Developmental regulation of mitogen-activated protein kinase-activated kinases-2 and-3 (MAPKAPK-2/-3) in vivo during corpus luteum formation in the rat

The current study investigates the activation in vivo and regulation of the expression of components of the p38 mitogen-activated protein kinase (MAPK ) pathway during gonadotropin-induced formation and development of the rat corpus luteum, employing a sequential PMSG/human CG (hCG) treatment paradig m. We postulated that the p38 MAPK pathway could serve to promote phosphory lation of key substrates during luteal maturation, since maturing luteal ce lls, thought to be cAMP-nonresponsive, nevertheless maintain critical phosp hoproteins. Both p38 MAPK and its upstream activator MAPK kinase-6 (MKKG) w ere found to be chronically activated during the luteal maturation phase, w ith activation detected by 24 h post hCG and maintained through 4 days post hCG. The p38 MAPK downstream protein kinase target termed MAPK-activated p rotein kinase-3 (MAPKAPK-3) was newly induced at both mRNA and protein leve ls during luteal formation and maturation, while mRNA and protein expressio n of the closely related MAPKAPK-2 diminished. Two potential substrates for MAPKAPKs, the small heat shock protein HSP-27 and the cAMP regulatory elem ent binding protein CREB, were monitored in vivo for phosphorylation. HSP-2 7 phosphorylation was not modulated during luteal maturation. In contrast, we observed sustained luteal-phase CREB phosphorylation in vivo, consistent with upstream MKK6/p38 MAPK activation and MAPKAPK-3-induction. MAPKAPK-3- specific immune complex kinase assays provided direct evidence that MAPKAPK -3 was in an activated state during luteal maturation in vivo. Cellular inh ibitor studies indicated that an intact p38 MAPK path was required for CREB phosphorylation in a cellular model of luteinization, as treatment of lute inized granulosa cells with the p38 MAPK inhibitor SE 203580 strongly inhib ited CREB phosphorylation. Transient transfection studies provided direct e vidence that MAPKAPK-3 was capable of signaling to activate CREB transcript ional activity, as assessed by means of GAL4-CREB fusion protein construct coexpressed with GAL4-luciferase reporter construct. Introduction of wild-t ype, but not kinase-dead mutant, MAPKAPK-3 cDNA, into a mouse ovarian cell line stimulated GAL4-CREB-dependent transcriptional activity approximately 3-fold. Thus MAPKAPK-3 is indeed uniquely poised to support luteal maturati on through the phosphorylation and activation of the nuclear transcription factor CREB.