Keratin 17 as a Marker of UVB-Induced Stress in Human Epidermis and Modulation by Vitis vinifera Extract.

Human epidermis responds to ultraviolet (UV)B-induced damage by tolerating it, restoring it, or undergoing programmed cell death when the damage is massive. Recently, compounds rich in polyphenols, such as Vitis vinifera L. leaf extract (VVLe), have attracted a lot of interest for skin protection. We investigated the effect of VVLe pre-treatment (1 h) in a 2D model of HaCaT cells and in 3D organotypic cultures of normal human skin exposed to a single UVB dose to study the immediate specific events 1 h and the response orchestrated in the epidermal layer 24 h after irradiation, respectively. In both models, transmission electron microscopy analysis was carried out. The expression of the inducible keratin K17, the activation of both pSTAT3 and Nuclear Factor (NF)-κB signalling pathways, and the epidermal distribution of Toll-Like Receptor (TLR) 4 were assessed by immunofluorescence in the 2D and 3D model. In 3D organotypic cultures, thanks to the preservation of a multi-layered structure, the epidermal distribution of the differentiation biomarkers K10 and K14 as well as of K16 was analysed by immunofluorescence, while the release of interleukin (IL)-8 was evaluated by ELISA. In skin bioptic fragments, cytotoxicity and genotoxicity were investigated by LDH assay and Alkaline Comet assay, respectively, and then compared to cell proliferation. The epidermal distribution of the histone γ-H2AX, indicating the fragmented DNA, was analysed by immunofluorescence. In both experimental models, VVLe tuned UVB-induced K17 expression to a different extent in HaCaT cells and in the skin. In HaCaT cells, pSTAT3 activation was induced by UVB and reverted by VVLe pre-treatment. TLR4 expression was triggered by UVB in both models, but VVLe pre-treatment abolished this event only in HaCaT cells. NF-κB immunostaining increased both in the nucleus and in the cytoplasm only in HaCaT cells after UVB irradiation. In all irradiated skin samples, VVLe pre-treatment was not able to revert the inhibition of epidermal proliferation, K16 expression, and IL-8 secretion. The effectiveness of VVLe in contrasting the irradiation-induced genotoxicity still remains unclear. In conclusion, our study clearly shows that K17 is a robust marker induced in keratinocytes upon UVB stimulation and that this event can be reverted by a pre-treatment with VVLe. On the whole, these observations represent a novelty in the scenario of the complex relationships between the effects exerted by UVB rays on human skin and significantly improve the knowledge regarding the modulation of the early epidermal response induced by a single exposure to UVB in the presence of VVLe.

Anti-psoriatic properties of paeoniflorin: suppression of the NF-kappaB pathway and Keratin 17.

BACKGROUND: Psoriasis is a common inflammatory skin disease characterized by hyperproliferation of epidermal keratinocytes, however, there is still no curative therapy for psoriasis. Paeoniflorin (PF), a Chinese herbal medicine, has shown anti-inflammatory effects. OBJECTIVES: We aimed to illustrate the effect and associated mechanism of PF on keratinocyte proliferation using the IMQ-induced psoriasis mouse model. MATERIALS AND METHODS: The anti-psoriatic effect of PF in vivo and in vitro was assessed by western blot, RT-PCR, immunofluorescence, cell counting kit-8 and haematoxylin/eosin staining. RESULTS: In vivo, epidermal thickness, dermal infiltrated lymphocytes and the level of several antimicrobial peptides, pro-inflammatory cytokines, and K17 were significantly reduced in mice with topical application of PF. In vitro, PF inhibited the proliferation of HaCaT cells in a dose-dependent manner, down-regulated K17 expression, and suppressed NF-kappaB activation. CONCLUSION: Our findings indicate that PF may inhibit the proliferation of keratinocytes by targeting K17, suggesting that PF might be a potential target in the treatment of psoriasis.

Randomized, split-body, single-blinded clinical trial of topical broccoli sprout extract: Assessing the feasibility of its use in keratin-based disorders.

BACKGROUND: Epidermolysis bullosa simplex is a skin-blistering disorder caused by mutations in keratin (K)14 or K5. Treatment with nuclear factor (erythroid-derived 2)-like 2 inducer sulforaphane ameliorated skin blistering in Krt14-null mice, correlating with induction of K17. To be therapeutically useful for epidermolysis bullosa simplex, topical broccoli sprout extract (BSE), enriched for sulforaphane, would ideally induce the expression of homologous keratins (eg, K6, K17, K16) in the basal layer of human epidermis without impacting expression of defective keratins (K5/K14). OBJECTIVE: The purpose of this 1-week, randomized, split-body, single-blinded, placebo-controlled trial was to assess the impact of BSE on keratin expression. METHODS: Five subjects (34-71 years old) applied BSE (500 nmol of sulforaphane/mL) or vehicle alone to the inner aspect of the arm daily. Expression of keratin, nuclear factor (erythroid-derived 2)-like 2, and other markers was assessed using reverse transcription-polymerase chain reaction and indirect immunofluorescence. RESULTS: One subject (age 71 years) was excluded a posteriori because of poor tissue quality. Topical BSE activated nuclear factor (erythroid-derived 2)-like 2 and up-regulated K17 in the epidermis of all subjects, had variable effects on K16 and K6 expression, and did not alter expression of K14 or K5. LIMITATIONS: Small sample size is a limitation. CONCLUSION: BSE represents an attractive therapeutic candidate for K14-associated epidermolysis bullosa simplex.

Proteomics Analysis Reveals Involvement of Krt17 in Areca Nut-Induced Oral Carcinogenesis.

The areca nut is a known carcinogen that causes oral cancer in individuals in Southeast Asia, but the molecular mechanism that leads to this malignancy is still unclear. To mimic the habit of areca nut chewing, our laboratory has established four oral cancer cell sublines (SAS, OECM1, K2, C9), which have been chronically exposed to areca nut extract (ANE). To elucidate the molecular basis of areca nut-induced oral carcinogenesis, the differential proteomes between oral cancer cells and the ANE-treated sublines were determined using isobaric mass tag (iTRAQ) labeling and multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Over 1000 proteins were identified in four sublines, and 196 proteins were found to be differentially expressed in at least two ANE-treated sublines. A bioinformatic analysis revealed that these proteins participate in several pathways, and one of the most prominent pathways was the regulation of epithelial to mesenchymal transition (EMT). In all, 24 proteins including Krt17 were confirmed to be differentially expressed in the ANE-treated sublines. To reveal additional information on the mechanism of ANE-induced carcinogenesis, Krt17 was further investigated. Krt17 knockdown significantly suppressed ANE-induced cell migration and invasion and modulated the EMT process. Furthermore, in a murine model of carcinogen-induced (arecoline cocktail, an active compound of ANE) oral cancer, Krt17 was significantly up-regulated in all hyperplastic tissues and in carcinoma tissues (p < 0.001). In conclusion, we have identified a proteome of oral cancer cells that is associated with chronic areca nut exposure. Krt17 was demonstrated to contribute to areca nut-induced oral malignancy. The results of this study contribute to risk assessment, disease prevention and other clinical applications associated with areca nut-induced oral cancer.