Staurosporine rapidly commits 3T3-F442A cells to the formation of adipocytes by activation of GSK-3β and mobilization of calcium

Pre-adipose 3T3-F442A cells exposed to fetal bovine serum or human growth hormone (adipogenic medium) become irreversibly committed to differentiation into adipocytes within 24-36 h. We show now that the action of the serine-threonine kinase inhibitor staurosporine is much more rapid since its addition in non-adipogenic medium resulted in commitment to adipocyte differentiation within 4-6 h. During this period, glycogen synthase kinase 3 beta was activated. Commitment depended on an increase in the intracellular calcium concentration that was modulated in part by a T-type calcium channel since mibefradil, amiloride, and NiCl2, which are selective blockers of the T-type channels, partially inhibited adipose differentiation. Studies of the inhibitory action of retinoic acid showed that a period of time after exposure to St was required in order to stabilize the commitment to adipose differentiation. It was concluded that the commitment of the cells consists of two stages. Commitment: is promoted during the first one, and during the second there is a stabilization which still can be destabilized by the addition of retinoic acid or other drugs. The commitment becomes stable after 40 h of staurosporine treatment, and can no longer be prevented by retinoic acid. The identification of these two stages of commitment makes it possible to analyze in further detail early molecular events of the process and the nature of any other participating genes.