Stephan Immenschuh

Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes.

Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress stimuli including sodium arsenite. Since mitogen-activated protein kinases (MAPKs) are involved in stress signaling we investigated the role of arsenite and MAPKs for HO-1 gene regulation in primary rat hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA expression. HO-1 protein and luciferase activity of reporter gene constructs with −754 bp of the HO-1 promoter were induced by overexpression of kinases of the JNK pathway and MKK3. By contrast, overexpression of Raf-1 and ERK2 did not affect expression whereas overexpression of p38α, β, and δ decreased and p38γ increased HO-1 expression. Electrophoretic mobility shift assays (EMSA) revealed that a CRE/AP-1 element (−668/−654) bound c-Jun, a target of the JNK pathway. Deletion or mutation of the CRE/AP-1 obliterated the JNK- and c-Jun-dependent up-regulation of luciferase activity. EMSA also showed that an E-box (−47/−42) was bound by a putative p38 target c-Max. Mutation of the E-box strongly reduced MKK3, p38 isoform-, and c-Max-dependent effects on luciferase activity. Thus, the HO-1 CRE/AP-1 element mediatesHO-1 gene induction via activation of JNK/c-Jun whereas p38 isoforms act through a different mechanism via the E-box.

Inhibition and Genetic Deficiency of p38 MAPK Up-Regulates Heme Oxygenase-1 Gene Expression via Nrf2

Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. The HO products carbon monoxide and bilirubin not only provide antioxidant cytoprotection, but also have potent anti-inflammatory and immunomodulatory functions. Although HO-1 has previously been shown to be induced by various stimuli via activation of the p38 MAPK signaling pathway, the role of this protein kinase for HO-1 gene regulation is largely unknown. In the present study, it is demonstrated that pharmacological inhibitors of p38 induced HO-1 expression in monocytic cells. Moreover, basal HO-1 gene expression levels were markedly higher in untreated murine embryonic fibroblasts (MEF) from p38α−/− mice compared with those from wild-type mice. Transfection studies with luciferase reporter gene constructs indicate that increased HO-1 gene expression via inhibition of p38 was mediated by the transcription factor Nrf2, which is a central regulator of the cellular oxidative stress response. Accordingly, inhibitors of p38 induced binding of nuclear proteins to a Nrf2 target sequence of the HO-1 promoter, but did not affect HO-1 protein expression and promoter activity in Nrf2−/− MEF. Genetic deficiency of p38 led to enhanced phosphorylation of ERK and increased cellular accumulation of reactive oxygen species. In addition, pharmacological blockage of ERK and scavenging of reactive oxygen species with N-acetylcysteine reduced HO-1 gene expression in p38−/− MEF, respectively. Taken together, it is demonstrated that pharmacological inhibition and genetic deficiency of p38 induce HO-1 gene expression via a Nrf2-dependent mechanism in monocytic cells and MEF.

Nitric oxide mediates the lipopolysaccharide dependent upregulation of the heme oxygenase-1 gene expression in cultured rat Kupffer cells

BACKGROUND/AIMS Heme oxygenase catalyzes the rate-limiting enzymatic step of heme degradation. The inducible isoform of heme oxygenase, heme oxygenase-1, is expressed at a low level in most tissues and is upregulated by its substrate heme and various stress stimuli. Kupffer cells which represent the largest population of the bodys tissue macrophages serve physiological functions in the defense against various pathogens such as lipopolysaccharide. The goal of the present study was to investigate the heme oxygenase-1 gene expression in Kupffer cells of rat liver and in isolated Kupffer cell cultures during treatment with lipopolysaccharide. METHODS Cryostat sections of normal rat liver were investigated by immunofluorescence double-staining using specific antibodies for rat heme oxygenase-1 and ED2. Isolation and cell culture of Kupffer cells and primary hepatocytes from rat liver, as well as Northern and Western blot analysis, were performed with standard protocols. RESULTS Heme oxygenase-1 protein was highly expressed in large sinusoidal cells of normal rat liver, which were identified as Kupffer cells by staining with the macrophage surface marker ED2. By contrast, no expression of heme oxygenase-1 was detected in liver parenchymal cells. High expression of heme oxygenase-1 was also found in isolated Kupffer cells in culture by immunocytochemical staining as well as by Western and Northern blot analysis. After treatment of Kupffer cells cultures with lipopolysaccharide, heme oxygenase-1 was upregulated on the protein and mRNA level in a time- and dose-dependent manner. This increase in heme oxygenase-1 expression by lipopolysaccharide was prevented by the nitric oxide inhibitor N(G)-monomethyl-L-arginine which was reversed by an excess of L-arginine. Various nitric oxide donors up-regulated heme oxygenase-1 mRNA expression in Kupffer cells. CONCLUSIONS The lipopolysaccharide-dependent upregulation of the heme oxygenase-1 gene which is highly expressed in Kupffer cells is mediated by a nitric oxide-dependent mechanism.

Differential Cellular and Subcellular Localization of Heme-Binding Protein 23/Peroxiredoxin I and Heme Oxygenase-1 in Rat Liver

Heme-binding protein 23 (HBP23), also termed peroxiredoxin (Prx) I, and heme oxygenase-1 (HO-1) are distinct antioxidant stress proteins that are co-ordinately induced by oxidative stress. HBP23/Prx I has thioredoxin-dependent peroxidase activity with high binding affinity for the pro-oxidant heme, while HO-1 is the inducible isoform of the rate-limiting enzyme of heme degradation. We investigated the cellular and subcellular localization of both proteins in rat liver. Whereas by immunohistochemistry (IHC) a uniformly high level of HBP23/Prx I expression was observed in liver parenchymal and different sinusoidal cells, HO-1 expression was restricted to Kupffer cells. By immunoelectron microscopy using the protein A–gold technique, HBP23/Prx I immunoreactivity was detected in cytoplasm, nuclear matrix, mitochondria, and peroxisomes of parenchymal and non-parenchymal liver cell populations. In contrast, the secretory pathway, i.e., the endoplasmic reticulum and Golgi complex, was free of label. As determined by immunocytochemical (ICC) studies in liver cell cultures and by Western and Northern blotting analysis, HBP23/Prx I was highly expressed in cultures of isolated hepatocytes and Kupffer cells. In contrast, HO-1 was constitutively expressed only in Kupffer cell cultures but was also inducible in hepatocytes. These data suggest that HBP23/Prx I and HO-1 may have complementary antioxidant functions in different cell populations in rat liver.

Biology of Heme in Health and Disease

Heme is an essential molecule with contradictory biological functions. In hemoproteins such as hemoglobin and cytochromes protein-bound heme is a prosthetic group serving physiological functions as a transporter for oxygen and electrons. On the other hand free heme can have deleterious effects by generating reactive oxygen species that cause oxidative stress. Consequently, heme homeostasis of the cell must be tightly controlled. The biosynthesis of heme is catalyzed by eight enzymes that are differentially regulated in liver and erythroid cells. Recent findings on proinflammatory functions of heme and its role in the pathogenesis of diseases, such as rhabdomyolysis or atherosclerosis are summarized. The regulation of gene expression by heme in yeast and mammalian cells and the underlying molecular mechanisms are presented. Finally, we discuss the functional significance of the heme-degrading enzyme heme oxygenase and heme-binding proteins for the regulation of heme homeostasis.

Heme Oxygenase-1 and Iron in Liver Inflammation: A Complex Alliance

Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. HO-1 has potent antioxidant and also anti-inflammatory functions, the underlying mechanisms of which are not well understood. Together with antioxidant carbon monoxide and biliverdin, HO produces reactive iron, which unambiguously connect this enzyme with the iron metabolism and its potential toxicity. A link between HO-1 and iron homeostasis has been demonstrated in HO-1 knockout mice, which develop major hemosiderosis in solid organs such as liver and kidney. Moreover, genetic HO-1 deficiency causes a chronic inflammatory condition in these animals. As the liver plays a crucial role for the bodys iron homeostasis (eg. via secretion of the iron regulatory hormone hepcidin) and also for systemic inflammation, hepatic HO-1 may be important for the regulation of both systems. In particular, cell-specific functions of HO-1 in liver tissue macrophages (Kupffer cells) might be of major significance, because these cells play a key role in iron recycling during erythrophagocytosis and also in the control of hepatic and systemic inflammatory responses. This review discusses the current knowledge on interactions of HO-1 with iron metabolism in the context of systemic as well as hepatic inflammatory disorders. Recent advances in the understanding of the functional role of HO-1 in inflammatory liver diseases, namely viral hepatitis, alcoholic liver disease and non-alcoholic steatohepatitis are summarized. Finally, it is highlighted how targeted modulation of HO-1 may provide specific protection in these inflammatory disorders.

An Atypical NF-κB-Regulated Pathway Mediates Phorbol Ester-Dependent Heme Oxygenase-1 Gene Activation in Monocytes

Heme oxygenase (HO)-1 catalyzes the rate-limiting step of heme degradation and plays an important anti-inflammatory role via its enzymatic products carbon monoxide and biliverdin. In this study it is reported that the HO-1 gene is transcriptionally induced by the phorbol ester PMA in cell cultures of monocytic cells with a regulatory pattern that is different from that of LPS-dependent HO-1 induction in these cells. Activation of HO-1 by PMA was mediated via a newly identified κB element of the proximal rat HO-1 gene promoter region (−284 to −275). This HO-κB element was a nuclear target for the NF-κB subunit p65/RelA as determined by nuclear binding assays and transfection experiments with luciferase reporter gene constructs in RAW264.7 monocytes. Moreover, PMA-dependent induction of endogenous HO-1 gene expression and promoter activity was abrogated in embryonic fibroblasts from p65−/− mice. PMA-dependent HO-1 gene activation was reduced by an overexpressed dominant negative mutant of IκBα, but not by dominant negative IκB kinase-2, suggesting that the classical NF-κB pathway was not involved in this regulation. The antioxidant N-acetylcysteine and inhibitors of p38 MAPK or serine/threonine kinase CK2 blocked PMA-dependent HO-1 gene activation. Finally, it is demonstrated by luciferase assays with a Gal4-CHOP fusion protein that the activation of p38 MAPK by PMA was independent of CK2. Taken together, induction of HO-1 gene expression by PMA is regulated via an IκB kinase-independent, atypical NF-κB pathway that is mediated via the activation of p38 MAPK and CK2.

Regulation of heme oxygenase-1 gene expression by anoxia and reoxygenation in primary rat hepatocyte cultures

Heme oxygenase (HO) catalyzes the rate-limiting enzymatic step of heme degradation and regulates the cellular heme content. Gene expression of the inducible isoform of HO, HO-1, is upregulated in response to various oxidative stress stimuli. To investigate the regulatory role of anoxia and reoxygenation (A/R) on hepatic HO-1 gene expression, primary cultures of rat hepatocytes were exposed after an anoxia of 4 hr to normal oxygen tension for various lengths of time. For comparison, gene expression of the noninducible HO isoform, HO-2, and that of the heat-shock protein 70 (HSP70) were determined. During reoxygenation, a marked increase of HO-1 and HSP70 steady-state mRNA levels was observed, whereas no alteration of HO-2 mRNA levels occurred. Corresponding to HO-1 mRNA, an increase of HO-1 protein expression was determined by Western blot analysis. The anoxia-dependent induction of HO-1 was prevented by pretreatment with the transcription inhibitor, actinomycin D, but not by the protein synthesis inhibitor, cycloheximide, suggesting a transcriptional regulatory mechanism. After exposure of hepatocytes to anoxia, the relative levels of oxidized glutathione increased within the first 40 min of reoxygenation. Pretreament of cell cultures with the antioxidant agents, β-carotene and allopurinol, before exposure to A/R led to a marked decrease of HO-1 and HSP70 mRNA expression during reoxygenation. An even more pronounced reduction of mRNA expression was observed after exposure to desferrioxamine. Taken together, the data demonstrate that HO-1 gene expression in rat hepatocyte cultures after A/R is upregulated by a transcriptional mechanism that may be, in part, mediated via the generation of ROS and the glutathione system.

Opposite expression of the antioxidant heme oxygenase-1 in primary cells and tumor cells: regulation by interaction of USF-2 and Fra-1.

Heme oxygenase-1 is the rate-limiting enzyme for the degradation of the prooxidant heme. Previously, we showed that an E-box within the HO-1 promoter is crucial for the regulation of HO-1 expression in primary hepatocytes. Further to investigate the importance of this E-box, we determined the regulatory capacity of the E-box-binding factor USF-2 in primary cells in comparison with transformed cell lines. We found that HO-1 expression was inhibited by USF-2 in primary cells, whereas it was induced in tumor cell lines. Mutation of either the E-box or the AP-1 site within the HO-1 promoter only partially affected the USF-dependent regulation. However, this regulation was dramatically reduced in tumor cells and completely abolished in primary cells transfected with an HO-1 promoter construct containing mutations in both the E-box and the AP-1 site, suggesting that AP-1 factors and USF-2 may act in a cooperative manner. Indeed, protein-protein interaction studies revealed that USF proteins interacted with Fra-1. Further, the USF-dependent HO-1 promoter activity was not detectable with an USF-2 mutant lacking residues of the USF-specific region (USR) or the transactivation domain encoded by exon 4. Together, these data suggest that USF-2 has opposite regulatory roles for HO-1 gene expression in primary cells and tumor cell lines.

Heme oxygenase-1 is a novel target and antioxidant mediator of S-adenosylmethionine.

The sulfur compound and dietary supplement S-adenosylmethionine (SAM) has been reported to have cytoprotective and antioxidant properties. However, the underlying mechanisms remain unresolved. The present study investigates the effect of SAM on the expression of the antioxidant stress proteins heme oxygenase-1 (HO-1) and ferritin in endothelial cells. Induction of the HO-1/ferritin-system leads to protection of tissues against several inflammatory stimuli. SAM increased the protein and mRNA levels of HO-1 in cultured endothelial cells. Induction of HO-1 gene expression was associated with elevated ferritin protein levels and regulated at the transcriptional level via increased promoter activity. HO-1 upregulation by SAM was causally related to a decrease in NADPH-mediated production of oxygen radicals. Our results demonstrate that the HO-1/ferritin-system is a novel target of the antioxidant compound SAM.