Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor that regulates glucose and lipid metabolism, endothelial function and inflammation. Rosiglitazone (RGZ) and other thiazolidinedione (TZD) synthetic ligands of PPARγ are insulin sensitizers that have been used for the treatment of type 2 diabetes. However, undesirable side effects including weight gain, fluid retention, bone loss, congestive heart failure, and a possible increased risk of myocardial infarction and bladder cancer, have limited the use of TZDs. Therefore, there is a need to better understand PPARγ signaling and to develop safer and more effective PPARγ-directed therapeutics. In addition to PPARγ itself, many PPARγ ligands including TZDs bind to and activate G protein-coupled receptor 40 (GPR40), also known as free fatty acid receptor 1. GPR40 signaling activates stress kinase pathways that ultimately regulate downstream PPARγ responses. Recent studies in human endothelial cells have demonstrated that RGZ activation of GPR40 is essential to the optimal propagation of PPARγ genomic signaling. RGZ/GPR40/p38 MAPK signaling induces and activates PPARγ co-activator-1α, and recruits E1A binding protein p300 to the promoters of target genes, markedly enhancing PPARγ-dependent transcription. Therefore in endothelium, GPR40 and PPARγ function as an integrated signaling pathway. However, GPR40 can also activate ERK1/2, a proinflammatory kinase that directly phosphorylates and inactivates PPARγ. Thus the role of GPR40 in PPARγ signaling may have important implications for drug development. Ligands that strongly activate PPARγ, but do not bind to or activate GPR40 may be safer than currently approved PPARγ agonists. Alternatively, biased GPR40 agonists might be sought that activate both p38 MAPK and PPARγ, but not ERK1/2, avoiding its harmful effects on PPARγ signaling, insulin resistance and inflammation. Such next generation drugs might be useful in treating not only type 2 diabetes, but also diverse chronic and acute forms of vascular inflammation such as atherosclerosis and septic shock.
Regulation of gene transcription is an incompletely understood function of nitric oxide (NO).
Nitric oxide (NO) increases tumor necrosis factor (TNF) synthesis in human peripheral blood mononuclear cells by a cGMP-independent mechanism. NO has been shown to inhibit adenylate cyclase in cell membranes. Since cAMP down-regulates TNF transcription, we examined the possibility that NO enhances TNF synthesis by decreasing cAMP. U937 cells were induced to differentiate using phorbol myristate acetate (100 nM for 48 h) and then were incubated for 24 h with sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP). These NO donors increased TNF production (7.0-and 15.6-fold, respectively, at 500 M) in a dose-dependent manner (p ؍ 0.002). However, SNP and SNAP did not elevate cGMP levels in U937 cell cultures, and the cGMP analog, 8-bromo-cGMP, had no effect on TNF production. In contrast, SNP (p ؍ 0.001) and SNAP (p ؍ 0.009) decreased intracellular cAMP levels by up to 51.5% over 24 h and, in the presence of a phosphodiesterase inhibitor, blunted isoproterenol-stimulated increases in cAMP by 21.8% (p ؍ 0.004) and 27.6% (p ؍ 0.008), respectively. H89, an inhibitor of cAMP-dependent protein kinase, dose dependently increased TNF production in phorbol myristate acetate-differentiated U937 cells in the absence (6.5-fold at 30 M; p ؍ 0.035), but not in the presence (p ؍ 0.77) of SNAP. Conversely, the cAMP analog dibutyryl cAMP (Bt 2 cAMP) blocked SNAPinduced TNF production (p ؍ 0.001). SNP and SNAP (500 M) increased relative TNF mRNA levels by 57.5% (p ؍ 0.045) and 66.2% (p ؍ 0.001), respectively. This effect was prevented by Bt 2 cAMP. These results indicate that NO up-regulates TNF production by decreasing intracellular cAMP. Nitric oxide (NO)1 is a free-radical gas produced by many cell types (1-4). NO has a diverse repertoire of important functions (5-9) including neurotransmission (5, 10), vasodilatation (11), antiplatelet activity, and immune modulation (12)(13)(14). Most of these effects are mediated through a unique cGMP signaling pathway. NO covalently attacks the heme moiety of soluble guanylate cyclase, activating the enzyme, and thereby elevating intracellular cGMP concentrations (15)(16)(17). This increase in cGMP subsequently activates certain protein kinases, which phosphorylate target proteins involved in regulation of cell function (17)(18)(19). Although the role of cGMP as a NO second messenger is undisputed, some findings have led to speculation about the existence of cGMP-independent signal transduction pathways for NO.First, NO is a free radical with the ability to react with a variety of enzymes besides soluble guanylate cyclase. NO has been shown to catalyze the covalent binding of NAD to glyceraldehyde-3-phosphate dehydrogenase (20), oxidize iron-containing proteins such as aconitase or ribonucleotide reductase (21-23), and nitrosylate tyrosine and cysteine residues in a variety of proteins (24 -26). Second, some effects of NO cannot be reproduced with cell permeable cGMP analogs. For example, the synthesis of tumor necrosis factor ␣ (TNF␣), a proinflammatory cyt...
Background: Regulatory functions of nitric oxide (NO • ) that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO • on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1) were exposed to S-nitrosoglutathione, a NO • donor, or glutathione alone (control) for 6 h without or with dibutyryl-cAMP (Bt 2 cAMP), and then harvested to extract total RNA for microarray analysis. Bt 2 cAMP was used to block signaling attributable to NO • -induced decreases in cAMP.
Reactive oxygen species can function as intracellular messengers, but linking these signaling events with specific enzymes has been difficult.
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