Metabolic Regulation and Transgenic Mouse.

Abstract

The ornithine-urea cycle is the major pathway for detoxication of ammonia formed in amino acid metabolism and is responsible for nitrogen balance of the body. The cycle is also involved in biosynthesis of arginine. Ornithine transcarbamylase (OTC) catalyzes the second among the five enzymatic steps of the cycle. The enzyme is present in the liver and to a lesser extent in the small intestine. In the fetal liver, the level of the enzyme increases late in gestation, coordinately with other urea cycle enzymes. OTC deficiency is known in humans and mice, as an inborn error of metabolism that results in protein intolerance and hyperammonemia. The rat and human OTC genes span a region of 70-75 kb on the X chromosome. Transient expression analysis showed that the promoter of this gene is much more active in a hepatoma cell line (HepG2) than in a non-hepatic cell line (CHO) . Two sites of the promoter region are recognized by both HNF-4 (hepa-tocyte nuclear factor-4) and COUP-TF (chicken ovalbumin upstream promoter-transcription factor) that are members of steroid receptor superfamily. In cotransfection analysis, HNF-4 activated expression from the OTC promoter, whereas COUP-TF repressed it. In transgenic mice, the promoter of the rat OTC gene directed liver-and small intestine-specific expression, but the expression of the transgene in the liver was very low. We found a hepatoma-specific enhancer region of -110 by located 11 kb upstream of the transcription start site. There are four protein binding sites in this region; two were recognized by HNF-4 and the other two by a factor (s) related to C/EBP (CCAAT/enhancer binding protein) . Both HNF-4 and C/EBP sites were required for the enhancer activity. In transgenic mice, an OTC enhancer/OTC promoter/OTC cDNA transgene was expressed more strongly in the liver than in the small intestine, thus resembling the expression of the endogenous gene. Introduction of the OTC promoter/OTC cDNA transgene into the OTC-deficient spf-ash mouse apparently corrected the OTC deficiency under normal dietary conditions. However, this transgenic spf-ash mouse was not completely healthy under stressed conditions. Introduction of the OTC enhancer-containing transgene into the OTC deficiency mouse is underway.