PROLACTIN AND BILE SECRETORY FUNCTION

本提案的目的是表征两个关键转运蛋白的激素调节机制，这两个转运蛋白对维持肝脏胆汁分泌功能至关重要。这项研究建立在我们的新的和令人兴奋的发现，在产后期间，垂体前叶激素催乳素（PRL）增加的活性Na+/牛磺胆酸盐（TC）共转运蛋白（ntcp）在基底外侧域，和ATP依赖性TC转运蛋白，最近被确定为P-糖蛋白（spgp）的妹妹在小管域的肝细胞。 PRL诱导的ntcp转录增加由长型PRL受体介导，并由Jak 2-Stat 5信号转导途径和ntcp启动子中的两个Stat 5识别序列（GLE）转导。目的1将确定其他Stat蛋白（1，3，5a，5 b）和该激素家族的其他成员（即，生长激素和胎盘催乳素）调节HepG 2细胞培养物中和/或哺乳期母鼠体内ntcp和spgp表达。 目的2将确定雌二醇抑制PRL介导的ntcp表达增加的机制，并确定spgp表达是否受到类似的调节。具体地说，我们将检验以下假设：雌二醇通过雌激素受体起作用，并且1）结合ntcp启动子中的半雌激素反应元件以防止PRL介导的信号转导，或2）竞争有限的核辅激活因子库，例如，CBP/P300是转录起始所必需的。 目的3探讨泌乳素在产后分泌、输注、泌乳素或生长激素是否通过增加基因转录而增加ntcp和spgp mRNA和蛋白的表达。 用于实现这些目标的方法包括在HepG 2细胞培养模型系统中的转染研究、在非妊娠对照和产后大鼠中的体内研究、在用绵羊PRL和/或雌二醇治疗的卵巢切除大鼠中的体内研究以及用生长激素治疗的hypophysectomized大鼠中的体内研究。 催乳素是唯一的生理机制，确定增加胆汁酸转运蛋白的表达。 这些PRL作用机制的表征为开发用于治疗人类胆汁淤积性肝病的选择性治疗干预提供了机会，例如由雌激素和早产儿诱导的胆汁淤积性肝病。

The objectives of the present proposal are to characterize the mechanisms of hormonal regulation of two key transporters essential for the maintenance of hepatic bile secretory function. The research proposed builds on our novel and exciting finding that in the postpartum period, the anterior pituitary hormone prolactin (PRL) increases the activity of both the Na+/taurocholate (TC) cotransporter (ntcp) in the basolateral domain, and the ATP-dependent TC transporter, recently identified as the sister of P-glycoprotein (spgp) in the canalicular domain of the hepatocyte. The PRL-induced increase in ntcp transcription is mediated by the long form of the PRL receptor and is transduced by the Jak2-Stat5 signal transduction pathway and two Stat5 recognition sequences (GLEs) in the ntcp promoter. Aim 1 will determine the role of other Stat proteins (1, 3, 5a, 5b) and other members of this hormone family (i.e., growth hormone and placental lactogen) in regulating ntcp and spgp expression in HepG2 cell culture and/or in vivo in lactating dams. Aim 2 will identify the mechanism(s) by which estradiol inhibits the PRL-mediated increase in ntcp expression, and determine if spgp expression is similarly regulated. Specifically, we will test the hypotheses that estradiol acts via the estrogen receptor and 1) binds the half-estrogen response elements in the ntcp promoter to prevent PRL-mediated signal transduction, or 2) competes for a limited pool of nuclear coactivators, e.g., CBP/P300, which are essential for initiation of transcription. Aim 3 will determine if PRL secretion in the postpartum period or infusion or PRL or growth hormone increase expression of ntcp and spgp mRNA and protein by increasing gene transcription. Approaches used to achieve these goals include transfection studies in a HepG2 cell culture model system, studies in vivo in nonpregnant control and postpartum rats, in ovariectomized rats treated with ovine PRL and/or estradiol and hyphophysectomized rats treated with growth hormone. PRL is the only physiological mechanism identified which increases expression of bile acid transporters. Characterization of the mechanism of these PRL actions presents the opportunity for development of selective therapeutic interventions for the treatment of human cholestatic liver disease, such as that induced by estrogens and in prematurity.