Heparin-Binding EGF-Like Growth Factor Down Regulates Proinflammatory Cytokine-Induced Nitric Oxide and Inducible Nitric Oxide Synthase Production in Intestinal Epithelial Cells

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Many cells upon injury mount extensive, compensatory responses that increase cell survival; however, the intracellular signals that regulate these responses are not completely understood. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated as a cytoprotective agent. We have previously demonstrated that pretreatment of human intestinal epithelial cells with HB-EGF significantly decreased cytokine-induced activation of inducible NO synthase mRNA expression and NO production and protected the cells from apoptosis and necrosis. However, the mechanisms by which HB-EGF exerts these effects are not known. Here we show that cytokine exposure (IL-1β and IFN-γ) induced NF-κB activation and IL-8 and NO production in DLD-1 cells. Transient expression of a dominant negative form of IκBα decreased NO production, suggesting that the cytokines stimulated NO production in part through activation of NF-κB. HB-EGF dramatically suppressed NF-κB activity and IL-8 release and decreased NO production in cells pretreated with HB-EGF. HB-EGF blocked NF-κB activation by inhibiting IκB kinase activation and IκB phosphorylation and degradation, thus interfering with NF-κB nuclear translocation, DNA-binding activity, and NF-κB-dependent transcriptional activity. The data demonstrate that HB-EGF decreases inflammatory cytokine and NO production by interfering with the NF-κB signaling pathway. Inhibition of NF-κB may represent one of the mechanisms by which HB-EGF exerts its potent anti-inflammatory and cytoprotective effects.

Section: Discussionsupporting

confidence: 52%

Section: Inhibition Of Nf-b Activation and Its Target Genes By Heparimentioning

confidence: 99%

Section: Il-8 and No Measurementsmentioning

confidence: 99%

See 1 more Smart Citation

Inhibition of NF-κB Activation and Its Target Genes by Heparin-Binding Epidermal Growth Factor-Like Growth Factor

Mehta¹,

Besner²

2003

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“…The Human GAPDH Real-time RT-PCR 5Ј-GAA GGT GAA GGT CGG AGT C-3Ј Forward ϩ321 to ϩ339 5Ј-GAA GAT GGT GAT GGG ATT TC-3Ј Reverse ϩ2270 to ϩ2251 VIC 5Ј-CAA GCT TCC CGT TCT CAG CC-3Ј TAMRA Probe ϩ2241 to ϩ2222 primers and the TaqMan probes for the quantitative analysis of the human iNOS and GAPDH mRNA as well as the SYBR Green primers for the quantification of human VCAM-1 mRNA are listed in Table II. The human iNOS primers and probe set spans intron 4 (38). The human GAPDH primers and probe set spans introns 2 and 3.…”

Section: Quantitative Taqman Real-time Rt-pcrmentioning

confidence: 99%

Epigenetic Basis for the Transcriptional Hyporesponsiveness of the Human Inducible Nitric Oxide Synthase Gene in Vascular Endothelial Cells

Chan¹,

Fish

Mawji³

et al. 2005

126

103

A marked difference exists in the inducibility of inducible NO synthase (iNOS) between humans and rodents. Although important cis and trans factors in the murine and human iNOS promoters have been characterized using episomal-based approaches, a compelling molecular explanation for why human iNOS is resistant to induction has not been reported. In this study we present evidence that the hyporesponsiveness of the human iNOS promoter is based in part on epigenetic silencing, specifically hypermethylation of CpG dinucleotides and histone H3 lysine 9 methylation. Using bisulfite sequencing, we demonstrated that the iNOS promoter was heavily methylated at CpG dinucleotides in a variety of primary human endothelial cells and vascular smooth muscle cells, all of which are notoriously resistant to iNOS induction. In contrast, in human cell types capable of iNOS induction (e.g., A549 pulmonary adenocarcinoma, DLD-1 colon adenocarcinoma, and primary hepatocytes), the iNOS promoter was relatively hypomethylated. Treatment of human cells, such as DLD-1, with a DNA methyltransferase inhibitor (5-azacytidine) induced global and iNOS promoter DNA hypomethylation. Importantly, 5-azacytidine enhanced the cytokine inducibility of iNOS. Using chromatin immunoprecipitation, we found that the human iNOS promoter was basally enriched with di- and trimethylation of H3 lysine 9 in endothelial cells, and this did not change with cytokine addition. This contrasted with the absence of lysine 9 methylation in inducible cell types. Importantly, chromatin immunoprecipitation demonstrated the selective presence of the methyl-CpG-binding transcriptional repressor MeCP2 at the iNOS promoter in endothelial cells. Collectively, our work defines a role for chromatin-based mechanisms in the control of human iNOS gene expression.

“…We have previously shown that HB-EGF pretreatment decreases IL-1␤-and IFN-␥-stimulated iNOS expression and production of NO and IL-8 in HT-29 and DLD-1 colonic epithelial cells by interfering with the NF-B signaling pathway (11,27). In view of the inhibitory effects exerted by HB-EGF on NF-B activation, we have now investigated the signaling mechanisms of HB-EGF-induced inhibition of NF-B activation and NO production.…”

mentioning

confidence: 99%

Heparin-Binding Epidermal Growth Factor-Like Growth Factor Inhibits Cytokine-Induced NF-κB Activation and Nitric Oxide Production via Activation of the Phosphatidylinositol 3-Kinase Pathway

Mehta

Besner

2005

Self Cite

NO produced by inducible NO synthase (iNOS) has been implicated in various pathophysiological processes including inflammation. Therefore, inhibitors of NO synthesis or iNOS gene expression have been considered as potential anti-inflammatory agents. We have previously demonstrated that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) decreases proinflammatory cytokine IL-8 and NO production in cytokine-stimulated intestinal epithelial cells by interfering with the NF-κB signaling pathway. However, the upstream signaling mechanisms involved in these responses have not yet been defined. In this report, we show that in intestinal epithelial cells, HB-EGF triggered PI3K-dependent phosphorylation of Akt. Inhibition of PI3K reversed the ability of HB-EGF to block NF-κB activation, expression of iNOS, and NO production. Small interfering RNA of PI3K also reversed the inhibitory effect of HB-EGF on iNOS expression. Alternatively, transient expression of constitutively active PI3K decreased NO production by ∼2-fold more than treatment with HB-EGF alone. This PI3K effect was HB-EGF dependent. Thus, activation of PI3K is essential but not sufficient for decreased NO synthesis. PI3K and HB-EGF act synergistically to decrease NO synthesis. Neither overexpression or inhibition of MEK, Ras, or Akt affected HB-EGF-mediated inhibition of NF-κB activation. These data demonstrate that HB-EGF decreases proinflammatory cytokine-stimulated NF-κB activation and NO production via activation of the PI3K signaling pathway. These results also suggest that inhibition of NF-κB and activation of the PI3K-dependent signaling cascade by HB-EGF may represent key signals responsible for the anti-inflammatory effects of HB-EGF.