Analysis of Myc/Max/Mad network members in adipogenesis: inhibition of the proliferative burst and differentiation by ectopically expressed Mad1.

Transcription factors of the Myc/Max/Mad network affect multiple aspects of cellular behavior, including proliferation, differentiation, and apoptosis. Recent studies have shown that Mad proteins can inhibit cellular growth and transformation and thus antagonize the function of Myc proteins. To define further the contribution of these proteins to cellular growth control, we have studied the expression of the respective genes and proteins in 3T3-L1 cells, both upon serum stimulation of quiescent cells and during adipocytic differentiation in response to insulin, dexamethasone, and isobutylmethylxanthine. We found distinct expression patterns for the mad genes. Mad4 was induced when cells exit the cell cycle and, together with mad1, during the late phase of differentiation. In contrast, mad3 expression was associated with progression through S phase and the proliferative burst of differentiating preadipocytes, overlapping in part c-myc expression. DNA binding analyses revealed that the most prominent network complex both in cycling and in differentiating cells was Mnt/Max, whereas c-Myc/Max complexes were detectable only during peak c-Myc expression periods. Ectopic expression of Mad1 in preadipocytes resulted in the inhibition of S phase and the proliferation associated with the proliferative burst; as a consequence, adipocytic differentiation was significantly inhibited. Our findings suggest that the precise temporal regulation of Myc/Max/Mad network proteins is critical for determining cellular behavior.