REGULATION OF FUNCTION OF THE MURINE LUTEINIZING-HORMONE RECEPTOR PROMOTER BY CIS-ACTING AND TRANS-ACTING ELEMENTS IN MOUSE LEYDIG TUMOR-CELLS

The transcriptional activity of various lengths of the 5'-untranslated region (UTR) of the murine LH receptor (R) gene were studied using th e luciferase reporter gene in transiently transfected mouse Leydig tum or cells (mLTC-1). Chinese hamster ovarian (CHO) and HeLa cells were u sed as controls. The basal transcriptional promoter activity in mLTC-1 cells resided within the first 173 base pairs (bp) of the 5'-UTR. Pla cing an LHR promoter fragment (bases - 715/ - 56) in front of the Herp es simplex virus thymidine kinase (TK) minimal promoter resulted in a 7-fold increase in luciferase activity. Deletion of bases - 56 to - 17 3 of the above construct totally abolished the increased luciferase ac tivity, brought about by the LHR promoter sequences. Basically similar results on LHR promoter function were observed using CHO cells. In co ntrast, no LHR promoter activity was detected in HeLa cells. indicatin g a cell specific nature of its function. The first 173 bp promoter do main is GC-rich, with several SP-1 binding domains, and it bound speci fically nuclear proteins isolated from mLTC-1 and HeLa cells. RNAse pr otection assays reconfirmed the presence of several transcription init iation sites within the first 310 bp of the 5'-UTR, also in the absenc e of the cognate LHR coding sequences. The most distal site at bp - 31 0 did not function in the absence of the first 173 bp of the 5'-UTR. O ther transcription initiation sites were identified closer to the tran slation initiation site, hCG (50 mu g/l), 8-bromo (Br)-cAMP (100 mu mo l/l) and cholera toxin (100 mu g/l) displayed qualitatively similar ne gative 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 decrease d in constructs containing less than or equal to 304 bp of the promote r region. Hence, the hCG/cAMP associated inhibitory effects interact w ith region(s) located mainly between bp - 565 and - 305 of the LHR pro moter. The inhibitory role of cAMP on LHR gene expression was also con firmed at the level of hCO-binding and hCG stimulated cAMP production of mLTC-1 cells. In conclusion, the current results elucidate the cis- and traits-acting elements in the regulation of expression of the mur ine LHR gene in cultured mouse Leydig cells. The minimal basal promote r activity is within the first 173 nucleotides of the 5'-UTR and the s tructural elements of the negative LHR regulation by the cognate hormo ne and elevated cAMP levels are mainly located within nucleotides - 30 5 to - 565 of the 5'-UTR. The function of the murine LHR promoter is s imilar to, though not identical with that of the rat, but at variance with that of the human LHR gene.