The Role of Reactive Oxygen Species in Integrin and Matrix Metalloproteinase Expression and Function

Cell adhesion and migration is largely dependent on integrin binding to extracellular matrix, and several signalling pathways involved in these processes have been shown to be modified by reactive oxygen species (ROS). In fact, integrin activation is linked to increased ROS production by NADPH-oxidases, 5-lipoxygenase, and release from mitochondria. Cell migration is intimately linked to degradation of the extracellular matrix, and activated matrix metalloproteinases (MMPs) are a prerequisite for cancer cell invasion and metastasis. In this minireview, we focus on the interplay between integrin-mediated ROS production and MMP expression as well as its biological and pathobiological significance.     

Integrin-mediated cell adhesion and spreading engage different sources of reactive oxygen species

The tightly regulated production of intracellular reactive oxygen species (ROS) participates in several biologic processes such as cellular growth, programmed cell death, senescence, and adhesion. It is increasingly evident that the same enzymatic processes that were originally linked to ROS generation during host defence or apoptosis execution are also involved in redox-mediated signal transduction. We investigated in murine NIH3T3 fibroblasts the contribution of a variety of redox-dependent events during signal transduction initiated by integrin engagement due to fibronectin stimulation and report that a mitochondrial ROS release occurs, strictly confined to the early phase of extracellular matrix (ECM) contact (10 min). Besides, 5-lipoxygenase (5-LOX) is engaged by integrin receptor ligation as another ROS source, contributing to the more-intense, second ROS burst (45 min), possibly orchestrating the spreading of cells in response to ECM contact. To define a potential mechanism for ROS signaling, we demonstrate that on integrin recruitment, the Src homology-2 domain-containing phosphatase 2 (SHP-2) undergoes a reversible oxidization/inactivation to which mitochondrial and 5-lipoxygenase ROS contribute differentially. In keeping with a key role of oxidants during integrin signaling, the inactivation of SHP-2 prevents the dephosphorylation and inactivation of SHP-2 substrates (p125FAK and SHPS-1), thus enabling the continued propagation of the signal arising by integrin engagement.     

CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize β1 integrin-independent strategies for polarization,interaction with collagen fibers and locomotion

Cell migration may depend on integrin-mediated adhesion to and deadhesion from extracellular matrix ligands. This concept, however, has not yet been confirmed for T lymphocytes migrating in three-dimensional extracellular matrices. We investigated receptor involvement in T cell migration combining a three-dimensional collagen matrix model with time-lapse videomicroscopy, computer-assisted cell tracking and confocal microscopy. In collagen lattices, the migration of CD4⁺ T cells (1) involved interactions with collagen fibers at the leading edge and uropod likewise, (2) occurred independently of the co-clustering of β1, β2, or β3 integrins with F-actin, focal adhesion kinase, and phosphotyrosine at interactions with collagen fibers, (3) was counteracted by high-affinity β1 integrin binding induced by antibody TS2/16; however, (4) the migration could not be blocked by a combination of adhesion-perturbing anti-β1, -β2, -β3, and αv integrin antibodies. Integrin blocking neither affected cell polarization, interaction with fibers, β1 integrin distribution, migration velocity, path structure, nor the number of locomoting cells in spontaneously migrating or concanavalin A-activated cells. Hence, T lymphocytes migrating in three-dimensional collagen matrices may utilize highly transient interactions with collagen fibers of low adhesivity, thereby differing from focal adhesion-dependent migration strategies employed by other cells.     

Integrins in regulation of tissue development and function

Cell adhesion is indispensable for embryonic development and for proper tissue function. In metazoans, integrins are the major adhesion receptors that connect cells to components of the extracellular matrix. Integrins are implicated in assembly of extracellular matrices, cell adhesion and migration on extracellular matrices, and in vertebrates (in which the integrin family has expanded) they can also mediate cell-cell adhesion. Furthermore, integrin-mediated adhesion can modulate many different signal transduction cascades and support cell survival, proliferation, and influence the expression of differentiation-related genes. In this review we briefly explain how integrins can affect so many different aspects of cell behavior and discuss evidence for roles of integrins in tissue development, function, and disease.     

Differential integrin expression by cardiac fibroblasts from hypertensive and exercise-trained rat hearts.

The cardiac fibroblast is the principal cell type responsible for extracellular matrix (ECM) synthesis in the heart during growth and pathophysiological conditions. A dynamic interaction exists between the cardiac ECM and fibroblasts that is sensitive to the local mechanical and chemical tissue environment. We propose here that cardiac fibroblasts structurally and functionally adapt to changing local environments by altering their expression of receptor integrins. Changes in the extracellular environment are communicated in part by integrins, which link the ECM to the cell and regulate phenotype and function. In this report, we analyze integrin protein expression, migration and gel contraction by cardiac fibroblasts from rats subjected to 10 weeks of treadmill exercise (XTR), experimental hypertension (HYP) or controls (CONT). Immunoprecipitation shows that beta1 protein increases in XTR and HYP. Also, alpha1 and alpha2 integrins are lower in XTR and HYP, and alpha5 integrin is higher in XTR and lower in HYP. Functional assays show that XTR and HYP migrate slower on collagen, while XTR migrate faster and HYP slower on fibronectin. Cell isolation procedure, population expansion number or a general adaptation to culture conditions does not explain the differences observed. No significant differences in collagen gel contraction are detected. These results indicate that cardiac fibroblasts retain their in vivo patterns in vitro for a limited number of population expansions. This tissue-specific phenotype is exhibited in early passage (< or =6). However, by late passage (>8), cells begin to show adaptation to the in vitro conditions. These results show that cardiac fibroblasts respond to changing environments in pathophysiological conditions by modulating integrin expression, which is associated with changes in cell migration. They also suggest a pragmatic use for primary cardiac fibroblasts as a model to study the cardiac matrix remodeled by physiological (exercise) and pathological (hypertension) stressors.     

GM-CSF activates RhoA, integrin and MMP expression in human monocytic cells

Monocyte migration is one of the key events occurring in the early stage of atherosclerosis. This process includes monocytic adhesion to and penetration through the arterial intima. In such an environment, many factors stimulate the monocytes to enhance integrin activation and extracellular matrix degradation. To investigate the coordinative operation of these two events in relation to monocyte migration, we paid particular attention to the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on monocytes in terms of RhoA activation and matrix metalloproteinase (MMP) expression. RhoA and integrin clustering were activated by GM-CSF, monocyte chemoattractant protein-1 (MCP-1) and platelet-derived growth factor-BB (PDGF-BB) in human monocytic cell lines. Furthermore, enhancement of migration was observed with stimulation by MCP-1 and PDGF-BB. Granulocyte-macrophage colony-stimulating factor did not enhance the migration, even though it activated RhoA and integrin. However, GM-CSF is known to stimulate monocytes to express MCP-1, suggesting the presence of an indirect mechanism for GM-CSF-mediated migratory activity. In contrast, only GM-CSF enhanced the expression of MMP-1 and MMP-9. These results provide evidence that GM-CSF has multiple functions enhancing monocytic migration via RhoA and integrin activation, and via MMP expression.     

Lack of integrin alpha1beta1 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 alpha1-null mice develop more severe glomerulosclerosis than wild-type mice. Moreover, primary alpha1-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 alpha1beta1 as a modulator of glomerulosclerosis. In addition, we showed a novel pathway where integrin alpha1beta1 modulates ROS production, which in turn controls collagen turnover and ultimately fibrosis. Because integrin alpha1beta1 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.     

Integrin-Mediated Preadipocyte Adhesion and Migration on Laminin-1

Cell adhesion and migration are key events in many biological processes and depend on extracellular matrix proteins. Understanding these cellular events in adipogenesis is paramount to elucidating the biological mechanisms underlying preadipocyte-specific aspects of obesity, diabetes, and adipose tissue development for the design of pharmaceutical screening and tissue engineering strategies. We quantitatively investigated preadipocyte adhesion and migration on laminin-1 surfaces and identified candidate cognate preadipocyte receptors for laminin-1. In adhesion studies, we found that preadipocytes readily adhered to laminin-1 as compared with other extracellular matrix proteins. In addition, immunocytochemical analysis demonstrated that an array of integrin molecules was present on the surface of preadipocytes. Preadipocyte adhesion on laminin-1 was quantitatively assessed using a sedimentation adhesion assay, and results suggested that preadipocyte adhesion to laminin-1 was mediated by the ₁₁ integrin. In addition, digital time-lapse microscopy and quantitative cell tracking revealed that inhibition of the ₁₁ integrin resulted in abrogation of preadipocyte migration on laminin-1 surfaces. These results strongly support the hypothesis that preadipocyte adhesion to and migration on laminin-1 substrata are regulated, in part, by integrins. © 2003 Biomedical Engineering Society. PAC2003: 8717Jj, 8715Kg, 8764Rr, 8714Ee     

CD44 and β1 integrins mediate ovarian carcinoma cell migration toward extracellular matrix proteins

Epithelial cancer of the ovary spreads by implantation of tumor cells onto the mesothelial cells that line the peritoneal cavity. The aim of this study was to identify the cell–matrix interactions that mediate ovarian carcinoma cell migration toward components of the mesothelial cell-associated extracellular matrix. The human ovarian carcinoma cell lines NIH:OVCAR5 and SKOV3 were analyzed by flow cytometry for the expression of cell surface receptors. The ability of those receptors to mediate ovarian carcinoma cell migration toward fibronectin, type IV collagen, and laminin was determined. A monoclonal antibody against the β1 integrin subunit abrogated the migration of both cell lines toward the extracellular matrix proteins. Blocking antibodies against alpha integrin subunits suggest that ovarian carcinoma cell migration toward fibronectin is primarily mediated by the ∝5β1 integrin, type IV collagen by the ∝2β1 integrin, and laminin by the ∝6β1 integrin. These results suggest that ovarian carcinoma cell migration is regulated by multiple β1 integrin–matrix interactions. Significant reduction of cell migration was observed with a monoclonal antibody against CD44 that blocks the hyaluronan-binding site of CD44, but not with an antibody that binds at an alternate site on CD44. Intact hyaluronan and/or hyaluronan oligomers also inhibited cell migration, suggesting that the CD44–hyaluronan interaction provides an integrin-independent mechanism of control for ovarian carcinoma cell migration. These results suggest that ovarian carcinoma cell migration is regulated by both integrin-dependent mechanisms, involving the interaction of β1 integrins with extracellular matrix proteins, and an integrin-independent mechanism that involves the interaction of CD44 and hyaluronan.     

Human hepatocellular carcinoma (HCC) cells require both alpha3beta1 integrin and matrix metalloproteinases activity for migration and invasion

Hepatocellular carcinoma (HCC) is the most frequent malignant tumor of the liver; prognosis depends on the tendency to metastasize. Cancer cell invasion is regulated by proteolytic remodeling of extracellular matrix components and by integrin expression. We have shown that matrix metalloproteinase-2 (MMP-2) and membrane-type-1 matrix metalloproteinase (MT1-MMP) cleave Laminin-5 (Ln-5), stimulating cell migration. Here we report that all HCC cells express MT1-MMP, migrate on Ln-1 and Collagen IV, whereas only HCC cells that express alpha3beta1 integrin secrete detectable levels of gelatinases, migrate on Ln-5, and invade through a reconstituted basement membrane (BM). Migration on Ln-5 is blocked by BB-94, an MMP inhibitor, and by MIG1, a monoclonal antibody that hinders migration on MMP-2-cleaved Ln-5. Invasion through a reconstituted BM is also inhibited by BB-94. HCC alpha3beta1-negative cells migrate on Ln-1 and Collagen IV, but not on Ln-5, and do not invade through a reconstituted BM, although they express MT1-MMP. Anti-alpha3beta1 blocking antibodies inhibit gelatinase activation, cell motility, and cell invasion through MATRIGEL: In vivo, alpha3beta1 integrin and Ln-5 are expressed in HCC tissue but not in normal liver. In conclusion, our data suggest that both alpha3beta1 integrin and gelatinase activity are required for HCC migration and invasion.     

Integrin subunit alpha V promotes growth,migration, and invasion of gastric cancer cells

Integrin subunit alpha V (ITGAV), a member of integrin family of extracellular matrix receptors, is involved in many types of cancer. In this study, the expression levels, clinical features and prognosis of ITGAV in gastric cancer (GC) patients were investigated, and the functional roles of ITGAV were also investigated. Cell Counting Kit-8 (CCK-8) assay was performed to examine the proliferation of GC cells. Transwell assays and wound-healing assays were conducted to explore the effect of ITGAV expression on GC cell migration and invasion. We found that ITGAV was overexpressed in both GC tissues and GC cells. ITGAV expression was positively correlated with lymph node metastasis and TNM stage of GC. High expression of ITGAV was associated with shorter overall survival (OS) and disease-free survival (DFS). Interestingly, the downregulation of ITGAV resulted in suppression of proliferation, migration, and invasion in GC cells. In conclusion, ITGAV is overexpressed in gastric cancer and is associated with poorer prognostic outcomes. ITGAV may serve as an important prognostic marker for GC staging and progression.     

Enhanced expression of lumican inhibited the attachment and growth of human embryonic kidney 293 cells

Lumican is a member of a small leucine-rich proteoglycan (SLRP) family and it regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. Lumican expression was reported in various kinds of tumor cells. Lumican inhibits the growth of melanoma cells, but the lumican in pancreatic cancer correlated with an advanced stage and retroperitoneal and duodenal invasion. In this study, we clarified whether the enhanced expression of lumican contributes to cellular attachment, growth, colony formation, migration and invasion. HEK 293 cell, stably transfected with lumican cDNA synthesized and secreted a 50 kDa lumican protein at high levels in culture medium. The cells showed a polygonal appearance with long projections and the degree of adhesion of the cells to fibronectin was lower than that of empty vector transfected control cells (mock cells). In contrast, the degree of adhesion of the cells to type I collagen was not different from that of mock cells. The expression levels of α5 integrin, the major integrin subunit for fibronectin, were lower in lumican-transfected HEK cells than in mock cells. Furthermore, lumican-transfected HEK cells showed reduced growth rates in vitro and did not form colonies in soft agar. Phosphorylation of AKT, extracellular signal-regulated kinase (ERK) 1/2 and mammalian target of rapamycin (mTOR) decreased in the lumican-transfected HEK cells. Cell migration and invasion were not altered in lumican-transfected HEK cells and mock cells. These findings indicate that the 50 kDa lumican protein plays important roles in the inhibition of HEK cell attachment and growth, and it might inhibit the activation of integrin pathways.     

整合素在烧伤创面愈合中的作用研究进展

整合素家族为跨膜糖蛋白受体家族,其作用是介导细胞与细胞外基质之间的相互作用,参与细胞相互之间的黏附功能。整合素通过双向信号转导,影响细胞的分化、增生与凋亡;整合素信号系统还可以通过调控细胞周期,产生细胞凋亡,影响瘢痕的形成。本文就整合素家族参与烧伤创面成纤维细胞分化、促进创面收缩,增强细胞外基质的收缩能力以及整合素连接激酶与创面愈合之间的关系作一综述。     

Role of extracellular matrix-mediated interactions in thymocyte migration

Cell adhesion, migration, differentiation and survival or death is amongst a large spectrum of biological responses that can be elicited by ligation of extracellular matrix components to their corresponding receptors. As regards the physiology of the thymus, cell migration is a crucial event in the general process of T cell differentiation. Studies on the intrathymic distribution of ECM components revealed that fibronectin, laminin and type IV collagen, are not restrictedly located at typical basement membrane sites, also forming a thick network in the medullary region of the thymic lobules, whereas very thin ECM fibers are found within the cortex. These ECM components are essentially produced by thymic microenvironmental cells, which also drive thymocyte differentiation. Signals triggered by ECM are conveyed into thymocytes or microenvironmental cells through specific membrane receptors, and most of them belong to the integrin type, such as the VLA-3, VLA-4, VLA-5 and VLA-6. In vitro studies revealed that adhesion of thymocytes to thymic microenvironmental cells is mediated by extracellular matrix. Such an adhesion is preferentially done by immature thymocytes. Importantly, ECM-mediated interactions also govern the entrance and exit of thymocytes in the lymphoepithelial complexes named thymic nurse cells. Lastly, pathological conditions, including infectious and autoimmune diseases, in which changes of ECM ligands and receptors are observed, course with alterations in thymocyte migration and death. In conclusion, the fact that ECM can modulate traffic, differentiation, death and survival of normal thymocytes adds clues for understanding how ECM-mediated interactions behave in the thymus, not only in normal, but also in pathological conditions.     

Integrin-dependent pathologies

Cell adhesion and migration are essential for embryonic development, tissue regeneration, and immune defence. The physical link between the extracellular substrate and the actin cytoskeleton is mediated by receptors of the integrin family and a large set of adaptor proteins. During cell migration this physical link is dynamically modified, allowing the cell to sense and adapt to the microenvironment. This includes the formation of integrin clusters at the cell front, their stabilization in the cell body and subsequent disassembly of these clusters at the rear of the cell. The modulation of the adhesion strength of the cell to the substrate is regulated by the affinity switch of integrin molecules and increased avidity through clustering of integrins. Here we explain how integrins mediate cell migration and how genetic defects of integrins and their adaptors lead to cellular dysfunction and generate pathological situations.     

Effects of anti-alpha1 integrin subunit antibody on anti-Thy-1 glomerulonephritis

alpha1beta1 integrin is a potential collagen-binding extracellular matrix receptor that mediates collagen-dependent cell adhesion, proliferation, migration, and collagen matrix assembly and thereby may participate in the wound healing and pathologic scarring observed in some damaged organs. To clarify the role of alpha1beta1 integrin predominantly expressed on the mesangial cell (MC) surface in nephritic glomeruli, we investigated the involvement of MC-alpha1beta1 integrin in rat anti-Thy-1 glomerulonephritis (GN) by administering function-blocking monoclonal mouse anti-rat alpha1 integrin subunit antibody (anti-alpha1 Ab). Assay of collagen types I and IV mixed gel contraction, an in vitro model of pathologic collagen matrix remodeling, with function-blocking anti-alpha1 Ab and anti-beta1 Ab, revealed that collagen I and IV matrix reorganization is mediated by MC-alpha1beta1 integrin. In addition, conditioned medium from isolated Day 3 anti-Thy-1 nephritic glomeruli showed increased activity of MC-alpha1beta1 integrin-induced mixed collagen gel contraction as compared with that from isolated normal rat glomeruli. Treatment of Day 3 conditioned medium with anti-platelet-derived growth factor-BB antibody significantly inhibited conditioned media-induced gel contraction, whereas treatment with anti-transforming growth factor-beta antibody did not have a significant effect. Rats that received anti-alpha1 Ab from the left renal artery 3 days after anti-Thy-1 GN induction showed significant decreases of glomerular hypercellularity and mesangial matrix accumulation, including collagen I and IV in the left kidney, compared with those rats in which the left kidney received control mouse IgG1. These results suggest that MC-alpha1beta1 integrin is an important extracellular matrix receptor mediating mesangial remodeling characterized by MC proliferation and mesangial matrix reorganization in anti-Thy-1 GN. Platelet-derived growth factor-BB may be involved in early collagen matrix reorganization leading to pathologic mesangial remodeling in this GN model.     

Reactive oxygen species-mediated PKC and integrin signaling promotes tumor progression of human hepatoma HepG2

The poor prognosis and recurrence of HCC are majorly caused by intrahepatic metastasis. Delineating the molecular pathways mediating these processes may benefit developing effective targeting therapies. Using human hepatoma HepG2 as a model, we have found reactive oxygen species (ROS) may cooperate with protein kinase C (PKC) for sustained ERK phosphorylation and migration of HepG2 induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We further investigated whether integrin signaling is involved. Various antagonists of integrin signaling prevented TPA-induced activation of ERK and PKC, ROS generation and migration of HepG2. On the other hand, TPA-induced phosphorylation of integrin signaling components including focal adhesion kinase (FAK), Src (Tyr416) and paxillin (Tyr31 and Ser178) can be prevented by PKC inhibitor Bisindolylmaleimides (BIS) and antioxidant dithiotheritol (DTT). HepG2 overexpressing PKCα contained elevated phosphorylated paxillin. Also, ROS generator phenazine methosulfate and tert-Butyl hydroperoxide may induce phosphorylation of paxillin and activation of PKC. Taken together, ROS mediated cross talk of PKC and integrin for migration of HepG2 induced by TPA. Furthermore, TPA induced intrahepatic metastasis of HepG2 in SCID mice, which was prevented by BIS or (BIS plus DTT). Elevated phosphorylation of paxillin was observed in tumor of mice treated with TPA as compared with those co-treated with TPA/BIS. In summary, the signal pathways for tumor progression of hepatoma induced by TPA can be established both in vitro and in vivo.     

Ras-transfection up-regulated HaCaT cell migration: Inhibition by Marimastat

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-ras clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines. This revised version was published online in July 2006 with corrections to the Cover Date.