THE ROLE OF PLACENTAL LACTOGEN IN FETAL DEVELOPMENT

描述（由申请人提供）：催乳素（PRL）和胎盘乳原（PL）刺激啮齿动物和人类的脂肪沉积、体重增加和瘦素产生，表明乳原在母胎脂质代谢和肥胖中发挥作用。乳原促进脂肪储存和体重增加的机制尚不清楚。我们假设：(a)乳原通过诱导脂肪生成（脂肪细胞从基质前体分化的过程）促进脂肪沉积；(b)乳原的致脂作用是通过诱导包括PPARkhi-2在内的转录因子介导的；(c) PPARkhi-2的诱导是通过激活Stat5介导的；(d)乳原的成脂作用是由胰岛素、IGF-1、糖皮质激素和性类固醇调节的。为了验证这些假设，我们将研究外源性PRL和PL对3T3-L1前脂肪细胞、原代小鼠前脂肪细胞和胚胎成纤维细胞脂肪形成的影响，以及PL在一种新型3T3-L1细胞系中组成性表达的影响。为了确定前脂肪细胞分化是否需要乳源性信号，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幼崽细胞的脂肪形成率。为了表征乳原对PPARkhi表达的影响，我们将研究PRL和PL对3T3-L1细胞、原代前脂肪细胞、小鼠胚胎成纤维细胞中PPARkhi1和2 mRNA和蛋白水平的影响，并将PRLR缺陷小鼠组织中的PPARkhi1和2 mRNA和蛋白水平与野生型幼崽的PPARkhi表达进行比较。我们将研究乳酸原对人类和小鼠3T3-L1细胞中表达的PPARkhi1I和pparkhi2启动子转录激活的影响，并比较prl处理细胞中PPARkhi1I和pparkhi2 mrna与ADD1和c/ ebp β， δ和α的表达时间过程。为了验证PPARkhi-2的诱导是通过激活Stat5介导的假设，我们将确定乳原是否诱导Stat5与人类和mPPAR c-2启动子的一致结合位点结合。我们将研究突变PPARkhi-2 Stat5一致序列对PPAR gamma2转录的乳酸依赖激活的影响，以及在3T3-L1细胞中，显性阴性Stat5结构对诱导PPARkhi mRNA和蛋白水平的影响。最后，为了验证乳原的成脂作用是由性类固醇调节的假设，我们将研究孕酮和雌二醇对3t3 - l1细胞中PRL的成脂作用的影响，以及孕酮补充对prlr缺陷小鼠脂肪沉积和瘦素产生的影响。我们的研究结果将为垂体和胎盘激素在脂肪发育和功能中的作用提供新的坚持。

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.