Expression of heme oxygenase and its RNA in mouse liver after injection of heme and splenectomy

Biochimica et Biophysica Acta, 1132 (1992) 255-258 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

255

BBAEXP 92416

Expression of heme oxygenase and its RNA in mouse liver after injection of heme and splenectomy Shun-ichi K u r a t a a and Midori M a t s u m o t o

b

a Department of Biochemical Genetics Medical Research Institute, Tokyo Medical and Dental University, Tokyo (Japan) and t, Department of Life Science, Tokyo Institute of Technology, Kanagawa (Japan)

(Received 11 February 1992)

Key words: Heme oxygenase;Gene expression; RNA Heme is known to activate the HO (heme oxygenase) gene in cultured cells, but little is known about the effect of heme on the HO gene in intact organisms. The expressions of HO and its RNA in mouse liver were measured using mouse HO cDNA and HO antibody after injection of heme or splenectomy. The antibody was prepared against a /3-galactosidase-HO hybrid protein made in Escherichia coli. The HO mRNA level increased to a maximum 15 h after heme injection. In contrast, expression of HO was maximal about 45 h after heme injection. Essentially the same results were obtained in mice after splenectomy. These results suggest that the HO gene in mouse liver was activated by the injection of heme and splenectomy.

Introduction In the intact organism, red cell hemoglobin is converted almost quantitatively to bile pigment [1]. The red cells are removed and metabolized primarily in the spleen, but other tisses including the liver and bone marrow can share this function [2], after injection of heme and splenectomy, these secondary sites assume the major role in heine catabolism [3]. Heine oxygenase catalyzes the rate-limiting step in heme catabolism, the oxidative degradation of heine to biliverdin [4]. Under physiological conditions, the H O activity of the liver is one-tenth of that in the spleen [4], but it after intravenous administration of heme and splenectomy [3]. The H O activity in cultured cells is also induced by treatment with heine [5,6], and recent investigations showed that this was due to activation of the H O gene and accumulation of its m R N A [7-9]. In the present study, we measured the amounts of H O m R N A and the expression of H O in the liver of mice after splenectomy and injection of heine. For examination of the expression of HO, the H O protein was produced as a /3-galactosidase-HO hybrid protein in Escherichia coli, and specific antibody to the fusion protein was produced by injecting this protein into rabbits [10]. We found that H O expression in mouse liver increased

Correspondence to: S.-i. Kurata, Department of Biochemical Genetics Medical Research Institute, Tokyo Medical and Dental University, Tokyo ll3, Japan.

after injection of heme and splenectomy, and that this active expression was due to increase of the H O m R N A levels. Materials and Methods Mice All experiments were carried out in female D D Y mice of about 7 weeks old. They were given free access to water, but food was withheld for the last 24 h before they were killed. T e n mice were subjected to splenectomy, 10 control mice to a shamoperation with similar. Injection o f heme H e m i n were injected into mice at a dose of 6/~mol per 100 g body weight (about 1.5 /zmol/mouse). For injection, heroin was dissolved in 10 m M sodium phosphate buffer (pH 8.0) at a final concentration of 5 raM. Control mice received 10 mM sodium phosphate buffer (pH 8.0) only. Expression and purification o f recombinant mouse H O protein in E. coli The 339 bp PstI fragment of a mouse H O e D N A clone [12] containing the C-end of the coding region was purified and ligated into the Pst I site of p U R 292 at the unique site near the 3' end of the lacZ gene. The D N A from a colony-purified plasmid was prepared and screened for insertion of the fragment in the correct orientation. This recombinant plasmid was

256 transformed into E. coli JM109, and the transformants were plated on LB plates with 25/.tg/ml ampicillin and 1 mM IPTG (isopropyl-/3-D-thio-galactopyranoside) and cultured at 37°C for 18 h. The cells were collected and solubilized in 0.1 ml of sample buffer (25 mM Tris-HC1 (pH 6.8), 2% SDS, 5% glycerol, 2%/3-mercaptoethanol and Bromophenol blue) per 10 cm culture dish, and then they were heated at 100°C for 5 rain. The lysate was centrifuged and the supernatant was loaded onto 7.5% SDS-polyacrylamide gel. The/3-galactosidase-HO fusion protein was then eluted from the gel, dialyzed and freeze-dried.

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Western blot analysis Samples of 20 p.g of protein from the liver of mice after treatment with heme and splenectomy were subjected to 7.5% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to Millipore lmmobilon polyvinylidene difluoride membranes in a Bio-Rad transblot system. The electroblotted filters were first blocked with 10% FCS in "I-TBS (0.05% Tween 20, 50 mM Tris-HC1, 150 mM NaC1, p H 7.5) and then the blot was treated with a primary antibody diluted appropriately in TTBS for 18 h at room temprature. The blot was then treated with ABC kit reagents (Vectastain USA) according to the supplier's recommended protocol. Immunoreactive H O proteins were visualized by the peroxidase reaction substrate (4-chloro-l-naphthol with imidazole). Signals that appeared on the filters were photographed and the amount of H O was measured by densitometry. Northern blot hybridization After injection of heine or splenectomy, mice were decapitated at 15 h intervals and the liver was promptly frozen in liquid N 2. R N A was extracted from the liver by a guanidium method [11], denatured, separated by electrophoresis on formaldehyde-agarose gel and transferred to a nylon membrane. The filters were incubated with S2p-labelled H O c D N A for 18 h at 45°C in hybridization buffer (0.45 M NaCI, 3 mM EDTA, 30 mM sodium-phosphate buffer, p H 7.4), containing 40% formamide, 7% SDS and 200/.Lg/ml of salmon sperm DNA. Then the filters were washed first with 2 × SSC (0.15 M NaCI, 15 mM sodium citrate) at 30°C for 30 rain, and then twice with 0.1 × SSC, 0.1% SDS at 65°C for 30 min. The filters were then exposed to X-ray film (Kodak XAR) at - 7 0 ° C for about 3 days. After au-

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Preparation of HO antiserum After preimmune bleeding, three rabbits were given a series of eight bi-weekly subcutaneous injections (50 /zg/injection) of purified /3-galactosidase-HO fusion protein emulsified in 4 ml of complete Freund's adjuvant. The rabbits were bled weekly and the serum was stored at - 80°C.

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Fig. 1. Construction of the lacZ-HO fusion gene in pUR292. The 3' end of the 339 bp Pstl fragment of HO cDNA was inserted into the same site of the pUR292 expression vector. The subcloned fragment in the correct orientation was established in E. coli.

toradiography the amount of H O RNA was measured by densitometry. Results

Generation of antibodies against the HO protein For generation of antibodies directed against the H O protein, a fused gene was constructed in pUR292 to produce the protein in E. coli. To express H O as hybrid protein, we ligated a 339bp Pst I fragment from the H O c D N A into the same site of pUR292 (Fig. 1). The ligated plasmid, established in E. coli, produced a hybrid protein containing 113 amino acids of C-terminal region of H O protein on treatment with I P T G (Fig. 2). On induction with IPTG, a novel protein with an apparent molecular weight of 124000 was produced. The antiserum for used in immunoblotting analyses was raised by immunization of a rabbit with the /~galactosidase-HO fusion protein purified by elution of the 124 kDa protein from SDS-polyacrylamide gel. The specificity of the antiserum for the H O encoded portion of the fusion protein was demonstrated by its reaction with the H O fusion protein but not with any other E. coli proteins except /3-galactosidase. Expression of HO mRNA after treatment with heme and splenectomy RNA was extracted from the liver of mice killed at 15 h intervals after injection of heme or splenectomy. The extracted R N A was separated by electrophoresis, transferred to a nylon membrane, and hybridized with the H O c D N A and /3-actin probe. H O m R N A was

257

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detectable immediately after both treatments (heme injection and splenectomy), and reached a maximum level after 15 h (Fig. 3A). In sham operated animals, the amount of HO RNA remained low throughout the observation period. As the level of/3-actin RNA in the liver did not change during the experimental period in either test or control mice (Fig. 3A), changes in the amount of HO mRNA reflected actual changes. The relative amounts of HO mRNA at various times after the two treatments are shown in Fig. 3B. In both cases the level was maximal 15 h after treatment. In this connection, it is interesting that in cultured ceils the effect of heme treatment was maximal after 3 h [3,7].

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Expression of riO after treatment with heme and splenectomy After injection of heme or splenectomy, the expression of HO in mouse liver were examined at 15 h intervals by Western blotting. Protein samples were separated by electrophoresis and blotted onto a Immoblon filter, and the filters were immunostained with HO antibody. HO was detectable immediately after both, treatments (0 h), and then gradually increased to a maximum after 45 h with subsequent decrease to a low level at 75 h (Fig. 4A). In sham operated animals, the expression of HO remained low level throughout the experimental period. The relative amounts of HO at various time after the two treatments are shown in Fig. 4B.

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Fig. 2. Expression of a /3-galactosidase-HO fusion protein in E. coil and production of specific antibody to the HO-encoded domain. Recombinant pUR292 transcripts were grown for 5 h on LB plates with ( + ) or without ( - ) IPTG as described in the text. Then the the cells were collected and sonicated in polyacrylamide gel buffer, and aliquots of whole cell lysates were subjected to SDS-PAGE (7.5%). Part of the gel was stained with Coomassie Blue (lanes l, 3) and replicate lanes were transferred to a filter and probed with antiserum against fl-galactosidase-HO fusion protein (lanes 2, 4). The molecular mass markers used were myosin (200 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa) and ovalbumin (45 kDa). The arrowhead indicates the position of the fusion protein.

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Fig. 3. Northern blot analysis of mouse liver HO m R N A after heme injection and splenectomy. (A) Northern blot analysis of RNAs. RNAs were isolated from the livers of mice at intervals after heme injection, splenectomy or a sham operation. Isolated RNAs were subjected to blot hybridization with 32p labelled HO cDNA and /3-actin probe, a, injection of heme; a', injection of buffer only; b, splenectomy; b', sham operation. Specimens in lanes 1-6 were obtained 0, 15, 30, 45, 60 and 75 h, respectively, after treatments. (B) Relative amounts of HO mRNA. HO mRNA was measured by densitometry and values were normalized by the amount of/3-actin. The amounts of transcripts are shown relative to those 0 h after treatment (lane 1).

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Fig. 4. Western blot analysis of mouse liver HO after heme injection and splenectomy. (A) Mouse liver protein samples were withdrawn and processed as described in the Materials and Methods. Aliquots of the whole liver cell lysates were then subjected to SDS-PAGE and probed with HO antiserum by the ABC method, a, injection of heme; a', injection of buffer only; b, splenectomy; b', sham operation. Samples in lanes 1-6 were obtained 0, 15, 30, 45, 60 and 75 h, respectively, after treatments. (B) Relative amounts of HO. Amounts of HO were measured by densitometry and values were normalized by the amount 0 h after treatment (lane 1).

Discussion HO, the rate-limiting enzyme in the oxidative breakdown of hemin to bile pigment, has been characterized [1-3]. Heme has been shown to activate the H O gene in cultured ceils [7-9], but its effect on the H O gene in intact organisms has not been reported. Tenhunen et al. [3] found that the rats H O activity was higher in the spleen, followed by the bone marrow, liver, brain and kidney, and that splenectomy and injection of heme increased the H O activity in the liver. Therefore, in this work we examined activation of the H O gene and expression of H O in mouse liver after these two treatments. We found that the H O m R N A level was increased 15-30 h after, and that the amount o f H O was increased 30-60 h after these treatments. These exchange were not observed in sham-operated animals. Activation of the H O gene in mouse liver was also obseved at 15 hours after splenectomy. In contrast, in cultured cells, the H O gene is reported to be activated after treatment with heme for about 3 h. The reason of this difference in the rate of activation of the H O gene in cultured cells and in intact organisms is unknown. Possibly, in intact organisms, the homeostatic effects may suppressed the rapid activation of the H O gene. The expressions of HO transcription activating factors in the nuclei of cultured cells after various stress treatments have been reported, i.e., HOTF, heine oxyge-

nase transcription factors in rat glioma cells [5], and los and jun in mouse M1 cells [9]. Further experiments are required on expression of the H O gene in intact organisms. References 10strow, J.D., Jandl, J.H. and Schmid, R. (1962) J. Clin. Invest. 41, 1628-164l. 2 Jandl, J.H., Jones, A.R. and Castle, W.B. (1957) J. Clin. Invest. 36, 1428-1443. 3 Tenhunen, R., Marver, H.S. and Schmid, R. (1970) J. Lab. Clin. Med. 75, 410-421. 4 Tenhunen, R., Marver, H.S. and Schimid, R. (1969) J. Biol. Chem. 244, 6338-6349. 5 Shibahara, S., Yoshida, T. and Kikuchi, G. (1979) Arch. Biochem. Biophys. 197, 607-617. 6 Bardana, M.K., Sassa, S. and Kappas, A. (1985) Biochem. Pharmacol. 34, 2937-2944. 7 Shibahara, S., Muller, R.M. and Taguchi, H. (1987) J. Biol. Chem. 262, 12889-12892. 8 Alam, J., Shibahara, S. and Smith, A. (1989) J. Biol. Chem. 264, 6371-6375. 9 Kurata, S. and Nakajima, H. (1990) Exp. Cell Res. 191, 89-94. 10 Carroll, S.B. and Scott, M.P. (1985) Cell 43, 47-57. 11 Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 12 Kageyama, H., Hiwasa, T., Tokunaga, K. and Sakiyama, S. (1988) Cancer Res. 48, 4798-4798. 13 Sato, M., Fukushi, Y., Ishizawa, S., Okinaga, S., Muller, R.M. and Shibahara, S. (1989) J. Biol. Chem. 264, 10251-10260.