Transforming growth factor β1 mediates hypopigmentation of B16 mouse melanoma cells by inhibition of melanin formation and melanosome maturation

Abstract

Transforming growth factor-β1 (TGFβ1) downregulates tyrosinase in B16 melanoma cells by decreasing gene expression and the intracellular half-life of the enzyme, but does not block tyrosinase stimulation by α-melanocyte stimulating hormone (αMSH). In the presence of both agents, the enzymatic activity is intermediate between the one of cells treated with either agent alone. Here we show that TGFβ1 equally inhibits the melanogenic activities of melan-a melanocytes and B16 melanoma cells, thus validating the B16 model. In both cell types, TGFβ1 (10 −10 M, 48 h) inhibited to comparable levels tyrosine hydroxylation and melanin formation from L-tyrosine. Thus, the inhibitory effect is exerted mainly at the rate limiting step of the pathway. By means of quantitative image analysis techniques, we also studied the effects of TGFβ1 and αMSH on melanosome number, volume density and maturation degree. αMSH (10 −7 M, 48 h) increased 7-fold melanosome volume density, whereas TGFβ1 by itself had no significant effect. However, melanosomal volume density was intermediate in cells treated with both agents, as compared to control or αMSH-treated cells. Moreover, TGFβ1 blocked the αMSH-elicited increase in the number of melanosomes. Control and αMSH-treated melanocytes contained more stage I+II premelanosomes and stage IV, fully melanized organelles than partially melanized stage III melanosomes. TGFβ1 increased the percentage of stage III melanosomes. This trend was even more marked in cells treated with αMSH and TGFβ1. The accumulation of incompletely melanized melanosomes is consistent with the inhibition of melanin formation activity by TGFβ1 and with its hypopigmenting effect.