Differential responsiveness and signaling pathways for prostaglandin F2a on chemokine mRNA in bovine corpus luteum and luteinized granulosa cells

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.