Mahin D. Maines

Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice.

Abstract : Heme oxygenase‐1 (HO‐1, HSP32) is an early gene that is responsive to an array of pathological conditions including, but not limited to, hypoxia and cerebral ischemia. HO‐1 cleaves the heme molecule and produces carbon monoxide (CO) and biliverdin (an antioxidant) and is essential for iron homeostasis. The purpose of this study was to investigate, using transgenic (Tg) mice, whether overexpression of HO‐1 in the brain augments or attenuates cellular injury caused by ischemic stroke. Homozygous HO‐1 Tg mice that overexpress HO‐1 under the control of the neuron‐specific enolase promoter (characterized previously) were used. Under halothane anesthesia and normothermic conditions, wildtype nontransgenic (nTg ; n = 22) and HO‐1 Tg (n = 24) mice were subjected to middle cerebral artery occlusion (MCAo). Six hours after induction of ischemia, Tg and nTg mice developed infarcts that were 39 ± 6 and 63 ± 9 mm3, respectively (p < 0.01). No significant difference between the two strains was observed in the values of brain edema (11.3 ± 4% in Tg vs. 14.6 ± 5% in nTg ; p < 0.1). At 24 h after MCAo, Tg mice exhibited significant neuroprotection as determined by the stroke volumes (41 ± 2 mm3 in Tg vs. 74 ± 5 mm3 in nTg ; p < 0.01) and values of ischemic cerebral edema (21 ± 6% in Tg vs. 35 ± 11% in nTg ; p < 0.01). Data suggest that neuroprotection in Tg mice was, at least in part, related to the following findings : (a) constitutively up‐regulated cyclic GMP and bcl‐2 levels in neurons ; (b) inhibition of nuclear localization of p53 protein ; and (c) antioxidant action of HO‐1, as detected by postischemic neuronal expression of ferritin, and decreases in iron staining and tissue lipid peroxidation. We suggest that pharmacological stimulation of HO‐1 activity may constitute a novel therapeutic approach in the amelioration of ischemic injury during the acute period of stroke.

Neurons overexpressing heme oxygenase-1 resist oxidative stress-mediated cell death.

Abstract : This is the first report on the protective effect of heme oxygenase‐1 (HO‐1) overexpression against oxidative stress‐mediated neuronal cell death and demonstration of a decreased production of oxygen free radicals when HO‐1 levels are increased. HO‐1 is the heat shock/stress cognate of the heat shock protein 32 family of proteins. A known function of these proteins is α‐meso bridge‐specific cleavage of the heme molecule. For the present study, we used cerebellar granular neurons (CGNs) isolated from homozygous transgenic (Tg) mice that overexpress HO‐1 under neuron‐specific enolase control and nontransgenic (Ntg) littermates. The Tg mouse CGNs were characterized by increased levels of HO‐1 mRNA and protein, a lower resting intracellular calcium concentration, and a reduced HO‐1 transcriptional response to glutamate‐mediated oxidative stress. Compared with the Ntg neurons, when exposed to glutamate (30 μM or 3 mM), the magnitude of cell viability was increased and the number of cells exhibiting membrane permeability and chromatin condensation were significantly decreased in the Tg CGN cultures. The population of neurons surviving glutamate toxicity decreased when HO‐1 activity was inhibited by a peptide inhibitor. The neuroprotective effect by HO‐1 was extended to H2O2‐induced cell death. The mechanism of protection may involve in part a reduced production of reactive oxygen species upon exposure to glutamate. We suggest that induction of HO‐1 by pharmacological means may be a novel approach to amelioration of oxidative insults to neurons.

In situ hybridization and immunohistochemical localization of heme oxygenase-2 mRNA and protein in normal rat brain: Differential distribution of isozyme 1 and 2.

Heme oxygenase isozymes, HO-1 (HSP32) and HO-2, stereospecifically bind and degrade the potent prooxidant, the heme molecule, and convert it to the effective antioxidant, biliverdin, and the potential cellular messenger, carbon monoxide. In the present study we have examined the pattern of expression of the two HO-2 transcripts and protein in normal rat brain by in situ hybridization and immunochemical analysis, respectively. We have found by Northern blot analysis that HO-2 isozyme is by far the most prevalent form in the brain. Analysis of HO-2 1.3- and 1.9-kb mRNAs by in situ hybridization histochemistry showed that these transcripts are abundantly expressed in many neuronal and nonneuronal cell populations in forebrain, diencephalon, cerebellum, and brain stem regions. Furthermore, the pattern of expression of HO-2 transcripts, as detected by oligonucleotide probes, is in good agreement with that of immunoreactive protein detected by immunohistochemical analysis. Impressive levels of HO-2 transcripts and immunoreactive protein were observed in Purkinje cells of cerebellum, red nucleus, superior and inferior colliculus, nucleus of the trapezoid body, cochlear neurons, and facial nucleus of brain stem. Furthermore, in certain select brain cell populations the pattern of expression of HO-1- and HO-2-immunoreactive proteins overlapped. We suggest that the high levels of heme degradation activity and the localization of HO-2 transcripts and protein in the brain may reflect the functions of this enzyme in processes such as production of cellular messenger, regulation of the activity of heme-dependent enzymes catalyzing intracellular signaling molecule synthesis, and production of antioxidants.

Glutathione depletion induces heme oxygenase-1 (HSP32) mRNA and protein in rat brain

Abstract: In mammalian systems, the heme oxygenase (HO) isozymes HO‐1 (HSP32) and HO‐2 oxidatively cleave the heme molecule to produce bile pigments and carbon monoxide. Although HO‐1 is inducible by various chemicals in systemic organs and cell culture systems, this communication reports for the first time the induction of this stress protein and its transcript by a chemical in the brain. In addition, this study demonstrates expression of HO‐1 in select populations of cells in the brain in response to GSH depletion. Specifically, treatment of adult rats with diethyl maleate (DEM; 4.7 mmol/kg) caused a pronounced decrease in brain GSH content within 1 h. GSH levels remained significantly depressed for at least 24 h postinjection. Northern blot analysis of brain poly(A)+ mRNA following DEM treatment revealed on the average a sixfold increase in the 1.8‐kb HO‐1 mRNA level compared with that of controls; concomitant with this change was a decrease in GSH levels. Total brain HO activity was not significantly altered along with the increase in HO‐1 mRNA level. The increase in transcription of HO‐1 was a direct response to GSH depletion, as judged by the observation that treatment of neonatal rats with L‐buthionine‐(S,R)‐sulfoximine (BSO) (3 mmol/kg, twice daily, for 2 days), a selective inhibitor of GSH synthesis, caused a marked depression in total brain GSH level and a concomitant increase in brain 1.8‐kb HO‐1 mRNA content. The magnitude of the increase was up to ∼ 11.5‐fold that of the control level, as evidenced by northern blot analysis. In contrast, the level of two homologous HO‐2 transcripts (1.3 and 1.9 kb) did not increase in response to either DEM or BSO treatment. Analysis of brain HO‐1‐immunoreactive protein following DEM treatment for 9 h indicated induction of HO‐1 protein in only select nonneuronal cell populations. In particular, the ependymal cells lining ventricles throughout brain, Bergmann glia of cerebellum, and leptomeninges lining brain and glia throughout brain responded to treatment by increasing the level of HO‐1‐like protein. We suggest that when GSH is depleted, an increase in HO‐1 protein content, resulting in increased capacity to form bile pigments, may be of significance to cells with compromised antioxidant capability. Bile pigments are potent antioxidants in biological systems.

Zinc · protoporphyrin is a selective inhibitor of heme oxygenase activity in the neonatal rat

The present study was undertaken to examine the liver, spleen and kidney heme oxygenase activity in the rat, and also to investigate the response of the enzyme to a variety of metalloporphyrin complexes. The enzyme activity in the liver and the kidney of 3--4 day-old rats was several-fold greater than the corresponding values in the adult animals; however, the splenic enzyme activity was markedly depressed in comparison to that of adult rats. During the first 2--3 weeks post-parturation period, the activity of heme oxygenase in the spleen progressively increased, and in 4 weeks approached the adult values. The treatment of the newborn animals with the metalloporphyrin complex. Zn . protoporphyrin-IX, inhibited heme oxygenase activity in the spleen, liver and the kidney. Sn . protoporphyrin treatment also inhibited the activity of the enzyme in the liver and the spleen. The mechanism of the inhibition appeared to be competitive in nature. In contrast, the treatment of the newborn animals with Co . protoporphyrin increased the activity of the enzyme in the tested organs. The treatment of newborn animals with Fe . protoporphyrin (heme) also increased heme oxygenase activity in the spleen and the kidney. In addition, Co . and Fe . protoporphyrin complexes inhibited the activity of delta-aminolevulinate synthetase in the spleen; Sn . protoporphyrin and Zn . protoporphyrin, however, did not alter the activity of this enzyme. The effects of Co . protoporphyrin and Zn. protoporphyrin on the microsomal contents of cytochromes P-450, b5, the total heme, and the microsomal drug metabolism activity in the liver were compared. Zn . protoporphyrin was ineffective in altering the indicated cellular variables. According to these findings Zn . protoporphyrin may be useful as an experimental tool for the selective suppression of heme degradation activity.

Corticosterone Regulates Heme Oxygenase-2 and NO Synthase Transcription and Protein Expression In Rat Brain

Abstract: Heme oxygenase (HO)‐1 and ‐2 produce carbon monoxide, which is suspected, as is nitric oxide (NO), to function as a neuronal messenger. We report on glucocorticoid‐mediated modulation of HO‐2 and NO synthase expression in brain and the differential response of the two proteins to corticosterone in different brain regions. Corticosterone treatment (40 mg/kg, 20 days) had opposing effects on HO‐2 and NO synthase transcript levels: increasing the 1.3‐ and 1.9‐kb HO‐2 mRNAs and decreasing that of the brain‐specific 10.5‐kb NO synthase. Corticosterone did not uniformly affect HO‐2 protein expression in all regions, but appeared to cause a universal reduction in NO synthase, e.g., HO‐2 was decreased in hippocampus (CA1 and dentate gyrus), but not in cerebellum. In contrast, NADPH diaphorase staining was reduced in hippocampus and in molecular and granule layers of cerebellum (not detected in Purkinje cells). Striking deficits in neuronal morphology and number of diaphorase‐staining neurons were observed in the lateral tegmental area, paraventricular nucleus, and frontal cortex; HO‐2 expression was only selectively affected. In cerebellum, activity of NO synthase, but not that of HO, was reduced. Consistent with the possibility that carbon monoxide can generate cyclic GMP, the change in cyclic GMP level did not mirror the decrease in NO synthase. We suggest that glucocorticoid‐mediated deficits in hippocampal functions may reflect their negative effect on messenger‐generating systems.

Expression of heme oxygenase-1 (HSP32) in human prostate: normal, hyperplastic, and tumor tissue distribution

OBJECTIVES Heme oxygenase isozymes, HO-1 and HO-2, are members of the stress/heat shock (HSP) family of proteins, with the known function of cleaving the heme molecule to biliverdin, iron, and carbon monoxide. The aim of this study was to examine the pattern of tissue expression of HO-1 in the human prostate under different states of proliferation and differentiation and to investigate whether the pattern differs between these states. METHODS Presently, we have determined the pattern of tissue expression of the stress-inducible isozyme, HO-1 (HSP32), in human prostate under normal and pathologic conditions, by immunohistochemistry, using polyclonal antibodies, and have measured HO-1 and HO-2 mRNA levels in normal prostate and benign prostatic hyperplasia (BPH) by Northern blotting. The activity of prostate to catalyze heme degradation was also assessed. RESULTS In normal and BPH tissue, columnar epithelial cells of acini and ducts and cells in stroma displayed HO-1 immunoreactivity; in all cells, perinuclear staining was prominent. In BPH tissue, however, a more intense staining of the epithelial cells occurred, with notable staining of the basal cells. In undifferentiated malignant tumors, intense HO-1 staining was manifest in nearly all tumor cells, and also in the epithelial lining of blood vessels. HO-1 in the prostate tissue was found catalytically active and oxidatively cleaved the heme molecule (Fe-protoporphyrin IX) to biliverdin. Northern blot analysis shows that two forms of HO are present in the human prostate. Compared with normal tissue, predominantly hyperplastic tissue demonstrates a pronounced increase in the approximately 1.8 kb mRNA that hybridizes to the rat HO-1 probe. The levels of two transcripts, approximately 1.3 and approximately 1.7 kb, that hybridize to the rat HO-2 probe are not increased in BPH tissue. CONCLUSIONS The finding that HO-1 expression is increased in BPH and malignant prostate tissue is consistent with a role for this stress protein in the pathogenesis of BPH and prostate cancer; in the context of iron metabolism, an argument is made in support of this possibility.

Brain heme oxygenase isoenzymes and nitric oxide synthase are co-localized in select neurons.

Two isoforms of the enzyme heme oxygenase are expressed in distinct populations of neurons in the brain. These enzymes catalyse the oxidative cleavage of heme to the cellular antioxidant biliverdin resulting in the release of carbon monoxide in the process. Both heme and carbon monoxide may play important roles in regulating the nitric oxide-cyclic guanosine monophosphate signal transduction system. Thus we have examined the distributions of both isoforms of heme oxygenase in the rat brain, and compared their localizations with that of nitric oxide synthase determined with the NADPH-diaphorase histochemical technique. Heme oxygenase-1 is highly expressed in a few select populations of neurons including cells in the hilus of the dentate gyrus, in the hypothalamus, cerebellum and brainstem. This enzyme appears to be coexpressed with nitric oxide synthase only in a few cells in the dentate gyrus. Heme oxygenase-2 is much more widely expressed. It is present in mitral cells in the olfactory bulb, pyramidal cells in the cortex and hippocampus, granule cells in the dentate gyrus, many neurons in the thalamus, hypothalamus, cerebellum and caudal brainstem. However, only some of these labelled neurons also displayed nitric oxide synthase. Instead, many neurons expressing heme oxygenase-2 correspond to those known to express high levels of the hemoprotein soluble guanylyl cyclase. These results suggest that heme oxygenase may play a role in modulating guanylyl cyclase independent of nitric oxide synthase. This may result from regulation of intracellular heme and carbon monoxide levels by the heme oxygenase system.

New Developments in the Regulation of Heme Metabolism and Their Implications

Various endogenous and exogenous chemicals, such as hormones, drugs, and carcinogens and other environmental pollutants are enzymatically converted to polar metabolites as a result of their oxidative metabolism by the mixed-function oxidase system. This enzyme complex constitutes the major detoxifying system of man and utilizes the hemoprotein--cytochrome P-450--as the terminal oxidase. Recent studies with trace metals have revealed the potent ability of these elements to alter the synthesis and to enhance the degradation of heme moiety of cytochrome P-450. An important consequence of these metal actions is to greatly impair the ability of cells to oxidatively metabolize chemicals because of the heme dependence of this metabolic process. In this report the effects of exposure to trace metals on drug oxidations is reviewed within the framework of metal alterations of heme metabolism, including both its synthesis and degradation, since these newly discovered properties of metals have made it possible to define a major dimension of metal toxicity in terms of a unified cellular mechanism of action.

REGULATION OF HEME OXYGENASE-2 BY GLUCOCORTICOIDS IN NEONATAL RAT BRAIN : CHARACTERIZATION OF A FUNCTIONAL GLUCOCORTICOID RESPONSE ELEMENT

Heme oxygenase-2 (HO-2) is constitutively expressed in mammalian tissues; together with HO-1 (HSP32) it catalyzes the cleavage of heme to produce biliverdin IX alpha, CO and Fe. Detection of a consensus sequence of the glucocorticoid response element (GRE) in the promoter region of the HO-2 gene prompted the present study which has investigated the role of glucocorticoids (Gcs) in the regulation of HO-2 protein and transcript development in the newborn rat brain and has examined the promoter activity of the GRE in HeLa cells. Using in situ hybridization histochemistry, we noted a pronounced increase in signal for HO-2 mRNA in the brain of 14-day-old rats postnatally treated with corticosterone (5 microg/g, 4 x, starting 24-36 h after birth). And, using immunohistochemistry, a striking increase in neuronal HO-2 immunostaining in treated brains was detected. The HO-2 GRE was tested for responsiveness to dexamethasone (DX) using both a promoterless CAT expression vector, and a heterologous promoter containing luciferase expression vector in HeLa cells. The HO-2 promoter containing the GRE and transcription start site induced CAT reporter gene activity in response to DX, whereas mutation or deletion in the GRE abolished hormone responsiveness. Similarly, constructs containing the GRE conferred responsiveness to DX in an orientation-independent manner and increased relative luciferase activity. Further, specific binding of glucocorticoid receptor protein to the GRE was observed; binding could be competed out only by excess cold GRE and not by mutated HO-2 GRE, or AP1. HO-2 mRNAs (approximately 1.3 and approximately 1.9 kb) increased in HeLa cells treated with DX (5 microM), the level reached a maximum at 24 h. DX did not effect HO-1 mRNA level. The increase in the HO-2 transcript was accompanied by an increase in HO-2 protein, as assessed by Western blot analysis, and an increase in HO activity, as measured by bilirubin formation. Also, an increase in intensity of immunostaining was noted in DX-treated HeLa cells. We conclude that the GRE present in the HO-2 gene promoter region is functional, and propose the direct involvement of the adrenal glucocorticoids in modulation of HO-2 gene expression. In the context of biological functions of heme degradation products, we suggest that this regulation may be of significance, particularly to the neurons.