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Ultraviolet Light-Induced Gasdermin C Expression Is Mediated Via TRPV1/Calcium/Calcineurin/Nfatc1 Signaling

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Ultraviolet Light-Induced Gasdermin C Expression Is Mediated Via TRPV1/Calcium/Calcineurin/Nfatc1 Signaling INTERNATIONAL JOURNAL OF MOleCular meDICine 42: 2859-2866, 2018 Ultraviolet light-induced gasdermin C expression is mediated via TRPV1/calcium/calcineurin/NFATc1 signaling NOVI KUSUMANINGRUM1-4,7, DONG HUN LEE1-4, HYun‑SUN YOON2-5, CHI-HYUN PARK2-4 and JIN HO CHUNG1-4,6 1Department of Biomedical Sciences, Seoul National University Graduate School; 2Department of Dermatology, Seoul National University College of Medicine; 3Institute of Human-Environment Interface Biology, Medical Research Center, Seoul National University; 4Laboratory of Cutaneous Aging Research, Biomedical Research Institute, Seoul National University Hospital, Seoul 110-744; 5Department of Dermatology, Seoul National University Boramae Hospital, Seoul 156-707; 6Institute on Aging, Seoul National University, Seoul, 110-744, Republic of Korea Received April 26, 2018; Accepted August 20, 2018 DOI: 10.3892/ijmm.2018.3839 Abstract. Gasdermin (GSDM)‑C is a member of the GSDM demonstrated that extracellular calcium and calcineurin gene family and is expressed in the epithelial cells of various activity may be necessary for UV-induced GSDMC expression tissue types, including skin. GSDMC expression is induced in HaCaT cells. In addition, UV-induced GSDMC expression by ultraviolet (UV) irradiation and contributes to UV-induced was either decreased or increased following knockdown or over- matrix metalloproteinase 1 expression in human skin kera- expression of nuclear factor of activated T-cells, cytoplasmic 1 tinocytes. However, how UV irradiation induces GSDMC (NFATc1), respectively. These data suggested that TRPV1 may expression remains unclear. The present study aimed to serve an important role in the induction of GSDMC expres- investigate the role of transient receptor potential cation sion by UV and that UV-induced GSDMC expression may be channel subfamily V member 1 (TRPV1) and a calcium/calci- mediated via a calcium/calcineurin/NFATc1 pathway. neurin-signaling pathway in UV-induced GSDMC expression in human skin keratinocytes. Suppression of TRPV1 activity Introduction by treatment with the TRPV1 antagonists capsazepine and ruthenium red significantly reduced UV-induced GSDMC Ultraviolet (UV) radiation is an important environ- expression, whereas direct activation of TRPV1 by capsaicin, mental factor; exposure of skin to UV irradiation may a TRPV1 agonist, increased GSDMC expression. The results induce various kinds of skin damage, including sunburn, premature aging, cancer and inflammation. The effects of UV may be mediated by various types of cellular-level changes, including autophagy, cell cycle control and cell Correspondence to: Professor Jin Ho Chung, Department of death, as well as molecular-level changes, including signal Dermatology, Seoul National University College of Medicine, Seoul transduction and gene expression. UV irradiation may also National University Hospital, 101 Daehak, Jongno, Seoul 110-744, serve a role as a broad activator of cell surface growth Republic of Korea factor receptors and cytokine receptors (1,2). Activation of E-mail: [email protected] ligand-independent receptors stimulates various downstream Dr Chi-Hyun Park, Laboratory of Cutaneous Aging Research, signaling pathways that control the expression of a number Biomedical Research Institute, Seoul National University Hospital, of genes (1-3). Matrix metalloproteinases (MMPs) are group 101 Daehak, Jongno, Seoul 110-744, Republic of Korea of zinc-dependent endopeptidases that degrade extracellular E-mail: [email protected] matrix proteins (4-6). Certain MMPs have been reported to be induced by UV irradiation, which contributes to skin Present address: 7Department of Dermatology, Diponegoro damage by UV irradiation in vivo (2-4). University, Semarang 50244, Indonesia Gasdermin (GSDM)‑C belongs to the Gasdermin super- Abbreviations: UV, ultraviolet; GSDMC, gasdermin C; NFATc1, family, a novel group of genes that include GSDMA, GSDMB, nuclear factor of activated T-cells, cytoplasmic 1; TRPV1, transient GSDMC and GSDMD, as well as the Gasdermin-related receptor potential cation channel subfamily V member 1 genes (GSDME and pejvakin) in humans (5-7). GSDM family members were reported to be differentially expressed in the Key words: gasdermin C, UV, calcium, calcineurin, NFATc1, epithelial cells of various tissue types, including the skin (5,8). TRPV1 Previous studies have suggested that GSDMC may serve a role in the course of carcinogenesis, such as colorectal cancer cell proliferation and increased metastatic potential in malignant 2860 KUSUMANINGRUM et al: GASDERMIN C IS INDUCED BY ULTRAVIOLET LIGHT melanoma cells (9-11). However, the functions of GSDMC in For treatments, HaCaT cells were cultured to 80% conflu- the skin remain poorly understood. ence and serum-starved for 24 h in DMEM without FBS, and Our previous study reported that GSDMC is induced by UV primary human skin keratinocytes were serum-starved for irradiation and contributes to MMP-1 expression through the 24 h in MCDB 153. HaCaT cells and primary human skin kera- activation of extracellular signal-regulated kinase (ERK) and tinocytes were washed with phosphate-buffered saline (PBS) c-Jun N-terminal kinase (JNK) pathways in human skin kerati- two times; subsequently, HaCaT cells were irradiated with nocytes (6). However, how UV modulates GSDMC expression UV at 60 mJ/cm2 and primary human skin keratinocytes were has remained unclear. In the present study, the signaling irradiated with UV at 100 mJ/cm2 in PBS. UV irradiation was pathways involved in UV-induced GSDMC expression in performed with Philips TL 20W/12RS fluorescent sun lamps human skin keratinocytes were examined by determining the (Philips Medical Systems B.V., Eindhoven, The Netherlands) role of transient receptor potential cation channel subfamily with an emission spectrum between 275 and 380 nm (peak, V member 1 (TRPV1) on UV-induced GSDMC expression. 310‑315 nm); a Kodacel filter TA401/407 (Kodak, Rochester, TRPV1 is a capsaicin receptor and functions as a non-selective NY, USA) was used to block UVC of wavelength below cation channel that may lead to calcium influx (12‑14). TRPV1 290 nm. UV irradiation intensity was measured with a Model and GSDMC have been previously reported to serve crucial 585100 UV meter from Herbert Waldmann GmbH & Co. KG roles in UV-induced MMP-1 expression in human skin (Villingen-Schwenningen, Germany). Following UV irradia- keratinocytes (6,12). However, TRPV1 activation appears to tion, PBS was removed and replaced with DMEM without FBS occur at relatively early time points (12), whereas GSDMC for HaCaT cells and keratinocyte basal medium for primary expression increases at relatively late time points, following human skin keratinocytes, and cells were further incubated for UV irradiation (6). Therefore, whether TRPV1 may serve any 24 h. When required, specific TRPV1 antagonist (capsazepine role in UV-induced GSDMC expression was examined. The or ruthenium red) or specific TRPV1 agonist (capsaicin) was results demonstrated that TRPV1 serves an important role in added 30 min prior to UV irradiation, and treated again with UV-induced GSDMC expression. Through additional studies, specific TRPV1 antagonist (capsazepine or ruthenium red) for UV-induced GSDMC expression was determined to be depen- 24 h following UV irradiation; calcineurin inhibitor (cyclo- dent on calcium and calcineurin; it was also demonstrated that sporine A) was added immediately following UV irradiation nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and cells were incubated for 24 h. To investigate the role of mediated UV-induced GSDMC expression. extracellular calcium in UV irradiation, HaCaT cells were serum-starved for 24 h and cultured in either calcium-free Materials and methods DMEM or calcium-containing DMEM for 30 min prior to UV irradiation. Fresh corresponding culture medium was added, Materials. Dulbecco's modified Eagle's medium (DMEM), was and the cells were further incubated for 24 h. Each experiment purchased from Welgene, Inc. (Gyeongsan, Gyeongsangbuk, was repeated three times. The medical ethical committee at Korea). Calcium-free DMEM was obtained from Invitrogen Seoul National University approved the study protocol, and (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fetal written informed consent was received from the guardians Bovine Serum (FBS) was purchased from HyClone (GE of participants. The study was conducted according to the Healthcare Life Sciences, Logan, UT, USA). Keratinocyte Declaration of Helsinki principles. basal medium MCDB 153 was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) and keratinocyte growth Transfection with NFATc1 small interfering (si)RNA. For medium was purchased from Clonetics Corp. (San Diego, CA, knockdown of NFATc1, cultured HaCaT cells were seeded USA). Antibiotics (penicillin and streptomycin) and TRIzol and maintain until approximately 80% confluency, and subse- reagent were obtained from Thermo Fisher Scientific, Inc. quently transfected with the scrambled negative control siRNA Capsazepine, ruthenium red and capsaicin were purchased (siNC) or a NFATc1‑specific siRNA (siNFATc1; 5'‑CCA AGG from Sigma-Aldrich; Merck KGaA. Expression plasmids for UCA UUU UCG UGG A-3'; Bioneer Corporation, Daejeon, the wild-type (pMX-NFATc1-WT) or a constitutively active Korea) at 100 pmol using Lipofectamine® 2000 Reagent form of NFATc1 (pMSCV-NFATc1‑CA) were kindly provided (Invitrogen: Thermo Fisher Scientific, Inc.) in a humidified by Dr Hong-Hee Kim (Department of Cell and
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