Cytokine induction of NO synthase II in human DLD‐1 cells: roles of the JAK‐STAT, AP‐1 and NF‐κB‐signaling pathways

Abstract

1 In human epithelial-like DLD-1 cells, nitric oxide synthase (NOS) II expression was induced by interferon-g (100 u ml 71 ) alone and, to a larger extent, by a cytokine mixture (CM) consisting of interferon-g, interleukin-1b (50 u ml 71 ) and tumor necrosis factor-a (10 ng ml 71 ). 2 CM-induced NOS II expression was inhibited by tyrphostin B42 (mRNA down to 1%; nitrite production down to 0.5% at 300 mM) and tyrphostin A25 (mRNA down to 24%, nitrite production down to 1% at 200 mM), suggesting the involvement of janus kinase 2 (JAK-2). Tyrphostin B42 also blocked the CM-induced JAK-2 phosphorylation (kinase assay) and reduced the CM-stimulated STAT1a binding activity (gel shift analysis). 3 CM reduced the nuclear binding activity of transcription factor AP-1. A heterogenous group of compounds, that stimulated the expression of c-fos/c-jun, enhanced the nuclear binding activity of AP-1. This group includes the protein phosphatase inhibitors calyculin A, okadaic acid, and phenylarsine oxide, as well as the inhibitor of translation anisomycin. All of these compounds reduced CM-induced NOS II mRNA expression (to 9% at 50 nM calyculin A; to 28% at 500 nM okadaic acid; to 18% at 10 mM phenylarsine oxide; and to 19% at 100 ng ml 71 anisomycin) without changing NOS II mRNA stability. In cotransfection experiments, overexpression of c-Jun and c-Fos reduced promoter activity of a 7 kb DNA fragment of the 5'-¯anking sequence of the human NOS II gene to 63%. 4 Nuclear extracts from resting DLD-1 cells showed signi®cant binding activity for transcription factor NF-kB, which was only slightly enhanced by CM. The NF-kB inhibitors dexamethasone (1 mM), 3,4-dichloroisocoumarin (50 mM), panepoxydone (5 mg ml 71 ) and pyrrolidine dithiocarbamate (100 mM) produced no inhibition of CM-induced NOS II induction. 5 We conclude that in human DLD-1 cells, the interferon-g ± JAK-2-STAT1a pathway is important for NOS II induction. AP-1 (that is downregulated by CM) seems to be a negative regulator of NOS II expression. NF-kB, which is probably important for basal activity of the human NOS II promoter, is unlikely to function as a major e ector of CM in DLD-1 cells.

Amplification of IL-1β-Induced Matrix Metalloproteinase-9 Expression by Superoxide in Rat Glomerular Mesangial Cells Is Mediated by Increased Activities of NF-κB and Activating Protein-1 and Involves Activation of the Mitogen-Activated Protein Kinase Pathways

“…Most surprisingly, we found that both radicals have opposing effects on MMP-9 mRNA levels, with O 2 Ϫ amplifying and NO attenuating the response. By contrast, we have identified superoxide and NO as costimulatory factors amplifying cytokineinduced inducible NO synthase gene expression in rat MC (32,34). These apparently inconsistent observations are unexpected, as a common set of transcription factors, including AP-1 and NF-B is critically involved in the regulation of both proinflammatory genes inducible NO synthase (19,34) and MMP-9 (13).…”

JAK2/STAT3, Not ERK1/2, Mediates Interleukin-6-induced Activation of Inducible Nitric-oxide Synthase and Decrease in Contractility of Adult Ventricular Myocytes

“…Studies using human DLD-1 cells, a colon epithelial-derived cell line, showed that a cytokine mixture of INF-␥, IL-1␤, and tumor necrosis factor-␣ induced iNOS expression via activation of the JAK2/STAT1␣ pathway, which was blocked by AG490 (43). INF-␥-induced iNOS expression has also been shown to be paralleled by STAT1␣ activation in adult rat ventricular myocytes (44).…”

Tissue Inhibitor of Metalloproteinase-1 Prevents Cytokine-Mediated Dysfunction and Cytotoxicity in Pancreatic Islets and β-cells

“…One study evaluated a similar cytokine mix (IL-1␤ 50 U/ml, IFN-␥ 100 U/ml, and TNF-␣ 10 ng/ml) on iNOS gene transcription in human epithelial DLD-1 cells (33). Cytokine-induced iNOS gene transcription was inhibited with a number of tyrosine kinase inhibitors, including the Janus kinase (JAK)-2 inhibitor tyrphostin B42, whereas a variety of NF-B inhibitors (dexamethasone, 3,4-dichloroisocoumarin, panepoxydone, and pyrrolidine dithiocarbamate) did not affect iNOS gene transcription.…”