Zhang F P, El-Hefnawy T, Huhtaniemi I
Department of Physiology, University of Turku, FIN-20520 Turku, Finland.
Biol Reprod. 1998 Nov;59(5):1116-23. doi: 10.1093/biolreprod/59.5.1116.
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.
据推测，胰岛素样生长因子-I（IGF-I），一种含70个氨基酸的促有丝分裂多肽，可调节睾丸间质细胞的类固醇生成。在本研究中，我们评估了IGF-I对永生化小鼠睾丸间质细胞瘤细胞系（BLT-1）中促黄体生成素受体（LHR）基因表达的影响。在IGF-I（0.1 - 100 ng/ml）存在的情况下培养BLT-1细胞24或48小时，其[125I]碘 - hCG结合能力以剂量依赖方式增加，最高可达对照水平的275%。Northern杂交分析显示BLT-1细胞中有四种主要的LHR mRNA转录本（6.9、2.6、1.7和1.2千碱基），用10 - 100 ng/ml的IGF-I处理可使LHR mRNA的稳态水平协同增加，最高可达2.2倍。当研究长达48小时时，IGF-I（30 ng/ml）在2 - 6小时延迟期后诱导[125I]hCG结合呈时间依赖性增加，随后下降。在总LHR mRNA中观察到类似的反应，直至72小时持续稳定增加。为阐明IGF-I对LHR mRNA表达作用的分子机制，我们通过核转录分析测量了LHR基因的转录速率，并通过放线菌素D阻断法评估转录本稳定性。结果表明，IGF-I处理对LHR基因的转录速率无影响，而LHR mRNA的半衰期（t1/2）显著延长（IGF-I处理的细胞为30 ± 3.8小时；对照为17 ± 2.5小时）。此外，30 ng/ml和100 ng/ml的IGF-I增加了BLT-1细胞中LHR启动子驱动的荧光素酶和巨细胞病毒启动子驱动的β - 半乳糖苷酶活性；然而，前者仅比后者略有增加。这表明IGF-I使睾丸间质细胞中LHR mRNA增加主要是由于mRNA稳定性增加。
