Induction of relaxin messenger RNA expression in response to prolactin receptor activation requires protein kinase C delta signaling

The ability of PRL or rat placental lactogen (rPL)-1 to induce relaxin mRNA expression was analyzed in a luteinized rat granulosa cell culture model. PRL receptor activation induced relaxin mRNA expression in a concentration- and time-dependent manner. High concentrations of PRL receptor agonist, eq uivalent to those of the second half of pregnancy in rats, were required to elicit relaxin mRNA expression. A 40-fold induction of relaxin mRNA was ob served in cells treated 24 h with 1 mu g/ml of rPL-1. Estrogen enhanced rel axin expression induced by PRL but did not affect relaxin expression on its own. PRL/rPL-1 induction of relaxin expression was independent of the extr acellular regulated kinase (ERK) members of the mitogen-activated protein k inase (MAPK) pathway, based on the inability of the ERK kinase inhibitor PD 98059 to block induction of relaxin expression. PRL/rPL-1 induction of rela xin expression required protein kinase C (PKC) delta, based on the ability of the preferential PKC delta inhibitor rottlerin to abolish induction of r elaxin expression. Direct activation of PKC by phorbol myristate acetate, h owever, was not sufficient to promote induction of relaxin mRNA expression. Stats (signal transducers and activators of transcription) 3 and 5 DNA bin ding activities were induced by PRL/rPL-1 treatment of luteinized granulosa cells but only Stat 3 DNA binding was reduced by rottlerin. PRL/rPL-1 trea tment of luteinized granulosa cells resulted in increased phosphorylation o n tyrosine-705 and serine-727 of Stat 3, and these responses were reduced a nd blocked, respectively, by rottlerin. Tyrosine and serine phosphorylation s of Stat 3 in the corpus luteum were also increased in the second half of pregnancy when pi, levels are highest. Stat 3, but not Stat 1 or 5, coimmun oprecipitated with luteal PKC delta during pregnancy; Stat 3 transiently co immunoprecipitated with PKC delta from luteinized granulosa cells in respon se to PRL receptor activation; and Stat 3/PKC delta complex formation requi red PKC delta kinase activity. Taken together, these results show that PKC delta is obligatory for PRL/rPL-1-dependent relaxin expression, that PKC de lta complexes with Stat 3 in response to PRL receptor activation, and that PKC delta is involved in the regulation of Stat 3 phosphorylation downstrea m of the PRL receptor. These results demonstrate that PRL/rPL-1 promotes re laxin expression in luteal cells and that this event is mediated, at least in part, via PKC delta.