Mechanism of regulation of the 422(aP2) gene by cAMP during preadipocyte differentiation.

Abstract

During differentiation of 3T3-L1 preadipocytes into adipocytes, expression of the gene encoding adipocyte 422(aP2) protein is activated. We have shown that the first 248 base pairs of the 422(aP2) gene promoter (which lacks a consensus cAMP response element) are sufficient to confer inducibility of a reporter gene by cAMP in preadipocytes. We now demonstrate by deletion analysis that this DNA segment contains overlapping negative and positive regulatory elements. The positive regulatory element contains a consensus activator protein 1 (AP-1) binding sequence. The effect of the negative regulatory element is observed in preadipocytes but not in fully differentiated adipocytes, suggesting that it is an important component of the regulatory mechanism governing expression of the 422(aP2) gene during differentiation. cAMP activates the 422(aP2) promoter in confluent preadipocytes but not in proliferating preadipocytes or fully differentiated adipocytes. The stimulatory effect of cAMP is abolished by deletions that enter the negative element, suggesting that cAMP increases expression by relieving the inhibitory effect of the negative regulatory element.Under appropriate cell culture conditions, 3T3-L1 preadipocytes (1-3) differentiate into cells possessing the morphological and biochemical properties of adipocytes (1)(2)(3)(4)(5)(6). This differentiation is accompanied by the acquisition of enzymatic and regulatory functions that characterize the adipocyte phenotype. Several differentiation-induced genes and their corresponding cDNAs have been isolated and characterized (7-10). One of these genes encodes the adipocyte 422(aP2) protein that has extensive homology to myelin P2 protein and the family of intracellular fatty acid-binding proteins (7)(8)(9). Expression of this gene is transcriptionally activated during adipose conversion, leading to high cellular levels of its mRNA in the adipocyte (11)(12)(13). The 422(aP2) gene including an extensive region of its flanking sequence has been cloned and characterized (14-16). It was demonstrated that the first 168 base pairs of 5' flanking sequence of the gene are sufficient to drive differentiation-dependent expression of a heterologous reporter gene in 3T3-F422A preadipocytes (17). This region of the gene contains a consensus activator protein 1 (AP-1) binding sequence with which a complex of two protooncoproteins, Fos and Jun (AP-1), appears to interact (17-19).Differentiation of 3T3-L1 preadipocytes and expression of adipocyte-specific genes are induced by methylisobutylxanthine, dexamethasone, and insulin (4-6). Since methylisobutylxanthine, an inhibitor of cAMP phosphodiesterase in 3T3-Li preadipocytes (20), can be replaced by 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) (14, 21), it is evident that cAMP is an important agent for induction of the differentiation process. Previous studies showed that the first 248 base pairs of5' flanking sequence ofthe 422(aP2)

Adipocyte fatty acid binding protein: a novel adipokine involved in the pathogenesis of metabolic and vascular disease?

“…Afabp mRNA and protein production are significantly induced during adipocyte differentiation [7]. AFABP has frequently been used as an adipocyte differentiation marker [8][9][10], based on these observations. The DNA-binding protein CCAAT/enhancerbinding protein interacts with the AFABP promoter and elevates expression of the AFABP gene [11].…”

“…The DNA-binding protein CCAAT/enhancerbinding protein interacts with the AFABP promoter and elevates expression of the AFABP gene [11]. Furthermore, cyclic adenosine monophosphate induces AFABP gene expression during adipocyte differentiation by relieving the inhibitory effect of a negative regulatory element in the AFABP promoter [10]. Moreover, insulin-sensitising and pro-adipogenic peroxisome proliferator-activated receptor (PPAR)γ agonists induce AFABP production and secretion [12,13].…”

Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes.

“…These results, taken together with those described above, indicate that the differentiation-specific factor is C/EBP. Moreover, the sites at which C/EBP (and nuclear extracts from differentiated 3T3-L1 adipocytes) interacts with the 422(aP2) and SCDl promoters are within the regions that have been shown to be essential for the activation of both genes during preadipocyte differentiation (Distel et al 1987;Ntambi et al 1988;Yang et al 1989). …”

“…Transfection experiments with chimeric 422(aP2) promoter-CAT constructs show that the gene is activated by glucocorticoid and cAMP (Cook et al 1988;Yang et al 1989), two agents used to induce differentiation of 3T3-L1 preadipocytes. It has been demonstrated that the first 168 bp of the 5'-flanking sequence of this gene are sufficient for differentiation-dependent expression in 3T3-442A preadipocytes [Distel et al 1987).…”

“…This region contains a consensus AP-1 (Jun)-binding sequence with which a complex of two proto-oncogenes, (i.e., c-/os and c-jun or their related gene products), interact (Distel et al 1987;Rauscher et al 1988;Franza et al 1988;Nakabeppu et al 1988;Cohen et al 1989). A DNA-protem mteraction in the region of the AP-1 (Jun) site appears to inhibit expression of the 422(aP2) gene in 3T3 preadipocytes (Distel et al 1987;Yang et al 1989). Transfection experiments with chimeric SCDI promoter-CAT constructs in 3T3-L1 preadipocytes have shown that 363 bp of the SCDI 5'-flanking region contain sequences that promote CAT expression; this expression is activated by cAMP, one of the agents known to induce differentiation of 3T3-L1 preadipocytes (Ntambi et al 1988).…”