Importance of cAMP-response element-binding protein in regulation of expression of the murine cyclic nucleotide phosphodiesterase 3B (Pde3b) gene in differentiating 3T3-L1 preadipocytes.
Incubation of 3T3-L1 preadipocytes with isobutylmethylxanthine (IBMX), dexamethasone, and insulin, alone or in combination, demonstrated that IBMX, which increased cAMP-response element-binding protein (CREB) phosphorylation, was the predominant regulator of Pde3b expression. Real time PCR and immunoblotting indicated that in 3T3-L1 preadipocytes, IBMX-stimulated induction of Pde3b mRNA and protein was markedly inhibited by dominant-negative CREB proteins. By transfecting preadipocytes, differentiating preadipocytes, and HEK293A cells with luciferase reporter vectors containing different fragments of the 5'-flanking region of the Pde3b gene, we identified a distal promoter that contained canonical cis-acting cAMP-response elements (CRE) and a proximal, GC-rich promoter region, which contained atypical CRE. Mutation of the CRE sequences dramatically reduced distal promoter activity; H89 inhibited IBMX-stimulated CREB phosphorylation and proximal and distal promoter activities. Distal promoter activity was stimulated by IBMX and phorbol ester (PMA) in Raw264.7 monocytes, but only by IBMX in 3T3-L1 preadipocytes. Chromatin immunoprecipitation analyses with specific antibodies against CREB, phospho-CREB, and CBP/p300 (CREB-binding protein) showed that these proteins associated with both distal and proximal promoters and that interaction of phospho-CREB, the active form of CREB, with both Pde3b promoter regions was increased in IBMX-treated preadipocytes. These results indicate that CRE in distal and proximal promoter regions and activation of CREB proteins play a crucial role in transcriptional regulation of Pde3b expression during preadipocyte differentiation.
Liu
Hanguan
H
Pulmonary/Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Tang
Jing Rong
JR
Pulmonary/Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Choi
Young Hun
YH
Pulmonary/Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Napolitano
Maria
M
Pulmonary/Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Hockman
Steven
S
Pulmonary/Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Taira
Masato
M
Pulmonary/Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Degerman
Eva
E
Section for Molecular Signaling, Department of Cell and Molecular Biology, University of Lund, S-22100 Lund, Sweden.
Manganiello
Vincent C
VC
Pulmonary/Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892. Electronic address: manganiv@nhlbi.nih.gov.
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000502:physiology*; D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology*; D000818:Animals; D002454:Cell Differentiation; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008055:Lipids; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology
D017362:Cyclic AMP Response Element-Binding Protein; D008055:Lipids; D010726:Phosphodiesterase Inhibitors; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517291:PDE3B protein, human; C517293:Pde3b protein, mouse; D015056:1-Methyl-3-isobutylxanthine