Prolactin (PRL) is an anterior pituitary hormone that regulates a variety of processes including lactation, immune response and reproduction. In the ovary PRL causes an increase in protein synthesis in the corpus luteum and sustains progesterone secretion. Although PRL stimulates the overall synthesis of proteins in the corpus luteum, it has a rather drastic inhibitory effect on few specific proteins within the 37-kD range. The 37 kD plays an important role in the tropic action of PRL in the corpus luteum. This protein with activity of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) has a pivotal role in the termination of pregnancy and luteolysis. The overall goal of this investigation is to clarify the mechanism by which PRL transduces its message and inhibits the 20alpha-HSD gene activity.
Aim 1. To examine the time course and dose response of PRL action on 20alpha-HSD gene expression. Luteinized granulosa cell culture will best suit these purposes. These cells express both 20alpha-HSD and the PRL receptor genes. Cells will be treated with different concentrations of PRL. Northern and Western analysis will be performed on the cells after different treatments to examine levels of 20alpha-HSD mRNA and protein.
Aim 2. To examine whether JAK2 and Stat 91 are respectively the kinase and the transcriptional factor(s) associated with PRL signaling. JAK kinases and Stat 91 have been shown to be involved in the signal transduction of interferon-alpha, gamma and other members in the cytokine/erythropoietin family. PRL receptor (PRL-R) is also a member of this family, it is quite possible that it transduces its signal through similar pathway.
The aims of these experiments will be: a) To find if PRL transduces its signal through JAK2 kinase, luteinized cells will be cultured in the absence or presence of PRL. Cell lysates will be immunoprecipitated with anti-JAK2. b) To further examine the relationship between PRL-R and JAK2, cell lysates obtained from cells treated +/- PRL will be immunoprecipitated with either anti-PRL-R or anti-JAK2. c) Phosphorylation of the PRL receptor and the non-receptor tyrosine kinases and other cellular proteins can be further demonstrated by in vivo phosphorylation. Cells will be incubated with PRL and [gamma- 32P]ATP, cell lysates will be subjected to immunoprecipitation with PRL-R antibody, JAK2 and Stat 91 antibodies and electrophoresis and autoradiography.
