Expression of the differentiation-induced gene for fatty acid-binding protein is activated by glucocorticoid and cAMP.

Abstract

We have isolated and characterized a fragment of the gene encoding adipose fatty acid-binding protein (gene 422) from a 3T3-L1 adipocyte genomic library. The 5'-flanking sequence of the 422 gene contains potential regulatory regions for adipose-specific expression. At position -120 there is a fat-specific element that occurs in several genes expressed as preadipocytes differentiate, and at position -393 there is a glucocorticoid regulatory element core sequence. Chimeric constructs were prepared by ligating 858 base pairs or 248 base pairs of 5'-flanking sequence and 22 nucleotides of 5'-untranslated sequence of the 422 gene to the bacterial gene encoding chloramphenicol acetyltransferase (CAT); these constructs (A858.CAT and A248.CAT) were transfected into 3T3-L1 preadipocytes. When differentiation was initiated by the adipogenic agents methylisobutylxanthine (a cAMP phosphodiesterase inhibitor), dexamethasone, and insulin, expression of both constructs increased, reaching maximal levels within 24 hr. Both constructs were maximally induced 48 hr before appreciable accumulation of the endogenous 422 mRNA. Expression of A858.CAT, but not of A248.CAT, was induced by dexamethasone, which correlates with deletion of the potential glucocorticoid regulatory element. Expression of both constructs was induced by 8-bromoadenosine 3',5'-cycic monophosphate, thus implicating the first 248 base pairs of 5'-flanking sequence of the 422 gene in the response to cAMP. Indirect effects by the adipogenic factors on CAT protein or mRNA synthesis and turnover were ruled out, since replacing the 5'-flanking region of the 422 gene constructs with viral promoters abolished the effects of dexamethasone and 8-bromoadenosine 3',5'-cyclic monophosphate on CAT expression. We conclude that the first 858 base pairs of 5'-flanking sequence of the 422 gene contains elements that mediate activation by dexamethasone and cAMP.Green and Kehinde (1, 2) have established several 3T3 cell lines that differentiate into adipocytes in response to appropriate stimuli. The expression of adipocyte morphology by these cells is accompanied by the specific reprogramming of enzymatic and regulatory functions to those typical of adipose cells. The accumulation of cytoplasmic triglyceride that occurs during differentiation is correlated with a coordinate rise in the activity of every enzyme of the pathways of de novo fatty acid and triglyceride synthesis (3-7). In addition, 3T3 preadipocytes acquire the enzymatic capacity to mobilize triglyceride (8) and respond with great sensitivity to lipogenic (9-11) and lipolytic hormones (12). There is now compelling evidence (13-15) that the increase in the levels of the specific translatable mRNAs for these differentiationinduced proteins is responsible for the observed increases in their expression during 3T3 preadipocyte differentiation, which in all cases thus far examined depends on increased transcription of the corresponding genes (16-18).We (19) Cell Culture. 3T3-L1 preadipocytes were maintained and induce...

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

“…Therefore, although the transfections do not indicate the exact mechanism of C/EBP under normal physiological levels, the fact that C/EBP or certain agents used to induce differentiation of 3T3-L1 preadipocytes (Cook et al 1988; J.M. Ntambi and M.D.…”

“…Transfection experiments with chimeric promoter-CAT constructs of the 422(aP2) (Cook et al 1988;Yang et al 1989) and SCDl (Ntambi et al 1988) genes, which contain the C/EBP-binding site, are expressed during differentiation of 3T3-L1 preadipocytes. To determine whether C/EBP can activate expression driven by the promoters of these genes, 422(aP2) promoter-CAT and SCDl promoter-CAT genes were cotransfected with pMSV-C/EBP-wt, an expression vector containing the rat C/EBP cDNA linked to the MSV LTR (Friedman et al 1989).…”

“…The coding strand of the 248-bp Nsil-Pstl fragment (Cook et al 1988) of the 422(aP2) promoter was subjected to DNase I footprinting. Figure lA shows that two regions, between nucleotides -147 and -128 and between nucleotides -124 and -107, are protected from DNase I digestion by the nuclear extract.…”

“…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).…”

“…The 422(aP2) and SCDl genes have been cloned and their 5'-flanking sequences characterized (Hunt et al 1986;Phillips et al 1986;Cook et al 1988;Ntambi et al 1988). 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.…”