Biomedical subjects

J Alam

Publications and source records attributed to J Alam.

At least 55 records · Page 3Linked to original sources

PubMed1997

Regulation of heme oxygenase-1 gene expression in vascular smooth muscle cells by nitric oxide.

Heme oxygenase (HO)-mediated heme degradation is the primary mechanism for production of cellular carbon monoxide (CO). Analogous to nitric oxide (NO), CO mediates physiological and cellular functions such as vasodilation, stimulation of guanylate cyclase, and neuronal transmission. In view of accumulating data demonstrating a correlation between the activity of these two gaseous molecules and that the predominant source of CO is via HO catalysis, we hypothesized that NO regulates HO expression. We demonstrate that the NO donor spermine NONOate (SNN) increases steady-state levels of HO-1 mRNA in aortic vascular smooth muscle cells (aSMC) in both a time- and dose-dependent manner. The accumulation of HO-1 mRNA that correlated with increased HO-1 protein synthesis resulted from both an increased rate of gene transcription and a decreased rate of mRNA turnover. Inhibition of the NO-induced HO-1 mRNA expression by cycloheximide suggests that new protein synthesis is required for increased HO-1 gene expression. Induction of HO-1 expression by SNN occurs in a guanosine 3',5'-cyclic monophosphate (cGMP)-independent manner because exposure of cells to 8-bromoguanosine 3',5'-cyclic monophosphate, a cGMP analog, did not increase HO-1 mRNA levels, and pretreatment of cells with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective guanylate cyclase inhibitor, did not prevent SNN-induced HO-1 mRNA accumulation. The antioxidant N-acetyl-L-cysteine markedly inhibited SNN-induced HO-1 mRNA expression, whereas peroxynitrite did not induce HO-1 expression in aSMC. Interestingly, CO did not attenuate NO-induced HO-1 expression through an autocrine negative feedback mechanism as had been observed for hypoxia-induced HO-1 expression. These data provide evidence for an important regulatory network between NO and CO via HO-1.

Animals↗

PubMed1996

Glomerular inflammation induces resistance to tubular injury in the rat. A novel form of acquired, heme oxygenase-dependent resistance to renal injury.

Considerable attention is directed to a surprising biologic phenomenon wherein tissues exposed to one insult acquire resistance to another. We identify a novel example of acquired resistance to acute renal failure and a mechanism that contributes to such resistance. Nephrotoxic serum, administered to rats 24 h before the induction of glycerol-induced acute renal failure, reduces functional and structural injury that occurs in this model. Since heme oxygenase, the rate-limiting enzyme in heme degradation, protects against heme protein-induced renal injury, we questioned whether induction of heme oxygenase underlies the protection afforded by nephrotoxic serum. Kidney heme oxygenase (HO-1) mRNA was induced 6 h after nephrotoxic serum and renal tubules were identified as the site of expression of heme oxygenase protein. Induction of heme oxygenase was accompanied by increased renal content of ferritin but not by induction of other antioxidant enzymes. Inhibition of heme oxygenase prevented the protection afforded by nephrotoxic serum. Nephrotoxic serum did not protect against ischemic acute renal failure, a model in which heme oxygenase is not induced. Thus, nephrotoxic serum protects against glycerol-induced acute renal failure by inducing heme oxygenase in tubules. This study provides the first demonstration of resistance to tubular injury acquired from glomerular inflammation, uncovers a mechanism for such resistance, and exposes the dialogue that occurs between glomeruli and tubules.

Acute Kidney Injury↗

PubMed1996

Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia.

Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme to biliverdin, which is reduced by biliverdin reductase to bilirubin. Heme oxygenase-1 (HO-1) is inducible not only by its heme substrate, but also by a variety of agents causing oxidative stress. Although much is known about the regulation of HO-1 expression, the functional significance of HO-1 induction after oxidant insult is still poorly understood. We hypothesize and provide evidence that HO-1 induction serves to protect cells against oxidant stress. Human pulmonary epithelial cells (A549 cells) stably transfected with the rat HO-1 cDNA exhibit marked increases of HO-1 mRNA levels which were correlated with increased HO enzyme activity. Cells that overexpress HO-1 (A549-A4) exhibited a marked decrease in cell growth compared with wild-type A549 (A549-WT) cells or A549 cells transfected with control DNA (A549-neo). This slowing of cell growth was associated with an increased number of cells in G0/G1 phase during the exponential growth phase and decreased entry into the S phase, as determined by flow cytometric analysis of propidium iodide-stained cells and pulse experiments with bromodeoxyuridine. Furthermore, the A549-A4 cells accumulated at the G2/M phase and failed to progress through the cell cycle when stimulated with serum, whereas the A549-neo control cells exhibited normal cell cycle progression. Interestingly, the A549-A4 cells also exhibited marked resistance to hyperoxic oxidant insult. Tin protoporphyrin, a selective inhibitor of HO, reversed the growth arrest and ablated the increased survival against hyperoxia observed in the A549-A4 cells overexpressing HO-1. Taken together, our data suggest that overexpression of HO-1 results in cell growth arrest, which may facilitate cellular protection against non-heme-mediated oxidant insult such as hyperoxia.

Animals↗

PubMed1996

The heme-responsive element of the mouse heme oxygenase-1 gene is an extended AP-1 binding site that resembles the recognition sequences for MAF and NF-E2 transcription factors.

Jun and Fos (AP-1) transcription factors were recently proposed to mediate induction of the mouse heme oxygenase-1 gene by different agents including heme and cadmium. In this report we show that the AP-1 binding sequence, TGAGTCA, is necessary but insufficient for gene activation in response to heme or cadmium. The minimal heme response element was identified as an extended AP-1 binding site, (T/C)GCTGAGTCA. In addition to the AP-1 heptad, this element also contains an interdigitated antioxidant response element, GCnnnGTCA. Specific antioxidant response elements from the NAD(P)H:quinone oxidoreductase-1 and the glutathione S-transferase Ya subunit genes were in fact responsive to heme but not all sequences that conform to the consensus antioxidant response element were activated by this agent. The heme response element resembles the consensus binding sites for the product of the maf oncogene and for the transcription factor NF-E2. The potential role of these and related transcription factors and the implication of the composite heme response element in heme oxygenase-1 gene regulation are discussed.

Animals↗

PubMed1996

Gas-generating systems in acute renal allograft rejection in the rat. Co-induction of heme oxygenase and nitric oxide synthase.

Gases are now viewed as biologic messengers, and in this regard, carbon monoxide and nitric oxide are incriminated in signaling processes in neural tissue. Carbon monoxide is generated by heme oxygenase (HO), an enzyme inducible by heme, cytokines, and oxidative stress and considered an antioxidant response; nitric oxide is generated by nitric oxide synthase, an enzyme also inducible by cytokines. Since mononuclear cells infiltrate the acutely rejecting kidney, and foster within the kidney oxidative stress and a cytokine-enriched milieu, we examined the expression of these enzymes in acute renal allograft rejection (AR) (Brown Norway kidney to a Lewis rat; n = 17) and in control isografts (Lewis kidney to a Lewis rat; n = 17). No immunosuppressives were used. We found marked induction of HO mRNA and protein in renal allografts at day 5 after transplantation. Prominent expression of HO protein, as detected by immunofluorescence, was observed in the mononuclear cells infiltrating the renal allograft. More than 80% of these cells were macrophages, as identified by positive staining with ED1 antibody. ED1+ cells were rare in isografts and did not stain for HO. We also found co-expression of mRNA and protein for the inducible isoform of nitric oxide synthase (iNOS) in AR at day 5 after transplantation. Induction of HO and iNOS may reflect the cellular effect of diverse cytokines elaborated in the rejecting kidney. HO may enable the macrophage to degrade heme-containing proteins released from erythrocytes and other damaged cells; alternatively, induction of HO may defend the macrophage against oxidant injury. Increased nitric oxide, as a result of iNOS activity, may antagonize the vasoconstrictive effects of a number of mediators (i.e., thromboxane and endothelin) present in acute rejection; conversely, nitric oxide may prove cytotoxic through a number of recognized effects. Our studies provide the first demonstration of the induction of HO in the rejecting renal allograft as well as the first demonstration in vivo for the induction of HO in macrophages at the site of an inflammatory response. Such expression, linked as it is to the expression of iNOS, indicates that the macrophage mimics the behavior of neural cells by generating these gaseous messengers; thus, neural cells are not alone in deploying these mediators. Through a number of effects, these products of HO and iNOS may influence the nature and severity of tissue injury in AR.

Animals↗

PubMed1996

Interferon beta induces interleukin-10 expression: relevance to multiple sclerosis.

Interferon-beta decreases the relapse rate, relapse severity, progression of neurological disability, and development of new brain lesions observed with brain magnetic resonance imaging in relapsing-remitting multiple sclerosis patients. The mechanism of action of this effect is presently unknown. This study was based on the hypothesis that immunoregulatory effects of interferon-beta may underlie its demonstrated clinical efficacy. The objective of the study was to determine the effect of interferon-beta-1a on the expression of interleukin-10, a cytokine that strongly inhibits cell-mediated immune responses. Interferon-beta-1a induced accumulation of interleukin-10 messenger RNA and protein secretion by cultured peripheral blood mononuclear cells. The observed in vitro effects were similar for healthy control subjects and multiple sclerosis patients. Intramuscular injections of interferon-beta-1a increased serum levels of interleukin-10 at 12 and 24 hours following the injection. Greater increases were induced with 12 x 10(6)-IU than 6 x 10(6)-IU injections. The effect of interferon-beta-1a was relatively specific for interleukin-10, as treatment with interferon-beta-1a did not result in accumulation of transforming growth factor-beta messenger RNA. Upregulation of interleukin-10 represents a possible mechanism of action of interferon-beta's therapeutic effect in relapsing-remitting multiple sclerosis, and has implications for therapy of other autoimmune diseases.

Antibodies, Monoclonal↗

PubMed1996

Regulation of heme oxygenase-1 expression in vivo and in vitro in hyperoxic lung injury.

Using hyperoxia as a model of oxidant-induced lung injury in the rat, we explored the regulation of heme oxygenase-1 (HO-1) expression in vivo and in vitro. We demonstrate marked increase of HO-1 messenger ribonucleic acid (mRNA) levels in rat lungs after hyperoxia. Increased HO-1 mRNA expression correlated with increased HO-1 protein and enzyme activity. Immunohistochemical studies of the rat lung after hyperoxia showed increased HO-1 expression in a variety of cell types, including the bronchoalveolar epithelium and interstitial and inflammatory cells. We then examined the regulation of HO-1 expression in vitro after hyperoxia and observed increased HO-1 gene expression in various cultured cells including epithelial cells, fibroblasts, macrophages, and smooth muscle cells. Increased HO-1 mRNA expression correlated with increased HO-1 protein in vitro, and resulted from increased gene transcription and not from increased mRNA stability. We show that transcriptional activation of the HO-1 gene by hyperoxia requires cooperation between the HO-1 promoter and an enhancer fragment located 4 kb upstream from its transcription site. Increased HO-1 gene transcription was associated with increased activator protein-1 (AP-1) binding activity and supershift of the AP-1 complex by antibodies to c-Fos and c-Jun after hyperoxia. Taken together, our data suggest that AP-1 activation may represent one mechanism mediating hyperoxia-induced HO-1 gene transcription.

Amino Acid Sequence↗

PubMed1996

Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury.

Accumulating evidence suggests that oxidative stress plays a central role in the pathogenesis of many pulmonary diseases including adult respiratory distress syndrome, emphysema, asthma, bronchopulmonary dysplasia, and interstitial pulmonary fibrosis. The morbidity and mortality of these diseases remain high even with optimal medical management. In our attempts to devise new therapies for these disorders, it is crucial to improve our understanding of the basic mechanism(s) of oxidant-induced lung injury. A major line of investigation seeks to characterize the cellular and molecular responses of the lung to oxidant insults. Much progress has been made in our understanding of the role of the "classic" antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione peroxidase) in mediating the lung's resistance against oxidant lung injury. However, it is becoming clear that other oxidant-induced gene products may also play vital roles in the lung's adaptive and/or protective response to oxidative stress. One such stress-response protein is heme oxygenase-1, HO-1. Since the identification of HO-1 in 1968, many of the studies involving this enzyme were understandably focused on the regulation and function of HO-1 in heme metabolism. This emphasis is self-evident as HO-1 catalyzes the first and rate-limiting step in heme degradation. Interestingly, however, evidence accumulated over the past 25 years demonstrates that HO-1 is induced not only by the substrate heme but also by a variety of non-heme inducers such as heavy metals, endotoxin, heat shock, inflammatory cytokines, and prostaglandins. The chemical diversity of HO-1 inducers led to the speculation that HO-1, besides its role in heme degradation, may also play a vital function in maintaining cellular homeostasis. Further support for this hypothesis was provided by Tyrrell and colleagues who showed in 1989 that HO-1 is also highly induced by a variety of agents causing oxidative stress. Subsequently, many investigators have focused their attention on the function and regulation of HO-1 in various in vitro and in vivo models of oxidant-mediated cellular and tissue injury. The magnitude of HO-1 induction after oxidative stress and the wide distribution of this enzyme in systemic tissues coupled with the intriguing biological activities of the catalytic byproducts, carbon monoxide, iron, and bilirubin, makes HO-1 a highly attractive and interesting candidate stress-response protein which may play key role(s) in mediating protection against oxidant-mediated lung injury. This review will focus on the current understanding of the physiological significance of HO-1 induction and the molecular regulation of HO-1 gene expression in response to oxidative stress. We hope that this discussion will stimulate interest and investigations into a field which is still largely uncharted in the pulmonary research community.

Animals↗

PubMed1995

Distance constraints and stereospecific alignment requirements characteristic of p53 DNA-binding consensus sequence homologies.

We present evidence in favor of the position that some mutant p53 proteins retain the ability to trans-activate downstream genes through p53 DNA-binding consensus sequence (CS) homologies. We tested one cell line possessing high levels of mutant p53 and found that this mutant p53 is highly active in trans-activating one CS homology, moderately active in trans-activating a second sequence and inactive in modulating a third sequence. We tested a second cell line, also possessing high levels of mutant p53 and found the same pattern of activation. In addition we find that inter-motif distance [represented by N in RRRCWWGYYY(N)RRRCWWGYYY] is very important in determining the relative binding affinity of a given CS homology for wild-type or mutant p53. Our studies suggest that stereospecific alignment of the DNA-binding motifs within the CS may favor binding of wild-type p53 while misalignment may favor binding of mutant p53. Furthermore, we find that the maximum distances at which p53 DNA-binding CS homologies are functionally active vary for different sequences. Introduction of as few as 200 bp between one CS homology and the downstream TATA box can eliminate a 45-fold p53-mediated transactivation. We present evidence that the composition of the DNA which flanks a p53 DNA-binding consensus sequence may also modulate trans-activation.

Amino Acid Sequence↗

PubMed1995

Phase I study of high-dose, intravenous rsCD4 in subjects with advanced HIV-1 infection.

In vitro, recombinant soluble CD4 (rsCD4) attaches to and inactivates human immunodeficiency virus (HIV). To determine if prolonged therapy with high-dose intravenous rsCD4 provides an in vivo benefit, we gave three HIV-1-infected patients with AIDS, whose isolates were susceptible in vitro to rsCD4, 10 mg/kg of rsCD4 for 4 weeks, 5 mg/kg for 4 weeks, and 1 mg/kg for 2 weeks. Single-dose pharmacokinetic studies performed prior to this showed transient in vivo decreases of HIV-1 plasma viremia in all three subjects. Surrogate markers of HIV activity, clinical status, HIV-1 p24 antigen, plasma HIV-1 titers, and peripheral blood mononuclear cell (PBMC) intracellular titers of virus were measured at entry, and every other week after onset of therapy. All subjects demonstrated rsCD4 concentration-dependent reduction in plasma viremia, with two subjects having complete neutralization of cell-free virus. The third subject's isolate was relatively resistant to the in vivo effects of rsCD4 and only partial reduction in plasma virus titers was obtained, even at the highest dose of 10 mg/kg. There was no change in the PBMC intracellular viral titer or surrogate markers of HIV-1 activity (including CD4 cell count and beta 2-microglobulin). There was subjective improvement in clinical symptoms, and all subjects gained weight with the highest doses of rsCD4. rsCD4 exhibited linear pharmacokinetics over the dose range studied. We conclude that high-dose intravenous rsCD4 can be safely given for up to 10 weeks and that it has a stable pharmacokinetic profile.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗

PubMed1995

Identification of a second region upstream of the mouse heme oxygenase-1 gene that functions as a basal level and inducer-dependent transcription enhancer.

A 161-base pair fragment (AB1) approximately 10 kilobase pairs upstream of the transcription start site of the mouse heme oxygenase-1 gene functions as a basal level and inducer-dependent enhancer. AB1/chloramphenicol acetyltransferase fusion genes stably transfected into mouse hepatoma (Hepa) cells or L929 fibroblasts were activated 7-8- or 17-22-fold, respectively, after treatment of the cells with either CdCl2 or heme. The AB1 fragment is composed largely of three tandem repeats containing two conserved core elements, A and B. Part of core element A (TCCGGAGCTGTG) resembles the consensus-binding site for transcription factor AP-4, whereas core element B (GCTGAGTCANGG) includes the consensus-binding site (TGAGTCA) for the AP-1 family of transcription factors. Nuclear proteins from Hepa cells did not bind to any of the core A elements, but bound to all three copies of the core B element. AB1 derivatives with one or two mutant AP-1-binding elements exhibited reduced but measurable inducer-dependent enhancer activity, but mutation of all three AP-1-binding sites abolished activation by CdCl2 and heme and also by mercury chloride, zinc chloride, H2O2, sodium arsenate, and 12-O-tetradecanoylphorbol-13-acetate. Pretreatment of stably transfected L929 cells with protein kinase C inhibitors, but not with tyrosine kinase inhibitors or N-acetylcysteine, abrogated 12-O-tetradecanoylphorbol-13-acetate-dependent activation of the AB1/chloramphenicol acetyltransferase fusion gene. Induction by H2O2 was unaffected by the kinase inhibitors, but completely abolished by N-acetylcysteine. Heme-dependent induction was not significantly affected by any of these chemicals.

Acetylcysteine↗

PubMed1995

Urinary Tamm-Horsfall protein increased after potassium citrate therapy in calcium stone formers.

OBJECTIVES: To evaluate the effect of oral potassium citrate therapy on urinary excretion rates of citrate. Tamm-Horsfall protein (THP), and on calcium oxalate monohydrate crystal agglomeration inhibition [tm], in patients with recurrent calcium stone formation. METHODS: To evaluate the effect of oral therapy with potassium citrate on urinary citrate, THP, and [tm], 24-hour urine samples were collected before and at least 2 months after initiation of oral potassium citrate therapy in 33 calcium stone-forming patients who had no dietary restrictions. The citrate concentration was measured by an adaptation of a citrate lyase method. Urinary disaggregated THP concentration was determined with a quantitative enzyme-linked immunosorbent assay. The [tm] was determined by observing the effects of patients' urine, before and after oral potassium citrate therapy, on the uptake of 45Ca2+ onto the surfaces of added preformed calcium oxalate crystals in a supersaturated solution of calcium oxalate, using the in vitro kinetic method described by other investigators. RESULTS: We observed an increased urinary excretion rate of citrate from a mean of 1.9 mmol/24 h prealkali to 2.6 mmol/24 h postalkali (P < 0.0004) and of THP from a mean of 94.0 mg/24 h prealkali to 199.3 mg/24 h postalkali (P < 0.0016). A corresponding increase in [tm] from a mean of 177.1 minutes prealkali to 221.0 minutes postalkali (P < 0.024) was also observed. CONCLUSIONS: To our knowledge this is the first report correlating increased urinary citrate with THP excretion rate following oral alkalinization with potassium citrate in calcium stone formers. Of clinical importance is the corresponding increase in [tm], which was previously shown to be inversely related to stone-forming activity. Moreover, urinary citrate and THP are known to have a synergistic effect on [tm]. Our data suggest that the effectiveness of potassium citrate therapy in calcium stone-forming patients may, at least in part, be due to increased levels of THP.

Adult↗

PubMed1995

Induction of heme oxygenase in toxic renal injury: a protective role in cisplatin nephrotoxicity in the rat.

Cellular content of heme is regulated by heme oxygenase, the rate limiting enzyme in the degradation of heme. Induction of heme oxygenase is a protective response in an in vivo model of heme protein mediated renal injury, the glycerol model of acute renal failure. In addition to heme, heme oxygenase is induced by diverse forms of oxidative stress, the functional significance of which is currently unknown. We examined whether heme oxygenase is induced, and the functional significance of such induction, in two in vivo models of oxidant-induced toxic nephropathy, namely, cisplatin and gentamicin nephropathies; nephrotoxicity in these models is not dependent on the delivery of a burden of heme proteins to the kidney as occurs in the glycerol model. We demonstrate induction of heme oxygenase mRNA and protein in the kidney as early as 6 and 12 hours after a single dose of cisplatin (6 mg/kg i.v.). Pretreatment with tin protoporphyrin, a competitive inhibitor of heme oxygenase, led to higher serum creatinine values on days 3 through 5 and lower inulin clearances on day 5; tin protoporphyrin also exacerbated renal injury in this model. Renal hemodynamics studied at day 2 after cisplatin demonstrate reduced renal blood flow rates, increased renal vascular resistance and increased fractional excretion of sodium in rats treated with tin protoporphyrin. Tin protoporphyrin alone had no significant effect on serum creatinine and renal hemodynamics in rats with intact, disease-free kidneys. We confirmed that tin protoporphyrin prevented the increase in heme oxygenase activity induced by cisplatin. Induction of heme oxygenase by cisplatin was associated with increased kidney heme content and ferritin content.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation.

Gram-negative sepsis is the most common cause of the adult respiratory distress syndrome (ARDS). Lipopolysaccharide (LPS) when administered in vivo produces pathophysiologic changes similar to those seen in ARDS. The pathogenesis of these changes is mediated in part by oxidative stress. We demonstrate that LPS induces high mRNA levels of the stress-inducible gene heme oxygenase-1 (HO-1) in the rat lung. Increased HO-1 mRNA levels correlate with increased HO-1 protein and enzyme activity. Immunohistochemical analyses of lung tissues from rats treated with LPS reveal abundant HO-1 expression in inflammatory and bronchoalveolar epithelial cells. We further examined the molecular regulation of HO-1 gene expression after exposure of RAW 264.7 macrophage cells to LPS in vitro. These cells respond to LPS with increased HO-1 mRNA expression and HO-1 gene transcription. Transcriptional activation of the mouse HO-1 gene by LPS is mediated by a 5' distal enhancer fragment located approximately 4 kbp upstream from the transcription site. Electrophoretic mobility shift assays show increased activator protein-1 (AP-1) binding activity in RAW 264.7 cells after LPS treatment. Mutation of the AP-1 binding site in this enhancer fragment completely abolishes HO-1 gene activation while mutation of CCAAT/enhancer-binding protein (C/EBP) binding site exerts negligible effect, suggesting that the AP-1 family of transcription factors plays a critical role in regulating HO-1 gene activation following LPS treatment. Furthermore, upstream phosphorylation events modulate this AP-1-dependent expression of the HO-1 gene after LPS treatment.

Animals↗

PubMed1995

Acquired resistance to acute oxidative stress. Possible role of heme oxygenase and ferritin.

BACKGROUND: Prior administration of endotoxin has conferred resistance to tissue damage in a number of models of organ injury. The mechanisms by which this resistance is conferred are enigmatic. Recognizing that enhanced tissue oxidative stress may be a feature of endotoxin-associated injury and is present in many models of tissue injury, we questioned whether the beneficial effect conferred by endotoxin is dependent on the up-regulation of antioxidant defenses. EXPERIMENTAL DESIGN: We employed the glycerol model of acute renal failure (Gly-ARF), a model in which oxidant damage occurs in the kidney and other organs as a result of rhabdomyolysis and hemolysis. Rats were pretreated with endotoxin 24 hours before, or at the time of, induction of Gly-ARF. Renal functional studies and assessment of renal antioxidant status were performed. The effect of prior treatment with endotoxin was also examined in models of methemoglobin-induced and ischemic ARF. RESULTS: Renal function was improved in rats pretreated with endotoxin but worsened in rats subjected to Gly-ARF and endotoxin simultaneously. Endotoxin induced heme oxygenase activity and ferritin content in the kidney but did not induce other antioxidant systems such as catalase and glutathione peroxidase. Treatment with a competitive inhibitor of heme oxygenase blocked endotoxin-induced protection on days 2 and 3, while markedly attenuating the protective effect on day 1. Pretreatment with endotoxin reduced renal injury induced by methemoglobin, but not ischemia. CONCLUSIONS: The resistance to injury conferred by endotoxin in Gly-ARF involves induction of an antioxidant response, consisting of increased heme oxygenase and ferritin synthesis. This coupled response allows degradation of heme as well as chelation of iron, thus decreasing oxidant-mediated tissue injury. This novel mechanism of endotoxin-induced resistance may be applicable not only to Gly-ARF but also to other models of tissue injury in which enhanced oxidative stress is implicated.

Acute Kidney Injury↗

PubMed1995

Parallel induction of heme oxygenase-1 and chemoprotective phase 2 enzymes by electrophiles and antioxidants: regulation by upstream antioxidant-responsive elements (ARE).

BACKGROUND: Heme oxygenase (HO; EC 1.14.99.3) catalyzes the conversion of heme to biliverdin, which is reduced enzymatically to bilirubin. Since bilirubin is a potent antioxidant and heme a pro-oxidant, HO may protect cells against oxidative damage. HO-1 is highly inducible by diverse chemical agents, resembling those evoking induction of phase 2 enzymes (i.e., Michael reaction acceptors, heavy metals, trivalent arsenicals, and sulfhydryl reagents). Phase 2 enzymes (glutathione transferases; NAD (P)H:quinone reductase; glucuronosyltransferases) are regulated by antioxidant-responsive elements (ARE), and their induction protects against chemical carcinogenesis. Is HO-1 regulated by chemical agents and enhancer elements similar to those controlling phase 2 enzymes? MATERIALS AND METHODS: Induction of HO-1 by phorbol ester and heavy metals is transcriptionally controlled through a 268-bp SX2 fragment, containing two phorbol ester-responsive (TRE) sites (TGAC/GT C/AA) which overlap ARE consensus sequences (TGACNNNGC). Therefore, mutations of the SX2 element designed to distinguish ARE from TRE were inserted into chloramphenicol acetyltransferase (CAT) reporter plasmids, and the response of the CAT activity of murine hepatoma cells stably transfected with these constructs was examined with a wide range of inducers of phase 2 enzymes. RESULTS: All compounds raised HO-1 mRNA and CAT expression constructs containing wild-type SX2. When the SX2 region was mutated to alter TRE consensus sequences without destroying the ARE consensus, full inducibility was preserved. Conversely, when the ARE consensus was disturbed, inducibility was abolished. CONCLUSION: Induction of heme oxygenase-1 is regulated by several chemically distinct classes of inducers (mostly electrophiles), which also induce phase 2 enzymes, and these inductions are mediated by similar AREs. These findings support the importance of HO-1 as a protector against oxidative damage and suggest that HO-1 induction is part of a more generalized protective cellular response that involves phase 2 enzymes.

Antioxidants↗

PubMed1994

Multiple elements within the 5' distal enhancer of the mouse heme oxygenase-1 gene mediate induction by heavy metals.

A 268-base pair 5' distal fragment, SX2, which mediates basal level and inducer-dependent activation of the mouse heme oxygenase-1 gene, contains two activator protein-1 (AP-1) binding sites (Alam, J., and Zhining, D. (1992) J. Biol. Chem. 267, 21894-21900). Mutation of both AP-1 binding elements diminishes (by 50-70%), but does not abolish, the enhancer activity of SX2 in transient expression assays, suggesting that other sequences contribute to enhancer function. Directly upstream of the AP-1 binding sites are two copies of a sequence motif, TGAGGAAAT, which resemble elements found in cellular and viral genes that are known to interact with the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. These SX2 sequences bind specifically to liver-enriched, heat-stable nuclear proteins and confer C/EBP alpha-dependent transactivation of the heterologous chloramphenicol acetyltransferase (CAT) gene. Site-directed mutagenesis of these 9-base pair elements abolishes protein binding and transactivation, establishing these sequences as functional C/EBP binding sites. Stably transfected SX2/CAT fusion genes are induced between 37- and 44-fold in mouse hepatoma, Hepa, cells and between 52- and 111-fold in mouse fibroblast L929 cells in response to CdCl2 treatment. Subfragments of SX2 lacking the AP-1 binding elements do not mediate cadmium-dependent activation of the CAT gene, whereas subfragments containing the AP-1 binding elements, but lacking the C/EBP binding sites, exhibit only partial transcriptional activity. Site-directed mutagenesis of one or more of the C/EBP and AP-1 binding sites indicates that each of these elements is required for optimal activity of the SX2 enhancer fragment. The AP-1 binding elements, however, appear to be more important for induction as constructs containing multiple copies of either of the AP-1 binding elements, but not the C/EBP binding sequences, are readily activated by CdCl2. Treatment of Hepa cells with cadmium or heme does not alter the nuclear concentration of AP-1 or C/EBP binding activity.

Animals↗

PubMed1994

Isolation and characterization of the mouse heme oxygenase-1 gene. Distal 5' sequences are required for induction by heme or heavy metals.

Mouse genomic fragments encoding heme oxygenase-1 (HO-1) were isolated from a recombinant lambda library by in situ plaque hybridization. The mouse HO-1 gene, approximately 7 kilobase pairs (kbp) in length, is organized into 5 exons and 4 introns. The primary structure of the exons and 1287 base pairs (bp) of the 5'-flanking region was determined. The deduced amino acid sequence of the mouse HO-1 gene is identical to that of p32, initially identified as a stress-induced protein in mouse BALBc/3T3 cells. A single, major transcription initiation site is utilized for constitutive and heme- or metal-induced expression of the HO-1 gene in mouse hepatoma (Hepa) cells. The transcriptional activity of the 5'-flanking region was examined by transient expression assays using the chloramphenicol acetyltransferase gene as the reporter gene. Basal promoter activity in several cell lines was localized to within 149 bp of the upstream sequence by deletion analysis. This proximal promoter region of the mouse HO-1 gene contains several sequence elements that are not only conserved in both the rat and human HO-1 genes but also resemble consensus binding sites of various transcription factors including AP-1, AP-4, C/EBP and c-Myc:Max/USF. Heavy metals activate HO-1 gene transcription and the rat gene contains a putative metal regulatory element (Müller, R. M., Taguchi, H., and Shibahara, S. (1987) J. Biol. Chem. 262, 6795-6802) that is completely conserved in the mouse gene. Transient expression analyses, however, indicate that this sequence, which contains a core heptanucleotide, TGCACTC, identical to that of the strongest metal regulatory element of the mouse metallothionein-1 gene, is not responsive to Cd2+ or Zn2+. Stable transfection of constructs containing the entire mouse HO-1 gene and various portions of the 5'-flanking region into rat C6 glioma cells and simultaneous, quantitative analysis of the mouse and rat HO-1 mRNAs indicate that distal 5' sequences, between positions -3.5 and -12.5 kbp, are required for induction of mouse HO-1 gene transcription by both heme and heavy metals. A 5-7-fold difference in the levels of induction between stably integrated and transiently expressed mouse HO-1 gene constructs is observed in this cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗