Regulation of Heme Oxygenase-1 Gene Expression by Anoxia and Reoxygenation in Primary Rat Hepatocyte Cultures

Abstract

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.

Heme oxygenase (HO) is the first and rate-limiting enzyme of heme degradation and controls cellular heme availability (1). HO breaks down the pro-oxidant heme and produces equimolar amounts of carbon monoxide (CO), iron, and biliverdin, which is converted by biliverdin reductase into the antioxidant bilirubin (2). At least two distinct isoforms of HO, which are the products of different genes, are known. In contrast with the constitutive isozyme HO-2 (3), HO-1 is the inducible isozyme, which is highly upregulated by various stress stimuli, including its substrate heme, heavy metals, UV light, lipopolysaccharide, heat shock, and hyperoxia (4–9). The exact functional role of HO-1 induction, however, is not fully understood, but it has been shown that HO-1 provides protection against oxidative stress in various cell culture and in vivo models (10, 11). Overexpression of HO-1 protects coronary endothelial cells against the toxic effects of heme proteins (10) and pulmonary epithelial cells against hyperoxia (11). Moreover, it has been shown that HO-1-deficient mice develop an anemia with low serum iron levels along with an overload of iron in the liver and kidney causing oxidative damage and chronic inflammation (12, 13). More recently, these observations in HO-1-deficient mice were essentially confirmed in a case report of human genetic HO-1 deficiency (14). Therefore, upregulation of HO-1 is considered a potential therapeutic target.

Liver injury caused by ischemia–reperfusion is a key clinical problem in liver transplantation and in hepatic failure after shock or during liver surgery (15). Ischemia–reperfusion injury of the liver is caused by the toxic effects of an excess of reactive oxygen species (ROS) that are released from invading neutrophils or resident liver tissue macrophages (Kupffer cells; refs. 16–18). Much less, however, is known about the cellular mechanisms of anoxia and reoxygenation (A/R) in hepatocytes, which are the parenchymal cells of the liver. The goal of the present study was to investigate the direct effect of A/R on the gene expression of HO-1 in primary cultured rat hepatocytes.

Experimental Procedures

Materials.

Media M199 was obtained from Gibco-BRL (Karlsruhe, Germany). The chemiluminescent detection systems for Western blot, radioisotopes and nitrocellulose filters were from Amersham-Buchler (Braunschweig, Germany). The nucleotide removal kit was from Quiagen (Düsseldorf, Germany). The multiprime labeling kit and restriction endonucleases were from New England Biolabs (Cambridge, MA). The Falcon tissue culture dishes were purchased from Becton Dickinson (Heidelberg, Germany). All other chemicals were purchased from Sigma (Munich, Germany).

Antibodies.

The commercial polyclonal rabbit anti-rat HO-1, HO-2, and HSP70 antibodies were obtained from StressGen (Victoria, Canada).

Animals.

Male Wistar rats (2 months old, body weight 200 g) were used throughout the study. All experiments were approved by the local animal experiment review committee.

Cell Isolation and Culture.

Primary rat hepatocytes were isolated and cultivated as previously described (19). They were cultured under 95% air/ 5% CO₂ in medium M199 with Earle’s salts containing 2 g/l bovine serum albumin, 20 mM NaHCO₃, 10 mM HEPES, 100 U penicillin/ml, 100 μg streptomycin/ml, and 1 nM insulin. 5% fetal calf serum and 10 nM dexamethasone were present during the plating phase up to 4 hr, and cell cultures were incubated in serum-free medium for another 18 hr before treatment. After changing the medium, the hepatocytes were then exposed to anoxia (95% N₂/5% CO₂) in an anaerobic container at 37°C.

RNA Isolation, Northern Blot Analysis, and Hybridization.

Total RNA for Northern blotting was isolated as previously described (20). Equal quantities of RNA were separated on 1.2% agarose/2.2 M formaldehyde gels. After electrophoresis, RNA was blotted onto nitrocellulose membranes and baked at 80°C for 4 hr. After prehybridization for 3–4 hr at 42°C, blots were hybridized at 42°C overnight with radiolabeled cDNA probes for rat HO-1, HO-2 and HSP70 (5, 21). The cDNA fragment was radioactively labeled with α[³²P]-dCTP by random priming using the multiprime DNA labeling kit according to the manufacturer’s instructions. To correct for differences in RNA loading of Northern blots the nitrocellulose filters were stripped and were rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) cDNA. The hybridization solution contained 6× SSC, 5× Denhardt’s solution (0.2% Ficoll 400, 0.2% polyvinylpyrrolidone and 0.2% bovine serum albumin), 0.5% sodium dodecyl sulfate (SDS), 50% formamide and 100 μg/ml denatured salmon sperm DNA. Blots were washed subsequently with 2 × SSC/0.1% SDS (once) and 0.1× SSC/0.1% SDS (twice) at 65°C. Filters were exposed to a phosphorimager.

Western Blot Analysis.

After washing cell cultures twice with 0.9% NaCl, cytosol was prepared essentially as previously described (20). After the addition of 1 ml of lysis buffer (0.1% SDS, 10 mM Tris, pH 7.4), cells were boiled for 5 min and homogenized by passing through a 25-gauge needle. The homogenate was centrifuged for 5 min at 4°C and the protein content was determined in the supernatant using the Bradford method. Total protein (50 μg) was loaded onto a 12% SDS polyacrylamide gel and was blotted onto nitrocellulose membranes. Membranes were blocked with Tris-buffered saline containing 1% bovine serum albumin, 10 mM Tris/HCl (ph 7.5) and 0.1% Tween, for 1 hr at room temperature. The primary antibodies for HO-1, HSP70 or HO-2 were added at 1:1000 dilution, respectively, and the blot was incubated for 12 hr at 4°C. The secondary anti-rabbit IgG was diluted 1:8000 and a chemiluminescent detection system for Western blotting was used according to the manufacturer’s instructions.

Measurement of Glutathione.

Total glutathione and oxidized glutathione were measured with an enzymatic method as described by Tietze (22). The relative content of oxidized glutathione was calculated as the ratio of absorbance of oxidized and total glutathione.

Results

Steady-state levels of HO-1 mRNA were examined after anoxia of 4 hr followed by 6 hr of reoxygenation in primary cultured rat hepatocytes. As determined by Northern blot analysis, HO-1 mRNA levels were induced about 9-fold (Fig. 1A). For comparison, the expression of HSP70 and HO-2 mRNA levels were also detected. Whereas HSP70 mRNA was induced to an even higher level than HO-1, HO-2 mRNA levels were not affected by A/R (Fig. 1A ). A time response curve of HO-1 and HSP70 mRNA levels after reoxygenation showed that mRNA expression levels for both genes reached a peak after 6 hr of reoxygenation. Further reoxygenation, up to 12 hr, resulted in a decrease of mRNA with values slightly above the level before reoxygenation. The upregulation of HSP70 mRNA occurred slightly faster compared with that of HO-1. By contrast, the mRNA levels of HO-2 were slightly decreased during prolonged reoxygenation (data not shown). The gene regulation of HO-1, HSP70 and HO-2 was also determined on the protein level. As shown in Figure 2 , a representative Western blot, both HO-1 and HSP70 protein levels were increased after 14 hr (Fig. 2 ). When quantified, a more prominent induction was observed for HSP70 compared to that of HO-1, whereas HO-2 did not exhibit any alteration in the protein level.

Induction of HO-1 by most stimuli occurs on the transcriptional level. To investigate the mechanism of HO-1 induction by A/R primary rat hepatocytes were pretreated with the transcription inhibitor actinomycin D (1 μg/ml) before exposure to anoxia. As shown in Figure 3 , pretreatment with actinomycin D completely prevented the induction of HO-1 mRNA levels after A/R. Similarly, the induction of HSP70 mRNA levels was prevented by pretreatment with actinomycin D. By contrast, pretreament of hepatocytes with the protein synthesis inhibitor, cycloheximide (1.5 μg/ml), did not change the level of HO-1 mRNA, while that of HSP70 mRNA was enhanced by cycloheximide (Fig. 3 ). The apparent half-life of HO-1 mRNA was 2.2 hr and that of HSP70 mRNA was 2.9 hr as measured by Northern blot analysis in the presence of actinomycin D (data not shown).

We also detected the relative amount of oxidized glutathione after 4 hr of anoxia in primary rat hepatocytes. The relative levels of oxidized glutathione were increased after 4 hr of anoxia as compared to control and were further increased after reoxygenation for another 40 min (Fig. 4 ). In addition, during the reoxygenation phase, an increase of ROS was observed as determined by the formation of the fluorescent dye rhodamine 123 from dihydrorhodamine 123 (data not shown).

To study the potential significance of the generation of ROS during A/R for the induction of HO-1 and HSP70 mRNA, the effects of pretreatment with the antioxidant agents, desferrioxamine (DFO), β-carotene, and allopurinol were examined. The most prominent effect was detected in the presence of DFO, which decreased HO-1 mRNA levels to 27% and HSP70 levels to 21% as compared to control cultures without DFO (Fig. 5 ). A significant decrease of mRNA expression levels was also seen in the presence of β-carotene (1 μM) for both HO-1 and for HSP70 mRNA levels. Allopurinol (20 μM) reduced steady state mRNA levels during reoxygenation for HSP70, while the inhibition of HO-1 mRNA levels was not statistically significant (Fig. 5 ).

Discussion

The major findings of the present study are as follows: i) HO-1 gene expression is upregulated by A/R in a time-dependent manner on the mRNA and protein level in rat hepatocyte cultures; ii) the A/R-dependent induction of HO-1 occurs on the transcriptional level; iii) oxidized glutathione levels are increased after exposure of hepatocytes to A/R; and iv) treatment with antioxidant agents attenuates the induction of HO-1 mRNA expression after A/R.

Upregulation of HO-1 after exposure of primary rat hepatocytes to A/R occurs in parallel to that of HSP70 (Figs. 1 and 2 ) suggesting a common mechanism of induction. A similar parallel upregulation of HO-1 and HSP70 has previously been reported for various stress stimuli in human hepatoma cell lines (23). By contrast, differential patterns of induction have been demonstrated in a rat liver model by treatment with isoflurane and halothane under hypoxic conditions (24). The latter finding may suggest that distinct mechanisms could also be involved in the regulation of these two genes. The induction of HO-1 by A/R was regulated transcriptionally as indicated by inhibition with actinomycin D, but not with cycloheximide (Fig. 3). Most stimuli of HO-1 gene induction occur on the transcriptional level and are mediated by a variety of cis-regulatory elements localized in the promoter region of the mouse, rat and human HO-1 genes (for a review, see Ref. 7). A prominent role for the regulation of a host of genes under hypoxic conditions has been ascribed to the transcription factor (TF) hypoxia inducible factor (HIF) that belongs to the group of the basic helix-loop-helix proteins (25). Contradictory observations, however, have been reported as to the regulatory role of HIF for gene regulation of HO-1. Wood et al. have demonstrated a regulatory role of HIF for the HO-1 gene (26). Moreover, an important regulatory role for HIF has previously been demonstrated in lung pulmonary cells (27). In contrast, Alam and colleagues have recently shown that HO-1 is induced during hypoxia via a HIF-independent in Chinese hamster ovary cells (28). Whether the induction of HO-1 gene expression by A/R in our system of primary rat hepatocytes is mediated by HIF remains to be established and experiments to clarify this issue are underway in our laboratory. Another TF candidate that may participate in HO-1 induction by A/R is Nrf2, which has previously been shown to be involved in stress-dependent transcriptional regulation of the HO-1 gene (29). Tacchini et al. (30) have recently shown that the TFs C/ EBP, STAT and HNF1 are upregulated during postischemic liver injury in the rat and could be responsible for the regulation of a number of liver genes.

What are the signaling pathways that mediate the A/R-dependent induction of HO-1 gene expression in primary rat hepatocytes? Since exposure to A/R increased oxidized glutathione levels (Fig. 4) and antioxidative agents markedly reduced the A/R-dependent induction of HO-1 gene expression (Fig. 5 ), the generation of ROS appears to be involved in the induction of HO-1 in hepatocyte cultures. ROS have previously been shown to activate multiple signaling cascades such as various mitogen-activated protein kinases (MAPKs)(for a review, see Ref. 31). Therefore, MAPKs are likely involved in the A/R-dependent induction of HO-1 gene expression. Accordingly, it has recently been demonstrated that HO-1 gene expression is induced by the MAPK extracellular regulated kinase, c-jun N-terminal kinase, and p38 after ischemia reperfusion injury in the lung (32).

The generation of excessive ROS plays an important role for the tissue damage by ischemia-reperfusion injury. In general, during ischemia-reperfusion injury of the liver, ROS are released from invading activated neutrophils or Kupffer cells during the reperfusion of the organ (16–18). In contrast to this in vivo situation, ROS appear to be also generated by A/R per se in primary rat hepatocytes and induce the stress proteins HO-1 and HSP70. Similar observations as to the induction of HO-1 gene expression by A/R have recently been reported for a model of hypoxia-reoxygenation in cultured cardiomyocytes (33). Because a protective role for HO-1 during liver transplantation has previously been demonstrated in a rat model (34), it is conceivable that induction of HO-1 may provide a useful therapeutic target for protection against ischemia-reperfusion injury.

Figure 1.

Induction of HO-1 and HSP70 mRNA levels in primary rat hepatocytes exposed to anoxia-reoxygenation (A/R). (A) Primary rat hepatocytes, after an anoxia of 4 hr, were reoxygenated for an additional 6 h. Total RNA (10 μg) was isolated, subjected to Northern blot analysis, and probed sequentially with ³²P-labeled cDNAs of HO-1, HSP-70, HO-2, and GAP-DH. Results were quantitated with a phosphorimager and normalized with GAP-DH. Numbers show the fold induction rate relative to the basal mRNA expression. Open bars, control; solid bars, exposure to A/R. The values represent means ± SEM of three independent experiments. Statistics, Student’s t test for paired values: *significant difference control vs. A/R, P ≤ 0.05. (B) After anoxia for 4 hr cells were reoxygenated for the times indicated and were processed as described in A. Numbers show the fold induction rate relative to the basal mRNA expression levels normalized to GAP-DH at corresponding time points. Numbers shown are from a representative experiment.

Figure 2.

HO-1, HO-2 and HSP70 protein expression after A/R. Primary rat hepatocytes, after anoxia for 4 hr, were reoxygenated for 14 hr. Cellular protein was subjected to Western blot analysis and probed with antibodies against HO-1, HO-2 and HSP70 as detailed in Experimental Procedures section. Audioradiographs of enhanced chemoluminescence-developed immunoblots are presented. Cells exposed to A/R are demonstrated in Lane 2 and control cells are in Lane 1.

Figure 3.

Effect of actinomycin D and cycloheximide on HO-1 and HSP70 mRNA expression levels after A/R. Primary rat hepatocytes were treated with actinomycin D (1 μg/ml; lane 2) or cycloheximide (1.5 μg/ml; lane 3) before exposure to anoxia for 4 hr and subsequent reoxygenation for another 3 hr. Total RNA (10 μg) was subjected to Northern blot analysis and probed with ³²P-labeled cDNA for HO-1, HSP70 and GAP-DH. Reoxygenated cells without treatment are demonstrated in Lane 1.

Figure 4.

Time course of oxidized glutathione formation in primary rat hepatocyte cultures after A/R. Primary rat hepatocytes, after exposure to anoxia for 4 hr, were reoxygenated for various times. Cellular oxidized and total glutathione were determined by an enzymatic method as detailed in Experimental Procedures. The content of oxidized glutathione is shown as ratio of oxidized to total glutathione. Values represent means ± SEM of at least five independent experiments. Statistics, Student’s t test for paired values: *significant difference control vs A/R, P ≤ 0.01.

Figure 5.

Effect of desferrioxamine (DFO), β-carotene (Caro), and allopurinol (Allo) on HO-1 and HSP70 mRNA expression in primary rat hepatocyte cultures. Before exposure to anoxia for 4 hr, primary rat hepatocytes were incubated with DFO (1 mM; 8 hr before anoxia), allopurinol (20 μM; 30 min before anoxia), or β-carotene (10 μM; 30 min before anoxia). After reoxygenation for 6 hr, 10 μg of total RNA was isolated, subjected to Northern blot analysis, and probed with ³²P-labeled cDNA of HO-1, HSP70, and GAP-DH. Blots were quantified by a phosphorimager and normalized with GAP-DH. Values represent the relative expression of mRNA in presence of the antioxidants as compared to mRNA levels from untreated reoxygenated cells. Statistics, Student t test for paired values: *significant difference control vs. A/R, P ≤ 0.05.

Footnotes

S.I. was supported by a grant from the Deutsche Forschungsgemeinschaft (Bonn, Germany) Grant SFB402 (A8).

1

To whom requests for reprints should be addressed at Institut für Klinische Chemie und Pathobiochemie Justus-Liebig-Universität Giessen Gaffkystr. 11C 35392 Giessen Germany. E-mail:

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