Haem oxygenase-1 in inflammation - CiteSeerX

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it heterodimerizes with members of the small Maf protein family and binds to the ARE. Although several kinases have been suggested to play a role in the ...
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Haem oxygenase-1 in inflammation S.A. Rushworth and M.A. O’Connell1 MRC Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge CB1 9NL, U.K.

Abstract HO-1 (haem oxygenase-1) is a stress-inducible enzyme that plays a protective role in inflammation. Pro-inflammatory mediators, including lipopolysaccharide and cytokines, induce HO-1 expression. The 5 -flanking region of the HO-1 gene contains binding sites for the transcription factors that regulate inflammation, including nuclear factor-κB and activator protein 1. However, these do not appear to mediate lipopolysaccharide-induced HO-1 gene expression. In response to haem and antioxidants, murine HO-1 is regulated by the transcription factor Nrf2 (NF-E2-related factor 2). This transcription factor may also be important in the regulation of HO-1 by pro-inflammatory stimuli.

HO-1 (haem oxygenase-1) Haem oxygenase is the rate-limiting enzyme in the oxidative degradation of haem to biliverdin, free iron and carbon monoxide. Biliverdin is rapidly metabolized to bilirubin, a potent antioxidant, in mammalian cells. Three isoforms of HO have been identified (HO-1, HO-2 and HO-3), which differ according to their tissue distribution and modes of activation. In contrast with HO-2 and HO-3, HO-1 gene expression is induced in various cell types by a diverse range of stimuli, including haem, heavy metals, antioxidants and proinflammatory mediators [1].

the anti-inflammatory effects of HO-1 are mediated by its metabolites. Although preinduction of HO-1 inhibits inflammation, pro-inflammatory mediators themselves also induce HO-1 expression. Tumour necrosis factor α and interleukin-1α stimulate HO-1 expression in human endothelial cells [7]. Furthermore, LPS has been reported to induce HO-1 expression in rodent macrophages [8] and we have found that LPS stimulates HO-1 expression in human monocytes (Figure 1). Other pro-inflammatory mediators, including oxidized low-density lipoprotein and shear stress, induce HO-1 expression in macrophages and endothelial cells [1,9].

HO-1 and inflammation Studies in knockout mice and HO-1 deficiency in humans have pointed to a protective role for HO-1 in inflammation. HO-1 deficiency in humans is associated with susceptibility to oxidative stress and an increased pro-inflammatory state with severe endothelial damage [2]. HO-1 knockout mice have a similar phenotype and have higher mortality rates and end-organ damage after exposure to endotoxin compared with wild-type controls [3], suggesting that HO-1 is a potent anti-inflammatory mediator in vivo. Indeed, the antiinflammatory cytokine interleukin-10 up-regulates HO-1 in vivo and protects against endotoxic shock in mice [4]. In in vitro studies, HO-1 induction by interleukin-10 or 15-deoxy-prostaglandin J2 inhibited the production of LPS (lipopolysaccharide)-induced tumour necrosis factor α in mouse macrophages. These effects depended on the presence of carbon monoxide [4,5]. HO-1 up-regulation also inhibits micovascular endothelial cell–leucocyte adhesion through the action of bilirubin [6]. These studies suggest that

Key words: antioxidant response element, haem oxygenase-1 (HO-1), inflammation, NF-E2related factor 2 (Nrf2), lipopolysaccharide (LPS), cytokine. Abbreviations used: AP-1, activator protein 1; ARE, antioxidant response element; HO-1, haem oxygenase-1; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; Nrf2, NF-E2-related factor 2; PKC, protein kinase C. 1

To whom correspondence should be addressed (email [email protected]. ac.uk).

HO-1 regulation during inflammation Regulation of the HO-1 gene is cell-specific and differs among species [10]. So far, most work has focused on the murine gene. The 5 -flanking region of the human HO-1 gene contains a number of putative binding sites for transcription factors that regulate pro-inflammatory gene expression. These include NF-κB (nuclear factor-κB), AP-1 (activator protein 1), AP-2 and CCAAT/enhancer-binding protein sites (Figure 2). NF-κB plays a major role in gene transcription during inflammation. The presence of NF-κBbinding sites in the human and mouse HO-1 promoters implicate this transcription factor in pro-inflammatory induction of HO-1 in these species. Lavrovsky et al. [11] reported that haem induced NF-κB binding to the human HO-1 κB promoter sequence. However, deletion analysis of the mouse HO-1 promoter demonstrated that the promoter that contains this site is not crucial for LPS-mediated HO-1 up-regulation [8]. AP-1 also responds to pro-inflammatory stimuli. Camhi et al. [8] reported that two distal enhancers in the mouse HO-1 5 -flanking region were responsible for LPS-induced HO-1 expression in mouse macrophages using deletion analysis. These enhancers also regulated HO-1 expression by other stimuli [12]. As these regions contained AP-1-binding elements and LPS induces AP-1 binding to these sites, this transcription factor was considered to play  C 2004

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Figure 1 LPS induction of HO-1 in primary human monocytes Monocytes were isolated from peripheral blood of healthy volunteers by Optiprep density-gradient centrifugation and left unstimulated or stimulated with LPS (100 ng/ml) for the indicated times. mRNA was extracted, and HO-1 mRNA expression measured by real-time PCR. Gene expression was normalized to 18S ribosomal subunit mRNA expression (mean ± S.D., n = 3).

Figure 2 Schematic representation of the 5 -flanking region of the human HO-1 gene

a major role in LPS-induced HO-1 expression. AP-1 sites are also present in similar regions in the human 5 -flanking region (http://www.genomatix.de/products/MatInspector). However, recent reports have failed to confirm that AP-1 proteins play a major role in mediating HO-1 up-regulation [12]. These AP-1 sites overlap with other transcriptional regulatory sequences termed AREs (antioxidant response elements), and deletion of the AP-1 sites also abolishes AREmediated HO-1 transcription. The ARE is a cis-acting DNA regulatory element with a core sequence of 5 -RTGACnnnGC-3 (R = G or C). The ARE plays a key role in the transcriptional regulation of a wide range of cytoprotective and detoxification genes, including ferritin, NAD(P)H quinone reductase and glutathione S-transferase [9]. Three putative ARE sites are present upstream (within 4.5 kb) of the human HO-1 start codon (http://www.genomatix.de/products/MatInspector). Alignment of the mouse and human 5 -flanking regions demonstrates that there is a high degree of similarity in the immediate vicinity of ARE sequences. These are present in the LPS regulatory regions in the mouse gene [12] and, therefore, may

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be important in the regulation of the human HO-1 gene by LPS and other pro-inflammatory mediators.

Nrf2 (NF-E2-related factor 2) and HO-1 expression The principal transcription factor that regulates ARE-dependent gene expression appears to be Nrf2. In resting cells, Nrf2 resides in the cytosol bound to an inhibitor, Keap1. In response to heavy metals, antioxidants and other stimuli, Nrf2 is released by Keap1 and translocates to the nucleus, where it heterodimerizes with members of the small Maf protein family and binds to the ARE. Although several kinases have been suggested to play a role in the activation of Nrf2, PKC (protein kinase C) appears to be particularly important, since two independent studies reported that PKC phosphorylation of S40 on Nrf2 is required for its dissociation from Keap1 [13]. The role of Nrf2 and kinases such as PKC in HO-1 upregulation by LPS and other pro-inflammatory stimuli in human cells remains to be determined.

Conclusion In summary, HO-1 plays a key role in inflammation. Both anti-inflammatory and pro-inflammatory mediators activate HO-1 to regulate the inflammatory response. Induction of HO-1 by LPS and other pro-inflammatory mediators in immune cells is poorly understood, especially in humans. Further studies are required to examine the role of the Nrf2/ARE pathway in the regulation of HO-1 by proinflammatory mediators.

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