Molecular mechanism for transcriptional activation of ganglioside GM3 synthase and its function in differentiation of HL-60 cells

Previous studies have demonstrated that the activity of GM3-synthase and GM3 content are increased during the differentiation of human promyelocytic leukemia HL-60 cells into the monocyte/macrophage lineage after phorbol 12-myristate 13-acetate (PMA) treatment. However, the molecular mechanisms involved in transcriptional activation of GM3 synthase during differentiation of PMA-induced HL-60 cells are not well understood. As evidenced by-western blot analysis, PMA induced the marked activation of protein kinase. C (PKC)/ extracellular regulated kinases (ERKs) signal transduction pathway during the differentiation of HL-60 cells. In addition, PKC/ERKs activation induced by PMA in HL-60 cells led to the phosphorylation of cAMP-responsive element binding protein (CREB) as a transcription factor. In PMA-stimulated HL-60 cells, the PKC/ERKs-dependent CREB activation regulated expression of GM3 synthase, inducing a synthesis of ganglioside GM3 product. On the other hand, although ganglioside GM3 was shown to be able to induce the differentiation of HL-60 cells into the monocyte/macrophage lineage, effect of ganglioside GM3 on.eipressio'n of CD11b as a differentiation marker in HL-60 cells has been not reported yet. Interestingly, the increased ganglioside GM3 through PKC/ERKs/CREB-dependent pathway by PMA resulted in an increase of CD11b,surface antigen-expression and induction of HL-60 cells adherence. Treatment with L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), glucosylceramide synthase inhibitor, decreased induction of not only CD11b expression but also cellular adherence by reduction of PMA-induced ganglioside GM3. Furthermore, treatment of HL,-60 cells with, exogenous ganglioside GM3 induced CD11b expression. These results show that the enhanced expression of,GM3 synthase through PKC/ERKs-dependent CREB activation by,PMA is associated with the differentiation of HL-60 cells by inducing expression of CD11b known as. a monocyte/macrophage differentiation maker.