Zinc chelation decreases IFN-beta-induced STAT1 upregulation and iNOS expression in RAW 264.7 macrophages

One consequence of lipopolysaccharide (LPS)-induced stimulation of macrophages is the release of Interferon (IFN)-beta, and subsequently the activation of the JAK-STAT1 pathway, resulting in the expression of inducible nitric oxide synthase (iNOS). Free intracellular zinc'ions (Zn2+) have a profound impact as a second messenger in LPS-dependent gene expression. Previous work had indicated a Zn2+-dependent upregulation of STAT1 mRNA in response to LPS and IFN-beta, potentially affecting STAT1-dependent downstream signaling upon pre-incubation with these agents. The aim of the present study was to investigate the long-term influence of Zn2+ chelation on cellular STAT1 levels and their effect on protein levels and activity of iNOS. The LPS- and IFN-beta-mediated increase of STAT1 mRNA and protein levels was abrogated by chelation of Zn2+ with the membrane permeable chelator N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) in RAW 264.7 macrophages. After 48 h pre-incubation together with IFN-beta, TPEN also led to reduced nitric monoxide formation in response to a second stimulation with LPS. Nonetheless, the latter was observed regardless of any pre-incubation with IFN-beta, suggesting that the effect of treatment with TPEN negatively affects iNOS induction independently from cellular STAT1 levels. In conclusion, long term Zn2+ chelation does affect STAT1 protein expression, but interferes with NO production by a different, yet unknown pathway not involving STAT1. However, as there are many additional STAT1-dependent genes, there might still be effects on targets other than iNOS.