EARLY RESPONSES OF TRANSACTIVATING FACTORS TO GROWTH-HORMONE IN PREADIPOCYTES - DIFFERENTIAL REGULATION OF CCAAT ENHANCER-BINDING PROTEIN-BETA (C EBP-BETA) AND C/EBP-DELTA/

Using the 3T3-F442A preadipocyte line as a model of on-dependent diffe rentiation, early changes in the DNA-binding affinity of transcription factors in response to on addition were investigated. Addition of 50 ng/ml human on to cells in chemically defined medium led to a rapid in crease in binding activity of activator protein 1 (AP-1) and CCAAT enh ancer-binding protein (C/EBP), which was significant at 30 min and rea ched maximal induction by 2 h (3-fold for AP-1, 2.5-fold for C/EBP). I nduction in AP-1 DNA binding correlates with a concomitant on trans-ac tivation of c-jun and c-fos genes described previously. Using specific antibodies in electrophoretic mobility shift assays and Western blots , it was shown that the increase in activity of C/EBP is the result of an increase in synthesis of two alternatively translated forms of C/E BP beta: 40-C/EBP beta and 23-C/EBP beta. This increase in protein was not accompanied by alteration in mRNA level and could be blocked by a Janus kinase 2 tyrosine kinase inhibitor and a C kinase inhibitor at concentrations shown to inhibit on-dependent activation of microtubule -associated protein (MAP) kinases. Concomitant with the translationall y activated increase in C/EBP beta, a on-dependent increase was observ ed in C/EBP delta transcription. This was accompanied by an increase i n mRNA for C/EBP delta, which was superinduced by cycloheximide and, u nlike the increase in C/EBP beta protein, was not observed with insuli n. Thus on exerts its effects on C/EBP isoforms at two levels: transcr iptional activation of C/EBP delta and translational activation of C/E BP beta. It is proposed that on-dependent phosphorylation results in t he efficient translation of 40-C/EBP beta and 23-C/EBP beta (the mouse homolog of the inhibitor liver-enriched inhibitory protein), and that together with the induction of C/EBP delta, these may be involved in initiating the adipocyte differentiation program.