Shuibang Wang

A SP1 BINDING SITE OF THE TUMOR NECROSIS FACTOR ALPHA PROMOTER FUNCTIONS AS A NITRIC OXIDE RESPONSE ELEMENT

Regulation of gene transcription is an incompletely understood function of nitric oxide (NO). Human leukocytes produce increased amounts of tumor necrosis factor α (TNF-α) in response to NO. This effect is associated with decreases in intracellular cAMP, suggesting that NO might regulate gene transcription through promoter sequences sensitive to cAMP such as cAMP response elements (CRE) and Sp1 binding sites. Here we report that a Sp1 binding site in the TNF-α promoter conveys NO responsiveness. Human U937 cells were differentiated for TNF-α production with phorbol 12-myristate 13-acetate. NO donors and H89, an inhibitor of cAMP-dependent protein kinase increased, while dibutyryl cAMP (Bt2cAMP) decreased TNF-α promoter activity. Deletion or mutation of the proximal Sp1 site, but not the CRE site, abolished the activating effects of NO donors and H89. Further, NO- and H89-mediated increases in TNF-α promoter activity were associated with decreased Sp1 binding. The insertion of Sp1 sites into a minimal cytomegalovirus promoter conferred NO responsiveness, an effect blocked by Bt2cAMP. Mutation of these inserted Sp1 sites prevented this heterologous promoter from responding to NO, H89 and Bt2cAMP. These results identify the Sp1 binding site as a promoter motif that allows NO to control gene transcription.

NITRIC OXIDE INCREASES TUMOR NECROSIS FACTOR PRODUCTION IN DIFFERENTIATED U937 CELLS BY DECREASING CYCLIC AMP

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 μM) 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 μM; p = 0.035), but not in the presence (p = 0.77) of SNAP. Conversely, the cAMP analog dibutyryl cAMP (Bt2cAMP) blocked SNAP-induced TNF production (p = 0.001). SNP and SNAP (500 μM) increased relative TNF mRNA levels by 57.5% (p = 0.045) and 66.2% (p = 0.001), respectively. This effect was prevented by Bt2cAMP. These results indicate that NO up-regulates TNF production by decreasing intracellular cAMP.

Nitric oxide activation of peroxisome proliferator-activated receptor gamma through a p38 MAPK signaling pathway

Both nitric oxide (NO·) and peroxisome proliferator‐activated receptors (PPARs) protect the endothelium and regulate its function. Here, we tested for crosstalk between these signaling pathways. Human umbilical vein and hybrid EA.hy926 endothelial cells were exposed to S‐nitrosoglutathione (GSNO) or dieth‐ylenetriamine NONOate (DETA NONOate). Electro‐phoretic mobility shift assays using PPAR‐response element (PPRE) probe showed that NO· caused a rapid dose‐dependent increase in PPARγ binding, an effect that was confirmed in vivo by chromatin immunopre‐cipitation. Conversely, NGmonomethyl‐L‐arginine, a NOS inhibitor, decreased PPARγ binding. NO·‐medi‐ated PPARγ binding and NO· induction of cyclooxy‐genase‐2 (COX‐2), diacylglycerol (DAG) kinase alpha (DGKx), and heme oxygenase‐1 (HO‐1), genes with well‐characterized PPRE motifs, were cGMP independent. NO· dose dependently activated p38 MAPK, and p38 MAPK inhibition with SB202190 or knockdown with siRNA was shown to block NO· activation of PPARγ. Likewise, p38 MAPK and PPARγ inhibitors or knockdown of either transcript all significantly blocked NO· induction of PPRE‐regulated genes. PPARγ activation by p38 MAPK may contribute to the anti‐inflammatory and cytoprotective effects of NO· in the vascu‐lature. This crosstalk mechanism suggests new strategies for preventing and treating vascular dysfunction.Ptasinska, A., Wang, S., Zhang, J., Wesley, R. A., Danner, R. L. Nitric oxide activation of peroxisome proliferator‐activated receptor gamma through a p38 MAPK signaling pathway. FASEB J. 21, 950–961 (2007)

cGMP-independent nitric oxide signaling and regulation of the cell cycle

BackgroundRegulatory 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 (Bt2cAMP), and then harvested to extract total RNA for microarray analysis. Bt2cAMP was used to block signaling attributable to NO•-induced decreases in cAMP.ResultsNO• regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43%) and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE). Bt2cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO•, Bt2cAMP was associated with ARE-containing transcripts. A comparison of NO• and Bt2cAMP effects showed that NO• regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO• annotated to G1/S (7/8) and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27); 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO•, as was E2F1 binding to E2F promoter elements. NO• activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript) and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M.ConclusionNO• coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO• may rely substantially on cGMP-independent mechanisms. Stress kinase signaling and alterations in mRNA stability appear to be major pathways by which NO• regulates the transcriptome.

Nitric oxide post-transcriptionally up-regulates LPS-induced IL-8 expression through p38 MAPK activation

Nitric oxide (NO·) contributes to vascular collapse in septic shock and regulates inflammation. Here, we demonstrate in lipopolysaccharide (LPS)‐stimulated human THP‐1 cells and monocytes that NO· regulates interleukin (IL)‐8 and tumor necrosis factor α (TNF‐α) by distinct mechanisms. Dibutyryl‐cyclic guanosine 5′‐monophosphate (cGMP) failed to simulate NO·‐induced increases in TNF‐α or IL‐8 production. In contrast, dibutyryl‐cyclic adenosine monophosphate blocked NO·‐induced production of TNF‐α (P=0.009) but not IL‐8. NO· increased IL‐8 (5.7‐fold at 4 h; P=0.04) and TNF‐α mRNA levels (2.2‐fold at 4 h; P=0.037). However, nuclear run‐on assays demonstrated that IL‐8 transcription was slightly decreased by NO· (P=0.08), and TNF‐α was increased (P=0.012). Likewise, NO· had no effect on IL‐8 promoter activity (P=0.84) as measured by reporter gene assay. In THP‐1 cells and human primary monocytes treated with actinomycin D, NO· had no effect on TNF‐α mRNA stability (P>0.3 for both cell types) but significantly stabilized IL‐8 mRNA (P=0.001 for both cell types). Because of its role in mRNA stabilization, the p38 mitogen‐activated protein kinase (MAPK) pathway was examined and found to be activated by NO· in LPS‐treated THP‐1 cells and human monocytes. Further, SB202190, a p38 MAPK inhibitor, was shown to block NO·‐induced stabilization of IL‐8 mRNA (P<0.02 for both cell types). Thus, NO· regulates IL‐8 but not TNF‐α post‐transcriptionally. IL‐8 mRNA stabilization by NO· is independent of cGMP and at least partially dependent on p38 MAPK activation.

Carbon Monoxide Blocks Lipopolysaccharide-Induced Gene Expression by Interfering with Proximal TLR4 to NF-κB Signal Transduction in Human Monocytes

Carbon monoxide (CO) is an endogenous messenger that suppresses inflammation, modulates apoptosis and promotes vascular remodeling. Here, microarrays were employed to globally characterize the CO (250 ppm) suppression of early (1 h) LPS-induced inflammation in human monocytic THP-1 cells. CO suppressed 79 of 101 immediate-early genes induced by LPS; 19% (15/79) were transcription factors and most others were cytokines, chemokines and immune response genes. The prototypic effects of CO on transcription and protein production occurred early but decreased rapidly. CO activated p38 MAPK, ERK1/2 and Akt and caused an early and transitory delay in LPS-induced JNK activation. However, selective inhibitors of these kinases failed to block CO suppression of LPS-induced IL-1β, an inflammation marker. Of CO-suppressed genes, 81% (64/79) were found to have promoters with putative NF-κB binding sites. CO was subsequently shown to block LPS-induced phosphorylation and degradation of IκBα in human monocytes, thereby inhibiting NF-κB signal transduction. CO broadly suppresses the initial inflammatory response of human monocytes to LPS by reshaping proximal events in TLR4 signal transduction such as stress kinase responses and early NF-κB activation. These rapid, but transient effects of CO may have therapeutic applications in acute pulmonary and vascular injury.

Nitric oxide-p38 MAPK signaling stabilizes mRNA through AU-rich element-dependent and -independent mechanisms

Regulation of mRNA stability by p38 MAPK has been linked to adenosine‐uridine‐rich elements (AURE) within the 3′‐untranslated region (3′UTR) of mRNA. Using microarrays, we previously found that AURE‐containing mRNA is over‐represented among transcripts up‐regulated by NO•, an activator of p38 MAPK. Here, we investigated NO•‐induced mRNA stabilization of specific AURE‐containing genes to determine the sequence specificity and protein‐binding interactions associated with this effect. IL‐8, TNF‐α, and p21/Waf1 3′UTRs were inserted into a luciferase (LUC) reporter gene system and found to decrease LUC activity and mRNA half‐life in transfected THP‐1 cells. The inhibitory effect of these 3′UTRs on LUC expression inversely correlated with the number of AUUUA motifs. Sequence truncation of the IL‐8 3′UTR revealed that two segments, one with AURE sites and another without, contributed to mRNA destabilization. NO• activation of p38 MAPK increased LUC activity and mRNA half‐life for reporter constructs that contained either of these IL‐8 3′UTR segments. AURE‐dependent and ‐independent NO• effects were blocked by p38 MAPK inhibition, and AURE‐dependent effects were also blocked by site‐directed mutagenesis of AUUUA sites. Two proteins, HuR and heterogeneous nuclear ribonucleoprotein A0, were identified, which bound to the AURE‐containing region of exogenous and endogenous IL‐8 mRNA in a NO•‐p38 MAPK‐dependent manner. These results demonstrate that NO•‐p38 MAPK signaling can stabilize mRNA via AURE‐dependent and ‐independent mechanisms.

PPARγ signaling and emerging opportunities for improved therapeutics.

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.

G Protein-coupled Receptor 40 (GPR40) and Peroxisome Proliferator-activated Receptor γ (PPARγ): AN INTEGRATED TWO-RECEPTOR SIGNALING PATHWAY*

Background: PPARγ ligands are used to treat type 2 diabetes mellitus, but signaling by these drugs is incompletely understood. Results: Rosiglitazone activation of GPR40 markedly enhanced PPARγ-dependent transcription through downstream effects on p38 MAPK, PGC1α, and EP300. Conclusion: GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway. Significance: Future drug development should consider the effects of prospective ligands at both receptors. Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been widely used to treat type 2 diabetes mellitus. However, knowledge of PPARγ signaling remains incomplete. In addition to PPARγ, these drugs also activate G protein-coupled receptor 40 (GPR40), a Gαq-coupled free fatty acid receptor linked to MAPK networks and glucose homeostasis. Notably, p38 MAPK activation has been implicated in PPARγ signaling. Here, rosiglitazone (RGZ) activation of GPR40 and p38 MAPK was found to boost PPARγ-induced gene transcription in human endothelium. Inhibition or knockdown of p38 MAPK or expression of a dominant negative (DN) p38 MAPK mutant blunted RGZ-induced PPARγ DNA binding and reporter activity in EA.hy926 human endothelial cells. GPR40 inhibition or knockdown, or expression of a DN-Gαq mutant likewise blocked activation of both p38 MAPK and PPARγ reporters. Importantly, RGZ induction of PPARγ target genes in primary human pulmonary artery endothelial cells (PAECs) was suppressed by knockdown of either p38 MAPK or GPR40. GPR40/PPARγ signal transduction was dependent on p38 MAPK activation and induction of PPARγ co-activator-1 (PGC1α). Silencing of p38 MAPK or GPR40 abolished the ability of RGZ to induce phosphorylation and expression of PGC1α in PAECs. Knockdown of PGC1α, its essential activator SIRT1, or its binding partner/co-activator EP300 inhibited RGZ induction of PPARγ-regulated genes in PAECs. RGZ/GPR40/p38 MAPK signaling also led to EP300 phosphorylation, an event that enhances PPARγ target gene transcription. Thus, GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development.

Nitric Oxide Down-regulates Polo-like Kinase 1 through a Proximal Promoter Cell Cycle Gene Homology Region

Polo-like kinase 1 (PLK1) is an evolutionarily conserved serine/threonine kinase essential for cell mitosis. As a master cell cycle regulator, p21/Waf1 plays a critical role in cell cycle progression. Nitric oxide (NO·) has been shown to down-regulate PLK1 and up-regulate p21/Waf1 independent of cGMP. Here, the respective roles of p38 MAPK and p21/Waf1 in NO·-mediated PLK1 repression were investigated using differentiated U937 cells that lack soluble guanylate cyclase. NO· was shown to down-regulate both PLK1 mRNA and protein. Nuclear run-on assays and mRNA stability studies demonstrated that the effect of NO· on PLK1 expression was associated with decreased transcription without changes in transcript stability. SB202190, a p38 MAPK inhibitor, prevented transcriptional repression of PLK1 by NO·. Transfection with dominant-negative p38 MAPK mutant eliminated the NO· effect on both p21/Waf1 and PLK1 gene expression. Knockdown of p21/Waf1 with siRNA also substantially reduced the regulatory effect of NO· on PLK1. Reporter gene experiments showed that NO· decreased activity of the PLK1 proximal promoter, an effect that was blocked by p38 MAPK inhibitor. Deletion or mutation of the CDE/CHR promoter site, an element regulated by p21/Waf1, increased base-line promoter activity and abolished NO· repression of the PLK1 promoter. Likewise, electrophoretic mobility shift assays with CDE/CHR probe revealed a NO·-mediated change in protein-probe complex formation. Competition with various unlabeled CDE/CHR mutant sequences showed that NO· increased nuclear protein binding to intact CHR. These results demonstrate that a NO·-p38 MAPK-p21/Waf1 signal transduction pathway represses PLK1 through a canonical CDE/CHR promoter element.