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p63 is a transcriptional regulator of ectodermal development that is required for basal cell proliferation and stem cell maintenance. p73 is a closely related p53 family member that is expressed in select p63-positive basal cells and can heterodimerize with p63. p73-/- mice lack multiciliated cells and have reduced numbers of basal epithelial cells in select tissues; however, the role of p73 in basal epithelial cells is unknown. Herein, we show that p73-deficient mice exhibit delayed wound healing despite morphologically normal-appearing skin. The delay in wound healing is accompanied by decreased proliferation and increased levels of biomarkers of the DNA damage response in basal keratinocytes at the epidermal wound edge. In wild-type mice, this same cell population exhibited increased p73 expression after wounding. Analyzing single-cell transcriptomic data, we found that p73 was expressed by epidermal and hair follicle stem cells, cell types required for wound healing. Moreover, we discovered that p73 isoforms expressed in the skin (ΔNp73) enhance p63-mediated expression of keratinocyte genes during cellular reprogramming from a mesenchymal to basal keratinocyte-like cell. We identified a set of 44 genes directly or indirectly regulated by ΔNp73 that are involved in skin development, cell junctions, cornification, proliferation, and wound healing. Our results establish a role for p73 in cutaneous wound healing through regulation of basal keratinocyte function.
Psoriasis is a chronic inflammatory skin disease characterized mainly by epidermal hyperplasia, scaling, and erythema; T helper 17 cells have a role in its pathogenesis. Although IL-26, known as a T helper 17 cytokine, is upregulated in psoriatic skin lesions, its precise role is unclear. We investigated the role of IL-26 in the imiquimod-induced psoriasis-like murine model using human IL-26 transgenic mice. Erythema symptoms induced by daily applications of imiquimod increased dramatically in human IL-26 transgenic mice compared with controls. Vascularization and immune cell infiltration were prominent in skin lesions of human IL-26 transgenic mice. Levels of fibroblast growth factor (FGF) 1, FGF2, and FGF7 were significantly upregulated in the skin lesions of imiquimod-treated human IL-26 transgenic mice and psoriasis patients. In vitro analysis demonstrated that FGF1, FGF2, and FGF7 levels were elevated in human keratinocytes and vascular endothelial cells following IL-26 stimulation. Furthermore, IL-26 acted directly on vascular endothelial cells, promoting proliferation and tube formation, possibly through protein kinase B, extracellular signal-regulated kinase, and NF-κB pathways. Moreover, similar effects of IL-26 were observed in the murine contact hypersensitivity model, indicating that these effects are not restricted to psoriasis. Altogether, our data indicate that IL-26 may be a promising therapeutic target in T cell-mediated skin inflammation, including psoriasis.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Emerging evidence indicates that even subtle changes in the expression of key genes of signalling pathways can have profound effects. MicroRNAs (miRNAs) are masters of subtlety and generally have only mild effects on their target genes. The microRNA miR-31 is one of the major microRNAs in many cutaneous conditions associated with activated keratinocytes, such as the hyperproliferative diseases psoriasis, non-melanoma skin cancer and hair follicle growth. miR-31 is a marker of the hair growth phase, and in our miR-31 transgenic mouse model it impairs the function of keratinocytes. This leads to aberrant proliferation, apoptosis, and differentiation that results in altered hair growth, while the loss of miR-31 leads to increased hair growth. Through in vitro and in vivo studies, we have defined a set of conserved miR-31 target genes, including LATS2 and STK40, which serve as new players in the regulation of keratinocyte growth and hair follicle biology. LATS2 can regulate growth of keratinocytes and we have identified a function of STK40 that can promote the expression of key hair follicle programme regulators such as HR, DLX3 and HOXC13.
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
How metabolic pathways required for epidermal tissue growth and remodeling influence the ability of keratinocytes to survive stressful conditions is still largely unknown. The mechanistic target of rapamycin complex 2 (mTORC2) regulates growth and metabolism of several tissues, but its functions in epidermal cells are poorly defined. Rictor is an adaptor protein essential for mTORC2 activity. To explore the roles of mTORC2 in the epidermis, we have conditionally deleted rictor in mice via K14-Cre-mediated homologous recombination and found that its deficiency causes moderate tissue hypoplasia, reduced keratinocyte proliferation and attenuated hyperplastic response to TPA. Noteworthy, rictor-deficient keratinocytes displayed increased lifespan, protection from senescence, and enhanced tolerance to cellular stressors such as growth factors deprivation, epirubicin and X-ray in vitro and radioresistance in vivo. Rictor-deficient keratinocytes exhibited changes in global gene expression profiles consistent with metabolic alterations and enhanced stress tolerance, a shift in cell catabolic processes from glycids and lipids to glutamine consumption and increased production of mitochondrial reactive oxygen species (ROS). Mechanistically, the resiliency of rictor-deficient epidermal cells relies on these ROS increases, indicating stress resistance via mitohormesis. Thus, our findings reveal a new link between metabolic changes and stress adaptation of keratinocytes centered on mTORC2 activity, with potential implications in skin aging and therapeutic resistance of epithelial tumors.
PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Lims1) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN.
© 2015. Published by The Company of Biologists Ltd.
Scarring is a significant medical burden; financially to the health care system and physically and psychologically for patients. Importantly, there have been numerous case reports describing the occurrence of cancer in burn scars. Currently available therapies are not satisfactory due to their undesirable side-effects, complex delivery routes, requirements for long-term use and/or expense. Radix Arnebiae (Zi Cao), a perennial herb, has been clinically applied to treat burns and manage scars for thousands of years in Asia. Shikonin, an active component extracted from Radix Arnebiae, has been demonstrated to induce apoptosis in cancer cells. Apoptosis is an essential process during scar tissue remodelling. It was therefore hypothesized that Shikonin may induce apoptosis in scar-associated cells. This investigation presents the first detailed in vitro study examining the functional responses of scar-associated cells to Shikonin, and investigates the mechanisms underlying these responses. The data obtained suggests that Shikonin inhibits cell viability and proliferation and reduces detectable collagen in scar-derived fibroblasts. Further investigation revealed that Shikonin induces apoptosis in scar fibroblasts by differentially regulating the expression of caspase 3, Bcl-2, phospho-Erk1/2 and phospho-p38. In addition, Shikonin down-regulates the expression of collagen I, collagen III and alpha-smooth muscle actin genes hence attenuating collagen synthesis in scar-derived fibroblasts. In summary, it is demonstrated that Shikonin induces apoptosis and decreases collagen production in scar-associated fibroblasts and may therefore hold potential as a novel scar remediation therapy.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
The TGFβ signaling pathway is essential to epithelial homeostasis and is often inhibited during progression of esophageal squamous cell carcinoma. Recently, an important role for TGFβ signaling has been described in the crosstalk between epithelial and stromal cells regulating squamous tumor cell invasion in mouse models of head-and-neck squamous cell carcinoma (HNSCC). Loss of TGFβ signaling, in either compartment, leads to HNSCC however, the mechanisms involved are not well understood. Using organotypic reconstruct cultures (OTC) to model the interaction between epithelial and stromal cells that occur in dysplastic lesions, we show that loss of TGFβ signaling promotes an invasive phenotype in both fibroblast and epithelial compartments. Employing immortalized esophageal keratinocytes established to reproduce common mutations of esophageal squamous cell carcinoma, we show that treatment of OTC with inhibitors of TGFβ signaling (A83-01 or SB431542) enhances invasion of epithelial cells into a fibroblast-embedded Matrigel/collagen I matrix. Invasion induced by A83-01 is independent of proliferation but relies on protease activity and expression of ADAMTS-1 and can be altered by matrix density. This invasion was associated with increased expression of pro-inflammatory cytokines, IL1 and EGFR ligands HB-EGF and TGFα. Altering EGF signaling prevented or induced epithelial cell invasion in this model. Loss of expression of the TGFβ target gene ROBO1 suggested that chemorepulsion may regulate keratinocyte invasion. Taken together, our data show increased invasion through inhibition of TGFβ signaling altered epithelial-fibroblasts interactions, repressing markers of activated fibroblasts, and altering integrin-fibronectin interactions. These results suggest that inhibition of TGFβ signaling modulates an array of pathways that combined promote multiple aspects of tumor invasion.
Copyright © 2014 Elsevier Inc. All rights reserved.
Transforming growth factor-β1 (TGF-β1) regulates the tissue response to injury and is the principal driver of excessive scarring leading to fibrosis and eventual organ failure. The TGF-β1 effectors SMAD3 and p53 are major contributors to disease progression. While SMAD3 is an established pro-fibrotic factor, the role of p53 in the TGF-β1-induced fibrotic program is not clear. p53 gene silencing, genetic ablation/subsequent rescue, and pharmacological inhibition confirmed that p53 was required for expression of plasminogen activator inhibitor-1 (PAI-1), a major TGF-β1 target gene and a key causative element in fibrotic disorders. TGF-β1 regulated p53 activity by stimulating p53(Ser15 and 9) phosphorylation and acetylation, promoting interactions with activated SMADs and subsequent binding of p53/SMAD3 to the PAI-1 promoter in HK-2 human renal tubular epithelial cells and HaCaT human keratinocytes. Immunohistochemistry revealed prominent co-induction of SMAD3, p53 and PAI-1 in the tubular epithelium of the obstructed kidney consistent with a potential in vivo role for p53 and SMADs in TGF-β1-driven renal fibrosis. TGF-β1-initiated phosphorylation of p53(Ser15) and up-regulation of expression of several pro-fibrotic genes, moreover, was dependent on the rapid generation of reactive oxygen species (ROS). shRNA silencing of the p22(Phox) subunit of NADP(H) oxidases in HK-2 cells partially attenuated (over 50%) p53(Ser15) phosphorylation and PAI-1 induction. These studies highlight the role of free radicals in p53 activation and subsequent pro-fibrotic reprogramming by TGF-β1 via the SMAD3-p53 transcriptional axis. Present findings provide a rationale for therapeutic targeting of SMAD3-p53 in aberrant TGF-β1 signaling associated with renal fibrosis.
Copyright © 2014 Elsevier Inc. All rights reserved.
To increase our understanding of psoriasis, we used high-throughput complementary DNA sequencing (RNA-seq) to assay the transcriptomes of lesional psoriatic and normal skin. We sequenced polyadenylated RNA-derived complementary DNAs from 92 psoriatic and 82 normal punch biopsies, generating an average of ∼38 million single-end 80-bp reads per sample. Comparison of 42 samples examined by both RNA-seq and microarray revealed marked differences in sensitivity, with transcripts identified only by RNA-seq having much lower expression than those also identified by microarray. RNA-seq identified many more differentially expressed transcripts enriched in immune system processes. Weighted gene coexpression network analysis (WGCNA) revealed multiple modules of coordinately expressed epidermal differentiation genes, overlapping significantly with genes regulated by the long noncoding RNA TINCR, its target gene, staufen-1 (STAU1), the p63 target gene ZNF750, and its target KLF4. Other coordinately expressed modules were enriched for lymphoid and/or myeloid signature transcripts and genes induced by IL-17 in keratinocytes. Dermally expressed genes were significantly downregulated in psoriatic biopsies, most likely because of expansion of the epidermal compartment. These results show the power of WGCNA to elucidate gene regulatory circuits in psoriasis, and emphasize the influence of tissue architecture in both differential expression and coexpression analysis.
The skin interstitium sequesters excess Na+ and Cl- in salt-sensitive hypertension. Mononuclear phagocyte system (MPS) cells are recruited to the skin, sense the hypertonic electrolyte accumulation in skin, and activate the tonicity-responsive enhancer-binding protein (TONEBP, also known as NFAT5) to initiate expression and secretion of VEGFC, which enhances electrolyte clearance via cutaneous lymph vessels and increases eNOS expression in blood vessels. It is unclear whether this local MPS response to osmotic stress is important to systemic blood pressure control. Herein, we show that deletion of TonEBP in mouse MPS cells prevents the VEGFC response to a high-salt diet (HSD) and increases blood pressure. Additionally, an antibody that blocks the lymph-endothelial VEGFC receptor, VEGFR3, selectively inhibited MPS-driven increases in cutaneous lymphatic capillary density, led to skin Cl- accumulation, and induced salt-sensitive hypertension. Mice overexpressing soluble VEGFR3 in epidermal keratinocytes exhibited hypoplastic cutaneous lymph capillaries and increased Na+, Cl-, and water retention in skin and salt-sensitive hypertension. Further, we found that HSD elevated skin osmolality above plasma levels. These results suggest that the skin contains a hypertonic interstitial fluid compartment in which MPS cells exert homeostatic and blood pressure-regulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3-mediated modification of cutaneous lymphatic capillary function.