PDE3B Although white adipose tissue (WAT) affects body fat and energy utilization, it also contributes to metabolic dysregulation, and obesity is linked to many disease states. The idea of inducing white adipose tissue (WAT) to assume characteristics of brown adipose tissue (BAT), and thus gearing it to fat burning instead of storage, is receiving consideration as potential treatment for obesity and related disorders. We therefore utilized PDE3B knockout (KO) mice to elucidate mechanisms involved in formation of BAT in epididymal WAT (EWAT). Gene expression profiles in EWAT from C57BL/6 PDE3B KO mice revealed increased expression of several genes blocking white and promoting brown adipogenesis, such as C-terminal binding protein (Ctbp), bone morphogenetic protein 7 (Bmp7) and PR domain containing 16 (Prdm16), but a clear BAT-like phenotype was not induced. However, acute treatment of KO mice with the β3-adrenergic agonist, CL316243, markedly increased expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin synthesis, together with elongation of very long chain fatty acids 3 (Elovl3) (linked to BAT recruitment), and caused a clear shift toward fat-burning and induction of BAT in KO EWAT. Thus, in C57BL/6 background, an increase in cAMP, caused by ablation of PDE3B and administration of CL316243, may promote differentiation of prostaglandin-responsive progenitor cells residing in the EWAT stromal vascular fraction into functional brown adipocytes. Development of obesity and insulin resistance is also associated with chronic inflammation and increased infiltration of macrophages in WAT. As discussed in a previous Annual Report, targeted disruption of SvJ129 PDE3B was associated with decreased expression of pro-inflammatory genes and macrophage markers in KO EWAT, including MCP-1, MIP1 (Ccl3), F4/80 (Emr1), TNFα, caspase1, Ccr2 and Ccr5. Inflammasomes are molecular platforms that are comprised of intracellular innate immune sensors (typically a Nod-like receptor, NLR), precursor procaspase-1 and the adaptor ASC (apoptosis-associated speck-like protein). They are activated upon stress or cellular infection and trigger generation of proinflammatory cytokines. In adipose tissue, activation of the NLRP3 inflammasome (Nod-like receptor ,leucine rich, pyrin domain-containing-3), leading to activation of caspase1 and generation/release of the pro-inflammatory cytokines interleulin-1and IL-18, is important in inducing obesity and insulin resistance. Our current findings indicate that targeted inactivation of the SvJ129 PDE3B gene resulted in decreased inflammasome activation in KO EWAT, as reflected in decreased protein expression of NLRP3, procaspase-1, apoptosis-associated speck-like protein (ASC), interferon-inducible protein AIM2, and the proinflammatory cytokine TNFα. These results correlated with the observations that, after lipopolysaccaride (LPS) injection in intact mice, plasma levels of IL-1, TNFα, IL-12, MCP-1, and MIP-1were lower in PDE3B KO mice than WT. Understanding the mechanisms for these beneficial phenotypic changes in the PDE3B KO mice, i.e., the conversion of WAT to BAT and the reduced pro-inflammatory state, is important, since conversion of fat-storing EWAT to healthy fat-burning BAT represents a potential strategy in treatment of obesity and diabetes. This work might not only increase our understanding of the pathophysiology of these disorders, but also could identify important therapeutic targets. PDE3A Intracellular concentrations of cyclic nucleotides are tightly regulated, and their signaling pathways are exquisitely compartmentalized. PDEs are major contributors to this compartmentation, and assume specific functional roles by regulating discrete cyclic nucleotide signaling pathways at distinct subcellular locations. Individual PDEs are targeted to specific macromolecular complexes or signalosomes at different subcellular locations via their interactions with molecular scaffolds such as A Kinase Anchoring Proteins (AKAPs). We are currently very interested in identifying PDE3-containing signalosomes and PDE3-interacting partners, to better understand their roles in regulating cyclic nucleotide concentrations and complex cyclic nucleotide signaling pathways. BIG1 and BIG2 proteins catalyze activation of class I ARFs (ADP-ribosylation factors) by accelerating exchange of bound GDP for GTP. They contain AKAP sequences which may act as scaffolds for the assembly of PKA with other enzymes, substrates and regulators. PKA-catalyzed phosphorylation of BIG inhibits its GEF activity. In Hela cells, PDE3A interacts with BIG proteins (PNAS 106, 6158, 2009). Three PDE3A isoforms (PDE3A1-3), with identical sequences except for N-terminal deletions of different lengths, are present in human myocardium. Non-phosphorylated cytosolic PDE3A2-3 eluted from Superose 6 (S6) in LMW (low molecular weight) fractions (800 kD). Incubation of cytosol with PKA and ATP induced assembly of macromolecular signalosomes that eluted from S6 as HMW (high molecular weight) complexes (>3000 kD) and contained phosphorylated (p)PDE3A (predominantly pPDE3A2) and proteins involved in cAMP signaling (e.g., PKA-RII, PP2A, 14-3-3, BIG1). These molecules co-immunoprecipitated with pPDE3A in HMW, but not with non-phosphorylated PDE3A in LMW fractions. These data suggest that, in cardiomyocytes, pPDE3A is incorporated into BIG1-organized signalosomes, and in these spatially restricted compartments, pPDE3A may modulate cAMP/PKA signaling and thereby contribute to regulation of BIG1 activity and ARF function, including vesicular trafficking. In searching for interacting partners a yeast-two hybrid system was used, and 15 novel PDE3A-interacting partners, including two E3 ubiquitin ligases (RNF125 and RNM5DA) were identified. To begin to study the interactions between the E3 ligases and PDE3A, potential ubiquination sites in PDE3A were mutated. Expression of the various mutants in yeast, however, indicated that, compared to control (mock-transfected) yeast, growth of a yeast strain expressing the PDE3A mutation of lysine 13 to arginine (K13R) was dramatically reduced. On the other hand, growth was slightly accelerated in yeast expressing WT PDE3A. In response to oxidative stress induced by H2O2, yeast expressing WT PDE3A were slightly more resistant than mock-transfected yeast, whereas yeast expressing K13R were more sensitive, indicating that WT PDE3A, but not K13R, facilitated recovery from oxidative stress. Expression of PDE3A was associated with a marked increase in the effect of H2O2 on expression of YAP1 and YAP1-dedendent oxidative stress-response genes, including GLR1, ZWF1, TSA1, and SRX1, and with an increase in sulfiredoxin Srx 1 protein expression and in the reduced form of the peroxiredoxin Tsa1. K13R expression blocked the effect of H2O2 on expression of these oxidative stress-response genes and proteins. Both WT PDE3A and K13R PDE3A were expressed at similar levels, based on Western blots and PDE3 enzymatic activity measurements in yeast homogenates. H2O2 induced phosphorylation/activation and ubiquination of WT, but not K13R, PDE3A. These and other results suggest that PDE3A is involved in thiol redox regulation and protection during oxidative stress, and that its ubiquination may be important in this effect. Given that these are highly conserved reactions it will be important to examine mechanisms whereby heterologous PDE3A regulates expression of oxidative stress-response genes in yeast, as well as to examine effects of PDE3A on the oxidative stress response in mammalian systems. We will attempt to assess the interactions between hPDE3A and E3 ligases, and functional sequellae of these interactions and of PDE3A ubiquination, in mammalian cells.

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