Mouse Epidermis Research Articles

A G1 Sizer Coordinates Growth and Division in the Mouse Epidermis.

Cell size homeostasis is often achieved by coupling cell-cycle progression to cell growth. Growth has been shown to drive cell-cycle progression in bacteria and yeast through "sizers," wherein cells of varying birth size divide at similar final sizes [1-3], and "adders," wherein cells increase in size a fixed amount per cell cycle [4-6]. Intermediate control phenomena are also observed, and even the same organism can exhibit different control phenomena depending on growth conditions [2, 7, 8]. Although studying unicellular organisms in laboratory conditions may give insight into their growth control in the wild, this is less apparent for studies of mammalian cells growing outside the organism. Sizers, adders, and intermediate phenomena have been observed invitro [9-12], but it is unclear how this relates to mammalian cell proliferation invivo. To address this question, we analyzed time-lapse images of the mouse epidermis taken over 1week during normal tissue turnover [13]. We quantified the 3D volume growth and cell-cycle progression of single cells within the mouse skin. In dividing epidermal stem cells, we found that cell growth is coupled to division through a sizer operating largely in the G1 phase of the cell cycle. Thus, although the majority of tissue culture studies have identified adders, our analysis demonstrates that sizers are important invivo and highlights the need to determine their underlying molecular origin.

Skin permeability barrier formation by the ichthyosis-causative gene FATP4 through formation of the barrier lipid ω-O-acylceramide

The epidermis-specific lipid acylceramide plays a pivotal role in the formation of the permeability barrier in the skin; abrogation of its synthesis causes the skin disorder ichthyosis. However, the acylceramide synthetic pathway has not yet been fully elucidated: Namely, the acyl-CoA synthetase (ACS) involved in this pathway remains to be identified. Here, we hypothesized it to be encoded by FATP4/ACSVL4, the causative gene of ichthyosis prematurity syndrome (IPS). In vitro experiments revealed that FATP4 exhibits ACS activity toward an ω-hydroxy fatty acid (FA), an intermediate of the acylceramide synthetic pathway. Fatp4 knockout (KO) mice exhibited severe skin barrier dysfunction and morphological abnormalities in the epidermis. The total amount of acylceramide in Fatp4 KO mice was reduced to ∼10% of wild-type mice. Decreased levels and shortening of chain lengths were observed in the saturated, nonacylated ceramides. FA levels were not decreased in the epidermis of Fatp4 KO mice. The expression levels of the FA elongase Elovl1 were reduced in Fatp4 KO epidermis, partly accounting for the reduction and shortening of saturated, nonacylated ceramides. A decrease in acylceramide levels was also observed in human keratinocytes with FATP4 knockdown. From these results, we conclude that skin barrier dysfunction observed in IPS patients and Fatp4 KO mice is caused mainly by reduced acylceramide production. Our findings further elucidate the molecular mechanism governing acylceramide synthesis and IPS pathology.

Phosphatase Regulator NIPP1 Restrains Chemokine-Driven Skin Inflammation

Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a ubiquitously expressed nuclear protein that regulates functions of protein serine/threonine phosphatase-1 in cell proliferation and lineage specification. The role of NIPP1 in tissue homeostasis is not fully understood. This study shows that the selective deletion of NIPP1 in mouse epidermis resulted in epidermal hyperproliferation, a reduced adherence of basal keratinocytes, and a gradual decrease in the stemness of hair follicle stem cells, culminating in hair loss. This complex phenotype was associated with chronic sterile skin inflammation and could be partially rescued by dexamethasone treatment. NIPP1-deficient keratinocytes massively expressed proinflammatory chemokines and immunomodulatory proteins in a cell-autonomous manner. Chemokines subsequently induced the recruitment and activation of immune cells, in particular conventional dendritic cells and Langerhans cells, accounting for the chronic inflammation phenotype. The data identifies NIPP1 as a key regulator of epidermal homeostasis and as a potential target for the treatment of inflammatory skin diseases.

The Vitamin D Receptor as Tumor Suppressor in Skin.

Cutaneous malignancies including melanomas and keratinocyte carcinomas (KC) are the most common types of cancer, occurring at a rate of over one million per year in the United States. KC, which include both basal cell carcinomas and squamous cell carcinomas, are substantially more common than melanomas and form the subject of this chapter. Ultraviolet radiation (UVR), both UVB and UVA, as occurs with sunlight exposure is generally regarded as causal for these malignancies, but UVB is also required for vitamin D synthesis in the skin. Keratinocytes are the major cell in the epidermis. These cells not only produce vitamin D but contain the enzymatic machinery to metabolize vitamin D to its active metabolite, 1,25(OH)2D, and express the receptor for this metabolite, the vitamin D receptor (VDR). This allows the cell to respond to the 1,25(OH)2D that it produces. Based on our own data and that reported in the literature, we conclude that vitamin D signaling in the skin suppresses UVR-induced epidermal tumor formation. In this chapter we focus on four mechanisms by which vitamin D signaling suppresses tumor formation. They are inhibition of proliferation/stimulation of differentiation with discussion of the roles of hedgehog, Wnt/β-catenin, and hyaluronan/CD44 pathways in mediating vitamin D regulation of proliferation/differentiation, regulation of the balance between oncogenic and tumor suppressor long noncoding RNAs, immune regulation, and promotion of DNA damage repair (DDR).

Keratinocyte-specific ablation of Mcpip1 impairs skin integrity and promotes local and systemic inflammation.

MCPIP1 (Regnase-1, encoded by the ZC3H12A gene) regulates the mRNA stability of several inflammatory cytokines. Due to the critical role of this RNA endonuclease in the suppression of inflammation, Mcpip1 deficiency in mice leads to the development of postnatal multiorgan inflammation and premature death. Here, we generated mice with conditional deletion of Mcpip1 in the epidermis (Mcpip1EKO). Mcpip1 loss in keratinocytes resulted in the upregulated expression of transcripts encoding factors related to inflammation and keratinocyte differentiation, such as IL-36α/γ cytokines, S100a8/a9 antibacterial peptides, and Sprr2d/2h proteins. Upon aging, the Mcpip1EKO mice showed impaired skin integrity that led to the progressive development of spontaneous skin pathology and systemic inflammation. Furthermore, we found that the lack of epidermal Mcpip1 expression impaired the balance of keratinocyte proliferation and differentiation. Overall, we provide evidence that keratinocyte-specific Mcpip1 activity is crucial for the maintenance of skin integrity as well as for the prevention of excessive local and systemic inflammation. KEY MESSAGES: Loss of murine epidermal Mcpip1 upregulates transcripts related to inflammation and keratinocyte differentiation. Keratinocyte Mcpip1 function is essential to maintain the integrity of skin in adult mice. Ablation of Mcpip1 in mouse epidermis leads to the development of local and systemic inflammation.

Hyaluronic Acid-Based Dissolving Microneedle Patch Loaded with Methotrexate for Improved Treatment of Psoriasis.

Methotrexate (MTX) is one of the first-line treatments for moderate to severe psoriasis, while the side effects caused by injection and oral administration of MTX greatly restrict its clinical application. Transdermal drug delivery offers a desirable alternative to the conventional approaches, but the performances of the currently available skin penetration enhancement techniques are not so satisfactory. To address these limitations, we developed a dissolving microneedle (MN) patch made of hyaluronic acid (HA) with excellent water solubility, biocompatibility, biodegradability, and mechanical properties. The amount of MTX encapsulated in the needles of the patch could be controlled during the fabrication process for precise dosage. Interestingly, the MTX-loaded MNs successfully penetrated imiquimod (IMQ)-induced thickened epidermis in mice and delivered the drug intralesionally. Meanwhile, fast dissolution of HA endowed the MNs with operability for patients. We found that the MTX-loaded MNs not only showed well-maintained inhibitory effect in vitro but also alleviated the psoriasis-like skin inflammation in mice. Moreover, the MTX-loaded MNs were significantly more efficacious than taking the same dose of drug orally. Consequently, a higher oral dose of MTX was required for a comparable amelioration, which in turn increased its systemic toxicity. Taken together, the proposed MTX-loaded dissolving MN patch strategy provides a new opportunity for efficient and safe treatment of psoriasis.

Homeostatic Function of Dermokine in the Skin Barrier and Inflammation

Dermokine is a chiefly skin-specific secreted glycoprotein localized in the upper epidermis, and its family consists of three splice variants in mice and five in humans. To investigate the pathophysiological impact of dermokine, we generated mice deficient for two (βγ) or all dermokine isoforms (αβγ). Both variants, especially dermokine αβγ-deficient mice exhibited scale and wrinkle formation resembling ichthyosis accompanied by transepidermal water imbalance at the neonatal stage. Several dermokine αβγ-deficient mice died by postnatal day 21 when reared under low humidity. Moreover, the cornified envelope was vulnerable, and skin barrier lipid ceramides were reduced in the epidermis of dermokine αβγ-deficient mice. cDNA microarray and quantitative reverse transcriptase-PCR assays of the epidermis revealed the upregulation of small proline-rich protein and late cornified envelope family members, as well as antimicrobial peptides in the dermokine αβγ-deficient mice. These barrier gene signatures were similar to that seen in psoriasis, whereas recent studies demonstrated that congenital ichthyosis has gene profiles resembling psoriasis. In line with these findings, adult dermokine αβγ-deficient mice exhibited aggravated phenotypes in psoriasis-like dermatitis models but not in allergic dermatitis models. Dermokine may play a regulatory role in inflammatory dyskeratotic diseases, such as congenital ichthyosis and psoriasis, in the crosstalk between barrier dysfunction and inflammation.

Nitrogen mustard prevents transport of Fra-1 into the nucleus to promote c-Fos- and FosB-dependent IL-8 induction in injured mouse epidermis

Transcription factor activator protein (AP)-1 can be activated in nitrogen-mustard-injured mouse skin, and is thought to participate in the inflammatory response. AP-1 consists of homo- or heterodimers of Fos [c-Fos, Fos-B, fos-related antigen (Fra)-1 and Fra-2] and Jun (c-Jun, JunB and JunD) family members, and information about their expression, location and function are still unclear. In nitrogen-mustard-exposed mouse skin, we found p-ERK activation increased Fra-1 and FosB. Unlike the nucleus location of c-Fos and FosB, Fra-1 and Fra-2 were mainly expressed in the cytoplasm. In nitrogen-mustard-exposed cultured immortalized human keratinocytes (HaCaT cells), Fra-1 in the nucleus functioned as an inhibitor of inflammatory cytokine interleukin (IL)-8. Co-immunoprecipitation showed that Fra-1 formed dimers with IL-8 transcription factors c-Jun, JunB and JunD. Fra-1 depletion increased c-Fos and FosB in the nucleus, accompanied by increased heterodimers of c-Fos/c-Jun, c-Fos/JunB, c-Fos/JunD, and FosB/JunB. In conclusion, Fra-1 trapped in the cytoplasm after nitrogen mustard exposure might be a driving force for IL-8 over-expression in injured skin.

Wavelength- and Tissue-dependent Variations in the Mutagenicity of Cyclobutane Pyrimidine Dimers in Mouse Skin.

The cyclobutane pyrimidine dimer (CPD) is a main mutagenic photolesion in DNA produced by UVR. We previously studied the wavelength-dependent kinetics of mutation induction efficiency using monochromatic UVR sources and transgenic mice developed for mutation assay and established the action spectra of UVR mutagenicity in the mouse epidermis and dermis. Here, we further established the action spectra of CPD and pyrimidine(6-4)pyrimidone photoproduct formation in the same tissues and in naked DNA using the same sources and mouse strain. Quantitative ELISA helped us estimate the photolesion formation efficiencies on a molecule-per-nucleotide basis. Using these action spectra, we confirmed that the UVR mutation mostly depends on CPD formation. Moreover, the mutagenicity of a CPD molecule (CPD mutagenicity) was found to vary by wavelength, peaking at approximately 313nm in both the epidermis and dermis with similar wavelength-dependent patterns. Thus, the CPD formation efficiency is a main determinant of UVR mutagenicity in mouse skin, whereas a wavelength-dependent variation in the qualitative characteristics of CPD molecules also affects the mutagenic consequences of UVR insults. In addition, the CPD mutagenicity was always higher in the epidermis than in the dermis, suggesting different cellular responses to UVR between the two tissues irrespective of the wavelength.

Preliminary Results of Betulinic Acid-Loaded Magnetoliposomes - a Potential Approach to Increase Therapeutic Efficacy in Melanoma

Magnetoliposomes were placed into the biomedical spotlight due to their unique structure, preserving also magnetic features valuable for biomedical applications. The present study provides a preliminary evidence of the cytotoxicity induced by betulinic acid-loaded magnetoliposomes (BA-Fe3O4@Lip) on two melanoma cell lines - A375 and B164A5 cells and one healthy cell line - mice epidermis JB6 Cl 41-5a cells, augmented by references to potential biomedical applications. BA-Fe3O4@Lip showed significant cytotoxicity on the melanoma cell lines, compared with the blank liposomal structures. In addition, the healthy cell line displayed good viability rate after exposure to BA-Fe3O4@Lip.

Aryl Hydrocarbon Receptor Directly Regulates Artemin Gene Expression.

Transgenic mice expressing a constitutively active form of the aryl hydrocarbon receptor in keratinocytes (AhR-CA mice) develop severe dermatitis that substantially recapitulates the pathology of human atopic dermatitis. The neurotrophic factor artemin (Artn) is highly expressed in the epidermis of AhR-CA mice and causes hypersensitivity to itch (alloknesis) by elongating nerves into the epidermis. However, whether the Artn gene is regulated directly by AhR or indirectly through complex regulation associated with AhR remains unclear. To this end, we previously conducted chromatin immunoprecipitation-sequencing analyses of the Artn locus and found a xenobiotic response element (XRE) motif located far upstream (52 kb) of the gene. Therefore, in this study, we addressed whether the XRE actually regulates the Artn gene expression by deleting the region containing the motif. We generated two lines of ArtnΔXRE mice. In the mouse epidermis, inducible expression of the Artn gene by the AhR agonist 3-methylcholanthrene was substantially suppressed compared to that in wild-type mice. Importantly, in AhR-CA::ArtnΔXRE mice, Artn expression was significantly suppressed, and alloknesis was improved. These results demonstrate that the Artn gene is indeed regulated by the distal XRE-containing enhancer, and alloknesis in AhR-CA mice is provoked by the AhR-mediated direct induction of the Artn gene.

Fatty acid transport protein 4 is required for incorporation of saturated ultralong-chain fatty acids into epidermal ceramides and monoacylglycerols

Fatty acid transport protein 4 (FATP4) is an acyl-CoA synthetase that is required for normal permeability barrier in mammalian skin. FATP4 (SLC27A4) mutations cause ichthyosis prematurity syndrome, a nonlethal disorder. In contrast, Fatp4−/− mice die neonatally from a defective barrier. Here we used electron microscopy and lipidomics to characterize defects in Fatp4−/− mice. Mutants showed lamellar body, corneocyte lipid envelope, and cornified envelope abnormalities. Lipidomics identified two lipids previously speculated to be present in mouse epidermis, sphingosine β-hydroxyceramide and monoacylglycerol; mutants displayed decreased proportions of these and the two ceramide classes that carry ultralong-chain, amide-linked fatty acids (FAs) thought to be critical for barrier function, unbound ω-O-acylceramide and bound ω-hydroxyceramide, the latter constituting the major component of the corneocyte lipid envelope. Other abnormalities included elevated amounts of sphingosine α-hydroxyceramide, phytosphingosine non-hydroxyceramide, and 1-O-acylceramide. Acyl chain length alterations in ceramides also suggested roles for FATP4 in esterifying saturated non-hydroxy and β-hydroxy FAs with at least 25 carbons and saturated or unsaturated ω-hydroxy FAs with at least 30 carbons to CoA. Our lipidomic analysis is the most thorough such study of the Fatp4−/− mouse skin barrier to date, providing information about how FATP4 can contribute to barrier function by regulating fatty acyl moieties in various barrier lipids.

Pathogenic CD8+ Epidermis-Resident Memory T Cells Displace Dendritic Epidermal T Cells in Allergic Dermatitis

The skin is our interface with the outside world, and consequently it is exposed to a wide range of microbes and allergens. Recent studies have indicated that allergen-specific skin-resident memory T (TRM) cells play a role in allergic contact dermatitis (ACD). However, the composition and dynamics of the epidermal T-cell subsets during ACD are not known. Here we show that exposure of the skin to the experimental contact allergen DNFB results in a displacement of the normally occurring dendritic epidermal T cells (DETC) concomitant with an accumulation of epidermal CD8+CD69+CD103+ TRM cells in mice. By studying knockout mice, we provide evidence that CD8+ T cells are required for the displacement of the DETC and that DETC are not required for recruitment of CD8+ TRM cells to the epidermis following allergen exposure. We demonstrate that the magnitude of the allergic reaction correlates with the number of CD8+ epidermal TRM cells, which again correlates with allergen dose and number of allergen exposures. Finally, in an attempt to elucidate why CD8+ epidermal TRM cells persist in the epidermis, we show that CD8+ epidermal TRM cells have a higher proliferative capability and are bioenergetically more stable, displaying a higher spare respiratory capacity than DETC.

463 Hypoxia-inducible factor-1α play a critical role in UVB-induced tumorigenesis by affecting DNA repair capacity and oxidative stress

Hypoxia inducible factor (HIF) 1 is a heterodimeric transcription factor consisting of α and β subunits. The β subunit is constitutively expressed, while the expression of the α subunit is regulated by the relative concentration of oxygen. When stabilized, HIF-1α play a pivotal role in many biological processes by affecting the expression of hundreds of genes involved in angiogenesis, cell growth and apoptosis, cell adhesion and migration, the energy metabolism and DNA repair. Others and we showed that HIF-1α is constitutively expressed in mouse and human epidermis and that HIF-1α plays an important role in the skin, especially in local and systemic adaptation to environmental stresses including ultraviolet (UV) radiation. However, little information is available about its role in photocarcinogenesis. Using a multistage model of UVB radiation-induced skin cancer, we show that the knockout of Hif-1α in the epidermis prevents tumorigenesis, but at the same time triggers the formation of hyperkeratotic plaques. Our results indicate that the absence of oncogenic transformation in Hif-1α-ablated mice is related to increased DNA repair in keratinocytes. Indeed, Hif-1α ablation in the epidermis leads to more proficient removal of UV-induced DNA damage via boosting DNA repair machinery. Consequently, impairing the DNA repair machinery by ablating xeroderma pigmentosum C restores the UVB-induced neoplastic transformation of Hif-1α-ablated keratinocytes. Concerning the formation of hyperkeratotic plaques in irradiated K-Hif-1-/- mice, a causative role of the disturbance of the epidermal redox balance was found. Accordingly, the development of hyperkeratotic plaques is blocked by chronic antioxidant treatment. Altogether, our results show that the deletion of Hif-1α in keratinocytes has two profound effects on the skin’s responses to UVB exposure: it leads to decreased tumorigenesis by promoting DNA repair efficiency; and it causes skin hyperkeratosis by affecting the redox regulation machinery in keratinocytes.

348 Homeostatic functions of dermokine in skin barrier and innate immunity

Dermokine (DMKN) is a secreted glycoprotein and its family members consist of five splicing variants (α, β, γ, δ and ε)in human and three variants (α, β and γ) in mice. Especially, α, β and γ isoforms are expressed in the upper epidermis. DMKN α represents the C-terminal end of β isoform, which is responsible for cytokine-like action and inhibits phosphorylation of ERK, leading to suppression of aberrant keratinocyte proliferation in vitro. To investigate the DMKN function in vivo, we generated mice deficient for all the three DMKN α, β and γ. The mice exhibited dry scaly skinand deep wrinkle formation with tail constriction and autoamputation in the neonatal stage, contributing to increased TEWL. Some of them showed growth retardation and death at low humidity. The skin pathology revealed thickening of corneal and granular layers with increased proteolytic activity in the corneal layers and cornified envelope fragility. cDNA microarray and quantitative RT-PCR assays using the mouse epidermis disclosed upregulation of small proline-rich protein and late CE family members, clustering genes for CE assembly, in parallel with increased levels of antimicrobial peptides. DMKN deficiency did not affect the development of DNFB- or FITC-induced contact hypersensitivity models, as well as OVA- or MC903-induced atopic dermatitis models in adult mice. However, DMKN-deficient mice showed exacerbation of psoriasiform dermatitis models induced by topical imiquimod application or intradermal IL-23 injection. Augmented expression profile of TNF-α/IL-17/IL-23 and ELR+ CXC chemokines were characteristic for psoriasis. Thus, DMKN possesses dual biological properties for skin barrier maintenance and regulatory innate immune response associated with psoriasis.

Epidermal mammalian target of rapamycin complex 2 controls lipid synthesis and filaggrin processing in epidermal barrier formation

Perturbation of epidermal barrier formation will profoundly compromise overall skin function, leading to a dry and scaly, ichthyosis-like skin phenotype that is the hallmark of a broad range of skin diseases, including ichthyosis, atopic dermatitis, and a multitude of clinical eczema variants. An overarching molecular mechanism that orchestrates the multitude of factors controlling epidermal barrier formation and homeostasis remains to be elucidated. Here we highlight a specific role of mammalian target of rapamycin complex 2 (mTORC2) signaling in epidermal barrier formation. Epidermal mTORC2 signaling was specifically disrupted by deleting rapamycin-insensitive companion of target of rapamycin (Rictor), encoding an essential subunit of mTORC2 in mouse epidermis (epidermis-specific homozygous Rictor deletion [RicEKO] mice). Epidermal structure and barrier function were investigated through a combination of gene expression, biochemical, morphological and functional analysis in RicEKO and control mice. RicEKO newborns displayed an ichthyosis-like phenotype characterized by dysregulated epidermal de novo lipid synthesis, altered lipid lamellae structure, and aberrant filaggrin (FLG) processing. Despite a compensatory transcriptional epidermal repair response, the protective epidermal function was impaired in RicEKO mice, as revealed by increased transepidermal water loss, enhanced corneocyte fragility, decreased dendritic epidermal T cells, and an exaggerated percutaneous immune response. Restoration of Akt-Ser473 phosphorylation in mTORC2-deficient keratinocytes through expression of constitutive Akt rescued FLG processing. Our findings reveal a critical metabolic signaling relay of barrier formation in which epidermal mTORC2 activity controls FLG processing and de novo epidermal lipid synthesis during cornification. Our findings provide novel mechanistic insights into epidermal barrier formation and could open up new therapeutic opportunities to restore defective epidermal barrier conditions.

Conditional Alox12b Knockout: Degradation of the Corneocyte Lipid Envelope in a Mouse Model of Autosomal Recessive Congenital Ichthyoses

Conditional Alox12b Knockout: Degradation of the Corneocyte Lipid Envelope in a Mouse Model of Autosomal Recessive Congenital Ichthyoses

Abstract 4647: Keratinocyte specific deletion of TWIST1 promotes differentiation and inhibits UVB-induced hyperproliferation

Abstract Non-melanoma skin cancers (NMSC) have one of the highest incidences in the United States with over 5 million diagnoses every year. The major risk factor for the development of NMSC is solar ultraviolet radiation consisting of both UVA and UVB. In epidermal keratinocytes, UV radiation results in DNA damage as well as deregulation of both proliferative and survival signaling pathways that contribute to tumorigenesis. The transcription factor TWIST1 has been reported to be essential for the formation and invasiveness of chemically-induced tumors in skin. TWIST1 deletion in mouse epidermis results in arrested cell cycle progression in response to treatment with the tumor promoter, TPA, which in turn prevents skin tumor growth in two-stage skin carcinogenesis assays. However, the impact of the keratinocyte specific TWIST1 deletion on skin carcinogenesis caused by UV radiation has not been clarified. In preliminary experiments, we found that primary mouse keratinocytes with TWIST1 knockout (KO) displayed an increase in the G2/M phase population after a single exposure to 20mJ/cm2 of UVB radiation. Accordingly, protein levels of cell cycle regulator p21 increased while those of pMDM2 decreased after UVB exposure. Moreover, we found that primary mouse keratinocytes with TWIST1 KO displayed an increase in apoptosis as shown by augmented Annexin V staining that corresponded with the reduction in levels of anti-apoptotic regulators Bcl-2 and Bcl-XL and concomitant upregulation of cleaved PARP and cleaved caspase-7 after UVB exposure. Furthermore, deletion of TWIST1 in skin keratinocytes in vivo (using K5-Cre x TWIST1flox/flox mice) led to a significant reduction in UVB-induced epidermal hyperproliferation compared to wild-type mice assessed by both Ki67 immunofluorescence staining as well as BrdU incorporation. Additionally, the protein level for p21 was increased while the levels of c-myc, and Cyclin B1 were reduced in the TWIST1 deficient keratinocytes in vivo. Interestingly, expression of differentiation markers such as Loricrin, Keratin-10, Sca-1, and transglutaminase-1 were also increased in the epidermis of TWIST1 deficient mice. These results from the in vivo model show that TWIST1 deletion results in an interruption of UVB-induced hyperproliferation via cell cycle arrest and induction of keratinocyte differentiation. Collectively, our findings indicate that the deficiency of TWIST1 has significant effects on the proliferation, survival and differentiation of keratinocytes; processes important for UVB skin tumor development. Future experiments will continue to assess the changes in oncogenic signaling in epidermal keratinocytes in response to UVB exposure and further investigate the impact of the deletion of TWIST1 on UV-induced skin carcinogenesis. Research supported by CPRIT 2018 Grant RP150247 and by CONACYT Fellowship 671661. Citation Format: Fernando Eguiarte-Solomon, Okkyung Rho, John DiGiovanni. Keratinocyte specific deletion of TWIST1 promotes differentiation and inhibits UVB-induced hyperproliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4647.

Abstract 2355: IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis

Abstract Our previous studies have shown that the absence of tumor suppressor IKKα leads to development of spontaneous mouse squamous cell carcinomas (SCCs) in the skin, lungs, and esophagus, which is partially mediated by aberrant epigenetic alterations and inflammation. However, it remains unknown whether there is a crosstalk between the above-mentioned mechanisms. As a part of the epigenetic machinery, miRNAs regulate many genes post-transcriptionally. Here we show that IKKα deletion in the mouse epidermis results in impaired miRNA processing with markedly reduced expression of ribonuclease Dicer, an enzyme required for miRNA maturation. We found that the lack of IKKα causes Dicer gene silencing through DNA methylation. As a result, the maturation of miR-203-3p, which is the most abundant miRNA in keratinocytes, is blocked in Ikkα-null keratinocytes. Next, we identified Myd88, an important component of innate immunity and inflammation, as one of miR-203-3p targets. The level of Myd88 expression is robustly increased in Ikkα-null keratinocytes due to the lack of miR-203-3p. We also observed increased activity of caspase 1 cleavage, an indicator of inflammasome activation, in Ikkα-null keratinocytes. Interestingly, Myd88 deletion or 5-aza-2’-deoxycytidine (DNA methyltransferase inhibitor) treatment rescued the survival of Ikkαf/f;K5Cre mice, which usually die within three weeks after birth. Ikkαf/f;Myd88f/f;K5Cre mice also develop much fewer spontaneous skin tumors. By analyzing a cohort of human head and neck SCCs from TCGA database, we found that Dicer expression is concurrently downregulated among 32% of patients with low IKKα expression. Taken together, our finding illustrated how IKKα loss mediates Dicer gene silencing, which turns off miRNA processing, including miR-203-3p maturation, and eventually triggers Myd88-dependent inflammation. Thus, our study revealed a novel IKKα/Dicer/miR-203-3p/Myd88 signaling loop, in which IKKα serves as a molecular switch linking microRNA biogenesis and inflammation in skin tumorigenesis. Citation Format: Feng Zhu, Jian Zhang, Jami Willette-Brown, Ling Su, Xiaolin Wu, Yinling Hu. IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2355.

Activation of a protumorigenic IFNγ/STAT1/IRF‐1 signaling pathway in keratinocytes following exposure to solar ultraviolet light

In this study, we evaluated the role of signal transducer and activator of transcription 1 (STAT1) in response to acute solar ultraviolet (SUV) radiation in mouse epidermis. Analysis of the epidermis from SUV-irradiated mice revealed rapid phosphorylation of STAT1 (pSTAT1) on both tyrosine (tyr701) and serine (ser727) residues and increased levels of IRF-1 while later timepoints showed increased levels of unphosphorylated STAT1 (uSTAT1). STAT1 activation led to upregulation of several proinflammatory chemokine mRNAs in epidermis including Cxcl9, Cxcl10, and Ccl2, as well as, the immune checkpoint inhibitor Pd-l1. In addition, mRNA and protein levels of cyclooxygenase-2 (Cox-2/COX2) were upregulated in epidermis following exposure to SUV. Mice with keratinocyte-specific STAT1 deletion did not exhibit increased IRF-1 or proinflammatory gene expression in epidermis. Furthermore, epidermal COX-2 induction after SUV exposure was significantly reduced in mice with keratinocyte-specific deletion of STAT1. Additionally, SUV irradiation rapidly upregulated interferon gamma (IFNγ) mRNA in the epidermis and that skin resident epidermal CD3 + T-cells were the source of IFNγ production. IFNγ receptor-deficient mice confirmed dependency of STAT1 activation, proinflammatory gene expression and COX-2 upregulation in the epidermis on paracrine IFNγ signaling. Furthermore, keratinocyte-specific STAT1-deficiency reduced proliferation and hyperplasia due to SUV irradiation and this was associated with decreased immune infiltration of mast cells in the dermis. Collectively, the current results demonstrate that exposure to SUV leads to upregulation of IFNγ and downstream pSTAT1/IRF-1/uSTAT1 signaling in the epidermis. Further study of this pathway could lead to identification of novel targets for the prevention of nonmelanoma skin cancer.