Regulation of function of the murine luteinizing hormone receptor promoter by cis- and trans-acting elements in mouse Leydig tumor cells

Abstract

The transcriptional activity of various lengths of the 5′-untranslated region (UTR) of the murine LH receptor (R) gene were studied using the luciferase reporter gene in transiently transfected mouse Leydig tumor cells (mLTC-1). Chinese hamster ovarian (CHO) and HeLa cells were used as controls. The basal transcriptional promoter activity in mLTC-1 cells resided within the first 173 base pairs (bp) of the 5′-UTR. Placing and LHR promoter fragment (bases −715 −56 ) in front of the Herpes simplex virus thymidine kinase (TK) minimal promoter resulted in a 7-fold increase in luciferase activity. Deletion of bases −56 to −173 of the above construct totally abolished the increased luciferase activity, brought about by the LHR promoter sequences. Basically similar results on LHR promoter function were observed using CHO cells. In contrast, no LHR promoter activity was detected in HeLa cells, indicating a cell specific nature of its function. The first 173 bp promoter domain is GC-rich, with several SP-1 binding domains, and it bound specifically nuclear proteins isolated from mLTC-1 and HeLa cells. RNAse protection assays reconfirmed the presence of several transcription initiation sites within the first 310 bp of the 5′-UTR, also in the absence of the cognate LHR coding sequences. The most distal site at bp −310 did not function in the absence of the first 173 bp of the 5′-UTR. Other transcription initiation sites were identified closer to the translation initiation site. hCG (50 μg/l), 8-bromo (Br)-cAMP (100 μmol/l) and cholera toxin (100 μg/l) displayed qualitatively similar negative effects on the LHR promoter activity in the transfected mLTC-1 cells when the constructs containing at least the first 565 bp of the LHR 5′-UTR were used, but the inhibitory effects were greatly decreased in constructs containing ≤304 bp of the promoter region. Hence, the hCG cAMP associated inhibitory effects interact with region(s) located mainly between bp −565 and −305 of the LHR promoter. The inhibitory role of cAMP on LHR gene expression was also confirmed at the level of hCO-binding and hCG stimulated cAMP production of mLTC-1 cells. In conclusion, the current results elucidate the cis- and trans-acting elements in the regulation of expression of the murine LHR gene in cultured mouse Leydig cells. The minimal basal promoter activity is within the first 173 nucleotides of the 5′-UTR and the structural elements of the negative LHR regulation by the cognate hormone and elevated cAMP levels are mainly located within nucleotides −305 to −565 of the 5′-UTR. The function of the murine LHR promoter is similar to, though not identical with that of the rat, but at variance with that of the human LHR gene.