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.

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.

Regulation of myoepithelial differentiation.

The salivary gland can be permanently impaired by radiation treatment for head and neck cancers. Efforts at tissue regeneration have focused on saliva-producing acinar cells. However, myoepithelial cells are also critical to gland function, but mechanisms that regulate their differentiation are poorly defined. To study myoepithelial differentiation, we employed mSG-PAC1 murine salivary gland epithelial cells. We demonstrate that mSG-PAC1 spheroids exhibit phenotypic plasticity between pro-acinar and myoepithelial cell fates. Increased expression of pro-acinar/acinar or myoepithelial RNAs was identified from spheroids cultured under different media conditions by microarray followed by gene-set enrichment analysis. Spheroids cultured with different medium components expressed proteins typical of either acinar or myoepithelial cells, as detected by immunocytochemistry. We demonstrate that the pattern of TAZ expression in the epithelial compartment of the differentiating murine salivary gland correlates with the expression of the myoepithelial marker alpha-SMA, as is the case for TAZ expression in mSG-PAC1 spheroids. Our analysis also indicates that YAP/TAZ target genes are upregulated together with myoepithelial markers. Importantly, siRNA targeting of TAZ expression in mSG-PAC1 spheroids diminished the expression of myoepithelial markers. Our results in this in vitro cell model implicate TAZ signaling in myoepithelial differentiation.

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.

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.

Serological analysis reveals an imbalanced IgG subclass composition associated with COVID-19 disease severity.

Coronavirus disease 2019 (COVID-19) is associated with a wide spectrum of disease presentation, ranging from asymptomatic infection to acute respiratory distress syndrome (ARDS). Paradoxically, a direct relationship has been suggested between COVID-19 disease severity and the levels of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, including virus-neutralizing titers. A serological analysis of 536 convalescent healthcare workers reveals that SARS-CoV-2-specific and virus-neutralizing antibody levels are elevated in individuals that experience severe disease. The severity-associated increase in SARS-CoV-2-specific antibody is dominated by immunoglobulin G (IgG), with an IgG subclass ratio skewed toward elevated receptor binding domain (RBD)- and S1-specific IgG3. In addition, individuals that experience severe disease show elevated SARS-CoV-2-specific antibody binding to the inflammatory receptor FcÉ£RIIIa. Based on these correlational studies, we propose that spike-specific IgG subclass utilization may contribute to COVID-19 disease severity through potent Fc-mediated effector functions. These results may have significant implications for SARS-CoV-2 vaccine design and convalescent plasma therapy.

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.

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.

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.