REGULATION OF 3-HYDROXY-3-METHYLGLUTARYL COENZYME-A REDUCTASE GENE-EXPRESSION IN FRTL-5 CELLS .1. IDENTIFICATION AND CHARACTERIZATION OF A CYCLIC AMP-RESPONSIVE ELEMENT IN THE RAT REDUCTASE PROMOTER

Thyrotropin (TSH) increases 3-hydroxy-3-methylglutaryl coenzyme A (HMG -CoA) reductase gene transcription in FRTL-5 rat thyroid cells, and th e effect of TSH can be mimicked by cAMP. Sequence analysis of the rat reductase promoter has revealed a hitherto unnoticed cAMP-responsive e lement (CRE)-like octamer. This octamer is located between 53 and 60 n ucleotides downstream of the sterol regulatory element 1; its first 6 nucleotides are identical to the consensus somatostatin CRE, and the e ntire octamer is identical to the fos CRE. A synthetic oligonucleotide containing the HMG-CoA reductase CRE-like octamer (RED CRE) formed pr otein-DNA complexes with nuclear extracts from FRTL-5 cells, which cou ld be prevented by unlabeled CRE-containing oligonucleotides whose fla nking sequences were otherwise nonidentical. The complexes were specif ically supershifted by anti-CREE antibodies. FRTL-5 cells transfected with a fusion plasmid carrying the bacterial chloramphenicol acetyl tr ansferase (CAT) under the control of the HMG-CoA reductase promoter di splayed CAT activity, which was specifically stimulated by TSH. In con trast, CAT activity in FRTL-5 cells transfected with similar construct s carrying mutations in the reductase CRE was significantly lower and did not increase after TSH challenge. We suggest that the HMG-CoA redu ctase gene contains a functional CRE, important for TSH regulation of transcription. The data presented provide the molecular basis for a no vel regulatory mechanism for HMG-CoA reductase gene expression in rat thyroid cells, which involves the direct effect of cAMP.