Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in FRTL-5 cells. I. 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 the effect of TSH can be mimicked by cAMP. Sequence analysis of the rat reductase promoter has revealed a hitherto unnoticed cAMP-responsive element (CRE)-like octamer. This octamer is located between 53 and 60 nucleotides downstream of the sterol regulatory element 1; its first 6 nucleotides are identical to the consensus somatostatin CRE, and the entire octamer is identical to the fos CRE. A synthetic oligonucleotide containing the HMG-CoA reductase CRE-like octamer (RED CRE) formed protein-DNA complexes with nuclear extracts from FRTL-5 cells, which could be prevented by unlabeled CRE-containing oligonucleotides whose flanking sequences were otherwise nonidentical. The complexes were specifically supershifted by anti-CREB antibodies. FRTL-5 cells transfected with a fusion plasmid carrying the bacterial chloramphenicol acetyl transferase (CAT) under the control of the HMG-CoA reductase promoter displayed CAT activity, which was specifically stimulated by TSH. In contrast, CAT activity in FRTL-5 cells transfected with similar constructs carrying mutations in the reductase CRE was significantly lower and did not increase after TSH challenge. We suggest that the HMG-CoA reductase gene contains a functional CRE, important for TSH regulation of transcription. The data presented provide the molecular basis for a novel regulatory mechanism for HMG-CoA reductase gene expression in rat thyroid cells, which involves the direct effect of cAMP.
Bifulco
M
M
Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Università Federico II, Napoli, Italy.
Perillo
B
B
Saji
M
M
Laezza
C
C
Tedesco
I
I
Kohn
L D
LD
Aloj
S M
SM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000378:metabolism*; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015698:Genomic Library; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D013961:Thyroid Gland / Q000201:enzymology; D013972:Thyrotropin / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D013972:Thyrotropin; D000242:Cyclic AMP; D006903:Hydroxymethylglutaryl CoA Reductases; D015500:Chloramphenicol O-Acetyltransferase