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

Abstract

A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid-and cyclic AMPinducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor-and C/EBP␣-deficient mice, whereas the CPS mRNA was normally expressed in C/EBP␤ knockout mice and in HNF3␣ and -␥ double-knockout mice. (The role of HNF␤ could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway.

DNA Methylation Suppresses Expression of the Urea Cycle Enzyme Carbamoyl Phosphate Synthetase 1 (CPS1) in Human Hepatocellular Carcinoma

“…11 Previous studies [12][13][14][15] have shown that CPS1 transcription is subject to physiological regulation and that CPS1 activity can be inhibited by a hepatotoxic dose of acetaminophen. Studies 16,17 have also suggested that an 80-bp glucocorticoid response unit located 6.3 kb upstream of the transcription start site mediates hormone responsiveness and liver-specific expression of CPS1 in rats. A recent study 18 revealed that Y-box binding protein-1 negatively regulates CPS1 expression via suppression of CCAAT enhancer-binding protein-␣ function in fetal and injured adult mouse liver but not in normal adult liver.…”

A Single Regulatory Module of the Carbamoylphosphate Synthetase I Gene Executes Its Hepatic Program of Expression

“…20). Mutation of the C/EBP, HNF3, or GR recognition site within this unit almost completely abolishes the hormonal response, showing the strict requirement of the intact GRU for such a response (20).…”

“…The promoter of construct 4 extends from Ϫ38 to ϩ138, that is, additionally lacks the GAG box (19,25). Constructs 5-7 are comparable with constructs 2-4, respectively, except that the URF is replaced by the 102-bp GRU fragment (20). * The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…In NIH 3T3 fibroblasts and CHO cells, all constructs do show basal activity and no response to hormones (Ref. 20 and not shown).…”

“…This upstream regulatory fragment (URF) contains multiple recognition sites for HNF3 and C/EBP family members, the glucocorticoid receptor (GR) and several unknown factors. The GR recognition site is present within a 102-bp fragment, together with a C/EBP and a HNF3 site, and forms a functional response unit (20). Here, we present an experimental analysis of the regulatory functions of the 469-bp URF and the 0.3-kbp promoter in transient transfection assays and in transgenic mice.…”