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

Abstract

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 3beta 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 NiCl(2), 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.

The transient expression ofKlf4andKlf5during adipogenesis depends on GSK3β activity

“…11 This adipogenesis cellular model induced with St-Dex has the advantage of not requiring adipogenic serum interfering factors which can affect data interpretation, and it allows the study of some of the early events during induction and stabilization of adipose commitment. 1,4 The main transcription factors C/EBPb, SREBP1a, PPARg2, C/EBPa, and SREBP1c also participate in adipose differentiation in this cellular model. 1,12 There are several factors that affect adipogenesis during commitment, 4,13,14 among them, all-trans retinoic acid (RA) that inhibits adipose differentiation.…”

“…1,4 The main transcription factors C/EBPb, SREBP1a, PPARg2, C/EBPa, and SREBP1c also participate in adipose differentiation in this cellular model. 1,12 There are several factors that affect adipogenesis during commitment, 4,13,14 among them, all-trans retinoic acid (RA) that inhibits adipose differentiation. 4,[15][16][17][18] In this paper, we studied the KLF4-C/EBPb-KLF5 part of the transcriptional cascade during early adipogenesis of 3T3-F442A cells induced by St-Dex.…”

“…2,3 The adipogenic program is comprised of commitment, clonal expansion, and phenotype expression. 4 An important transcription factor during adipose commitment is C/EBPb, whose activity depends on specific phosphorylation at Thr188 by either GSK3b or MEK1. 5 A loss in the activity of GSK3b blocks the adipogenic program.…”

“…5 A loss in the activity of GSK3b blocks the adipogenic program. 4,6 C/EBPb is involved in the clonal expansion of committed cells 7 and in the expression of Pparg2 and adiponectin gene. 1,5 The expression of Pparg2 is delayed several hours after the expression and activation of C/EBPb.…”

“…We have already demonstrated that in non-adipogenic conditions, Staurosporine (St) at low concentrations, rapidly induced adipogenesis of 3T3-F442A cells, 4 while Dexamethasone (Dex) enhanced St-induced adipose conversion; however, Dex alone, without St, did not induce adipose conversion under the nonadipogenic conditions. 10 It was reported that treatment with staurosporine leads to GSK3b activation as shown by its reduced phosphorylation on Ser21/9.…”