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

Abstract

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.

An internal ribosome entry segment in the 5′ untranslated region of the mnt gene

“…Moreover, Mnt ± Max heterodimers appear to be amongst the most abundant Maxcontaining complexes (Sommer et al, 1998;Pulverer et al, 2000). Therefore, we have begun to characterize the regulation of mnt gene expression.…”

“…Such an element may allow the e cient translation of mnt transcripts under conditions when the activity of the cap binding complex eIF4F is reduced, as has been observed for the VEGF IRES during hypoxia and the XIAP and c-myc IRESs during apoptosis (Stein et al, 1998;Holcik et al, 1999;Stoneley et al, 2000). In this regard, it is noteworthy that Mnt is synthesized in quiescent ®broblasts and in terminally di erentiated adipocytes (Pulverer et al, 2000). Given the ability of Mnt to suppress Mycmediated cellular transformation, the function and mechanism of the mnt IRES warrant further investigation.…”

Coordinated regulation of c-Myc and Max in rat liver development

“…In many in vitro systems, the regulation of the c-myc/max/ mad network has been shown to rely on the opposing expression patterns of c-myc and mad family members, whereas max expression is constitutive (22,25). We (29) have reported previously that c-Myc protein content is not changed even though c-myc RNA expression is decreased as proliferating hepatocytes growth arrest during liver development, but c-Myc localization switches from a diffuse nuclear to a nucleolar pattern (29).…”

“…We also examined the expression of mad4 and mad3 in the fetal and adult liver. In agreement with previous studies in other systems, we found mad4 to be induced in the quiescent adult, whereas mad3 was elevated in the fetus (12,25).…”

“…This repression is mediated through binding to the same E boxes as c-Myc and recruitment of the Sin3 repressor complex through the Sin3 interaction domain of Mad (2). In contrast to c-Myc, Mad members (Mad1 and Mad4) are expressed during growth arrest and in differentiated cells, whereas Mad3 is expressed in proliferating cells, where it plays a role in S phase progression (11,12,25).…”