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Activation of TRPV3 Inhibits Lipogenesis and Stimulates Production of Inflammatory Mediators in Human Sebocytes—A Putative Contributor to Dry Skin Dermatoses

2018; Elsevier BV; Volume: 139; Issue: 1 Linguagem: Inglês

10.1016/j.jid.2018.07.015

1523-1747

Magdolna Szántó, Attila Oláh, Attila Gábor Szöllősi, Kinga Tóth, Edit Páyer, Nóra Czakó, Ágnes Pór, Ilona Kovács, Christos C. Zouboulis, Lajos Kemény, Tamás Bı́ró, Balázs I. Tóth,

Exercise and Physiological Responses

TRPV3 was cloned from keratinocytes and described as a thermosensitive member of the transient receptor potential ion channel family. Although its role in thermosensation is currently under debate, it is highly expressed in the epidermis and functions as a nonselective, Ca2+-permeable cation channel (Huang et al., 2011Huang S.M. Li X. Yu Y. Wang J. Caterina M.J. TRPV3 and TRPV4 ion channels are not major contributors to mouse heat sensation.Mol Pain. 2011; 7: 37Crossref PubMed Scopus (131) Google Scholar, Nilius and Bíró, 2013Nilius B. Bíró T. TRPV3: a "more than skinny" channel.Exp Dermatol. 2013; 22: 447-452Crossref PubMed Scopus (59) Google Scholar, Peier et al., 2002Peier A.M. Reeve A.J. Andersson D.A. Moqrich A. Earley T.J. Hergarden A.C. et al.A heat-sensitive TRP channel expressed in keratinocytes.Science. 2002; 296: 2046-2049Crossref PubMed Scopus (727) Google Scholar). Despite its high abundance in the skin, its genetic deletion causes only moderate, often strain- or sex-dependent, cutaneous phenotypic modifications, such as formation of curly whiskers and wavy hair, defect in epidermal barrier functions, and alterations in epidermal nitrate homeostasis (Cheng et al., 2010Cheng X. Jin J. Hu L. Shen D. Dong X.-P. Samie M.A. et al.TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation.Cell. 2010; 141: 331-343Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, Miyamoto et al., 2011Miyamoto T. Petrus M.J. Dubin A.E. Patapoutian A. TRPV3 regulates nitric oxide synthase-independent nitric oxide synthesis in the skin.Nat Commun. 2011; 2: 369Crossref PubMed Scopus (93) Google Scholar). In contrast, gain-of-function mutations of TRPV3 result in dramatic cutaneous alterations associated with severely dry skin, dermatitis, and hairless phenotype in both mice and rats (Asakawa et al., 2006Asakawa M. Yoshioka T. Matsutani T. Hikita I. Suzuki M. Oshima I. et al.Association of a mutation in TRPV3 with defective hair growth in rodents.J Invest Dermatol. 2006; 126: 2664-2672Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, Xiao et al., 2008Xiao R. Tian J. Tang J. Zhu M.X. The TRPV3 mutation associated with the hairless phenotype in rodents is constitutively active.Cell Calcium. 2008; 43: 334-343Crossref PubMed Scopus (72) Google Scholar). Moreover, similar gain-of-function mutations of TRPV3 were found to play an etiological role in a rare human genodermatosis, Olmsted syndrome, characterized by periorificial hyperkeratosis, hypotrichosis, alopecia, and severe pruritus (He et al., 2015He Y. Zeng K. Zhang X. Chen Q. Wu J. Li H. et al.A gain-of-function mutation in TRPV3 causes focal palmoplantar keratoderma in a Chinese family.J Invest Dermatol. 2015; 135: 907-909Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Lin et al., 2012Lin Z. Chen Q. Lee M. Cao X. Zhang J. Ma D. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am J Hum Genet. 2012; 90: 558-564Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, Ni et al., 2016Ni C. Yan M. Zhang J. Cheng R. Liang J. Deng D. et al.A novel mutation in TRPV3 gene causes atypical familial Olmsted syndrome.Sci Rep. 2016; 6: 21815Crossref PubMed Scopus (27) Google Scholar). Encouraged by these findings, we verified the role of TRPV3 in human hair growth control by inducing catagen in mechanistic studies (Borbíró et al., 2011Borbíró I. Lisztes E. Tóth B.I. Czifra G. Oláh A. Szöllosi A.G. et al.Activation of transient receptor potential vanilloid-3 inhibits human hair growth.J Invest Dermatol. 2011; 131: 1605-1614Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and, most recently, we also described the proinflammatory action of TRPV3 activation in human epidermal keratinocytes (Szöllősi et al., 2018Szöllősi A.G. Vasas N. Angyal Á. Kistamás K. Nánási P.P. Mihály J. et al.Activation of TRPV3 regulates inflammatory actions of human epidermal keratinocytes.J Invest Dermatol. 2018; 138: 365-374Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), whereas others reported its role in dry skin-associated itching (Yoshioka et al., 2009Yoshioka T. Imura K. Asakawa M. Suzuki M. Oshima I. Hirasawa T. et al.Impact of the Gly573Ser substitution in TRPV3 on the development of allergic and pruritic dermatitis in mice.J Invest Dermatol. 2009; 129: 714-722Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). However, the extended inflammatory symptoms and the disrupted lipid barrier found both in rodents and humans suggested that TRPV3-expressing skin cells other than keratinocytes might also be involved in the development of inflammatory skin conditions induced by TRPV3 hyperfunction. Therefore, in this study, we investigated the expression and activation of TRPV3 in human sebocytes, which are important regulators of cutaneous homeostasis (Tóth et al., 2011Tóth B.I. Oláh A. Szöllosi A.G. Czifra G. Bíró T. "Sebocytes' makeup": novel mechanisms and concepts in the physiology of the human sebaceous glands.Pflugers Arch. 2011; 461: 593-606Crossref PubMed Scopus (57) Google Scholar). Immunohistochemical analysis showed that, like epidermal keratinocytes, human sebaceous glands (SG) express TRPV3 in situ (Figure 1a). Peripheral undifferentiated cells show stronger immunopositivity than centrally located terminally differentiated cells. We also showed the presence of TRPV3 protein and mRNA transcripts in human SG-derived SZ95 sebocytes (Zouboulis et al., 1999Zouboulis C.C. Seltmann H. Neitzel H. Orfanos C.E. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).J Invest Dermatol. 1999; 113: 1011-1020Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar), a widely accepted model cell line to study SG biology in vitro (Figure 1b–d). We observed that TRPV3 expression is decreased in post-confluent, more differentiated cultures compared with the highly proliferating pre-confluent cultures (Figure 1c and d). The synthetic TRPV3 activator 2-APB, as well as the plant-derived carvacrol, evoked marked elevation of the intracellular Ca2+ concentration (Figure 1e), suggesting that TRPV3 is, indeed, functionally expressed in human sebocytes. The evoked Ca2+ signals were practically abolished in the presence of the general TRP channel blocker ruthenium red but were not affected by either AMG9810 or HC067047, selective antagonists of the closely related channels TRPV1 and TRPV4, respectively (Figure 1f), channels that are also functionally expressed by sebocytes (Oláh et al., 2014Oláh A. Tóth B.I. Borbíró I. Sugawara K. Szöllõsi A.G. Czifra G. et al.Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes.J Clin Invest. 2014; 124: 3713-3724Crossref PubMed Scopus (152) Google Scholar, Tóth et al., 2009Tóth B.I. Géczy T. Griger Z. Dózsa A. Seltmann H. Kovács L. et al.Transient receptor potential vanilloid-1 signaling as a regulator of human sebocyte biology.J Invest Dermatol. 2009; 129: 329-339Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Because highly specific TRPV3 activators and inhibitors are not available commercially, we then investigated the effect of RNA interference-based silencing of TRPV3 expression on Ca2+ signals. Transfection of the sebocytes with small interfering RNA (siRNA) targeting TRPV3 resulted in a partial, yet marked decrease of the channel expression (see Supplementary Figure S1 online) compared with the scrambled RNA-transfected cells, and significantly suppressed the amplitude and the rate of rise of the agonist-evoked Ca2+ signals (Figure 1g and h); these data provided strong evidence for the activation of TRPV3 by the applied compounds. Higher concentrations of the activators reduced the living cell number in 24 hours, but lower concentrations (still able to evoke Ca2+ signals) did not influence the viability of sebocytes (see Supplementary Figure S2 online), confirming previous results on Ca2+ signaling-induced sebocyte apoptosis (Zouboulis et al., 2017Zouboulis C.C. Seltmann H. Abdel-Naser M.B. Hossini A.M. Menon G.K. Kubba R. Effects of extracellular calcium and 1,25 dihydroxyvitamin D3 on sebaceous gland cells in vitro and in vivo.Acta Derm Venereol. 2017; 97: 313-320Crossref PubMed Scopus (19) Google Scholar). Because sebaceous lipids essentially contribute to the epidermal barrier functions, we also investigated the influence of TRPV3 on lipid synthesis in SZ95 cells. Activation of TRPV3 with non-cytotoxic concentrations of 2-APB and carvacrol decreased lipid synthesis during arachidonic acid (AA)-induced differentiation of scrambled RNA-transfected cells used as control. TRPV3 agonists were less effective at suppressing AA-induced lipid synthesis in cells transfected with siRNA targeting TRPV3 (Figure 2a). Moreover, AA was slightly but significantly more effective in TRPV3-silenced cells, suggesting that basal activity of TRPV3 also negatively regulates AA-induced lipid synthesis. The effect of TRPV3 agonists was not restricted to AA-induced lipid synthesis: they also inhibited the lipogenic effect of the endocannabinoid anandamide and of the combination of linoleic acid and testosterone; and they slightly decreased basal lipid synthesis of nontransfected sebocytes, as well (see Supplementary Figure S3 online). Moreover, carvacrol did not induce cellular differentiation because it did not affect cellular granulation either under control conditions or in AA-treated cells. However, carvacrol selectively inhibited the AA-induced accumulation of cellular lipids, the effect of which was prevented by ruthenium red, indicating that indeed TRPV3 mediates the effect (see Supplementary Figure S4a online). Simultaneously, carvacrol down-regulated PPAR-γ and NRIP1, important positive regulators of sebaceous lipid synthesis (Dozsa et al., 2014Dozsa A. Dezso B. Toth B.I. Bacsi A. Poliska S. Camera E. et al.PPARγ-mediated and arachidonic acid-dependent signaling is involved in differentiation and lipid production of human sebocytes.J Invest Dermatol. 2014; 134: 910-920Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, Oláh et al., 2014Oláh A. Tóth B.I. Borbíró I. Sugawara K. Szöllõsi A.G. Czifra G. et al.Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes.J Clin Invest. 2014; 124: 3713-3724Crossref PubMed Scopus (152) Google Scholar). These results suggest that TRPV3-mediated Ca2+ signaling generally inhibits lipid synthesis of sebocytes independently of the activated lipogenic pathways, confirming a recent study of our group on the role of Ca2+ signaling in human sebocytes using another experimental setting (Zouboulis et al., 2017Zouboulis C.C. Seltmann H. Abdel-Naser M.B. Hossini A.M. Menon G.K. Kubba R. Effects of extracellular calcium and 1,25 dihydroxyvitamin D3 on sebaceous gland cells in vitro and in vivo.Acta Derm Venereol. 2017; 97: 313-320Crossref PubMed Scopus (19) Google Scholar). Our data are consistent with our previous findings because TRPV1 and TRPV4 activation by capsaicin and cannabidiol, respectively, inhibited sebaceous lipid synthesis (Oláh et al., 2014Oláh A. Tóth B.I. Borbíró I. Sugawara K. Szöllõsi A.G. Czifra G. et al.Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes.J Clin Invest. 2014; 124: 3713-3724Crossref PubMed Scopus (152) Google Scholar, Tóth et al., 2009Tóth B.I. Géczy T. Griger Z. Dózsa A. Seltmann H. Kovács L. et al.Transient receptor potential vanilloid-1 signaling as a regulator of human sebocyte biology.J Invest Dermatol. 2009; 129: 329-339Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Beyond lipid synthesis, SGs play an important role in the regulation of cutaneous immune functions (Tóth et al., 2011Tóth B.I. Oláh A. Szöllosi A.G. Czifra G. Bíró T. "Sebocytes' makeup": novel mechanisms and concepts in the physiology of the human sebaceous glands.Pflugers Arch. 2011; 461: 593-606Crossref PubMed Scopus (57) Google Scholar). Therefore, we also assessed the effect of TRPV3 activation on cytokine expression of SZ95 sebocytes. Our findings showed that transcription of several proinflammatory cytokines was unambiguously triggered by the TRPV3 agonist carvacrol within 6 hours (Figure 2b), although during this time, 2-APB was ineffective. To assess the TRPV3 specificity of the carvacrol treatment, we repeated the experiments on TRPV3-silenced SZ95 sebocytes. In this condition, we found a reduced effect of the activator compared with scrambled RNA-transfected cells (Figure 2c), again arguing for the involvement of TRPV3 in mediating the effect of carvacrol. Moreover, we found that not only the expression but also the release of some proinflammatory cytokines was decreased by TRPV3-specific RNA interference in carvacrol-treated sebocytes (Figure 2c). Taken together, our findings suggest that sebocytes might be involved in the pathogenesis of dry skin-associated inflammatory dermatoses linked to TRPV3 hyperactivity. Furthermore, our preclinical findings introduce TRPV3 as a previously unreported negative regulator of sebaceous lipid synthesis with a marked proinflammatory effect. Further clinical studies are urged to assess the clinical efficacy of TRPV3 inhibitors on the therapeutic management of certain inflammatory skin conditions. Attila Oláh: http://orcid.org/0000-0003-4122-5639 Balázs István Tóth: http://orcid.org/0000-0002-4103-4333 CCZ owns an international patent on the SZ95 sebaceous gland cell line (WO2000046353). The other authors state no conflict of interest. This work was supported through the New National Excellence Program of the Ministry of Human Capacities, by a grant from the University of Debrecen to MS, and by other Hungarian research grants (NKFI K_105369, K_120187, PD_121138, PD_121360, FK_125055, and GINOP-2.3.2-15-2016-00050). MS, OA, and BIT are recipients of the János Bolyai research scholarship of the Hungarian Academy of Sciences. Download .pdf (.49 MB) Help with pdf files Supplementary Data