Primary Mouse Keratinocytes Research Articles

Keratinocyte-Derived Exosomes in Painful Diabetic Neuropathy.

Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Exosomes are small extracellular vesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived exosomes (KDEs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEs in a well-established high-fat diet (HFD) mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEs through size exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of HFD KDEs compared to KDEs obtained from control mice on a regular diet (RD), including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle (EV) reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEs are retrogradely trafficked into the DRG neuron cell body. Overall, our study presents a potential novel mode of communication between keratinocytes and DRG neurons in the skin, revealing a possible role for KDEs in contributing to the axonal degeneration that underlies neuropathic pain in PDN. Moreover, this study presents potential therapeutic targets in the skin for developing more effective, disease-modifying, and better-tolerated topical interventions for patients suffering from PDN, one of the most common and untreatable peripheral neuropathies.

NETs contribute to psoriasiform skin inflammation: A novel therapeutic approach targeting IL-36 cytokines by a small molecule tetrahydroxystilbene glucoside

BackgroundPsoriasis, a chronic immune-mediated skin disease with pathological features such as aberrant differentiation of keratinocytes, dermal-epidermal inflammation, and angiogenesis. 2,3,5,4′-Tetrahydroxy stilbene 2-Ο-β-d-glucoside (2354Glu) is a natural small molecule polyhydrostilbenes isolated from Polygonum multiglorum Thunb. The regulation of IL-36 subfamily has led to new pharmacologic strategies to reverse psoriasiform dermatitis. PurposeHere we investigated the therapeutic potential of 2354Glu and elucidated the underlying mechanism in psoriasis. MethodsThe effects of 2354Glu on IL-36 signaling were assessed by psoriasiform in vivo, in vitro and ex vivo model. The in vivo mice model of psoriasis-like skin inflammation was established by applying imiquimod (IMQ), and the in vitro and ex vitro models were established by stimulating mouse primary keratinocyte, human keratinocytes cells (HaCaT) and ex vivo skin tissue isolated from the mice back with Polyinosine-polycytidylic acid (Poly(I:C)), IMQ, IL-36γ and Lipopolysaccharide (LPS) respectively. Moreover, NETs formation was inhibited by Cl-amidine to evaluate the effect of NETs in psoriatic mouse model. The effects of 2354Glu on skin inflammation were assessed by western blot, H&E, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay and real-time quantitative PCR. ResultsIn Poly(I:C)-stimulated keratinocytes, the secretion of IL-36 was inhibited after treatment with 2354Glu, similar to the effects of TLR3, P2X7R and caspase-1 inhibitors. In aldara (imiquimod)-induced mice, 2354Glu (100 and 25 mg/kg) improved immune cell infiltration and hyperkeratosis in psoriasis by directly targeting IL-36 in keratinocytes through P2X7R-caspase-1. When treatment with 2354Glu (25 mg/kg) was insufficient to inhibit IL-36γ, NETs reduced pathological features and IL-36 signaling by interacting with keratinocytes to combat psoriasis like inflammation. Conclusion: These results indicated that NETs had a beneficial effect on psoriasiform dermatitis. 2354Glu alleviates psoriasis by directly targeting IL-36/P2X7R axis and NET formation, providing a potential candidate for the treatment of psoriasis.

Abstract 5899: Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy

Abstract Recent research into prostate cancer treatment has unveiled a promising contender known as Up284. In laboratory experiments, this compound has exhibited a remarkable preference for targeting prostate cancer cells as opposed to normal cells. Specifically, in vitro cell viability assays using MTT have shown that prostate cancer cells display high sensitivity to Up284 at low nanomolar IC50 values, while primary human prostate epithelial cells and mouse keratinocytes exhibit minimal sensitivity. This establishes a notable therapeutic window between cancerous and non-cancerous cells, with Up284 significantly inhibiting colony formation in PC3 cells. Additional functional assays have confirmed Up284's potential, including the 4UbFL Reporter Gene assay, which has revealed robust, dose-dependent proteasome inhibition. This effect is supported by the accumulation of polyubiquitinated proteins in various prostate cancer cell lines following Up284 treatment, suggesting a distinct mechanism of toxicity compared to bortezomib. Furthermore, Up284 has been found to induce apoptosis in PC3 cells, surpassing the efficacy of bortezomib. Competitive binding assays have shown that Up284 exhibits a stronger binding affinity to the proteasomal receptor RPN13 than the prototype inhibitor RA190B. This is further supported by a Cellular Thermo Stabilization Assay (CETSA), which indicates that Up284 stabilizes RPN13 in cells. A preliminary in vivo study utilizing the bone metastatic prostate cancer genetically engineered mouse model(BMPC GEMM) model for metastatic prostate adenocarcinoma has demonstrated that Up284 leads to significant tumor regression and increased levels of poly-ubiquitinated proteins in residual tumor tissues. Moreover, when Up284 is conjugated with an E3 ligase ligand, it successfully degrades RPN13, resulting in the death of PC3 cells. Up284 has also shown favorable pharmacokinetics/pharmacodynamics (PK/PD) profiles and safety in animal models. It is well-tolerated in mice and holds promise for oral administration, demonstrating a proteasome-dependent reporter protein stabilization effect superior to ixazomib. Importantly, Up284 has induced tumor regression and improved survival rates in various cancer models, including ovarian and breast cancers. In conclusion, Up284 emerges as a lead candidate for targeted prostate cancer therapy due to its high specificity, substantial therapeutic index, and potent anti-cancer activity both in vitro and in vivo. Further development and clinical investigation of Up284 are warranted to fully explore its therapeutic potential. Citation Format: Balasubramanyam Karanam, Ravi Anchoori, Yung Chang. Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5899.

Interferon lambda restricts herpes simplex virus skin disease by suppressing neutrophil-mediated pathology.

Type III interferons (IFN-λ) are antiviral and immunomodulatory cytokines that have been best characterized in respiratory and gastrointestinal infections, but the effects of IFN-λ against skin infections have not been extensively investigated. We sought to define the skin-specific effects of IFN-λ against the highly prevalent human pathogen, herpes simplex virus (HSV). We infected mice lacking the IFN-λ receptor (Ifnlr1-/-), both the IFN-λ and the IFN-α/β receptors (Ifnar1-/-Ifnlr1-/-), or IFN-λ cytokines (Ifnl2/3-/-) and found that IFN-λ restricts the severity of HSV-1 and HSV-2 skin lesions without affecting viral loads. We used RNAseq to define IFN-λ- and IFN-β-induced transcriptional responses in primary mouse keratinocytes. Using conditional knockout mice, we found that IFN-λ signaling in both keratinocytes and neutrophils was necessary to control HSV-1 skin lesion severity and that IFN-λ signaling in keratinocytes suppressed CXCL9-mediated neutrophil recruitment to the skin. Furthermore, depleting neutrophils or blocking CXCL9 protected against severe HSV-1 skin lesions in Ifnlr1-/- mice. Altogether, our results suggest that IFN-λ plays an immunomodulatory role in the skin that restricts neutrophil-mediated pathology during HSV infection and suggests potential applications for IFN-λ in treating viral skin infections.IMPORTANCEType III interferons (IFN-λ) have been shown to have antiviral and immunomodulatory effects at epithelial barriers such as the respiratory and gastrointestinal tracts, but their effects on the skin have not been extensively investigated. We used mice lacking IFN-λ signaling to investigate the skin-specific effects of IFN-λ against the herpes simplex virus (HSV), which targets epithelial tissues to cause cold sores and genital herpes. We found that IFN-λ limited the severity of HSV skin lesions without affecting viral load and that this protective effect required IFN-λ signaling in both keratinocytes and neutrophils. We found that IFN-λ signaling in keratinocytes suppressed neutrophil recruitment to the skin and that depleting neutrophils protected against severe HSV skin lesions in the absence of IFN-λ. Altogether, our results suggest that IFN-λ plays an immunomodulatory role in the skin that restricts neutrophil-mediated pathology during HSV infection and suggests potential applications for IFN-λ in treating viral skin infections.

Involvement of skin TRPV3 in temperature detection regulated by TMEM79 in mice

TRPV3, a non-selective cation transient receptor potential (TRP) ion channel, is activated by warm temperatures. It is predominantly expressed in skin keratinocytes, and participates in various somatic processes. Previous studies have reported that thermosensation in mice lacking TRPV3 was impaired. Here, we identified a transmembrane protein, TMEM79, that acts as a negative regulator of TRPV3. Heterologous expression of TMEM79 was capable of suppressing TRPV3-mediated currents in HEK293T cells. In addition, TMEM79 modulated TRPV3 translocalization and promoted its degradation in the lysosomes. TRPV3-mediated currents and Ca2+ influx were potentiated in primary mouse keratinocytes lacking TMEM79. Furthermore, TMEM79-deficient male mice preferred a higher temperature than did wild-type mice due to elevated TRPV3 function. Our study revealed unique interactions between TRPV3 and TMEM79, both in vitro and in vivo. These findings support roles for TMEM79 and TRPV3 in thermosensation.

Characterization of a Chimeric MmuPV1 Genome with HPV-16E6E7

Background/Objectives: Papilloma Viruses (PVs) are double-stranded DNA viruses that infect cutaneous and mucosal epithelium. In humans, there are over 100 types of PVs. HPV-16 is a high-risk type that causes ~50% of cervical cancers and ~70% of oropharyngeal cancers by expressing viral oncogenes E6 and E7 in replicating keratinocytes. The mouse PV, MmuPV1, displays species tropism and causes neoplastic lesions. Its discovery in 2011 introduced the opportunity to study PVs from early stages of infection to cancer development. Such an in vivo model of HPV-16 could uncover novel mechanisms for treatment intervention and disease prevention. Our study aims to characterize a small-animal infection model using a chimeric MmuPV1 genome with HPV-16 E6 and E7 in place of mouse E6 and E7 (MmuPV1-16E6E7). The goal of our study is to investigate the genome’s ability to express the HPV-16 oncogenes in vitro and to cause tumors in vivo. Methods: To increase selective pressure in vitro, we replaced the MmuPV1 L1 and L2 genes with a neomycin cassette (MmuPV1-16E6E7neo). We packaged these genomes into infectious quasiviruses using a HEK 293TTF viral packing cell line. After isolating quasiviruses, we infected two donor human foreskin keratinocytes (HFK) cell lines. Forty-eight hours post infection, we isolated mRNA for qPCR to quantify HPV-16 E6 expression. To investigate whether MmuPV1-16E6E7 can cause tumors in in vivo, we infected athymic nude (N/J) mice orally with quasiviruses to evaluate infection and viable genome persistence. Results: HPV-16 E6 cDNA was detected in three HFK isolates. Conclusions/Future Directions: The presence of HPV-16 E6 cDNA indicates that the gene can be successfully transcribed from MmuPV1-16E6E7neo in HFKs. Future studies will assess this ability in primary mouse keratinocytes. If tumors are observed, we can use this model to study the efficacy of antiviral compounds to inhibit HPV16 E6 in vivo.

Advanced Glycation End Products and Activation of Toll-like Receptor-2 and -4 Induced Changes in Aquaporin-3 Expression in Mouse Keratinocytes.

Prolonged inflammation and impaired re-epithelization are major contributing factors to chronic non-healing diabetic wounds; diabetes is also characterized by xerosis. Advanced glycation end products (AGEs), and the activation of toll-like receptors (TLRs), can trigger inflammatory responses. Aquaporin-3 (AQP3) plays essential roles in keratinocyte function and skin wound re-epithelialization/re-generation and hydration. Suberanilohydroxamic acid (SAHA), a histone deacetylase inhibitor, mimics the increased acetylation observed in diabetes. We investigated the effects of TLR2/TLR4 activators and AGEs on keratinocyte AQP3 expression in the presence and absence of SAHA. Primary mouse keratinocytes were treated with or without TLR2 agonist Pam3Cys-Ser-(Lys)4 (PAM), TLR4 agonist lipopolysaccharide (LPS), or AGEs, with or without SAHA. We found that (1) PAM and LPS significantly upregulated AQP3 protein basally (without SAHA) and PAM downregulated AQP3 protein with SAHA; and (2) AGEs (100 µg/mL) increased AQP3 protein expression basally and decreased AQP3 levels with SAHA. PAM and AGEs produced similar changes in AQP3 expression, suggesting a common pathway or potential crosstalk between TLR2 and AGEs signaling. Our findings suggest that TLR2 activation and AGEs may be beneficial for wound healing and skin hydration under normal conditions via AQP3 upregulation, but that these pathways are likely deleterious in diabetes chronically through decreased AQP3 expression.

Topical application of imatinib mesylate suppresses vitamin D3 analog-induced dermatitis in Balb/c mice.

Atopic dermatitis (AD) is an allergic disease mediated by Th2 cells. In AD, externally stimulated keratinocytes release inflammatory cytokines, such as IL-33 and TSLP. Inflammatory cells infiltrate skin tissue and increase vascular permeability. Therefore, we hypothesized that imatinib mesylate (IMT), which suppresses vascular permeability, may be a candidate therapeutic agent for AD. A vitamin D3 analog (MC903) was administered daily to both ears of Balb/c mice to create a murine AD model to which IMT was applied. The skin lesions were evaluated histopathologically and by immunostaining. Cytokine expression in the skin was assessed by using real-time polymerase chain reaction (PCR) and immunostaining and was investigated using Evans Blue to determine whether IMT suppressed vascular permeability due to histamine. The suppressive effect of TNF-α/IL-4-induced TSLP expression in primary mouse keratinocytes (MKCs) treated with IMT was then investigated. Tslp gene and protein expression in the lesion was measured using real-time PCR and ELISA. The activation of signal transduction was analysed by western blotting. Topical application of IMT significantly reduced ear thickness, Evans blue leakage, and scratch onset. IMT suppressed the number of infiltrating cells (CD4+ T cells, eosinophils, and basophils), and the expression of IL-13, IL-33, and TSLP in a MC903-induced, murine AD model and inhibited TNF-α/IL-4-induced TSLP expression via downregulation of ERK phosphorylation in MKCs. IMT reduced the skin symptoms in a MC903-induced, murine AD model, suggesting that it may have potential as a new treatment for AD.

Scutellaria baicalensis Georgi regulates REV-ERBα/BMAL1 to protect against skin aging in mice.

Scutellaria baicalensis Georgi (SBG) is a traditional Chinese medicine widely used to treat disorders such as hypertension, dysentery and hemorrhaging. Here, we aimed to assess the pharmacological effects of SBG on skin aging and to investigate the underlying mechanisms. Mice with skin aging were established by treatment with D-galactose and ultraviolet-B. SBG (topical application) showed a protective effect on skin aging in mice, as evidenced by less formation of skin wrinkles, higher levels of SOD (superoxide dismutase) and HYP (hydroxyproline) as well as a lower level of MDA (malondialdehyde). In the meantime, skin MMP-1 and p53 expression were lower, epidermis was thinner and collagen amount was higher in SBG-treated mice. Anti-skin aging effects of SBG were also confirmed in NIH3T3 and HaCaT cells, as well as in mouse primary dermal fibroblasts and human primary epidermal keratinocytes. Furthermore, we found that loss of Rev-erbα (a known repressor of Bmal1) up-regulated skin BMAL1 (a clock component and a known anti-aging factor) and ameliorated skin aging in mice. Moreover, SBG dose-dependently increased the expression of BMAL1 in the skin of aged mice and in senescent NIT3H3 cells. In addition, based on a combination of Gal4 chimeric, luciferase reporter and expression assays, SBG was identified as an antagonist of REV-ERBα and thus an inducer of BMAL1 expression. In conclusion, SBG antagonizes REV-ERBα to up-regulate BMAL1 and to protect against skin aging in mice.

SerpinB7 deficiency contributes to development of psoriasis via calcium-mediated keratinocyte differentiation dysfunction

Defective execution of proteases and protease inhibitors that mediate abnormal signaling cascades is emerging as a key contributor to skin diseases, such as psoriasis. SerpinB7 is identified as a skin-specific endogenous protease inhibitor, but the role and underlying mechanism in psoriasis are poorly understood. Here we found that SerpinB7 is highly expressed in psoriatic keratinocytes of patients and imiquimod-induced psoriatic lesions in mice. SerpinB7-/- mice showed abnormal epidermal barrier integrity and skin architecture in homeostasis, and aggravated psoriatic lesion with inhibiting terminal differentiation and increasing inflammatory cells infiltration compared to SerpinB7+/+ mice after Imiquimod treatment. Mechanistically, SerpinB7 deficiency results in excessive proliferation and impaired differentiation, as well as increased chemokines and antimicrobial peptide expression in normal human epidermal keratinocyte and mouse primary keratinocyte. Transcriptomics and proteomics results showed that the SeprinB7 deficiency affected keratinocyte differentiation and proinflammatory cytokines, possibly by affecting the calcium ion channel-related proteins. Notably, we demonstrated that SerpinB7 deficiency prevented the increase in intracellular Ca2+ influx, which was partly eliminated by the intracellular Ca2+ chelator BAPTA-AM. Our findings first described the critical role of SerpinB7 in the regulation of keratinocyte differentiation and psoriatic microenvironment mediated via keratinocytes' intracellular calcium flux, proposing a new candidate for therapeutic targets in psoriasis.

Aging-Conferred SIRT7 Decline Inhibits Rosacea-Like Skin Inflammation by Modulating Toll-Like Receptor 2‒NF-κB Signaling

Rosacea is a chronic inflammatory skin disorder that manifests abnormal enhanced sensitivity to environmental stimuli. The decreased prevalence of rosacea in aged population has been reported, but the underlying mechanism is unclear. In this study, we confirm that the rosacea-like skin inflammation induced by cathelicidin LL37 is alleviated in aged mice and mice with progeria. Primary mouse keratinocytes isolated from aged mice and human dermal fibroblasts that undergo senescence present a much lower sensitivity to proinflammatory stimuli. Mechanistically, toll-like receptor 2 (TLR2) is downregulated in the skin of both aged population and mice. Knockdown of TLR2 in young human dermal fibroblasts mimics the attenuated immune response to LL37 and TNF-α evidenced in aged human dermal fibroblasts, whereas overexpression of TLR2 in aged human dermal fibroblasts rescued this attenuation. At the molecular level, in response to inflammatory stimuli, SIRT7 mediates the upregulation of TLR2, which promotes the activation of NF-κB signaling. The decay of SIRT7 confers an age-related decline of TLR2‒NF-κB signaling. Although the overexpression of exogenous Sirt7 abrogates skin immune reactivity reduction in aged mice, loss of Sirt7 alleviates the rosacea-like features in mice. Thus, we reveal a SIRT7‒TLR2‒NF-κB axis that can be targeted for the improvement of rosacea.

Coordinated post-transcriptional control of oncogene-induced senescence by UNR/CSDE1.

Oncogene-induced senescence (OIS) is a form of stable cell-cycle arrest arising in response to oncogenic stimulation. OIS must be bypassed for transformation, but the mechanisms of OIS establishment and bypass remain poorly understood, especially at the post-transcriptional level. Here, we show that the RNA-binding protein UNR/CSDE1 enables OIS in primary mouse keratinocytes. Depletion of CSDE1 leads to senescence bypass, cell immortalization, and tumor formation, indicating that CSDE1 behaves as a tumor suppressor. Unbiased high-throughput analyses uncovered that CSDE1 promotes OIS by two independent molecular mechanisms: enhancement of the stability of senescence-associated secretory phenotype (SASP) factor mRNAs and repression of Ybx1 mRNA translation. Importantly, depletion of YBX1 from immortal keratinocytes rescues senescence and uncouples proliferation arrest from the SASP, revealing multilayered mechanisms exerted by CSDE1 to coordinate senescence. Our data highlight the relevance of post-transcriptional control in the regulation of senescence.

BMAL1 regulates Propionibacterium acnes-induced skin inflammation via REV-ERBα in mice.

Acne vulgaris is a common skin disease, affecting over 80% of adolescents. Inflammation is known to play a central role in acne development. Here, we aimed to investigate the role of the central clock gene Bmal1 in acne-associated inflammation in mice. To this end, mice were injected intradermally with Propionibacterium acnes (P. acnes) to induce acne-associated skin inflammation. We found that Bmal1 and its target genes Rev-erbα, Dbp, Per1 and Cry2 were down-regulated in the skin of P. acnes-treated mice, suggesting a role of Bmal1 in the condition of acne. Supporting this, Bmal1-deleted or jet-lagged mice showed exacerbated P. acnes-induced inflammation in the skin. Regulation of P. acnes-induced inflammation by Bmal1 was further confirmed in RAW264.7 cells and primary mouse keratinocytes. Transcriptomic and protein expression analyses suggested that Bmal1 regulated P. acnes-induced inflammation via the NF-κB/NLRP3 axis, which is known to be repressed by REV-ERBα (a direct target of BMAL1). Moreover, loss of Rev-erbα in mice exacerbated P. acnes-induced inflammation. In addition, Rev-erbα silencing attenuated the inhibitory effects of Bmal1 on P. acnes-induced inflammation. Bmal1 knockdown failed to modulate P. acnes-induced inflammation in Rev-erbα-silenced cells. It was thus proposed that Bmal1 restrained P. acnes-induced skin inflammation via its target REV-ERBα, which acts on the NF-κB/NLRP3 axis to repress inflammation. In conclusion, Bmal1 disruption is identified as a potential pathological factor of acne-associated inflammation. The findings increase our understanding of the crosstalk between skin clock and acne and suggest targeting circadian rhythms as a promising approach for management of acne.

SerpinB7 deficiency contributes to development of psoriasis via Calcium-mediated keratinocyte differentiation dysfunction

Abstract Defective execution of protease and protease inhibitor, which meditates abnormal signaling cascade, is merging as key contributors to autoimmune disease. SerpinB7, is identified as a skin-specific endogenous protease inhibitor, the role and underlying mechanism in psoriasis is poorly understood. Here we found that SerpinB7 is highly expressed in psoriatic keratinocytes of patient and imiquimod-induced psoriatic lesion in mice. SerpinB7−/− mice showed abnormal epidermal barrier integrity and skin architecture in homeostasis, and aggravated psoriatic lesion with inhibiting terminal differentiation and increasing inflammatory cells infiltration compared to WT after IMQ treatment. Mechanistically, SerpinB7 deficiency resulted in excessive proliferation and impaired differentiation, as well as increased chemokines and antimicrobial peptide expression in human epidermal keratinocyte and mouse primary keratinocyte. Transcriptome analysis demonstrate that SerpinB7 has a significant correlation with keratinocyte differentiation, consistent with WGCNA analysis from GTEx. Notably, we demonstrated that SerpinB7 deficiency prevented the increase of intracellular Ca2+ influx and the generation of mitochondria-derived reactive oxygen species (ROS), which were partly eliminated by antioxidant NAC and intracellular Ca2+ chelator BAPTA-AM. More importantly, iTraq and iTraq-tail proteomics demostrated SerpinB7 deficiency attenuated proteolytic networks that control calcium flux in keratinocytes. Our findings first described the critical role of SerpinB7 in the regulation of keratinocytes differentiation and psoriatic microenvironment, implying a new candidate for therapeutic targets in psoriasis.

Functional Specificity of the Members of the Sos Family of Ras-GEF Activators: Novel Role of Sos2 in Control of Epidermal Stem Cell Homeostasis.

Simple SummaryThe Sos Ras-GEFs are known to participate in a wide range of skin-related diseases including cutaneous cancers, cardio-facio-cutaneous syndromes, or hirsutism. However, the specific functional roles played by the Sos1 and/or Sos2 family members in specific skin compartments remain largely unknown. This report aimed at precisely characterizing the specific functions played by Sos1 and/or Sos2 in keratinocytes, an essential cellular component of the skin. Our data show that Sos1 and Sos2 make overlapping contributions to both keratinocyte proliferation and survival. However, Sos1 seems to have a preferential involvement in regulating the ERK axis, whereas Sos2 seems to control the signaling output from the PI3K axis. We also uncovered an essential role of Sos2 in the control of the population of epidermal stem cells.Prior reports showed the critical requirement of Sos1 for epithelial carcinogenesis, but the specific functionalities of the homologous Sos1 and Sos2 GEFs in skin homeostasis and tumorigenesis remain unclear. Here, we characterize specific mechanistic roles played by Sos1 or Sos2 in primary mouse keratinocytes (a prevalent skin cell lineage) under different experimental conditions. Functional analyses of actively growing primary keratinocytes of relevant genotypes—WT, Sos1-KO, Sos2-KO, and Sos1/2-DKO—revealed a prevalent role of Sos1 regarding transcriptional regulation and control of RAS activation and mechanistic overlapping of Sos1 and Sos2 regarding cell proliferation and survival, with dominant contribution of Sos1 to the RAS-ERK axis and Sos2 to the RAS-PI3K/AKT axis. Sos1/2-DKO keratinocytes could not grow under 3D culture conditions, but single Sos1-KO and Sos2-KO keratinocytes were able to form pseudoepidermis structures that showed disorganized layer structure, reduced proliferation, and increased apoptosis in comparison with WT 3D cultures. Remarkably, analysis of the skin of both newborn and adult Sos2-KO mice uncovered a significant reduction of the population of stem cells located in hair follicles. These data confirm that Sos1 and Sos2 play specific, cell-autonomous functions in primary keratinocytes and reveal a novel, essential role of Sos2 in control of epidermal stem cell homeostasis.

SUV39H2 epigenetic silencing controls fate conversion of epidermal stem and progenitor cells.

Epigenetic histone trimethylation on lysine 9 (H3K9me3) represents a major molecular signal for genome stability and gene silencing conserved from worms to man. However, the functional role of the H3K9 trimethylases SUV39H1/2 in mammalian tissue homeostasis remains largely unknown. Here, we use a spontaneous dog model with monogenic inheritance of a recessive SUV39H2 loss-of-function variant and impaired differentiation in the epidermis, a self-renewing tissue fueled by stem and progenitor cell proliferation and differentiation. Our results demonstrate that SUV39H2 maintains the stem and progenitor cell pool by restricting fate conversion through H3K9me3 repressive marks on gene promoters encoding components of the Wnt/p63/adhesion axis. When SUV39H2 function is lost, repression is relieved, and enhanced Wnt activity causes progenitor cells to prematurely exit the cell cycle, a process mimicked by pharmacological Wnt activation in primary canine, human, and mouse keratinocytes. As a consequence, the stem cell growth potential of cultured SUV39H2-deficient canine keratinocytes is exhausted while epidermal differentiation and genome stability are compromised. Collectively, our data identify SUV39H2 and potentially also SUV39H1 as major gatekeepers in the delicate balance of progenitor fate conversion through H3K9me3 rate-limiting road blocks in basal layer keratinocytes.

Wwox Deficiency Causes Downregulation of Prosurvival ERK Signaling and Abnormal Homeostatic Responses in Mouse Skin.

Deficiency of tumor suppressor WW domain-containing oxidoreductase (WWOX) in humans and animals leads to growth retardation and premature death during postnatal developmental stages. Skin integrity is essential for organism survival due to its protection against dehydration and hypothermia. Our previous report demonstrated that human epidermal suprabasal cells express WWOX protein, and the expression is gradually increased toward the superficial differentiated cells prior to cornification. Here, we investigated whether abnormal skin development and homeostasis occur under Wwox deficiency that may correlate with early death. We determined that keratinocyte proliferation and differentiation were decreased, while apoptosis was increased in Wwox–/– mouse epidermis and primary keratinocyte cultures and WWOX-knockdown human HaCaT cells. Without WWOX, progenitor cells in hair follicle junctional zone underwent massive proliferation in early postnatal developmental stages and the stem/progenitor cell pools were depleted at postnatal day 21. These events lead to significantly decreased epidermal thickness, dehydration state, and delayed hair development in Wwox–/– mouse skin, which is associated with downregulation of prosurvival MEK/ERK signaling in Wwox–/– keratinocytes. Moreover, Wwox depletion results in substantial downregulation of dermal collagen contents in mice. Notably, Wwox–/– mice exhibit severe loss of subcutaneous adipose tissue and significant hypothermia. Collectively, our knockout mouse model supports the validity of WWOX in assisting epidermal and adipose homeostasis, and the involvement of prosurvival ERK pathway in the homeostatic responses regulated by WWOX.

127 ROCK inhibition reduces Ras-induced senescence in primary mouse keratinocytes

The Rho-associated protein kinase (ROCK) is responsible for the regulation of cell adhesion, shape and migration by altering cytoskeletal structure. The ROCK inhibitor, Y27632, protects cells from anoikis and enhances embryonic stem cell viability in culture and cryopreservation. Y27632 prolongs the life span of cultured primary mouse keratinocytes. Oncogenic RAS transduced primary mouse keratinocytes (RAS-keratinocytes) become senescent in vitro. Here, we show that chronic treatment of Ras transduced primary mouse keratinocytes (RAS-keratinocytes) with Y27632 reduces the number of senescence associated β-galactosidase positive cells which are flat and enlarged.

114 Analyzing the metabolic demands of genetic alterations observed in squamous cell carcinoma

Squamous cell carcinoma (SCC) of various organ sites is frequently characterized by amplification of TP63, leading to elevated levels of the ΔNp63α isoform. In a murine orthotopic grafting model of multistage carcinogenesis, overexpression of ΔNp63α cooperates with oncogenic ras (v-rasHa) to drive malignant progression in vivo. Metabolic changes are a hallmark of cancer, including SCC, and preliminary gene array data from our lab revealed altered mRNA levels of metabolic genes in primary murine keratinocytes expressing elevated levels of ΔNp63α and/or v-rasHa. Furthermore, both v-rasHa and ΔNp63α are known to play a role in regulating glycolysis and oxidative phosphorylation. As such, we evaluated the metabolic characteristics of primary murine keratinocytes using Seahorse technology to test if v-rasHa and elevated ΔNp63α levels cooperate to meet the energetic demands of SCC. v-rasHa and/or ΔNp63α were introduced by retro/lentiviral gene transduction and analyzed for basal metabolic activity using the Seahorse XFe96 Analyzer with CyQUANT data normalization. v-rasHa led to a dramatic increase in basal glycolysis and respiration, while ΔNp63α slightly decreased respiration. In combination, ΔNp63α mitigated the v-rasHa-dependent metabolic effects. We next tested if ΔNp63α imparts energy source flexibility to control or v-rasHa-expressing primary mouse keratinocytes and impacts their maximal energy output during stress. v-rasHa-expressing mouse keratinocytes displayed higher maximal respiration and glycolytic rates with increased spare energy capacity over control and ΔNp63α-expressing mouse keratinocytes during chemically induced stress, while co-expression of exogenous ΔNp63α and v-rasHa partially reversed the v-rasHa-dependent metabolic effects. Given that high metabolic activity can lead to excess reactive oxygen species, these data suggest the role of ΔNp63α may be multifaceted; e.g., by controlling metabolic flux to a level that is permissive for cell survival in stressful environments associated with hallmarks of cancer.

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice.

BackgroundNumerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as “leaky” hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels.MethodsWe employed the antibody to treat Cx30A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca2+ imaging and ATP release assay in vitro.FindingsTwo weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels.InterpretationOur results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases.FundFondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.