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. 2013 Jan 10;49(1):145-57.
doi: 10.1016/j.molcel.2012.10.027. Epub 2012 Nov 29.

PGC-1 coactivators regulate MITF and the tanning response

Jonathan Shoag  1 , Rizwan Haq, Mingfeng Zhang, Laura Liu, Glenn C Rowe, Aihua Jiang, Nicole Koulisis, Caitlin Farrel, Christopher I Amos, Qingyi Wei, Jeffrey E Lee, Jiangwen Zhang, Thomas S Kupper, Abrar A Qureshi, Rutao Cui, Jiali Han, David E Fisher, Zoltan Arany
Affiliations

PGC-1 coactivators regulate MITF and the tanning response

Jonathan Shoag et al. Mol Cell. .

Abstract

The production of pigment by melanocytes tans the skin and protects against skin cancers. UV-exposed keratinocytes secrete α-MSH, which then activates melanin formation in melanocytes by inducing the microphthalmia-associated transcription factor (MITF). We show that PPAR-γ coactivator (PGC)-1α and PGC-1β are critical components of this melanogenic system in melanocytes. α-MSH signaling strongly induces PGC-1α expression and stabilizes both PGC-1α and PGC-1β proteins. The PGC-1s in turn activate the MITF promoter, and their expression correlates strongly with that of MITF in human melanoma cell lines and biopsy specimens. Inhibition of PGC-1α and PGC-1β blocks the α-MSH-mediated induction of MITF and melanogenic genes. Conversely, overexpression of PGC-1α induces pigment formation in cell culture and transgenic animals. Finally, polymorphism studies reveal expression quantitative trait loci in the PGC-1β gene that correlate with tanning ability and protection from melanoma in humans. These data identify PGC-1 coactivators as regulators of human tanning.

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Figures

Figure 1
Figure 1. PGC-1α Induces Melanogenesis
(A) B16-F10 cells were infected with retrovirus overexpressing PGC-1α or vector control. Cell pellets (left panel), and light microscopy (right panel) are shown. (B) Tyrosinase activity in extracts from cells, as in(A). (C) Relative mRNA expression of the indicated genes in cells, as in (A). (D) Cell pellets of B16-F10 cells infected with lentivirus expressing shPGC-1α or shGFP. (E) Tyrosinase activity in extracts from cells, as in(D). (F) Relative mRNA expression of the indicated genes in cells, as in (D). *p < .05. n ≥ 3 for all groups. Error bars represent mean ± SD.
Figure 2
Figure 2. PGC-1 Coactivators Induce MITF-M Expression
(A–D) mRNA expression of the indicated genes in B16-F10 cells stably overexpressing PGC-1α versus vector control (A) and lentiviral knockdown of PGC-1α in B16-F10 (B), UACC-257 (C), and normal human melanocytes (D). (E) Relative activity of luciferase expressed from the MITF-M promoter, cotransfected into UACC-257 cells with plasmids expressing the indicated proteins. (F) Relative luciferase activity, as in (E). (G) Occupancy by PGC-1α on the MITF-M promoter at the indicated sites. Black bars correspond to binding near the SOX and CRE sites. IgG, immunoglobulin G. (H) Relative MITF-M mRNA in B16-F10 cells stably overexpressing PGC-1α or PGC-1β. CTRL, control. (I) mRNA of indicated genes in UACC-257 cells following lentiviral knockdown of PGC-1β. (J) MITF-M promoter activity in UACC-257 cells expressing the indicated proteins. *p < 0.05. Error bars represent mean ± SD.
Figure 3
Figure 3. PGC-1 and MITF Expression Correlates in Multiple Melanoma Cell Lines and Biopsies
(A–D) Relative expression of PGC-1α and MITF in (A) the NCI-60 cell lines (GSE5846), (B) 88 melanoma cell lines (Lin et al., 2008; Lee et al., 2007), (C) 60 melanoma cell lines (Broad Institute CCLE), and (D) 45 melanoma biopsies (GSE3189) (Talantov et al., 2005). (E) Relative expression of PGC-1β and MITF in 60 melanoma cell lines (Broad Institute CCLE). p values for Spearman’s rho are as indicated.
Figure 4
Figure 4. Regulation of PGC-1α and MITF by α-MSH
(A) mRNA expression of the indicated genes in B16-F10 cells treated with α-MSH (left panels) and normal human melanocytes treated with forskolin + IBMX (right panels) for the indicated times. (B) Relative activity of the two PGC-1α promoters in B16-F10 cells treated with α-MSH, as assessed by luciferase activity. Schematic of PGC-1α promoters and the alternatively spliced exon 1* is shown below. (C) PGC-1α protein in B16-F10 cells treated with α-MSH for 16 hr. NS, nonspecific band. (D) MITF-M and PGC-1α induction in C57Bl6e/e K14 SCF mice topically treated with vehicle or forskolin + Rolipram (FSK + Ro) for 6 hr. (E) Expression of the indicated genes in B16-F10 cells after 5 hr incubation in medium that had been conditioned for 16 hr by PAM212s exposed to UV or mock treatment. The experimental outline is schematized on the left. (F) MITF-M and TRPM1 mRNA in UACC-257 cells expressing shPGC-1α and treated with forskolin + IBMX. (G) MITF-M and Tyrosinase induction in response to forskolin + IBMX (F+IB) in primary mouse melanocytes lacking PGC-1α and PGC-1β. AD, adenovirus. *p < .05. Error bars represent mean ± SD.
Figure 5
Figure 5. Stabilization of PGC-1α Protein by α-MSH
(A) Time course of induction of PGC-1α and PGC-1β mRNA (top) and PGC-1α protein (bottom) in B16-F10 cells treated with forskolin and IBMX (Fsk + IBMX) for the indicated times. (B) Stabilization of PGC-1α by α-MSH. WB, western blot. (C) The PKA inhibitor (H89) blocks stabilization of PGC-1α protein. (D) PGC-1α protein stabilization in cells treated with the adenylate cyclase activator (forskolin). GFP (lower panel) is expressed by the same promoter (CMV). (E) Half-life of PGC-1α in B16-F10 cells treated with forskolin + IBMX. CHX, cycloheximide. (F) Schematic of murine PGC-1α and predicted PKA target sites. Sites mutated to alanine in the 7A mutant are indicated below. (G) Absence of stabilization of PGC-1α mutated at PKA target sites. The quantification of five experiments is shown below. WT, wild-type. (H) Stabilization of PGC-1β in B16-F10 cells treated with forskolin + IBMX or α-MSH. NS, nonspecific band. *p < 0.05. Error bars represent mean ± SD.
Figure 6
Figure 6. Evidence in Humans and Rodents for Regulation of Pigmentation by PGC-1 Coactivators
(A) Melanocytic overexpression of PGC-1α in mice darkens the fur of Ay/a mice (top panel) and increases melanin in hair follicles (bottom panels). Representative of n > 4 for each group. (B) Manhattan plot of the 100 kb region surrounding the human PGC-1β gene (PPARGC1B), and the association of SNPs within this region with tanning ability. (C) Relative expression of PGC-1β in 87 HapMap human cell lines according to their genotype at the rs32579 SNP. Interquartile ranges (boxes) and mean values (lines in boxes) are shown. (D) The PGC-1 coactivators regulate MITF and melanin production.
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