SMILE Downregulation during Melanogenesis Induces MITF Transcription in B16F10 Cells

Abstract

SMILE (small heterodimer partner-interacting leucine zipper protein) is a transcriptional corepressor that potently regulates various cellular processes such as metabolism and growth in numerous tissues. However, its regulatory role in skin tissue remains uncharacterized. Here, we demonstrated that SMILE expression markedly decreased in human melanoma biopsy specimens and was inversely correlated with that of microphthalmia-associated transcription factor (MITF). During melanogenesis, α-melanocyte-stimulating hormone (α-MSH) induction of MITF was mediated by a decrease in SMILE expression in B16F10 mouse melanoma cells. Mechanistically, SMILE was regulated by α-MSH/cAMP/protein kinase A signaling and suppressed MITF promoter activity via corepressing transcriptional activity of the cAMP response element-binding protein. Moreover, SMILE overexpression significantly reduced α-MSH-induced MITF and melanogenic genes, thereby inhibiting melanin production in melanocytes. Conversely, SMILE inhibition increased the transcription of melanogenic genes and melanin contents. These results indicate that SMILE is a downstream effector of cAMP-mediated signaling and is a critical factor in the regulation of melanogenic transcription; in addition, they suggest a potential role of SMILE as a corepressor in skin pigmentation.

Keywords: MITF; SMILE; cAMP; melanogenesis; skin pigmentation.

Figure 1

SMILE inversely correlates with MITF in human melanoma biopsies. (a) Expression level of CREBZF, MITF, TYR, TRP1, and TRP2 in human normal (n = 7) and melanoma (n = 34) biopsies (GSE3189). The boxes represent the 25th to 75th percentiles, the line is at the median, and the whiskers represent the range. *** p < 0.0005 vs. Normal. (b) Negative correlation between CREBZF and melanogenic genes in human melanoma (n = 34) biopsies (GSE3189). p = 0.0004 vs. MITF-M; p < 0.0001 vs. TYR; p = 0.0002 vs. TRP1; p = 0.5080 vs. TRP2.

Figure 2

SMILE and MITF are reciprocally regulated by α-MSH in B16F10 mouse melanoma cells. Cells were treated with 1 µM α-MSH in a time-dependent manner. (a) Protein levels of MITF, melanogenic enzymes, and SMILE. (b) mRNA levels of genes encoding SMILE and MITF. * p < 0.05 vs. no treatment of α-MSH by one-way ANOVA.

Figure 3

SMILE overexpression downregulates the expression of melanogenic genes in B16F10 cells. Cells were infected with Ad-GFP or Ad-SMILE for 48 h and then, treated with 1 µM α-MSH for 1 h or 24 h. (a) Melanin contents. (b) Cellular tyrosinase activity. (c) mRNA levels of melanogenic genes. (d) Protein levels of SMILE and melanogenic genes. Data are mean ± SEM. * p < 0.05 vs. Ad-GFP; ^# p < 0.05 vs. Ad-GFP + α-MSH by one-way ANOVA (a). * p < 0.05 vs. Ad-GFP (b,c) by Student’s t-test.

Figure 4

SMILE knockdown upregulates the expression of melanogenic genes in B16F10 cells. Cells were transfected with siRNA negative control (NCi, 50 nM) or siRNA targeting SMILE (50 nM) for 48 h. (a) Melanin contents. (b) mRNA and (c) protein levels of SMILE and melanogenic genes. Data are mean ± SEM. * p < 0.05 vs. NCi (a,b) by Student’s t-test.

Figure 5

SMILE suppresses transcription of MITF via binding to CREB. (a) Cis-regulatory elements bound by indicated transcription factors on moue Mitf promoter. (b) B16F10 cells were co-transfected with pGL3-Mitf-M (wtCRE or mtCRE) and/or pcNDA3.1 Flag-SMILE for 48 h, after which the cells were treated with 1 µM α-MSH for 1 h. Luciferase reporter assays were performed for measuring promoter activity. (c) ChIP assay for SMILE binding to the CRE site of the Mitf promoter. IgG was used as the negative control. (d) HA-SMILE and Flag-CREB were co-transfected into 293T cells for 48 h. IP was performed with Flag antibodies and analyzed with HA antibodies. Data are mean ± SEM. * p < 0.05 vs. control; ^# p < 0.05 vs. α-MSH by one-way ANOVA (b). * p < 0.05 vs. Ad-GFP (c) by Student’s t-test.

Figure 6

SMILE and MITF are reciprocally regulated by cAMP/PKA signaling. (a) B16F10 cells were treated with 10 µM forskolin in a time-dependent fashion. mRNA levels of genes encoding SMILE and MITF. (b,c) B16F10 cells were treated with 5 µM H89 for 24 h and 1 µM α-MSH for 1 h. (b) mRNA level of Smile and (c) protein levels of MITF and CREB. Data are mean ± SEM. * p < 0.05 vs. no treatment of α-MSH by one-way ANOVA (a). * p < 0.05 vs. control; ^# p < 0.05 vs. α-MSH by one-way ANOVA (b).