This report describes studies designed to study roles of PDE3A and PDE3B in regulation of inflammation, myocardial function, and oxidative stress. Phosphodiesterase 3B (PDE3B) regulates the NLRP3 inflammasome and metabolic cross talk in adipose tissue: Activation of inflammation in white adipose tissue (WAT), which includes infiltration/expansion of WAT macrophages, contributes to pathogenesis of obesity, insulin resistance, type 2 diabetes, and metabolic syndrome. The NLRP3 inflammasome protein complex comprises an intracellular sensor (typically a Nod-like receptor, NLR), caspase-1 (a cysteine protease) and the adaptor ASC (apoptosis-associated speck-like protein). Inflammasome activation leads to the maturation of caspase-1 and processing of IL1β, contributing to inflammation and many metabolic disorders,and directing adipocytes to a more insulin-resistant phenotype. Ablation of PDE3B in SVJ129 mice epididymal white adipose tissue (EWAT) prevents inflammasome activation by reducing the expressions of NLRP3, caspase-1, ASC, interferon-inducible protein AIM2, TNFα, IL1β and proinflammatory genes (i.e, cyclooxygenase2). Following IP (intraperitoneal) injection of lipopolysaccharide (LPS), serum levels of IL1β and TNFα were reduced in PDE3B-/- mice compared to WT. The activation of signaling cascades that mediate inflammasome responses were modulated in PDE3B-KO mice EWAT, including smad, NFAT, NFkB, and MAP kinases. Moreover, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein-1 (MCP-1) and its receptor CCR2, which play an important role in the macrophage chemotaxis, were less highly expressed in EWAT of PDE3B-/- mice. In addition, atherosclerotic plaque formation was significantly reduced in the aorta of apoE-/-/PDE3B-/- and LDL-R-/-/PDE3B-/- double KO mice compared to apoE-/- and LDL-R-/- mice, respectively. Ablation of PDE3B in mice prevents obesity-induced changes in serum-cholestrol. Thus, in SvJ129 PDE3B KO EWAT expression of pro-inflammatory markers was reduced, compared to WT, as were components of the NLRP3 inflammasome (activation of NLRP3 inflammasomes may be related to insulin resistance and obesity-related inflammation). Collectively, these data establish a role for PDE3B in modulating the inflammatory response which may contribute to the decreased inflammatory state in KO EWAT. Regulation of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) activity by phosphodiesterase 3A (PDE3A) in human myocardium: phosphorylation-dependent interaction of PDE3A1 with SERCA2: Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in mouse sarcoplasmic reticulum (SR) (Circ Res 112:289-97, 2013). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity. Effects of heterologous expression of human Cyclic Nucleotide Phosphodiesterase 3A (hPDE3A) on redox regulation in yeast: Yeast expressing WT human (h)PDE3A or K13R hPDE3A (putative ubiquitinylation site mutant) exhibited resistance or sensitivity to exogenous H2O2, respectively. H2O2-stimulated ROS production was markedly increased in yeast expressing K13R hPDE3A, named OxiS1, compared to yeast expressing WT hPDE3A, named OxiR1. In OxiR1, YAP1 and YAP1-dependent anti-oxidant genes involved in recovery from oxidative stress, including SRX1, were upregulated, and accompanied by reduction of Tsa1p. However, in OxiS1, expression of YAP1 and YAP1-dependent genes was greatly reduced, resulting in failure to recover from oxidative stress. H2O2 increased ubiquitinylation, phosphorylation, and hydrolytic activity of WT hPDE3A, but not K13R hPDE3A. The changes in anti-oxidant gene expression did not directly correlate with differences in cAMP/PKA signaling, since despite differences in their capacities to hydrolyze cAMP, cAMP levels in the three strains were similar, and PKA activity was lower in OxiS1 than in OxiR1 or mock cells. During exposure to H2O2, however, the activity of Sch9p, a TORC1-regulated rpS6 kinase and negative regulator of PKA in S. cerevisiae, was rapidly reduced in OxiR1, and Tpk1p, a PKA catalytic subunit, was diffusely spread throughout the cytosol. This distribution of Tpk1 is associated with PKA activation. On the other hand, in OxiS1, Sch9 activity was maintained during exposure to H2O2, consistent with reduced activation of PKA and reduced phosphorylation of PKA substrates. These results suggest that, during oxidative stress, PKA activity may be regulated by TOR-Sch9 signaling, and that post-translational modifications of hPDE3A are critical in its regulation of cellular recovery from oxidative stress.

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21
Fiscal Year
2015
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U.S. National Heart Lung and Blood Inst
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