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