Expression, Structure/function And Regulation Of Phospho

Heart, Lung, And Blood Institute

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Abstract

By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases (PDEs) are critical regulators of intracellular concentrations and biological effects of cyclic nucleotides. 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. PDE3B appears (or greatly increases in activity) during differentiation of cultured 3T3-L1 adipocytes or cultured human monocyte-derived macrophages. To study regulation of PDE3B expression, a genomic fragment of the 5'-flanking region of the mouse(M) PDE3B gene was isolated; two promoter regions were identified, a distal promoter located ~4 kb upstream, and a proximal region, ~500 bp upstream of the translation initiation ATG codon. Transfection of 3T3-L1 fibroblasts and differentiating 3T3-L1 adipocytes with a series of reporter plasmids containing the luciferase gene coupled to different fragments of the 5'-UTR of the MPDE3B gene indicated that the distal region induced about 50-fold higher promoter activity than did the proximal region. Transfection with distal and proximal promoters indicated that (1) a strong negative regulatory region was present between the distal and proximal promoter regions; and (2) transfection with constructs containing CRE elements of the distal promoter markedly increased promoter activity in differentiating adipocytes. Differentiation of 3T3-L1 adipocytes is induced by medium containing DMI (dexamethasone, isobutylmethylxanthine and insulin). Induction of PDE3B was markedly enhanced by M (which presumably increased cAMP, leading to phosphorylation/activation of CREB proteins)and was dissociated from lipid accumulation, which was more pronounced in the presence of DI. Taken together, these results suggest that activation of CREB proteins might play a crucial role in the expression of MPDE3B in 3T3-L1 differentiating adipocytes. Studies with 3T3-L1 adipocytes and FDCP2 cells indicated that insulin- and IGF-1-induced phosphorylation/activation of PDE3B was mediated via PI3-K- and PKB-dependent signals, and that PDE3B is most likely a direct substrate of PKB. MPDE3B contains a consensus phosphorylation site (Ser 273) for PKB; after mutation of Ser 273 Ala, PKB does not activate MPDE3B in vitro. To study activation of microsomal MPDE3B by insulin in 3T3-L1 cells, we examined the subcellular distribution of components of the insulin-signaling pathway involved in activation of PDE3B. In 3T3-L1 adipocytes, PDE3B is primarily, but not exclusively, associated with intracellular membranes (LDM and HDM fractions). Incubation with insulin results in tyrosine phosphorylation of IRS-1 and activation of PI3-K associated with intracellular membranes. In addition, a portion of the intracellular pool of PKB is also found in association with intracellular membranes, co-elutes with PDE3B during size exclusion chromatography on AcA34, and co-immunoprecipitates with MPDE3B. The structural determinants for this later interaction reside in the N-terminal portion of MPDE3. These results suggest that signals to activate membrane-associated PDE3B are directed via IRS/PI3-K to intracellular membranes where activation of PI3-K and production of phosphoinositides results in activation of PDK, PKB and, consequently, PDE3B. To further examine the functional role of PDE3B isoforms, null PDE3A and PDE3B mice were generated. As PDE3B null mice age, the males demonstrate abnormal glucose and insulin tolerance tests. Adipocytes from PDE3B null mice are smaller than those from WT animals, and they are less responsive to lipolytic stimuli, including isoproterenol and adenosine deaminase. Studies are in progress to understand the role of PDE3B in these and other phenotypic changes.