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 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 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.

Adaptation of the group A Streptococcus adhesin Scl1 to bind fibronectin type III repeats within wound-associated extracellular matrix: implications for cancer therapy.

The human-adapted pathogen group A Streptococcus (GAS) utilizes wounds as portals of entry into host tissue, wherein surface adhesins interact with the extracellular matrix, enabling bacterial colonization. The streptococcal collagen-like protein 1 (Scl1) is a major adhesin of GAS that selectively binds to two fibronectin type III (FnIII) repeats within cellular fibronectin, specifically the alternatively spliced extra domains A and B, and the FnIII repeats within tenascin-C. Binding to FnIII repeats was mediated through conserved structural determinants present within the Scl1 globular domain and facilitated GAS adherence and biofilm formation. Isoforms of cellular fibronectin that contain extra domains A and B, as well as tenascin-C, are present for several days in the wound extracellular matrix. Scl1-FnIII binding is therefore an example of GAS adaptation to the host's wound environment. Similarly, cellular fibronectin isoforms and tenascin-C are present in the tumor microenvironment. Consistent with this, FnIII repeats mediate GAS attachment to and enhancement of biofilm formation on matrices deposited by cancer-associated fibroblasts and osteosarcoma cells. These data collectively support the premise for utilization of the Scl1-FnIII interaction as a novel method of anti-neoplastic targeting in the tumor microenvironment.

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.

Streptococcal Collagen-like Protein 1 Binds Wound Fibronectin: Implications in Pathogen Targeting.

Group A Streptococcus (GAS) infections are responsible for significant morbidity and mortality worldwide. The outlook for an effective global vaccine is reduced because of significant antigenic variation among GAS strains worldwide. Other challenges in GAS therapy include the lack of common access to antibiotics in developing countries, as well as allergy to and treatment failures with penicillin and increasing erythromycin resistance in the industrialized world. At the portal of entry, GAS binds to newly deposited extracellular matrix, which is rich in cellular fibronectin isoforms with extra domain A (EDA, also termed EIIIA) via the surface adhesin, the streptococcal collagen-like protein 1 (Scl1). Recombinant Scl1 constructs, derived from diverse GAS strains, bind the EDA loop segment situated between the C and C' ß-strands. Despite the sequence diversity in Scl1 proteins, multiple sequence alignments and secondary structure predictions of Scl1 variants, as well as crystallography and homology modeling studies, point to a conserved mechanism of Scl1-EDA binding. We propose that targeting this interaction may prevent the progression of infection. A synthetic cyclic peptide, derived from the EDA C-C' loop, binds to recombinant Scl1 with a micromolar dissociation constant. This review highlights the current concept of EDA binding to Scl1 and provides incentives to exploit this binding to treat GAS infections and wound colonization.

Surface-exposed loops and an acidic patch in the Scl1 protein of group A Streptococcus enable Scl1 binding to wound-associated fibronectin.

Keratinized epidermis constitutes a powerful barrier of the mucosa and skin, effectively preventing bacterial invasion, unless it is wounded and no longer protective. Wound healing involves deposition of distinct extracellular matrix (ECM) proteins enriched in cellular fibronectin (cFn) isoforms containing extra domain A (EDA). The streptococcal collagen-like protein 1 (Scl1) is a surface adhesin of group A Streptococcus (GAS), which contains an N-terminal variable (V) domain and a C-terminally located collagen-like domain. During wound infection, Scl1 selectively binds EDA/cFn isoforms and laminin, as well as low-density lipoprotein (LDL), through its V domain. The trimeric V domain has a six-helical bundle fold composed of three pairs of anti-parallel α-helices interconnected by hypervariable loops, but the roles of these structures in EDA/cFn binding are unclear. Here, using recombinant Scl (rScl) constructs to investigate structure-function determinants of the Scl1-EDA/cFn interaction, we found that full-length rScl1, containing both the globular V and the collagen domains, is necessary for EDA/cFn binding. We established that the surface-exposed loops, interconnecting conserved α-helices, guide recognition and binding of Scl1-V to EDA and binding to laminin and LDL. Moreover, electrostatic surface potential models of the Scl1-V domains pointed to a conserved, negatively charged pocket, surrounded by positively charged and neutral regions, as a determining factor for the binding. In light of these findings, we propose an updated model of EDA/cFn recognition by the Scl1 adhesin from GAS, representing a significant step in understanding the Scl1-ECM interactions within the wound microenvironment that underlie GAS pathogenesis.

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.

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.

Hic-5 is required for myofibroblast differentiation by regulating mechanically dependent MRTF-A nuclear accumulation.

How mechanical cues from the extracellular environment are translated biochemically to modulate the effects of TGF-ß on myofibroblast differentiation remains a crucial area of investigation. We report here that the focal adhesion protein, Hic-5 (also known as TGFB1I1), is required for the mechanically dependent generation of stress fibers in response to TGF-ß. Successful generation of stress fibers promotes the nuclear localization of the transcriptional co-factor MRTF-A (also known as MKL1), and this correlates with the mechanically dependent induction of α smooth muscle actin (α-SMA) and Hic-5 in response to TGF-ß. As a consequence of regulating stress fiber assembly, Hic-5 is required for the nuclear accumulation of MRTF-A and the induction of α-SMA as well as cellular contractility, suggesting a crucial role for Hic-5 in myofibroblast differentiation. Indeed, the expression of Hic-5 was transient in acute wounds and persistent in pathogenic scars, and Hic-5 colocalized with α-SMA expression in vivo. Taken together, these data suggest that a mechanically dependent feed-forward loop, elaborated by the reciprocal regulation of MRTF-A localization by Hic-5 and Hic-5 expression by MRTF-A, plays a crucial role in myofibroblast differentiation in response to TGF-ß.

Matrix compliance and the regulation of cytokinesis.

Integrin-mediated cell adhesion to the ECM regulates many physiological processes in part by controlling cell proliferation. It is well established that many normal cells require integrin-mediated adhesion to enter S phase of the cell cycle. Recent evidence indicates that integrins also regulate cytokinesis. Mechanical properties of the ECM can dictate entry into S phase; however, it is not known whether they also can affect the successful completion of cell division. To address this issue, we modulated substrate compliance using fibronectin-coated acrylamide-based hydrogels. Soft and hard substrates were generated with approximate elastic moduli of 1600 and 34,000 Pascals (Pa) respectively. Our results indicate that dermal fibroblasts successfully complete cytokinesis on hard substrates, whereas on soft substrates, a significant number fail and become binucleated. Cytokinesis failure occurs at a step following the formation of the intercellular bridge connecting presumptive daughter cells, suggesting a defect in abscission. Like dermal fibroblasts, mesenchymal stem cells require cell-matrix adhesion for successful cytokinesis. However, in contrast to dermal fibroblasts, they are able to complete cytokinesis on both hard and soft substrates. These results indicate that matrix stiffness regulates the successful completion of cytokinesis, and does so in a cell-type specific manner. To our knowledge, our study is the first to demonstrate that matrix stiffness can affect cytokinesis. Understanding the cell-type specific contribution of matrix compliance to the regulation of cytokinesis will provide new insights important for development, as well as tissue homeostasis and regeneration.

Control of cell migration through mRNA localization and local translation.

Cell migration plays an important role in many normal and pathological functions such as development, wound healing, immune defense, and tumor metastasis. Polarized migrating cells exhibit asymmetric distribution of many cytoskeletal proteins, which is believed to be critical for establishing and maintaining cell polarity and directional cell migration. To target these proteins to the site of function, cells use a variety of mechanisms such as protein transport and messenger RNA (mRNA) localization-mediated local protein synthesis. In contrast to the former which is intensively investigated and relatively well understood, the latter has been understudied and relatively poorly understood. However, recent advances in the study of mRNA localization and local translation have demonstrated that mRNA localization and local translation are specific and effective ways for protein localization and are crucial for embryo development, neuronal function, and many other cellular processes. There are excellent reviews on mRNA localization, transport, and translation during development and other cellular processes. This review will focus on mRNA localization-mediated local protein biogenesis and its impact on somatic cell migration.

The α4ß1 integrin and the EDA domain of fibronectin regulate a profibrotic phenotype in dermal fibroblasts.

Prompt deposition of fibronectin-rich extracellular matrix is a critical feature of normal development and the host-response to injury. Fibronectin isoforms that include the EDA and EDB domains are prominent in these fibronectin matrices. We now report using human dermal fibroblast cultures that the EDA domain of fibronectin or EDA-derived peptides modeled after the C-C' loop promote stress fiber formation and myosin-light chain phosphorylation. These changes are accompanied by an increase in fibronectin synthesis and fibrillogenesis. These effects are blocked by pretreating cells with either siRNA or blocking antibody to the α4 integrin. Our data indicate that the interaction between the α4ß1 integrin and the EDA domain of fibronectin helps to drive tissue fibrosis by promoting a contractile phenotype and an increase in fibronectin synthesis and deposition.

The group A streptococcal collagen-like protein-1, Scl1, mediates biofilm formation by targeting the extra domain A-containing variant of cellular fibronectin expressed in wounded tissue.

Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen-like protein-1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA-containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C-C' loop region recognized by the α(9)ß(1) integrin. The extracellular 2-D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization.

Mechanoregulation of the Myofibroblast in Wound Contraction, Scarring, and Fibrosis: Opportunities for New Therapeutic Intervention.

SIGNIFICANCE: Myofibroblasts are responsible for wound closure that occurs in healed acute wounds. However, their actions can result in disfiguring scar contractures, compromised organ function, and a tumor promoting stroma. Understanding the mechanisms regulating their contractile machinery, gene expression, and lifespan is essential to develop new therapies to control their function. RECENT ADVANCES: Mechanical stress and transforming growth factor beta-1 (TGF-ß1) regulate myofibroblast differentiation from mesenchymal progenitors. As these precursor cells differentiate, they assemble a contractile apparatus to generate the force used to contract wounds. The mechanisms by which mechanical stress promote expression of contractile genes through the TGF-ß1 and serum response factor pathways and offer therapeutic targets to limit myofibroblast function are being elucidated. CRITICAL ISSUES: Emerging evidence suggests that the integration of mechanical cues with intracellular signaling pathways is critical to myofibroblast function via its effects on gene expression, cellular contraction, and paracrine signaling with neighboring cells. In addition, while apoptosis is clearly one pathway that can limit myofibroblast lifespan, recent data suggest that pathogenic myofibroblasts can become senescent and adopt a more beneficial phenotype, or may revert to a quiescent state, thereby limiting their function. FUTURE DIRECTIONS: Given the important role that myofibroblasts play in pathologies as disparate as cutaneous scarring, organ fibrosis, and tumor progression, knowledge gained in the areas of intracellular signaling networks, mechanical signal transduction, extracellular matrix biology, and cell fate will support efforts to develop new therapies with a wide impact.

Dynamic Regulation of Integrin α6ß4 During Angiogenesis: Potential Implications for Pathogenic Wound Healing.

OBJECTIVE: Angiogenesis is an essential component of normal cutaneous wound repair, but is altered in pathogenic forms of wound healing, such as chronic wounds and fibrosis. We previously reported that endothelial expression of integrin α6ß4 is developmentally regulated, with α6ß4 expression correlating with tissue maturation and further showed that endothelial α6ß4 is downregulated in explant angiogenesis assays. These data support the hypothesis that dynamic regulation of α6ß4 may play an important role during new vessel formation in healing wounds. APPROACH: To test this hypothesis, we examined the endothelial expression of α6ß4 using a murine model of cutaneous wound healing and in vitro cultures of primary human dermal microvascular endothelial cells (HDMECs). RESULTS: Expression of α6ß4 is downregulated during early stages of wound healing; angiogenic vessels in day 7 wounds do not express α6ß4. Endothelial expression of α6ß4 is resumed in day 14 wounds. Moreover, explanted HDMECs do not express α6ß4, but expression is induced by treatment with histone deacetylase inhibitors. INNOVATION: We provide in vivo data supporting a role for the dynamic regulation of α6ß4 during vessel formation and remodeling during cutaneous wound repair and in vitro findings that suggest endothelial ß4 expression is regulated transcriptionally, providing an important foundation for future studies to understand the transcriptional mechanisms involved in endothelial cell maturation during normal wound repair. CONCLUSION: Our data indicate that α6ß4 is dynamically regulated during angiogenesis and vessel maturation and suggest that disruption of this regulation may contribute to defective angiogenesis associated with diabetic wounds or cutaneous fibrosis.

Transforming growth factor-ß1-induced transcript 1 protein, a novel marker for smooth muscle contractile phenotype, is regulated by serum response factor/myocardin protein.

Serum response factor (SRF) plays a central role in regulating expression of smooth muscle-specific genes partly by associating with the potent tissue-specific cofactor myocardin. Previous studies have shown that transforming growth factor-ß1-induced transcript 1 (TGFB1I1, also known as Hic-5) is a TGF-ß-responsive gene and is involved in the cellular response to vascular injury, but the regulation of TGFB1I1 expression remains elusive. In this report, we demonstrated that TGFB1I1 is a novel marker for the smooth muscle contractile phenotype and is regulated by SRF/myocardin. We found that TGFB1I1 is specifically expressed in smooth muscle cells (SMCs) and in smooth muscle-rich tissues. Furthermore, TGFB1I1 expression is significantly down-regulated in a variety of models for smooth muscle phenotypic modulation. The TGFB1I1 promoter contains an evolutionarily conserved CArG element, and this element is indispensible for myocardin-induced transactivation of TGFB1I1 promoter. By oligonucleotide pulldown and chromatin immunoprecipitation assays, we found that SRF binds to this CArG element in vitro and in vivo. Ectopic expression of myocardin is sufficient to induce endogenous TGFB1I1 expression in multiple cell lines whereas knocking-down myocardin or SRF significantly attenuated TGFB1I1 expression in SMCs. Furthermore, our data demonstrated that SRF is essential for TGF-ß-mediated induction of TGFB1I1. Finally, silencing of TGFB1I1 expression significantly promotes SMC proliferation. Collectively, this study provides the first evidence that TGFB1I1 is not only an SRF/myocardin-regulated smooth muscle marker but also critical for maintaining smooth muscle contractile phenotype by inhibiting smooth muscle proliferation.