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

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

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 (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. 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). Bt(2)cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO(•), Bt(2)cAMP was associated with ARE-containing transcripts. A comparison of NO(• )and Bt(2)cAMP 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