THE ROLE OF PLACENTAL LACTOGEN IN FETAL DEVELOPMENT

描述（由申请方提供）：催乳素（PRL）和胎盘催乳素（PL）刺激啮齿动物和人类的脂肪沉积、体重增加和瘦素产生，表明催乳素在母胎脂质代谢和肥胖中发挥作用。催乳素促进脂肪储存和体重增加的机制尚不清楚。我们假设：（a）催乳素通过诱导脂肪形成促进脂肪沉积，所述脂肪形成是脂肪细胞从基质前体分化的过程;（B）催乳素的脂肪形成作用是通过诱导包括PPARkhi-2的转录因子介导的;（c）PPARkhi-2诱导是通过激活Stat 5介导的;和（d）催乳素的成脂作用受胰岛素、IGF-1、糖皮质激素和性类固醇调节。为了验证这些假设，我们将研究外源性PRL和PL对3 T3-L1前脂肪细胞、原代小鼠前脂肪细胞和胚胎成纤维细胞中脂肪形成的影响，以及PL在一种新的3 T3-L1细胞系中组成性表达的影响。为了确定催乳信号是否是前脂肪细胞分化所必需的，我们将比较PRL受体（PRLR）缺陷小鼠前脂肪细胞的脂肪形成率与野生型同窝仔细胞的脂肪形成率。为了表征催乳素对PPARkhi表达的影响，我们将检测PRL和PL对3 T3-L1细胞、原代前脂肪细胞、小鼠胚胎成纤维细胞中PPARkhi 1和2 mRNA和蛋白水平的影响，并将比较PRLR缺陷小鼠组织中PPARkhi的表达与野生型同窝小鼠组织中PPARkhi的表达，我们将研究催乳素对在3 T3-T4细胞中表达的人和小鼠PPARkhi-2启动子的转录激活的影响。L1细胞，并将比较PRL处理细胞中PPARkhi 1 I和2 mRNA表达的时间过程与ADD 1和c/EBP β、δ和α的表达时间过程。为了检验PPARkhi-2的诱导是通过激活Stat 5介导的这一假设，我们将确定催乳素是否诱导STAT 5与人和mPPAR c-2启动子中的共有结合位点结合。我们将研究PPARkhi-2 Stat 5共有序列突变对催乳素依赖性激活的PPAR γ 2转录的影响，以及显性负性STAT 5构建体对3 T3-L1细胞中PPARkhi mRNA和蛋白水平诱导的影响。最后，为了检验催乳素的成脂作用受性类固醇调节的假设，我们将研究孕酮和雌二醇对3 T3-L 1细胞中PRL的成脂作用的影响，以及补充孕酮对PRL缺乏小鼠脂肪沉积和瘦素产生的影响。本研究结果为垂体和胎盘激素在脂肪发育和功能中的作用提供了新的理论依据。

DESCRIPTION (provided by applicant): Prolactin (PRL) and placental lactogen (PL) stimulate fat deposition, weight gain, and leptin production in rodents and humans, suggesting that lactogens play roles in maternofetal lipid metabolism and obesity. The mechanisms by which lactogens promote fat storage and weight gain are unknown. We hypothesize: (a) that lactogens promote fat deposition through induction of adipogenesis, the process of differentiation of adipocytes from stromal precursors; (b) that adipogenic effects of lactogens are mediated through induction of transcription factors including PPARkhi-2; (c) that PPARkhi-2 induction is mediated through activation of Stat5; and (d) that adipogenic effects of lactogens are modulated by insulin, IGF-1, glucocorticoids, and sex steroids. To test these hypotheses we will examine effects of exogenous PRL and PL on adipogenesis in 3T3-L1 preadipocytes, primary mouse preadipocytes and embryonic fibroblasts and the effects of constitutive expression of PL in a novel 3T3-L1 cell line. To determine if lactogenic signaling is required for preadipocyte differentiation, we will compare rates of adipogenesis in preadipocytes of PRL receptor (PRLR)-deficient mice with rates of adipogenesis in cells of wild-type littermates. To characterize effects of lactogens on PPARkhi expression we will examine effects of PRL and PL on PPARkhi1 and 2 mRNA and protein levels in 3T3-L1 cells, primary preadipoyctes, mouse embryonic fibroblasts and will compare PPARkhi expression in tissues of PRLR deficient mice with that in wild-type littermates, We will examine effects of lactogens on transcriptional activation of the human and mouse PPARkhi-2 promoters expressed in 3T3-L1 cells and will compare the time course of expression of PPARkhi1I and 2 mRNAs in PRL-treated cells with that of ADD1 and c/EBPs beta, delta, and alpha. To test the hypothesis that induction of PPARkhi-2 is mediated through activation of Stat5, we will determine if lactogens induce binding of STAT 5 to consensus binding sites in the human and mPPAR c-2 promoters. We will examine the effect of mutating PPARkhi-2 Stat5 consensus sequences on lactogen-dependent activation of PPAR gamma2 transcription, and the effect of a dominant-negative STAT5 construct on the induction of PPARkhi mRNA and protein levels in 3T3-L1 cells. Finally, to test the hypothesis that the adipogenic effects of the lactogens are modulated by sex steroids, we will examine the effects of progesterone and estradiol on the adipogenic effects of PRL in 3T3-L 1 cells and the effects of progesterone supplementation on fat deposition and leptin production in PRLR-deficient mice. The results of our studies should provide new insist into the roles of pituitary and placental hormones in adipose development and function.