UV-induced Expression of Key Component of the Tanning Process, the POMC and MC1R Genes, Is Dependent on the p-38-activated Upstream Stimulating Factor-1 (USF-1)

Sébastien Corre,Aline Primot,Elena Sviderskaya,Dorothy C Bennett,Sophie Vaulont,Colin R Goding,Marie-Dominique Galibert

doi:10.1074/jbc.m409768200

Abstract

Protection against UV-mediated DNA damage and the onset of oncogenesis is afforded by the tanning response in which UV irradiation triggers melanocytes to increase production of melanin that is then transferred to keratinocytes. A key component of the tanning process is the UV-mediated induction of the pro-opiomelanocortin (POMC) and MC1R genes encoding the alpha-melanocyte-stimulating hormone and its receptor, respectively, which play a crucial role in pigmentation by regulating the intracellular levels of cAMP. How these genes are regulated in response to UV irradiation is not known. Here we have shown that UV-induced activation of the POMC and MC1R promoters is mediated by p38 stress-activated kinase signaling to the transcription factor, upstream stimulating factor-1 (USF-1). Importantly, melanocytes derived from USF-1 -/- mice exhibit a defective UV response and fail to activate POMC and MC1R expression in response to UV irradiation. The results define USF-1 as a critical UV-responsive activator of genes implicated in protection from solar radiation.

Highlights

Solar ultraviolet (UV)1 radiation [1] is a major environmental hazard that can generate reactive oxygen species, inducing DNA damage and protein oxidation [2] that subsequently lead to skin inflammation, photo-aging, and skin cancer
In silico analysis of the POMC and MC1R promoters revealed that both proximal promoters displayed several E-box motifs (29 –31) with only a small number conserved in humans, mouse, and rat species that could potentially interact with the microphthalmia-associated transcription factor (Mitf) and upstream stimulating factor-1 (USF-1) transcription factors to mediate the response of POMC and MC1R to UV irradiation
We examined constitutive and UVinduced POMC and MC1R expression levels in different skin cell types implicated in the pigmentation process: mouse keratinocytes (XB2), mouse melanocytes, and human melanoma (501 mel)

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Tissue Culture—Melan-a and melan-usf mouse melanocytes and 501 mel human melanoma cell lines were maintained in RPMI 1640 medium (catalogue number 21875– 034; Invitrogen), supplemented with 10% fetal calf serum and 1% penicillin-streptomycin antibiotics (Invitrogen) in a controlled atmosphere (10% CO2). Transient Transfection Assays—For in vivo gene expression analysis, cells were plated in supplemented medium in 10-cm diameter Petri dishes and allowed to grow overnight to 80% confluence. Cells were transfected for 1 h using the specific transfection medium OptiMEM (catalogue number 51985– 026; Invitrogen) containing up to 5 ␮g of total plasmid DNA, including 4 ␮g of USF-1 constructs (WT pCMV-USF-1 or T153E and T153A mutants), 500 ng each of p38 and MKK6(b)E expression vector (pCDNA p38; pCDNA MKK6(b)E), or empty vector mixed with the TransfastTM reagent (Promega) according to the manufacturer’s instructions. 24 h posttransfection, cells were washed twice with phosphate-buffered saline, harvested, and subjected to RNA extraction as previously described. The RNA was subjected to POMC and MC1R gene expression analysis using real-time PCR. The p38 expression vector and MKK6(b)E were provided by Dr Jiahan Han and have been described previously [34]

DNA Binding Assays

UVB inducibility

RESULTS

DISCUSSION