Inducible nitric oxide synthase can be induced in the absence of active nuclear factor-kappaB in rat mesangial cells: involvement of the Janus kinase 2 signaling pathway.

The contribution of nuclear factor-kappaB (NF-kappaB) and interferon-gamma (IFN-gamma) signaling to nitric oxide generation is not completely understood. The effect of NF-kappaB release and its inhibition on nitrite production and the involvement of Janus kinase 2 (JAK2) in inducible nitric oxide synthase (iNOS) induction were investigated. The following assays were performed. (1) Nitrite produced by rat mesangial cells in primary culture was measured in incubations with tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS), with or without IFN-gamma. Cells were stimulated with TNF-alpha or LPS plus IFN-gamma in the presence of NF-kappaB inhibitors, herbimycin A (HerA), or the more specific JAK2 inhibitor AG490. (2) Immunoblotting was performed against the p65 and p50 subunits of NF-kappaB and iNOS. (3) Electrophoretic mobility shift assays were performed against NF-kappaB in the presence of NF-kappaB inhibitors or AG490. (4) iNOS promoter activity was measured in the presence of AG490 or JAK2 antisense oligonucleotides. TNF-alpha or LPS alone did not induce nitrite production, but with IFN-gamma these compounds did induce nitrite production. Pyrrolidine dithiocarbamate (PDTC), N-acetyl-L-cysteine, dexamethasone (Dex), HerA, and AG490 partially inhibited LPS/ IFN-gamma- or TNF-alpha/IFN-gamma-induced nitrite production. p65 was inhibited by the three NF-kappaB inhibitors described above, whereas p50 was not. PDTC and Dex completely inhibited the p65/p50 heterodimer, but HerA and AG490 had little effect on p65/p50. AG490 and JAK2 antisense oligonucleotides suppressed iNOS promoter activity. It can be concluded that (1) iNOS can be induced without active NF-kappaB; (2) Dex, acetylsalicylic acid, and PDTC inhibit only p65; and (3) JAK2 is involved in iNOS induction, and the contribution of JAK2 to nitrite production is greater than that of NF-kappaB.