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 nontransformed rat uterine stromal cell line (U(III)) that decidualizes spontaneously in culture. In this report, we have established by immunocytochemistry, RT-PCR, Western blot analysis, labeled amino acid incorporation and RIA that 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 fragment whose sequence is identical with that of rat pituitary PRL. In addition, U(III) cells express the PRL receptor (PRL-R) long form, all the components involved in the PRL signal transduction pathway, estrogen receptor beta (ER beta) 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 regulation of these genes was observed. Moreover, in these cells, the PRL signaling components: the tyrosine kinase Jak2 and the transcription factor Stat5 were endogenously phosphorylated and their phosphorylation states were not enhanced in the presence of exogenous PRL. To examine whether the endogenously secreted PRL affects the expression of PRL-regulated genes, U(III) cells were treated with either an anti-PRL receptor antibody or a Jak2 inhibitor, AG490. The anti-PRL receptor antibody decreased alpha(2)-MG expression. AG490 inhibited Jak2 and Stat5 phosphorylation, prevented Stat5 binding to its DNA consensus sequence, and also caused a dose-dependent down-regulation of alpha(2)-MG and ER beta expression. In contrast, AG490 enhanced PRL mRNA levels. In summary, we have established that the U(III) stromal cells of uterine origin produce PRL. Furthermore, we have shown for the first time that decidual PRL may act locally to activate the Jak2/Stat5 pathway and up-regulate important genes involved in decidual growth and placentation.
Prigent-Tessier
A
A
Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.
Barkai
U
U
Tessier
C
C
Cohen
H
H
Gibori
G
G
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000378:metabolism; D003656:Decidua / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047629:Estrogen Receptor beta; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D053614:Janus Kinase 2; D008894:Milk Proteins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011388:Prolactin / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000235:genetics; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000502:physiology; D015534:Trans-Activators / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology; D014599:Uterus / Q000166:cytology* / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000235:genetics
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D047629:Estrogen Receptor beta; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D011960:Receptors, Estrogen; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D020032:Tyrphostins; D000511:alpha-Macroglobulins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D011388:Prolactin; D011505:Protein-Tyrosine Kinases; C507927:Jak2 protein, rat; D053614:Janus Kinase 2