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Shuibang Wang

Publications and source records attributed to Shuibang Wang.

9 recordsLinked to original sources
PubMed2006

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.

Cells, Cultured↗
PubMed2006

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.

Cell Cycle Proteins↗
PubMed2006

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.

3' Untranslated Regions↗
PubMed2005

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.

Blotting, Western↗
PubMed2004

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.

Blotting, Western↗
PubMed2004

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.

Animals↗
PubMed2003

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.

Base Sequence↗
PubMed2002

Within the fold: assessing differential expression measures and reproducibility in microarray assays.

BACKGROUND: 'Fold-change' cutoffs have been widely used in microarray assays to identify genes that are differentially expressed between query and reference samples. More accurate measures of differential expression and effective data-normalization strategies are required to identify high-confidence sets of genes with biologically meaningful changes in transcription. Further, the analysis of a large number of expression profiles is facilitated by a common reference sample, the construction of which must be carefully addressed. RESULTS: We carried out a series of 'self-self' hybridizations in which aliquots of the same RNA sample were labeled separately with Cy3 and Cy5 fluorescent dyes and co-hybridized to the same microarray. From this, we can analyze the intensity-dependent behavior of microarray data, define a statistically significant measure of differential expression that exploits the structure of the fluorescent signals, and measure the inherent reproducibility of the technique. We also devised a simple procedure for identifying and eliminating low-quality data for replicates within and between slides. We examine the properties required of a universal reference RNA sample and show how pooling a small number of samples with a diverse representation of expressed genes can outperform more complex mixtures as a reference sample. CONCLUSION: Analysis of cell-line samples can identify systematic structure in measured gene-expression levels. A general procedure for analyzing cDNA microarray data is proposed and validated. We show that pooled reference samples should be based not only on the expression of individual genes in each cell line but also on the expression levels of genes within cell lines.

Brain Neoplasms↗