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.

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.

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.

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.

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.

Targeted Inhibition of PAI-1 Activity Impairs Epithelial Migration and Wound Closure Following Cutaneous Injury.

Objective: Aberrant plasminogen activator inhibitor-1 (PAI-1) expression and activity have been implicated in bleeding disorders, multiorgan fibrosis, and wound healing anomalies. This study details the physiological consequences of targeted PAI-1 functional inhibition on cutaneous injury repair. Approach: Dorsal skin wounds from FVB/NJ mice, created with a 4 mm biopsy punch, were treated topically with the small-molecule PAI-1 antagonist tiplaxtinin (or vehicle control) for 5 days and then analyzed for markers of wound repair. Results: Compared to controls, tiplaxtinin-treated wounds displayed dramatic decreases in wound closure and re-epithelialization. PAI-1 immunoreactivity was evident at the migratory front in all injury sites indicating these effects were due to PAI-1 functional blockade and not PAI-1 expression changes. Stimulated HaCaT keratinocyte migration in response to recombinant PAI-1 in vitro was similarly attenuated by tiplaxtinin. While tiplaxtinin had no effect on keratinocyte proliferation, cell cycle progression, or apoptosis, it effectively reduced collagen deposition, the number of Ki-67+ fibroblasts, and incidence of differentiated myofibroblasts (i.e., smooth muscle α-actin immunoreactive cells), but not fibroblast apoptosis. Innovation: The role for PAI-1 in hemostasis and fibrinolysis is established; involvement of PAI-1 in cutaneous wound healing, however, remains unclear. This study tests the effect of a small-molecule PAI-1 inhibitor in a murine model of skin wound repair. Conclusion: Loss of PAI-1 activity significantly impaired wound closure. Re-epithelialization and fibroblast recruitment/differentiation were both reduced in tiplaxtinin-treated mice. Therapies directed at manipulation of PAI-1 expression and/or activity may have applicability as a treatment option for chronic wounds and scarring disorders.

Integrin Regulation of Epidermal Functions in Wounds.

Significance: Integrins are bidirectional signaling receptors for extracellular matrix that regulate both inside-out signaling that controls keratinocyte-mediated changes to the wound microenvironment and outside-in signaling that controls keratinocyte responses to microenvironmental changes. As such, integrins represent attractive therapeutic targets for treatment of chronic wounds or general promotion of wound healing. Advances in wound management are particularly important as the elderly and diabetic populations within the United States continue to grow. Recent Advances: Although integrins are best known for mediating cell adhesion and migration, integrins in wound epidermis also control cell survival, proliferation, matrix remodeling, and paracrine crosstalk to other cellular compartments of the wound. Importantly, the concept of targeting integrins in the clinic has been established for treatment of certain cancers and other diseases, laying the groundwork for similar exploitation of integrins as targets to treat chronic wounds. Critical Issues: Despite their attractiveness as therapeutic targets, integrins have complex roles in wound healing that are impacted by both their own expression and a highly dynamic wound microenvironment that determines ligand availability. Therefore, identifying relevant integrin ligands in the wound and understanding both distinct and overlapping functions that different integrins play in the epidermis will be critical to determine their precise roles in wound healing. Future Directions: Future research should focus on gaining a thorough understanding of the highly coordinated functions of different integrins in wound epidermis, and on determining which of these functions go awry in pathological wounds. This focus should facilitate development of integrin-targeting therapeutics for treating chronic wounds.

Reduced fibulin-2 contributes to loss of basement membrane integrity and skin blistering in mice lacking integrin α3ß1 in the epidermis.

Deficient epidermal adhesion is a hallmark of blistering skin disorders and chronic wounds, implicating integrins as potential therapeutic targets. Integrin α3ß1, a major receptor in the epidermis for adhesion to laminin-332 (LN-332), has critical roles in basement membrane (BM) organization during skin development. In the current study we identify a role for α3ß1 in promoting stability of nascent epidermal BMs through induction of fibulin-2, a matrix-associated protein that binds LN-332. We demonstrate that mice lacking α3ß1 in the epidermis display ruptured BM beneath neo-epidermis of wounds, characterized by extensive blistering. This junctional blistering phenocopies defects reported in newborn α3-null mice, as well as in human patients with α3 gene mutations, indicating that the developmental role of α3ß1 in BM organization is recapitulated during wound healing. Mice lacking epidermal α3ß1 also have reduced fibulin-2 expression, and fibulin-2-null mice display perinatal skin blisters similar to those in α3ß1-deficient mice. Interestingly, α3-null wound epidermis or keratinocytes also show impaired processing of the LN-332 γ2 chain, although this defect was independent of reduced fibulin-2 and did not appear to cause blistering. Our findings indicate a role for integrin α3ß1 in BM stability through fibulin-2 induction, both in neonatal skin and in adult wounds.

Suppression of integrin alpha3beta1 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.