EXPRESSION/REGULATION OF PHOSPHODIESTERASE 3 ISOFORMS

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 of, and biological responses mediated by, cyclic nucleotides, including immune/inflammatory responses.Understanding cellular regulation of PDE isoforms [which belong to eleven gene families(PDE1-11)] will be of increasing importance for targeting specific PDEs in treating pulmonary disorders. Although individual cells usually contain representatives of several PDE gene families, little is known of signalling pathways involved in cytokine and growth factor regulation of different PDEs in a single cell.In murine FDCP2 promyeloid cells, IL-4 and IGF-1 activate PDE3 and PDE4, whereas IL-3 activates only PDE4. Studies with TNFalpha and inhibitors of JAK, PI3-K, PKC, and MAPK kinases indicate that both IGF-1 and IL-3 activate PDE3 and PDE4 via PI3-K-dependent signals. Downstream of PI3-K, regulatory pathways diverge; PDE4, but not PDE3, is activated by MEK/MAPK-dependent signals. FDCP2 cells were, therefore, permanently transfected with wild type (wt), constitutively active (CA), or kinase inactive (KI) forms of MEK and PKB. Studies with these transfected cells indicated that PDE4 was activated by MEK/MAPK-dependent signals, and that PDE3 was phosphorylated and activated by PKB-dependent signals. Recombinant mouse (M) PDE3B was phosphorylated and activated in vitro by PKB; a truncated MPDE3B mutant lacking consensus PKB phosphorylation sites was not phosphorylated or activated. Serine-alanine mutations were introduced into MPDE3B at S272(PKB consensus phosphorylation site),and at Ser296 and 421(PKA sites).Experiments in intact FDCP2 cells and Sf21 cell lysates indicated that although Ser 272 was involved in activation of MPDE3B by PKB,and Ser296 by PKA, Ser421 seemed to be important in regulation of activation of MPDE3B by both PKA and PKB.In FDCP2 cells expressing WT PKB, the proapoptotic protein BAD was phosphorylated in response to IGF-1; phosphorylation was blocked by 8-Br-cAMP or the PDE3 inhibitor cilostamide. These and other data suggest that PDE3B is a downstream target, if not substrate, of PKB and may function as an effector of PKB in regulation of cAMP pools that modulate, at least in part, effects of IGF-1 and insulin on survival/proliferation of FDCP2 cells and lipolysis in adipocytes. Two promoter regions in the 5'-flanking region of the MPDE3B gene have been identified,a distal promoter region located ~4kb upstream from the translation initiation site and a proximal TATA-less region.Transfection of 3T3-L1 fibroblasts and differentiating 3T3-L1 adipocytes with various luciferase reporter plasmid vectors suggested that:(1)a strong negative regulatory region was present between the distal and proximal promoter regions;(2)CRE cis-elements and CREB proteins might play a crucial regulatory role in the induction of PDE3B that occurs during differentiation of 3T3-L1 adipocytes.In collaborative experiments we found that the stimulatory effects of TNF alphaon lipolysis may in part be related to down-regulation of PDE3B expression in 3T3-L1 adipocytes.Whether downregulation of PDE3B is involved in the effects of TNF alphaon the development of insulin-resistance is not known.