Xiwu Chen

Integrin α1β1 Controls Reactive Oxygen Species Synthesis by Negatively Regulating Epidermal Growth Factor Receptor-Mediated Rac Activation

ABSTRACT 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 α1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by α1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin α1-null mesangial cells produce excessive ROS. Integrin α1-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 α1-null mesangial cells. Thus, our study demonstrates that integrin α1β1-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.

Lack of Integrin α1β1 Leads to Severe Glomerulosclerosis after Glomerular Injury

Severity of fibrosis after injury is determined by the nature of the injury and host genetic susceptibility. Metabolism of collagen, the major component of fibrotic lesions, is, in part, regulated by integrins. Using a model of glomerular injury by adriamycin, which induces reactive oxygen species (ROS) production, we demonstrated that integrin α1-null mice develop more severe glomerulosclerosis than wild-type mice. Moreover, primary α1-null mesangial cells produce more ROS both at baseline and after adriamycin treatment. Increased ROS synthesis leads to decreased cell proliferation and increased glomerular collagen IV accumulation that is reversed by antioxidants both in vivo and in vitro. Thus, we have identified integrin α1β1 as a modulator of glomerulosclerosis. In addition, we showed a novel pathway where integrin α1β1 modulates ROS production, which in turn controls collagen turnover and ultimately fibrosis. Because integrin α1β1 is expressed in many cell types this may represent a generalized mechanism of controlling matrix accumulation, which has implications for numerous diseases characterized by fibrosis.

Analysis of Host- and Tumor-Derived Proteinases Using a Custom Dual Species Microarray Reveals a Protective Role for Stromal Matrix Metalloproteinase-12 in Non–Small Cell Lung Cancer

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.

Inhibition of 11β–hydroxysteroid dehydrogenase type II selectively blocks the tumor COX-2 pathway and suppresses colon carcinogenesis in mice and humans

Colorectal cancer (CRC) is a leading cause of cancer death, yet primary prevention remains the best approach to reducing overall morbidity and mortality. Studies have shown that COX-2-derived PGE2 promotes CRC progression, and both nonselective COX inhibitors (NSAIDs) and selective COX-2 inhibitors (such as glucocorticoids) reduce the number and size of colonic adenomas. However, increased gastrointestinal side effects of NSAIDs and increased cardiovascular risks of selective COX-2 inhibitors limit their use in chemoprevention of CRC. We found that expression of 11beta-hydroxysteroid dehydrogenase type II (11betaHSD2), which converts active glucocorticoids to inactive keto-forms, increased in human colonic and Apc+/min mouse intestinal adenomas and correlated with increased COX-2 expression and activity. Furthermore, pharmacologic inhibition or gene silencing of 11betaHSD2 inhibited COX-2-mediated PGE2 production in tumors and prevented adenoma formation, tumor growth, and metastasis in mice. Inhibition of 11betaHSD2 did not reduce systemic prostacyclin production or accelerate atherosclerosis in mice, thereby avoiding the major cardiovascular side effects seen with systemic COX-2 inhibitors. Therefore, 11betaHSD2 inhibition represents what we believe to be a novel approach for CRC chemoprevention and therapy by increasing tumor glucocorticoid activity, which in turn selectively blocks local COX-2 activity.

Increased plasma MMP9 in integrin α1-null mice enhances lung metastasis of colon carcinoma cells

Inhibitors of matrix metalloproteinases (MMPs) were developed as anticancer agents based on the observation that MMPs facilitate local tumor spread and metastasis by promoting matrix degradation and cell migration. Unfortunately, these inhibitors were unsuccessful in the clinical treatment of several cancers, including lung cancer. A possible reason contributing to their failure is that MMP activity is critical for the generation of inhibitors of tumor angiogenesis, including angiostatin. Thus, MMPs might play opposing roles in tumor vascularization and invasion. To determine which effect of elevated MMP levels dominates in the progression of metastatic cancer, experimental lung metastasis assays were performed in integrin α1‐null mice, a genetic model for increased plasma levels of MMP9 and MMP9‐generated angiostatin (Pozzi et al., Proc. Natl. Acad. Sci. USA 2000;97:2202–7). We show that while the number of lung colonies in integrin α1‐null mice was significantly increased compared to their wild‐type counterparts, tumor volume was markedly reduced. In vivo treatment with the MMP inhibitor doxycycline resulted in a significant decrease in the number of lung colonies in both genotypes, but the tumors that formed were bigger and more vascularized. Increased tumor vascularization paralleled decreased plasma levels of MMP9 and consequent decreased angiostatin synthesis. These results demonstrate that while inhibition of MMPs prevents and/or reduces tumor invasion and lung metastasis, it has the paradoxical effect of increasing the size and vascularization of metastatic tumors due to decreased generation of inhibitors of endothelial cell proliferation. The continued growth of these large well‐vascularized tumors may explain the poor efficacy of MMP inhibitors in lung cancer clinical trials.

Inhibition of Integrin α2β1 Ameliorates Glomerular Injury

Mesangial cells and podocytes express integrins α1β1 and α2β1, which are the two major collagen receptors that regulate multiple cellular functions, including extracellular matrix homeostasis. Integrin α1β1 protects from glomerular injury by negatively regulating collagen production, but the role of integrin α2β1 in renal injury is unclear. Here, we subjected wild-type and integrin α2-null mice to injury with adriamycin or partial renal ablation. In both of these models, integrin α2-null mice developed significantly less proteinuria and glomerulosclerosis. In addition, selective pharmacological inhibition of integrin α2β1 significantly reduced adriamycin-induced proteinuria, glomerular injury, and collagen deposition in wild-type mice. This inhibitor significantly reduced collagen synthesis in wild-type, but not integrin α2-null, mesangial cells in vitro, demonstrating that its effects are integrin α2β1-dependent. Taken together, these results indicate that integrin α2β1 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.

Integrin-mediated type II TGF-β receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling

Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-β mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-β receptor (TβRII), which in turn promotes a TβRI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within TβRII is considered the principal regulatory mechanism of TβRII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate TβRII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the TβRII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated TβRII tail tyrosine residues, resulting in inhibition of TβR-dependent fibrotic signaling. The collagen-binding receptor integrin α1β1 was required for recruitment of TCPTP to the TβRII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of TβRII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin α1β1 and TβRII that is essential for TβRII-mediated SMAD activation and fibrotic signaling pathways.

Integrin α1β1 Regulates Epidermal Growth Factor Receptor Activation by Controlling Peroxisome Proliferator-Activated Receptor γ-Dependent Caveolin-1 Expression

ABSTRACT Integrin α1β1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin α1β1-mediated EGFR activation. Integrin α1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin α1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin α1-null MCs decreases EGFR-mediated ROS production. We further show that integrin α1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPARγ or inhibition of ERK increases Cav-1 levels in the integrin α1-null MCs. Finally, we show that glomeruli of integrin α1-null mice have reduced levels of Cav-1 and activated PPARγ but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin α1β1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPARγ axis plays a key role in regulating integrin α1β1-dependent Cav-1 expression and consequent EGFR-mediated ROS production.

Integrins in renal development

The kidney develops from direct interactions between the ureteric bud and the metanephric mesenchyme. The ureteric bud gives rise to the collecting system and the metanephric mesenchyme to the nephrons. The complex process of renal development which occurs between these embryologically distinct structures is mediated by numerous factors, including the communication of cells with their surrounding extracellular matrix. Integrins are the principal cellular receptors for extracellular matrix proteins, and they play a role in organ and tissue development. In this review we focus on how integrins regulate renal development.

Integrin α1β1 Regulates Matrix Metalloproteinases via P38 Mitogen-Activated Protein Kinase in Mesangial Cells: Implications for Alport Syndrome

Previous work has shown that integrin alpha1-null Alport mice exhibit attenuated glomerular disease with decreased matrix accumulation and live much longer than strain-matched Alport mice. However, the mechanism underlying this observation is unknown. Here we show that glomerular gelatinase expression, specifically matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14, was significantly elevated in both integrin alpha1-null mice and integrin alpha1-null Alport mice relative to wild-type mice; however, only MMP-9 was elevated in glomeruli of Alport mice that express integrin alpha1. Similarly, cultured mesangial cells from alpha1-null mice showed elevated expression levels of all three MMPs, whereas mesangial cells from Alport mice show elevated expression levels of only MMP-9. In both glomeruli and cultured mesangial cells isolated from integrin alpha1-null mice, activation of the p38 and ERK branches of the mitogen-activated protein kinase pathway was also observed. The use of small molecule inhibitors demonstrated that the activation of the p38, but not ERK, pathway was linked to elevated MMP-2, -9, and -14 expression levels in mesangial cells from integrin alpha1-null mice. In contrast, elevated MMP-9 levels in mesangial cells from Alport mice were linked to ERK pathway activation. Blockade of gelatinase activity using a small molecule inhibitor (BAY-12-9566) ameliorated progression of proteinuria and restored the architecture of the glomerular basement membrane in alpha1 integrin-null Alport mice, suggesting that elevated gelatinase activity exacerbates glomerular disease progression in these mice.