ORP150 protects against hypoxia/ischemia-induced neuronal death.

:Oxygen-regulated protein 150 kD (ORP150) is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. Although ORP150 was sparingly upregulated in neurons from human brain undergoing ischemic stress, there was robust induction in astrocytes. Cultured neurons overexpressing ORP150 were resistant to hypoxemic stress, whereas astrocytes with inhibited ORP150 expression were more vulnerable. Mice with targeted neuronal overexpression of ORP150 had smaller strokes compared with controls. Neurons with increased ORP150 demonstrated suppressed caspase-3-like activity and enhanced brain-derived neurotrophic factor (BDNF) under hypoxia signaling. These data indicate that ORP150 is an integral participant in ischemic cytoprotective pathways.

Tamatani M ，Matsuyama T ，Yamaguchi A ，Mitsuda N ，Tsukamoto Y ，Taniguchi M ，Che YH ，Ozawa K ，Hori O ，Nishimura H ，Yamashita A ，Okabe M ，Yanagi H ，Stern DM ，Ogawa S ，Tohyama M ... -  《NATURE MEDICINE》

Expression of 150-kd oxygen-regulated protein in the hippocampus suppresses delayed neuronal cell death.

ORP150-150-kd oxygen-regulated protein-is a novel stress protein localized in the endoplasmic reticulum (ER). To investigate the role of ORP150 in delayed neuronal cell death, the authors examined its expression in the gerbil brain after an ischemic insult. The expression of ORP150 antigen, as well as its transcripts, was observed in the CA1 region after the occlusion of the common carotid artery, and the preconditioning enhanced this expression. In cultured neurons, exposure either to hypoxia or to glutamate induced the expression of ORP150, and this effect was also observed by treating the culture with breferdin A or thapsigargin, indicating that both glutamate and hypoxia can cause stress in the ER (ER stress). Neurons became more vulnerable to these stresses following treatment with cycloheximide or after infection with an adenovirus carrying the ORP150-antisense structure. In contrast, the overexpression of ORP150 by an adenovirus suppressed neuronal cell death, and this was accompanied by the suppression of Ca2+ elevation and proteolytic activity induced by glutamate. Further, overexpression of ORP150 in CA1 neurons by an adenovirus carrying the ORP150-sense structure suppressed delayed neuronal cell death after ischemia. These data suggest a possible function of ORP150 as an intracellular apparatus that participates in a protective response in ischemic tolerance.

Miyazaki M ，Ozawa K ，Hori O ，Kitao Y ，Matsushita K ，Ogawa S ，Matsuyama T ... -  《JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM》

150-kDa oxygen-regulated protein attenuates myocardial ischemia-reperfusion injury in rat heart.

Early contractile dysfunction and the later death of cardiomyocytes are two major problems that can follow myocardial infarction or major cardiovascular surgery that demands ischemic arrest of the heart. Here, we found that 24 h of hypoxia and 1 h of reoxygenation induced the expression of the chaperone ORP150 in cultured rat cardiomyocytes. Inhibition of its induction using an adenovirus to express anti-sense ORP150 significantly enhanced the hypoxia-reoxygenation-induced cardiomyocyte death; cell death was reduced by overexpressing ORP150. Decreased levels of ORP150 expression also enhanced caspase-3 and -8 activation, cytochrome-c release, and DNA fragmentation, suggesting that this chaperone regulates apoptotic cell death. In contrast, increasing the expression of ORP150 in the cardiomyocytes had the opposite effect on the expression of these molecules. Moreover, apoptotic cell death initiated by myocardial ischemia-reperfusion (I/R) was significantly inhibited in vivo by transfecting an ORP150 expression plasmid into whole rat heart using the hemagglutinating virus of Japan (HVJ)-liposome method. Interestingly, ORP150 seemed to preserve calcium homeostasis in cardiomyocytes that underwent ischemia-reoxygenation in vitro. Calpain activity in the cardiomyocytes was enhanced by anti-sense ORP150 and suppressed by sense ORP150. Finally, we examined the functional recovery of rat hearts that overexpressed ORP150 or GFP protein and were subjected to I/R; we found that ORP150 preserved early contractile function after transient ischemia. Our results indicated cytoprotective roles for ORP150 in rat heart and suggested a therapeutic role for the protein both in preventing cardiomyocyte death and in preserving contractile function after ischemic damage.

Aleshin AN ，Sawa Y ，Kitagawa-Sakakida S ，Bando Y ，Ono M ，Memon IA ，Tohyama M ，Ogawa S ，Matsuda H ... -  《JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY》

ORP150 ameliorates ischemia/reperfusion injury from middle cerebral artery occlusion in mouse brain.

The 150-kDa oxygen-regulated protein (ORP150), a novel stress protein localized to the endoplasmic reticulum (ER), is induced by hypoxia/ischemia. To determine the role of ORP150 in cerebral infarction following ischemia/reperfusion, ORP150 transgenic (TG) and knockout (KO) mice were subjected to 1 or 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion for 24 h. At 24 h after 1 h of occlusion, significantly less infarct volume was evident in cerebral cortex, but not in striatum, in ORP150TG than ORP150KO mice (P<0.001). Infarct volume did not differ significantly between these groups at 24 h after 3 h of occlusion. Immunohistochemical reactivity for microtubule-associated protein (MAP)2 in the MCA territory was lost in ORP150KO mice at 24 h after 1 h of occlusion. In contrast, MAP2 staining still was present in the affected cortex of ORP150TG mice, where markedly enhanced ORP150 immunoreactivity was demonstrated. MAP2 staining had disappeared from the affected area at 24 h after 3 h of occlusion in both groups, but astrocytic ORP150 reactivity was preserved in the ORP150TG group. At 6 h after 1-h occlusion, when MAP2 staining was evident in the affected cortex, some cortical neurons of the TG mice were reactive for Bcl-xS/L. Thus, ORP150 may be cytoprotective against ischemia/reperfusion injury via reduction of ER stress and probably also inhibition of apoptosis.

Kitano H ，Nishimura H ，Tachibana H ，Yoshikawa H ，Matsuyama T ... -  《BRAIN RESEARCH》

Regulation of tumor angiogenesis by oxygen-regulated protein 150, an inducible endoplasmic reticulum chaperone.

Expression of angiogenic factors such as vascular endothelial growth factor (VEGF) under conditions of cell stress involves both transcriptional and translational events, as well as an important role for inducible endoplasmic reticulum (ER) chaperones. Coexpression of VEGF and 150-kDa oxygen-regulated protein (ORP), a novel ER chaperone, in human glioblastoma suggested a link between angiogenesis and ORP150. C6 glioma cells stably transfected with ORP150 antisense displayed selectively reduced ORP150 expression. Tumors raised after inoculation of immunocompromised mice with ORP150 antisense C6 glioma transfectants demonstrated an initial phase of growth comparable to wild-type C6 glioma cells which was followed by marked regression within 8 days. Decreased density of platelet/endothelial cell adhesion molecule 1-positive structures within the tumor bed was consistent with reduced angiogenesis in C6 gliomas expressing ORP150 antisense, compared with tumors derived from C6 cells overexpressing ORP150 sense or vector controls. In vitro, inhibition of ORP150 expression decreased release of VEGF into culture supernatants; in ORP150 antisense transfectants, VEGF accumulated intracellularly within the ER. These findings demonstrate a critical role for the inducible ER chaperone ORP150 in tumor-mediated angiogenesis via processing of VEGF, and, thus, highlight a new facet of angiogenic mechanisms amenable to therapeutic manipulation in tumors.

Ozawa K ，Tsukamoto Y ，Hori O ，Kitao Y ，Yanagi H ，Stern DM ，Ogawa S ... -  《CANCER RESEARCH》