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TGFβ2 Upregulates Tyrosinase Activity through Opsin-3 in Human Skin Melanocytes In Vitro

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    2 These authors contributed equally to this work.
    Yu Wang
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    2 These authors contributed equally to this work.
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    Department of Dermatology, Affiliated Hospital of Guizhou Medical University, Guiyang, China
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    2 These authors contributed equally to this work.
    Yinghua Lan
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    2 These authors contributed equally to this work.
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    Department of Dermatology, Affiliated Hospital of Guizhou Medical University, Guiyang, China
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    2 These authors contributed equally to this work.
    Xincun Yang
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    2 These authors contributed equally to this work.
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    Department of Dermatology, Affiliated Hospital of Guizhou Medical University, Guiyang, China
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  • Yangguang Gu
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    Department of Dermatology, Affiliated Hospital of Guizhou Medical University, Guiyang, China
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  • Hongguang Lu
    Correspondence
    Correspondence: Hongguang Lu, Department of Dermatology, Affiliated Hospital of Guizhou Medical University, Guiyang 550001, China.
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    Department of Dermatology, Affiliated Hospital of Guizhou Medical University, Guiyang, China
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  • Author Footnotes
    2 These authors contributed equally to this work.
Open ArchivePublished:May 21, 2021DOI:https://doi.org/10.1016/j.jid.2021.01.040
      Opsin-3 (OPN3) is a potential key regulator of human melanocyte melanogenesis. How OPN3-mediated regulation of melanocyte melanogenesis is triggered is largely unclear. TGFβ can inhibit the growth of human melanocytes and reduce melanin synthesis in melanocytes. However, whether TGFβ2 can modulate pigmentation in normal human primary melanocytes through OPN3 is entirely unknown. In this study, we constructed a coculture model with human epidermal melanocytes and keratinocytes. OPN3, tyrosinase (TYR), tyrosinase-related protein (TRP)-1, and TRP-2 expression and TYR activity were detected to be higher in cocultured cells than in monocultured cells. Moreover, elevated levels of TGFβ2 were detected in the culture supernatant of melanocytes cocultured with keratinocytes. OPN3 inhibition in melanocytes decreased TYR, TRP-1, and TRP-2 expression and downregulated TYR activity. Our findings indicate that TGFβ2 upregulates TYR activity and TRP-1 and TRP-2 expression in human melanocytes through OPN3 and downstream calcium-dependent G-protein coupled signaling pathways to induce melanogenesis. Interestingly, treatment with the TGFβ2 receptor inhibitor LY2109761 (10 μM) did not inhibit TGFβ2-induced melanocyte melanogenesis though OPN3. Collectively, our data suggest that TGFβ2 upregulates TYR activity through OPN3 through a TGFβ2 receptor–independent and calcium-dependent G-protein coupled signaling pathway.

      Graphical abstract

      Abbreviations:

      GPCR (G-protein coupled receptor), KC (keratinocyte), NHEK (normal human epidermal keratinocyte), NHEM (normal human epidermal melanocyte), OPN3 (opsin-3), p-CAMKII (phosphorylated CAMKII), p-CREB (phosphorylated CREB), p-MITF (phosphorylated MITF), siRNA (small interfering RNA), TGFβ2R (TGFβ2 receptor), TRP (tyrosinase-related protein), TYR (tyrosinase)

      Introduction

      G-protein coupled receptors (GPCRs) can mediate numerous physiological signaling processes in humans and are the most important targets of drugs that are currently in clinical use (
      • Davenport A.P.
      • Scully C.C.G.
      • de Graaf C.
      • Brown A.J.H.
      • Maguire J.J.
      Advances in therapeutic peptides targeting G protein-coupled receptors.
      ). Signaling through GPCRs, including histamine (
      • Lee H.J.
      • Park M.K.
      • Lee E.J.
      • Kim Y.L.
      • Kim H.J.
      • Kang J.H.
      • et al.
      Histamine receptor 2-mediated growth-differentiation factor-15 expression is involved in histamine-induced melanogenesis.
      ) and leukotriene receptors (
      • Moore A.R.
      • Ceraudo E.
      • Sher J.J.
      • Guan Y.
      • Shoushtari A.N.
      • Chang M.T.
      • et al.
      Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma.
      ), can directly regulate melanin production through Gs and Gi subunits (
      • Natale C.A.
      • Duperret E.K.
      • Zhang J.
      • Sadeghi R.
      • Dahal A.
      • O'Brien K.T.
      • et al.
      Sex steroids regulate skin pigmentation through nonclassical membrane-bound receptors.
      ).
      Opsin is classified as a member of the class A (rhodopsin-like) family of light-sensitive GPCRs (
      • Rodríguez-Espigares I.
      • Torrens-Fontanals M.
      • Tiemann J.K.S.
      • Aranda-García D.
      • Ramírez-Anguita J.M.
      • Stepniewski T.M.
      • et al.
      GPCRmd uncovers the dynamics of the 3D-GPCRome.
      ). Opsin-3 (OPN3) was first cloned in 1999 from mouse brain (
      • Blackshaw S.
      • Snyder S.H.
      Encephalopsin: a novel mammalian extraretinal opsin discretely localized in the brain.
      ). Previous research has shown that OPN3 induces photopigment formation in various nonvisual tissues by activating GPCR signaling (
      • Koyanagi M.
      • Takada E.
      • Nagata T.
      • Tsukamoto H.
      • Terakita A.
      Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue.
      ;
      • Ozdeslik R.N.
      • Olinski L.E.
      • Trieu M.M.
      • Oprian D.D.
      • Oancea E.
      Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor.
      ;
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      ). Recently, our team and others have detected high expression of OPN3 in human epidermal melanocytes in vitro (
      • Haltaufderhyde K.
      • Ozdeslik R.N.
      • Wicks N.L.
      • Najera J.A.
      • Oancea E.
      Opsin expression in human epidermal skin.
      ;
      • Wang Y.
      • Lu H.
      739 opsin expression in human epidermis melanocytes co-cultured with keratinocytes in vitro.
      ;
      • Wang Y.
      • Lan Y.
      • Lu H.
      Opsin3 downregulation induces apoptosis of human epidermal melanocytes via mitochondrial pathway.
      ). OPN3 may participate in human pigmentation and in the apoptosis of normal melanocytes (
      • Ozdeslik R.N.
      • Olinski L.E.
      • Trieu M.M.
      • Oprian D.D.
      • Oancea E.
      Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor.
      ;
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      ;
      • Wang Y.
      • Lan Y.
      • Lu H.
      Opsin3 downregulation induces apoptosis of human epidermal melanocytes via mitochondrial pathway.
      ). However, the exact function of OPN3 in human epidermal melanocytes needs further study.
      Interestingly, two independent research groups have drawn contradictory conclusions about OPN3-mediated regulation of melanocyte pigmentation in epidermal melanocytes exposed to different light doses or culture medium ingredients (
      • Ozdeslik R.N.
      • Olinski L.E.
      • Trieu M.M.
      • Oprian D.D.
      • Oancea E.
      Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor.
      ;
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      ).
      • Ozdeslik R.N.
      • Olinski L.E.
      • Trieu M.M.
      • Oprian D.D.
      • Oancea E.
      Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor.
      cultured melanocytes in a rich medium (Medium 254, Cascade Biologics, Gibco, Waltham, MA) that enhanced cell activation, whereas
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      maintained the cells in a physiological medium (MCDB 153 Medium). The expression of opsins can be regulated by different culture conditions and cell–cell contact (
      • Jacob V.
      • Rothermel A.
      • Wolf P.
      • Layer P.G.
      Rhodopsin, violet and blue opsin expressions in the chick are highly dependent on tissue and serum conditions.
      ;
      • Ueda K.
      • Onishi A.
      • Ito S.I.
      • Nakamura M.
      • Takahashi M.
      Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells.
      ). The difference in findings may be explained by the different expression levels of OPN3 in melanocytes under unstimulated culture conditions (
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      ). These results prompted us to investigate whether OPN3 regulates melanogenesis in human epidermal melanocytes directly and how this OPN3-mediated signal is triggered under physiological conditions.
      Melanocyte pigmentation is controlled by the regulation of the balance between MITF-mediated promelanogenic pathways and TGFβ2 receptor (TGFβ2R)-mediated antimelanogenic pathways (
      • Dai X.
      • Rao C.
      • Li H.
      • Chen Y.
      • Fan L.
      • Geng H.
      • et al.
      Regulation of pigmentation by microRNAs: MITF-dependent microRNA-211 targets TGF-β receptor 2.
      ). Previous studies have shown that some paracrine factors secreted by adjacent keratinocytes (KCs) can regulate melanogenesis in melanocytes (
      • Yuan X.H.
      • Jin Z.H.
      Paracrine regulation of melanogenesis.
      ); for example, TGFβ secreted from human KCs plays an important role in regulating melanocyte differentiation (
      • Yuan X.H.
      • Jin Z.H.
      Paracrine regulation of melanogenesis.
      ). TGFβ targets PAX3 to negatively regulate the proliferation and differentiation of normal melanocytes through the induction of SMAD signaling (
      • Yang G.
      • Li Y.
      • Nishimura E.K.
      • Xin H.
      • Zhou A.
      • Guo Y.
      • et al.
      Inhibition of PAX3 by TGF-beta modulates melanocyte viability.
      ). Recent studies have shown that TGFβ2R may be a potential target of some melanin synthesis signaling molecules (
      • Dai X.
      • Rao C.
      • Li H.
      • Chen Y.
      • Fan L.
      • Geng H.
      • et al.
      Regulation of pigmentation by microRNAs: MITF-dependent microRNA-211 targets TGF-β receptor 2.
      ). However, TGFβ2, another representative member of the TGFβ family, may not affect the melanogenesis in cultured uveal melanocytes at physiological concentrations (
      • Hu D.N.
      • McCormick S.A.
      • Lin A.Y.
      • Lin J.Y.
      TGF-beta2 inhibits growth of uveal melanocytes at physiological concentrations.
      ).
      Recent research has found that the TGFβ/SMAD signaling pathway engages in cross-talk with other signaling pathways regulating cell proliferation, survival, and differentiation at multiple levels (
      • Luo K.
      Signaling cross talk between TGF-β/Smad and other signaling pathways.
      ). The expression of opsin has been found to be induced by growth hormone in the retinas of a salmonid fish (
      • Novales Flamarique I.
      • Sayed Ahmed A.
      • Cheng C.L.
      • Molday R.S.
      • Devlin R.H.
      Growth hormone regulates opsin expression in the retina of a salmonid fish.
      ). However, whether OPN3 is regulated by TGFβ superfamily members has yet to be determined.
      In this study, we successfully constructed a coculture model with human epidermal melanocytes and KCs. We found that the expression of OPN3 in melanocytes is upregulated by the elevated levels of TGFβ2 in the supernatant in this coculture model. We present evidence that TGFβ2 upregulates tyrosinase (TYR) activity and tyrosinase-related protein (TRP)-1 and TRP-2 expression in human melanocytes through OPN3 and downstream calcium-dependent G-protein coupled signaling pathways to induce melanogenesis. This TGFβ2-induced melanocyte melanogenesis is independent of TGFβ2R. To our knowledge, our data have identified a previously unreported melanogenic regulatory mechanism and a key function of OPN3 in human skin.

      Results

      OPN3 was upregulated in human epidermal melanocytes cocultured with KCs with or without cell chambers compared with that seen in monocultured human epidermal melanocytes

      To investigate the differences in OPN3 expression in human epidermal melanocytes under different culture conditions, we constructed a coculture model with normal human epidermal melanocytes (NHEMs) and normal human epidermal KCs (NHEKs). Successful model establishment was confirmed by double immunocytochemical staining of Melan-A and Pan Cytokeratin (Figure 1a). The expression of OPN3 in epidermal melanocytes was higher in the cocultured group than in the monocultured group (P < 0.05) (Figure 1b and c).
      Figure thumbnail gr1
      Figure 1OPN3 was upregulated in human epidermal melanocytes cocultured with keratinocytes with or without cell chambers compared with that in monocultured human epidermal melanocytes. (a) Cell culture and establishment of cocultured system of NHEMs and NHEKs in vitro. Bar = 20 μm. (b) Relative expression level of OPN3 were analyzed by qPCR. OPN3 level was normalized to GAPDH level (n = 3) in monocultured human epidermal melanocytes and cocultured human epidermal melanocytes and keratinocytes. ∗P < 0.05. (c) The cell lysate in monocultured human epidermal melanocytes and cocultured human epidermal melanocytes and keratinocytes was analyzed by western blot using anti-OPN3 antibody and β-actin. The relative protein level was quantified using Quantity-One software (Bio-Rad, Hercules, CA) (n = 3). (d) The cell lysate in monocultured human epidermal melanocytes and coculture model in cell chambers of human epidermal melanocytes and keratinocytes were analyzed by western blot using anti-OPN3 antibody and β-actin. The relative protein level was quantified using Quantity-One software (n = 3). Bar = 20 μm. IM, light microscope; IHC, immunohistochemistry; NHEK, normal human epidermal keratinocytes; NHEM, normal human epidermal melanocytes; OPN3, opsin-3.
      To eliminate the effects of direct cell–cell interactions of NHEMs and NHEKs on the expression of OPN3 (
      • Chung H.
      • Jung H.
      • Jho E.H.
      • Multhaupt H.A.B.
      • Couchman J.R.
      • Oh E.S.
      Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation.
      ;
      • Kumar R.
      • Parsad D.
      • Kanwar A.
      • Kaul D.
      Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes.
      ), coculture models were also constructed in cell chambers with NHEM-to-NHEK ratios of 1:2, 1:5, and 1:10 (Figure 1d). We found that the protein expression of OPN3 was higher in the NHEM–NHEK cocultured group than in the NHEM monocultured group, especially in the cocultured group with a ratio of 1:5 (Figure 1d).
      Further analysis showed that the melanocytes in the cocultured group were actively proliferating, as indicated by the increased cell volumes and increased numbers of dendrites (Figure 1a and d).

      OPN3 upregulation increased active proliferation, upregulated TYR activity, and upregulated the expression of TRPs and calcium channel signaling proteins in the NHEMs of the cocultured group

      Flow cytometry analysis further indicated that higher percentages of cells were in G2 phase and S phase in the NHEM–NHEK co-cultured group than in the NHEM monocultured group (Figure 2a and b). At 48 hours after transfection with RNA interference of OPN3 to inhibit OPN3, the NHEMs in the monoculture group exhibited increased numbers of melanocytes in G1 phase and decreased numbers of melanocytes in G2 phase and S phase (Figure 2c, d, and e).
      Figure thumbnail gr2ae
      Figure 2OPN3 upregulation increased active proliferation, upregulated TYR activity, and upregulated the expression of TRPs and calcium channel signaling proteins in the NHEMs of the cocultured group. (a, b) Cell cycle of NHEMs was determined using flow cytometry analysis in (a) monocultured NHEM group and (b) cocultured NHEM and NHEK group in vitro. Percentage of G1-phase and percentage of G2-phase and S-phase cells detected in the NHEM–NHEK cocultured group are lower and higher, respectively, than those detected in the NHEM monocultured group. (c, d, e) At 48 hours after the transfection of RNAi-OPN3, (c) cell cycle of NHEMs of the control group, RNAi-control group, and (e) RNAi-OPN3 group was determined using flow cytometry analysis in monocultured NHEMs in vitro. OPN3 inhibition in NHEMs increased the number of melanocytes in G1 phase and decreased the number of melanocytes in G2 and S phases. (f, g, h) The intracellular Ca2+ level of NHEMs was detected through fluorometric Ca2+ imaging in (f) monocultured NHEMs group and (g) NHEM–NHEK cocultured group in vitro. (h) The intracellular Ca2+ concentration of melanocytes of NHEM–NHEK cocultured group was higher than that seen in the NHEM monocultured group, and this was detected using flow cytometry. Bar = 20 μm. (i, j) At 48 hours after the transfection of RNAi-OPN3, the intracellular calcium levels of NHEMs control group, RNAi-control group, and RNAi-OPN3 group were detected through fluorometric Ca2+ imaging in (i) monocultured NHEMs in vitro. (i, j) The intracellular Ca2+ concentration of melanocytes of RNAi-OPN3 group was lower than those in the control group and the RNAi-control group, and this was detected by flow cytometry. Bar = 20 μm. (k, l, m) The relative protein levels of melanocyte lysates in monocultured NHEM group and NHEM–NHEK cocultured group were analyzed by western blot. The relative protein level was quantified using Quantity-One software (n = 3). (k) The protein content of OPN3, TYR, TRP-1, and TRP-2 in the NHEM–NHEK cocultured group was higher than those in the NHEM monocultured group. (l) Similar results were found in the expression of p-MITF, p-CAMKⅡ, and p-CREB in the NHEM–NHEK co-cultured group. (m) In addition, higher TYR activity was detected in NHEM–NHEK cocultured group. (n, o, p) At 48 hours after the transfection of RNAi-OPN3, the relative protein levels of melanocyte lysates in the control group, RNAi-control group, and RNAi-OPN3 group were analyzed by western blot. The relative protein level was quantified using Quantity-One software (n = 3). (n) The protein content of OPN3, TYR, TRP-1, and TRP-2 in the RNAi-OPN3 group was lower than those in the control group and RNAi-control group. (o) Similar results were found in the expression of p-MITF, p-CAMKⅡ, and p-CREB in the RNAi-OPN3 group. (p) In addition, lower TYR activity was detected in the RNAi-OPN3 group. ∗P < 0.05; ∗∗P < 0.01. Ca2+, calcium ion, NHEK, normal human epidermal keratinocytes; NHEM, normal human epidermal melanocytes; OPN3, opsin-3; p-CAMKII, phosphorylated CAMKII; p-CREB, phosphorylated CREB; p-MITF, phosphorylated MITF; RNAi, RNA interference; TYR, tyrosinase.
      Figure thumbnail gr2fp
      Figure 2OPN3 upregulation increased active proliferation, upregulated TYR activity, and upregulated the expression of TRPs and calcium channel signaling proteins in the NHEMs of the cocultured group. (a, b) Cell cycle of NHEMs was determined using flow cytometry analysis in (a) monocultured NHEM group and (b) cocultured NHEM and NHEK group in vitro. Percentage of G1-phase and percentage of G2-phase and S-phase cells detected in the NHEM–NHEK cocultured group are lower and higher, respectively, than those detected in the NHEM monocultured group. (c, d, e) At 48 hours after the transfection of RNAi-OPN3, (c) cell cycle of NHEMs of the control group, RNAi-control group, and (e) RNAi-OPN3 group was determined using flow cytometry analysis in monocultured NHEMs in vitro. OPN3 inhibition in NHEMs increased the number of melanocytes in G1 phase and decreased the number of melanocytes in G2 and S phases. (f, g, h) The intracellular Ca2+ level of NHEMs was detected through fluorometric Ca2+ imaging in (f) monocultured NHEMs group and (g) NHEM–NHEK cocultured group in vitro. (h) The intracellular Ca2+ concentration of melanocytes of NHEM–NHEK cocultured group was higher than that seen in the NHEM monocultured group, and this was detected using flow cytometry. Bar = 20 μm. (i, j) At 48 hours after the transfection of RNAi-OPN3, the intracellular calcium levels of NHEMs control group, RNAi-control group, and RNAi-OPN3 group were detected through fluorometric Ca2+ imaging in (i) monocultured NHEMs in vitro. (i, j) The intracellular Ca2+ concentration of melanocytes of RNAi-OPN3 group was lower than those in the control group and the RNAi-control group, and this was detected by flow cytometry. Bar = 20 μm. (k, l, m) The relative protein levels of melanocyte lysates in monocultured NHEM group and NHEM–NHEK cocultured group were analyzed by western blot. The relative protein level was quantified using Quantity-One software (n = 3). (k) The protein content of OPN3, TYR, TRP-1, and TRP-2 in the NHEM–NHEK cocultured group was higher than those in the NHEM monocultured group. (l) Similar results were found in the expression of p-MITF, p-CAMKⅡ, and p-CREB in the NHEM–NHEK co-cultured group. (m) In addition, higher TYR activity was detected in NHEM–NHEK cocultured group. (n, o, p) At 48 hours after the transfection of RNAi-OPN3, the relative protein levels of melanocyte lysates in the control group, RNAi-control group, and RNAi-OPN3 group were analyzed by western blot. The relative protein level was quantified using Quantity-One software (n = 3). (n) The protein content of OPN3, TYR, TRP-1, and TRP-2 in the RNAi-OPN3 group was lower than those in the control group and RNAi-control group. (o) Similar results were found in the expression of p-MITF, p-CAMKⅡ, and p-CREB in the RNAi-OPN3 group. (p) In addition, lower TYR activity was detected in the RNAi-OPN3 group. ∗P < 0.05; ∗∗P < 0.01. Ca2+, calcium ion, NHEK, normal human epidermal keratinocytes; NHEM, normal human epidermal melanocytes; OPN3, opsin-3; p-CAMKII, phosphorylated CAMKII; p-CREB, phosphorylated CREB; p-MITF, phosphorylated MITF; RNAi, RNA interference; TYR, tyrosinase.
      Previous work has demonstrated that intracellular calcium plays a central role in determining the fate of the cell (
      • Belote R.L.
      • Simon S.M.
      Ca2+ transients in melanocyte dendrites and dendritic spine-like structures evoked by cell-to-cell signaling.
      ;
      • La Rovere R.M.
      • Roest G.
      • Bultynck G.
      • Parys J.B.
      Intracellular Ca(2+) signaling and Ca(2+) microdomains in the control of cell survival, apoptosis and autophagy.
      ;
      • Scorrano L.
      • Oakes S.A.
      • Opferman J.T.
      • Cheng E.H.
      • Sorcinelli M.D.
      • Pozzan T.
      • et al.
      BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis.
      ;
      • Wang Y.
      • Lan Y.
      • Lu H.
      Opsin3 downregulation induces apoptosis of human epidermal melanocytes via mitochondrial pathway.
      ). In addition, calcium flux increases after the upregulation of OPN3 induced by blue light (
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      ). Therefore, intracellular calcium levels in NHEMs in the monocultured NHEM group and the NHEM–NHEK cocultured group (Figure 2f, g, and h) were detected in vitro through fluorometric calcium imaging and flow cytometry. The intracellular calcium concentrations of melanocytes in the NHEM–NHEK cocultured group were increased (Figure 2f, g, and h). At 48 hours after transfection of RNA interference of OPN3, the intracellular calcium concentrations were decreased significantly in the NHEM monocultured group (Figure 2i and j).
      Our results also indicated that the protein content of TYR, TRP-1, and TRP-2 in the NHEM–NHEK coculture group was higher than that in the NHEM monocultured group (Figure 2k). Similar results were found for the expression of phosphorylated MITF (p-MITF), phosphorylated CAMKII (p-CAMKII), and phosphorylated CREB (p-CREB) in the NHEM–NHEK cocultured group (Figure 2l). Elevated TYR activity was also detected in the NHEM–NHEK cocultured group (Figure 2m).
      To determine whether OPN3 induces TRP and calcium channel signaling protein expression and increases TYR activity, we knocked down OPN3 in the NHEM monocultured group using small interfering RNA (siRNA). OPN3 inhibition in NHEMs decreased TYP, TRP-1, TRP-2, p-MITF, p-CAMKII, and p-CREB protein expression (Figure 2n and o) and reduced TYR activity (Figure 2p). These results show that highly expressed OPN3 upregulates the activity of TYR and the expression of related proteins and calcium channel signaling proteins in melanocytes.

      TGFβ2 upregulated TRP expression and TYR activity in the NHEMs of the cocultured group through OPN3

      In this study, we detected the levels of TGFβ, TGFβ1, and TGFβ2 in the supernatant of the cocultured model by ELISA. Our results revealed higher levels of TGFβ2 (Figure 3a), higher levels of TGFβ (Figure 3b), and lower levels of TGFβ1 (Figure 3a) in the supernatant in the cocultured group than seen in the monoculture group. This observation led us to further investigate the mechanisms of interaction between TGFβ2 derived from KCs and OPN3 in melanocytes.
      Figure thumbnail gr3
      Figure 3TGFβ2 upregulates TRP expression and TYR activity in the NHEMs of cocultured group through OPN3. (a) The levels of TGFβ, TGFβ1, and TGFβ2 in the supernatant of NHEM–NHEK cocultured group were detected by ELISA. Levels of TGFβ2 and TGFβ were higher and level of TGFβ1 was lower in the supernatant of cocultured group than the levels seen in the monocultured group. ∗P < 0.05. (b) At various time points after adding 10 ng/ml TGFβ2 to cultured NHEMs, the expression of OPN3 was analyzed by western blot. The relative protein level was quantified using Quantity-One software (n = 3). At 24 h, 48 h, and 72 h after adding 10 ng/ml TGFβ2, the expression of OPN3 increased at the protein level and peaked at 48 h in vitro. At various time points after adding 10 ng/ml TGFβ2 to cultured NHEMs, the expression of TYR, TRP-1, and TRP-2 was analyzed by western blot. The relative protein level was quantified using Quantity-One software (n = 3). Similar results as OPN3 were found related to the expression of TYR, TRP-1 and TRP-2 at the protein level. (c) At various time points after adding 10 ng/ml TGFβ2 to cultured NHEMs, the activity of TYR upregulated and peaked at 48 h in vitro. (d) At various time points after adding 10 ng/ml TGFβ2 to cultured NHEMs, the melanin content increased and peaked at 48 h in vitro. h, hour; NHEK, normal human epidermal keratinocytes; NHEM, normal human epidermal melanocytes; OD, optical density; OPN3, opsin-3; TYR, tyrosinase.
      To explore the possible mechanisms, we added 10 ng/ml TGFβ2 to cultured NHEMs and then detected the expression of OPN3, TYR, TRP-1, and TRP-2 at various time points. OPN3 protein levels were increased 24 hours, 48 hours, and 72 hours after the addition of 10 ng/ml TGFβ2 and peaked at 48 hours in vitro (Figure 3b). Similar results were found regarding the protein expression of TYR, TRP-1, and TRP-2 (Figure 3b). TYR activity and melanin content showed trends similar to that of the expression of OPN3 (Figure 3c and d). The results suggest that TGFβ2 upregulates TRP expression and TYR activity in NHEMs likely through OPN3.

      TGFβ2 regulated TYR activity in human epidermal melanocytes through a calcium ion pathway independent of TGFβ2R

      In this study, we uncovered the possible signaling pathway induced by TGFβ2. After incubation with 10 ng/ml TGFβ2 for 48 hours, using a laser scanning confocal microscope, we found that TGFβ2 can increase the nuclear localization of p-MITF (Figure 4a). In addition, we found that after incubation with 10 ng/ml TGFβ2 for 48 hours, the protein expression of OPN3, p-MITF, p-CAMKII, and p-CREB was upregulated in melanocytes (Figure 4b). After simultaneous incubation with 10 ng/ml TGFβ2 and 9 mM U73122 for 48 hours, the protein expression of OPN3, p-MITF, p-CAMKII, and p-CREB was downregulated (Figure 4b). TYR activity (Figure 4c) and melanin content (Figure 4d) showed trends similar to those of the expression of OPN3, p-MITF, p-CAMKII, and p-CREB. These data suggest that TGFβ2 regulates TYR activity in NHEMs through a calcium-dependent pathway.
      Figure thumbnail gr4
      Figure 4TGFβ2 regulated TYR activity in human epidermal melanocytes through a calcium ion pathway independent of TGFβ2R. (a) The cultured human epidermal melanocytes were treated with 10 ng/ml TGFβ2 for 48 hours. Under a laser scanning confocal microscope, we detected the increase of the nuclear localization of p-MITF in the cells. p-MITF (green), Melan-A (red), and DAPI (blue) were shown using immunofluorescence. Bar = 20 μm). (b) The relative protein levels of melanocyte lysates in monoculture of NHEMs was analyzed by western blot. The relative protein level was quantified using Quantity-One software (n = 3). After incubation with 10 ng/ml TGFβ2 for 48 hours, the expressions of p-MITF, p-CAMKⅡ, and p-CREB at the protein level was upregulated in melanocytes. After simultaneous incubation with 10 ng/ml TGFβ2 and 9 mM U73122 for 48 hours, the expression of p-MITF, p-CAMKⅡ, and p-CREB at the protein level was downregulated. (c) After incubation with 10 ng/ml TGFβ2 for 48 hours, the activity of TYR was upregulated in melanocytes. After simultaneous incubation with 10 ng/ml TGFβ2 and 9 mM U73122 for 48 hours, the activity of TYR was downregulated. (d) After incubation with 10 ng/ml TGFβ2 for 48 hours, the melanin content increased in melanocytes. After simultaneous incubation with 10 ng/ml TGFβ2 and 9 mM U73122 for 48 hours, the melanin content decreased. (e) The relative protein levels of melanocyte lysates in monocultured NHEM group was analyzed by western blot. The relative protein level was quantified using Quantity-One software (n = 3). At 48 hours after adding 10 μM LY2109761, there was no significant change in the expression of OPN3 and TGFβ2R between the experimental group and the blank control group. The expression of SMAD2, p-SMAD2, SMAD3, and p-SMAD3 decreased at the protein level in vitro. After simultaneous incubation with 10 ng/ml TGFβ2 and 10 μM LY2109761 for 48 hours, the expression of OPN3 and TGFβ2R at the protein level increased compared with that seen in the blank control group. Similar results were found in the expression of SMAD2 and p-SMAD2 at the protein level. However, there was no significant change in the expression of SMAD3 and p-SMAD3 between the experimental group and the blank control group. ∗P < 0.05; ∗∗P < 0.01. OD, optical density; OPN3, opsin-3; p-CAMKII, phosphorylated CAMKII; p-CREB, phosphorylated CREB; p-MITF, phosphorylated MITF; p-SMAD, phosphorylated SMAD; TYR, tyrosinase.
      Interestingly, treatment with the TGFβ2R inhibitor LY2109761 (10 μM) (
      • Zhang Z.H.
      • Miao Y.Y.
      • Ke B.L.
      • Liu K.
      • Xu X.
      LY2109761, transforming growth factor β receptor type I and type II dual inhibitor, is a novel approach to suppress endothelial mesenchymal transformation in human corneal endothelial cells.
      ) did not inhibit TGFβ2-induced melanocyte melanogenesis through OPN3. At 48 hours after the addition of 10 μM LY2109761, there were no significant differences in the expression of OPN3 and TGFβ2R between the experimental group and the blank control group (Figure 4e). The protein expression of SMAD2, p-SMAD2, SMAD3, and p-SMAD3 was decreased in vitro (Figure 4e). Simultaneous incubation with 10 ng/ml TGFβ2 and 10 μM LY2109761 for 48 hours increased the protein expression of OPN3 and TGFβ2R in the experimental group compared with that in the blank control group. Similar results were found in the case of protein expression of SMAD2 and p-SMAD2 (Figure 4e). However, there were no significant differences in the expression of SMAD3 and p-SMAD3 between the experimental group and the blank control group (Figure 4e). These results indicate that TGFβ2 upregulates TYR activity through OPN3 through a TGFβ2R-independent and calcium-dependent G-protein coupled signaling pathway (Figure 5).
      Figure thumbnail gr5
      Figure 5The signal pathway map of our key finding in this study. OPN3, opsin-3; TYR, tyrosinase.

      Discussion

      In human skin, cell-to-cell interaction between NHEMs and NHEKs or signaling through adjacent KC-derived soluble factors regulates melanocyte behaviors, including melanogenesis (
      • Kumar R.
      • Parsad D.
      • Kanwar A.
      • Kaul D.
      Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes.
      ). Previous studies have exhibited the function of OPN3 in the regulation of melanogenesis in human epidermal melanocytes in vitro (
      • Ozdeslik R.N.
      • Olinski L.E.
      • Trieu M.M.
      • Oprian D.D.
      • Oancea E.
      Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor.
      ;
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      ). In addition, the expression of opsins can be regulated by different culture conditions and cell–cell contact (
      • Jacob V.
      • Rothermel A.
      • Wolf P.
      • Layer P.G.
      Rhodopsin, violet and blue opsin expressions in the chick are highly dependent on tissue and serum conditions.
      ;
      • Ueda K.
      • Onishi A.
      • Ito S.I.
      • Nakamura M.
      • Takahashi M.
      Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells.
      ). However, in two-dimensional culture systems of NHEMs and NHEKs, the role of OPN3 in the regulation of the melanogenic pathway in epidermal melanocytes is still unknown. To faithfully replicate the conditions found in actual skin (
      • Kumar R.
      • Parsad D.
      • Kanwar A.
      • Kaul D.
      Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes.
      ), we successfully established a coculture model of NHEMs and NHEKs using suspended epidermal cells, which were characterized by a double-staining method. Our results showed that the mRNA and protein expression of OPN3 was higher in the NHEM–NHEK cocultured group than in the NHEM monocultured group (P < 0.05). This finding indicates that the expression of OPN3 can be upregulated by cell-to-cell interaction between NHEMs and NHEKs or by signaling through adjacent KC-derived soluble factors.
      To eliminate the effect of direct cell–cell interaction between NHEMs and NHEKs on the expression of OPN3 (
      • Belote R.L.
      • Simon S.M.
      Ca2+ transients in melanocyte dendrites and dendritic spine-like structures evoked by cell-to-cell signaling.
      ;
      • Chung H.
      • Jung H.
      • Jho E.H.
      • Multhaupt H.A.B.
      • Couchman J.R.
      • Oh E.S.
      Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation.
      ), a coculture model was also constructed with NHEMs and NHEKs in cell chambers at different ratios. Higher protein expression of OPN3 was detected in the NHEM–NHEK coculture than that detected in the NHEM monoculture, especially at an NHEM-to-NHEK ratio of 1:5. These findings suggest that some neighboring KC-derived soluble factors may upregulate the expression of OPN3 during NHEM–NHEK coculture.
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      found that OPN3 is the key blue light sensor responsible for the melanogenesis in melanocytes and that it regulates this process in a calcium-dependent and MITF-dependent manner.
      • Ozdeslik R.N.
      • Olinski L.E.
      • Trieu M.M.
      • Oprian D.D.
      • Oancea E.
      Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor.
      revealed that OPN3 acts as a negative regulator of MC1R-mediated melanogenic signaling induced by UVR in human epidermal melanocytes. Related studies have focused on the function of OPN3 under light radiation. The role of OPN3 in melanogenesis in melanocytes under physiological conditions is not completely understood.
      Our results further demonstrated that the increase in OPN3 expression upregulated the activity of TYR and the expression of TRPs and calcium channel signaling proteins in the NHEM–NHEK cocultured group. In addition, OPN3 upregulation in NHEMs induced active proliferation characterized by increased cell volumes, increased numbers of dendrites, and increased percentages of G2-phase and S-phase cells in the cocultured group. Inhibiting OPN3 in NHEMs in the monocultured group with RNA interference of Opn3 increased the number of melanocytes in G1 phase; decreased the numbers of melanocytes in G2 phase and S phase; downregulated TYR, TRP-1, TRP-2, p-MITF, p-CAMKII, and p-CREB protein expression; and reduced TYR activity. These results indicate that OPN3 induces cell proliferation and enhances TYR activity in NHEMs through a calcium-dependent G-protein coupled signaling pathway, consistent with the findings of previous studies (
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      ;
      • Wang Y.
      • Lan Y.
      • Lu H.
      Opsin3 downregulation induces apoptosis of human epidermal melanocytes via mitochondrial pathway.
      ).
      Which KC-derived soluble factors upregulate OPN3 in NHEMs? α-Melanocyte stimulating hormone, ACTH/ACTH fragments, TGFβ, and nerve GF released from KCs can regulate the melanogenesis of NHEMs through multiple signaling pathways, including the protein kinase C, protein kinase A, MK, and phospholipase C pathways, in primary culture (
      • Hirobe T.
      Role of keratinocyte-derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes.
      ;
      • Yuan X.H.
      • Jin Z.H.
      Paracrine regulation of melanogenesis.
      ). As the OPNs are a family of class A light-sensitive GPCRs, their expression can also be induced by some cytokines and growth hormones, such as endothelin (
      • Moraes M.N.
      • Lima L.H.
      • Ramos B.C.
      • Poletini Mde O.
      • Castrucci A.M.
      Endothelin modulates the circadian expression of non-visual opsins.
      ) and thyroid hormone (
      • Novales Flamarique I.
      • Sayed Ahmed A.
      • Cheng C.L.
      • Molday R.S.
      • Devlin R.H.
      Growth hormone regulates opsin expression in the retina of a salmonid fish.
      ). However, in human skin, whether OPN3 is regulated by neighboring KC-derived soluble factors remains to be clarified.
      TGFβ-family proteins play vital roles in cellular physiology by regulating cell proliferation, survival, and differentiation (
      • Luo K.
      Signaling cross talk between TGF-β/Smad and other signaling pathways.
      ). TGFβ acts as a negative modulator of melanocyte proliferation (
      • Yang G.
      • Li Y.
      • Nishimura E.K.
      • Xin H.
      • Zhou A.
      • Guo Y.
      • et al.
      Inhibition of PAX3 by TGF-beta modulates melanocyte viability.
      ;
      • Yuan X.H.
      • Jin Z.H.
      Paracrine regulation of melanogenesis.
      ). This impelled us to explore whether TGFβ-family proteins trigger the OPN3-mediated regulatory effects on NHEMs.
      In this study, a higher level of TGFβ2 was detected in the supernatant of the cocultured group than in that of the monocultured group. TGFβ2 (10 ng/ml) upregulated the expression of OPN3 in monocultured melanocytes in a time-dependent manner. Similar results related to the protein expression of TYR, TRP-1, and TRP-2 were found. The TYR activity and melanin content showed trends similar to those of the expression of OPN3. These results demonstrate that TGFβ2 can upregulate TRP expression and TYR activity in NHEMs likely through OPN3. Previous reports have shown that TGFβ2 may not affect the melanogenesis in cultured uveal melanocytes at physiological concentrations (
      • Hu D.N.
      • McCormick S.A.
      • Lin A.Y.
      • Lin J.Y.
      TGF-beta2 inhibits growth of uveal melanocytes at physiological concentrations.
      ). However, in this study, we drew the opposite conclusion: TGFβ2 can upregulate TYR activity and promote melanin synthesis through OPN3.
      How does TGFβ2 upregulate the activity of TYR through OPN3 in epidermal melanocytes in vitro? Previous studies have shown that calcium flux increases after upregulation of OPN3 induced by blue light (
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      ). We found in this study that after incubation with 10 ng/ml TGFβ2 for 48 hours, the protein expression of p-MITF, p-CAMKII, and p-CREB was upregulated in melanocytes. These effects were blocked after simultaneous incubation with 10 ng/ml TGFβ2 and 9 mM U73122 (an inhibitor of calcium flux) for 48 hours.
      It has been demonstrated that MITF acts as the master regulator of melanocyte proliferation, migration, survival, and differentiation (
      • Fock V.
      • Gudmundsson S.R.
      • Gunnlaugsson H.O.
      • Stefansson J.A.
      • Ionasz V.
      • Schepsky A.
      • et al.
      Subcellular localization and stability of MITF are modulated by the bHLH-Zip domain.
      ;
      • Goding C.R.
      • Arnheiter H.
      MITF-the first 25 years.
      ;
      • Ngeow K.C.
      • Friedrichsen H.J.
      • Li L.
      • Zeng Z.
      • Andrews S.
      • Volpon L.
      • et al.
      BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export.
      ). As MITF is a transcription factor, the subcellular localization of MITF is important for its function (
      • Fock V.
      • Gudmundsson S.R.
      • Gunnlaugsson H.O.
      • Stefansson J.A.
      • Ionasz V.
      • Schepsky A.
      • et al.
      Subcellular localization and stability of MITF are modulated by the bHLH-Zip domain.
      ;
      • Goding C.R.
      • Arnheiter H.
      MITF-the first 25 years.
      ;
      • Lu S.Y.
      • Wan H.C.
      • Li M.
      • Lin Y.L.
      Subcellular localization of Mitf in monocytic cells.
      ;
      • Ngeow K.C.
      • Friedrichsen H.J.
      • Li L.
      • Zeng Z.
      • Andrews S.
      • Volpon L.
      • et al.
      BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export.
      ).
      • Regazzetti C.
      • Sormani L.
      • Debayle D.
      • Bernerd F.
      • Tulic M.K.
      • De Donatis G.M.
      • et al.
      Melanocytes sense blue light and regulate pigmentation through opsin-3.
      further confirmed that OPN3 can induce the phosphorylation of MITF and increase the expression of p-MITF in nuclear extracts on blue light exposure. After incubation with 10 ng/ml TGFβ2, we found that TGFβ2 increased the nuclear localization of p-MITF. Our results indicate that TGFβ2 mediates p-MITF nuclear import signal through OPN3.
      Interestingly, treatment with the TGFβ2R inhibitor LY2109761 (10 μM) (
      • Zhang Z.H.
      • Miao Y.Y.
      • Ke B.L.
      • Liu K.
      • Xu X.
      LY2109761, transforming growth factor β receptor type I and type II dual inhibitor, is a novel approach to suppress endothelial mesenchymal transformation in human corneal endothelial cells.
      ) did not inhibit TGFβ2-induced melanocyte melanogenesis through OPN3. These results indicate that TGFβ2 regulates TYR activity in human epidermal melanocytes through the calcium ion pathway in a manner independent of TGFβ2R.
      Collectively, the findings of this study reveal a possible signaling pathway induced by TGFβ2. We conclude that TGFβ2 promotes melanin synthesis in a calcium-dependent and TGFβ2R-independent manner by upregulating OPN3 in human skin melanocytes. To our knowledge, this study shows a previously unreported finding that TGFβ2 produced by adjacent KCs modulates OPN3 expression in epidermal melanocytes in human skin.

      Materials and Methods

      Cell culture and establishment of a coculture system of NHEMs and NHEKs in vitro

      Epidermal cells were obtained from human children’s foreskin with a two-step enzyme digestion method as described previously (
      • Wang Y.
      • Lan Y.
      • Lu H.
      Opsin3 downregulation induces apoptosis of human epidermal melanocytes via mitochondrial pathway.
      ). Human children’s foreskin was obtained from healthy children aged 3–12 years at the Department of Dermatology, Affiliated Hospital of Guizhou Medical University, Guiyang, China. The study was approved by the ethics committee of the Affiliated Hospital of Guizhou Medical University. All subjects' parents or guardians provided written informed consent.
      Using suspended cells, a coculture model with NHEMs and NHEKs was immediately established in Medium 254 supplemented with a human melanocyte growth supplement (Gibco), L-glutamine (Solarbio Life Science, Beijing, China), and antibiotics (Solarbio Life Science). Monocultured NHEMs were also amplified in the above medium and used at the third passage.
      NHEKs were amplified in EpiLife Medium (Gibco) supplemented with a human KC growth supplement (Gibco), L-glutamine (Solarbio Life Science), and antibiotics (Solarbio Life Science). The third-passage NHEMs and NHEKs were cultured in two cell chambers without direct interaction.
      All cells were cultured at 37 °C in 5% carbon dioxide and grown to 70–80% confluence a day before the start of the experiment.

      Immunohistochemistry

      The immunohistochemistry protocol used was described previously (
      • Yang G.
      • Li Y.
      • Nishimura E.K.
      • Xin H.
      • Zhou A.
      • Guo Y.
      • et al.
      Inhibition of PAX3 by TGF-beta modulates melanocyte viability.
      ). Cells were double-labeled with a horseradish peroxidase–conjugated antibody (1:50, MDL, Beijing, China) against the melanocyte-specific protein S-100 (1:50, MDL) and the KC-specific marker Pan Cytokeratin (1:50, MDL).

      Fluorescent quantitative real-time reverse transcriptase–PCR

      Total RNA was isolated from cultured NHEMs using TRIzol (Invitrogen, Carlsbad, CA) and reverse-transcribed with SuperScript II (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time reverse transcriptase–PCR was performed using a Mastercycler ep realplex Real-time PCR System (Eppendorf, Hamburg, Germany) with SYBR Green PCR Master Mix (Tiangen Biotech, Beijing, China). The relative RNA expression of opsins was calculated using the 2 ΔΔCt method, and human GAPDH was used as an internal control. The following human primers were used in this study: OPN3, forward 5′-CAATCCAGTGATTTATGTCTTCATGATCAGAAAG-3′ and reverse 5′-GCATTTCACTTCCAGCTGCTGGTAGGT-3′ and GAPDH, forward 5′- GACATCCGCAAAGACCTG-3′ and reverse 5′-GGAAGGTGGACAGCGAG -3′.

      Antibodies and western blotting

      Protein extracts were obtained by cell lysis in radioimmunoprecipitation assay lysis buffer (Solarbio Life Science) containing 1 mM phenylmethylsulfonyl fluoride (Solarbio Life Science). Western blotting on polyvinylidene difluoride membranes was performed as described previously. Rabbit polyclonal anti–human OPN3 (1:1,000, MDL), TYP (1:1,000, MDL), TRP-1 (1:1,000, MDL), TRP-2 (1:1,000, MDL), MITF (1:1,000, MDL), CAMKII (1:1,000, MDL), CREB (1:1,000, MDL), p-MITF (1:1,000, MDL), p-CAMKII (1:1,000, MDL), p-CREB (1:1,000, MDL), SMAD2 (1:1,000, Cell Signaling Technology, Danvers, MA), p-SMAD2 (1:1,000, Cell Signaling Technology), SMAD3 (1:1,000, Cell Signaling Technology), and p-SMAD3 (1:1,000, Cell Signaling Technology) primary antibodies were used. A rabbit monoclonal anti–human β-actin (1:2000; MDL) antibody was used as an internal control. Horseradish peroxidase-labeled antirabbit and antimouse antibodies (1:2000; MDL) were used as secondary antibodies.

      TYR activity determination in cultured cells

      The TYR activity of melanocytes was estimated by measuring the oxidation rate of DL-dopa (Invitrogen) according to the manufacturer’s recommendations.

      Flow cytometry

      The cell cycle phases of human primary melanocytes were assessed by flow cytometry. Briefly, 2 × 105 cells were collected and fixed in 70% alcohol overnight at 4 °C, washed once with PBS (Solarbio Life Science), and stained with propidium iodide and RNase A (7sea Biotech, Shanghai, China) according to the kit instructions. After staining, flow cytometric analysis was performed with a FACSCalibur benchtop flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data analysis was performed using ModFit LT software (BD Biosciences).

      Calcium mobilization and calcium imaging

      Human primary melanocytes were incubated with Fluo-3-AM (2.5 mM) for 20 minutes at 37 °C in 5% carbon dioxide. Then, the cells were collected, and the intracellular calcium concentration was detected by flow cytometry.

      Measurement of TGFβ, TGFβ1, and TGFβ2 levels in culture supernatants

      TGFβ, TGFβ1, and TGFβ2 levels in culture supernatants were determined using ELISA kits (Chenglin Biotech, Beijing, China) according to the manufacturer’s recommendations.

      siRNA transfection

      Knock down of OPN3 was performed using siRNA. Three pooled siRNA oligos targeting OPN3 and negative control siRNAs were obtained from ViewSolid Biotech (Beijing, China). The OPN3 siRNA sequences were as follows: 5′-ACCUCCUCCUGGUCAACAUTT-3′, 5′-GUCACCUUUACCUUCGUGUTT-3′, 5′-CAAUUCAAGUGAUCAAGAUTT-3′, and 5′-UUCUCCGAACGUGUCACGUTT-3′. Transfection was carried out with Lipofectamine 2000 (ViewSolid Biotech) according to the manufacturer’s guidelines. After siRNA transfection, the cells were cultured for 48 hours and subjected to further detection. The controls included samples not transfected with siRNA and samples transfected with negative control siRNA.

      Immunofluorescence assay

      Human primary melanocytes were inoculated on cell climbing sheets at a density of 104 cells at 37 °C with 5% carbon dioxide for 24 hours. When the cells reached 30–50% confluence, they were fixed with 95% ethanol at room temperature for 15 minutes and dried at room temperature. After being blocked with BSA for 30 minutes at 37 °C and washed for 5 minutes three times with a 0.1 M PBS buffer solution, the cells were incubated with the primary antibody (rabbit polyclonal anti–human p-MITF, 1:100, AF3027, Affinity Biosciences, Cincinnati, OH) at 4 °C overnight. Then, the cells were washed three times with a 0.1 M PBS buffer solution for 5 minutes and incubated with rabbit polyclonal anti–human Melan-A (1:50, sc-20032, Santa Cruz Biotechnology, Dallas, Texas) for 8 hours. After being washed again with the 0.1 M PBS buffer solution (P1010-2L, Solarbio Life Science), the cells were covered with a mixture of a goat anti–mouse IgG FITC-labeled fluorescent antibody (S0017, Affinity Biosciences) and a goat anti–rabbit IgG FITC-labeled fluorescent antibody (S0013, Affinity Biosciences) for 1 hour at 37 °C and finally stained with DAPI (D21490, Gibco). The expression of p-MITF in human primary melanocytes was visualized under a confocal microscope (FV3000, Olympus, Tokyo, Japan).

      Statistics

      Statistical analysis was performed using paired two-tailed Student’s t-test and one-way ANOVA. The results were considered significant at P < 0.05. The data are presented as mean ± SD for at least three independent experiments.

      Data availability statement

      No datasets were generated or analyzed during this study.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      Jishi Wang’s (Key Laboratory of Hematological Disease Diagnostic & Treat Centre of Guizhou Province, Guiyang, China) research team provided valuable technical assistance. This work is supported by National Natural Science Foundation of China, Beijing, China (numbers: 81660616,81972920).

      Author Contributions

      Conceptualization: HL; Data Curation: HL, YW, YL; Formal Analysis: HL, YW, YL, XY, YG; Funding Acquisition: HL; Investigation: YW, YL, XY, YG; Methodology: HL, YW; Project Administration: HL, YW; Resources: HL, YW, YL, XY, YG; Supervision: HL; Validation: HL, YW, YL; Visualization: HL, YW, YL; Writing - Original Draft Preparation: HL, YW, YL; Writing - Review and Editing: HL, YW, YL

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