Regulation of luteinizing hormone receptor gene expression by insulin-like growth factor-I in an immortalized murine Leydig tumor cell line (BLT-1)
It is postulated that insulin-like growth factor-I (IGF-I), a 70-amino acid mitogenic polypeptide, regulates Leydig cell steroidogenesis. In the present study, we assessed the effect of IGF-I on LH receptor (LHR) gene expression in an immortalized murine Leydig tumor cell line (BLT-1). Culture of BLT-1 cells in the presence of IGF-I (0.1-100 ng/ml) for 24 or 48 h increased their [125I]iodo-hCG binding in a dose-dependent manner up to 275% of the control level. Northern hybridization analysis revealed four major transcripts of LHR mRNA in BLT-1 cells (6.9, 2.6, 1.7, and 1.2 kilobases), and treatment at 10-100 ng/ml of IGF-I increased steady-state levels of LHR mRNAs in coordinate fashion up to 2. 2-fold. IGF-I (30 ng/ml) induced a time-dependent increase in [125I]hCG binding after a lag period of 2-6 h when studied up to 48 h, with a subsequent decrease. A similar response with steady increase up to 72 h was observed in total LHR mRNA. To elucidate the molecular mechanism of IGF-I action on LHR mRNA expression, we measured the transcription rate of the LHR gene by nuclear run-off assay and assessed transcript stability by the actinomycin D blocking method. The results showed that IGF-I treatment had no effect on the transcription rate of the LHR gene, whereas the half-life (t1/2) of LHR mRNA was significantly prolonged (IGF-I-treated cells, 30 +/- 3.8 h; controls, 17 +/- 2.5 h). Furthermore, IGF-I at 30 ng/ml and 100 ng/ml increased the expression of LHR promoter-driven luciferase and cytomegalovirus-promoter driven ss-galactosidase activities in BLT-1 cells; however, the former increased only marginally more than the latter. This suggests that the increase of LHR mRNA by IGF-I in Leydig cells is mainly due to increased mRNA stability.
Zhang
F P
FP
Department of Physiology, University of Turku, FIN-20520 Turku, Finland.
El-Hefnawy
T
T
Huhtaniemi
I
I
El-Hafnawy
T
T
D000818:Animals; D015152:Blotting, Northern; D006063:Chorionic Gonadotropin / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007457:Iodine Radioisotopes; D007700:Kinetics; D007984:Leydig Cell Tumor / Q000378:metabolism*; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011974:Receptors, LH / Q000235:genetics*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D006063:Chorionic Gonadotropin; D007457:Iodine Radioisotopes; D012333:RNA, Messenger; D011974:Receptors, LH; D007334:Insulin-Like Growth Factor I