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

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 beta-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.     

人体血红素加氧酶-1的研究进展

血红素加氧酶（heme oxygenase,HO)是哺乳动物中血红素代谢的限速酶,HO－1是HO同功酶之一,主要分布在肝、脾、肺等多种脏器,具有调节和保护功能。作者拟从人体HO－1蛋白的晶体结构、HO－1的功能和HO－1表达的诱导因素,以及HO－1基因的表达与调控等研究进展做一综述。     

Heme-Oxygenases during Erythropoiesis in K562 and Human Bone Marrow Cells

In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity.     

Time course of increased heme oxygenase activity and expression after experimental intracerebral hemorrhage: correlation with oxidative injury

Heme oxygenase (HO) activity in tissue adjacent to an intracerebral hematoma may modulate cellular vulnerability to heme-mediated oxidative injury. Although HO-1 is induced after experimental intracerebral hemorrhage (ICH), the time course of this induction, its effect on tissue HO activity, and its association with oxidative injury markers has not been defined. We therefore quantified HO activity, HO-1 expression, tissue heme content, and protein carbonylation for 8 days after injection of autologous blood into the mouse striatum. Increased striatal HO-1 protein was observed within 24 h, peaked on day 5 at a level that was 10-fold greater than baseline, and returned to baseline by day 8; HO-2 expression was not altered. HO activity increased by only 1.6-fold at its peak on day 5, and had also returned to baseline by day 8. A significant increase in protein carbonylation was observed at 3–5 days, which also was markedly attenuated by 8 days, concomitant with a return of tissue heme to near-normal levels. These results suggest that the increase in HO activity in tissue surrounding an experimental ICH is considerably less than would be predicted based on an analysis of HO-1 expression per se . As HO-1 expression is temporally associated with increased tissue heme and increased protein carbonylation, it may be more useful as a marker of heme-mediated oxidative stress in ICH models, rather than as an index of HO activity.     

Effect of heme oxygenase-1 on the vulnerability of astrocytes and neurons to hemoglobin

The heme oxygenase (HO) enzymes catalyze the rate-limiting step of heme breakdown. Prior studies have demonstrated that the vulnerability of neurons and astrocytes to hemoglobin is modified in cells lacking HO-2, the constitutive isoform. The present study assessed the effect of the inducible isoform, HO-1. Wild-type astrocytes treated for 3-5 days with 3-30 microM hemoglobin sustained no loss of viability, as quantified by LDH and MTT assays. The same treatment resulted in death of 25-50% of HO-1 knockout astrocytes, and a 4-fold increase in protein oxidation. Cell injury was attenuated by transfer of the HO-1 gene, but not by bilirubin, the antioxidant heme breakdown product. Conversely, neuronal protein oxidation and cell death after hemoglobin exposure were similar in wild-type and HO-1 knockout cultures. These results suggest that HO-1 induction protects astrocytes from the oxidative toxicity of Hb, but has no effect on neuronal injury.     

Regulation of endothelial heme oxygenase activity during hypoxia is dependent on chelatable iron

The regulation of heme oxygenase (HO) activity and its dependence on iron was studied in bovine aortic endothelial cells (BAEC) subjected to hypoxia-reoxygenation (H/R). HO activity was induced by hypoxia (10 h) and continued to increase during the reoxygenation phase. HO-1 protein levels were strongly induced by hypoxia from undetectable levels and remained elevated at least 8 h postreoxygenation. Addition of the Fe(3+) chelator desferrioxamine mesylate (DFO) or the Fe(2+) chelator o-phenanthroline during hypoxia alone or during the entire H/R period inhibited the induction of HO activity and HO-1 protein levels. However, DFO had no effect and o-phenanthroline had a partial inhibitory effect on HO activity and protein levels when added only during reoxygenation. Loading of BAEC with Fe(3+) enhanced the activation of the HO-1 gene by H/R, whereas loading with L-aminolevulinic acid, which stimulates heme synthesis, had little effect. These results suggest that chelatable iron participates in regulating HO expression during hypoxia.     

Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilization by cadmium

Nrf2 mediates inducer-dependent activation of the heme oxygenase-1 (HO-1) gene (Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A. M., and Cook, J. L. (1999) J. Biol. Chem. 274, 26071-26078), but the mechanism by which HO-1 inducers regulate Nrf2 function is not known. Treatment of mouse hepatoma (Hepa) cells with 50 microm CdCl(2) increased the amount of Nrf2 protein in a time-dependent manner; induction was observed within 30 min, prior to the accumulation of HO-1 mRNA. Cadmium did not significantly affect the steady-state level of Nrf2 mRNA or the initial rate of Nrf2 protein synthesis but increased the half-life of Nrf2 from approximately 13 to 100 min. Proteasome inhibitors, but not other protease inhibitors, enhanced the expression of Nrf2, and ubiquitinylated Nrf2 was detected after proteasome inhibition. Cycloheximide inhibited cadmium-stimulated Nrf2 expression and DNA binding activity and attenuated HO-1 mRNA accumulation. Conversely, proteasome inhibitors enhanced HO-1 mRNA and protein accumulation by a Nrf2-dependent mechanism. Together, these results indicate that Nrf2 is targeted for rapid degradation by the ubiquitin-proteasome pathway and that cadmium delays the rate of Nrf2 degradation leading to ho-1 gene activation.     

Effects of hemoglobin on heme oxygenase gene expression and viability of cultured smooth muscle cells

Ferrous Hb contributes to cerebral vasospasm after subarachnoid hemorrhage, although the mechanisms involved are uncertain. The hypothesis that cytotoxic effects of ferrous Hb on smooth muscle cells contribute to vasospasm was assessed. Cultured rat basilar artery smooth muscle cells were exposed to pure Hb, dog erythrocyte hemolysate, or Hb breakdown products; and heme oxygenase (HO-1 and HO-2) and ferritin mRNA and protein were measured. Cytotoxicity was assessed by lactate dehydrogenase release and fluorescence assays. Pure Hb or hemolysate caused dose- and time-dependent increases in HO-1 mRNA and protein. Hemin was the component of Hb that increased HO-1 mRNA. Cycloheximide inhibited the increase in HO-1 mRNA in response to hemin. Ferritin protein heavy chain but not mRNA increased upon exposure of cells to Hb. Hemin and ferric but not ferrous Hb were toxic to smooth muscle cells. Toxicity was increased by exposure to Hb plus tin protoporphyrin IX. In conclusion, exposure of smooth muscle cells to Hb induces HO-1 mRNA and protein through pathways that involve new protein synthesis. Hemin is the component of Hb that induces HO-1. Hemin and ferric but not ferrous Hb are toxic.