Impaired proteolysis of collagen I inhibits proliferation of hepatic stellate cells: implications for regulation of liver fibrosis

Myofibroblastic-activated hepatic stellate cells are the major source of the collagen I-rich extracellular matrix in liver fibrosis but also produce matrix metalloproteinases, which remodel this protein. We have investigated the role of collagen I proteolysis in both regulating proliferation and maintaining the activated myofibroblastic phenotype of stellate cells in vitro. Compared with stellate cells plated on normal collagen I, those plated on a collagenase-resistant form of collagen I (r/r collagen) had reduced thymidine incorporation and proliferating cell nuclear antigen expression but increased p21 expression. Collagen I was shown to be rendered resistant to matrix metalloproteinases by artificial cross-linking in vitro using tissue transglutaminase exerted similar antiproliferative effects on stellate cells to r/r collagen. Of the stellate cell activation markers examined (tissue inhibitor of metalloproteinases-1, alpha-smooth muscle actin, matrix metalloproteinases-2 and -9, and procollagen I) only the last was decreased by culture on r/r collagen relative to normal collagen I. Antagonists of integrin alphavbeta3, an integrin reported to stimulate stellate cell proliferation, significantly inhibited adhesion, proliferation, and procollagen I synthesis of stellate cells plated on normal collagen I but had reduced effectiveness on these parameters in cells on r/r collagen. We conclude that proliferation of stellate cells is promoted by pericellular collagen I proteolysis acting via alphavbeta3 integrin. Cross-linking of collagen I by tissue transglutaminase, a process known to occur in chronic liver fibrosis, might not only increase its resistance to matrix metalloproteinases thereby inhibiting resolution of fibrosis but also functions to constrain the fibroproliferative process.     

肝纤维化发生发展及逆转过程中肝巨噬细胞亚群分类

肝纤维化是由持续性损伤修复反应引起的,导致肝组织内细胞外基质异常沉积,进一步引发肝脏结构和肝功能异常改变的一种病理过程.已有大量研究表明,肝纤维化在去除损伤因素后是可以逆转的.肝星状细胞作为主要的效应细胞,合成和分泌各种胶原和细胞外基质,一直被认为是肝纤维化发生发展的中心环节.最近的研究发现,巨噬细胞作为主要的调节细胞,能同时调节肝星状细胞的功能和基质胶原的降解,促进肝纤维化的形成.而在肝纤维化逆转过程中,促使活化的肝星状细胞凋亡和纤维胶原的降解,促进肝纤维化的逆转.目前已有研究表明,巨噬细胞亚群在肝纤维化发生发展及逆转中具有双向调控作用,但是对于动物模型体内还没有系统的研究巨噬细胞亚群的分类.本文对巨噬细胞亚群的分类研究做一个全面的综述,对肝脏巨噬细胞在肝纤维化中分子机制的进一步研究具有一定的参考价值和借鉴意义.     

牛磺酸上调基因1(TUG1)对肝纤维化的作用及其机制*

目的:探讨牛磺酸上调基因1(TUG1)在肝纤维化中的作用机制。方法:按照文献建立TGF-β1(5 ng/ml)刺激的活化肝星状细胞模型和经典的1%DMN(1 ml/kg/d)致大鼠肝纤维化模型,将肝纤维化大鼠和活化肝星状细胞(HSC)均分为模型对照组、阴性对照组(沉默TUG1阴性对照)、siRNA干扰组(TUG1基因沉默组)。实验结束后利用苏木精-伊红(HE)染色检测大鼠肝脏组织病理变化;采用逆转录-聚合酶链反应(RT-PCR)法、蛋白免疫印记(Western blot)分别测定大鼠肝组织及活化肝星状细胞中α-平滑肌肌动蛋白(α-SMA)、TUG1、I型胶原蛋白(collagenI)、基质金属蛋白酶-2(MMP-2)、金属蛋白酶组织抑制因子(TIMP-1)、Smad2、Smad3表达水平。结果:肝组织病理学检查显示,沉默TUG1能够明显缓解肝脏纤维化病理改变,Western blot结果显示,沉默TUG1能够显著降低大鼠肝组织和活化肝星状细胞中TUG1、α-SMA、collagenI、MMP-2、TIMP-1、Smad2、Smad3基因与蛋白表达水平(P＜0.05)。与模型对照组相比,阴性对照组的TUG1、α-SMA的蛋白与基因水平明显升高(P＜0.05)。与模型对照组和阴性对照组相比,siRNA干扰组中TUG1, α-SMA, collagenI, MMP-2, TIMP-1, Smad2 and Smad3的蛋白和基因水平显著降低(P＜0.05),而在模型对照组和阴性对照组中TUG1, α-SMA, collagenI, MMP-2, TIMP-1, Smad2 and Smad3的蛋白和基因表达水平之间差异无显著性。结论:TUG1在肝纤维化组织和活化的肝星状细胞中显著上调,沉默TUG1可能通过抑制转化生长因子-β1(TGF-β1)/Smad信号通路改善1%DMN致大鼠肝纤维化病理损伤,降低活化肝星状细胞中纤维化相关蛋白水平,发挥抗肝纤维化的作用。     

Reduced intimal thickening following alpha(v)beta3 blockade is associated with smooth muscle cell apoptosis

The adhesion integrin alpha(v)beta3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of alpha(v)beta3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of alpha(v)beta3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of alpha(v)beta3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of alpha(v)beta3. At both 2 and 4 wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of alpha(v)beta3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing alpha(v)beta3. These findings may explain the reduction in restenosis observed clinically following beta3 integrin blockade.     

Fas activation induces renal tubular epithelial cell beta 8 integrin expression and function in the absence of apoptosis

Cell fate following Fas (CD95) ligand or agonistic anti-Fas antibody stimulation is determined by multiple factors, including Fas expression level, microdomain localization, and modulating cytokines. Highly expressed Fas clusters and activates a canonical apoptosis signaling pathway. In less susceptible cells, Fas transduces apoptosis-independent signals, which are not well defined, but have been linked to inflammation, angiogenesis, and fibrosis. To identify apoptosis-independent Fas pathways, cultured renal tubular epithelial cells were stimulated with agonistic anti-Fas antibodies under conditions that did not cause cell death. Analysis of filter cDNA microarrays revealed beta(8) integrin subunit mRNA induction in Fas-stimulated cells. beta(8) integrin mRNA expression increased within 3-6 h of Fas ligation due to enhanced mRNA stabilization, and mRNA increases were sustained for 48-72 h. Expression of plasma membrane beta(8) integrin, as well as its heterodimer partner alpha(v), was increased by Fas activation with a similar kinetic pattern. Fas-induced alpha(v)beta(8) expression correlated with increased migration to vitronectin, the ligand for alpha(v)beta(8). Results from studies with function-blocking antibodies against other alpha(v)beta integrins or suppression of beta(8) integrin expression by RNA interference demonstrated that induced beta(8) integrin expression mediated Fas-stimulated migration. We conclude that alpha(v)beta(8) integrin induction defines an unexpected role for Fas in cell migration, rather than as a cell death receptor.     

The activin axis in liver biology and disease

Activins are a closely related subgroup within the TGFbeta superfamily of growth and differentiation factors. They consist of two disulfide-linked beta subunits. Four mammalian activin beta subunits termed beta(A), beta(B), beta(C), and beta(E), respectively, have been identified. Activin A, the homodimer of two beta(A) subunits, has important regulatory functions in reproductive biology, embryonic development, inflammation, and tissue repair. Several intra- and extracellular antagonists, including the activin-binding proteins follistatin and follistatin-related protein, serve to fine-tune activin A activity. In the liver there is compelling evidence that activin A is involved in the regulation of cell number by inhibition of hepatocyte replication and induction of apoptosis. In addition, activin A stimulates extracellular matrix production in hepatic stellate cells and tubulogenesis of sinusoidal endothelial cells, and thus contributes to restoration of tissue architecture during liver regeneration. Accumulating evidence from animal models and from patient data suggests that deregulation of activin A signaling contributes to pathologic conditions such as hepatic inflammation and fibrosis, acute liver failure, and development of liver cancer. Increased production of activin A was suggested to be a contributing factor to impaired hepatocyte regeneration in acute liver failure and to overproduction of extracellular matrix in liver fibrosis. Recent evidence suggests that escape of (pre)neoplastic hepatocytes from growth control by activin A through overexpression of follistatin and reduced activin production contributes to hepatocarcinogenesis. The role of the activin subunits beta(C) and beta(E), which are both highly expressed in hepatocytes, is still quite incompletely understood. Down-regulation in liver tumors and a growth inhibitory function similar to that of beta(A) has been shown for beta(E). Contradictory results with regard to cell proliferation have been reported for beta(C). The profound involvement of the activin axis in liver biology and in the pathogenesis of severe hepatic diseases suggests activin as potential target for therapeutic interventions.     

In vitro interactions between rat bone marrow-derived endothelial progenitor cells and hepatic stellate cells

Transplantation of bone marrow (BM)-derived endothelial progenitor cells (EPCs) has been reported to improve liver fibrosis, but there is no direct evidence for the mechanism of improvement. We investigated the mechanism in vitro by coculturing BM-derived EPCs with activated hepatic stellate cells (HSCs) to mimic the hepatic environment. EPCs and HSCs were cultured alone and indirectly cocultured at a 1:1 ratio in a Transwell system. The characteristics of HSCs and EPCs were examined at different time points. An invasion assay showed the time-dependent effect on degradation of the extracellular matrix (ECM) layer in EPCs cultured alone. Real-time PCR and enzyme-linked immunosorbent assay analysis revealed that EPCs served as a source of matrix metalloproteinase-9 (MMP-9), and MMP-9 expression levels significantly increased during the 2 d of coculture. CFSE labeling showed that EPCs inhibited proliferation of HSCs. Annexin-V/PI staining, erminal deoxynucleotidyl transferase X-dUTP nick end labeling analysis, and (cleaved) caspase-3 activity revealed that EPCs promoted HSC apoptosis. However, the proliferation and apoptosis of EPCs were unaffected by cocultured HSCs. Coculturing increased the expression of inducible nitric oxide synthase, vascular endothelial growth factor, and hepatocyte growth factor (HGF) in EPCs, promoted differentiation of EPCs, and reduced the expression of types I and III collagens and transforming growth factor beta 1. Knockdown of HGF expression attenuated EPC-induced activation of HSC apoptosis and profibrotic ability. These findings demonstrated that BM-derived EPCs could degrade ECM, promoting activated HSC apoptosis, suppressing proliferation and profibrotic ability of activated HSCs. HGF secretion by EPCs plays a key role in inducing activated HSC apoptosis and HSC profibrotic ability.     

Combination of integrin siRNA and irradiation for breast cancer therapy

Up-regulation of integrin alpha(v)beta(3) has been shown to play a key role in tumor angiogenesis and metastasis. In this study, we evaluated the role of integrin alpha(v)beta(3) in breast cancer cell resistance to ionizing irradiation (IR) and tested the anti-tumor efficacy of combining integrin alpha(v) siRNA and IR. Colonogenic survival assay, cell proliferation, apoptosis, and cell cycle analysis were carried out to determine the treatment effect of siRNA, IR, or combination of both on MDA-MB-435 cells (integrin alpha(v)beta(3)-positive). Integrin alpha(v)beta(3)-negative MCF-7 cells exert more radiosensitivity than MDA-MB-435 cells. IR up-regulates integrin alpha(v)beta(3) expression in MDA-MB-435 cells and integrin alpha(v) siRNA can effectively reduce both alpha(v) and alpha(v)beta(3) integrin expression, leading to increased radiosensitivity. Integrin alpha(v) siRNA also promotes IR-induced apoptosis and enhances IR-induced G2/M arrest in cell cycle progression. This study, with further optimization, may provide a simple and highly efficient treatment strategy for breast cancer as well as other integrin alpha(v)beta(3)-positive cancer types.     

Role of endoproteolytic processing in the adhesive and signaling functions of alphavbeta5 integrin

Some integrin alpha subunits undergo a post-translational cleavage in their extracellular domain. However, the role of this cleavage in integrin function is unclear. Enzymes involved in this maturation belong to the subtilisin-like endoprotease family (convertases). To understand the role of the alpha subunit cleavage in integrin function, we have designed stable transfectants (PDX39P cells) expressing alpha(1)-PDX, a convertase inhibitor. Immunoprecipitation of cell surface proteins from PDX39P showed that alpha(3), alpha(6) and alpha(v) integrins lack endoproteolytic cleavage. We have compared adhesion between PDX39P cells and mock-transfected cells on different extracellular matrix proteins. No difference in adhesion could be observed on laminin-1 and type I collagen, while attachment of PDX39P cells to vitronectin (ligand of the alpha(v)beta(5) integrin) was dramatically reduced. The reduced adhesion of PDX39P cells was not due to changes in integrin affinity as determined by solid-phase receptor assay in a cell-free environment. Intracellular signaling pathways activated by alpha(v) integrin ligation were also affected in PDX39P cells. It thus seems that the absence of endoproteolytic cleavage of alpha(v) integrins has important consequences on signal transduction pathways leading to alterations in integrin function such as cell adhesion.     

Senescence of activated stellate cells limits liver fibrosis

Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to noncancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell-cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix-degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage.     

Unique ability of integrin alpha(v)beta 3 to support tumor cell arrest under dynamic flow conditions

Shear-resistant arrest of circulating tumor cells is required for metastasis from the blood stream. Arrest during blood flow can be supported by tumor cell interaction with attached, activated platelets. This is mediated by tumor cell integrin alpha(v)beta3 and cross-linking plasma protein ligands. To analyze the mechanism of tumor cell ligand interactions under dynamic flow conditions, we used real-time video microscopy and tested human melanoma cell binding to fibrinogen, von Willebrand Factor, or fibronectin matrices in a buffer perfusion system. When perfused at venous flow, melanoma cells arrested abruptly and began to spread immediately. This was uniquely mediated by integrin alpha(v)beta3 on all tested ligands, and required alpha(v)beta3 activation and actin polymerization. Under static conditions, alpha(v)beta3 cooperated with alpha(v)beta1 and alpha5beta1 in supporting melanoma cell adhesion to fibronectin. But even when activated, beta1 integrins did not contribute to melanoma cell arrest during flow. Soluble ligand served as a cross-linker between attached and circulating tumor cells and enhanced melanoma cell arrest. Cohesion of activated melanoma cells was restricted to the matrix surface and did not occur in suspension. We conclude that the presence of alpha(v)beta3 in a functionally activated state provides a unique advantage for circulating tumor cells by promoting tumor cell arrest in the presence of flow-dependent shear forces.     

Different effects of rat interferon alpha, beta and gamma on rat hepatic stellate cell proliferation and activation

Background  

Liver fibrosis is the common sequel of chronic liver diseases. Recent studies have identified hepatic stellate cells as the primary cell type mediating hepatic fibrogenesis. It has been demonstrated that hepatic stellate cells undergo a process of activation during the development of liver fibrosis. During the activation process, hepatic stellate cells acquire myofibroblast-like phenotype featuring the expression of smooth muscle alpha actin. Interferons have been employed for the treatment of viral hepatitis. However, it is unclear what is the effect of interferons on the prevention and treatment of liver fibrosis. Moreover, it is not clear whether there are any differences among interferon alpha, interferon beta, and interferon gamma in the treatment of liver fibrosis. Therefore, our objective in current study is to investigate the effects of rat interferon-α, interferon-β, and interferon-γ on the proliferation and activation of rat hepatic stellate cells.     

Essential role of matrix metalloproteinases in interleukin-1-induced myofibroblastic activation of hepatic stellate cell in collagen

Located within the perisinusoidal space and surrounded by extracellular matrix, hepatic stellate cells (HSC) undergo phenotypic trans-differentiation called "myofibroblastic activation" in liver fibrogenesis. This study investigated the regulation of interleukin-1 (IL-1alpha) on expression of matrix metalloproteinases (MMPs) by HSC grown in three-dimensional extracellular matrix and the role of MMPs in HSC activation. To recapitulate the in vivo "quiescent" state of HSC, the isolated rat HSC were grown in three-dimensional Matrigel or type I collagen. Stimulation with IL-1alpha caused robust induction of pro-MMP-9 (the precursor of matrix metalloproteinase-9) when HSC were cultured in these matrices. IL-1alpha induced a conversion of the pro-MMP-9 to the active form only when the cells were in type I collagen. In collagen lattices, IL-1alpha provoked activation of HSC with induction of MMP-13, MMP-3, and breakdown of the matrix. The HSC activation was completely prevented by a treatment of the cells with tissue inhibitor of metalloproteinase-1 or deprivation of MMP-9. Once fully activated, HSC failed to express MMP-9 and showed attenuated induction of MMP-13 and MMP-3. Further, we demonstrated colocalization of alpha-smooth muscle actin and MMP-9 in a subpopulation of HSC in human fibrotic liver tissues. Thus, this study provides a novel model to enlighten the role of MMPs, particularly that of MMP-9, in HSC activation regulated by a specific cytokine in liver fibrogenesis.     

Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: implications for reversibility of liver fibrosis.

The activated hepatic stellate cell (HSC) is central to liver fibrosis as the major source of collagens I and III and the tissue inhibitors of metalloproteinase-1 (TIMP-1). During spontaneous recovery from liver fibrosis, there is a decrease of TIMP expression, an increase in collagenase activity, and increased apoptosis of HSC, highlighting a potential role for TIMP-1 in HSC survival. In this report, we use tissue culture and in vivo models to demonstrate that TIMP-1 directly inhibits HSC apoptosis. TIMP-1 demonstrated a consistent, significant, and dose-dependent antiapoptotic effect for HSC activated in tissue culture and stimulated to undergo apoptosis by serum deprivation, cycloheximide exposure, and nerve growth factor stimulation. A nonfunctional mutated TIMP-1 (T2G mutant) in which all other domains are conserved did not inhibit apoptosis, indicating that inhibition of apoptosis was mediated through MMP inhibition. Synthetic MMP inhibitors also inhibited HSC apoptosis. Studies of experimental liver cirrhosis demonstrated that persistent expression of TIMP-1 mRNA determined by PCR correlated with persistence of activated HSC quantified by alpha smooth muscle actin staining, while in fibrosis, loss of activated HSC correlated with a reduction in TIMP-1 mRNA. We conclude that TIMP-1 inhibits apoptosis of activated HSC via MMP inhibition.     

Unoccupied alpha(v)beta3 integrin regulates osteoclast apoptosis by transmitting a positive death signal

Cell/matrix detachment is a general inducer of programmed cell death, an event mediated by loss of integrin/ligand association. Because alpha(v)beta3 is the major integrin expressed by the osteoclast, we asked whether its occupancy promotes survival of the resorptive cell. Thus, we generated wild-type preosteoclasts and placed them on selective matrix proteins. Consistent with the posture that alpha(v)beta3 occupancy promotes survival, preosteoclasts plated on native collagen, a matrix not recognized by the integrin, undergo apoptosis 4-fold faster than those on the alpha(v)beta3 ligand, vitronectin. To further explore the role of alpha(v)beta3 in osteoclast apoptosis, wild-type and beta3-/- preosteoclasts were suspended and apoptosis determined, with time. Beta3-/- preosteoclasts, in suspension, undergo a rate of apoptosis only 40-60% of that of their wild-type counterparts, indicating that unoccupied alpha(v)beta3 transmits a positive death signal that we find regulated by caspase-8. Attesting to specificity of the unoccupied integrin-transmitted death signal, apoptosis in the absence of alpha(v)beta3 is mediated by capsase-9. We have shown that the resorptive defect of beta3-/- osteoclasts is rescued by wild-type beta3 cDNA but not by one bearing a S752P mutation. To determine whether the same holds true regarding osteoclast apoptosis, we constructed lentivirus vectors encoding green fluorescent protein, wild-type beta3, or beta3S752P. Once again, native beta3-/- preosteoclasts were protected against apoptosis. Similar to its effect on bone resorption, transduced wild-type beta3 normalizes the apoptotic rate of beta3-/- preosteoclasts. Unexpectedly, however, beta3S752P transductants also die at a rate indistinguishable from wild type. Thus, unoccupied alpha(v)beta3 integrin regulates osteoclast apoptosis via a component of the integrin that is different than that regulating resorption.     

Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A

Recent studies indicate that angiogenesis depends, in part, on ligation of integrin alpha(5)beta(1) by fibronectin. Evidence is now provided that integrin alpha(5)beta(1) regulates the function of integrin alpha(v)beta(3) on endothelial cells during their migration in vitro or angiogenesis in vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin alpha(5)beta(1), which potentiates alpha(v)beta(3)-mediated migration on vitronectin without influencing alpha(v)beta(3)-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-alpha(5)beta(1) restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or alpha(5)beta(1) antagonists, suppress endothelial cell migration on vitronectin in vitro or angiogenesis in vivo. In contrast, inhibitors of PKA reverse the anti-migratory or anti-angiogenic effects mediated by alpha(5)beta(1) antagonists. Therefore, alpha(v)beta(3)-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin alpha(5)beta(1).     

The pattern of enhancement of Src kinase activity on platelet-derived growth factor stimulation of glioblastoma cells is affected by the integrin engaged

Enhanced expression of both integrin alpha v beta 3 and platelet-derived growth factor receptor (PDGFr) has been described in glioblastoma tumors. We therefore explored the possibility that integrin alpha v beta 3 cooperates with PDGFr to promote cell migration in glioblastoma cells, and extended the study to identify the Src family members that are activated on PDGF stimulation. Glioblastoma cells utilize integrins alpha v beta 3 and alpha v beta 5 to mediate vitronectin attachment. We found that physiologic PDGF stimulation (83 pm, 10 min) of vitronectin-adherent cells promoted the specific recruitment of integrin alpha v beta 3-containing focal adhesions to the cell cortex and alpha v beta 3-mediated cell motility. Analysis of PDGFr immunoprecipitates indicated an association of the PDGFr beta with integrin alpha v beta 3, but not integrin alpha v beta 5. Cells plated onto collagen or laminin, which engage different integrins, exhibited significantly less migration on PDGF stimulation, indicating a cooperation of alpha v beta 3 and the PDGFr beta in glioblastoma cells that promotes migration. Further analysis of the cells plated onto vitronectin indicated that PDGF stimulation caused an increase in Src kinase activity, which was associated with integrin alpha v beta 3. In the vitronectin-adherent cells, Lyn was associated preferentially with alpha v beta 3 both in the presence and absence of PDGF stimulation. In contrast, Fyn was associated with both alpha v beta 3 and alpha v beta 5. Moreover, PDGF stimulation increased the activity of Lyn, but not Fyn, in vitronectin-adherent cells, and the activity of Fyn, but not Lyn, in laminin-adherent cells. Using cells attached to mAb anti-alpha v beta 3 or mAb anti-integrin alpha 6, we confirmed the activation of specific members of the Src kinase family with PDGF stimulation. Down-regulation of Lyn expression by siRNA significantly inhibited the cell migration mediated by integrin alpha v beta 3 in PDGF-stimulated cells, demonstrating the PDGFr beta cooperates with integrin alpha v beta 3 in promoting the motility of vitronectin-adherent glioblastoma cells through a Lyn kinase-mediated pathway. Notably, the data indicate that engagement of different integrins alters the identity of the Src family members that are activated on stimulation with PDGF.