Concerted regulation of low density lipoprotein receptor gene expression by follicle-stimulating hormone and insulin-like growth factor I in porcine granulosa cells: Promoter activation, messenger ribonucleic acid stability, and sterol feedback
Concerted regulation of low density lipoprotein receptor gene expression by follicle-stimulating hormone and insulin-like growth factor I in porcine granulosa cells: Promoter activation, messenger ribonucleic acid stability, and sterol feedback
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DOI：
10.1210/en.140.1.178
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发表时间：
1999-01-01
期刊：
影响因子：
4.8
通讯作者：
Veldhuis, JD
中科院分区：
文献类型：
--
作者：
LaVoie, HA;Garmey, JC;Veldhuis, JD
胰岛素样生长因子I(TGF-I)和促性腺激素FSH可以协同刺激成熟颗粒细胞原代培养中孕酮的产生。这些营养激素增加了颗粒细胞对低密度脂蛋白(LDL)受体的结合和内化，以及对低密度脂蛋白携带的胆固醇的利用。为了确定IGF-I和FSH是否以及如何控制低密度脂蛋白受体的基因组表达，我们评估了它们在第一代猪颗粒细胞单层(无血清)培养中对低密度脂蛋白受体信使RNA(MRNA)积累、稳定性和基因启动子活性的单独和协同作用。核糖核酸酶保护实验显示，重组人IGF-I(100 ng/ml)、FSH(25 ng/mlNIDDK sFSH-20)或二者合用可使低密度脂蛋白受体基因表达增加2.2倍、2.6倍和4.6倍(P<；0.01)。同时加入低密度脂蛋白底物(50微克/毫升)，可抑制激素刺激的低密度脂蛋白受体基因表达的54-75%。即使在低密度脂蛋白存在的情况下，FSH和IGF-I的组合也显著延长了消息的半衰期。采用快速扩增cDNA5‘-末端、连接接头基因组DNA的PCB板、Southern杂交和DNA测序相结合的方法，分离了猪低密度脂蛋白受体基因编码区上游的1076个碱基。在瞬时转染法中，用pLDLR1076/荧光素酶质粒构建，FSH、FSH加IGF-I或8-bromo-cAMP(1 MM)处理(但不是单独的IGF-I)可使猪颗粒细胞的荧光素酶报告基因活性增加10-23倍。在无血清培养中，随着时间的推移，低密度脂蛋白受体启动子的基础活性增加，并最终超过激素刺激的作用，但在24小时被LBL底物抑制(75%)。上述刺激激素作用和类固醇抑制被定位在猪启动子的-255至-139之间的116个碱基区。我们得出结论，FSH和IGF-I联合应用可以诱导培养的颗粒细胞中低密度脂蛋白受体mRNA的积聚，即使在激素负反馈的情况下也是如此，并且可以通过激活启动子和增加mRNA的稳定性来实现这一机制。
Insulin-like growth factor I(TGF-I) and the gonadotropin, FSH, can synergize to stimulate progesterone production in primary cultures of maturing granulosa cells. These trophic hormones increase law density lipoprotein (LDL) receptor binding and internalization, and the utilization of LDL-borne cholesterol by granulosa cells. To determine whether and how IGF-I and FSH control the genomic expression of the LDL receptor, we evaluated their individual and concerted effects on LDL receptor messenger RNA (mRNA) accumulation, stability, and gene promoter activity in first passage monolayer (serum-free) cultures of porcine granulosa cells. Ribonuclease protection assays revealed that LDL receptor mRNA accumulation was increased by human recombinant IGF-I(100 ng/ml), FSH (25 ng/ml NIDDK sFSH-20), or their combination by 2.2-, 2.6-, and 4.6-fold, respectively (P < 0.01). Hormonally stimulated LDL receptor mRNA accumulation was suppressed by 54-75% by the concurrent addition of LDL substrate (50 mu g/ml). The combination of FSH and IGF-I significantly prolonged the message half-life, even in the presence of LDL. Using a combination of rapid amplification of cDNA 5'-ends, PCB with adapter-ligated genomic DNA, Southern hybridization, and DNA sequencing, we isolated 1076 bp of the porcine LDL receptor gene upstream of the coding region. In transient transfection assays, with a pLDLR1076/luciferase plasmid construct, FSH, FSH plus IGF-I, or 8-bromo-cAMP (1 mM) treatment (but not IGF-I alone) increased luciferase reporter gene activity by 10- to 23-fold in porcine granulosa cells. Over time in serum-free culture, the basal activity of the LDL receptor gone promoter increased and eventually surpassed hormone-stimulated effects, but was suppressed by LBL substrate (by 75%) at 24 h. The foregoing stimulatory hormone effects and sterol repression were localized to a 116-bp region in the porcine promoter between -255 and -139 upstream of the translational start site. We conclude that the combination of FSH and IGF-I can induce accumulation of LDL receptor mRNA in cultured granulosa cells Even in the presence of sterol negative feedback and can do so mechanistically by a combination of promoter activation and increased mRNA stability.