhance tumorigenesis and, thus, offers new insights into the pathophysiological mechanisms of tumor development.     

Negative regulation of chemokine receptor signaling and B-cell chemotaxis by p66Shc

Shc (Src homology 2 domain containing) adaptors are ubiquitous components of the signaling pathways triggered by tyrosine kinase-coupled receptors. In lymphocytes, similar to other cell types, the p52 and p66 isoforms of ShcA/Shc participate in a self-limiting loop where p52Shc acts as a positive regulator of antigen receptor signaling by promoting Ras activation, whereas p66Shc limits this activity by competitively inhibiting p52Shc. Based on the fact that many signaling mediators are shared by antigen and chemokine receptors, including p52Shc, we have assessed the potential implication of p66Shc in the regulation of B-cell responses to chemokines, focusing on the homing receptors CXCR4 (C-X-C chemokine receptor type 4) and CXCR5 (C-X-C chemokine receptor type 5). The results identify p66Shc as a negative regulator of the chemotactic responses triggered by these receptors, including adhesion, polarization and migration. We also provide evidence that this function is dependent on the ability of p66Shc to interact with the chemokine receptors and promote the assembly of an inhibitory complex, which includes the phosphatases SHP-1 (Src homology phosphatase-1) and SHIP-1 (SH2 domain-containing inositol 5''-phosphatase-1), that results in impaired Vav-dependent reorganization of the actin cytoskeleton. This function maps to the phosphorylatable tyrosine residues in the collagen homology 1 (CH1) domain. The results identify p66Shc as a negative regulator of B-cell chemotaxis and suggest a role for this adaptor in the control of B-cell homing.     

Serine/threonine phosphorylation of ShcA. Regulation of protein-tyrosine phosphatase-pest binding and involvement in insulin signaling

Serine phosphorylation of the ShcA signaling molecule has been reported recently. In this work, we have identified 12-O-tetradecanoylphorbol-13-acetate (TPA)- and growth factor-induced serine/threonine phosphorylation sites in p52(Shc) and p66(Shc). Among them, Ser(29) in p52(Shc) (equivalent to Ser(138) in p66(Shc)) was phosphorylated only after TPA stimulation. Phosphorylation of this site together with the intact phosphotyrosine-binding domain was essential for ShcA binding to the protein-tyrosine phosphatase PTP-PEST. TPA-induced ShcA phosphorylation at this site (and hence, its association with PTP-PEST) was inhibited by a protein kinase C-specific inhibitor and was induced by overexpression of constitutively active mutants of protein kinase Calpha, -epsilon, and -delta isoforms. Insulin also induced ShcA/PTP-PEST association, although to a lesser extent than TPA. Overexpression of a PTP-PEST binding-defective mutant of p52(Shc) (S29A) enhanced insulin-induced ERK activation in insulin receptor-overexpressing HIRc-B cells. Consistent with this, p52(Shc) S29A was more tyrosine-phosphorylated than wild-type p52(Shc) after insulin stimulation. Thus, we have identified a new mechanism whereby serine phosphorylation of ShcA controls the ability of its phosphotyrosine-binding domain to bind PTP-PEST, which is responsible for the dephosphorylation and down-regulation of ShcA after insulin stimulation.     

p66shc inhibits pro-survival epidermal growth factor receptor/ERK signaling during severe oxidative stress in mouse renal proximal tubule cells

The fully executed epidermal growth factor receptor (EGFR)/Ras/MEK/ERK pathway serves a pro-survival role in renal epithelia under moderate oxidative stress. We and others have demonstrated that during severe oxidative stress, however, the activated EGFR is disconnected from ERK activation in cultured renal proximal tubule cells and also in renal proximal tubules after ischemia/reperfusion injury, resulting in necrotic death. Studies have shown that the tyrosine-phosphorylated p46/52 isoforms of the ShcA family of adaptor proteins connect the activated EGFR to activation of Ras and ERK, whereas the p66(shc) isoform can inhibit this p46/52(shc) function. Here, we determined that severe oxidative stress (after a brief period of activation) terminates activation of the Ras/MEK/ERK pathway, which coincides with ERK/JNK-dependent Ser(36) phosphorylation of p66(shc). Isoform-specific knockdown of p66(shc) or mutation of Ser(36) to Ala, but not to Asp, attenuated severe oxidative stress-mediated ERK inhibition and cell death in vitro. Also, severe oxidative stress (unlike ligand stimulation and moderate oxidative stress, both of which support survival) increased binding of p66(shc) to the activated EGFR and Grb2. This binding dissociated the SOS1 adaptor protein from the EGFR-recruited signaling complex, leading to termination of Ras/MEK/ERK activation. Notably, Ser(36) phosphorylation of p66(shc) and its increased binding to the EGFR also occurred in the kidney after ischemia/reperfusion injury in vivo. At the same time, SOS1 binding to the EGFR declined, similar to the in vitro findings. Thus, the mechanism we propose in vitro offers a means to ameliorate oxidative stress-induced cell injury by either inhibiting Ser(36) phosphorylation of p66(shc) or knocking down p66(shc) expression in vivo.     

Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.     

Rac1-MKK3-p38-MAPKAPK2 pathway promotes urokinase plasminogen activator mRNA stability in invasive breast cancer cells

We reported previously that down-regulating or functionally blocking alphav integrins inhibits endogenous p38 mitogen-activated protein kinase (MAPK) activity and urokinase plasminogen activator (uPA) expression in invasive MDA-MB-231 breast cancer cells whereas engaging alphav integrins with vitronectin activates p38 MAPK and up-regulates uPA expression (Chen, J., Baskerville, C., Han, Q., Pan, Z., and Huang, S. (2001) J. Biol. Chem. 276, 47901-47905). Currently, it is not clear what upstream and downstream signaling molecules of p38 MAPK mediate alphav integrin-mediated uPA up-regulation. In the present study, we found that alphav integrin ligation activated small GTPase Rac1 preferentially, and dominant negative Rac1 inhibited alphav integrin-mediated p38 MAPK activation. Using constitutively active MAPK kinases, we found that both constitutively active MKK3 and MKK6 mutants were able to activate p38 MAPK and up-regulate uPA expression, but only dominant negative MKK3 blocked alphav integrin-mediated p38 MAPK activation and uPA up-regulation. These results suggest that MKK3, rather than MKK6, mediates alphav integrin-induced p38 MAPK activation. Among the potential downstream effectors of p38 MAPK, we found that only MAPK-activated protein kinase 2 affects alphav integrin-mediated uPA up-regulation significantly. Finally, using beta-globin reporter gene constructs containing uPA mRNA 3'-untranslated region (UTR) and adenosine/uridine-rich elements-deleted 3'-UTR, we demonstrated that p38 MAPK/MAPK-activated protein kinase 2 signaling pathway regulated uPA mRNA stability through a mechanism involving the adenosine/uridine-rich elements sequence in 3'-UTR of uPA mRNA.     

Protein kinase C modulates negatively the hepatocyte growth factor-induced migration, integrin expression and phosphatidylinositol 3-kinase activation

Previously, we reported that, in hepatocyte growth factor (HGF)-induced HepG2 cells, protein kinase C (PKC) decreased the duration of intensive Erk1/Erk2 MAP kinase activation. This study shows that the inhibition of PKC enhanced significantly the HGF-induced integrin expression. Beside the prolonged activation of Erk1/Erk2, the activity of phosphatidylinositol 3-kinase (PI 3K) was required for growth factor-induced integrin expression. PI 3-kinase was activated to a higher extent in response to HGF than to epidermal growth factor (EGF), though the activation was transient in both cases. In EGF-induced cells, PI 3K activation was terminated by the loss of phosphotyrosine docking sites for PI 3K. To the contrary, the decrease of PI 3K activation, which followed the HGF-induced increase was not accompanied by the loss of phosphotyrosine docking sites and was prevented by the inhibition of PKC. The negative modulator effects of PKC on integrin expression and PI 3-kinase activation correlated with its ability to limit the HGF-induced motogen response.     

Requirement for tyrosine kinase-ERK1/2 signaling in alpha 1 beta 1 integrin-mediated collagen matrix remodeling by rat mesangial cells

Abnormal mesangial extracellular matrix remodeling by mesangial cells (MCs) is the hallmark of progressive glomerulonephritis (GN). We recently showed, using a type I collagen gel contraction assay, that alpha 1 beta 1 integrin-dependent MC adhesion and migration are necessary cell behaviors for collagen matrix remodeling. To further determine the mechanism of alpha 1 beta 1 integrin-mediated collagen remodeling, we studied the signaling pathways of MCs that participate in the regulation of collagen gel contraction. Immunoprecipitation and phosphotyrosine detection revealed that gel contraction is associated with the enhanced activity and phosphorylation of ERK1/2 by MCs. The tyrosine kinase inhibitors herbimycin and genistein inhibited collagen gel contraction dose dependently. Furthermore, targeting ERK1/2 activity with a MEK inhibitor, PD98059, and antisense ERK1/2 hindered gel contraction in a dose-dependent manner. Similar inhibitory effects on gel contraction and ERK1/2 phosphorylation were observed when MC-mediated gel contraction was performed in the presence of function-blocking anti-alpha1 or anti-beta1 integrin antibodies. However, cell adhesion and migration assays indicated that PD98059 and antisense ERK1/2 blocked alpha 1 beta 1 integrin-dependent MC migration, but did not interfere with collagen adhesion, although there was a marked decrease in ERK1/2 phosphorylation and ERK1/2 protein expression in cell adhesion on type I collagen. None of the above could affect membrane expression of alpha 1 beta 1 integrin. These results suggested that ERK1/2 activation is critical for the alpha 1 beta 1 integrin-dependent MC migration necessary for collagen matrix reorganization. We therefore conclude that ERK1/2 may serve as a possible target for pharmacological inhibition of pathological collagen matrix formation in GN.