Keratinocyte-Specific SOX2 Overexpression Suppressed Pressure Ulcer Formation after Cutaneous Ischemia-Reperfusion Injury via Enhancement of Amphiregulin Production.

Ischemia-reperfusion (I/R) injury is a key player in the pathogeneses of pressure ulcer formation. Our previous work demonstrated that inducing the transcription factor SOX2 promotes cutaneous wound healing through EGFR signaling pathway enhancement. However, its protective effect on cutaneous I/R injury was not well-characterized. We aimed to assess the role of SOX2 in cutaneous I/R injury and the tissue-protective effect of SOX2 induction in keratinocytes (KCs) in cutaneous I/R injury. SOX2 was transiently expressed in KCs after cutaneous I/R injury. Ulcer formation was significantly suppressed in KC-specific SOX2-overexpressing mice. SOX2 in skin KCs significantly suppressed the infiltrating inflammatory cells, apoptotic cells, vascular damage, and hypoxic areas in cutaneous I/R injury. Oxidative stress-induced mRNA levels of inflammatory cytokine expression were suppressed, and antioxidant stress factors and amphiregulin were elevated by SOX2 induction in skin KCs. Recombinant amphiregulin administration suppressed pressure ulcer development after cutaneous I/R injury in mice and suppressed oxidative stress-induced ROS production and apoptosis in vitro. These findings support that SOX2 in KCs might regulate cutaneous I/R injury through amphiregulin production, resulting in oxidative stress suppression. Recombinant amphiregulin can be a potential therapeutic agent for cutaneous I/R injury.

CD271 activation prevents low to high-risk progression of cutaneous squamous cell carcinoma and improves therapy outcomes.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, showing a rapid increasing incidence worldwide. Although most cSCC can be cured by surgery, a sizeable number of cases are diagnosed at advanced stages, with local invasion and distant metastatic lesions. In the skin, neurotrophins (NTs) and their receptors (CD271 and Trk) form a complex network regulating epidermal homeostasis. Recently, several works suggested a significant implication of NT receptors in cancer. However, CD271 functions in epithelial tumors are controversial and its precise role in cSCC is still to be defined. METHODS: Spheroids from cSCC patients with low-risk (In situ or Well-Differentiated cSCC) or high-risk tumors (Moderately/Poorly Differentiated cSCC), were established to explore histological features, proliferation, invasion abilities, and molecular pathways modulated in response to CD271 overexpression or activation in vitro. The effect of CD271 activities on the response to therapeutics was also investigated. The impact on the metastatic process and inflammation was explored in vivo and in vitro, by using zebrafish xenograft and 2D/3D models. RESULTS: Our data proved that CD271 is upregulated in Well-Differentiated tumors as compared to the more aggressive Moderately/Poorly Differentiated cSCC, both in vivo and in vitro. We demonstrated that CD271 activities reduce proliferation and malignancy marker expression in patient-derived cSCC spheroids at each tumor grade, by increasing neoplastic cell differentiation. CD271 overexpression significantly increases cSCC spheroid mass density, while it reduces their weight and diameter, and promotes a major fold-enrichment in differentiation and keratinization genes. Moreover, both CD271 overexpression and activation decrease cSCC cell invasiveness in vitro. A significant inhibition of the metastatic process by CD271 was observed in a newly established zebrafish cSCC model. We found that the recruitment of leucocytes by CD271-overexpressing cells directly correlates with tumor killing and this finding was further highlighted by monocyte infiltration in a THP-1-SCC13 3D model. Finally, CD271 activity synergizes with Trk receptor inhibition, by reducing spheroid viability, and significantly improves the outcome of photodynamic therapy (PTD) or chemotherapy in spheroids and zebrafish. CONCLUSION: Our study provides evidence that CD271 could prevent the switch between low to high-risk cSCC tumors. Because CD271 contributes to maintaining active differentiative paths and favors the response to therapies, it might be a promising target for future pharmaceutical development.

Chromatin Landscape Governing Murine Epidermal Differentiation.

Chromatin landscape and regulatory networks are determinants in lineage specification and differentiation. To define the temporospatial differentiation axis in murine epidermal cells in vivo, we generated datasets profiling expression dynamics (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin using sequencing), architecture (Hi-C), and histone modifications (chromatin immunoprecipitation followed by sequencing) in the epidermis. We show that many differentially regulated genes are suppressed during the differentiation process, with superenhancers controlling differentiation-specific epigenomic changes. Our data shows the relevance of the Dlx/Klf/Grhl combinatorial regulatory network in maintaining correct temporospatial gene expression during epidermal differentiation. We determined differential open compartments, topologically associating domain score, and looping in the basal cell and suprabasal cell epidermal fractions, with the evolutionarily conserved epidermal differentiation complex region showing distinct suprabasal cell-specific topologically associating domain and loop formation that coincided with superenhancer sites. Overall, our study provides a global genome-wide resource of chromatin dynamics that define unrecognized regulatory networks and the epigenetic control of Dlx3-bound superenhancer elements during epidermal differentiation.

Molecular mechanisms regulating wound repair: Evidence for paracrine signaling from corneal epithelial cells to fibroblasts and immune cells following transient epithelial cell treatment with Mitomycin C.

In this paper, we use RNAseq to identify senescence and phagocytosis as key factors to understanding how mitomyin C (MMC) stimulates regenerative wound repair. We use conditioned media (CM) from untreated (CMC) and MMC treated (CMM) human and mouse corneal epithelial cells to show that corneal epithelial cells indirectly exposed to MMC secrete elevated levels of immunomodulatory proteins including IL-1α and TGFß1 compared to cells exposed to CMC. These factors increase epithelial and macrophage phagocytosis and promote ECM turnover. IL-1α supplementation can increase phagocytosis in control epithelial cells and attenuate TGFß1 induced αSMA expression by corneal fibroblasts. Yet, we show that epithelial cell CM contains factors besides IL-1α that regulate phagocytosis and αSMA expression by fibroblasts. Exposure to CMM also impacts the activation of bone marrow derived dendritic cells and their ability to present antigen. These in vitro studies show how a brief exposure to MMC induces corneal epithelial cells to release proteins and other factors that function in a paracrine way to enhance debris removal and enlist resident epithelial and immune cells as well as stromal fibroblasts to support regenerative and not fibrotic wound healing.

Intrinsic Networks Regulating Tissue Repair: Comparative Studies of Oral and Skin Wound Healing.

Wound repair is a systematic biological program characterized by four overlapping phases: hemostasis, inflammation, proliferation, and remodeling. Notwithstanding differences between species and distinct anatomical sites, the fundamental phases in the wound healing process are conserved among mammalian species. Oral wound healing is defined as an ideal wound healing model because it resolves rapidly and without scar formation. Understanding the regulation and contribution of the different molecular events that drive rapid wound healing in oral mucosa compared with those of the skin will help us define how these lesions heal more efficiently and may provide new therapeutic strategies that can be translated to the clinical settings for patients with chronic nonhealing wounds. Although all epithelial tissues have remarkable ability for tissue repair, the efficiency of such repair differs between epithelia (oral mucosa vs. cutaneous). This prompts the long-standing, fundamental biological and clinically relevant questions as to why and how does the oral mucosa achieve its enhanced wound healing capacity. In this review, we focus on (1) distinct innate wound healing capabilities of the oral mucosa, (2) lessons learned from comparative transcriptomic studies of oral mucosa versus skin, and (3) translation of findings to therapeutics for enhanced wound healing.

FOXM1 network in association with TREM1 suppression regulates NET formation in diabetic foot ulcers.

Diabetic foot ulcers (DFU) are a serious complication of diabetes mellitus and associated with reduced quality of life and high mortality rate. DFUs are characterized by a deregulated immune response with decreased neutrophils due to loss of the transcription factor, FOXM1. Diabetes primes neutrophils to form neutrophil extracellular traps (NETs), contributing to tissue damage and impaired healing. However, the role of FOXM1 in priming diabetic neutrophils to undergo NET formation remains unknown. Here, we found that FOXM1 regulates reactive oxygen species (ROS) levels in neutrophils and inhibition of FOXM1 results in increased ROS leading to NET formation. Next generation sequencing revealed that TREM1 promoted the recruitment of FOXM1+ neutrophils and reversed effects of diabetes and promoted wound healing in vivo. Moreover, we found that TREM1 expression correlated with clinical healing outcomes of DFUs, indicating TREM1 may serve as a useful biomarker or a potential therapeutic target. Our findings highlight the clinical relevance of TREM1, and indicates FOXM1 pathway as a novel regulator of NET formation during diabetic wound healing, revealing new therapeutic strategies to promote healing in DFUs.

Elucidating the role of keratin 75 in enamel using Krt75tm1Der knock-in mouse model.

Keratin 75 (K75) was recently discovered in ameloblasts and enamel organic matrix. Carriers of A161T substitution in K75 present with the skin condition Pseudofollicullitis barbae. This mutation is also associated with high prevalence of caries and compromised structural and mechanical properties of enamel. Krt75tm1Der knock-in mouse (KI) with deletion of Asn159, located two amino acids away from KRT75A161T, can be a potential model for studying the role of K75 in enamel and the causes of the higher caries susceptibility associated with KRT75A161T mutation. To test the hypotheses that KI enamel is more susceptible to a simulated acid attack (SAA), and has altered structural and mechanical properties, we conducted in vitro SAA experiments, microCT, and microhardness analyses on 1st molars of one-month-old WT and KI mice. KI and WT hemimandibles were subjected to SAA and contralateral hemimandibles were used as controls. Changes in enamel porosity were assessed by immersion of the hemimandibles in rhodamine, followed by fluorescent microscopy analysis. Fluorescence intensity of KI enamel after SSA was significantly higher than in WT, indicating that KI enamel is more susceptible to acid attack. MicroCT analysis of 1st molars revealed that while enamel volumes were not significantly different, enamel mineral density was significantly lower in KI, suggesting a potential defect of enamel maturation. Microhardness tests revealed that in KI enamel is softer than in WT, and potentially less resilient to damages. These results suggest that the KI enamel can be used as a model to study the role of K75 in enamel.

Loss of DLX3 tumor suppressive function promotes progression of SCC through EGFR-ERBB2 pathway.

Cutaneous squamous cell carcinoma (cSCC) ranks second in the frequency of all skin cancers. The balance between keratinocyte proliferation and differentiation is disrupted in the pathological development of cSCC. DLX3 is a homeobox transcription factor which plays pivotal roles in embryonic development and epidermal homeostasis. To investigate the impact of DLX3 expression on cSCC prognosis, we carried out clinicopathologic analysis of DLX3 expression which showed statistical correlation between tumors of higher pathologic grade and levels of DLX3 protein expression. Further, Kaplan-Meier survival curve analysis demonstrated that low DLX3 expression correlated with poor patient survival. To model the function of Dlx3 in skin tumorigenesis, a two-stage dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA) study was performed on mice genetically depleted of Dlx3 in skin epithelium (Dlx3cKO). Dlx3cKO mice developed significantly more tumors, with more rapid tumorigenesis compared to control mice. In Dlx3cKO mice treated only with DMBA, tumors developed after ~16 weeks suggesting that loss of Dlx3 has a tumor promoting effect. Whole transcriptome analysis of tumor and skin tissue from our mouse model revealed spontaneous activation of the EGFR-ERBB2 pathway in the absence of Dlx3. Together, our findings from human and mouse model system support a tumor suppressive function for DLX3 in skin and underscore the efficacy of therapeutic approaches that target EGFR-ERBB2 pathway.

Deregulated immune cell recruitment orchestrated by FOXM1 impairs human diabetic wound healing.

Diabetic foot ulcers (DFUs) are a life-threatening disease that often result in lower limb amputations and a shortened lifespan. However, molecular mechanisms contributing to the pathogenesis of DFUs remain poorly understood. We use next-generation sequencing to generate a human dataset of pathogenic DFUs to compare to transcriptional profiles of human skin and oral acute wounds, oral as a model of "ideal" adult tissue repair due to accelerated closure without scarring. Here we identify major transcriptional networks deregulated in DFUs that result in decreased neutrophils and macrophages recruitment and overall poorly controlled inflammatory response. Transcription factors FOXM1 and STAT3, which function to activate and promote survival of immune cells, are inhibited in DFUs. Moreover, inhibition of FOXM1 in diabetic mouse models (STZ-induced and db/db) results in delayed wound healing and decreased neutrophil and macrophage recruitment in diabetic wounds in vivo. Our data underscore the role of a perturbed, ineffective inflammatory response as a major contributor to the pathogenesis of DFUs, which is facilitated by FOXM1-mediated deregulation of recruitment of neutrophils and macrophages, revealing a potential therapeutic strategy.

Interferon lambda promotes immune dysregulation and tissue inflammation in TLR7-induced lupus.

Type III IFN lambdas (IFN-λ) have recently been described as important mediators of immune responses at barrier surfaces. However, their role in autoimmune diseases such as systemic lupus erythematosus (SLE), a condition characterized by aberrant type I IFN signaling, has not been determined. Here, we identify a nonredundant role for IFN-λ in immune dysregulation and tissue inflammation in a model of TLR7-induced lupus. IFN-λ protein is increased in murine lupus and IFN-λ receptor (Ifnlr1) deficiency significantly reduces immune cell activation and associated organ damage in the skin and kidneys without effects on autoantibody production. Single-cell RNA sequencing in mouse spleen and human peripheral blood revealed that only mouse neutrophils and human B cells are directly responsive to this cytokine. Rather, IFN-λ activates keratinocytes and mesangial cells to produce chemokines that induce immune cell recruitment and promote tissue inflammation. These data provide insights into the immunobiology of SLE and identify type III IFNs as important factors for tissue-specific pathology in this disease.

Molecular Approach to Cutaneous Squamous Cell Carcinoma: From Pathways to Therapy.

Cutaneous squamous cell carcinoma (cSCC) represents the second most frequent skin cancer,recently showing a rapid increase in incidence worldwide, with around >1 million cases/year in theUnited States and 2500 deaths [1] [...].

Trafficking and secretion of keratin 75 by ameloblasts in vivo.

A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicles, nail beds, and lingual papillae, was recently discovered in dental enamel, the most highly mineralized hard tissue in the human body. Among many questions this discovery poses, the fundamental question regarding the trafficking and secretion of this protein, which lacks a signal peptide, is of an utmost importance. Here, we present evidence that K75 is expressed during the secretory stage of enamel formation and is present in the forming enamel matrix. We further show that K75 is secreted together with major enamel matrix proteins amelogenin and ameloblastin, and it was detected in Golgi and the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) but not in rough ER (rER). Inhibition of ER-Golgi transport by brefeldin A did not affect the association of K75 with Golgi, whereas ameloblastin accumulated in rER, and its transport from rER into Golgi was disrupted. Together, these results indicate that K75, a cytosolic protein lacking a signal sequence, is secreted into the forming enamel matrix utilizing portions of the conventional ER-Golgi secretory pathway. To the best of our knowledge, this is the first study providing insights into mechanisms of keratin secretion.

Do DLX3 and CD271 Protect Human Keratinocytes from Squamous Tumor Development?

Well-regulated epidermal homeostasis depends on the function of different classes of factors, such as transcription regulators and receptors. Alterations in this homeostatic balance may lead to the development of cutaneous squamous tumorigenesis. The homeobox transcription factor DLX3 is determinant for a p53-dependent regulation of epidermal differentiation and modulates skin carcinogenesis. The maintenance of skin homeostasis also involves the action of neurotrophins (NTs) and their receptors, Trk and CD271. While Trk receptor overexpression is a hallmark of cancer, there are conflicting data on CD271 expression and function in cutaneous SCC (cSCC). Previous studies have reported NT receptors expression in head and neck SSC (HNSCC). We show that CD271 is expressed at low levels in primary cSCC cells and the number of CD271+ cells correlates with cell cohesion in SCC spheroids. In normal epidermis, CD271 is expressed in proliferative progenitor cells and DLX3 in terminally differentiated keratinocytes. Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) increase DLX3 expression. In the absence of a functional BDNF receptor TrkB in keratinocytes, we hypothesize that the BDNF-dependent DLX3 response could be mediated via CD271. Altogether, our results support a putative CD271-DLX3 connection in keratinocytes, which might be crucial to preventing squamous skin cancer.

Correction: Genetic variants in pachyonychia congenita-associated keratins increase susceptibility to tooth decay.

[This corrects the article DOI: 10.1371/journal.pgen.1007168.].

The skin transcriptome in hidradenitis suppurativa uncovers an antimicrobial and sweat gland gene signature which has distinct overlap with wounded skin.

Hidradenitis suppurativa (HS) is a debilitating chronic inflammatory skin disease resulting in non-healing wounds affecting body areas of high hair follicle and sweat gland density. The pathogenesis of HS is not well understood but appears to involve dysbiosis-driven aberrant activation of the innate immune system leading to excessive inflammation. Marked dysregulation of antimicrobial peptides and proteins (AMPs) in HS is observed, which may contribute to this sustained inflammation. Here, we analyzed HS skin transcriptomes from previously published studies and integrated these findings through a comparative analysis with a published wound healing data set and with immunofluorescence and qPCR analysis from new HS patient samples. Among the top differently expressed genes between lesional and non-lesional HS skin were members of the S100 family as well as dermcidin, the latter known as a sweat gland-associated AMP and one of the most downregulated genes in HS lesions. Interestingly, many genes associated with sweat gland function, such as secretoglobins and aquaporin 5, were decreased in HS lesional skin and we discovered that these genes demonstrated opposite expression profiles in healing skin. Conversely, HS lesional and wounded skin shared a common gene signature including genes encoding for S100 proteins, defensins, and genes encoding antiviral proteins. Overall, our results suggest that the pathogenesis of HS may be driven by changes in AMP expression and altered sweat gland function, and may share a similar pathology with chronic wounds.

SOX2 Epidermal Overexpression Promotes Cutaneous Wound Healing via Activation of EGFR/MEK/ERK Signaling Mediated by EGFR Ligands.

Oral mucosa contains a unique transcriptional network that primes oral wounds for rapid resolution in humans. Our previous work identified genes that were consistently upregulated in the oral mucosa and demonstrated that induction of one of the identified genes, transcription factor SOX2, promoted cutaneous wound healing in mice. In this study, we investigated the molecular and cellular mechanisms by which SOX2 accelerates wound healing in skin. RNA-sequencing analysis showed that SOX2 induced a proliferative and wound-activated phenotype in skin keratinocytes prior to wounding. During wound healing, SOX2 induced proliferation of epithelial and connective tissue cells and promoted angiogenesis. Chromatin immunoprecipitation assay revealed that SOX2 directly regulates expression of EGFR ligands, resulting in activation of EGFR. In vitro, skin keratinocytes overexpressing SOX2 promoted cell migration via the EGFR/MEK/ERK pathway. We conclude that induction of SOX2 in skin keratinocytes accelerates cutaneous wound healing by promoting keratinocyte migration and proliferation, and enhancement of angiogenesis via upregulation of EGFR ligands and activation of EGFR/MEK/ERK pathway. Through the identification of putative cutaneous SOX2 targets, such as HBEGF, this study opens venues to determine clinical targets for treatment of skin wounds.

Ectodermal dysplasias: Classification and organization by phenotype, genotype and molecular pathway.

An international advisory group met at the National Institutes of Health in Bethesda, Maryland in 2017, to discuss a new classification system for the ectodermal dysplasias (EDs) that would integrate both clinical and molecular information. We propose the following, a working definition of the EDs building on previous classification systems and incorporating current approaches to diagnosis: EDs are genetic conditions affecting the development and/or homeostasis of two or more ectodermal derivatives, including hair, teeth, nails, and certain glands. Genetic variations in genes known to be associated with EDs that affect only one derivative of the ectoderm (attenuated phenotype) will be grouped as non-syndromic traits of the causative gene (e.g., non-syndromic hypodontia or missing teeth associated with pathogenic variants of EDA "ectodysplasin"). Information for categorization and cataloging includes the phenotypic features, Online Mendelian Inheritance in Man number, mode of inheritance, genetic alteration, major developmental pathways involved (e.g., EDA, WNT "wingless-type," TP63 "tumor protein p63") or the components of complex molecular structures (e.g., connexins, keratins, cadherins).

Molecular basis of Mitomycin C enhanced corneal sensory nerve repair after debridement wounding.

The ocular surface is covered by stratified squamous corneal epithelial cells that are in cell:cell contact with the axonal membranes of a dense collection of sensory nerve fibers that act as sentinels to detect chemical and mechanical injuries which could lead to blindness. The sheerness of the cornea makes it susceptible to superficial abrasions and recurrent erosions which demand continuous regrowth of the axons throughout life. We showed previously that topical application of the antibiotic and anticancer drug Mitomycin C (MMC) enhances reinnervation of the corneal nerves and reduces recurrent erosions in mice via an unknown mechanism. Here we show using RNA-seq and confocal imaging that wounding the corneal epithelium by debridement upregulates proteases and protease inhibitors within the epithelium and leads to stromal nerve disruption. MMC attenuates these effects after debridement injury by increasing serpine1 gene and protein expression preserving L1CAM on axon surfaces of reinnervating sensory nerves. These data demonstrate at the molecular level that gene expression changes in the corneal epithelium and stroma modulate sensory axon integrity. By preserving the ability of axons to adhere to corneal epithelial cells, MMC enhances sensory nerve recovery after mechanical debridement injury.