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Antibodies used for experiments. Supplementary Table S2: PCR primers used for genotyping of mouse lines.

Supplementary Figure S5: No change in infiltrating mast cells as determined by CEM staining is observed in melanocytic lesions from either Cdk4R24c or Tyr-NRASQ61K mice with epidermis-specific knockout of Rxra. Assay was performed on bleached 5 J.lm paraffin sections. Infiltrating mast cells are labeled with light blue staining. E=Epidermis, 0= Dermis. Scale bars= 50 micron

Supplementary Figure S8: Chronically UVB-irradiated tumor-adjacent normal skin from Tyr-NRASQBtK or Cdk4R24c mice with epidermis-specific knockout of Rxra show modest increase in CDK4 expression; downregulation of phosphorylated ERK, as well as increased E2F1 expression specifically in Rxraep-/- 1 Cdk4R24c mice. (A-D) Modest increase of CDK4 expression in both epidermis and dermis of Rxraep-/- I Tyr- NRASQ61K and Rxraep-/- 1 Cdk4R24c mice as compared to RxraL2/L2 controls, as determined by immunohistochemistry (5 IJm bleached paraffin sections). E=Epidermis, D= Dermis. Scale bars= 50 IJm. # = no statistical significance , *= p s 0.05, *** = p s 0.001 (E, F) Down regulation in phosphorylated ERK in tumor-adjacent normal skin of Rxraep-/- 1 Tyr-NRASQotK and Rxraep-/- 1 Cdk4R24cmice as compared to RxraL2/L2 controls; as shown by Western Blot (Two biological replicates for each group are shown). (G, H) Analysis of E2F1 protein levels in tumor-adjacent normal skin of Rxraep-/- 1 Tyr-NRASQotK and Rxraep-/- I Cdk4R24c mice as compared to RxraL2/L2 controls; as shown by Western Blot (Two biological replicates for each group are shown). No difference was observed in the Tyr-NRASQotK group, while increased E2F1 levels were observed in Rxraep-/- 1 Cdk4R'

Supplementary methods

Supplementary Figure S2: Chronic-UVB treated and parallel non-treated controls used in this study. (A,B) H&E staining of non-UVB treated skin from parallel age-matched mice (A) and TAN skin from chronic-UVB treated mice (B). In all groups, TAN skin has similar morphology to non-UVB treated skin, except for increased epidermal thickening; and dermal pigmentation which is enhanced further in the RXRaep-/- mice. Mice carrying Cdk4R24c in all groups show sebaceous gland hyperplasia within hair follicles (yellow boxes). E=Epidermis, D=Dermis, scale bar =100 IJm. (C) Epidermis measurements taken from H&E stained samples. In non-UVB treated mice, Tyr-NRAS0 61K or Cdk4R24c combined with Rxraep-/- mutation exhibited slightly thicker epidermis than RxraL2/L2 counterparts [7]. This is attenuated by chronic-UVB treatment, which results in a similarly thickened epidermis across all groups. ** = p

Supplementary Figure S1: Breeding/Genotyping of mouse lines used in this study. (A, B) Breeding crossings used. K14-Cre'g/O I RxraL2/L2 is also known as RXRaep-/-. (C) Semi-quantitative PCR was performed on biopsies containing both epidermal and non-epidermal tissue; to confirm genotypes with regard to presence/absence of K14-Cre recombinase (top), epidermis-specific ere-mediated excision of Rxra exon 4 (middle), and the mutant Cdk4R24c allele (bottom). Presence of K14-Cre recombinase is indicated by a single 349 bp band; animals negative for K14-Cre show no amplification. In K14-Cre positive animals, ere-mediated epidermis-specific excision of Rxra is indicated by a 110 bp (L-) band, while a 203 bp (L2) band is also amplified due to presence of non-epidermal tissue in the biopsy in which excision did not occur. Animals negative for K14-Cre show only the 203 bp (L2) band for Rxra, as no excision occurred in any tissue. Animals homozygous for the mutant Cdk4R24c allele show a single band at 530 bp, while animals homozygous for the WT Cdk4 allele show a single band at 440 bp. Primers used are detailed in the Supplementary Table S2. (D) Tyr-NRAS061K genotype is determined phenotypically 10-12 days postnatal by presence/absence of dark pigmentation of the foot pads.

Understanding the molecular mechanisms behind formation of melanoma, the deadliest form of skin cancer, is crucial for improved diagnosis and treatment. One key is to better understand the cross-talk between epidermal keratinocytes and pigment-producing melanocytes. Here, using a bigenic mouse model system combining mutant oncogenic NRASQ61K (constitutively active RAS) or mutant activated CDK4R24C/R24C (prevents binding of CDK4 by kinase inhibitor p16INK4A) with an epidermis-specific knockout of the nuclear retinoid X receptor alpha (RXRαep−/−) results in increased melanoma formation after chronic ultraviolet-B (UVB) irradiation compared with control mice with functional RXRα. Melanomas from both groups of bigenic RXRαep−/− mice are larger in size with higher proliferative capacity, and exhibit enhanced angiogenic properties and increased expression of malignant melanoma markers. Analysis of tumor adjacent normal skin from these mice revealed altered expression of several biomarkers indicative of enhanced melanoma susceptibility, including reduced expression of tumor suppressor p53 and loss of PTEN, with concomitant increase in activated AKT. Loss of epidermal RXRα in combination with UVB significantly enhances invasion of melanocytic cells to draining lymph nodes in bigenic mice expressing oncogenic NRASQ61K compared with controls with functional RXRα. These results suggest a crucial role of keratinocytic RXRα to suppress formation of UVB-induced melanomas and their progression to malignant cancers in the context of driver mutations such as activated CDK4R24C/R24C or oncogenic NRASQ61K.Implications: These findings suggest that RXRα may serve as a clinical diagnostic marker and therapeutic target in melanoma progression and metastasis. Mol Cancer Res; 13(1); 186–96. ©2014 AACR.

Supplementary Figure S6: Heat Map and Clustergram of dysregulated genes in melanocytic lesions from Rxraep-/- 1 Tyr-NRASQ61K mice_ Tissue from melanocytic lesions was captured using Laser Capture Microdissection (LCM) (See Figure 4 for details). Isolated RNA was converted to eDNA and gene expression in Rxraep-/- lesions relative to RxraL2/L2 lesions was assayed using an RT-qPCR array (SA Biosciences, PAMM-033Z (Mouse Cancer)). Mean fold change relative to Rxrcf21L2 group is represented by the heat map (A). Three replicate arrays were performed for both RxraL2/L2 and Rxraep-/- groups; magnitude of gene expression for each replicate is shown in the clustergram (B), which correlates genes with similar expression levels.

Supplementary Figure S3: Controls for IHC experiments used in this study. (A) For all IHC studies, a liquid-repellant slide marker pen was used to separate out a single section on each slide that does not receive primary antibody but otherwise carried through the same labeling protocol; for use as a negative control to demonstrate that signals observed were specific. (B) PEP1 antibody (anti-TYRP1) and PEPS antibody (anti-TYRP2) give similar specific staining patterns for melanocytic cells in RxraL2/L2 neonatal skin following irradiation with a single UVB dose. (C) Fluorescent IHC for p-AKT and PTEN on chronically-irradiated TAN skin showing similar staining pattern to chromogenic IHC shown in Figure 5. (D,E) Non-UVB treated skin showed reduced staining intensity of PTEN, p-AKT, and p53 compared to irradiated skin. Unlike skin treated with chronic UVB, no discernable difference in expression pattern between RxraL2/L2 and bi enic Rxraep-/- mice was observed. E=E idermis, D=Dermis, scale bars =1 00 J.Jm.

Supplementary Figure S4: An increase in infiltrating macrophages is observed in melanocytic lesions from Cdk4R24cmice with epidermal-specific knockout of Rxra, but not in Rxraep-/- Tyr-NRASQBtK mice. No change in infiltrating CD4+ T-cells observed in melanocytic lesions from either Cdk4R24c or Tyr-NRASQ61K mice with epidermal-specific knockout of Rxra. (A, B) No change in infiltrating macrophages, as determined by fluorescent immunohistochemistry (I HC) of of 5 J.lm paraffin sections (MAC1 antigen), was observed in Rxraep-/- 1 Tyr-NRAS061K mice as compared to RxraL2/L2 controls. (C, D) An increase in infiltrating macrophages was observed in Rxraep-/- 1 Cdk4R24c mice as compared to RxraL2/L2 controls. (E-H) No change in infiltrating CD4+ T-cells as determined by fluorescent IHC was observed in Rxraep-/- Tyr-NRAS061K or Rxraep-/- Cdk4R24c mice as compared to RxraL 21L2 controls. Scale bars= 50 J.lm. ** = p :5 0.01

Exosomes show great potential in diagnostic and therapeutic applications. Inspired by the human innate immune defense, herein, we report engineered exosomes derived from monocytic cells treated with immunomodulating compounds 1α,25-dihydroxyvitamin D

Models of skin diseases, such as psoriasis and scleroderma, must accurately recapitulate the complex microenvironment of human skin to provide an efficacious platform for investigation of skin diseases. Skin disease research has been shifting from less complex and less relevant 2D (two-dimensional) models to significantly more relevant 3D (three-dimensional) models. Three-dimensional modeling systems are better able to recapitulate the complex cell–cell and cell–matrix interactions that occur in vivo within skin. Three-dimensional human skin equivalents (HSEs) have emerged as an advantageous tool for the study of skin disease in vitro. These 3D HSEs can be highly complex, containing both epidermal and dermal compartments with integrated adnexal structures. The addition of adnexal structures to 3D HSEs has allowed researchers to gain more insight into the complex pathology of various hereditary and acquired skin diseases. One method of constructing 3D HSEs, 3D bioprinting, has emerged as a versatile and useful tool for generating highly complex HSEs. The development of commercially available 3D bioprinters has allowed researchers to create highly reproducible 3D HSEs with precise integration of multiple adnexal structures. While the field of bioengineered models for study of skin disease has made tremendous progress in the last decade, there are still significant efforts necessary to create truly biomimetic skin disease models. In future studies utilizing 3D HSEs, emphasis must be placed on integrating all adnexal structures relevant to the skin disease under investigation. Thorough investigation of the intricate pathology of skin diseases and the development of effective treatments requires use of highly efficacious models of skin diseases.

INTRODUCTION: COUP-TF INTERACTING PROTEIN 2 (CTIP2) is a crucial transcription factor exhibiting its control through coupled modulation of epigenetic modification and transcriptional regulation of key genes related to skin, immune, and nervous system development. Previous studies have validated the essential role of CTIP2 in skin development and maintenance, propagating its effects in epidermal permeability barrier (EPB) homeostasis, wound healing, inflammatory diseases, and epithelial cancers. Lipid metabolism dysregulation, on the other hand, has also established its independent emerging role over the years in normal skin development and various skin-associated ailments. This review focuses on the relatively unexplored connections between CTIP2-mediated control of lipid metabolism and alteration of EPB homeostasis, delayed wound healing, inflammatory diseases exacerbation, and cancer promotion and progression. AREAS COVERED: Here we have discussed the intricate interplay of various endogenous lipids and lipoproteins accompanying skin development and associated disease processes and the possible link to CTIP2-mediated regulation of lipid metabolism. EXPERT OPINION: Establishing the link between CTIP2 and lipid metabolism alterations in the context of skin morphogenesis and diverse types of skin diseases including cancer can help us identify novel targets for effective therapeutic intervention.

Treatment with vemurafenib, a potent and selective inhibitor of mitogen-activated protein kinase signaling downstream of the BRAFV600E oncogene, elicits dramatic clinical responses in patients with metastatic melanoma. Unfortunately, the clinical utility of this drug is limited by a high incidence of drug resistance. Thus, there is an unmet need for alternative therapeutic strategies to treat vemurafenib-resistant metastatic melanomas. We have conducted high-throughput screening of two bioactive compound libraries (Siga and Spectrum libraries) against a metastatic melanoma cell line (A2058) and identified two structurally analogous compounds, deguelin and rotenone, from a cell viability assay. Vemurafenib-resistant melanoma cell lines, A2058R and A375R (containing the BRAFV600E mutation), also showed reduced proliferation when treated with these two compounds. Deguelin, a mitochondrial complex I inhibitor, was noted to significantly inhibit oxygen consumption in cellular metabolism assays. Mechanistically, deguelin treatment rapidly activates AMPK signaling, which results in inhibition of mTORC1 signaling and differential phosphorylation of mTORC1's downstream effectors, 4E-BP1 and p70S6 kinase. Deguelin also significantly inhibited ERK activation and Ki67 expression without altering Akt activation in the same timeframe in the vemurafenib-resistant melanoma cells. These data posit that treatment with metabolic regulators, such as deguelin, can lead to energy starvation, thereby modulating the intracellular metabolic environment and reducing survival of drug-resistant melanomas harboring BRAF V600E mutations.

Skin barrier keeps the “inside-in” and the “outside-out” forming a protective blanket against external insults. Epidermal lipids such as ceramides, fatty acids, triglycerides and cholesterol are integral components in driving the formation and maintenance of epidermal permeability barrier (EPB). A breach in this lipid barrier sets the platform for the subsequent onset and progression of atopic dermatitis (AD). Such lipids are also important in the normal functioning of different organisms, both plants and animals and in diseases including cancer. A double increase in AD rate in the recent years and the chronic nature of this disorder emphasizes the need of this review to shed light on the multifaceted role of the diverse types of lipids in mediating AD pathogenesis.

Summary Inherent plasticity and various survival cues allow glioblastoma stem-like cells (GSCs) to survive and proliferate under intrinsic and extrinsic stress conditions. Here, we report that GSCs depend on the adaptive activation of ER stress and subsequent activation of lipogenesis and particularly stearoyl CoA desaturase (SCD1), which promotes ER homeostasis, cytoprotection, and tumor initiation. Pharmacological targeting of SCD1 is particularly toxic due to the accumulation of saturated fatty acids, which exacerbates ER stress, triggers apoptosis, impairs RAD51-mediated DNA repair, and achieves a remarkable therapeutic outcome with 25%–100% cure rate in xenograft mouse models. Mechanistically, divergent cell fates under varying levels of ER stress are primarily controlled by the ER sensor IRE1, which either promotes SCD1 transcriptional activation or converts to apoptotic signaling when SCD1 activity is impaired. Taken together, the dependence of GSCs on fatty acid desaturation presents an exploitable vulnerability to target glioblastoma., Graphical Abstract, Highlights • SCD1 is essential for GSC maintenance and confers tumor growth advantage in vivo • Therapeutic targeting of SCD1 is highly effective in GBM mouse models • SCD1 exerts a cytoprotective function in GSCs by regulating ER homeostasis • ER stress promotes lipogenesis through the activation of IRE1-SREBP1-SCD1 axis, In this article, Pinkham and colleagues demonstrate that the fatty acid desaturase stearoyl CoA desaturase (SCD1) is essential for the maintenance of glioblastoma cancer stem cells. SCD1 is activated by ER stress and exerts a cytoprotective function by regulating ER homeostasis, thus favoring survival and tumor growth. Pharmacological targeting of SCD1 exhibits potent therapeutic efficacy in brain tumor mouse models.

Pigment-producing melanocytes overcome frequent oxidative stress in their physiological role of protecting the skin against the deleterious effects of solar UV irradiation. This is accomplished by the activity of several endogenous antioxidant systems, including the thioredoxin antioxidant system, in which thioredoxin reductase 1 (TR1) plays an important part. To determine whether TR1 contributes to the redox regulation of melanocyte homeostasis, we have generated a selective melanocytic Txnrd1-knockout mouse model (Txnrd1

Wound healing process is the outcome of a series of actions and combined with collaborative process involving concerted efforts of multiple cell types. The dynamic series of events constituting each of these overlapping rather than discrete stages of wound healing increases its complexity and the necessity to understand it. The contrasting mechanisms of wound healing employed by mouse (via wound contraction) and humans (via reepithelialization) puts forth the need of a model closely mimicking human wound-healing and hence comes the applicability of the mouse excisional wound splinting model. Use of silicone-based splints has demonstrated their effectiveness in aptly resembling the human reepithelialization mediated wound healing by preventing contraction during healing. The rising popularity of nanofiber-based treatments for wound healing through sustained release of factors/molecules promoting wound closure can be potentially implemented in association with this model to determine its efficacy in wound management in a more humanized way.

Wound healing process is the outcome of a series of actions and combined with collaborative process involving concerted efforts of multiple cell types. The dynamic series of events constituting each of these overlapping rather than discrete stages of wound healing increases its complexity and the necessity to understand it. The contrasting mechanisms of wound healing employed by mouse (via wound contraction) and humans (via reepithelialization) puts forth the need of a model closely mimicking human wound-healing and hence comes the applicability of the mouse excisional wound splinting model. Use of silicone-based splints has demonstrated their effectiveness in aptly resembling the human reepithelialization mediated wound healing by preventing contraction during healing. The rising popularity of nanofiber-based treatments for wound healing through sustained release of factors/molecules promoting wound closure can be potentially implemented in association with this model to determine its efficacy in wound management in a more humanized way.

Aim: The aim of this study was to develop a nanofiber-based dressing capable of local sustained delivery of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and augmenting human CAMP induction. Materials & methods: Nanofibrous wound dressings containing 1,25(OH)2D3 were successfully prepared by electrospinning, which were examined in vitro, in vivo and ex vivo. Results: 1,25(OH)2D3 was successfully loaded into nanofibers with encapsulation efficiency larger than 90%. 1,25(OH)2D3 showed a sustained release from nanofibers over 4 weeks. Treatment of U937 and HaCaT cells with 1,25(OH)2D3-loaded poly(ϵ-caprolactone) nanofibers significantly induced hCAP18/LL37 expression in monocytes and keratinocytes, skin wounds of humanized transgenic mice and artificial wounds of human skin explants. Conclusion: 1,25(OH)2D3 containing nanofibrous dressings could enhance innate immunity by inducing antimicrobial peptide production.

Understanding the underlying molecular mechanisms involved in the formation of cutaneous malignant melanoma is critical for improved diagnosis and treatment. Keratinocytic nuclear receptor Retinoid X Receptor α (RXRα) has a protective role against melanomagenesis and is involved in the regulation of keratinocyte and melanocyte homeostasis subsequent acute ultraviolet (UV) irradiation. We generated a trigenic mouse model system (RXRα ep−/− | Tyr-NRAS Q61K | CDK4 R24C/R24C ) harboring an epidermal knockout of Retinoid X Receptor α (RXRα ep−/− ), combined with oncogenic NRAS Q61K (constitutively active RAS) and activated CDK4 R24C/R24C (constitutively active CDK4). Those mice were subjected to a single neonatal dose of UVB treatment and the role of RXR α was evaluated by characterizing the molecular and cellular changes that took place in the untreated and UVB treated trigenic RXRα ep−/− mice compared to the control mice with functional RXRα. Here we report that the trigenic mice develops spontaneous melanoma and exposure to a single neonatal UVB treatment reduces the tumor latency in those mice compared to control mice with functional RXRα. Melanomas from the trigenic RXRα ep−/− mice are substantial in size, show increased proliferation, exhibit increased expression of malignant melanoma markers and exhibit enhanced vascularization. Altered expression of several biomarkers including increased expression of activated AKT, p21 and cyclin D1 and reduced expression of pro-apoptotic marker BAX was observed in the tumor adjacent normal (TAN) skin of acute ultraviolet B treated trigenic RXRα ep−/− mice. Interestingly, we observed a significant increase in p21 and Cyclin D1 in the TAN skin of un-irradiated trigenic RXRα ep−/− mice, suggesting that those changes might be consequences of loss of functional RXRα in the melanoma microenvironment. Loss of RXRα in the epidermal keratinocytes in combination with oncogenic NRAS Q61K and CDK4 R24C/R24C mutations in trigenic mice led to significant melanoma invasion into the draining lymph nodes as compared to controls with functional RXRα. Our study demonstrates the protective role of keratinocytic RxRα in (1) suppressing spontaneous and acute UVB-induced melanoma, and (2) preventing progression of the melanoma to malignancy in the presence of driver mutations like activated CDK4 R24C/R24C and oncogenic NRAS Q61K .

In humans and other primates, 1,25(OH)(2)vitamin D3 regulates the expression of the cathelicidin antimicrobial peptide (CAMP) gene via toll-like receptor (TLR) signaling that activates the vitamin D pathway. Mice and other mammals lack the vitamin D response element (VDRE) in their CAMP promoters. To elucidate the biological importance of this pathway, we generated transgenic mice that carry a genomic DNA fragment encompassing the entire human CAMP gene and crossed them with Camp knockout (KO) mice. We observed expression of the human transgene in various tissues and innate immune cells. However, in mouse CAMP transgenic macrophages, TLR activation in the presence of 25(OH)D(3) did not induce expression of either CAMP or CYP27B1 as would normally occur in human macrophages, reinforcing important species differences in the actions of vitamin D. Transgenic mice did show increased resistance to colonization by Salmonella typhimurium in the gut. Furthermore, the human CAMP gene restored wound healing in the skin of Camp KO mice. Topical application of 1,25(OH)(2)vitamin D(3) to the skin of CAMP transgenic mice induced CAMP expression and increased killing of Staphylococcus aureus in a wound infection model. Our model can help elucidate the biological importance of the vitamin D-cathelicidin pathway in both pathogenic and non-pathogenic states.

INTRODUCTION-: Atopic dermatitis (AD) is a multifactorial ailment associated with barrier breach and intense systemic inflammation. Several studies over the years have shown the complex interplay of a large number of factors in governing the progression and outcome of AD. In addition to the diverse types of AD resulting due to variation in the intrinsic mechanisms giving rise to AD such as single nucleotide polymorphisms (SNPs), epigenetic alterations or transcriptional changes, extrinsic factors such as age, ancestry, ethnicity, immunological background of the subject, the interactions of the subject with environmental stimuli and existing microbiome in the periphery surrounding the subject account for further heterogeneity in the clinical manifestations of the disease. AREAS COVERED-: Here we have selectively discussed transcriptional regulation of genes associated with skin lipid metabolism in the context of AD. Transcriptional control and transcriptomic changes are just one face of this multifaceted disease known to affect humans and a detailed study concerning those will enable us to develop targeted therapies to deal with the disease. EXPERT OPINION-: Large-scale integration of different omics approaches (genomics, epigenomics, transcriptomics, lipidomics, proteomics, metabolomics, effect of exposome) will help identify the potential candidate gene(s) associated with the development of various endotypes of AD.

Introduction: Lipidomics is the large-scale profiling and characterization of lipid species in a biological system using mass spectrometry. The skin barrier is mainly comprised of corneocytes and a lipid-enriched extracellular matrix. The major skin lipids are ceramides, cholesterol and free fatty acids (FFA). Lipid compositions are altered in inflammatory skin disorders with disrupted skin barrier such as atopic dermatitis (AD).Areas covered: Here we discuss some of the recent applications of lipidomics in human skin biology and in inflammatory skin diseases such as AD, psoriasis and Netherton syndrome. We also review applications of lipidomics in human skin equivalent and in pre-clinical animal models of skin diseases to gain insight into the pathogenesis of the skin disease.Expert commentary: Skin lipidomics analysis could be a fast, reliable and noninvasive tool to characterize the skin lipid profile and to monitor the progression of inflammatory skin diseases such as AD.

AIM: The aim of this study was to develop a nanofiber-based dressing capable of local sustained delivery of 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and augmenting human CAMP induction. MATERIALS & METHODS: Nanofibrous wound dressings containing 1,25(OH)(2)D(3) were successfully prepared by electrospinning, which were examined in vitro, in vivo and ex vivo. RESULTS: 1,25(OH)(2)D(3) was successfully loaded into nanofibers with encapsulation efficiency larger than 90%. 1,25(OH)(2)D(3) showed a sustained release from nanofibers over 4 weeks. Treatment of U937 and HaCaT cells with 1,25(OH)(2)D(3)-loaded poly(ϵ-caprolactone) nanofibers significantly induced hCAP18/LL37 expression in monocytes and keratinocytes, skin wounds of humanized transgenic mice and artificial wounds of human skin explants. CONCLUSION: 1,25(OH)(2)D(3) containing nanofibrous dressings could enhance innate immunity by inducing antimicrobial peptide production.

ATP-dependent chromatin remodeling complexes are involved in chromatin remodeling thereby controlling gene expression. These multi-subunit containing complexes contain an ATPase of the SNF2 family that hydrolyzes ATP in order to modify or reshape histone-DNA interactions within nucleosomes. Several subfamilies of the SNF2 family have been identified in different species based on the status of their catalytic ATPase subunit. All of the SNF2 subfamily members, including SWI2/SNF2 (BRG1/BRM), ISWI and CHD/Mi-2β, play critical roles in the maintenance of epidermal homeostasis. The BRG1 chromatin remodeler is required for maintenance of bulge stem cells, hair cycling, normal skin homeostasis, and repair and regeneration processes. ACTL6a (actin-like 6a) modulates the SWI/SNF complex to suppress differentiation in the epidermis. Realignment of the epidermal differentiation complex (EDC) locus, which occurs before activation of EDC genes that drive keratinocyte terminal differentiation, is coordinated by p63 by directly regulating the expression of BRG1. The combined effects of p63 and BRG1 control higher order chromatin remodeling, 3D-genomic organization and efficient expression of EDC genes in epidermal precursor cells during epidermal morphogenesis. BRG1 also suppresses p27kip1, allowing self-renewal of hair follicle bulge cells, and recruits the transcription factor NF-kB, which in turn activates Shh in matrix cells, promoting proliferation. Finally, Shh signaling through Gli activates BRG1 in bulge cells. Hence, BRG1 is necessary for Shh expression in both matrix and bulge compartments, and for hair regeneration and skin repair post-wounding. Mice with deletion of Mi-2β of the SNF2 family in the epidermis die perinatally and display severalphenotypes that differ between dorsal and ventral skin, suggesting spatio-temporal control of gene expression by Mi-2β in the epidermis. Mi-2β is also important for hair follicle morphogenesis and is necessary to reprogram epidermal basal cells to a hair follicle fate. Non-melanoma skin cancers in humans display mutations in the Brm gene following ultraviolet (UV) irradiation, and BRM normally protects epidermal cells from UV irradiation-induced hyper-proliferation, even in the presence of a partial loss of p53, thereby establishing its role as a tumor suppressor.

The epidermal permeability barrier (EPB) prevents organisms from dehydration and infection. The transcriptional regulation of EPB development is poorly understood. We demonstrate here that transcription factor COUP-TF-interacting protein 1 (CTIP1/BCL11A; hereafter CTIP1) is highly expressed in the developing murine epidermis. Germline deletion of Ctip1 (Ctip1−/−) results in EPB defects accompanied by compromised epidermal differentiation, drastic reduction in profilaggrin processing, reduced lamellar bodies in granular layers and significantly altered lipid composition. Transcriptional profiling of Ctip1−/− embryonic skin identified altered expression of genes encoding lipid-metabolism enzymes, skin barrier-associated transcription factors and junctional proteins. CTIP1 was observed to interact with genomic elements within the regulatory region of the gene encoding the differentiation-associated gene, Fos-related antigen2 (Fosl2) and lipid-metabolism-related gene, Fatty acid elongase 4 (Elvol4), and the expression of both was altered in Ctip1−/− mice. CTIP1 appears to play a role in EPB establishment of via direct or indirect regulation of a subset of genes encoding proteins involved in epidermal differentiation and lipid metabolism. These results identify potential, CTIP1-regulated avenues for treatment of skin disorders involving EBP defects.

Table S1. Antibodies used for immuno-histochemical staining and immuno-blotting with details including the host, detection for-, the source, application and the dilution used. Table S2. PCR primers used for genotyping of mouse lines with the forward and reverse primers for K14-Cre, RXRα and CDK4, their sequences and the expected band sizes. Additional Methods. (DOCX 19 kb)

Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV-induced melanocyte homeostasis. Selective ablation of endothelin-1 (EDN1) in murine epidermis (EDN1(ep-/-) ) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non-cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte-derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV-induced melanocyte activation and recapitulated the phenotype seen in EDN1(ep-/-) mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV-induced melanocyte homeostasis in vivo.

Summary Epidermal morphogenesis results from a delicate balance between keratinocyte proliferation and differentiation, and this balance is perturbed upon deletion of transcription factor Ctip2. Here we demonstrate that Ctip2, in a cell autonomous manner, controls keratinocyte proliferation and cytoskeletal organization, and regulates the onset and maintenance of differentiation in keratinocytes in culture. Ctip2 integrates keratinocyte proliferation and the switch to differentiation by directly and positively regulating EGFR transcription in proliferating cells and Notch1 transcription in differentiating cells. In proliferative cells, the EGFR promoter is occupied by Ctip2, whereas Ctip2 is only recruited to the Notch1 promoter under differentiating conditions. Activation of EGFR signaling downregulates Ctip2 at the transcript level, whereas high calcium signaling triggers SUMOylation, ubiquitination and proteasomal degradation of Ctip2 at the protein level. Together, our findings demonstrate a novel mechanism(s) of Ctip2-mediated, coordinated control of epidermal proliferation and terminal differentiation, and identify a pathway of negative feedback regulation of Ctip2 during epidermal development.

Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part, through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. We show that absence of keratinocytic RXRα in combination with mutant Cdk4 generated cutaneous melanoma that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf2) were elevated in skin of bigenic mice, while Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes. Significance Malignant melanoma continues to evade modern curative efforts as a result of the complex and elusive nature of metastatic tumors. We demonstrated that RXRα expression is lost in epidermal keratinocytes during melanoma progression in humans. We have presented evidence for the association of keratinocytic nuclear receptor RXRα-mediated paracrine/juxtacrine signaling and the malignant transformation of melanocytic tumors. The present work highlights cooperative effects of aberrant keratinocytic signaling and activated Cdk4 in melanoma metastasis. This mouse model will allow greater understanding of the mechanisms responsible for transition towards a malignant phenotype. Manipulation of these receptor-mediated signaling pathways could hold therapeutic value when combating metastatic disease.

Understanding the molecular mechanisms behind formation of melanoma, the deadliest form of skin cancer, is crucial for improved diagnosis and treatment. One key is to better understand the cross-talk between epidermal keratinocytes and pigment-producing melanocytes. Here, using a bigenic mouse model system combining mutant oncogenic NRASQ61K (constitutively active RAS) or mutant activated CDK4R24C/R24C (prevents binding of CDK4 by kinase inhibitor p16INK4A) with an epidermis-specific knockout of the nuclear retinoid X receptor alpha (RXRαep−/−) results in increased melanoma formation after chronic ultraviolet-B (UVB) irradiation compared with control mice with functional RXRα. Melanomas from both groups of bigenic RXRαep−/− mice are larger in size with higher proliferative capacity, and exhibit enhanced angiogenic properties and increased expression of malignant melanoma markers. Analysis of tumor adjacent normal skin from these mice revealed altered expression of several biomarkers indicative of enhanced melanoma susceptibility, including reduced expression of tumor suppressor p53 and loss of PTEN, with concomitant increase in activated AKT. Loss of epidermal RXRα in combination with UVB significantly enhances invasion of melanocytic cells to draining lymph nodes in bigenic mice expressing oncogenic NRASQ61K compared with controls with functional RXRα. These results suggest a crucial role of keratinocytic RXRα to suppress formation of UVB-induced melanomas and their progression to malignant cancers in the context of driver mutations such as activated CDK4R24C/R24C or oncogenic NRASQ61K. Implications: These findings suggest that RXRα may serve as a clinical diagnostic marker and therapeutic target in melanoma progression and metastasis. Mol Cancer Res; 13(1); 186–96. ©2014 AACR.

Cutaneous wound healing assay is important to address many key questions including (1) migration ability of different cells; (2) communication between the different cell types such as keratinocytes, fibroblasts, and immune cells; (3) understanding the cell-autonomous and non-cell-autonomous function(s) of the different cells; and (4) gene regulation in healing processes. Wound healing studies can be used to test new treatment modalities, function of new drugs/compounds, and stem cell-based therapies on the different stages of healing and for accelerating wound healing in patients with compromised healing. In this chapter, we have described a simple step-by-step protocol to generate full-thickness cutaneous wounds in the dorsal skin of mice, followed by collecting the post-wounding biopsied materials on specific days for histological and immunohistochemical analyses and for RNA and protein extractions.

Cutaneous wound healing assay is important to address many key questions including (1) migration ability of different cells; (2) communication between the different cell types such as keratinocytes, fibroblasts, and immune cells; (3) understanding the cell-autonomous and non-cell-autonomous function(s) of the different cells; and (4) gene regulation in healing processes. Wound healing studies can be used to test new treatment modalities, function of new drugs/compounds, and stem cell-based therapies on the different stages of healing and for accelerating wound healing in patients with compromised healing. In this chapter, we have described a simple step-by-step protocol to generate full-thickness cutaneous wounds in the dorsal skin of mice, followed by collecting the post-wounding biopsied materials on specific days for histological and immunohistochemical analyses and for RNA and protein extractions.