Phosphatidylinositol 3-kinase/akt-dependent and -independent protection against apoptosis in normal human melanocytes

Abstract

Normal human melanocytes require the synergistic action of several growth-promoting agents for their growth in serum-free medium. The ability of four representative growth promoting agents including insulin, 12-O-tetradecanoylphorbol-13-acetate (TPA), basic fibroblast growth factor (bFGF), and 3-isobutyl-1-methylxanthine (IBMX), (iTbl) to protect melanocytes against apoptosis was examined. Also, the involvement of phosphatidylinositol (Pl) 3-kinase and Akt, one of the downstream targets of Pl 3-kinase, in the survival signaling pathway was examined. The percentage of apoptotic cells was negligible when the cells were grown in the presence of iTbl. Deprivation of iTbl from the culture medium for 72 h caused ∼30% of melanocytes to undergo apoptosis and this was suppressed to variable extents by the addition of one of the iTbl to the medium. Insulin and TPA protected against apoptosis almost completely, whereas bFGF and IBMX rescued melanocytes from apoptosis to a lesser extent. Wortmannin, an inhibitor of Pl 3-kinase, potently inhibited the protective effect of insulin on melanocytes, whereas it did not block the ability of TPA, bFGF, or IBMX to rescue the cells from apoptosis. Furthermore, apoptosis of melanocytes induced by deprivation of iTbl was prevented almost completely by infection with an adenovirus vector encoding a constitutively active mutant of either Pl 3-kinase or Akt. These results indicate that melanocytes can operate both Pl 3-kinase/Akt-dependent and -independent mechanisms for protection against apoptosis and that activation of the Pl 3-kinase/Akt pathway is sufficient for protection against apoptosis induced by deprivation of growth-promoting agents.