Expression of luteinising hormone-β subunit chloramphenicol acetyltransferase (LH-β-CAT) fusion gene in rat pituitary cells: Induction by cyclic 3′-adenosine monophosphate (cAMP)

Abstract

In this study we determined the activity of the rat luteinising hormone-β gene promoter in a heterologous rat pituitary cell line (GH 3 cells). 1.7 kb of LH-β 5′ flanking sequence and the first 5 bp of the 5′ untranslated region were ligated to the chloramphenicol acetyltransferase (CAT) receptor gene (LH-β-CAT) and transiently transfected by calcium phosphate precipitation into subconfluent cultures of GH 3 cells. Basal low-level CAT activity was only detected in GH 3 cells, being absent in two non-pituitary cell lines (BeWo and HeLa) RNase analysis revealed that mRNA from transfected GH 3 cells protected a fragment of labelled antisense probe of correct size for transcription initiation from the LH-β CAP site, confirming that promoter activity reflected correctly initiated LH-β-CAT fusion gene transcripts. CAT activity was consistently induced by an average of 3–5-fold from the full-length 1.7 kb promoter, in a dose- and time-dependent manner, by forskolin, dibutyryl cAMP, and 8-bromo cAMP implying presence of a cAMP-responsive cis-acting domain in the LH-β promoter region. Transfection of deletion mutants Δ-615-CAT, Δ-385-CAT and Δ-250-CAT each reduced forskolin inducibility to 1.7-fold but did not abolish induction completely suggesting a domain between −1.7 and −0.6 kb contained a cAMP-responsive element(s) (CRE). Further deletion of LH-β 5′ flanking sequences to Δ-85-CAT restored forskolin induction to wild-type levels (3–5-fold), suggesting the presence of a weak inhibitory element between −600 and −85 kb, and a cAMP-responsive domain in the proximal promoter region. The LH-β promoter does not contain perfect tandem repeat palindromic CRE DNA sequences, though there are several octanucleotide sequences differing by only 1 bp from AP-2 binding sites, the consensus CRE, and the vasointestinal peptide gene CRE. Although these data suggest that the LH-β gene is cAMP responsive this is likely mediated by several and complex protein interactions with multiple DNA sequences in the proximal and distal LH-β promoter enhancer.