Rat lung contains a developmentally regulated manganese superoxide dismutase mRNA-binding protein.

It has become increasingly clear that RNA-binding proteins play an important role in the regulation of gene expression. The presence in rat lung of a specific, redox-sensitive catalase RNA-binding protein was recently reported (Clerch, L. B., and D. Massaro, 1992. J. Biol. Chem. 267:2853). In order to determine if specific manganese superoxide dismutase (MnSOD) RNA-binding proteins exist, we tested whether protein in rat lung extract would bind to 32P-labeled MnSOD RNA. Using a gel mobility shift assay we show rat lung protein forms specific complexes with a 216 b fragment of the 3' untranslated region of MnSOD RNA and the binding requires the presence of free sulfhydryl groups. Competition studies indicate MnSOD RNA-binding protein is different from catalase RNA-binding protein. Furthermore, unlike catalase RNA-binding protein, rat lung MnSOD RNA-binding protein activity is developmentally regulated; there is less MnSOD RNA-protein binding activity in adult rat lung extract compared to prenatal or neonatal rat lung extracts. We conclude the lung contains developmentally regulated MnSOD mRNA-binding protein that is redox sensitive.

Tolerance of rats to hyperoxia. Lung antioxidant enzyme gene expression.

Tolerance to hyperoxia usually requires an increase of lung antioxidant enzyme (AOE) activity. We used rats with different degrees of tolerance to > 95% O2 to evaluate the importance of individual AOEs for tolerance; we also explored the regulation of AOE gene expression. During exposure of adult rats to > 95% O2, lung manganese superoxide dismutase (MnSOD) activity fell approximately 50% despite a threefold increase of MnSOD mRNA concentration; addition of a reducing agent to lung extracts from O2-exposed rats partially restored MnSOD activity. Endotoxin induced tolerance to O2 (a) without elevating Cu,Zn superoxide dismutase activity, (b) with increases of catalase and glutathione peroxidase (GP) activity of the same magnitude as occurred in O2-saline rats, but (c) with MnSOD activity 1.5-1.9-fold higher than in air-saline rats and 1.4-3.6-fold higher than in O2-saline rats. Endotoxin elevated the concentration of MnSOD and GP mRNAs without increasing their stability. O2 elevated MnSOD mRNA concentration, and increased its stability. O2 plus endotoxin increased the concentration and stability of MnSOD, catalase, and GP mRNAs. These data suggest that in adult rats tolerance to hyperoxia requires increased MnSOD activity; the data show gene expression and regulation vary among the AOEs, and that increased stability of the AOEs' mRNAs plays an important role in AOE gene expression and in tolerance to hyperoxia.

Rat lung Cu,Zn superoxide dismutase. Isolation and sequence of a full-length cDNA and studies of enzyme induction.

The synthesis of Cu,Zn SOD by rat lung increases spontaneously in the fetus in late gestation and during exposure of neonatal and adult rats to greater than 95% O2. To explore the regulation of these increases, we measured rat lung Cu,Zn SOD synthesis and activity. We also cloned and sequenced a rat lung Cu,Zn SOD cDNA that was used to measure Cu,Zn SOD mRNA concentration. We found that (a) under normal gestational and postgestational conditions the synthesis of this enzyme was regulated pretranslationally; (b) the increased synthesis that occurs under hyperoxia (greater than 95% O2), was pretranslationally mediated in otherwise unmanipulated neonatal rats but translationally controlled in hyperoxic adult rats; and (c) in lungs of rats made tolerant to greater than 95% O2 by allowing 24 h rest in air after an initial 48 h in greater than 95% O2, the increased Cu,Zn SOD synthesis that occurred during the second period of hyperoxia was regulated pretranslationally. We conclude Cu,Zn SOD gene expression in the lung is developmentally regulated under normal conditions and in response to an oxidant challenge. Tolerance, whether endogenous or induced, appears to require the accumulation of increased amounts of Cu,Zn SOD mRNA.

Pertussis toxin treatment alters manganese superoxide dismutase activity in lung. Evidence for lung oxygen toxicity in air-breathing rats.

Exposure of rats to hyperoxia or to treatment with endotoxin, increases lung manganese superoxide dismutase (MnSOD) gene expression. However, the paths by which these environmental signals are transduced into enhanced MnSOD gene expression are unknown. We now provide evidence that heterotrimeric G proteins are involved in the hyperoxia-induced increase in lung MnSOD gene expression but that pertussis toxin-sensitive G proteins are not involved in the endotoxin-induced elevation of lung MnSOD gene expression. We also show that treating rats with pertussis toxin decreased lung MnSOD activity approximately 50%. This decline in MnSOD activity occurred without a change in the lung activity of copper-zinc SOD, catalase, or glutathione peroxidase. In air-breathing rats, the pertussis toxin-induced decrease in MnSOD activity was associated with the development of lung edema, pleural effusion with a high concentration of protein, and biochemical evidence of lung oxygen toxicity. Compared to air-breathing rats, maintenance of pertussis toxin-treated rats under hypoxic or hyperoxic conditions respectively decreased or increased intrathoracic fluid. Endotoxin treatment elevated lung MnSOD activity and protected pertussis toxin-treated rats from an increase in intrathoracic fluid.

Retinoic acid receptor-beta: an endogenous inhibitor of the perinatal formation of pulmonary alveoli.

Pulmonary alveoli are formed, in part, by subdivision (septation) of the gas-exchange saccules of the immature lung. Septation is developmentally regulated, and failure to septate at the appropriate time is not followed by delayed spontaneous septation. We report retinoic acid receptor (RAR) beta knockout mice exhibit premature septation; in addition, they form alveoli twice as fast as wild-type mice during the period of septation but at the same rate as wild-type mice thereafter. Consistent with the perinatal effect of RARbeta knockout, RARbeta agonist treatment of newborn rats impairs septation. These results 1) identify RARbeta as the first recognized endogenous signaling that inhibits septation, 2) demonstrate premature onset of septation may be induced, and 3) show the molecular signaling regulating alveolus formation differs during and after the period of septation. Suppressing perinatal RARbeta signaling by RARbeta antagonists may offer a novel, nonsurgical, means of preventing, or remediating, failed septation in prematurely born children.

Estrogen regulates angiotensin AT1 receptor expression via cytosolic proteins that bind to the 5' leader sequence of the receptor mRNA.

Two of the most highly recognized factors implicated in the pathogenesis of hypertension, atherosclerosis, congestive heart failure and associated cardiovascular disease are the renin angiotensin system (RAS) and estrogen. A major effect of estrogen results from its influence on the RAS. Beta-estradiol (E2) replacement in ovariectomized (OVX) rats significantly decreased type 1 angiotensin (AT1) receptor expression in the pituitary and adrenal, whereas it significantly increased receptor expression in the uterus when compared to OVX controls. Additional evidence demonstrated an important influence of estrogen on a recently discovered post-transcriptional mechanism for regulating expression of the AT1 receptor. This mechanism consists of cytosolic RNA binding proteins (BPs) that recognize the 5' leader sequence (5'LS) of the receptor mRNA. The activities of these 5'LS BPs were modulated by estrogen in an inverse manner to AT1 receptor regulation. Moreover, in vitro translation assays in wheat germ lysates suggested that the 5'LS BPs inhibited AT1 receptor translation. Our data therefore indicate that hormonal regulation of AT1 receptors involves modulation of 5'LS BPs by estrogen. These findings may in part account for the observed protective effects of estrogen on cardiovascular disease.

Endotoxin induces rat lung ribonuclease activity.

Lipopolysaccharide (endotoxin), a component of Gram-negative bacteria, causes marked alterations in eukaryotic gene expression and cellular physiology. We show that within one hour of injection of endotoxin into adult rats there is an induction of ribonuclease activity in the lung. The degradation of RNA was prevented by treatment of the lung extract from endotoxin-injected rats with ribonuclease inhibitor (RNasin). We suggest that induction by endotoxin of ribonuclease activity is a novel mechanism by which cells could alter gene expression to meet an environmental challenge and caution that the presence of ribonuclease can hinder molecular biological analyses of tissue extracts from endotoxin-treated rats.

Post-transcriptional regulation of lung antioxidant enzyme gene expression.

It is an honor, and indeed fitting, to have a chapter on pulmonary oxygen toxicity included in a Festschrift for Dan Gilbert, whose contributions to the free radical theory of oxygen toxicity have been a catalyst to the last half-century of investigation in this field. There is cellular damage that results in pulmonary edema and even death if the increase in reactive oxygen species produced in the lung during exposure to hyperoxia is not counterbalanced by an increase in the cell's antioxidant defense systems. In this chapter experimental evidence will substantiate the importance of post-transcriptional regulation of antioxidant enzyme gene expression in animal models of pulmonary oxygen toxicity and tolerance to hyperoxia with special emphasis given to the role of manganese superoxide dismutase (MnSOD) synthesis, specific activity, and RNA half-life and to a proposed function of a MnSOD RNA-binding protein as a positive regulator in the control of translational efficiency.

A 3' untranslated region of catalase mRNA composed of a stem-loop and dinucleotide repeat elements binds a 69-kDa redox-sensitive protein.

Rat lung extract contains protein that forms redox-sensitive, specific complexes with a 1130-base catalase cRNA (J. Biol. Chem. 267, 2853-2855, 1992). The present paper reports studies aimed at delimiting the site of protein binding on the RNA and characterizing the protein. A 240-base sequence was identified as the 3' untranslated region of catalase mRNA that binds lung protein in a redox-sensitive manner. Two elements within this 240-base region bind protein; one is a 36-base element that has a computer-predicted stem-loop secondary structure and the other is a CA dinucleotide repeat. Competition studies indicate that both elements are required for specific binding. Cross-competition experiments demonstrated that catalase RNA-binding protein (CAT-BP) is not the iron-responsive element-binding protein. Ultraviolet light-induced cross-linking and two-dimensional electrophoresis showed that CAT-BP has an apparent molecular mass of 69 kDa and appears to be composed of four isoforms. Competition studies indicate that stem-loop cis element is directly involved in binding CAT-BP. In addition to rat, the 69-kDa catalase RNA-binding protein is present in mouse and human fibroblast cell lines.

Tyrosine phosphorylation regulates manganese superoxide dismutase (MnSOD) RNA-binding protein activity and MnSOD protein expression.

All cells tested contain a cytosolic protein that binds to a defined region in the 3' untranslated region of manganese superoxide dismutase (MnSOD) RNA; both the MnSOD RNA-binding protein (MnSOD-BP) and the cis element are required for efficient translation of MnSOD RNA [Chung, D. J., Wright, A. E., and Clerch, L. B. (1997) Biochemistry 37, 16298-16306]. This study was designed to test the hypothesis that MnSOD-BP activity is regulated by phosphorylation. When cell extracts from whole rat lung or a rat lung fibroblast cell line, RFL-6, were treated in vitro with a protein tyrosine phosphatase, there was a 4-fold increase in MnSOD-BP activity indicating that MnSOD-BP activity was upregulated by tyrosine dephosphorylation. RFL-6 cells treated in cell culture with herbimycin A or genistein, inhibitors of protein tyrosine kinase, had significantly more MnSOD-BP activity than cells treated with diluent. In RFL-6 cells treated with herbimycin A, the increase in MnSOD-BP activity was associated with an increase in the level of MnSOD protein without a change in MnSOD mRNA concentration. We propose that the modulation of MnSOD protein expression by the tyrosine phosphorylation state of MnSOD-BP is a potential therapeutic target for increasing MnSOD activity during periods of oxidative stress.

RNA in polysomes is an inhibitor of manganese superoxide dismutase RNA-binding protein activity.

A redox-sensitive protein in rat lung binds to the 3'-untranslated region (3'-UTR) of manganese superoxide dismutase (Mn-SOD) mRNA; the activity of this Mn-SOD RNA-binding protein (Mn-SOD-BP) is greater in 12,000-g supernatant fractions (S12) from neonates than in S12 from adults (H. Fazzone, A. Wangner, and L. B. Clerch. J. Clin. Invest. 92: 1278-1281, 1993). To determine the mechanism underlying this developmental difference, lung subcellular fractions were tested for their effect on Mn-SOD-BP activity. Protein in the 130,000-g supernatant (S130) of lung extracts bound the 3'-UTR. However, the developmental difference in binding was not present in S130. The 130,000-g pellet (P130) did not bind the 3'-UTR; rather, it contained an inhibitor of Mn-SOD-BP activity. Addition of P130 to S130 decreased RNA binding in a dose-dependent manner. Furthermore, adult P130 was a more potent inhibitor of RNA-binding activity than neonatal P130. These data indicate that the developmental difference in Mn-SOD-BP activity is due, in part, to an inhibitor in P130. Biochemical characterization revealed that the inhibitor is an RNA that may participate in the posttranscriptional control of Mn-SOD gene expression.

Oxidation-reduction-sensitive binding of lung protein to rat catalase mRNA.

Air-breathing organisms experience an elevated concentration of oxygen mainly under two conditions. One occurs at birth when the O2 tension in the lung increases from approximately 25 torr present in utero to approximately 100 torr. The lungs, in particular, are also exposed to hyperoxia when oxygen is administered for therapeutic reasons. Under hyperoxic conditions, increased lung antioxidant enzyme activity is important for survival. The molecular basis for the increase in antioxidant enzyme gene expression under these circumstances is not well understood; in hyperoxia-exposed neonatal rats the elevation of lung catalase activity is not due to an increased rate of transcription but is associated with an increased concentration of catalase mRNA due to enhanced stability of the mRNA (Clerch, L.B., Iqbal, J., and Massaro, D. (1991) Am. J. Physiol. 260, L428-L433). We now show that neonatal rat lung protein forms specific complexes with catalase mRNA; this binding is redox-sensitive since when oxidizing agents are added binding is abolished but is restored by reducing agents. Our data also indicate lungs from hyperoxia-exposed rats have a larger proportion of catalase RNA-binding protein in oxidized form than lungs from air-breathing rats. This redox-sensitive binding of protein to catalase mRNA may be important in the control of catalase gene expression.

Rat lung antioxidant enzymes: differences in perinatal gene expression and regulation.

The lung activity of the antioxidant enzymes (AOEs) copper, zinc superoxide dismutase (Cu,Zn SOD), catalase (CAT), and glutathione peroxidase (GP), but not manganese superoxide dismutase (Mn SOD), increases in rats during late gestation; the concentrations of Cu,Zn SOD mRNA and CAT mRNA also rise. During early postnatal exposure to > 95% O2, the lung activity of Cu,Zn SOD, CAT, and GP increases. We now show 1) the lung concentration of Mn SOD mRNA and GP mRNA does not increase in late gestation; 2) Mn SOD activity and the concentration of its mRNA and of GP mRNA increase during exposure of neonatal rats to > 95% O2; and 3) as previously shown for CAT mRNA, the increase in lung concentration of the mRNAs for Cu,Zn SOD, Mn SOD, and GP during early postnatal hyperoxia occurs with a 70-80% prolongation of the half-life of these mRNAs. We conclude that 1) in late gestation the level at which lung AOE gene expression is regulated differs among the enzymes, 2) the level at which lung AOE gene expression is regulated shortly after birth in response to > 95% O2 is uniform among the enzymes, and 3) the lung's AOE response to neonatal hyperoxia is not merely a step-up of its prenatal regulation but involves different regulatory mechanisms based on increased stability of AOE mRNAs.

A region in the 3' UTR of MnSOD RNA enhances translation of a heterologous RNA.

The studies reported in this paper were designed to test the hypothesis that a cis element located in the 3' UTR of manganese superoxide dismutase (MnSOD) RNA, designated MnSOD-response element (MnSOD-RE), is a translational enhancer in vivo. NIH/3T3 cells were transfected with a posttranscriptional reporter construct in which MnSOD-RE was placed 3' of the coding region of chloramphenicol acetyltransferase (CAT); this construct is designated CAT-RMS. Transient transfection of CAT-RMS did not change the concentration of CAT mRNA but increased CAT activity by approximately 400% compared to a control construct, CAT-V, which contains approximately the same size of non-MnSOD 3' UTR sequence. Transfection of CAT-RMS had no effect on endogenous MnSOD protein, mRNA, or MnSOD RNA-binding protein activity. Because of its ability to increase translation of a heterologous RNA, MnSOD-RE may be useful in designing expression vectors for in vitro expression systems and in vivo gene therapy.

Regulation of lung manganese superoxide dismutase: species variation in response to lipopolysaccharide.

Lipopolysaccharide (LPS) treatment increases survival of rats, but not of mice, during hyperoxia. Manganese superoxide dismutase (Mn SOD) in the lung plays a critical role in LPS-induced tolerance to hyperoxia in rats. Therefore, we now compared the response of lung Mn SOD with treatment of mice and rats with LPS. LPS treatment of rats increased Mn SOD activity and protein concentration, did not change its specific activity, increased Mn SOD mRNA concentration 35-fold, and elevated Mn SOD synthesis 50% without changing general protein synthesis. LPS treatment of mice did not alter any of these parameters except for a 16-fold increase in Mn SOD mRNA concentration. Mn SOD translational efficiency (synthesis/mRNA concentration) was diminished 93% in rat lung and 76% in mouse lung by treatment with LPS. However, the absolute translational efficiency was twofold higher in lungs of LPS-treated rats than in lungs of LPS-treated mice. The failure of LPS to raise Mn SOD activity in mouse lungs is due, at least in part, to a smaller increase in Mn SOD mRNA and lower translational efficiency in LPS-treated mice than in LPS-treated rats.

The 3' untranslated region of manganese superoxide dismutase RNA contains a translational enhancer element.

A redox-sensitive protein that binds to the 3' untranslated region (UTR) of manganese superoxide dismutase (MnSOD) RNA has been described previously [Fazzone, H., Wangner, A., and Clerch, L. B. (1993) J. Clin. Invest. 92, 1278-1281; Chung, D. J., and Clerch, L. B. (1997) Am. J. Physiol. 16, L714-L719]. In the present study, cross-competition gel retardation and RNase H assays were used to identify a 41-base region located 111 bases downstream of the stop codon as the 3' UTR cis element involved in protein binding. The base sequence of this region is approximately 75% conserved among the 3' UTRs of rat, mouse, cow, and human MnSOD mRNAs at approximately the same distance downstream of the stop codon. The role of this protein-binding region in RNA translation was assessed in an in vitro rabbit reticulocyte lysate system. Translation of MnSOD RNA from which the 3' UTR element was deleted decreased 60% compared with translation of MnSOD RNA containing the 3' UTR cis element. In the presence of a specific competitor oligoribonucleotide that inhibits MnSOD RNA protein-binding activity, translation of MnSOD RNA containing the 3' UTR was decreased by 65%. Thus, both the cis element and RNA protein-binding activity were required for more efficient translation of the MnSOD. An analysis of ribosomal profiles suggests the MnSOD RNA-binding protein participates in the formation of the translation initiation complex. When MnSOD RNA-binding activity was inhibited, initiation complex formation was decreased by 50%. From the data obtained in this study, we propose that the 3' UTR cis element of MnSOD through its interaction with MnSOD RNA-binding protein may function as a translational enhancer.

Lung manganese superoxide dismutase protein expression increases in the baboon model of bronchopulmonary dysplasia and is regulated at a posttranscriptional level.

The expression of lung manganese superoxide dismutase (MnSOD) mRNA and protein were examined in a premature baboon model of hyperoxia-induced bronchopulmonary dysplasia (BPD) and BPD superimposed with bacterial infection. When 140-d gestation baboons were delivered by hysterotomy and treated for 16 d with appropriate ventilatory and oxygen support (pro re nada controls), there was an increase in both MnSOD mRNA and protein compared with 140-d or 156-d gestation, nonventilated controls. The concentration of MnSOD protein was also elevated when the prematurely delivered baboons were ventilated with a high fraction of inspired O2 to produce a primate homolog of BPD, but there was a significant decrease in the concentration of MnSOD mRNA in BPD animals compared with pro re nada controls. In the lungs of premature baboons in which Escherichia coli infection was superimposed on hyperoxia-induced BPD, MnSOD mRNA was diminished to approximately the same extent as in BPD alone, but MnSOD protein was significantly increased compared with all other groups. Taken together these data indicate that the premature baboon is capable of mounting an antioxidant response and that increased MnSOD protein expression in BPD and BPD-infected premature baboons is regulated, at least in part, at a posttranscriptional level.

Rat lung antioxidant enzyme induction by ozone.

We exposed rats of different ages (weights approximately 45-300 g) to 0.7 ppm O3 for 1-5 days. At 5 days lungs of O3-exposed rats had higher activity of Cu,Zn superoxide dismutase (SOD), Mn SOD, catalase, and glutathione peroxidase than air-breathing rats; this greater activity was not due to blood-associated enzyme activity. The greater enzyme activity occurred with a higher concentration of the mRNA for each enzyme (Mn SOD not measured) without altered stability of these mRNAs. In adult rats the concentrations of these mRNAs were measured after 1, 3, and 5 days exposure to O3 and were elevated by day 3. The intergroup differences (air vs. O3) among antioxidant enzymes (AOEs) were unequal, and the intergroup differences in concentration of the specific AOE mRNA were greater than the differences in activity of their AOE. We conclude exposure to O3 led to greater expression of AOE genes; the increased expression was mediated pretranslationally probably at the level of transcription.

Molecular mechanisms of antioxidant enzyme expression in lung during exposure to and recovery from hyperoxia.

Manganese superoxide dismutase (MnSOD) activity falls approximately 50% in lung during 48 h of exposure of adult rats to > 95% O2 (L. B. Clerch and D. Massaro. J. Clin. Invest. 91: 499-508, 1993). We now show that hyperoxia also decreased MnSOD activity in lungs of adult baboons, making the phenomenon potentially more important to humans. In rats, a decrease in lung MnSOD activity during an initial 48 h of exposure to > 95% O2 and its increase during an immediately subsequent 24 h in air were due to decreases and increases, respectively, in MnSOD specific activity and synthesis rate; the latter was due to altered translational efficiency. The concentration in the lung of copper-zinc superoxide dismutase mRNA, catalase mRNA, and glutathione peroxidase mRNA, unchanged during the initial 48 h of exposure to O2, rose approximately twofold during reexposure to O2 after 24 h in air. The demonstration that the fall in MnSOD activity is translationally and posttranslationally regulated during the initial exposure to hyperoxia suggests that gene transfer to increase MnSOD activity in hyperoxic lungs may also require therapy that maintains translational efficiency and MnSOD specific activity.