Elsevier

Nitric Oxide

Volume 18, Issue 1, February 2008, Pages 19-27
Nitric Oxide

Pitavastatin up-regulates the induction of iNOS through enhanced stabilization of its mRNA in pro-inflammatory cytokine-stimulated hepatocytes

https://doi.org/10.1016/j.niox.2007.08.005Get rights and content

Abstract

Studies have indicated that protective effects of statins (HMG-CoA reductase inhibitor) are associated with the regulation of endothelial nitric oxide synthase (eNOS) or inducible NOS (iNOS) in heart and liver diseases. Statins have been reported to enhance hepatic NO production and decrease the vascular tone in patients with cirrhosis. However, it is unclear which NOS contributes to the increased NO production. We hypothesized that statins are involved in the up-regulation of iNOS in inflammatory liver, resulting in decreased hepatic resistance. Primary cultured rat hepatocytes were treated with pro-inflammatory cytokine interleukin (IL)-1β in the presence or absence of pitavastatin. Pretreatment of cells with pitavastatin resulted in up-regulation of iNOS induction by IL-1β, followed by increased NO production. Pitavastatin had no effects on the degradation of IκB or activation of NF-κB. However, pitavastatin super-induced the up-regulation of type I IL-1 receptor (IL-1RI), which is essential for iNOS induction in addition to the IκB/NF-κB pathway. Mevalonate and geranylgeranylpyrophosphate blocked the stimulatory effects of pitavastatin on iNOS and IL-1RI induction. Transfection experiments revealed that pitavastatin increased the stability of iNOS mRNA rather than its promoter transactivation. In support of this observation, pitavastatin increased the antisense-transcript corresponding to the 3′-UTR of iNOS mRNA, which stabilizes iNOS mRNA by interacting with the 3′-UTR- and RNA-binding proteins. These findings demonstrate that pitavastatin up-regulates iNOS by the stabilization of its mRNA, presumably through the super-induction of IL-1RI and antisense-transcript. This implies that statins may contribute to a novel potentiated treatment in liver injuries including cirrhosis.

Section snippets

Materials

Recombinant human IL-1β (2 × 107 units/mg protein) was provided by Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan). [γ-32P]Adenosine-5′-triphosphate (ATP, −222 TBq/mmol) and [α-32P]deoxycytidine-5′-triphosphate (dCTP, −111 TBq/mmol) were from DuPont-New England Nuclear Japan (Tokyo). Pitavastatin was provided by Kowa Co. Ltd. (Nagoya, Japan) and dissolved in Williams′ medium E (WE). All other chemicals were of reagent grade. Rats were kept at 22 °C under a 12 h light–dark cycle, and received food

Pitavastatin up-regulates the induction of iNOS in hepatocytes

The pro-inflammatory cytokine, IL-1β, stimulated the induction of iNOS gene expression, resulting in an increased NO production, in primary cultures of rat hepatocytes as reported previously [29], [32], [33], [34]. Pretreatment with pitavastatin (HMG-CoA reductase inhibitor, 50 μM) for 2 h or more (up to overnight) before the addition of IL-β markedly increased the production of NO with time (approximately 3- to 4-fold) compared with IL-β alone (Fig. 1a). Simultaneous addition of pitavastatin (50 

Discussion

In this study, we found that an HMG-CoA reductase inhibitor, pitavastatin, significantly up-regulated the induction of iNOS gene expression, as stimulated by pro-inflammatory cytokine IL-1β in cultured hepatocytes, followed by an increased production of NO (Fig. 1, Fig. 2). However, pitavastatin had no effects on either the degradation of IκBα and IκBβ or the activation of NF-κB (Fig. 3). In contrast, pitavastatin time- and dose-dependently enhanced the induction of IL-1RI mRNA and protein (

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan, and by grants from the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and from Amino Up Chemical Co., Ltd. (Sapporo, Japan).

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