Characterization of a rat uterine cell line, U-III cells: Prolactin (PRL) expression and endogenous regulation of PRL-dependent genes; estrogen receptor beta, alpha(2)-macroglobulin, and decidual PRL involving the Jak2 and Stat5 pathway

Decidualization of endometrial stroma in the rat induces the expression and secretion of rat decidual PRL (rdPRL). Recently, we have generated a nontr ansformed rat uterine stromal cell line (U-III) that decidualizes spontaneo usly in culture. In this report, we have established by immunocytochemistry , RT-PCR, Western blot analysis, labeled amino acid incorporation and RIA t hat these cells express the rat PRL messenger RNA as well as synthesize and secrete PRL. We have also cloned by RT-PCR a 403-bp complementary DNA frag ment whose sequence is identical with that of rat pituitary PRL. In additio n, U-III cells express the PRL receptor (PRL-R) long form, all the componen ts involved in the PRL signal transduction pathway, estrogen receptor beta (ERP) and alpha (2)-macroglobulin (alpha (2)-MG), which are known to be PRL -regulated genes. However, when U-III, cells were treated with PRL, no regu lation of these genes was observed. Moreover, in these cells, the PRL signa ling components: the tyrosine kinase Jak2 and the transcription factor Stat 5 were endogenously phosphorylated and their phosphorylation states were no t enhanced in the presence of exogenous PRL. To examine whether the endogen ously secreted PRL affects the expression of PRL-regulated genes, U-III cel ls were treated with either an anti-PRL receptor antibody or a Jak2 inhibit or, AG490. The anti-PRL receptor antibody decreased alpha (2)-MG expression . AG490 inhibited Jak2 and Stat5 phosphorylation, prevented Stat5 binding t o its DNA consensus sequence, and also caused a dose-dependent down-regulat ion of alpha (2)-MG and ERP expression. In contrast, AG490 enhanced PRL mRN A levels. In summary, we have established that the U-III stromal cells of u terine origin produce PRL. Furthermore, we have shown for the first time th at decidual PRL may act locally to activate the Jak2/Stat5 pathway and up-r egulate important genes involved in decidual growth and placentation.