Abstract

Maintenance of pregnancy in the rat requires the corpus luteum. At a time when rat placental lactogens (rPLs) are required to support progesterone production by the corpus luteum and when relaxin expression is initiated, expression of a specific protein kinase C (PKC) isoform, PKC delta, is dramatically increased. We therefore assessed whether prolactin (PRL) receptor activation promotes activation of PKC delta in a luteinized granulosa cell model. We also assessed the activation status of PKC delta in corpora lutea obtained when the corpus luteum is exposed to chronically high concentrations of rPLs. The activity of PKC delta was assessed by two means: an immune complex (IC) assay and Western blotting with a phospho-epitope-specific antibody that detects PKC delta phosphorylated on serine 662. PKC delta activation in the IC kinase assay was determined by the ability of immunoprecipitated PKC delta to phosphorylate the PKC delta-preferential substrate small heat shock protein (HSP-27). Treatment of luteinized rat granulosa cells with phorbol myristate acetate, a known activator of PKC, promoted a 7-fold increase in HSP-27 phosphorylation by PKC delta. Similarly, immunoreactivity with the phospho-epitope-specific PKC delta antibody was increased in extracts prepared from luteinized granulosa cells treated with phorbol myristate acetate or following in vitro activation of recombinant PKC delta. Using these assays, we assessed whether PRL receptor agonists were capable of activating PKC delta in luteinized granulosa cells. PRL receptor agonists induced translocation PKC delta from the cytosolic to the Triton-soluble membrane fraction and increased PKC delta activity assessed by both IC kinase assay and Western blotting with phospho-epitope-specific PKC delta antibody. Analysis of PKC delta activity in corpora lutea obtained during pregnancy by both the IC kinase assay and Western blotting with the phospho-epitope-specific PKC delta antibody revealed that PKC delta activity was increased throughout the second half of pregnancy. These results demonstrate that PRL receptor activation promotes the acute activation of PKC delta in luteinized rat granulosa cells. At a time when the rat is exposed to chronically high concentrations of rPLs, PKC delta is increasingly expressed and active.

Highlights

  • Maintenance of pregnancy in the rat requires the corpus luteum

  • Analysis of protein kinase C (PKC) ␦ activity in corpora lutea obtained during pregnancy by both the immune complex (IC) kinase assay and Western blotting with the phospho-epitope-specific PKC ␦ antibody revealed that PKC ␦ activity was increased throughout the second half of pregnancy

  • We have shown that PRL receptor activation promotes relaxin expression by luteinized rat granulosa cells and that PRL-dependent relaxin expression is abrogated by the PKC ␦-specific inhibitor rottlerin

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Summary

POTENTIAL ROLE OF PROLACTIN SIGNALING*

Analysis of PKC ␦ activity in corpora lutea obtained during pregnancy by both the IC kinase assay and Western blotting with the phospho-epitope-specific PKC ␦ antibody revealed that PKC ␦ activity was increased throughout the second half of pregnancy These results demonstrate that PRL receptor activation promotes the acute activation of PKC ␦ in luteinized rat granulosa cells. We have found that rPL-1 treatment of luteinized granulosa cells induces phosphorylation of Stat 3 on both tyrosine 705 and serine 727 and induction of relaxin mRNA expression, a major product of the rat corpus luteum in the second half of pregnancy [21]. RPL-1-induced Stat 3 serine phosphorylation and relaxin mRNA expression, we seek direct evidence (a) that PRL receptor activation by rPL-1 activates PKC ␦ in a luteinized granulosa cell model and (b) that PKC ␦ is active in an in vivo setting in the corpus luteum of pregnancy, coincident with high rPLs in serum of rats. These results implicate the PRL signaling pathway in the activation of PKC ␦ in corpora lutea of pregnancy

EXPERIMENTAL PROCEDURES
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