Hypoxemia and tissue hypoxia are common consequences of many acquired and inherited cardiorespiratory abnormalities. Because hypoxemic children are often able to maintain basic organ function and to sustain growth, it is reasonable to think that they develop effective cellular adaptations to the hypoxic state. The long range goal of this project is to characterize the mechanisms involved in these adaptations at the level of gene expression. Our initial studies have shown that expression of the gene encoding subunit e of the mitochondrial ATP synthase complex (subunit e) is regulated at a pretranslational level by changes in environmental oxygen tension and that this regulation depends on cell differentiation stage. The work contained in this proposal will test the hypothesis that hypoxia-mediated regulation of the gene encoding subunit e occurs through a novel gene regulatory pathway.
The specific aims of the research are: 1) To elucidate the contribution of transcriptional and post-transcriptional events to the hypoxia-induced regulation of subunit e gene expression. To this end, the effects of hypoxia on the transcription and stability of subunit e mRNA will be determined by nuclear runoff transcription assays and pulse-chase studies, respectively; the dependence of these effects on new protein synthesis will be assessed with protein synthesis inhibition studies. II) To determine the structural organization and regulatory regions of the murine subunit e gene by screening a mouse genomic library and characterizing the isolated clones using DNA sequencing analysis, restriction digest mapping, Southern blot analysis, primer extension analysis, and RNAse protection assays. III) To identify and characterize the cis-acting regulatory elements involved in the hypoxia-mediated regulation of subunit e gene expression by performing electrophoretic mobility shift assays and cell transfection studies with a construct containing the putative subunit e regulatory regions fused to a reporter gene. Completion of these aims will help identify the trans-acting regulatory proteins and corresponding upstream signaling pathways involved in the hypoxia-mediated regulation of the subunit e gene and perhaps other mitochondrial proteins. This work will serve as the basis for future studies to clarify the relationship between the regulation of oxidative energy metabolism and the tolerance to hypoxia and may help develop rational therapeutic strategies for hypoxic states.
| Huss, J M; Levy, F H; Kelly, D P (2001) Hypoxia inhibits the peroxisome proliferator-activated receptor alpha/retinoid X receptor gene regulatory pathway in cardiac myocytes: a mechanism for O2-dependent modulation of mitochondrial fatty acid oxidation. J Biol Chem 276:27605-12 |
