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

• Published in: The Journal of biological chemistry Vol. 281; no. 30; pp. 21096 - 21113
• Main Authors: Liu, Hanguan, Tang, Jing Rong, Choi, Young Hun, Napolitano, Maria, Hockman, Steven, Taira, Masato, Degerman, Eva, Manganiello, Vincent C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.07.2006; American Society for Biochemistry and Molecular Biology
• Subjects: 1-Methyl-3-isobutylxanthine - pharmacology; 3',5'-Cyclic-AMP Phosphodiesterases - physiology; 3T3-L1 Cells; Adipocytes - cytology; Animals; Cell Differentiation; Clinical Medicine; Cyclic AMP Response Element-Binding Protein - physiology; Cyclic Nucleotide Phosphodiesterases, Type 3; Endocrinology and Diabetes; Endokrinologi och diabetes; Gene Expression Regulation, Enzymologic; Humans; Klinisk medicin; Lipids; Medical and Health Sciences; Medicin och hälsovetenskap; Mice; Models, Genetic; Molecular Sequence Data; Phosphodiesterase Inhibitors - pharmacology
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M601307200

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.