Glucocorticoid receptor, C/EBP, HNF3, and protein kinase A coordinately activate the glucocorticoid response unit of the carbamoylphosphate synthetase I gene.
A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid- and cyclic AMP-inducible 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/EBPalpha-deficient mice, whereas the CPS mRNA was normally expressed in C/EBPbeta knockout mice and in HNF3alpha and -gamma double-knockout mice. (The role of HNFbeta 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.
Department of Anatomy and Embryology, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
V M
VM
Christoffels
T
T
Grange
K H
KH
Kaestner
T J
TJ
Cole
G J
GJ
Darlington
C M
CM
Croniger
W H
WH
Lamers
D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics*; D006224:Cricetinae; D000242:Cyclic AMP / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000502:physiology*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D051542:Hepatocyte Nuclear Factor 3-beta; D017403:In Situ Hybridization; D008099:Liver / Q000201:enzymology; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology*; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C496503:Foxa2 protein, mouse; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D003850:Deoxyribonuclease I; D002222:Carbamoyl-Phosphate Synthase (Ammonia)