The Upstream Regulatory Region of the Carbamoyl-phosphate Synthetase I Gene Controls Its Tissue-specific, Developmental, and Hormonal Regulation in Vivo

Abstract

The carbamoyl-phosphate synthetase I gene is expressed in the periportal region of the liver, where it is activated by glucocorticosteroids and glucagon (via cyclic AMP), and in the crypts of the intestinal mucosa. The enhancer of the gene is located 6.3 kilobase pairs upstream of the transcription start site and has been shown to direct the hormone-dependent hepatocytespecific expression in vitro. To analyze the function of the upstream region in vivo, three groups of transgenic mice were generated. In the first group the promoter drives expression of the reporter gene, whereas the promoter and upstream region including the far upstream enhancer drive expression of the reporter gene in the second group. In the third group the far upstream enhancer was directly coupled to a minimized promoter fragment. Reporter-gene expression was virtually undetectable in the first group. In the second group spatial, temporal, and hormonal regulation of expression of the reporter gene and the endogenous carbamoyl-phosphate synthetase gene were identical. The third group showed liver-specific periportal reporter gene expression, but failed to activate expression in the intestine. These results show that the upstream region of the carbamoyl-phosphate synthetase gene controls four characteristics of its expression: tissue specificity, spatial pattern of expression within the liver and intestine, hormone sensitivity, and developmental regulation. Within the upstream region, the far upstream enhancer at ؊6.3 kilobase pairs is the determinant of the characteristic hepatocyte-specific periportal expression pattern of carbamoyl-phosphate synthetase.The ornithine cycle converts ammonia, originating mainly from amino acid metabolism, into urea for excretion. This conversion is vital, because ammonia is toxic to vertebrates. All five ornithine cycle enzymes are active predominantly in the periportal hepatocytes (1, 2, 68). Parts of the cycle, however, are also found in the intestinal mucosa and the kidney, thereby creating an interorgan biosynthetic pathway for arginine biosynthesis (reviewed in Refs. 1, 3, and 4). The first and, under most conditions, major flux-determining step of the ornithine cycle, the conversion of ammonia, bicarbonate, and ATP into carbamoyl phosphate, is catalyzed by carbamoyl-phosphate synthetase I (CPS; EC 6.3.4.16) 1 (1, 5). The expression of CPS mRNA can be detected in the liver of the rat from embryonic day 15 (ED15) onwards. Expression is initially found only in a few hepatocytes, but toward the end of the fetal period all hepatocytes have been recruited to express CPS (6 -8). Subsequently, the expression gradually becomes confined to the hepatocytes surrounding the portal veins (8 -10). CPS enzyme and mRNA levels in liver change in parallel under experimental conditions that change circulating glucocorticosteroid and hepatic cyclic AMP levels (3,(11)(12)(13)(14), suggesting that the zonal restriction and hormonal modulation of CPS expression are regulated at the level of transcription. Accordingly, it was s...

Glucocorticoid Receptor, C/EBP, HNF3, and Protein Kinase A Coordinately Activate the Glucocorticoid Response Unit of the Carbamoylphosphate Synthetase I Gene

“…In the adult, CPS gene expression is regulated by intracellular levels of cyclic AMP (cAMP) and by glucocorticoids (33,34,45). During development, CPS expression is upregulated perinatally (6), presumably due to the late fetal rise in circulating free glucocorticoids and the neonatal rise in intracellular cAMP (19,23,34). CPS shares the periportal expression and hormonal regulation with other genes that encode key enzymes in amino acid breakdown, gluconeogenesis, and urea synthesis (27,28,42,44,66).…”

“…CPS shares the periportal expression and hormonal regulation with other genes that encode key enzymes in amino acid breakdown, gluconeogenesis, and urea synthesis (27,28,42,44,66). In vivo, the hepatic expression of CPS is controlled by a single farupstream enhancer (6), making the gene an attractive model to identify the minimal sequence that is necessary to bring about liver-specific, hormone-inducible periportal expression.…”

“…Using transgenic mouse studies, we and others have shown that many of these enzyme gradients are regulated at the transcriptional level (3,6,35,41,53). A genetic dissection of the DNA elements that confer periportal or pericentral expression upon a reporter gene should lead to insight into the signal transduction cascades that regulate gene expression in hepatocytes according to their position along the porto-central axis.…”

“…The carbamoylphosphate synthetase I (CPS) gene has proven to be a useful model for such an approach (6). CPS, the first enzyme of the ornithine cycle, converts ammonia, originating from amino acid degradation, into urea (42,44).…”

Position-dependent activity of α-fetoprotein enhancer element III in the adult liver is due to negative regulation

“…Other enzymes, such as carbamoylphosphate synthetase and ornithine transcarbamylase, are expressed in a broad band of hepatocytes encircling the portal triads (10,11). These periportal and pericentral regions of expression are nonoverlapping.…”