Microphthalmia-associated Transcription Factor Regulates RAB27A Gene Expression and Controls Melanosome Transport

Christine Chiaverini,Laurent Beuret,Enrica Flori,Roser Busca,Patricia Abbe,Karine Bille,Philippe Bahadoran,Jean-Paul Ortonne,Corine Bertolotto,Robert Ballotti

doi:10.1074/jbc.m800130200

Abstract

Melanosomes are lysosome-related organelles specialized in melanin synthesis and transport. In this study, we show that microphthalmia-associated transcription factor (MITF) silencing induces melanosome gathering around the nucleus and causes the relocalization of Rab27A, Slac2a-Mlph, and Myo5a that control the transport of melanosomes on the actin network. In an attempt to elucidate the mechanism by which MITF controls melanosome distribution, we identify RAB27A as a new MITF target gene. Indeed, MITF silencing leads to a dramatic decrease in Rab27A expression and blocks the stimulation of Rab27A expression evoked by cAMP. Further, forced expression of MITF increases Rab27A expression, indicating that MITF is required and sufficient for Rab27A expression in melanoma cells. MITF binds to two E-boxes in the proximal region of the Rab27A promoter and stimulates its transcriptional activity. Finally, re-expression of Rab27A, in MITF-depleted cells, restores the transport of melanosomes to the cell periphery. These results show that RAB27A is a new direct transcriptional target of MITF and link MITF to melanosome transport, another key parameter of melanocyte differentiation and skin pigmentation. Interestingly, Rab27A is involved in other fundamental physiological functions, such as the transport of lytic granules and insulin secretion. Thus our results, deciphering the mechanism of Rab27A transcriptional regulation, have an interest that goes beyond the skin pigmentation field.

Highlights

The Rab27A-melanophilin (Slac2a-Mlph) complex recruits the actin-dependent motor protein, myosin 5a (Myo5a), allowing melanosomes to move on the actin network (4 – 6)
We demonstrate that microphthalmia-associated transcription factor (MITF) regulates the peripheral distribution of melanosomes to the dendrite tips via the control of Rab27A expression
We demonstrate the involvement of MITF in the actin-dependent melanosome transport that is, together with melanin synthesis, a key functional trait of differentiated melanocytes underlying skin pigmentation

Summary

MATERIALS AND METHODS

Cell Culture, Transfection, and Infection—Mouse melanoma B16-F10 cells, human melanoma Mel501 cells, and A375 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 7% fetal bovine serum and penicillin/streptomycin (100 IU; 50 ␮g/ml) in a humidified atmosphere containing 5% CO2 at 37 °C. The membranes were saturated in a saline buffer, 2% bovine serum albumin and incubated with the corresponding primary antibody for 1 h at room temperature. After three 10-min washes in a saline buffer containing 0.05% Triton X-100, the blots were incubated for 1 h with the corresponding peroxidase-conjugated secondary antibody and washed again. Luciferase Assays—B16 melanoma cells were seeded in 24-well dishes, and transient transfections were performed the following day using 2 ␮l of Lipofectamine (Invitrogen) and 0.5 ␮g of total plasmid DNA in a 200-␮l final volume. Mitf silencing altered melanosome distribution and prevented melanosome docking at dendrite tips in forskolin-treated cells (Fig. 1B). Identification of the captured DNA fragments was performed by PCR analysis using the Rab27A or GAPDH promoter primers. 30 cycles of PCR were performed, and the amplified products were analyzed on a 2% agarose gel

RESULTS

Findings

DISCUSSION