Disruption of DLL4/NOTCH1 Causes Dysregulated PPARγ/AKT Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, activates NOTCH1 signaling and plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in PAECs activated AKT and decreased DLL4 expression. DLL4 loss was also seen in lungs of patients with IPAH and HPAH. Over-expression of DLL4 in PAECs induced BMPR2 promoter activity and exogenous DLL4 increased BMPR2 mRNA through NOTCH1 activation. Furthermore, DLL4/NOTCH1 signaling blocked AKT activation, decreased proliferation and reversed EndoMT in BMPR2-silenced PAECs and ECs from IPAH patients. PPARγ, suppressed by BMPR2 loss, was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH PAECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Finally, leniolisib, a well-tolerated oral PI3Kδ/AKT inhibitor, decreased cell proliferation, induced apoptosis and reversed markers of EndoMT in BMPR2-silenced PAECs. Restoring DLL4/NOTCH1/PPARγ signaling and/or suppressing AKT activation may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

BMPR2 Loss Activates AKT by Disrupting DLL4/NOTCH1 and PPARγ Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

Mineralocorticoid receptor antagonist treatment of established pulmonary arterial hypertension improves interventricular dependence in the SU5416-hypoxia rat model.

Treatment with mineralocorticoid receptor (MR) antagonists beginning at the outset of disease, or early thereafter, prevents pulmonary vascular remodeling in preclinical models of pulmonary arterial hypertension (PAH). However, the efficacy of MR blockade in established disease, a more clinically relevant condition, remains unknown. Therefore, we investigated the effectiveness of two MR antagonists, eplerenone (EPL) and spironolactone (SPL), after the development of severe right ventricular (RV) dysfunction in the rat SU5416-hypoxia (SuHx) PAH model. Cardiac magnetic resonance imaging (MRI) in SuHx rats at the end of week 5, before study treatment, confirmed features of established disease including reduced RV ejection fraction and RV hypertrophy, pronounced septal flattening with impaired left ventricular filling and reduced cardiac index. Five weeks of treatment with either EPL or SPL improved left ventricular filling and prevented the further decline in cardiac index compared with placebo. Interventricular septal displacement was reduced by EPL whereas SPL effects were similar, but not significant. Although MR antagonists did not significantly reduce pulmonary artery pressure or vessel remodeling in SuHx rats with established disease, animals with higher drug levels had lower pulmonary pressures. Consistent with effects on cardiac function, EPL treatment tended to suppress MR and proinflammatory gene induction in the RV. In conclusion, MR antagonist treatment led to modest, but consistent beneficial effects on interventricular dependence after the onset of significant RV dysfunction in the SuHx PAH model. These results suggest that measures of RV structure and/or function may be useful endpoints in clinical trials of MR antagonists in patients with PAH.

Type I interferon activation and endothelial dysfunction in caveolin-1 insufficiency-associated pulmonary arterial hypertension.

Interferonopathies, interferon (IFN)-α/ß therapy, and caveolin-1 (CAV1) loss-of-function have all been associated with pulmonary arterial hypertension (PAH). Here, CAV1-silenced primary human pulmonary artery endothelial cells (PAECs) were proliferative and hypermigratory, with reduced cytoskeletal stress fibers. Signal transducers and activators of transcription (STAT) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) were both constitutively activated in these cells, resulting in a type I IFN-biased inflammatory signature. Cav1-/- mice that spontaneously develop pulmonary hypertension were found to have STAT1 and AKT activation in lung homogenates and increased circulating levels of CXCL10, a hallmark of IFN-mediated inflammation. PAH patients with CAV1 mutations also had elevated serum CXCL10 levels and their fibroblasts mirrored phenotypic and molecular features of CAV1-deficient PAECs. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 frameshift mutations. While blocking JAK/STAT or AKT rescued aspects of CAV1 loss, only AKT inhibitors suppressed activation of both signaling pathways simultaneously. Silencing endothelial nitric oxide synthase (NOS3) prevented STAT1 and AKT activation induced by CAV1 loss, implicating CAV1/NOS3 uncoupling and NOS3 dysregulation in the inflammatory phenotype. Exogenous IFN reduced CAV1 expression, activated STAT1 and AKT, and altered the cytoskeleton of PAECs, implicating these mechanisms in PAH associated with autoimmune and autoinflammatory diseases, as well as IFN therapy. CAV1 insufficiency elicits an IFN inflammatory response that results in a dysfunctional endothelial cell phenotype and targeting this pathway may reduce pathologic vascular remodeling in PAH.

Spironolactone-induced degradation of the TFIIH core complex XPB subunit suppresses NF-κB and AP-1 signalling.

Aims: Spironolactone (SPL) improves endothelial dysfunction and survival in heart failure. Immune modulation, including poorly understood mineralocorticoid receptor (MR)-independent effects of SPL might contribute to these benefits and possibly be useful in other inflammatory cardiovascular diseases such as pulmonary arterial hypertension. Methods and results: Using human embryonic kidney cells (HEK 293) expressing specific nuclear receptors, SPL suppressed NF-κB and AP-1 reporter activity independent of MR and other recognized nuclear receptor partners. NF-κB and AP-1 DNA binding were not affected by SPL and protein synthesis blockade did not interfere with SPL-induced suppression of inflammatory signalling. In contrast, proteasome blockade to inhibit degradation of xeroderma pigmentosum group B complementing protein (XPB), a subunit of the general transcription factor TFIIH, or XPB overexpression both prevented SPL-mediated suppression of inflammation. Similar to HEK 293 cells, a proteasome inhibitor blocked XPB loss and SPL suppression of AP-1 induced target genes in human pulmonary artery endothelial cells (PAECs). Unlike SPL, eplerenone (EPL) did not cause XPB degradation and failed to similarly suppress inflammatory signalling. SPL combined with siRNA XPB knockdown further reduced XPB protein levels and had the greatest effect on PAEC inflammatory gene transcription. Using chromatin-immunoprecipitation, PAEC target gene susceptibility to SPL was associated with low basal RNA polymerase II (RNAPII) occupancy and TNFα-induced RNAPII and XPB recruitment. XP patient-derived fibroblasts carrying an N-terminal but not C-terminal XPB mutations were insensitive to both SPL-mediated XPB degradation and TNFα-induced target gene suppression. Importantly, SPL treatment decreased whole lung XPB protein levels in a monocrotaline rat model of pulmonary hypertension and reduced inflammatory markers in an observational cohort of PAH patients. Conclusion: SPL has important anti-inflammatory effects independent of aldosterone and MR, not shared with EPL. Drug-induced, proteasome-dependent XPB degradation may be a useful therapeutic approach in cardiovascular diseases driven by inflammation.

Dietary supplementation with long-chain monounsaturated fatty acid isomers decreases atherosclerosis and alters lipoprotein proteomes in LDLr-/- mice.

BACKGROUND AND AIMS: Concentrated fish oils, containing a mixture of long-chain monounsaturated fatty acids (LCMUFA) with aliphatic chains longer than 18 C atoms (i.e., C20:1 and C22:1), have been shown to attenuate atherosclerosis development in mouse models. It is not clear, however, how individual LCMUFA isomers may act on atherosclerosis. METHODS: In the present study, we used saury fish oil-derived concentrates enriched in either C20:1 or C22:1 isomer fractions to investigate their individual effect on atherosclerosis and lipoprotein metabolism. LDLR-deficient (LDLr-/-) mice were fed a Western diet supplemented with 5% (w/w) of either C20:1 or C22:1 concentrate for 12 wk. RESULTS: Compared to the control Western diet with no supplement, both LCMUFA isomers increased hepatic levels of LCMUFA by 2∼3-fold (p < 0.05), and decreased atherosclerotic lesion areas by more than 40% (p < 0.05), although there were no major differences in plasma lipoproteins or hepatic lipid content. Both LCMUFA isomers significantly decreased plasma CRP levels, improved Abca1-dependent cholesterol efflux capacity of apoB-depleted plasma, and enhanced Ppar transcriptional activities in HepG2 cells. LC-MS/MS proteomic analysis of lipoproteins (HDL, LDL and VLDL) revealed that both LCMUFA isomer diets resulted in similar potentially beneficial alterations in proteins involved in complement activation, blood coagulation, and lipid metabolism. Several lipoprotein proteome changes were significantly correlated with atherosclerotic plaque reduction. CONCLUSIONS: Dietary supplementation with the LCMUFA isomers C20:1 or C22:1 was equally effective in reducing atherosclerosis in LDLr-/-mice and this may partly occur through activation of the Ppar signaling pathways and favorable alterations in the proteome of lipoproteins.

Mineralocorticoid Receptor (MR) trans-Activation of Inflammatory AP-1 Signaling: DEPENDENCE ON DNA SEQUENCE, MR CONFORMATION, AND AP-1 FAMILY MEMBER EXPRESSION.

Glucocorticoids are commonly used to treat inflammatory disorders. The glucocorticoid receptor (GR) can tether to inflammatory transcription factor complexes, such as NFκB and AP-1, and trans-repress the transcription of cytokines, chemokines, and adhesion molecules. In contrast, aldosterone and the mineralocorticoid receptor (MR) primarily promote cardiovascular inflammation by incompletely understood mechanisms. Although MR has been shown to weakly repress NFκB, its role in modulating AP-1 has not been established. Here, the effects of GR and MR on NFκB and AP-1 signaling were directly compared using a variety of ligands, two different AP-1 consensus sequences, GR and MR DNA-binding domain mutants, and siRNA knockdown or overexpression of core AP-1 family members. Both GR and MR repressed an NFκB reporter without influencing p65 or p50 binding to DNA. Likewise, neither GR nor MR affected AP-1 binding, but repression or activation of AP-1 reporters occurred in a ligand-, AP-1 consensus sequence-, and AP-1 family member-specific manner. Notably, aldosterone interactions with both GR and MR demonstrated a potential to activate AP-1. DNA-binding domain mutations that eliminated the ability of GR and MR to cis-activate a hormone response element-driven reporter variably affected the strength and polarity of these responses. Importantly, MR modulation of NFκB and AP-1 signaling was consistent with a trans-mechanism, and AP-1 effects were confirmed for specific gene targets in primary human cells. Steroid nuclear receptor trans-effects on inflammatory signaling are context-dependent and influenced by nuclear receptor conformation, DNA sequence, and the expression of heterologous binding partners. Aldosterone activation of AP-1 may contribute to its proinflammatory effects in the vasculature.

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.

Raf/ERK drives the proliferative and invasive phenotype of BMPR2-silenced pulmonary artery endothelial cells.

A proliferative endothelial cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Bone morphogenetic protein type II receptor (BMPR2) loss-of-function is the most common cause of heritable PAH and has been closely linked to the formation of pathological plexiform lesions. Although some BMPR2 mutations leave ligand-dependent responses intact, the disruption of ligand-independent, noncanonical functions are universal among PAH-associated BMPR2 genotypes, but incompletely understood. This study examined the noncanonical signaling consequences of BMPR2 silencing in human pulmonary artery endothelial cells to identify potential therapeutic targets. BMPR2 siRNA silencing resulted in a proliferative, promigratory pulmonary artery endothelial cell phenotype and disruption of cytoskeletal architecture. Expression profiling closely reflected these phenotypic changes. Gene set enrichment and promoter analyses, as well as the differential expression of pathway components identified Ras/Raf/ERK signaling as an important consequence of BMPR2 silencing. Raf family members and ERK1/2 were constitutively activated after BMPR2 knockdown. Two Raf inhibitors, sorafenib and AZ628, and low-dose nintedanib, a triple receptor tyrosine kinase inhibitor upstream from Ras, reversed the abnormal proliferation and hypermotility of BMPR2 deficiency. Inhibition of dysregulated Ras/Raf/ERK signaling may be useful in reversing vascular remodeling in PAH.

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

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.

Carbon monoxide blocks lipopolysaccharide-induced gene expression by interfering with proximal TLR4 to NF-kappaB 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-1beta, an inflammation marker. Of CO-suppressed genes, 81% (64/79) were found to have promoters with putative NF-kappaB binding sites. CO was subsequently shown to block LPS-induced phosphorylation and degradation of IkappaBalpha in human monocytes, thereby inhibiting NF-kappaB 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-kappaB 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-alpha, 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.

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 diethylenetriamine NONOate (DETA NONOate). Electrophoretic mobility shift assays using PPAR-response element (PPRE) probe showed that NO* caused a rapid dose-dependent increase in PPARgamma binding, an effect that was confirmed in vivo by chromatin immunoprecipitation. Conversely, N(G)-monomethyl-L-arginine, a NOS inhibitor, decreased PPARgamma binding. NO*-mediated PPARgamma binding and NO* induction of cyclooxygenase-2 (COX-2), diacylglycerol (DAG) kinase alpha (DGKalpha), 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 PPARgamma. Likewise, p38 MAPK and PPARgamma inhibitors or knockdown of either transcript all significantly blocked NO* induction of PPRE-regulated genes. PPARgamma activation by p38 MAPK may contribute to the anti-inflammatory and cytoprotective effects of NO* in the vasculature. This crosstalk mechanism suggests new strategies for preventing and treating vascular dysfunction.

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.

Nitric oxide activation of Erk1/2 regulates the stability and translation of mRNA transcripts containing CU-rich elements.

Nitric oxide (NO*) can stabilize mRNA by activating p38 mitogen-activated protein kinase (MAPK). Here, transcript stabilization by NO* was investigated in human THP-1 cells using microarrays. After LPS pre-stimulation, cells were treated with actinomycin D and then exposed to NO* without or with the p38 MAPK inhibitor SB202190 (SB). The decay of 220 mRNAs was affected; most were stabilized by NO*. Unexpectedly, SB often enhanced rather than antagonized transcript stability. NO* activated p38 MAPK and Erk1/2; SB blocked p38 MAPK, but further activated Erk1/2. RT-PCR confirmed that NO* and SB could additively stabilize certain mRNA transcripts, an effect abolished by Erk1/2 inhibition. In affected genes, these responses were associated with CU-rich elements (CURE) in 3'-untranslated regions (3'-UTR). NO* stabilized the mRNA of a CURE-containing reporter gene, while repressing translation. Dominant-negative Mek1, an Erk1/2 inhibitor, abolished this effect. NO* similarly stabilized, but blocked translation of MAP3K7IP2, a natural CURE-containing gene. NO* increased hnRNP translocation to the cytoplasm and binding to CURE. Over-expression of hnRNP K, like NO*, repressed translation of CURE-containing mRNA. These findings define a sequence-specific mechanism of NO*-triggered gene regulation that stabilizes mRNA, but represses translation.

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

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 (Bt2cAMP), and then harvested to extract total RNA for microarray analysis. Bt2cAMP was used to block signaling attributable to NO*-induced decreases in cAMP. RESULTS: NO* 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. CONCLUSION: NO* 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 alpha (TNF-alpha) by distinct mechanisms. Dibutyryl-cyclic guanosine 5'-monophosphate (cGMP) failed to simulate NO(.-)-induced increases in TNF-alpha or IL-8 production. In contrast, dibutyryl-cyclic adenosine monophosphate blocked NO(.-)-induced production of TNF-alpha (P=0.009) but not IL-8. NO(.-) increased IL-8 (5.7-fold at 4 h; P=0.04) and TNF-alpha 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-alpha 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-alpha 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-alpha post-transcriptionally. IL-8 mRNA stabilization by NO(.-) is independent of cGMP and at least partially dependent on p38 MAPK activation.

Genetic regulation of endotoxin-induced airway disease.

To identify novel genes regulating the biologic response to lipopolysaccharide (LPS), we used a combination of quantitative trait locus (QTL) analysis and microarray-based gene expression studies of C57BL/6J x DBA/2J(BXD) F2 and recombinant inbred (RI) mice. A QTL affecting pulmonary TNF-alpha production was identified on chromosome 2, and a region affecting both polymorphonuclear leukocyte recruitment and TNF-alpha levels was identified on chromosome 11. Microarray analyses of unchallenged and LPS-challenged BXD RI strains identified approximately 500 genes whose expression was significantly changed by inhalation of LPS. Of these genes, 28 reside within the chromosomal regions identified by the QTL analyses, implicating these genes as high priority candidates for functional studies. Additional high priority candidate genes were identified based on their differential expression in mice having high and low responses to LPS. Functional studies of these genes are expected to reveal important molecular mechanisms regulating the magnitude of biologic responses to LPS.

Adjacent sequence controls the response polarity of nitric oxide-sensitive Sp factor binding sites.

Nitric oxide (NO*) and cAMP-dependent protein kinase (PKA) inhibitors up-regulate tumor necrosis factor alpha (TNFalpha) by decreasing Sp1 binding to a proximal GC box element. Here, elements flanking GC boxes were tested for their role in determining whether Sp sites act as activators or repressors. Promoter studies in receptive human cell lines demonstrated that NO* down-regulated endothelial NO* synthase (eNOS) but up-regulated TNFalpha. Like TNFalpha, Sp1 binding to the eNOS promoter was decreased by NO* and a PKA inhibitor, H89, and increased by a PKA activator, dibutyryl cAMP (Bt2cAMP). For either promoter, mutation of Sp sites abolished NO* responses. In contrast, mutation of an upstream AP1 site in the TNFalpha promoter (not present in eNOS) maintained NO* responsiveness, but reversed the direction of NO* and cAMP effects. Using artificial constructs, NO* increased transcription when Sp and AP1 sites were both present (TNFalpha-like response), but decreased it when the adjacent AP1 site was disrupted (eNOS-like response). NO*, H89, and Bt2cAMP were found to produce reciprocal protein binding changes at contiguous AP1 and Sp sites (p < 0.0001 for an interaction). Chromatin immunoprecipitation assays demonstrated that Sp1 and to a lesser extent Sp3 bound to the GC box regions of eNOS and TNFalpha in intact cells. Thus, this NO*- and cAMP-responsive regulatory module has a Sp site sensor variably coupled to an adjacent element that determines response polarity. These results define a composite element that can utilize secondary inputs to convert off signals to on, thereby conferring complex functionalities to the same DNA binding motif.