Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin ␣1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by ␣1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin ␣1-null mesangial cells produce excessive ROS. Integrin ␣1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in ␣1-null mesangial cells. Thus, our study demonstrates that integrin ␣1␤1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.Integrin ␣1␤1, a major collagen binding receptor, is expressed in different cell types, including fibroblasts (45), endothelial cells (8), and mesangial cells in the glomerulus of the kidney (30, 53). Integrin ␣1␤1 confers the ability of cells to bind to collagenous substrata, including collagens I and IV (4, 45), and to proliferate on these substrata (45). Moreover, cells expressing integrin ␣1␤1 sense extracellular levels of collagen and downregulate its synthesis at both transcriptional and translational levels (4,14). Finally, we demonstrated that integrin ␣1␤1 also downregulates the production of reactive oxygen species (ROS) (4, 58).Following renal injury, mice lacking integrin ␣1␤1 develop more extensive glomerular fibrosis characterized by excessive accumulation of collagen type IV compared to wild-type (WT) mice (4, 58). Increased fibrosis is due to both a direct effect of the lack of integrin ␣1␤1-mediated downregulation of collagen IV synthesis and excessive ROS production by ␣1-null mesangial cells.Constitutive production of ROS by mesangial cells, a major cell type found in the glomerulus of the kidney, originates from an intrinsic NADPH oxidase (26, 48) that normally functions at a low level and increases in response to inflammatory stimuli, high glucose, or stress (25,34,35,37,55). The NADPH oxidase, highly characterized in phagocytes, is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b 558 (p22 phox and gp91 phox ) and cytoplasmic proteins (p40 phox , p47 phox , p67 phox ) that translocate to the membrane following cellular stimulation to produce superoxide (3, 9, 47). A multicomponent phagocyte-like NADPH oxidase is also a major source of ROS in many nonphagocytic cells, including mesangial cells. In the phagocyte-like NADPH oxidase, the catalytic subunits are termed Nox proteins, with ...

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We used a customized Affymetrix protease microarray (Hu/Mu ProtIn chip) designed to distinguish human and mouse genes to analyze the expression of proteases and protease inhibitors in lung cancer. Using an orthotopic lung cancer model, we showed that murine matrix metalloproteinase (MMP)-12, MMP-13, and cathepsin K were up-regulated in tumor tissue compared with normal mouse lung. To determine the relevance of stromal proteases detected using this model system, we compared the results to an analysis of human lung adenocarcinoma specimens using the U133 Plus 2.0 Affymetrix microarray. MMP-12, MMP-13, and cathepsin K showed an increase in expression in human tumors compared with normal lung similar to that seen in the orthotopic model. Immunohistochemical analysis confirmed MMP-12 expression in the stroma of human lung tumor samples. To determine the biological relevance of stromal MMP-12, murine Lewis lung carcinoma cells were injected into the tail vein of syngeneic wild-type (WT) and MMP-12-null mice. MMP-12-null and WT mice developed equivalent numbers of lung tumors; however, there was a 2-fold increase in the number of tumors that reached >2 mm in diameter in MMP-12-null mice compared with WT controls. The increase in tumor size correlated with an increase in CD31-positive blood vessels and a decrease in circulating levels of the K1-K4 species of angiostatin. These results show a protective role for stromal MMP-12 in lung tumor growth. The use of the Hu/Mu ProtIn chip allows us to distinguish tumor-and host-derived proteases and guides the further analysis of the significance of these genes in tumor progression. (Cancer Res 2006; 66(16): 7968-75)

Mesangial cells and podocytes express integrins a1b1 and a2b1, which are the two major collagen receptors that regulate multiple cellular functions, including extracellular matrix homeostasis. Integrin a1b1 protects from glomerular injury by negatively regulating collagen production, but the role of integrin a2b1 in renal injury is unclear. Here, we subjected wild-type and integrin a2-null mice to injury with adriamycin or partial renal ablation. In both of these models, integrin a2-null mice developed significantly less proteinuria and glomerulosclerosis. In addition, selective pharmacological inhibition of integrin a2b1 significantly reduced adriamycin-induced proteinuria, glomerular injury, and collagen deposition in wildtype mice. This inhibitor significantly reduced collagen synthesis in wild-type, but not integrin a2-null, mesangial cells in vitro, demonstrating that its effects are integrin a2b1-dependent. Taken together, these results indicate that integrin a2b1 contributes to glomerular injury by positively regulating collagen synthesis and suggest that its inhibition may be a promising strategy to reduce glomerular injury and proteinuria. The most common cause of end stage kidney disease is glomerulosclerosis, which is characterized by excessive collagen deposition in the glomerulus. Although numerous disease processes can initiate glomerular injury, they all result in abnormal glomerular collagen homeostasis with progression. Glomerular collagen turnover is regulated by multiple factors, including growth factors and profibrotic reactive oxygen species (ROSs) as well as cell extracellular matrix interactions mediated by the matrix receptors integrins (reviewed in ref. 1). Of these factors, the mechanisms by which integrins regulate glomerulosclerosis are the most poorly understood.Integrins consist of two noncovalently associated a-and b-subunits that combine to form 24 different heterodimers in mammals (reviewed in ref.2). The integrin extracellular domains contain the ligand binding site and confer ligand specificity, whereas the cytoplasmic domain interacts with the cytoskeleton and regulates cell signaling. Integrins control critical cell functions, including proliferation, survival, migration, and matrix homeostasis (reviewed in refs. 1 and 2). Thus, it is not surprising that integrins regulate tissue responses to injury in whole organisms.The best-studied integrin in the context of glomerular injury is the major collagen IV receptor, integrin a1b1, which is expressed by mesangial cells 3 and podocytes. 4 We have shown that integrin

The role of matrix metalloproteinase (MMP)9 in lung cancer progression is controversial. MMP9 promotes local tumor progression and distant metastasis in mouse models by enhancing extracellular matrix degradation, releasing VEGF from extracellular matrix and promoting vascular pericyte recruitment. Furthermore, increased plasma MMP9 expression levels in human subjects with metastatic non-small cell lung cancer (NSCLC) inversely correlates with survival. In contrast, MMP9 can benefit the host by generating inhibitors of endothelial cell proliferation such as angiostatin and NC1 domains of collagen IV. To better understand the role of host MMP9 on the primary growth and metastatic potential of NSCLC, we performed an orthotopic model of NSLC in integrin alpha1-null mice (a genetic model for increased MMP9). In these mice we observed decreased number, size and vascularization of primary NSCLC tumors when compared to wild type controls. In addition, decreased number and size of NSCLC-derived metastases were evident in the alpha1-null mice. Furthermore, pharmacological inhibition of MMPs in the alpha1-null mice at the time of tumor cell injection resulted in an increase in the number of both primary and metastatic lung cancer as compared to untreated mice, suggesting that primary growth and metastases of NSCLC are worsened by the early inhibition of MMPs. In conclusion, although MMP9 may potentially promote tumor growth and metastasis, production of MMP-dependent anti-angiogenic factors seems to override these effects and protects the host from NSCL growth and progression.