Phosphodiesterase 3 Isoforms

Heart, Lung, And Blood Institute

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Abstract

Cyclic nucleotide phosphodiesterases (PDEs) are critical regulators of intracellular concentrations and biological effects of cAMP and cGMP. Understanding cellular expression and regulation of PDE isoforms [which belong to eleven gene families (PDEs 1-11)] will be of increasing importance for targeting specific PDEs in treating various diseases, including pulmonary disorders. Membrane-associated PDE3B appears (or greatly increases in activity) during differentiation of cultured human adipocytes or 3T3-L1 murine adipocytes in the presence of IBMX, dexamethasone (Dex), and insulin (ins). IBMX, a non-specific PDE inhibitor which increases CREB (cAMP Response Element Binding protein) phosphorylation, was the predominant regulator of PDE3B expression. IBMX-stimulated induction of PDE3B mRNA and protein was inhibited by overexpression of KCREB, a dominant-negative version of CREB. The 5'-flanking region of the murine PDE3B gene contains two promoter regions, a distal region (located ~5kb upstream of the translation initiation site) which include a canonical cis-acting CRE (cAMP Response Elements) sequence, and a more proximal region which includes atypical CRE elements. Mutation of CRE sequences markedly reduced IBMX-stimulated distal promoter activity; H89 (a PKA inhibitor)inhibited IBMX-stimulated proximal promoter activity. Chromatin immunoprecipitation assays indicated that CREB, phospho-CREB, and CBP/p300 associated with the proximal and distal promoters and that the interaction of phospho-CREB with both promoters was increased in IBMX-treated cells. Thus, cis-acting CRE elements and activation of CREB proteins seem to play a crucial regulatory role in PDE3B expression. Our results also indicate that insulin-mediated activation of membrane-associated PDE3B may involve formation of a signaling complex containing critical effector molecules, such as PKB, important in regulation of PDE3B activity. A portion of the intracellular pool of endogenous PKB (PKB2 more than PKB1) is found in association with intracellular membranes, co-elutes with endogenous PDE3B during gel filtration chromatography, and co-immunoprecipitates with PDE3B. Phosphorylated PKB seems to associate more effectively with PDE3B than non-phosphorylated PKB. The insulin-induced interactions between PDE3B and PKB are blocked by wortmannin, which blocks phosphorylation/activation of both PKB and PDE3B. To further examine the functional role of PDE3 isoforms, mice with targeted disruptions of PDE3A and B genes have been generated. Female (not male) PDE3A KO mice are sterile, most likely because the absence of oocyte PDE3A leads to increased cAMP and disruption of signals important in progression of meiosis, oocyte maturation, and subsequent fertilization. Recent results suggest that, in PDE3A KO oocytes, the failure of catalytic and regulatory subunits of PKA to translocate to the nucleus may be important in the inhibition of oocyte maturation. PDE3B KO mice seem to accumulate less fat and exhibit signs of insulin resistance and disruption of insulin-regulated homeostatic mechanisms. Insulin inhibits catecholamine-stimulated lipolysis in PDE3B WT but not PDE3B KO oocytes. In isolated pancreatic islets, glucose and cAMP-elevating agents stimulated insulin-secretion to a greater extent in PDE3B KO islets than WT islets. In addition, epididymal adipose tissue in PDE3B KO mice exhibit some phenotypic characteristics of brown adipose tissue, including increased expression of UCP-1 and increased fatty acid oxidation in PDE3B KO adipocytes. O2 consumption by intact mice in response to a Beta-3 receptor agonist was increaed to a greater extent in PDE3B KO than in WT mice.