Peg3/Pw1 Is Involved in p53-mediated Cell Death Pathway in Brain Ischemia/Hypoxia
2002; Elsevier BV; Volume: 277; Issue: 1 Linguagem: Inglês
10.1074/jbc.m107435200
ISSN1083-351X
AutoresAtsushi Yamaguchi, Manabu Taniguchi, Osamu Hori, Satoshi Ogawa, Nobuteru Tojo, Nobuya Matsuoka, Shinichi Miyake, Kousuke Kasai, Hisashi Sugimoto, Michio Tamatani, Toshihide Yamashita, Masaya Tohyama,
Tópico(s)Epigenetics and DNA Methylation
ResumoEmerging evidence has shown that tumor suppressor p53 expression is enhanced in response to brain ischemia/hypoxia and that p53 plays a critical role in the cell death pathway in such an acute neurological insult. However the mechanism remains unclear. Recently it was reported that Peg3/Pw1, originally identified as a paternally expressed gene, plays a pivotal role in the p53-mediated cell death pathway in mouse fibroblast cell lines. In this study, we found that Peg3/Pw1 expression is enhanced in peri-ischemic neurons in rat stroke model by in situ hybridization analysis, where p53 expression was also induced by immunohistochemical analysis. Moreover, we found that p53 was co-localized with Peg3/Pw1 in brain ischemia/hypoxia by double staining analysis. In human neuroblastoma-derived SK-N-SH cells, Peg3/Pw1 mRNA expression is enhanced remarkably at 24 h post-hypoxia, when p53 protein expression was also enhanced at high levels. Subcellular localization of Peg3/Pw1 was observed in the nucleus. Adenovirus-mediated high dose p53 overexpression induced Peg3/Pw1 mRNA expression. Overexpression of Peg3/Pw1 reduced cell viability under hypoxic conditions, whereas that of the C-terminal-deleted mutant and anti-sense Peg3/Pw1 inhibited hypoxia-induced cell death. These results suggest that Peg3/Pw1 is involved in the p53-mediated cell death pathway as a downstream effector of p53 in brain ischemia/hypoxia. Emerging evidence has shown that tumor suppressor p53 expression is enhanced in response to brain ischemia/hypoxia and that p53 plays a critical role in the cell death pathway in such an acute neurological insult. However the mechanism remains unclear. Recently it was reported that Peg3/Pw1, originally identified as a paternally expressed gene, plays a pivotal role in the p53-mediated cell death pathway in mouse fibroblast cell lines. In this study, we found that Peg3/Pw1 expression is enhanced in peri-ischemic neurons in rat stroke model by in situ hybridization analysis, where p53 expression was also induced by immunohistochemical analysis. Moreover, we found that p53 was co-localized with Peg3/Pw1 in brain ischemia/hypoxia by double staining analysis. In human neuroblastoma-derived SK-N-SH cells, Peg3/Pw1 mRNA expression is enhanced remarkably at 24 h post-hypoxia, when p53 protein expression was also enhanced at high levels. Subcellular localization of Peg3/Pw1 was observed in the nucleus. Adenovirus-mediated high dose p53 overexpression induced Peg3/Pw1 mRNA expression. Overexpression of Peg3/Pw1 reduced cell viability under hypoxic conditions, whereas that of the C-terminal-deleted mutant and anti-sense Peg3/Pw1 inhibited hypoxia-induced cell death. These results suggest that Peg3/Pw1 is involved in the p53-mediated cell death pathway as a downstream effector of p53 in brain ischemia/hypoxia. middle cerebral artery occlusion adenovirus cytomegalovirus multiplicity of infection phosphate-buffered saline reverse transcriptase green fluorescent protein promoter of enhanced GFP middle cerebral artery dominant negative TNF receptor-associated factor 2 Clarification of the molecular events underlying the cell death pathway in response to brain ischemia/hypoxia is an important step toward the development of fundamental treatment for such an acute neurological insult. However, the molecular mechanism in which neuronal cells undergo cell death in brain ischemia/hypoxia remains obscure. Previous reports have shown that p53 is activated under hypoxic conditions in vitro (1Stempien-Otero A. Karsan A. Cornejo C.J. Xiang H. Eunson T. Morrison R.S. Kay M. Winn R. Harlan J. J. Biol. Chem. 1999; 274: 8039-8045Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 2Long X. Boluyt M.O. Hipolito M.L. Lundberg M.S. Zheng J.S. O'Neill L. Cirielli C. Lakatta E.G. Crow M.T. J. Clin. Invest. 1997; 99: 2635-2643Crossref PubMed Scopus (275) Google Scholar) and also in peri-ischemic regions of the animal focal stroke model (3Li Y. Chopp M. Zhang Z.G. Zaloga C. Niewenhuis L. Gautam S. Stroke. 1994; 25: 849-855Crossref PubMed Scopus (176) Google Scholar, 4Chopp M. Li Y. Zhang Z.G. Freytag S.O. Biochem. Biophys. Res. Commun. 1992; 182: 1201-1207Crossref PubMed Scopus (117) Google Scholar). Moreover, it has also been shown that neuronal cells deficient in the p53 gene are significantly protected from excitotoxic and ischemic insult (5Xiang H. Hochman D.W. Saya H. Fujiwara T. Schwartzkroin P.A. Morrison R.S. J. Neurosci. 1996; 16: 6753-6765Crossref PubMed Google Scholar, 6Crumrine R.C. Thomas A.L. Morgan P.F. J. Cereb. Blood Flow Metab. 1994; 14: 887-891Crossref PubMed Scopus (278) Google Scholar), indicating that the p53-mediated signaling cascade contributes remarkably to neuronal cell death in such acute brain injuries. Peg3/Pw1, originally identified as a paternally expressed gene, is a large molecule containing 12 zinc finger-like domains and 2 proline-rich periodic repeat domains (7Kuroiwa Y. Kaneko-Ishino T. Kagitani F. Kohda T. Li L.L. Tada M. Suzuki R. Yokoyama M. Shiroishi T. Wakana S. Barton S.C. Ishino F. Surani M.A. Nat. Genet. 1996; 12: 186-190Crossref PubMed Scopus (217) Google Scholar). The expression of Peg3/Pw1 mRNA was detected ubiquitously in all tissues at low levels during mouse development, and in adult mouse, Peg3/Pw1 expression was observed abundantly in central nervous system and skeletal muscle (7Kuroiwa Y. Kaneko-Ishino T. Kagitani F. Kohda T. Li L.L. Tada M. Suzuki R. Yokoyama M. Shiroishi T. Wakana S. Barton S.C. Ishino F. Surani M.A. Nat. Genet. 1996; 12: 186-190Crossref PubMed Scopus (217) Google Scholar, 8Relaix F. Wei X.J. Wu X. Sassoon D.A. Nat. Genet. 1998; 18: 287-291Crossref PubMed Scopus (132) Google Scholar). In human, Peg3/Pw1 mRNA was observed at high levels in ovary, testis, and placenta and at modest levels in brain and pancreas (9Kim J. Ashworth L. Branscomb E. Stubbs L. Genome Res. 1997; 7: 532-540Crossref PubMed Scopus (99) Google Scholar). Its subcellular localization was observed in the nucleus (10Relaix F. Weng X. Marazzi G. Yang E. Copeland N. Jenkins N. Spence S.E. Sassoon D. Dev. Biol. 1996; 177: 383-398Crossref PubMed Scopus (116) Google Scholar), and Peg3/Pw1 was shown to be involved in the tumor necrosis factor-NF-κB signal transduction pathway through interacting with TRAF2 in some experimental system (8Relaix F. Wei X.J. Wu X. Sassoon D.A. Nat. Genet. 1998; 18: 287-291Crossref PubMed Scopus (132) Google Scholar). Recently it was reported that Peg3/Pw1 is up-regulated in the p53-mediated cell death pathway and involved in p53-mediated apoptosis through translocating Bax from cytosol to mitochondria (11Deng Y. Wu X. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12050-12055Crossref PubMed Scopus (177) Google Scholar) or interacting with members of Siah (seven inabsentia homology) family in mouse fibroblast cells (12Relaix F. Wei X.-J. Li W. Pan J. Lin Y. Bowtell D.D. Sassoon D.A. Wu X. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2105-2110Crossref PubMed Scopus (144) Google Scholar). Moreover, Peg3/Pw1 was specifically activated during p53/c-Myc-mediated and p53/E2F-1-mediated apoptosis but not during p53-dependent G1 arrest, and the inhibition of Peg3/Pw1 function blocked p53/c-Myc-mediated cell death, whereas Peg3/Pw1 expression alone did not induce apoptosis, suggesting that Peg3/Pw1 is involved in the cell death pathway downstream of p53 (12Relaix F. Wei X.-J. Li W. Pan J. Lin Y. Bowtell D.D. Sassoon D.A. Wu X. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2105-2110Crossref PubMed Scopus (144) Google Scholar). To investigate the molecular mechanism underlying the cell death pathway in response to brain ischemia/hypoxia, we identified the genes whose mRNA expressions are induced under hypoxic insult by a differential display technique using primary cultured astroglial cells. We found that Peg3/Pw1 mRNA expression was up-regulated in peri-ischemic neuronal cells in rat middle cerebral artery occlusion (MCA-O)1 model, where the induction of p53 protein expression was also observed by immunohistochemical analysis. In this study, we examined whether Peg3/Pw1 is involved in the p53-mediated cell death pathway under ischemic/hypoxic insult using rat MCA-O model and human neuroblastoma-derived SK-N-SH cells. Both the mouse full-length Peg3/Pw1 cDNA and C-terminal-deleted Peg3/Pw1 cDNA (gifts from Dr. Sassoon, The Mount Sinai Medical Center, New York, NY) were digested with BamHI and XhoI then directly subcloned into pEGFP plasmid (CLONTECH, Palo Alto, CA), which produces the N-terminal GFP-tagged protein under the control of CMV promoter. To generate antisense Peg3/Pw1 construct, Peg3/Pw1 cDNA was subcloned into pCDNA3(−) (Invitrogen) in reverse direction. The DNA was transfected into cells using LipofectAMINE reagent (Invitrogen) as described by the manufacturer. Stable cell lines were generated by selecting the transfected cells in neomycin (1200 μg/ml) for about 2 weeks. Rat cortex neurons or astroglial cells were prepared from Sprague-Dawley rat E17 embryo or postnatal day 1 pups, respectively, as described previously (13Tamatani M. Che Y.H. Matsuzaki H. Ogawa S. Okado H. Miyake S. Mizuno T. Tohyama M. J. Biol. Chem. 1999; 274: 8531-8538Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 14Maeda Y. Matsumoto M. Hori O. Kuwabara K. Ogawa S. Yan S.D. Ohtsuki T. Kinoshita T. Kamada T. Stern D.M. J. Exp. Med. 1994; 180: 2297-2308Crossref PubMed Scopus (185) Google Scholar). Briefly, cerebral hemispheres were dissected, and the meninges were carefully removed. Brain tissues were digested with collagenase B (Roche Molecular Biochemicals) at 37 °C for 15 min. Tissues were further dissociated by repeated trituration. The neuronal cells were seeded at a density of 1 × 10 6 cells/cm2 on poly-l-lysine (10 μg/ml)-coated plates (Falcon Lab and Maintainware, Lincoln Park, NJ) and grown in Dulbecco's modified Eagle's medium supplemented with B27 supplement (Invitrogen), 30 mm glucose, and 0.5% (v/v) penicillin-streptomycin at 37 °C in a humidified atmosphere of 5% CO2 and 95% room air. In experiments using primary cultured neurons, all experiments were performed in 8- to 10-day-old cultures. Astroglial cells were seeded at a density of 1 × 10 6cells/cm2 in 175-cm2 culture flasks and grown in α-modified Eagle's medium with 10% fetal calf serum for 10 days and agitated strongly on a shaking platform to separate astrocytes from microglial and oligodendroglial cells. Astroglial cells were then placed into 150-mm diameter dishes and grown for an additional 7 days. In experiments using astroglial cells, all experiments were performed when cultured astrocytes achieved 80% confluence. Hypoxia treatment was performed using an incubator attached to a hypoxia chamber (Coy Laboratory Products, Ann Arbor, MI) that maintained a humidified atmosphere with low oxygen tension (pO2, 12–14 torr) as described previously (15Ogawa S. Gerlach H. Esposito C. Pasagian-Macaulay A. Brett J. Stern D. J. Clin. Invest. 1990; 85: 1090-1098Crossref PubMed Scopus (276) Google Scholar). Before exposure to hypoxia, the growth medium was replaced with a serum-free medium that consisted of Dulbecco's modified Eagle's medium for neuronal cells, α-modified Eagle's medium for astroglial cells, and SK-N-SH cells supplemented with 30 mm glucose and 0.5% (v/v) penicillin-streptomycin. Where indicated, after exposure to hypoxia, cultures were returned to the ambient atmosphere (re-oxygenation) at which time the condition medium was rapidly exchanged for medium containing 10% fetal calf serum. In other experiments, cells were exposed to either calcium ionophoreA23187 (1 μm for 12 h) (Sigma) or tunicamycin (1 μg/ml for 12 h) (Sigma). When exposed to hydrogen peroxide, cells were returned to normal medium after a 5-min exposure to hydrogen peroxide (88 nm) (Wako Chemicals, Osaka, Japan) and incubated for an additional 2 h. Alternatively, cultures were subjected to heat shock at 42 °C for 1 h, then returned to 37 °C and incubated for an additional 2 h. AxCANLZ, which expresses LacZ under control of the CAG promoter, was generously provided by Dr. Saito (University of Tokyo, Tokyo, Japan). Another recombinant adenovirus vector, Ad5CMVp53, which expresses p53 protein under the control of CMV promoter, was generously provided by Dr. Roth (University of Texas, Houston, TX) (16Zhang W.W. Fang X. Mazur W. French B.A. Georges R.N. Roth J.A. Cancer Gene Ther. 1994; 1: 5-13PubMed Google Scholar). All viruses were grown in 293 cells. Virus titers were determined by plaque assay, and concentrated virus was stored at −80 °C. Infection was carried out by adding recombinant adenovirus to serum-containing medium. The cells were incubated with virus-containing medium at the indicated multiplicity of infection at 37 °C for 60 min with constant agitation. Cell viability was assessed by determining the release of lactate dehydrogenase into the culture medium thereby indicating a loss of membrane integrity and cell death. Lactate dehydrogenase activity was measured using a commercial kit (Kyokuto Chemical Co., Tokyo, Japan) in which a colorimetric assay measures the pyruvate-mediated conversion of 2,4-dinitrophenylhydrazine into a visible hydrazone precipitate. Percent neuronal viability was expressed as (1 − experimental value/maximum release) × 100, where the maximum release was obtained after exposure of untreated control cultures to 0.2% Triton X-100 for 15 min at 37 °C. Cell extracts for Western blot analysis were prepared by washing the cells three times with PBS and lysing them in sample buffer (50 mm Tris-HCl, pH 8.0, 20 mm EDTA, 1% SDS, and 100 mm NaCl). The samples were boiled for 5 min before subjecting 20-μl aliquots to electrophoresis on 7.5, 10, and 12% SDS-PAGE gels. After the proteins were transferred onto polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA), the membrane was incubated in blocking buffer (1× PBS, 5% non-fat dried milk) for 1 h at room temperature and then probed with a primary antibody in blocking buffer overnight at 4 °C. The membrane was washed four times in PBS containing 0.3% Tween 20, probed with the secondary antibody in blocking buffer for 1 h at room temperature, and washed again in PBS containing 0.05% Tween 20. Detection of signal was performed with an enhanced chemiluminescence detection kit (ECL; Amersham Biosciences, Inc.). The primary antibodies used were anti-p53 monoclonal antibody (DO-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Bax polyclonal antibody (number P-19; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-KDEL monoclonal antibody (StressGen Biotechnologies Corp., San Diego, CA), anti-β-tubulin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-GFP monoclonal antibody (CLONTECH, Palo Alto, CA). Total RNA derived from 2 × 107 cells was extracted from cells by the acid guanidium-thiocyanate/phenol chloroform method. For Northern blot analysis, total RNA (20 μg/lane) was separated by electrophoresis on 1.0% agarose/formide gels and transferred overnight onto polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The membrane was prehybridized for 1 h at 65 °C in hybridization buffer (0.9 m NaCl, 90 mm sodium citrate, pH 7.0) containing 5× Denhardt's solution, SDS (0.5%), and heat-denatured salmon sperm DNA (100 ng/ml). cDNA probe was radiolabeled with [32P]dCTP (NZ522; PerkinElmer Life Sciences) by a random labeling kit according to the manufacturer's manual (Takara Shuzo Co., Ltd., Shiga, Japan). After hybridization overnight at 65 °C in hybridization buffer containing radiolabeled cDNA probe (5 ng/ml), filters were washed twice with 2× SSC, 0.5% SDS and 0.2× SSC, 0.5% SDS for 60 min, respectively, at 65 °C, exposed to x-ray film (Fuji Photo Film), and subjected to autoradiography. For RT-PCR, 5 μg of total RNA was reverse-transcribed using oligo(dT) by reverse transcriptase from Moloney murine leukemia virus (Invitrogen) in a volume of 25 μl. For PCR amplification, specific oligonucleotide primer pairs (10 pmol each) were incubated with 1 μl of cDNA template in a 20-μl PCR reaction mixture containing 1.5 mm MgCl2, 25 mm KCl, 10 mm Tris, pH 9.2, mixed deoxynucleotides (1 mm each), and 1 unit of Taqpolymerase. The sequences of primers used in this study were as follows: rat Peg3/Pw1 sense primer, 5′-GAGAATCCTCCATTTATATC-3′ and rat Peg3/Pw1 antisense primer, 5′-TCATGAATCTTCTGGTGCTC-3′; human Peg3/Pw1 sense primer, 5′-TACGAATGTAAGGTGTGTGG-3′ and human Peg3/Pw1 antisense primer, 5′-TTGGCACGGAATACACTCTG-3′; human Bax sense primer, 5′-ATGGACGGGTCCGGGGAGCA-3′ and, human Bax antisense primer, 5′-CAGACACGTAAGGAAAACGC-3′; human GRP78 sense primer, 5′-TTATTTGGGAAAGAAGGTTAC-3′ and human GRP78 antisense primer, 5′-ATTGAAGAACTCTTTAACCAG-3′; rat GRP78 sense primer, 5′-GGTCTACGAAGGTGAACGAC-3′ and rat GRP78 antisense primer, 5′-CAATTTCTTCTAGTTCCTTC-3′; and as an internal control rat or human β-actin sense primer, 5′-TGCCCATCTATGAGGGTTACG-3′ and rat or human β-actin antisense primer, 5′-TAGAAGCATTTGCGGTGCACG-3′. Mus musculus Peg3, accession number AF038939; human Bax, accession number NM_004324; human Peg3, accession number XM_042345; human GRP78, accession number XM_044202; human β-actin, accession number XM_037235; rat β-actin, accession number NM_031144; rat GRP78, accession number S63521. Dilutions of cDNA were amplified for 23–32 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The amplified PCR products at each cycle number were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining. The product of constitutively expressed β-actin mRNA served as the internal standard. All the products were assayed in the linear response range of the RT-PCR amplification process; the cycle number used was determined by finding the midpoint of linear amplification on a sigmoid curve for amplification products with cycle numbers of 24–40 plotted against band density. The identity of each PCR product was confirmed by subcloning the amplified cDNAs into the pGEM-T vector (Promega) and sequencing. In Northern blot analysis, amplified cDNAs were used as probes to detect human and rat Peg3/Pw1 or human GRP78 transcripts. Unilateral permanent MCA-O was performed in 9-week-old male Sprague-Dawley rats (300–325 g) that were purchased from Japan SLC (Sizuoka, Japan) as described previously with minor modification (17Tamura A. Graham D.I. McCulloch J. Teasdale G.M. J Cereb. Blood Flow Metab. 1981; 1: 53-60Crossref PubMed Scopus (1245) Google Scholar, 18Yamashita T. Kohmura E. Yamauchi A. Shimada S. Yuguchi T. Sakaki T. Miyai A. Tohyama M. Hayakawa T. J Cereb. Blood Flow Metab. 1996; 16: 1203-1210Crossref PubMed Scopus (22) Google Scholar). Briefly, rats were anesthetized with halothane (4% for induction, 1.5% for maintenance) in a mixture of 70% nitrous oxide and 30% oxygen delivered through a close-fitting facemask during surgery. A vertical incision was made between the left orbit and the external auditory canal. The temporalis muscle was cut, and subtemporal craniotomy was performed without removing the zygomatic arch under an operation microscope. The main trunk of the MCA and olfactory tract were observed through the dura mater. The left MCA was exposed by a microsurgical approach, and the MCA was occluded by bipolar electrocoagulation. The MCA from proximal to the olfactory tract to the inferior cerebral vein and the lenticulostriate arteries were permanently occluded and transected to avoid recanalization. After occlusion of the MCA, the temporalis muscle and skin were closed in layers, and anesthesia was discontinued. Rectal temperature of rats was maintained at 38.0 ± 0.5 °C with a heating pad (TR-100, PS-100; Fine Science Tools, Belmont, CA) during the surgery. Brains were removed, and serial brain sections (10–15 μm) were obtained from the frozen brains with a cryostat and stored in a tightly closed case at −80 °C. Frozen sections (10–15-μm-thick) of adult mouse brain were cut in various planes and thaw-mounted onto poly-l-lysine-coated slides. [35S]-Labeled or digoxigenin-labeled cRNA probes (sense or antisense) were prepared by in vitro transcription using T7 or SP6 polymerase from the coding of rat Peg3/Pw1 cDNA subcloned into pGEM-T vector (Promega). All steps of in situhybridization reactions were carried out essentially according to Simmons et al. (19Simmons D.M. Arizza V.L. Swanson L.W. J. Histotechnol. 1989; 12: 169-181Crossref Google Scholar). Briefly, the sections were dried, fixed in 4% formaldehyde, treated with proteinase K (10 μg/ml, room temperature, 30 min), acetylated, dehydrated, and air-dried prior to hybridization overnight at 55 °C in a humidified chamber with 150–200 μl of hybridization buffer (10% sodium dextran sulfate, 20 mm Tris-HCl, pH 8.0, 0.3 m NaCl, 0.2% sarcosyl, 0.02% heat-denatured salmon sperm DNA, 1× Denhardt's solution, 50% formamide) with a cRNA probe. After being rinsed in 5× SSC at 60 °C for 20 min and washed in 50% formamide/2× SSC at 60 °C for 30 min, sections were subjected to RNase digestion for 20 min at 37 °C (1 μg/ml RNase A) and washed in 50% formamide/2× SSC at 60 °C for 30 min. For detection of hybridized digoxigenin-labeled cRNA probes, anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche Molecular Biochemicals) was reacted with 1:500 dilution, and color was developed by incubation with 4-nitro blue tetrazolium chloride. For detection of hybridized [35S]-labeled cRNA probes, sections were exposed to x-ray film (Fuji Photo Film) and subjected to autoradiography. For immunohistochemistry, frozen sections were dried, fixed in 4% paraformaldehyde solution, incubated in blocking buffer (1× PBS, 5% non-fat dried milk) for 1 h at room temperature, and reacted with anti-p53 monoclonal antibody (PharMingen) in blocking buffer for 1 h at room temperature, followed by incubation with Alexa Fluor 488 anti-mouse IgG conjugate (Molecular Probes, Eugene, OR) or biotinylated anti-mouse IgG secondary antibody (Vector Laboratories Inc., Burlingame, CA). For detection of fluorescent secondary antibody binding sites, sections were photographed under laser confocal microscopy (Carl Zeiss LSM510). Sites of biotinylated secondary antibody binding were visualized using a Vectastain Elite ABC kit according to manufacturer's manual (Vector Laboratories Inc., Burlingame, CA). For double staining of p53 protein by immunohistochemistry and rat Peg3/Pw1 mRNA by in situhybridization, frozen sections were reacted with anti-p53 monoclonal antibody (PharMingen) followed by incubation with Alexa Fluor 488 anti-mouse IgG conjugate (Molecular Probes, Eugene, OR). The sections were photographed under laser confocal microscopy (Carl Zeiss LSM510) subsequent to in situ hybridization analysis using digoxigenin-labeled cRNA probe for rat Peg3/Pw1. Using primary cultured astroglial cells exposed to hypoxia, we have identified the genes whose expressions are enhanced in hypoxic insult by differential display methods as described previously (20Yamaguchi A. Hori O. Stern D.M. Hartmann E. Ogawa S. Tohyama M. J. Cell Biol. 1999; 147: 1195-1204Crossref PubMed Scopus (100) Google Scholar). As shown in Fig. 1A, Peg3/Pw1 mRNA expression was induced 16–24 h after exposure to hypoxia and returned to basal levels upon re-oxygenation in rat primary cultured astroglial cells. Next, to investigate what kind of insults induce Peg3/Pw1 mRNA expression, we exposed primary cultured astroglial cells to various stresses and performed Northern blotting analysis using total RNAs extracted from those astroglial cells. Enhanced Peg3/Pw1 mRNA expressions were observed in hypoxia or heat shock stress (Fig. 1B). We then examined whether Peg3/Pw1 mRNA expression was up-regulated in primary cultured cortex neurons under hypoxic conditions by RT-PCR analysis. We exposed rat primary cultured cortex neurons to hypoxia or re-oxygenation for the indicated times, and performed RT-PCR analysis using first strand cDNAs synthesized from total RNAs extracted from those neurons as templates. Similar to the results in astroglial cells, Peg3/Pw1 mRNA expression was enhanced 12–24 h post-hypoxia and returned to basal levels upon re-oxygenation in primary cultured neurons (Fig.1C). Previous reports have shown that in human tissues Peg3/Pw1 expression is abundant in ovary, placenta, testis, and brain and that in adult mouse the highest expression levels are observed in brain and muscle (7Kuroiwa Y. Kaneko-Ishino T. Kagitani F. Kohda T. Li L.L. Tada M. Suzuki R. Yokoyama M. Shiroishi T. Wakana S. Barton S.C. Ishino F. Surani M.A. Nat. Genet. 1996; 12: 186-190Crossref PubMed Scopus (217) Google Scholar, 8Relaix F. Wei X.J. Wu X. Sassoon D.A. Nat. Genet. 1998; 18: 287-291Crossref PubMed Scopus (132) Google Scholar, 9Kim J. Ashworth L. Branscomb E. Stubbs L. Genome Res. 1997; 7: 532-540Crossref PubMed Scopus (99) Google Scholar, 10Relaix F. Weng X. Marazzi G. Yang E. Copeland N. Jenkins N. Spence S.E. Sassoon D. Dev. Biol. 1996; 177: 383-398Crossref PubMed Scopus (116) Google Scholar). We then examined tissue distributions of Peg3/Pw1 in adult rat tissues by Northern blotting analysis. We found that Peg3/Pw1 mRNA expression was remarkably abundant in brain, and we could not detect Peg3/Pw1 mRNA expression in other organs including heart, lung, liver, muscle, and kidney in our experiment (Fig. 1D). To address whether Peg3/Pw1 mRNA expression is enhanced in brain ischemia/hypoxia, we performed in situhybridization using [35S]-labeled Peg3/Pw1 cRNA probe in serial brain coronal sections derived from the rat permanent MCA-O model. Fig. 2, A–D shows that in the rat permanent 6-h MCA-O model, Peg3/Pw1 mRNA expression was enhanced in peri-ischemic lesions including caudate putamen, globus pallidus, cingulate cortex, and thalamus. The signals in the contralateral side to ischemia were also observed at high levels in septum, thalamus, hypothalamus, and hippocampus, at moderate levels in neocotex, and at low levels in basal ganglia (globus pallidus, caudate putamen), which is consistent with previous reports that Peg3/Pw1 expression is abundant throughout normal adult mouse and human brain and especially strong in hypothalamus (7Kuroiwa Y. Kaneko-Ishino T. Kagitani F. Kohda T. Li L.L. Tada M. Suzuki R. Yokoyama M. Shiroishi T. Wakana S. Barton S.C. Ishino F. Surani M.A. Nat. Genet. 1996; 12: 186-190Crossref PubMed Scopus (217) Google Scholar, 8Relaix F. Wei X.J. Wu X. Sassoon D.A. Nat. Genet. 1998; 18: 287-291Crossref PubMed Scopus (132) Google Scholar, 9Kim J. Ashworth L. Branscomb E. Stubbs L. Genome Res. 1997; 7: 532-540Crossref PubMed Scopus (99) Google Scholar, 10Relaix F. Weng X. Marazzi G. Yang E. Copeland N. Jenkins N. Spence S.E. Sassoon D. Dev. Biol. 1996; 177: 383-398Crossref PubMed Scopus (116) Google Scholar). Signals were only observed in sections hybridized with the antisense probe (data not shown). Microautoradiographies show that marked induction of Peg3/Pw1 mRNA signals was observed in the deep peri-ischemic cortex layer (Fig. 2E) and mainly in medium to large-sized cells (about 30–40 μm in diameter) with large nuclei (Fig. 2F), suggesting that this Peg3/Pw1 mRNA expression was enhanced morphologically in typical neuronal cells. These findings indicate that Peg3/Pw1 mRNA is abundant in adult rat brain and that its expression is enhanced in neuronal cells located in peri-ischemic lesions including basal ganglia, thalamus, hypothalamus, and cortex in the rat permanent MCA-O model. Previous studies have shown that p53 protein is enhanced under hypoxic conditions in various types of cells including neuronal cells (21Banasiak K.J. Haddad G.G. Brain Res. 1998; 797: 295-304Crossref PubMed Scopus (138) Google Scholar) and that Bax gene contains the p53 response element in its promoter region (22Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar), and Bax functions in the cell death pathway through activating caspase-3-like activity downstream of p53 in neurons (23Cregan S.P. MacLaurin J.G. Craig C.G. Robertson G.S. Nicholson D.W. Park D.S. Slack R.S. J. Neurosci. 1999; 19: 7860-7869Crossref PubMed Google Scholar). We then examined whether p53 and Bax expressions were enhanced under hypoxic conditions in human neuroblastoma-derived SK-N-SH cells by Western blotting analysis. As shown in Fig. 3A, both p53 and Bax protein expressions were induced 4 h after exposure to hypoxia, and its enhanced expression was sustained until 24 h after exposure to hypoxia in SK-N-SH cells. We performed Western blotting analysis using anti-KDEL antibody to detect both GRP78 (glucose-regulated protein 78) and GRP94 (glucose-regulated protein 94) protein as positive controls, which are reported to be enhanced in various stresses including hypoxia (24Hori O. Matsumoto M. Kuwabara K. Maeda Y. Ueda H. Ohtsuki T. Kinoshita T. Ogawa S. Stern D.M. Kamada T. J. Neurochem. 1996; 66: 973-979Crossref PubMed Scopus (86) Google Scholar, 25Giaccia A.J. Auger E.A. Koong A. Terris D.J. Minchinton A.I. Hahn G.M. Brown J.M. Int. J. Radiat. Oncol. Biol. Phys. 1992; 23: 891-897Abstract Full Text PDF PubMed Scopus (31) Google Scholar), and found that they were actually induced in a time-dependent manner under hypoxia (Fig. 3A). We next addressed whether Peg3/Pw1 mRNA expression is induced in SK-N-SH cells under hypoxic conditions and found that Peg3/Pw1 mRNA expression was enhanced in a time-dependent manner by RT-PCR analysis (Fig.3B). In contrast to GRP78 mRNA expression that is enhanced the most at 12 h and reduced at 24 h post-hypoxia, Peg3/Pw1 mRNA expression was enhanced at high levels at 24 h post-hypoxia, when p53 protein expression was also up-regulated at high levels (Fig. 3A). Recently it was reported that Peg3/Pw1 is induced during p53/c-Myc-mediated and p53/EF-1-mediated cell death pathways and plays an important role in making decisions between
Referência(s)