Roles for epithelial integrin α3ß1 in regulation of the microenvironment during normal and pathological tissue remodeling.

Integrin receptors for the extracellular matrix activate intracellular signaling pathways that are critical for tissue development, homeostasis, and regeneration/repair, and their loss or dysregulation contributes to many developmental defects and tissue pathologies. This review will focus on tissue remodeling roles for integrin α3ß1, a receptor for laminins found in the basement membranes (BMs) that underlie epithelial cell layers. As a paradigm, we will discuss literature that supports a role for α3ß1 in promoting ability of epidermal keratinocytes to modify their tissue microenvironment during skin development, wound healing, or tumorigenesis. Preclinical and clinical studies have shown that this role depends largely on ability of α3ß1 to govern the keratinocyte's repertoire of secreted proteins, or the "secretome," including 1) matrix proteins and proteases involved in matrix remodeling and 2) paracrine-acting growth factors/cytokines that stimulate other cells with important tissue remodeling functions (e.g., endothelial cells, fibroblasts, inflammatory cells). Moreover, α3ß1 signaling controls gene expression that helps epithelial cells carry out these functions, including genes that encode secreted matrix proteins, proteases, growth factors, or cytokines. We will review what is known about α3ß1-dependent gene regulation through both transcription and posttranscriptional mRNA stability. Regarding the latter, we will discuss examples of α3ß1-dependent alternative splicing (AS) or alternative polyadenylation (APA) that prevents inclusion of cis-acting mRNA sequences that would otherwise target the transcript for degradation via nonsense-mediated decay or destabilizing AU-rich elements (AREs) in the 3'-untranslated region (3'-UTR). Finally, we will discuss prospects and anticipated challenges of exploiting α3ß1 as a clinical target for the treatment of cancer or wound healing.

Resolvin D2-G-Protein Coupled Receptor 18 Enhances Bone Marrow Function and Limits Steatosis and Hepatic Collagen Accumulation in Aging.

Aging is associated with nonresolving inflammation and tissue dysfunction. Resolvin D2 (RvD2) is a proresolving ligand that acts through the G-protein-coupled receptor called GPR18. Unbiased RNA sequencing revealed increased Gpr18 expression in macrophages from old mice, and in livers from elderly humans, which was associated with increased steatosis and fibrosis in middle-aged (MA) and old mice. MA mice that lacked GPR18 on myeloid cells had exacerbated steatosis and hepatic fibrosis, which was associated with a decline in Mac2+ macrophages. Treatment of MA mice with RvD2 reduced steatosis and decreased hepatic fibrosis, correlating with increased Mac2+ macrophages, increased monocyte-derived macrophages, and elevated numbers of monocytes in the liver, blood, and bone marrow. RvD2 acted directly on the bone marrow to increase monocyte-macrophage progenitors. A transplantation assay further demonstrated that bone marrow from old mice facilitated hepatic collagen accumulation in young mice. Transient RvD2 treatment to mice transplanted with bone marrow from old mice prevented hepatic collagen accumulation. Together, this study demonstrates that RvD2-GPR18 signaling controls steatosis and fibrosis and provides a mechanistic-based therapy for promoting liver repair in aging.

Integrin α4ß1 is required for IL-1α- and Nrf2-dependent, Cox-2 induction in fibroblasts, supporting a mechanism that suppresses α-SMA expression.

Growth and repair processes, both normal and pathological, require reciprocal interactions between cells and their microenvironment. Integrins are bidirectional, cell surface receptors that transduce mechanical and chemical signals to and from the extracellular matrix. We recently reported that keratinocyte α3ß1 is required for interleukin (IL)-1α secretion. Importantly, IL-1α regulates fibroblast Cox-2 expression and prostaglandin E2 (PGE2 ) secretion, thereby linking keratinocyte integrin function to a paracrine signal that suppresses the myofibroblast phenotype. We now report that fibroblast integrin α4ß1 is required for this IL-1α-induced, Cox-2 expression. Moreover, Cox-2 induction by IL-1α requires Nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of redox homeostasis; and integrin α4ß1 is necessary to maintain IL-1α-dependent, Nrf2 levels. Treating fibroblasts with a Nrf-2 activating compound inhibits TGF-ß-dependent, alpha smooth muscle actin (α-SMA) expression and stress fibre formation. Our data suggest that fibroblast integrin α4ß1 regulates-depending on microenvironmental cues-the differentiated state of fibroblasts through a signalling network in which IL-1α, Cox-2 and Nrf2 participate.

Integrin α3ß1 controls mRNA splicing that determines Cox-2 mRNA stability in breast cancer cells.

It is unknown how cues from the tumor microenvironment can regulate post-transcriptional mechanisms, such as alternative splicing, that control genes that drive malignant growth. The induction of cyclooxygenase 2 (Cox-2) by integrin α3ß1 in breast cancer cells can promote tumor progression. We have used RNAi to suppress α3ß1 in human MDA-MB-231 breast cancer cells and then investigated changes in global gene expression. Numerous mRNAs, including Cox-2, show altered expression and/or alternative exon usage (AEU) in α3ß1-deficient cells. AEU included patterns predicted to render an mRNA susceptible to degradation, such as 3'-UTR variations or retention of elements that target an mRNA for nonsense-mediated decay (NMD). PCR-based analysis of α3ß1-deficient cells confirmed changes in Cox-2 mRNA that might target it for NMD, including retention of an intron that harbors premature termination codons and changes within the 3'-UTR. Moreover, Cox-2 mRNA has reduced stability in α3ß1-deficient cells, which is partially reversed by knockdown of the essential NMD factor UPF1. Our study identifies α3ß1-mediated AEU as a novel paradigm of integrin-dependent gene regulation that has potential for exploitation as a therapeutic target.

Integrin-mediated regulation of epidermal wound functions.

During cutaneous wound healing, keratinocyte proliferation and migration are critical for re-epithelialization. In addition the epidermis secretes growth factors, cytokines, proteases, and matricellular proteins into the wound microenvironment that modify the extracellular matrix and stimulate other wound cells that control the inflammatory response, promote angiogenesis and facilitate tissue contraction and remodeling. Wound keratinocytes express at least seven different integrins-the major cell adhesion receptors for the extracellular matrix-that collectively control essential cell-autonomous functions to ensure proper re-epithelialization, including migration, proliferation, survival and basement membrane assembly. Moreover, it has become evident in recent years that some integrins can regulate paracrine signals from wound epidermis that stimulate other wound cells involved in angiogenesis, contraction and inflammation. Importantly, it is likely that abnormal integrin expression or function in the epidermis contributes to wound pathologies such as over-exuberant healing (e.g., hypertrophic scar formation) or diminished healing (e.g., chronic wounds). In this review, we discuss current knowledge of integrin function in the epidermis, which implicates them as attractive therapeutic targets to promote wound healing or treat wound pathologies. We also discuss challenges that arise from the complex roles that multiple integrins play in wound epidermis, which may be regulated through extracellular matrix remodeling that determines ligand availability. Indeed, understanding how different integrin functions are temporally coordinated in wound epidermis and which integrin functions go awry in pathological wounds, will be important to determine how best to target them clinically to achieve maximum therapeutic benefit. Graphical abstract In addition to their well-characterized roles in keratinocyte adhesion, migration and wound re-epithelialization, epidermal integrins play important roles in modifying the wound microenvironment by regulating the expression and secretion of growth factors, extracellular proteases, and matricellular proteins that stimulate other wound cells, including vascular endothelial cells and fibroblasts/myofibroblasts.

Expression of integrin α3ß1 and cyclooxygenase-2 (COX2) are positively correlated in human breast cancer.

BACKGROUND: Expression of integrin α3ß1 is associated with tumor progression, metastasis, and poor prognosis in several cancers, including breast cancer. Moreover, preclinical studies have revealed important pro-tumorigenic and pro-metastatic functions for this integrin, including tumor growth, survival, invasion, and paracrine induction of angiogenesis. Our previously published work in a preclinical breast cancer model showed that integrin α3ß1 promotes expression of cyclooxygenase-2 (COX2/PTGS2), a known driver of breast cancer progression. However, the clinical significance of this regulation was unknown. The objective of the current study was to assess the clinical relevance of the relationship between integrin α3ß1 and COX2 by testing for their correlated expression among various forms of human breast cancer. METHODS: Immunohistochemistry was performed to assess co-expression of α3 and COX2 in specimens of human invasive ductal carcinoma (IDC), either on a commercial tissue microarray (n = 59 samples) or obtained from Albany Medical Center archives (n = 68 samples). Immunostaining intensity for the integrin α3 subunit or COX2 was scored, and Spearman's rank correlation coefficient analysis was performed to assess their co-expression across and within different tumor subtypes or clinicopathologic criteria. RESULTS: Although expression of integrin α3 or COX2 varied among clinical IDC samples, a statistically significant, positive correlation was detected between α3 and COX2 in both tissue microarrays (r(s) = 0.49, p < 0.001, n = 59) and archived samples (r(s) = 0.59, p < 0.0001, n = 68). In both sample sets, this correlation was independent of hormone receptor status, histological grade, or disease stage. CONCLUSIONS: COX2 and α3 are correlated in IDC independently of hormone receptor status or other clinicopathologic features, supporting the hypothesis that integrin α3ß1 is a determinant of COX2 expression in human breast cancer. These results support the clinical relevance of α3ß1-dependent COX2 gene expression that we reported previously in breast cancer cells. The findings also suggest that COX2-positive breast carcinomas of various subtypes might be vulnerable to therapeutic strategies that target α3ß1, and that α3 expression might serve as an independent prognostic biomarker.

Keratinocyte integrin α3ß1 induces expression of the macrophage stimulating factor, CSF-1, through a YAP/TEAD-dependent mechanism.

The development of wound therapy targeting integrins is hampered by inadequate understanding of integrin function in cutaneous wound healing and the wound microenvironment. Following cutaneous injury, keratinocytes migrate to restore the skin barrier, and macrophages aid in debris clearance. Thus, both keratinocytes and macrophages are critical to the coordination of tissue repair. Keratinocyte integrins have been shown to participate in this coordinated effort by regulating secreted factors, some of which crosstalk to distinct cells in the wound microenvironment. Epidermal integrin α3ß1 is a receptor for laminin-332 in the cutaneous basement membrane. Here we show that wounds deficient in epidermal α3ß1 express less epidermal-derived macrophage colony-stimulating factor 1 (CSF-1), the primary macrophage-stimulating growth factor. α3ß1-deficient wounds also have fewer wound-proximal macrophages, suggesting that keratinocyte α3ß1 may stimulate wound macrophages through the regulation of CSF-1. Indeed, using a set of immortalized keratinocytes, we demonstrate that keratinocyte-derived CSF-1 supports macrophage growth, and that α3ß1 regulates Csf1 expression through Src-dependent stimulation of Yes-associated protein (YAP)-Transcriptional enhanced associate domain (TEAD)-mediated transcription. Consistently, α3ß1-deficient wounds in vivo display a substantially reduced number of keratinocytes with YAP-positive nuclei. Overall, our current findings identify a novel role for epidermal integrin α3ß1 in regulating the cutaneous wound microenvironment by mediating paracrine crosstalk from keratinocytes to wound macrophages, implicating α3ß1 as a potential target of wound therapy.

ß1 integrins regulate cellular behavior and cardiomyocyte organization during ventricular wall formation.

AIMS: The mechanisms regulating the cellular behavior and cardiomyocyte organization during ventricular wall morphogenesis are poorly understood. Cardiomyocytes are surrounded by extracellular matrix (ECM) and interact with ECM via integrins. This study aims to determine whether and how ß1 integrins regulate cardiomyocyte behavior and organization during ventricular wall morphogenesis in the mouse. METHODS AND RESULTS: We applied mRNA deep sequencing and immunostaining to determine the expression repertoires of α/ß integrins and their ligands in the embryonic heart. Integrin ß1 subunit (ß1) and some of its ECM ligands are asymmetrically distributed and enriched in the luminal side of cardiomyocytes, and fibronectin surrounds cardiomyocytes, creating a network for them. Itgb1, which encodes the ß1, was deleted via Nkx2.5Cre/+ to generate myocardial-specific Itgb1 knockout (B1KO) mice. B1KO hearts display an absence of a trabecular zone but a thicker compact zone. The levels of hyaluronic acid and versican, essential for trabecular initiation, were not significantly different between control and B1KO. Instead, fibronectin, a ligand of ß1, was absent in the myocardium of B1KO hearts. Furthermore, B1KO cardiomyocytes display a random cellular orientation and fail to undergo perpendicular cell division, be organized properly, and establish the proper tissue architecture to form trabeculae. Mosaic clonal lineage tracing showed that Itgb1 regulates cardiomyocyte transmural migration and proliferation autonomously. CONCLUSIONS: ß1 is asymmetrically localized in the cardiomyocytes, and some of its ECM ligands are enriched along the luminal side of the myocardium, and fibronectin surrounds cardiomyocytes. ß1 integrins are required for cardiomyocytes to attach to the ECM network. This engagement provides structural support for cardiomyocytes to maintain shape, undergo perpendicular division, and establish cellular organization. Deletion of Itgb1 leads to loss of ß1 and fibronectin and prevents cardiomyocytes from engaging the ECM network, resulting in failure to establish tissue architecture to form trabeculae.

Loss of Integrin α9ß1 on Tumor Keratinocytes Enhances the Stromal Vasculature and Growth of Cutaneous Tumors.

Angiogenesis is critical to tumor progression, and the function of integrins in tumor angiogenesis is complex. In this study, we report that loss of integrin α9ß1 expression from epidermal tumor cells is critical to maintaining persistent stromal vessel density. Forced expression of α9 in transformed mouse keratinocytes dramatically reduces vessel density in allograft tumors in vivo compared with that in the same cells lacking α9ß1. Moreover, α9 mRNA expression is dramatically reduced in mouse and human epidermal tumors as is α9ß1-dependent gene regulation. Loss of tumor cell α9ß1 occurs through at least two mechanisms: (i) ITGA9 gene copy number loss in human tumors and (ii) epigenetic silencing in mouse and human tumors. Importantly, we show that reversal of epigenetic silencing of Itga9 restores α9 expression in mouse keratinocytes and that human tumors without ITGA9 copy number loss have increased promoter methylation. Our data suggest that for epidermal tumorigenesis to occur, tumor cells must avoid the tumor and angiogenic suppressive effects of α9ß1 by repressing its expression through deletion and/or epigenetic silencing, thereby promoting stromal development and tumor growth.

alpha3beta1 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.

Endothelial alpha3beta1-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.

Integrin α3ß1 Represses Reelin Expression in Breast Cancer Cells to Promote Invasion.

Integrin α3ß1, a cell adhesion receptor for certain laminins, is known to promote breast tumor growth and invasion. Our previous gene microarray study showed that the RELN gene, which encodes the extracellular glycoprotein Reelin, was upregulated in α3ß1-deficient (i.e., α3 knockdown) MDA-MB-231 cells. In breast cancer, reduced RELN expression is associated with increased invasion and poor prognosis. In this study we demonstrate that α3ß1 represses RELN expression to enhance breast cancer cell invasion. RELN mRNA was significantly increased upon RNAi-mediated α3 knockdown in two triple-negative breast cancer cell lines, MDA-MB-231 and SUM159. Modulation of baseline Reelin levels altered invasive potential, where enhanced Reelin expression in MDA-MB-231 cells reduced invasion, while RNAi-mediated suppression of Reelin in SUM159 cells increased invasion. Moreover, treatment of α3ß1-expressing MDA-MB-231 cells with culture medium that was conditioned by α3 knockdown MDA-MB-231 cells led to decreased invasion. RNAi-mediated suppression of Reelin in α3 knockdown MDA-MB-231 cells mitigated this effect of conditioned-medium, identifying secreted Reelin as an inhibitor of cell invasion. These results demonstrate a novel role for α3ß1 in repressing Reelin in breast cancer cells to promote invasion, supporting this integrin as a potential therapeutic target.

Comparative use of CRISPR and RNAi to modulate integrin α3ß1 in triple negative breast cancer cells reveals that some pro-invasive/pro-metastatic α3ß1 functions are independent of global regulation of the transcriptome.

Integrin receptors for the extracellular matrix play critical roles at all stages of carcinogenesis, including tumor growth, tumor progression and metastasis. The laminin-binding integrin α3ß1 is expressed in all epithelial tissues where it has important roles in cell survival, migration, proliferation, and gene expression programs during normal and pathological tissue remodeling. α3ß1 signaling and adhesion functions promote tumor growth and metastasis in a number of different types of cancer cells. Previously, we used RNA interference (RNAi) technology to suppress the expression of the ITGA3 gene (encoding the α3 subunit) in the triple-negative breast cancer cell line, MDA-MB-231, thereby generating variants of this line with reduced expression of integrin α3ß1. This approach revealed that α3ß1 promotes pro-tumorigenic functions such as cell invasion, lung metastasis, and gene regulation. In the current study, we used CRISPR technology to knock out the ITGA3 gene in MDA-MB-231 cells, thereby ablating expression of integrin α3ß1 entirely. RNA-seq analysis revealed that while the global transcriptome was altered substantially by RNAi-mediated suppression of α3ß1, it was largely unaffected following CRISPR-mediated ablation of α3ß1. Moreover, restoring α3ß1 to the latter cells through inducible expression of α3 cDNA failed to alter gene expression substantially, suggesting that use of CRISPR to abolish α3ß1 led to a decoupling of the integrin from its ability to regulate the transcriptome. Interestingly, both cell invasion in vitro and metastatic colonization in vivo were reduced when α3ß1 was abolished using CRISPR, as we observed previously using RNAi to suppress α3ß1. Taken together, our results show that pro-invasive/pro-metastatic roles for α3ß1 are not dependent on its ability to regulate the transcriptome. Moreover, our finding that use of RNAi versus CRISPR to target α3ß1 produced distinct effects on gene expression underlines the importance of using multiple approaches to obtain a complete picture of an integrin's functions in cancer cells.

Epidermal Integrin α3ß1 Regulates Tumor-Derived Proteases BMP-1, Matrix Metalloprotease-9, and Matrix Metalloprotease-3.

As the major cell surface receptors for the extracellular matrix, integrins regulate adhesion and migration and have been shown to drive tumor growth and progression. Previous studies showed that mice lacking integrin α3ß1 in the epidermis fail to form skin tumors during two-step chemical tumorigenesis, indicating a protumorigenic role for α3ß1. Furthermore, genetic ablation of α3ß1 in established skin tumors caused their rapid regression, indicating an essential role in the maintenance of tumor growth. In this study, analysis of immortalized keratinocyte lines and their conditioned media support a role for α3ß1 in regulating the expression of several extracellular proteases of the keratinocyte secretome, namely BMP-1, matrix metalloprotease (MMP)-9, and MMP-3. Moreover, immunofluorescence revealed reduced levels of each protease in α3ß1-deficient tumors, and RNA in situ hybridization showed that their expression was correspondingly reduced in α3ß1-deficient tumor cells in vivo. Bioinformatic analysis confirmed that the expression of BMP1, MMP9, and MMP3 genes correlate with the expression of ITGA3 (gene encoding the integrin α3 subunit) in human squamous cell carcinoma and that high ITGA3 and MMP3 associate with poor survival outcome in these patients. Overall, our findings identify α3ß1 as a regulator of several proteases within the secretome of epidermal tumors and as a potential therapeutic target.

Integrin α3ß1 on Tumor Keratinocytes Is Essential to Maintain Tumor Growth and Promotes a Tumor-Supportive Keratinocyte Secretome.

The development of integrin-targeted cancer therapies is hindered by incomplete understanding of integrin function in tumor cells and the tumor microenvironment. Previous studies showed that mice with epidermis-specific deletion of the α3 integrin subunit fail to form skin tumors during two-step chemical tumorigenesis, indicating a protumorigenic role for integrin α3ß1. Here, we generated mice with tamoxifen-inducible, epidermis-specific α3 knockout to determine the role of α3ß1 in the maintenance of established tumor cells and/or the associated stroma. Genetic ablation of α3 in established skin tumors caused their rapid regression, indicating that α3ß1 is essential to maintain tumor growth. Although reduced proliferation and increased apoptosis were observed in α3ß1-deficient tumor cells, these changes followed a robust increase in stromal apoptosis. Furthermore, macrophages and fibulin-2 levels were reduced in stroma following α3 deletion from tumor cells. Mass spectrometric analysis of conditioned medium from immortalized keratinocytes showed that α3ß1 regulates a substantial fraction of the keratinocyte secretome, including fibulin-2 and macrophage CSF1; RNA in situ hybridization showed that expression of these two genes was reduced in tumor keratinocytes in vivo. Our findings identify α3ß1 as a regulator of the keratinocyte secretome and skin tumor microenvironment and as a potential therapeutic target.

Integrin α3ß1 Promotes Invasive and Metastatic Properties of Breast Cancer Cells through Induction of the Brn-2 Transcription Factor.

In the current study, we demonstrate that integrin α3ß1 promotes invasive and metastatic traits of triple-negative breast cancer (TNBC) cells through induction of the transcription factor, Brain-2 (Brn-2). We show that RNAi-mediated suppression of α3ß1 in MDA-MB-231 cells caused reduced expression of Brn-2 mRNA and protein and reduced activity of the BRN2 gene promoter. In addition, RNAi-targeting of Brn-2 in MDA-MB-231 cells decreased invasion in vitro and lung colonization in vivo, and exogenous Brn-2 expression partially restored invasion to cells in which α3ß1 was suppressed. α3ß1 promoted phosphorylation of Akt in MDA-MB-231 cells, and treatment of these cells with a pharmacological Akt inhibitor (MK-2206) reduced both Brn-2 expression and cell invasion, indicating that α3ß1-Akt signaling contributes to Brn-2 induction. Analysis of RNAseq data from patients with invasive breast carcinoma revealed that high BRN2 expression correlates with poor survival. Moreover, high BRN2 expression positively correlates with high ITGA3 expression in basal-like breast cancer, which is consistent with our experimental findings that α3ß1 induces Brn-2 in TNBC cells. Together, our study demonstrates a pro-invasive/pro-metastatic role for Brn-2 in breast cancer cells and identifies a role for integrin α3ß1 in regulating Brn-2 expression, thereby revealing a novel mechanism of integrin-dependent breast cancer cell invasion.

Integrin alphaVbeta3 Binds to the RGD motif of glycoprotein B of Kaposi's sarcoma-associated herpesvirus and functions as an RGD-dependent entry receptor.

Kaposi's 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 alpha(3)beta(1) during virus entry. Although the ligand specificity of alpha(3)beta(1) has been controversial, current literature indicates that alpha(3)beta(1) ligand recognition is independent of RGD. We compared alpha(3)beta(1) to the RGD-binding integrin, alpha(V)beta(3), for binding to envelope-associated gB and a gB(RGD) peptide. Adhesion assays demonstrated that beta(3)-CHO cells overexpressing alpha(V)beta(3) specifically bound gB(RGD), whereas alpha(3)-CHO cells overexpressing alpha(3)beta(1) did not. Function-blocking antibodies to alpha(V)beta(3) inhibited the adhesion of HT1080 fibrosarcoma cells to gB(RGD), while antibodies to alpha(3)beta(1) did not. Using affinity-purified integrins and confocal microscopy, alpha(V)beta(3) bound to gB(RGD) and KSHV virions, demonstrating direct receptor-ligand interactions. Specific alpha(V)beta(3) antagonists, including cyclic and dicyclic RGD peptides and alpha(V)beta(3) function-blocking antibodies, inhibited KSHV infection by 70 to 80%. Keratinocytes from alpha(3)-null mice lacking alpha(3)beta(1) were fully competent for infection by KSHV, and reconstitution of alpha(3)beta(1) function by transfection with alpha(3) cDNA reduced KSHV infectivity from 74% to 55%. Additional inhibitory effects of alpha(3)beta(1) on the cell surface expression of alpha(V)beta(3) and on alpha(V)beta(3)-mediated adhesion of alpha(3)-CHO cells overexpressing alpha(3)beta(1) were detected, consistent with previous reports of transdominant inhibition of alpha(V)beta(3) function by alpha(3)beta(1). These observations may explain previous reports of an inhibition of KSHV infection by soluble alpha(3)beta(1). Our studies demonstrate that alpha(V)beta(3) is a cellular receptor mediating both the cell adhesion and entry of KSHV into target cells through binding the virion-associated gB(RGD).

Integrin Regulation of CAF Differentiation and Function.

Extensive remodeling of the extracellular matrix, together with paracrine communication between tumor cells and stromal cells, contribute to an "activated" tumor microenvironment that supports malignant growth and progression. These stromal cells include inflammatory cells, endothelial cells, and cancer-associated fibroblasts (CAFs). Integrins are expressed on all tumor and stromal cell types where they regulate both cell adhesion and bidirectional signal transduction across the cell membrane. In this capacity, integrins control pro-tumorigenic cell autonomous functions such as growth and survival, as well as paracrine crosstalk between tumor cells and stromal cells. The myofibroblast-like properties of cancer-associated fibroblasts (CAFs), such as robust contractility and extracellular matrix (ECM) deposition, allow them to generate both chemical and mechanical signals that support invasive tumor growth. In this review, we discuss the roles of integrins in regulating the ability of CAFs to generate and respond to extracellular cues in the tumor microenvironment. Since functions of specific integrins in CAFs are only beginning to emerge, we take advantage of a more extensive literature on how integrins regulate wound myofibroblast differentiation and function, as some of these integrin functions are likely to extrapolate to CAFs within the tumor microenvironment. In addition, we discuss the roles that integrins play in controlling paracrine signals that emanate from epithelial/tumor cells to stimulate fibroblasts/CAFs.

Keratinocyte Integrin α3ß1 Promotes Secretion of IL-1α to Effect Paracrine Regulation of Fibroblast Gene Expression and Differentiation.

After cutaneous injury, keratinocytes secrete paracrine factors that regulate wound cell functions; dysregulation of this signaling can lead to wound pathologies. Previously, we established that keratinocyte integrin α3ß1 promotes wound angiogenesis through paracrine stimulation of endothelial cells. We hypothesize here that α3ß1-dependent paracrine signaling from keratinocytes regulates the differentiation state of myofibroblasts. We report that epidermal α3-knockout mice exhibit more wound myofibroblasts and fewer cyclooxygenase 2 (Cox-2)-positive dermal cells than controls. We also found that conditioned medium from α3-expressing mouse keratinocytes (MKα3+), but not from α3-null MK cells (MKα3-), induces expression of Cox-2 in fibroblasts in a time- and dose-dependent manner and that this induction is mediated by IL-1α. Compared with MKα3- cells, MKα3+ cells secrete more IL-1α and less IL-1RA, a natural IL-1 receptor antagonist. Treatment with an IL-1α neutralizing antibody, recombinant IL-1RA, or IL-1 receptor-targeting small interfering RNA suppresses MKα3+ conditioned medium-dependent induction of Cox-2 expression in fibroblasts. Finally, active recombinant IL-1α is sufficient to induce Cox-2 in fibroblasts and to inhibit transforming growth factor-ß-induced α-SMA expression. Our findings support a role for keratinocyte integrin α3ß1 in controlling the secretion of IL-1α, a paracrine factor that regulates the wound myofibroblast phenotype.

Double duty for Rac1 in epidermal wound healing.

During cutaneous wound healing, increased proliferation and migration of epidermal keratinocytes is essential for efficient re-epithelialization of the wound and restoration of barrier function to the skin. Although numerous cell culture studies have identified intracellular signaling proteins that control proliferation and migration in response to extracellular cues from the wound microenvironment, confirming their importance in wound healing requires appropriate in vivo models. The Rho-family guanosine triphosphatase (GTPase) Rac1 is an effector of cellular responses to growth factors, cytokines, and adhesion proteins present in wounds, and it has long been suspected to be an important regulator of wound healing. Two different genetic models now confirm an essential role for Rac1 in wound healing and, further, identify a dual role for Rac1 in promoting keratinocyte migration and proliferation during wound re-epithelialization. This sets the stage for determining which of the known Rac1 pathways are critical for wound repair in vivo and for linking these pathways to specific integrin or growth factor receptors that mediate cellular responses to cues from the wound environment. Together with studies that implicate Rac1 in maintaining epidermal stem cell populations, these findings lay the foundation for identifying distinct epidermal compartments from which Rac1 controls different aspects of wound re-epithelialization.