El-Hefnawy T, Krawczyk Z, Nikula H, Viherä I, Huhtaniemi I
Department of Physiology, University of Turku, Finland.
Mol Cell Endocrinol. 1996 May 31;119(2):207-17. doi: 10.1016/0303-7207(96)03815-4.
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 an 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 micrograms/l), 8-bromo (Br)-cAMP (100 mumol/l) and cholera toxin (100 microgram/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 < or = 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.
利用荧光素酶报告基因，在瞬时转染的小鼠睾丸间质细胞瘤细胞（mLTC-1）中研究了小鼠促黄体生成素受体（R）基因不同长度5'-非翻译区（UTR）的转录活性。以中国仓鼠卵巢（CHO）细胞和人宫颈癌细胞（HeLa）作为对照。mLTC-1细胞中的基础转录启动子活性位于5'-UTR的前173个碱基对（bp）内。将促黄体生成素受体（LHR）启动子片段（碱基-715 / -56）置于单纯疱疹病毒胸苷激酶（TK）最小启动子之前，可使荧光素酶活性增加7倍。删除上述构建体中-56至-173的碱基，完全消除了LHR启动子序列引起的荧光素酶活性增加。使用CHO细胞观察到关于LHR启动子功能的基本相似结果。相反，在HeLa细胞中未检测到LHR启动子活性，表明其功能具有细胞特异性。前173 bp的启动子结构域富含GC，具有多个SP-1结合结构域，并且它与从mLTC-1和HeLa细胞中分离的核蛋白特异性结合。RNA酶保护试验再次证实，在5'-UTR的前310 bp内也存在多个转录起始位点，即使不存在同源LHR编码序列。在没有5'-UTR的前173 bp的情况下，bp -310处最远端的位点不起作用。在更靠近翻译起始位点处鉴定出其他转录起始位点。当使用包含至少LHR 5'-UTR的前565 bp的构建体时，表示定性地类似对转染的mLTC-1细胞中的LHR启动子活性的负面影响，但在含有≤304 bp启动子区域的构建体中，抑制作用大大降低。因此，hCG / cAMP相关的抑制作用与主要位于LHR启动子的bp -565和-305之间的区域相互作用。cAMP对LHR基因表达的抑制作用也在mLTC-1细胞的hCG结合和hCG刺激的cAMP产生水平上得到证实。总之，目前的结果阐明了培养的小鼠睾丸间质细胞中小鼠LHR基因表达调控中的顺式和反式作用元件。最小基础启动子活性在5'-UTR的前173个核苷酸内，同源激素和升高的cAMP水平对LHR负调控的结构元件主要位于5'-UTR的核苷酸-305至-565内。小鼠LHR启动子的功能与大鼠的相似，但不完全相同，与人LHR基因的功能不同。
