Early responses of trans-activating 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 GH-dependent differentiation, early changes in the DNA-binding affinity of transcription factors in response to GH addition were investigated. Addition of 50 ng/ml human GH to cells in chemically defined medium led to a rapid increase in binding activity of activator protein 1 (AP-1) and CCAAT enhancer-binding protein (C/EBP), which was significant at 30 min and reached maximal induction by 2 h (3-fold for AP-1, 2.5-fold for C/EBP). Induction in AP-1 DNA binding correlates with a concomitant GH trans-activation 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/EBP 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 GH-dependent activation of microtubule-associated protein (MAP) kinases. Concomitant with the translationally activated increase in C/EBP beta, a GH-dependent increase was observed in C/EBP delta transcription. This was accompanied by an increase in mRNA for C/EBP delta, which was superinduced by cycloheximide and, unlike the increase in C/EBP beta protein, was not observed with insulin. Thus GH exerts its effects on C/EBP isoforms at two levels: transcriptional activation of C/EBP delta and translational activation of C/EBP beta. It is proposed that GH-dependent phosphorylation results in the 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.
Clarkson
R W
RW
Centre for Molecular Biology and Biotechnology, University of Queensland St. Lucia, Australia.
Chen
C M
CM
Harrison
S
S
Wells
C
C
Muscat
G E
GE
Waters
M J
MJ
D016475:3T3 Cells / Q000187:drug effects; D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects*; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002454:Cell Differentiation / Q000187:drug effects; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D013006:Growth Hormone / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology; D053614:Janus Kinase 2; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D013997:Time Factors; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007328:Insulin; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D013006:Growth Hormone; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases