This FIRST Award proposal is to study the molecular mechanisms of retinoid (vitamin A) regulation of gene expression in vivo. This project is part of a long-term plan to study direct nutrient effects on eukaryotic gene expression, as they are involved in cellular differentiation, tissue specificity and intermediary metabolism. Current work in the area of retinoid regulation of gene expression is advancing at a rapid place and provides one of the best examples of the productivity of a bionutrition approach. The work proposed here involves altering vitamin A nutriture and measuring the molecular consequences of this dietary change in the adult and developing transgenic mouse. In order to do this, a retinoid- responsive gene, that for phosphoenolpyruvate carboxykinase (PEPCK), has been introduced into transgenic mice. Different segments of the 5' regulatory domain of the PEPCK gene, a prototype for differentiation- specific genes in the liver, are linked to a marker gene, that for bovine growth hormone (bGH). Transgenic mice are made vitamin A deficient and treated with either all-trans retinoic acid (RA) or its metabolite, 9-cis retinoic acid (RA). The molecular response to changes in vitamin A status is monitored by determining the expression of the marker transgene. This will be the first attempt to utilize altered nutritional status in transgenic mice to assess the effects of retinoids on gene expression in vivo. The ligand-activated transcription factors which mediate vitamin A action are retinoid receptors in the nucleus. These receptors are members of the steroid/thyroid/retinoid superfamily of nuclear proteins. There are different genes and mRNA isoforms for the all-trans RA receptor (RAR) and 9-cis RA receptor (RXR) which are involved in distinct intranuclear signaling pathways. One of the supporting objectives of this project is to measure the expression of the RAR and RXR alpha, beta and gamma subtypes and their isoforms in the liver in response to altered vitamin A status. The functional significance of alterations in receptor expression will be assessed by determining changes in hepatic nuclear protein binding to the PEPCK regulatory region in vitro. The nuclear signaling mechanisms of retinoids are made more complex by the interaction of retinoid receptors with other nuclear receptors of the same superfamily, including the thyroid hormone (T3) receptor. The PEPCK gene is also regulated by T3 and provides a prototype promoter for investigating the convergence of these two regulatory networks. The final objective of this project is to investigate T3 regulation of PEPCK/bGH transgene expression, and cross-signaling of the retinoid and T3 responses, in transgenic mice and in isolated transgenic hepatocytes.
