Abstract
Regulation of immune cells during luteolysis has been previously described, however inter- and intra-cellular pathways that mediate PGF(2α) induction of immune cells in the bovine corpus luteum (CL) have not been clearly defined. Real-time PCR was used to measure chemokine mRNA and Western blotting used to measure phosphorylation of signaling proteins after PGF(2α) treatment of early and mid-cycle CL and in similar-stage luteinized granulosa cells (LuGC). In Day 11 CL (with luteolytic capacity), PGF(2α) induced expression of CXCL8, CXCL2, CCL2, and CCL8 at 1 h after treatment and continued to stimulate the four chemokines at 10 h after treatment. In Day 4 CL (without luteolytic capacity), there was a less robust increase in chemokine mRNA at 1 h after PGF(2α) with no detectable effect at 10 h after PGF(2α). In luteinized granulosa cells (LuGC), PGF(2α) induced a dramatic increase in mRNA for CXCL8 and CXCL2 with no change in mRNA for CCL2 and CCL8. In granulosa cells incubated for a similar time in conditions that did not induce luteinization, PGF(2α) did not alter transcription of any of these chemokines. In mature LuGC, treatment with PGF(2α) rapidly phosphorylated a key protein in the NFκB pathway, NFKBIA, and in the MAP kinase pathway, MAPK3, with no change in total amounts of these proteins. Moreover, in LuGC, induction by PGF(2α) of CXCL2 was inhibited by a PKC inhibitor and a NFκB pathway inhibitor. In addition, PGF(2α)-regulated phosphorylation of NFKBIA was blocked by the PKC inhibitor. In contrast, induction of CXCL8 by PGF(2α) was inhibited by a MAP kinase inhibitor. Thus, PGF(2α) induction of chemokines is closely related to luteolytic capacity. Further, two key chemokines, CXCL8 and CXCL2, seem to originate from large luteal cells through distinct signaling pathways that are activated by PGF(2α).