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.

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.

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.

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.

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.

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.

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.

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 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.

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 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 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.

Opposing Roles of Epidermal Integrins α3ß1 and α9ß1 in Regulation of mTLD/BMP-1-Mediated Laminin-γ2 Processing during Wound Healing.

Proteolytic processing of the laminin-γ2 chain is a hallmark of basement membrane maturation in the skin. Integrin α3ß1, a major receptor for epidermal adhesion to laminin-332, is critical for proper basement membrane organization during skin development and wound healing. Previously, we identified a role for α3ß1 in promoting the processing of laminin-γ2 in cultured keratinocytes in vitro and in wound epidermis in vivo. In this study we identify the Bmp1 gene, which encodes variants of the mTLD/BMP-1 metalloproteases, as a critical regulator of α3ß1-dependent laminin-γ2 processing, thereby expanding the role of this integrin in controlling the secretion by the epidermis of factors that modulate the tissue microenvironment. Because our previous studies identified another epidermal integrin, α9ß1, as a suppressive regulator of α3ß1-dependent wound angiogenesis, we investigated whether α9ß1 has a similar cross-suppressive effect on the ability of α3ß1 to promote basement membrane organization. Here, we show that, rather than a cross-suppressive role, α9ß1 has an opposing role in basement membrane assembly/maturation through reduced laminin-γ2 processing via mTLD/BMP-1. Although α3ß1 promotes this process during wound healing, α9ß1 has an inhibitory role, suggesting that regulation of basement membrane assembly requires a complex interplay between these distinct epidermal integrins.

Beyond adhesion: emerging roles for integrins in control of the tumor microenvironment.

While integrins were originally discovered as cell adhesion receptors, recent studies have reinforced the concept that integrins have central roles in cancer that extend far beyond controlling cell adhesion and migration. Indeed, as transmembrane cell surface receptors that occupy a critical position at the interface of cellular and extracellular interactions and are capable of both "inside-out" and "outside-in" signaling, integrins are uniquely poised to regulate the cell's ability to promote, sense, and react to changes in the tumor microenvironment. Moreover, integrins are present on all cell types in the tumor microenvironment, and they have important roles in regulating intercellular communication. Decades of promising pre-clinical studies have implicated certain integrins as attractive therapeutic targets in the cancer clinic. Nevertheless, results of the few clinical trials that target integrins in cancer have thus far been disappointing. Importantly, these clinical failures likely reflect the emerging complexity of individual and combinatorial integrin function within both tumor cells and other cell types of the tumor microenvironment, together with a need to explore integrin-targeting agents not just as monotherapies but also as adjuvants to more conventional radiotherapies or chemotherapies. In this review, we will examine recent advances toward understanding how integrins regulate cancer progression, including their roles in intercellular communication and modulation of the tumor microenvironment. Additionally, we will discuss factors that underlie the limited efficacy of current efforts to target integrins in the cancer clinic as well as potential strategies to overcome these challenges.

An FAK-YAP-mTOR Signaling Axis Regulates Stem Cell-Based Tissue Renewal in Mice.

Tissue homeostasis requires the production of newly differentiated cells from resident adult stem cells. Central to this process is the expansion of undifferentiated intermediates known as transit-amplifying (TA) cells, but how stem cells are triggered to enter this proliferative TA state remains an important open question. Using the continuously growing mouse incisor as a model of stem cell-based tissue renewal, we found that the transcriptional cofactors YAP and TAZ are required both to maintain TA cell proliferation and to inhibit differentiation. Specifically, we identified a pathway involving activation of integrin α3 in TA cells that signals through an LATS-independent FAK/CDC42/PP1A cascade to control YAP-S397 phosphorylation and nuclear localization. This leads to Rheb expression and potentiates mTOR signaling to drive the proliferation of TA cells. These findings thus reveal a YAP/TAZ signaling mechanism that coordinates stem cell expansion and differentiation during organ renewal.

Suppression of integrin α3ß1 by α9ß1 in the epidermis controls the paracrine resolution of wound angiogenesis.

Development of wound therapies is hindered by poor understanding of combinatorial integrin function in the epidermis. In this study, we generated mice with epidermis-specific deletion of α3ß1, α9ß1, or both integrins as well as keratinocyte lines expressing these integrin combinations. Consistent with proangiogenic roles for α3ß1, α3-null keratinocytes showed reduced paracrine stimulation of endothelial cell migration and survival, and wounds of epidermis-specific α3 knockout mice displayed impaired angiogenesis. Interestingly, α9ß1 in keratinocytes suppressed α3ß1-mediated stimulation of endothelial cells, and wounds of epidermis-specific α9 knockout mice displayed delayed vascular normalization and reduced endothelial apoptosis, indicating that α9ß1 cross-suppresses α3ß1 proangiogenic functions. Moreover, α9ß1 inhibited α3ß1 signaling downstream of focal adhesion kinase (FAK) autoactivation at the point of Src-mediated phosphorylation of FAK Y861/Y925. Finally, α9ß1 cross-suppressed many α3ß1-dependent genes, including the gene that encodes MMP-9, which we implicated as a regulator of integrin-dependent cross talk to endothelial cells. Our findings identify a novel physiological context for combinatorial integrin signaling, laying the foundation for therapeutic strategies that manipulate α9ß1 and/or α3ß1 during wound healing.

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.

Integrin α3ß1 signaling through MEK/ERK determines alternative polyadenylation of the MMP-9 mRNA transcript in immortalized mouse keratinocytes.

Integrin α3ß1 is highly expressed in both normal and tumorigenic epidermal keratinocytes where it regulates genes that control cellular function and extracellular matrix remodeling during normal and pathological tissue remodeling processes, including wound healing and development of squamous cell carcinoma (SCC). Previous studies identified a role for α3ß1 in immortalized and transformed keratinocytes in the regulation of genes that promote tumorigenesis, invasion, and pro-angiogenic crosstalk to endothelial cells. One such gene, matrix metalloproteinase-9 (MMP-9), is induced by α3ß1 through a post-transcriptional mechanism of enhanced mRNA stability. In the current study, we sought to investigate the mechanism through which α3ß1 controls MMP-9 mRNA stability. First, we utilized a luciferase reporter assay to show that AU-rich elements (AREs) residing within the 3'-untranslated region (3'-UTR) of the MMP-9 mRNA renders the transcript unstable in a manner that is independent of α3ß1. Next, we cloned a truncated variant of the MMP-9 mRNA which is generated through usage of an alternative, upstream polyadenylation signal and lacks the 3'-UTR region containing the destabilizing AREs. Using an RNase protection assay to distinguish "long" (full-length 3'-UTR) and "short" (truncated 3'-UTR) MMP-9 mRNA variants, we demonstrated that the shorter, more stable mRNA that lacks 3'-UTR AREs was preferentially generated in α3ß1-expressing keratinocytes compared with α3ß1-deficient (i.e., α3-null) keratinocytes. Moreover, we determined that α3ß1-dependent alternative polyadenylation was acquired by immortalized keratinocytes, as primary neonatal keratinocytes did not display α3ß1-dependent differences in the long and short transcripts. Finally, pharmacological inhibition of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway in α3ß1-expressing keratinocytes caused a shift towards long variant expression, while Raf-1-mediated activation of ERK in α3-null keratinocytes dramatically enhanced short variant expression, indicating a role for ERK/MAPK signaling in α3ß1-mediated selection of the proximal polyadenylation site. These findings identify a novel mode of integrin α3ß1-mediated gene regulation through alternative polyadenylation.