Up-regulation of Protein-disulfide Isomerase in Response to Hypoxia/Brain Ischemia and Its Protective Effect against Apoptotic Cell Death

Abstract

We isolated and identified a stress protein that is up-regulated in response to hypoxia in primary-cultured glial cells. Protein-disulfide isomerase (PDI) was up-regulated not only by hypoxia in glia in vitro, but also by transient forebrain ischemia in rats in vivo. To determine whether newly synthesized PDI is involved in tolerance to ischemic stress, we carried out two procedures to induce PDI gene expression in human neuroblastoma SK-N-MC cells, as well as intrahippocampal injection following electroporation of an expression vector capable of overexpressing PDI in rats. Overexpression of this gene resulted in attenuation of the loss of cell viability induced by hypoxia in neuroblastoma SK-N-MC cells and a reduction in the number of DNA-fragmented cells in the CA1 area of the hippocampus in brain ischemic rats, respectively. These findings suggest that up-regulated PDI may play a critical role in resistance to ischemic damage, and that the elevation of levels of this protein in the brain may have beneficial effects against brain stroke. We isolated and identified a stress protein that is up-regulated in response to hypoxia in primary-cultured glial cells. Protein-disulfide isomerase (PDI) was up-regulated not only by hypoxia in glia in vitro, but also by transient forebrain ischemia in rats in vivo. To determine whether newly synthesized PDI is involved in tolerance to ischemic stress, we carried out two procedures to induce PDI gene expression in human neuroblastoma SK-N-MC cells, as well as intrahippocampal injection following electroporation of an expression vector capable of overexpressing PDI in rats. Overexpression of this gene resulted in attenuation of the loss of cell viability induced by hypoxia in neuroblastoma SK-N-MC cells and a reduction in the number of DNA-fragmented cells in the CA1 area of the hippocampus in brain ischemic rats, respectively. These findings suggest that up-regulated PDI may play a critical role in resistance to ischemic damage, and that the elevation of levels of this protein in the brain may have beneficial effects against brain stroke. fluoromethylketone heat shock protein glucose-regulated protein oxygen-regulated protein protein-disulfide isomerase, GFAP, glial fibrillary acidic protein deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling endoplasmic reticulum green fluorescent protein reverse transcription polymerase chain reaction wild type lactate dehydrogenase four-vessel occlusion 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside Two distinct phenomena are observed in the CA1 subfield of the hippocampus after transient forebrain ischemia in rodents: the death of neurons, which is referred to as “delayed neuronal death,” and the proliferation of glial cells, which is termed “gliosis” (1.Kirino T. Brain Res. 1982; 239: 57-69Crossref PubMed Scopus (2524) Google Scholar). Neurons are thus thought to be fragile and very sensitive to such stress, and, as a consequence, apoptotic cell death occurs (2.Ferrer I. Tortoise A. Macaya A. Sierra A. Moreno D. Munell F. Blanco R. Squier W. Brain Pathol. 1994; 4: 115-122Crossref PubMed Scopus (152) Google Scholar, 3.Nitatori T. Sato N. Waguri S. Karasawa Y. Araki H. Shibanai K. Kominami E. Uchiyama Y. J. Neurosci. 1995; 15: 1001-1011Crossref PubMed Google Scholar). Several studies have shown that caspases are involved in this neuronal apoptosis triggered by brain ischemia. Transgenic mice expressing dominant-negative mutants of caspase-1 and caspase-1-deficient mice show a reduced neuronal cell death induced by ischemic brain injury (4.Friedlander R.M. Gagliardini V. Hara H. Fink K.B. Li W. MacDonald G. Fishman M.C. Greenberg A.H. Moskowitz M.A. Yuan J. J. Exp. Med. 1997; 185: 933-940Crossref PubMed Scopus (326) Google Scholar, 5.Hara H. Fink K. Endres M. Friedlander R.M. Gagliardini V. Yuan J. Moskowitz M.A. J. Cereb. Blood Flow Metab. 1997; 17: 370-375Crossref PubMed Scopus (210) Google Scholar, 6.Schielke G.P. Yang G.-Y. Shivers B.D. Betz A.L. J. Cereb. Blood Flow Metab. 1998; 18: 180-185Crossref PubMed Scopus (286) Google Scholar). Caspase inhibitors such as Z-VAD-fmk,1 YVAD-fmk, and DEVD-fmk also significantly reduce the neuronal cell death induced by ischemia (7.Loddick S.A. MacKenzie A. Rothwell N.J. Neuroreport. 1996; 7: 1465-1468Crossref PubMed Scopus (194) Google Scholar, 8.Hara H. Friedlander R.M. Gagliardini V. Ayata C. Fink K. Huan Z. Shimizu-Sasamata M. Yuan J. Moskowitz M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2007-2012Crossref PubMed Scopus (802) Google Scholar, 9.Chen J. Nagayama T. Jin K. Stetler R.A. Zhu R.L. Graham S.H. Simon R.P. J. Neurosci. 1998; 18: 4914-4928Crossref PubMed Google Scholar). Furthermore, overexpression of Bcl-2 in transgenic mice as well as of neuronal apoptosis inhibitory protein, a member of the inhibitor of apoptosis protein family, by injection of adenovirus expression vectors has a protective effect against the neuronal cell death induced by focal cerebral or transient forebrain ischemia (10.Martinou J.-C. Dubois-Dauphin M. Staple J.K. Rodriguez I. Frankowski H. Missotten M. Albertini P. Talabot D. Catsicas S. Pietra C. Huarte J. Neuron. 1994; 13: 1017-1030Abstract Full Text PDF PubMed Scopus (1019) Google Scholar, 11.Xu D.G. Crocker S.J. Doucet J.-P. St-Jean M. Tamai K. Hakim A.M. Ikeda J.-E. Liston P. Thompson C.S. Korneluk R.G. MacKenzie A. Robertoson G.S. Nat. Med. 1997; 3: 997-1004Crossref PubMed Scopus (222) Google Scholar). These results suggest that caspases are involved in ischemia-induced neuronal death in an anti-apoptotic protein-dependent manner. On the other hand, glial cells show tolerance to ischemic stress, and their numbers are increased in areas originally containing neurons. Due to their abundance and ability to sustain environmental perturbations, glia play a critical role in maintaining neuronal function under both physiological and pathological conditions (12.Swanson R.A. Choi D.W. J. Cereb. Blood Flow Metab. 1993; 13: 162-169Crossref PubMed Scopus (192) Google Scholar). Glial cells subjected to hypoxia express stress proteins such as a 70-kDa heat shock protein (HSP70), a 78-kDa glucose-regulated protein (GRP78), a 150-kDa oxygen-regulated protein (ORP150), a 36-kDa putative RNA-binding protein (RA301), and a 70-kDa putative vesicle transport-related protein (RA410) (13.Hori O. Matsumoto M. Maeda M. Kuwabara K. Ueda H. Ohtsuki T. Kinoshita T. Ogawa S. Stern D.M. Kamada T. J. Neurochem. 1996; 66: 973-979Crossref PubMed Scopus (86) Google Scholar, 14.Kuwabara K. Matsumoto M. Ikeda J. Hori O. Ogawa S. Maeda Y. Kitagawa K. Imuta N. Kinoshita T. Stern D.M. Yanagi H. Kamada T. J. Biol. Chem. 1996; 271: 5025-5032Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 15.Matsuo N. Ogawa S. Imai Y. Takagi T. Tohyama M. Stern D.M. Wakana A. J. Biol. Chem. 1995; 270: 28216-28222Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16.Matsuo N. Ogawa S. Takagi T. Wakana A. Mori T. Matsuyama T. Pinsky D.J. Stern D.M. Tohyama M. J. Biol. Chem. 1997; 272: 16438-16444Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Hori et al. (17.Hori O. Matsumoto M. Maeda Y. Ueda H. Kinoshita T. Stern D.W. Ogawa S. Kamada T. J. Neurochem. 1994; 62: 1489-1495Crossref PubMed Scopus (63) Google Scholar) have reported that the inhibition of protein synthesis during early reoxygenation prevents effective astrocyte adaptation to hypoxia/reoxygenation, resulting in eventual cell death, indicating that several critical gene products are expressed in response to hypoxia/reoxygenation. Indeed, the inhibition of ORP150 using antisense results in reduced cell viability in hypoxia-treated cells, suggesting that ORP150 has a protective effect against hypoxia-stimulated cellular stress (18.Ozawa K. Kuwabara K. Tamatani M. Takatsuji K. Tsukamoto Y. Kaneda S. Yanagi H. Stern D.M. Eguchi Y. Tsujimoto Y. Ogawa S. Tohyama M. J. Biol. Chem. 1999; 274: 6397-6404Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). These observations indicate the possibility that the expression of several stress proteins in glial cells is partly implicated in the acquisition of tolerance against hypoxic or ischemic stress. In the present study, we attempted to isolate important molecules as protective factors against hypoxia in rat primary astrocytes. We found that protein-disulfide isomerase (PDI, EC 5.3.4.1) is up-regulated by hypoxia in rat primary astrocytes. Therefore, we assessed the protective potential of PDI by determining whether overexpression of this gene in vitro and in vivo renders neuronal cells more resistant to the damaging effects of hypoxic treatment and transient forebrain ischemia, respectively. Moreover, we attempted to characterize the possible critical sites in each thioredoxin-like domain. Primary astrocytes were prepared from the whole brain of neonatal Wistar rats as described previously (19.Simons M.L. Murphy S. J. Neurochem. 1992; 59: 897-905Crossref PubMed Scopus (481) Google Scholar) with some modifications (20.Uehara T. Kaneko M. Tanaka S. Okuma Y. Nomura Y. Brain Res. 1999; 823: 226-230Crossref PubMed Scopus (69) Google Scholar). Briefly, the cells were allowed to grow to confluence (12 days) in Dulbecco's modified Eagle's medium supplemented with heat-inactivated 10% fetal calf serum, 50 μg/ml penicillin, and 100 μg/ml streptomycin. Cultured cells were grown at 37 °C under an atmosphere of 5% CO2, 95% air. Subsequently, the cells were shaken at 120 rpm for 1 h at room temperature, and the adherent cells were cultured again for 6 days. The cells were then shaken to separate the astrocytes from the remaining microglia and oligodendrocytes, and they were then identified by immunohistochemical analysis with anti-glial fibrillary acidic protein (GFAP). The cell cultures were 95% positive for GFAP. Human neuroblastoma SK-N-MC cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum at 37 °C in humidified 5% CO2, 95% air. When the cells (astrocytes or SK-N-MC) became subconfluent, they were cultured in a mixture of 5% CO2 and the balance N2 in a humidified incubator (ANX-1, Hirasawa, Tokyo, Japan) at 37 °C within a sealed, anaerobic, gloved cabinet containing a catalyst to scavenge free oxygen as described previously (20.Uehara T. Kaneko M. Tanaka S. Okuma Y. Nomura Y. Brain Res. 1999; 823: 226-230Crossref PubMed Scopus (69) Google Scholar, 21.Araya R. Uehara T. Nomura Y. FEBS Lett. 1998; 439: 168-172Crossref PubMed Scopus (79) Google Scholar). The oxygen levels in the cabinet were measured with a monitor sensitive to oxygen concentrations of <10 ppm. Two-dimensional separation of proteins on small format gels was carried out as described (22.Young D.A. Voris B.P. Maytin E.V. Colbert R.A. Methods Enzymol. 1983; 91: 190-214Crossref PubMed Scopus (49) Google Scholar, 23.Voris B.P. Young D.A. Anal. Biochem. 1980; 104: 478-484Crossref PubMed Scopus (37) Google Scholar). Briefly, cytosolic proteins (10 μg) were first separated by isoelectric focusing in 1.5-mm × 7-cm rod gels containing 1.6% (w/v) ampholytes, pH 5–8 (Amersham Pharmacia Biotech) and 0.4% ampholytes, pH 3.5–10. First-dimension gels were attached to the top of 8 × 7 × 0.015-cm, 12% polyacrylamide gels. After electrophoresis, the gels were stained with silver. Cytosolic proteins from cells exposed to hypoxia were separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membranes. The protein bands on the membranes were stained in a solution of 0.25% Coomassie Brilliant Blue R250 in 7.5% methanol, 5% acetic acid in water and then rinsed in water. The ∼55-kDa protein was then cut out. The protein band was combined with its counterpart from three gels, transferred to 1.5-ml tubes, and then analyzed by Edman degradation using an automated protein sequencer (model 492, Applied Biosystems, CA). The sequences obtained were compared with those in the GenBank™ data base. Full-length PDI cDNA was isolated from rat astrocyte RNA by RT-PCR (24.Uehara T. Matsuno J. Kaneko M. Nishiya T. Fujimuro M. Yokosawa H. Nomura J. Biol. Chem. 1999; 274: 15875-15882Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The primers used were: rat PDI (upstream), 5′-CGA CGT CCG ACA TGC TGA GCC-3′; rat PDI (downstream), 5′-GGC TTC TGC ACT ACA GTT CAT-3′. The three mutants of PDI were generated by the overlapping PCR method. Adult male Wistar rats (250–300 g; Charles River Laboratories) were used for all of the experiments. All animal procedures conformed to the Guide for the Care and Use of Experimental Animals of the Medical Research Council of Japan. forebrain ischemia was by the four-vessel occlusion method. Animals that during for and after with those that not loss of during ischemia, were from the In the of the were exposed but not viability was by two viability was measured by the of stained cells for from of that Briefly, of of each PDI gene were SK-N-MC cells using and for After hypoxic cells were with for rinsed three with and stained in 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside 1 in pH at 37 °C for The number of stained cells in cultures by that in cultures was by two to the and the results are expressed as the of the viability was by the lactate dehydrogenase using a to the was measured as the at and was as the of in the medium to the remaining cellular were with intrahippocampal of was in of (10 1 pH The of the rat was then onto a The was cut and and a was The was the CA1 from in a of μg) using a by to a The injection was and the was in for an after Subsequently, a of in and in was the CA1 the were generated with a Tokyo, Japan) and were at (10 were in at and and the was PDI and were using a and a respectively. of PDI and were on (10 The apoptotic cells were by of generated by using a All were on a We electrophoresis and Coomassie Brilliant Blue R250 to proteins newly synthesized or up-regulated in response to hypoxia and reoxygenation in rat primary astrocytes. treatment with hypoxia/reoxygenation, the levels of several proteins were significantly after hypoxia not Therefore, we carried out two-dimensional electrophoresis and acid using On two-dimensional polyacrylamide gels of cell from astrocytes with hypoxia/reoxygenation, the of protein bands with of and were increased in cells protein of has reported to be up-regulated in response to in astrocytes to we its acid using the automated Edman The of the of the ∼55-kDa protein was to of rat PDI 1 PDI is in the has a from we that the sequences is to the ∼55-kDa protein as analysis of RNA from cultured astrocytes using a PDI cDNA that the PDI levels increased h after hypoxic the levels of PDI a at h after hypoxia and 6 h after analysis using a of PDI protein expression h after which a by h after reoxygenation We whether other induce The levels of PDI were by treatment with or with and heat which increased levels of effect on the expression of On the other hand, and which are to PDI and are in the were also up-regulated by treatment with We the in PDI levels following transient forebrain ischemia in rats using the we whether PDI levels are increased by brain ischemia in rats and the of this in vivo. the results of the PDI in each area of the ischemic brain by the RT-PCR method. analysis conditions of PDI was in the cerebral but not in the or other analysis that PDI expression to 1 after ischemic treatment occlusion by after ischemia, and to levels after ischemia. To whether the of PDI protein expression induced by brain ischemia in vivo with the of its we immunohistochemical analysis in brain using PDI was expressed in all areas of the brain In the was in the cerebral of the ischemic as shown in Moreover, with and that the PDI induced by brain ischemia is in cells In cells with those positive for 1 S. and Y. To the of PDI in neuronal cell death in vitro we the effects of this on cell death in human neuroblastoma SK-N-MC cells. PDI two thioredoxin-like the and the we mutants with in both of the sequences to determine the critical to the To the ability of or PDI to against cell death, we carried out two the of cells and the other a on cell In both the transient expression of PDI resulted in a of cell viability by hypoxia compared with and of the mutants both and in the loss of we an intrahippocampal injection of a PDI expression vector by electroporation to this in areas of the rat brain in vivo. To the of in vivo we green fluorescent protein or gene the CA1 number of or cells were in this area not Subsequently, we carried out in vivo overexpression of the PDI gene in the CA1 area of the forebrain ischemia is to induce neuronal apoptotic death in the CA1 area of the We whether apoptotic neuronal death occurs after brain ischemia. apoptotic cells were 7 after ischemia were in the cells rats with or and We whether the overexpression of PDI in by after of the vector or the PDI gene expression vector the hippocampus by was for To the possibility of the observed effects to electroporation we first the effects of with the vector were in the number of cells in this area the and rats not In the rats, a number of cells in the CA1 area were after with numbers In the numbers of DNA-fragmented cells in this area were reduced in the rats that cells were present after in rats apoptotic cells were at and 7 after in the rats, the number of cells was by compared with those of the rats The of this was to isolate and proteins capable of cells against ischemic stress using rat primary astrocytes. We found some in the hypoxia-treated astrocytes by two-dimensional electrophoresis and acid Several of molecules have a glucose-regulated protein the protein of ∼55-kDa not reported we analyzed this protein The of the of the ∼55-kDa protein was to the of rat PDI 1 a protein present within the of the is to the of both and N. Biochem. PubMed Scopus Google Scholar, R.A. PubMed Scopus Google and has a its R.A. PubMed Scopus Google Scholar). The ∼55-kDa protein identified this and we that this is to the protein as In the PDI is expressed in primary astrocytes. to hypoxia results in a of PDI and protein levels and and also the of PDI in the present study, the levels were very compared with those induced by heat which induce 70-kDa heat shock protein PDI These results suggest that the of PDI and are In which is to be an the levels of PDI and and ORP150 induced by are in the (13.Hori O. Matsumoto M. Maeda M. Kuwabara K. Ueda H. Ohtsuki T. Kinoshita T. Ogawa S. Stern D.M. Kamada T. J. Neurochem. 1996; 66: 973-979Crossref PubMed Scopus (86) Google Scholar, 14.Kuwabara K. Matsumoto M. Ikeda J. Hori O. Ogawa S. Maeda Y. Kitagawa K. Imuta N. Kinoshita T. Stern D.M. Yanagi H. Kamada T. J. Biol. Chem. 1996; 271: 5025-5032Abstract Full Text Full Text PDF PubMed Scopus (173) Google O. Matsumoto M. Maeda Y. Ueda H. Kinoshita T. Stern D.W. Ogawa S. Kamada T. J. Neurochem. 1994; 62: 1489-1495Crossref PubMed Scopus (63) Google suggesting that several proteins in the may be up-regulated in response to hypoxia in astrocytes. In with this both and which are in the and as were up-regulated by hypoxia on we that hypoxic stress the of several proteins in the such as and We whether PDI expression is in response to brain ischemia in vivo. transient forebrain ischemia PDI levels in the cerebral but not in the or other areas PDI was in the ischemic from 1 to after of with RT-PCR was observed only in the cerebral following brain ischemia. To the of we of brain with and cells were in cells a small of cells were for PDI and not suggesting that PDI expression is in astrocytes and only in forebrain ischemia results in to in the cerebral the CA1 area of the the and the is PDI is up-regulated by ischemic stress in the cerebral but not the hippocampus and other PDI in the ischemic may be involved at in the acquisition of tolerance against ischemic stress by an as To determine whether PDI a critical role in tolerance against ischemic stress, we its effects on cell death and its possible PDI is a protein that of and R.A. PubMed Scopus Google Scholar, T. T. K. R. J. J. PubMed Scopus Google Scholar, J. N.J. K. R.M. Sci. 1995; 4: PubMed Scopus (49) Google Scholar). The a and are to and both the two sites for PDI Biochem. J. PubMed Scopus Google Scholar, K. R. T. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, K. R. T. J. 1992; PubMed Scopus Google Scholar, M. 1993; Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, N.J. 1995; PubMed Scopus Google Scholar). To determine the protective of sites in cell death, were with in both the of the PDI gene human neuroblastoma SK-N-MC cells significantly the loss of cell viability triggered by hypoxia In of the mutants the loss of cell viability on hypoxic These results that PDI has a effect against hypoxia in neuronal cells, and that both thioredoxin-like are critical for this protective function against hypoxic stress. the overexpression of PDI in vivo was used to determine whether increased levels of this protein reduce the damaging effects in the CA1 of the hippocampus by transient brain ischemia. a with an vector was by electroporation may have for the observed effect against ischemic stress. the numbers of DNA-fragmented cells in the CA1 after in rats were and suggesting that in On the other hand, the numbers of CA1 were significantly reduced in the ischemic but not in These observations suggest that overexpression of the PDI gene significantly ischemia-induced apoptosis in the CA1 area of the In we have that PDI is up-regulated in response to hypoxia or transient forebrain ischemia in astrocytes. Furthermore, the overexpression of PDI in vitro and in vivo resistance to hypoxic or ischemic have some that PDI may as a A. J. Chem. 1994; Full Text PDF PubMed Google Scholar, J. Biochem. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google this effect may be partly on its stress results in the or of proteins and also several such as Furthermore, ORP150 in the is induced by hypoxia and ischemia in astrocytes K. Matsumoto M. Ikeda J. Hori O. Ogawa S. Maeda Y. Kitagawa K. Imuta N. Kinoshita T. Stern D.M. Yanagi H. Kamada T. J. Biol. Chem. 1996; 271: 5025-5032Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, K. Kuwabara K. Tamatani M. Takatsuji K. Tsukamoto Y. Kaneda S. Yanagi H. Stern D.M. Eguchi Y. Tsujimoto Y. Ogawa S. Tohyama M. J. Biol. Chem. 1999; 274: 6397-6404Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). These findings suggest that hypoxia or brain ischemia the expression of several stress proteins or in the which may in an acquisition of tolerance against that PDI expression in may be in brain ischemia. we a that to a gene in vivo using a has to the that the overexpression of the PDI gene in the CA1 area against the apoptotic cell death induced by brain ischemia. Therefore, the PDI in the may play a critical role in the against