Abstract
Melanins are synthesized in melanocytes within specialized organelles called melanosomes. Numerous studies have shown that the pH of melanosome plays a key role in the regulation of melanin synthesis. However, until now, acute regulation of melanosome pH by a physiological stimulus has never been demonstrated. In the present study, we show that the activation of the cAMP pathway by alphaMSH or forskolin leads to an alkalinization of melanosomes and a concomitant regulation of vacuolar ATPases and ion transporters of the solute carrier family. The solute carrier family members include SLC45A2, which is mutated in oculocutaneous albinism type IV, SLC24A4 and SLC24A5, proteins implicated in the control of eye, hair, and skin pigmentation, and the P protein, encoded by the oculocutaneous albinism type II locus. Interestingly, H89, a pharmacological inhibitor of protein kinase A (PKA), prevents the cAMP-induced pigmentation and induces acidification of melanosomes. The drastic depigmenting effect of H89 is not due to an inhibition of tyrosinase expression. Indeed, H89 blocks the induction of melanogenesis induced by LY294002, a potent inhibitor of the PI 3-kinase pathway, without any effect on tyrosinase expression. Furthermore, PKA is not involved in the inhibition of pigmentation promoted by H89 because LY294002 induces pigmentation independently of PKA. Also, other PKA inhibitors do not affect pigmentation. Taken together, our results strengthen the support for a key role of melanosome pH in the regulation of melanin synthesis and, for the first time, demonstrate that melanosome pH is regulated by cAMP and alphaMSH. Notably, these are both mediators of the response to solar UV radiation, the main physiological stimulus of skin pigmentation.
Highlights
Numerous clinical observations, animal models, and experimental data clearly demonstrate that ␣MSH and the cAMP pathway are key physiologic regulators of skin and hair pigmentation in mammals, including humans [4]
Some studies claim that this acidic pH is required for melanin synthesis [16], several other studies have demonstrated that vacuolar ATPase inhibitors, which increase melanosome pH, are able to stimulate melanin synthesis [17]
We demonstrated that the activation of the cAMP pathway by ␣MSH or forskolin led to alkalinization of melanosomes
Summary
After three washes of 10 min in 10 mM Tris, pH 7.4, 150 mM NaCl, and 1% Triton X-100, the secondary antibody conjugated with horseradish peroxidase (Dakopatts) was incubated for 1 h at 25 °C. Proteins were solubilized for 3 min at 4 °C in 0.1 M phosphate buffer, pH 7.4, and 1% Triton X-100, and 60 g of protein was incubated with 200 g of L-DOPA at 37 °C. Cells were washed with serum-free culture medium and incubated for 20 min in the presence of 30 M DAMP. Slides were incubated with a fluorescein isothiocyanate-labeled rabbit anti-2,4-dinitrophenol antibody (1/50 in PBS plus 1% bovine serum albumin). Melanosome labeling with primary monoclonal antibody (B8G3 anti-TYRP-1, 1/10; HMB45 anti-Pmel-17, 1/20) was performed in PBS plus 1% bovine serum albumin. After three washes with PBS, slides were incubated with Texas Red-labeled anti-mouse antibody (1/500). Tion and statistical analysis were performed using the limma package available from Bioconductor
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