C. Michael DiPersio

Integrin α3β1 directs the stabilization of a polarized lamellipodium in epithelial cells through activation of Rac1

Epithelial cell migration is a crucial event in wound healing, yet little is known about mechanisms whereby integrins regulate epithelial cell polarization and migration. In the present work, we demonstrate the importance of adhesion through the α3β1 integrin in promoting the stabilization of leading lamellipodia in migrating keratinocytes. We demonstrate that this integrin is found at the leading edge of migrating keratinocytes and that inhibition of α3β1 binding to laminin-5 prevents the formation of stable leading lamellipodia. Consistent with this observation, keratinocytes derived from α3β1-deficient mice fail to form stable leading lamellipodia but retain the ability to form actin-containing protrusions that rapidly extend and retract from the cell membrane. Formation of a leading lamellipodium also requires α3β1-dependent activation of Rac1, because α3β1-deficient keratinocytes show decreased activation of Rac1 compared with α3β1-expressing cells, and formation of stable leading lamellipodia can be inhibited in the latter cells by expression of the dominant negative Rac1 mutant Rac1N17. Furthermore, α3β1-deficient keratinocytes expressing constitutively active Rac1L61 failed to form stable lamellipodia when plated onto laminin-5, demonstrating that α3β1 is required for Rac1-mediated formation of a stable lamellipodium. These observations identify a crucial role for integrin-mediated adhesion and signaling in the formation of large, polarized, stable lamellipodia by migrating epithelial cells. To our knowledge, this study is the first to demonstrate that signal transduction through a specific integrin is required to direct the development of a lamellipodium from an initial protrusion and promote persistent epithelial cell migration.

α3β1 integrin promotes keratinocyte cell survival through activation of a MEK/ERK signaling pathway

Inadequate or inappropriate adhesion of epithelial cells to extracellular matrix leads to a form of apoptosis known as anoikis. During various tissue remodelling events, such as wound healing or carcinoma invasion, changes in the physical properties, and/or composition of the extracellular matrix, can lead to anoikis of epithelial cells that lack appropriate receptor-matrix interactions. Laminin-5 is the major ligand for keratinocyte adhesion in the epidermis, and it also promotes keratinocyte survival in vivo and in vitro. Integrins α3β1 and α6β4 are the major receptors for laminin-5; however, specific roles for these integrins in keratinocyte survival have not been determined. In the current study, we exploited keratinocyte cell lines derived from wild-type or α3 integrin knockout mice to reveal a critical role for α3β1 in protecting keratinocytes from apoptosis upon serum withdrawal. We show that α3β1-mediated adhesion to laminin-5 extracellular matrix inhibits proteolytic activation of caspase-3 and TUNEL-staining, both hallmarks of apoptosis. We also show that α3β1-mediated adhesion activates focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), and that inhibition of either FAK or ERK signaling leads to apoptosis of keratinocytes attached to laminin-5. α6β4-mediated adhesion to laminin-5 only partially protects cells from apoptosis in the absence of α3β1, and α6β4 is not necessary for cell survival in the presence of α3β1. These results suggest that α3β1 is necessary and sufficient for maximal keratinocyte survival on laminin-5. We propose a model to address the potential importance of α3β1-mediated survival for migrating keratinocytes at the leading edge of a cutaneous wound.

α3β1 integrin regulates MMP-9 mRNA stability in immortalized keratinocytes: a novel mechanism of integrin-mediated MMP gene expression

Matrix metalloproteinases facilitate cell migration and tumor invasion through their ability to proteolyse the extracellular matrix. The laminin-binding integrin α3β1 is expressed at high levels in squamous cell carcinomas and in normal keratinocytes during cutaneous wound healing. We showed previously that α3β1 is required for MMP-9/gelatinase B secretion in immortalized mouse keratinocytes (MK cells) and that this regulation was acquired as part of the immortalized phenotype, suggesting a possible role for α3β1 during malignant conversion. In the current study, we identify a novel mechanism whereby α3β1 regulates the induction of MMP-9 expression that occurs in response to activation of a MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Inhibition of MEK/ERK signaling in wild-type MK cells with a pharmacological inhibitor, U0126, showed that ERK activation was necessary for high levels of endogenous MMP-9 gene expression and activity of a transfected MMP-9 promoter. Furthermore, activation of MEK/ERK signaling in these cells with an oncogenic mutant of Ras, RasV12, increased both endogenous MMP-9 gene expression and MMP-9 promoter activity. Experiments with α3β1-deficient MK cells revealed that α3β1 was required for both baseline levels and RasV12-induced levels of MMP-9 mRNA expression. However, α3β1 was not required for RasV12-mediated activation of ERK or for ERK-dependent MMP-9 promoter activity. Direct comparison of mRNA turnover in the wild type and α3-null MK cells identified a requirement for α3β1 in stabilization of MMP-9 mRNA transcripts. These results identify a novel function for integrins in promoting mRNA stability as a mechanism to potentiate MAPK-mediated gene expression. They also suggest a role for α3β1 in maintaining high levels of MMP-9 mRNA expression in response to oncogenic activation of MEK/ERK signaling pathways.

Integrin αVβ3 Binds to the RGD Motif of Glycoprotein B of Kaposi's Sarcoma-Associated Herpesvirus and Functions as an RGD-Dependent Entry Receptor

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) envelope-associated glycoprotein B (gB) is involved in the initial steps of binding to host cells during KSHV infection. gB contains an RGD motif reported to bind the integrin α3β1 during virus entry. Although the ligand specificity of α3β1 has been controversial, current literature indicates that α3β1 ligand recognition is independent of RGD. We compared α3β1 to the RGD-binding integrin, αVβ3, for binding to envelope-associated gB and a gB(RGD) peptide. Adhesion assays demonstrated that β3-CHO cells overexpressing αVβ3 specifically bound gB(RGD), whereas α3-CHO cells overexpressing α3β1 did not. Function-blocking antibodies to αVβ3 inhibited the adhesion of HT1080 fibrosarcoma cells to gB(RGD), while antibodies to α3β1 did not. Using affinity-purified integrins and confocal microscopy, αVβ3 bound to gB(RGD) and KSHV virions, demonstrating direct receptor-ligand interactions. Specific αVβ3 antagonists, including cyclic and dicyclic RGD peptides and αVβ3 function-blocking antibodies, inhibited KSHV infection by 70 to 80%. Keratinocytes from α3-null mice lacking α3β1 were fully competent for infection by KSHV, and reconstitution of α3β1 function by transfection with α3 cDNA reduced KSHV infectivity from 74% to 55%. Additional inhibitory effects of α3β1 on the cell surface expression of αVβ3 and on αVβ3-mediated adhesion of α3-CHO cells overexpressing α3β1 were detected, consistent with previous reports of transdominant inhibition of αVβ3 function by α3β1. These observations may explain previous reports of an inhibition of KSHV infection by soluble α3β1. Our studies demonstrate that αVβ3 is a cellular receptor mediating both the cell adhesion and entry of KSHV into target cells through binding the virion-associated gB(RGD).

α3β1 integrin-controlled Smad7 regulates reepithelialization during wound healing in mice

Effective reepithelialization after injury is essential for correct wound healing. The upregulation of keratinocyte alpha3beta1 integrin during reepithelialization suggests that this adhesion molecule is involved in wound healing; however, its precise role in this process is unknown. We have shown here that retarded reepithelialization in Itga3(-/-) mouse skin wounds is due predominantly to repressed TGF-beta1-mediated responses. Specifically, expression of the inhibitor of TGF-beta1-signaling Smad7 was elevated in Itga3(-/-) keratinocytes. Indeed, in vivo blockade of Smad7 increased the rate of reepithelialization in Itga3(-/-) and WT wounds to similar levels. Our data therefore indicate that the function of alpha3beta1 integrin as a mediator of keratinocyte migration is not essential for reepithelialization but suggest instead that alpha3beta1 integrin has a major new in vivo role as an inhibitor of Smad7 during wound healing. Moreover, our study may identify a previously undocumented function for Smad7 as a regulator of reepithelialization in vivo and implicates Smad7 as a potential novel target for the treatment of cutaneous wounds.

Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Integrin receptors for cell adhesion to extracellular matrix have important roles in promoting tumor growth and progression. Integrin alpha3beta1 is highly expressed in breast cancer cells in which it is thought to promote invasion and metastasis; however, its roles in regulating malignant tumor cell behavior remain unclear. In the current study, we used short-hairpin RNA (shRNA) to show that suppression of alpha3beta1 in a human breast cancer cell line, MDA-MB-231, leads to decreased tumorigenicity, reduced invasiveness, and decreased production of factors that stimulate endothelial cell migration. Real-time PCR revealed that suppression of alpha3beta1 caused a dramatic reduction in expression of the cyclooxygenase-2 (COX-2) gene, which is frequently overexpressed in breast cancers and has been exploited as a therapeutic target. Decreased COX-2 was accompanied by reduced prostaglandin E2 (PGE(2)), a major prostanoid produced downstream of COX-2 and an important effector of COX-2 signaling. shRNA-mediated suppression of COX-2 showed that it has a role in tumor cell invasion and cross-talk to endothelial cells. Furthermore, treatment with PGE(2) restored these functions in alpha3beta1-deficient MDA-MB-231 cells. These findings identify a role for alpha3beta1 in regulating two properties of tumor cells that facilitate cancer progression: invasiveness and ability to stimulate endothelial cells. They also reveal a novel role for COX-2 as a downstream effector of alpha3beta1 in tumor cells, thereby identifying alpha3beta1 as a potential therapeutic target to inhibit breast cancer.

α3β1 integrin in epidermis promotes wound angiogenesis and keratinocyte-to-endothelial-cell crosstalk through the induction of MRP3

During cutaneous wound healing, epidermal keratinocytes play essential roles in the secretion of factors that promote angiogenesis. However, specific cues in the wound microenvironment that trigger the production of pro-angiogenic factors by keratinocytes, and the cellular receptors that mediate this response, remain unclear. In this study, we exploited a model of conditional integrin knockout to demonstrate impaired wound angiogenesis in mice that lack α3β1 integrin in epidermis. In addition, we used genetic and shRNA approaches to determine that α3β1-integrin deficiency in keratinocytes leads to reduced mRNA and protein expression of the pro-angiogenic factor mitogen-regulated protein 3 (MRP3; also known as PRL2C4), and to demonstrate that this regulation provides a mechanism of keratinocyte-to-endothelial-cell crosstalk that promotes endothelial-cell migration. Finally, we showed that the impaired wound angiogenesis in epidermis-specific α3-integrin-knockout mice is correlated with reduced expression of MRP3 in wounded epidermis. These findings identify a novel role for α3β1 integrin in promoting wound angiogenesis through a mechanism of crosstalk from epidermal to endothelial cells, and they implicate MRP3 in this integrin-dependent crosstalk. Such a mechanism represents a novel paradigm for integrin-mediated regulation of wound angiogenesis that extends beyond traditional roles for integrins in cell adhesion and migration.

Endothelial α3β1-Integrin Represses Pathological Angiogenesis and Sustains Endothelial-VEGF

Integrin alpha3beta1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of alpha3beta1 can affect angiogenesis either positively or negatively, either a direct in vivo role for alpha3beta1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of alpha3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where alpha3 is deleted specifically in endothelial cells (ec-alpha3-/-). Here we show that ec-alpha3-/- mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that alpha3beta1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial alpha3beta1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.

An immortalization-dependent switch in integrin function up-regulates MMP-9 to enhance tumor cell invasion.

Integrins, the major receptors for cell adhesion to the extracellular matrix, play important roles during tumor progression. However, it is still unclear whether genetic lesions that occur during carcinoma development can lead to altered integrin function, and how changes in integrin function contribute to subsequent carcinoma progression. Loss-of-function mutations in p53 and activating mutations in H-Ras, which immortalize and transform epithelial cells, respectively, are common causal events in squamous cell carcinoma (SCC). Phenotypes resulting from these two genetic lesions promote SCC progression and are, therefore, potential targets for anticancer therapies. We developed a model system of keratinocyte transformation that has allowed us to investigate the individual roles of p53 mutation and oncogenic Ras mutation in the acquisition of integrin alpha3beta1-regulated phenotypes that promote SCC progression. Using this model, we show that keratinocyte immortalization by p53-null mutation causes a switch in alpha3beta1 function that induces matrix metalloproteinase (MMP)-9 gene expression in tumorigenic cells. This acquired alpha3beta1-dependent regulation of MMP-9 was maintained during subsequent transformation by oncogenic Ras, and it promoted invasion of tumorigenic keratinocytes. Our results show that loss of p53 function leads to changes in integrin-mediated gene regulation that occur during SCC progression and play a critical role in tumor cell invasion.

Integrin α3β1 as a breast cancer target

Introduction: Integrin receptors for cell adhesion to the extracellular matrix have important roles in all stages of cancer progression and metastasis. Since the integrin family was discovered in the early 1980s, many studies have identified critical adhesion and signaling functions for integrins expressed on tumor cells, endothelial cells and other cell types of the tumor microenvironment, in controlling proliferation, survival, migration and angiogenesis. In recent years, the laminin-binding integrin α3β1 has emerged as a potentially promising anti-cancer target on breast cancer cells. Areas covered: Studies from the past decade that implicate integrins as promising anti-cancer targets and the development of integrin antagonists as anti-cancer therapeutics. Recent preclinical studies that have identified the laminin-binding integrin α3β1 as an appealing anti-cancer target and the knowledge gaps that must be closed to fully exploit this integrin as a therapeutic target for breast cancer. Expert opinion: Although the tumor-promoting functions of α3β1 implicate this integrin as a promising therapeutic target on breast cancer cells, successful exploitation of this integrin as an anti-cancer target will require a better understanding of the molecular mechanisms whereby it regulates specific tumor cell behaviors and the identification of the most appropriate α3β1 functions to antagonize on breast cancer cells.