Inhibition of small G proteins of the Rho family by statins or Clostridium difficile toxin B enhances cytokine‐mediated induction of NO synthase II

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Abstract
In order to investigate the involvement of Ras and/or Rho proteins in the induction of the inducible isoform of nitric oxide synthase (NOS II) we used HMG‐CoA reductase inhibitors (statins) and Clostridium difficile toxin B (TcdB) as pharmacological tools. Statins indirectly inhibit small G proteins by preventing their essential farnesylation (Ras) and/or geranylgeranylation (Rho). In contrast, TcdB is a glucosyltransferase and inactivates Rho‐proteins directly. Human A549/8‐ and DLD‐1 cells as well as murine 3T3 fibroblasts were preincubated for 18 h with statins (1–100 μM) or TcdB (0.01–10 ng ml−1). Then NOS II expression was induced by cytokines. NOS II mRNA was measured after 4–8 h by RNase protection assay, and NO production were measured by the Griess assay after 24 h. Statins and TcdB markedly increased cytokine‐induced NOS II mRNA expression and NO production. Statin‐mediated enhancement of NOS II mRNA expression was reversed almost completely by cotreatment with mevalonate or geranylgeranylpyrophosphate. It was only slightly reduced by farnesylpyrophosphate. Therefore, small G proteins of the Rho family are likely to be involved in NOS II induction. In A549/8 cells stably transfected with a luciferase reporter gene under the control of a 16 kb fragment of the human NOS II promoter (pNOS2(16)Luc), statins produced only a small increase in cytokine‐induced NOS II promoter activity. In contrast, statins had a considerable superinducing effect in DLD‐1 cells stably transfected with pNOS2(16)Luc. In conclusion, our studies provide evidence that statins and TcdB potentiate cytokine‐induced NOS II expression via inhibition of small G proteins of the Rho family. This in turn results in an enhanced NOS II promoter activity and/or a prolonged NOS II mRNA stability. British Journal of Pharmacology (2000) 131, 553–561; doi:10.1038/sj.bjp.0703607