SOX9 is a key player in ultraviolet B-induced melanocyte differentiation and pigmentation

Abstract

SOX (SRY type HMG box) proteins are transcription factors that are predominantly known for their roles during development. During melanocyte development from the neural crest, SOX10 regulates microphthalmia-associated transcription factor, which controls a set of genes critical for pigment cell development and pigmentation, including dopachrome tautomerase and tyrosinase. We report here that another SOX factor, SOX9, is expressed by melanocytes in neonatal and adult human skin and is up-regulated by UVB exposure. We demonstrate that this regulation is mediated by cAMP and protein kinase. We also show that agouti signal protein, a secreted factor known to decrease pigmentation, down-regulates SOX9 expression. In adult and neonatal melanocytes, SOX9 regulates microphthalmia-associated transcription factor, dopachrome tautomerase, and tyrosinase promoters, leading to an increase in the expression of these key melanogenic proteins and finally to a stimulation of pigmentation. SOX9 completes the complex and tightly regulated process leading to the production of melanin by acting at a very upstream level. This role of SOX9 in pigmentation emphasizes the poorly understood impact of SOX proteins in adult tissues.

Fig. 1.

TISH analysis of SOX9 expression in the skin in vivo. (A) Antisense probes directed against SOX9 are present in the skin with strong nuclear staining, whereas staining with the sense probe against SOX9 is negative and shows the presence of SOX9 RNA in the skin. (B) Coupling of TISH with antisense probes against SOX9 and immunohistochemistry with MART1 antibody as a melanocytic marker shows that SOX9 is expressed in keratinocytes and in melanocytes.

Fig. 2.

SOX9 expression is up-regulated after UVB exposure. (A) Expression levels of SOX9 mRNA using RT-PCR in untreated NHM (basal) or 1 h after 21 mJ/cm² UVB irradiation. β-actin expression served as a control. The histogram shows quantification of the data with the means ± SD of three independent experiments. Results are expressed as a ratio compared with the basal condition. (B) Protein expression levels of SOX9 using immunoblotting in a-LP NHM without treatment (basal) and after irradiation with 21 mJ/cm² UVB at 0, 1, 2, 4, 6, 8, and 24 h. (C) Expression levels of SOX9 by immunoblotting in n-DP NHM without treatment (basal) and after irradiation with 21 mJ/cm² UVB at 1, 2, and 4 h. Numbers above the gels indicate levels of intensity compared with GAPDH. (D) Comparison of the basal level expression of SOX9 in n-LP cells compared with n-DP cells using immunoblotting. The histogram shows quantification of the data with means ± SD in three independent experiments. Results are expressed as a ratio compared with the basal condition. SOX9 is expressed at higher levels in the more pigmented melanocytes.

Fig. 3.

The action of UVB on SOX9 is mediated through the cAMP pathway. (A) ³⁵S labeling of a-LP NHM treated for 3 h with 20 μM forskolin or 21 mJ/cm² UVB or untreated (Basal). Labeled proteins were immunoprecipitated with SOX9 or GAPDH antibodies. (B) UVB acts through the activation of PKA to up-regulate SOX9. ³⁵S labeling of a-LP NHM without treatment (Basal) or with 21 mJ/cm² UVB irradiation for 3 h with or without a PKA inhibitor (5 μM H89). Proteins were immunoprecipitated with antibodies against SOX9 and GAPDH.

Fig. 4.

UVB and cAMP up-regulate SOX9 and increase its nuclear concentration. (A) Immunocytochemical staining of a-LP NHM. Four hours after treatment without (Control) or with 20 μM forskolin (FSK), the cells were fixed and stained with SOX9 antibody (red). Counterstaining was performed with DAPI (blue) to visualize nuclei. An increased expression of SOX9 was noted in cells treated with forskolin. The transversal cuts shown beneath under the Control and FSK images confirmed the nuclear localization of SOX9. (B) Immunohistochemistry with MelanoDerm 8 h after 21 mJ/cm² UVB irradiation or without UVB (Control). Samples were stained with antibodies to SOX9 (green) and MART1 (red; as a melanocytic marker). An increased staining of SOX9 was observed after UVB irradiation both in keratinocytes and in melanocytes, with a stronger increase in melanocytes and a clear nuclear localization.

Fig. 5.

SOX9 is down-regulated by ASP. (A) Expression levels of SOX9 gene using RT-PCR in n-MP NHM untreated (basal) or treated with ASP (10 nM) for 4 h. β-actin expression served as a control. The histogram shows quantification of the data with the means ± SD of three independent experiments. Results are expressed as a ratio compared with the basal condition. The expression of SOX9 is down-regulated in melanocytes treated by ASP. (B) Proteins from n-MP NHM treated 48 h with 10 nM ASP were extracted and analyzed by immunoblotting. The histogram shows quantification of the data with the means ± SD of three independent experiments. Results are expressed as a ratio compared with the basal condition. SOX9 protein expression was decreased significantly in NHM after treatment with ASP.

Fig. 6.

SOX9 directly regulates the MITF promoter. ChIP of NHM was performed with SOX9 or IgG antibodies with or without 4-h treatment with 20 μM forskolin. The DNA recovered was subjected to amplification by PCR (30 cycles) using primers for human MITF promoter and human GAPDH promoter.

Fig. 7.

SOX9 regulates MITF expression. n-MP NHM were silenced for SOX9 using siRNA. After 24 h, they were fixed, and an immunocytochemistry was performed using SOX9 (red) and MITF (green) antibodies. DAPI (blue) served as a counterstaining. Cells silenced for SOX9 (white arrow) showed no or slight expression of MITF.

Fig. 8.

SOX9 does not regulate SOX10. SOX9 was silenced in n-DP NHM by using siRNA for 48 h, after which proteins were extracted and analyzed by immunoblotting. (Left) The silencing of SOX9 does not affect the expression of SOX10. SOX9 was overexpressed for 24 h in n-DP NHM, and again, after extraction, proteins were analyzed by immunoblotting. (Right) The expression of SOX10 was not affected when SOX9 was overexpressed.

Fig. 9.

SOX9 regulates proteins involved in melanogenesis and increases pigmentation of melanocytes. (A) SOX9 was silenced in n-LP NHM by using siRNA for 24 h; then, proteins were extracted and analyzed by immunoblotting. (Left) Concomitantly with the silencing of SOX9, the expression of DCT and tyrosinase proteins was decreased. SOX9 was overexpressed for 24 h in n-LP NHM. Again, after extraction, proteins were analyzed by immunoblotting. (Right) A parallel increase of DCT and tyrosinase proteins was observed when SOX9 was overexpressed. (B) n-MP NHM were silenced or overexpressed for SOX9 for 24 and 48 h. After protein extraction, the amount of melanin for each condition was calculated and adjusted against the total protein concentration. The histogram shows quantification of the data with the means ± SD of three independent experiments. Results are expressed as a ratio compared with the basal condition. Except after 24 h of SOX9 silencing, there was a significant decrease of melanin after 48 h of SOX9 silencing and a significant increase after overexpression of SOX9.

Fig. 10.

Schematic representation of the role of SOX9 in regulating pigmentation. UVB radiation, at least through the activation of cAMP via PKA, increases SOX9 and CREB expression. Those two transcription factors regulate the MITF promoter. SOX9 and MITF then act together to regulate the DCT promoter, whereas MITF also will act on the TYR promoter, finally leading to increased production of melanin within melanosomes.