Prokineticin 1 mediates fetal-maternal dialogue regulating endometrial leukemia inhibitory factor

Abstract

Implantation requires communication between a receptive endometrium and a healthy blastocyst. This maternal-embryonic crosstalk involves local mediators within the uterine microenvironment. We demonstrate that a secreted protein, prokineticin 1 (PROK1), is expressed in the receptive endometrium and during early pregnancy. PROK1 induces expression of leukemia inhibitory factor (LIF) in endometrial epithelial cells and first trimester decidua via a Gq-Ca(2+)-cSrc-mitogen-activated protein kinase kinase-mediated pathway. We show that human embryonic chorionic gonadotropin (hCG) induces sequential mRNA expression of PROK1 and LIF in an in vivo baboon model, in human endometrial epithelial cells, and in first-trimester decidua. We have used micro RNA constructs targeted to PROK1 to demonstrate that hCG-mediated LIF expression in the endometrium is dependent on prior induction of PROK1. Dual immunohistochemical analysis colocalized expression of the luteinizing hormone/chorionic gonadotropin receptor, PROK1, PROKR1, and LIF to the glandular epithelial cells of the first trimester decidual tissue. PROK1 enhances adhesion of trophoblast cells to fibronectin and laminin matrices, which are mediated predominantly via LIF induction. These data describe a novel signaling pathway mediating maternal-embryonic crosstalk, in which embryonic hCG via endometrial PROK1 may play a pivotal role in enhancing receptivity and maintaining early pregnancy.

Figure 1.

PROK1 induces LIF mRNA and protein expression. LIF mRNA (A) and protein (B) expression in WT and PROKR1 Ishikawa cells after treatment with 40 nM PROK1. LIF mRNA and protein expression peaked at 6 and 12 h, respectively, in PROKR1 Ishikawa cells. No elevation in LIF mRNA expression was observed in WT Ishikawa cells. Expression of LIF mRNA (C) and protein (D) was measured after treatment with 40 nM PROK1 (lane 1) in the absence or presence of YM254890 (Gq inhibitor, lane 2), BAPTA-AM (calcium inhibitor, lane 3), PKC inhibitor (Ro-318220, lane 4), cSrc inhibitor (PP2, lane 5), EGFR inhibitor (AG1478, lane 6), or MEK inhibitor (PD98059, lane 7). PROK1-induced LIF mRNA and protein expression was significantly inhibited in the presence of these inhibitors. Bars represent means ± se of at least 3 independent experiments. *P < 0.05. ns, not significant; −, absence of agent; +, presence of agent.

Figure 2.

hCG mediates expression of LIF via induction of PROK1. Expression of PROK1 in baboon endometrium was elevated after treatment with 1.25 IU of hCG/h for 5 d (A). Treatment of Ishikawa PROKR1 cells with 1 IU of hCG results in increased expression of PROK1 which peaks at 6 h (B). Similarly, treatment of Ishikawa PROKR1 cells with 1 IU of hCG results in increased expression of LIF, which peaks at 16 h (C). LIF mRNA expression in response to treatment with hCG is inhibited when PROKR1 Ishikawa cells are treated with 1 IU of hCG in the presence of cycloheximide (protein synthesis inhibitor) (D). PROKR1 Ishikawa cells were transiently transfected with miRNA constructs targeting PROK1 or scrambled sequences and subsequently treated with 1 IU of hCG. hCG-mediated PROK1 (E) and LIF (F) expression were significantly inhibited by PROK1 miRNA constructs. Bars represent means ± se of at least 3 independent experiments. *P < 0.05.

Figure 3.

Temporal expression and colocalization of signaling components. LH/CG receptor (A), PROK1 (B) and LIF (C) mRNA in human endometrium across the menstrual cycle and first trimester decidua. P, proliferative; ES, early secretory; MS, mid secretory, w7, w8, w9, w10, and w11 represent weeks of gestation (n=sample size from each stage of the cycle and early pregnancy). Immunofluorescent histochemical analysis of the LH/CG receptor (red panel) and PROK1 (green panel) show colocalization to the glandular epithelial cells of first trimester decidua (yellow panel, merge) (D). PROKR1 (green panel) and LIF (red panel) colocalize to glandular epithelial cells and a subset of stromal cells of first trimester decidua (yellow panel, merge) (E). Data are means ± se. *P < 0.05; ***P < 0.001.

Figure 4.

hCG mediates expression of LIF via induction of PROK1 in first trimester decidua. Treatment of first trimester decidua with 1 IU of hCG results in increased expression of PROK1 that peaks at 6 h (A). Similarly, treatment of first trimester decidua with 1 IU of hCG results in increased expression of LIF that peaks at 14 h (B). LIF mRNA expression in response to treatment with hCG is inhibited when first trimester decidua is treated with 1 IU of hCG for 14 h in the presence cycloheximide (protein synthesis inhibitor) (C). First trimester decidua tissue was infected with lentivirus encoding miRNA constructs targeting PROK1 or a scrambled sequence. Infected tissue was subjected to stimulation with 1 IU of hCG and subsequently analyzed for LIF mRNA expression. hCG-mediated LIF expression was significantly inhibited by PROK1 miRNA constructs compared with scrambled sequences (D). Bars represent means ± se of at least 3 independent experiments. *P < 0.05.

Figure 5.

PROK1 induces LIF expression in first trimester decidua. Treatment of first trimester decidua with 40 nM PROK1 induced LIF mRNA (A) and protein (B) expression that peaked at 6 and 12 h, respectively. Expression of LIF mRNA in first trimester decidua was measured after treatment with 40 nM PROK1 (lane 1) in the absence or presence of YM254890 (Gq inhibitor, lane 2), BAPTA-AM (calcium inhibitor, lane 3), PKC inhibitor (Ro-318220, lane 4), cSrc inhibitor (PP2, lane 5), EGFR inhibitor (AG1478, lane 6), or MEK inhibitor (PD98059, lane 7) (C). PROK1-induced LIF mRNA expression was significantly inhibited in the presence of these inhibitors. Bars represent means ± se of at least 3 independent experiments. *P < 0.05.

Figure 6.

PROK1 via LIF increases adhesion of trophoblast cells to fibronectin and laminin. Treatment of JEG-3 trophoblast cells with LIF increased adhesion to fibronectin (A, lane 6) and laminin (B, lane 6) compared with vehicle-treated cells (A, B; lane 5). Treatment of JEG-3 cells with conditioned medium collected from PROKR1 Ishikawa cells treated with 40 nM PROK1 for 24 h (PROK1 CM) also increased adhesion to fibronectin (A, lane 2) and laminin (B, lane 2) compared with conditioned medium collected from PROKR1 Ishikawa cells treated with vehicle for 24 h (vehicle CM; A, B; lane 1). This PROK1-mediated adhesion was abolished when LIF was immunoneutralized from the PROK1-conditioned medium (A, B; lane 4). Bars represent means ± se of at least 3 independent experiments. *P < 0.05. Ab, immunoneutralization.