GLUCOCORTICOID RECEPTOR, C EBP, HNF3, AND PROTEIN-KINASE-A COORDINATELY ACTIVATE THE GLUCOCORTICOID RESPONSE UNIT OF THE CARBAMOYLPHOSPHATESYNTHETASE-I GENE/

A single far-upstream enhancer is sufficient to confer hepatocyte-spec ific, glucocorticoid- and cyclic AMP-inducible periportal expression t o the carbamoylphosphate synthetase I (CPS) gene. To identify the mech anism 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 live r-enriched transcription factor families HNF3 and C/EBP. The in vivo r elevance of the transcription factors interacting,vith the enhancer in the regulation of CPS expression in the liver was assessed by the ana lysis of knockout mice. A strong reduction of CPS mRNA levels was obse rved in glucocorticoid receptor- and C/EBP alpha-deficient mice, where as the CPS mRNA was normally expressed in C/EBP beta knockout mice and in HNF3 alpha and -gamma double-knockout mice. (The role of HNF beta could not be assessed, because the corresponding knockout mice die at embryonic day 10), In hepatoma cells, most of the activity of the enha ncer is contained within a 103-bp fragment, which depends for its acti vity on the simultaneous occupation of the GRE, HNF3, acid C/EBP sites , thus meeting the requirement of a glucocorticoid response unit. In f ibroblast-like CHO cells, on the other hand, the GRE in the CPS enhanc er does not cooperate with the C/EBP and HNF3 elements in transactivat ion of the CPS promoter. In both hepatoma and CHO cells, stimulation o f expression by cyclic AMP depends mainly on the integrity of the gluc ocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway.