Linda Biadasz Clerch

Post‐Transcriptional Regulation of Lung Antioxidant Enzyme Gene Expression

Abstract: 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 cells 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.

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 whichEscherichia 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.

Expression of lung uncoupling protein-2 mRNA is modulated developmentally and by caloric intake.

Lung expresses a high concentration of uncoupling protein-2 (UCP-2) mRNA, but neither its pulmonary regulation nor function is known. We measured lung UCP-2 mRNA expression in two animal models: in neonatal rats when both the metabolic rate, as measured by oxygen consumption, and levels of serum free fatty acids (FFAs) increase and in adult mice during decreased food intake, when levels of serum FFAs increase but the metabolic rate decreases. In rat lung, the concentration of UCP-2 mRNA was low and unchanged during late gestation, increased approximately twofold within 6 hrs after birth, and, compared with late gestation, remained approximately threefold higher from day 1 to adulthood. The early postnatal rise in the lung UCP-2 mRNA concentration was partially blocked by an antithyroid drug and was increased by treatment with triiodothyronine. Unlike lung, heart UCP-2 mRNA levels were lower during adulthood than at day 15. In adult mice, lung UCP-2 mRNA concentrations increased approximately fivefold within 12 hrs of 67% calorie restriction (CR), remained elevated during 2 weeks of CR, fell to control levels within 24 hrs of refeeding (CR-RF), and positively correlated with serum FFA concentrations. Heart UCP-2 expression during CR and CR-RF was similar to that of lung; liver UCP-2 mRNA levels were slightly lower during CR and returned to control levels during CR-RF. These data suggest that the regulation of UCP-2 is at least partly tissue-specific and that, in the adult mouse, lung UCP-2 is regulated not by oxygen consumption but by FFAs. Moreover, lung UCP-2 mRNA levels in mice fed ad libitum was increased by the intraperitoneal administration of Intralipid, a 20% fat emulsion. On the basis of these data in adult mice, together with the findings of others that levels of FFAs increase by 2 hrs after birth, we propose lung UCP-2 is regulated by FFA.

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.

Molecular cloning and characterization of a novel gene upregulated early during postnatal rat lung development

Alveoli form, in part, by subdivision (septation) of saccules constituting the gas exchange region of the immature lung. Rat lungs septate from postnatal day 4 through 14 but alveoli in male rats continue to form, by other means, until about age 44 days. In rats, we sought to identify genes involved in septation. Using differential display reverse transcription polymerase chain reaction, we cloned a cDNA, rA5D3, that had 1270 bp with an open reading frame encoding a putative polypeptide of 165 amino acids containing potential leucine zipper motifs and phosphorylation sites. Database searches indicated rA5D3 was a novel gene. rA5D3 RNA relative concentration increased 1.7-fold between postnatal days 4 and 8, decreased 4-fold between days 8 and 14, declined significantly thereafter but was still detected post alveolus formation. If the expression of rA5D3 protein resembles its RNA, its peak (postnatal day 8) is well after the onset of septation, suggesting a role other than the initiation of septation.

Free Radical Mechanisms of Cellular Injury Symposium: Introduction

Free radical generation, including reactive nitrogen and reactive oxygen species, is known to participate in cell physiology in both a positive and negative manner. Moreover, alterations in their concentrations are implicated in a number of human diseases. Two of the talks in this mini symposium discussed research on nitroxides and the third talk addressed the clinical application of controlling free radical concentrations. Dr. David Wink eloquently discussed the chemistry of nitric oxide (NO) in the attempt to understand the mechanisms of NO reactivity during pathophysiological events. Such understanding may open the door to therapeutic applications. He specifically reviewed the indirect effects that are mediated by reactive nitrogen oxide species and talked about nitrosative and oxidative stress. Dr. James Mitchell presented data (in vitro as well as in vivo), and chemical explanations showing how non-toxic levels of nitroxides can actually act as protectors against oxidative damage. Dr. Mitchells research also involves testing the feasibility of using nitroxides and other paramagnetic probes for in vivo electron paramagnetic resonance imaging. The latter allows the study of probe uptake, oxygen content in tissues, and tissue redox reactivity. Dr. Christopher Wilcox discussed the clinical potential of controlling free radical reactions. He used the spontaneously hypertensive rat (SHR) as a model of oxidative stress. The data showed oxidative stress may adversely affect normal epithelial-vascular signaling between the macula densa and afferent arteriole of the kidneys in SHR, thereby inducing pre-glomerular vasoconstriction and salt sensitivity and hypertension. Long-term data using a superoxide dismutase agonist support the possibility of developing this category of compounds as a novel therapeutic approach to lower renal vascular resistance and blood pressure, as well as restoring normal blood vessel physiology.