Activation of PKC d in the Rat Corpus Luteum during Pregnancy

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 d, is dramatically increased. We therefore assessed whether prolactin (PRL) receptor activation promotes activation of PKC d in a luteinized granulosa cell model. We also assessed the activation status of PKC d in corpora lutea obtained when the corpus luteum is exposed to chronically high concentrations of rPLs. The activity of PKC d was assessed by two means: an immune complex (IC) assay and Western blotting with a phospho-epitope-specific antibody that detects PKC d phosphorylated on serine 662. PKC d activation in the IC kinase assay was determined by the ability of immunoprecipitated PKC d to phosphorylate the PKC d-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 d. Similarly, immunoreactivity with the phospho-epitopespecific PKC d antibody was increased in extracts prepared from luteinized granulosa cells treated with phorbol myristate acetate or following in vitro activation of recombinant PKC d. Using these assays, we assessed whether PRL receptor agonists were capable of activating PKC d in luteinized granulosa cells. PRL receptor agonists induced translocation PKC d from the cytosolic to the Triton-soluble membrane fraction and increased PKC d activity assessed by both IC kinase assay and Western blotting with phospho-epitope-specific PKC d antibody. Analysis of PKC d activity in corpora lutea obtained during pregnancy by both the IC kinase assay and Western blotting with the phospho-epitope-specific PKC d antibody revealed that PKC d activity was increased throughout the second half of pregnancy. These results demonstrate that PRL receptor activation promotes the acute activation of PKC d in luteinized rat granulosa cells. At a time when the rat is exposed to chronically high concentrations of rPLs, PKC d is increasingly expressed and active.