Figure 3. ALDH1A Regulates Melanogenesis by Converting 9-cis retinal to 9-cis retinoic acid.

(A–B) 9-cis retinal induces the accumulation of TYR and MITF mRNA independently of UVA. Primary melanocytes and MNT-1 cells were pre-incubated with 9-cis retinal at the indicated doses for 45 minutes. Relative accumulation of TYR (A) and MITF (B) mRNA was measured as described in the methods section. (C–E) 9-cis retinoic acid induces the accumulation of TYR and MITF mRNA. Primary melanocytes and MNT-1 cells were pre-incubated with 9-cis retinoic acid at the indicated concentrations for 45 minutes. Cells were harvested 20 hours post drug treatment, and the relative accumulation of TYR (C) and MITF (D, E) mRNA was quantified as described in methods. (F) 9-cis retinoic acid but not 9-cis retinal induces the accumulation of TYR protein in both primary melanocytes and MNT-1 melanoma cells. MNT-1 melanoma cells (upper panel) and primary melanocytes (lower panel) were pre-incubated with the indicated doses of 9-cis retinal or 9-cis retinoic acid for 45 minutes. 28 hours later, the relative accumulation of TYR protein was quantified by immunoblotting. The numerical values represent the relative intensity of the TYR band normalized to the Tubulin band (loading control) for each lane divided by the relative expression of the TYR band in the vehicle control. (G–H) 9-cis retinal induces the accumulation of TYR and MITF mRNAs in an ALDH1A1 dependent manner. MNT-1 melanoma cells transiently transfected with either mismatch control or ALDH1A1 siRNAs for 72 hours were briefly treated with the indicated doses of 9-cis retinal (G) or 9-cis retinoic acid (H). Cells were then harvested 20 hours post drug treatment and the relative accumulation of TYR and MITF mRNA was measured. All data are mean ± S.D. (n=3 as indicated by the error bars). *, p < 0.05 or **, p < 0.01 using a Student’s paired t-test with a two-tailed normal distribution versus vehicle-treated control.