PGF_2α modulates the output of chemokines and pro-inflammatory cytokines in myometrial cells from term pregnant women through divergent signaling pathways

Prostaglandin F_2α (PGF_2α) plays a critical role in the initiation and process of parturition. Since human labor has been described as an inflammatory event, we investigated the role of PGF_2α in the inflammatory process using cultured human uterine smooth muscle cells (HUSMCs) isolated from term pregnant women as a model. Using a multiplex assay, HUSMCs treated with PGF_2α changed their output of a number of cytokines and chemokines, with a distinct response pattern that differed between HUSMCs isolated from the upper and lower segment region of the uterus. Confirmatory enzyme-linked immunosorbent assays (ELISAs) showed that PGF_2α stimulated increased output of interleukin (IL) 1β, IL6, IL8 (CXCL8) and monocyte chemotactic protein-1 (MCP1, also known as chemokine (c-c motif) ligand 2, CCL2) by HUSMCs isolated from both upper and lower uterine segments. In contrast, PGF_2α inhibited tumor necrosis factor a (TNFa) release by HUMSCs from the lower uterine segment while the output of TNFa was undetectable in the upper segment. Small interfering (si) RNA mediated knockdown of the PGF_2α receptor prevented the changes in cytokine and chemokine output by the HUSMCs. Since the PGF_2α receptor (PTGFR) couples via the Gq protein and subsequently activates the phospholipase C (PLC) and protein kinase C (PKC) signaling pathways, we examined the role of these pathways in PGF_2α modulation of the cytokines. Inhibition of PLC and PKC reversed the effects of PGF_2α. PGF_2α activated multiple signaling pathways including extracellular signal-regulated kinases (ERK) 1/2, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), P38, calcineurin/nuclear factor of activated T-cells (NFAT) and NF-κB signaling. Inhibition of ERK reversed PGF_2α-induced IL1β, IL6 and CCL2 output, while inhibition of PI3K blocked the effect of PGF_2α on IL6, CXCL8 and CCL2 output and inhibition of NF-κB reversed PGF_2α-induced IL1β and CCL2 output. NFAT was involved in PGF_2α modulation of CCL2 and TNFa output. In conclusion, our results support a role of PGF_2α in creating an inflammatory environment during the late stage of human pregnancy.