Elsevier

Cellular Signalling

Volume 18, Issue 1, January 2006, Pages 32-39
Cellular Signalling

HMG–CoA reductase inhibitors upregulate heme oxygenase-1 expression in murine RAW264.7 macrophages via ERK, p38 MAPK and protein kinase G pathways

https://doi.org/10.1016/j.cellsig.2005.03.016Get rights and content

Abstract

Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme catabolism, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. HMG–CoA reductase inhibitors (statins) possess several anti-inflammatory mechanisms and may be beneficial in the treatment of inflammatory diseases. Our previous study has shown that statins can inhibit iNOS gene expression in murine RAW264.7 macrophages. In this study, we showed that lovastatin, fluvastatin, atorvastatin, simvastatin, mevastatin and pravastatin are able to upregulate the mRNA expression of HO-1 gene. This effect of lovastatin was attenuated by farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), a protein kinase G (PKG) inhibitor (KT5823), a soluble guanylyl cyclase inhibitor (ODQ), a p38 MAPK inhibitor (SB203580), and MEK inhibitors (U0126 and PD98059), but not by inhibitors of protein kinase C (PKC), protein kinase A (PKA), c-jun N-terminal kinase (JNK) and Rho kinase. Consistent with this notion, our previous study has reported the ability of statins to activate ERK and p38 MAPK in RAW264.7 macrophages. Here we further found the participation of cyclic guanosine monophosphate (cGMP)/PKG pathway for ERK activation in cells stimulated with statin and the ability of statin to induce AP-1 activity, which is an essential transcription factor in the regulation of HO-1 gene expression. In addition, a Ras inhibitor (manumycin A) treatment also caused a marked induction of HO-1 mRNA followed by a corresponding increase in HO-1 protein; instead, inhibition of Rho activity by toxin B only led to a transient and weak induction of HO-1. The involvement of signal pathways in manumycin A-induced HO-1 gene expression was associated with p38 MAPK, JNK and ERK activation. Taken together, these results demonstrate for the first time that statins might activate PKG to elicit activations of ERK and p38 MAPK pathways and finally induce HO-1 gene expression, which provides a novel anti-inflammatory mechanism in the therapeutic validity.

Introduction

Heme oxygenase (HO) is the rate-limiting enzyme in the oxidative degradation of heme into bilirubin, iron, and carbon monoxide (CO). While HO-2 and HO-3 are constitutively expressed, HO-1 is the inducible form. HO-1 is expressed with low level under basal conditions and can be highly induced in response to various agents causing oxidative stress including hyperthermia, UV irradiation [3], hydrogen peroxide [3], heavy metals [3], inflammatory cytokines [4], endotoxin [5], hypoxia [6], hyperoxia [7], and nitric oxide (NO) [8], [9]. HO-1 induction provides cytoprotection against oxidative stress and apoptosis and preserves cellular homeostasis [1], [2]. This action is demonstrated not only in cultured cell systems [10] but also in in vivo studies [11], [12]. Although the mediators and mechanisms by which HO-1 provides protection are not clear and depend on cell types and stimuli, accumulating lines of evidence point the important role of CO [13], [14]. A low concentration of CO can exert protection through a soluble guanylyl cyclase (sGC) and cyclic guanosine monophosphate (cGMP) pathway [15].

Statins are inhibitors of the 3-hydroxy-3-methyl-glutaryl–coenzyme A (HMG–CoA) reductase and are widely used as lipid-lowering agents [16]. Besides the therapeutic use in hyperlipidemia, the anti-inflammatory and immunomodulatory benefits of statins have been recently reported in many aspects, although mechanisms are not yet completely defined [17]. Most identified anti-inflammatory benefits of statins rely on the reduction of cellular levels of mevalonate, the direct product of HMG–CoA reductase, and mevalonate-derived isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are involved in post-translational modification of several small G proteins, such as Rho, Rac, Cdc42, and Ras [18], [19].

Since the understanding and evaluation of the pharmacological effects of statins are increasing and accelerating their clinical importance and validity, in this study we intended to identify the action of statins on HO-1 gene expression in murine RAW264.7 macrophages. Using this cell type we previously have demonstrated the abilities of statins to block inducible nitric oxide synthase (iNOS) induction caused by lipopolysaccharide (LPS) and interferon-γ [20]. Intriguingly in the present study we demonstrated that statins are capable of inducing HO-1 gene transcription in murine RAW264.7 macrophages and elucidated the mechanisms involved.

Section snippets

Materials

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco BRL (Grand Island, NY). Rabbit polyclonal antibodies specific for HO-1, β-actin, ERK, JNK and p38 mitogen activated protein kinase (MAPK) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific to the phosphorylated ERK, JNK and p38 MAPK were purchased from Cell Signaling Technology (Beverly, MA). The ECL detection agents were purchased from

Statins transcriptionally induce HO-1 gene expression in murine RAW264.7 macrophages

Murine RAW264.7 macrophages were chosen to investigate the signal pathways of statin in HO-1 expression, an anti-inflammatory gene. Treatment with lovastatin, fluvastatin and simvastatin (each at 30 μM) induced HO-1 protein expression. At basal state, a weak immunoreactivity of HO-1 protein was detected. The stimulating action of 30 μM lovastatin and fluvastatin displayed the time-dependency, occurring after 3 h exposure, peaking at 12 h and maintaining for up to 24 h (Fig. 1a). The HO-1

Discussion

Accumulating evidence has indicated HO-1 functions as a “therapeutic funnel”. Induction of HO-1 is suggested to have cytoprotective effect against oxidative injury and have the potent anti-inflammatory properties. Modulation of gene transcription is the principal mechanism by which HO-1 is regulated. Based on these results, induction of HO-1 is a therapeutic strategy for treating inflammatory diseases. In this aspect, animal studies and cell cultures have implicated the anti-inflammatory

Acknowledgement

This work was supported by research grants (NSC 93-2314-B-002-266 and NSC94-2314-B-002) from the National Science Council, ROC.

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