Prokineticin 1 signaling and gene regulation in early human pregnancy

Abstract

Prokineticin 1 (PROK1) is a recently described protein with a wide range of functions including tissue-specific angiogenesis, modulation of inflammatory responses, and regulation of hematopoiesis. The objective of this study was to investigate the role of PROK1 and prokineticin receptor 1 (PROKR1) in human endometrium during early pregnancy. PROK1 and PROKR1 expression is significantly elevated in first-trimester decidua, compared with nonpregnant endometrium. Expression of PROK1 and PROKR1 was localized in glandular epithelial and various cellular compartments within the stroma. To investigate the signaling pathways and target genes activated by PROK1, we generated an endometrial epithelial cell line stably expressing PROKR1 (Ishikawa PROKR1 cells). PROK1-PROKR1 interaction induced inositol phosphate mobilization and sequential phosphorylation of c-Src, epidermal growth factor receptor, and ERK 1/2. Gene microarray analysis on RNA extracted from Ishikawa PROKR1 cells treated with 40 nm PROK1 for 8 h revealed 49 genes to be differentially regulated. A number of these genes, including cyclooxygenase (COX)-2, leukemia inhibitory factor, IL-6, IL-8, and IL-11 are regulated in the endometrium during implantation and early pregnancy. We subsequently investigated the effect of PROK1 on expression of COX-2 in Ishikawa PROKR1 cells and first-trimester decidua. COX-2 mRNA and protein expression, and prostaglandin synthesis, were elevated in response to treatment with PROK1. Moreover, expression of COX-2 by PROK1 was dependent on activation of the Gq-phospholipase C-beta-cSrc-epidermal growth factor receptor-MAPK/ERK kinase pathway. These data demonstrate that PROK1 and PROKR1 expression is elevated in human decidua during early pregnancy and that PROK1-PROKR1 interaction regulates expression of a host of implantation-related genes.

FIG. 1

Temporal expression and localization of PROK1 and PROKR1 in human endometrium and first-trimester decidua. PROK1 (A) and PROKR1 (B) mRNA expression in endometrium and first-trimester decidua are shown. EP, Early proliferative, MP, mid proliferative, LP, Late proliferative; ES, early secretory; MS, midsecretory; LS, late secretory (sample size from each stage of the cycle is depicted in the figure). In first-trimester deciduas, PROK1 and PROKR1 expression (n = 7; representative sections shown in C and D, respectively) localize to the glandular epithelium (G), stromal cells (S), and vascular endothelial cells (E) of the microvasculature (×10 magnification, inset, black box, ×20 magnification). E, A population of PROK1-expressing stromal cells were identified as natural killer cells by immunofluorescent histochemistry and confocal microscopy for CD56 (natural killer cell marker, red panel) and PROK1 (green panel) by co-localization (yellow channel). This was confirmed by conventional PCR (G) conducted using natural killer cell RNA isolated from individual decidua tissue (n = 3). PROKR1 localization to endothelial cells was confirmed by double-immunofluorescent histochemistry (F) for CD31 (endothelial cell marker, red panel) and PROKR1 (green panel) by colocalization (yellow channel). Data are shown as mean ± sem (*, P < 0.05; **, P < 0.001). Negative control (−ve) is indicated in the figure.

FIG. 2

Expression of PROKR1 in PR OKR1-Ishikawa cells and signaling induced by PROKR1 via PROK1. A, PROKR1 RNA expression is significantly elevated in PROKR1-Ishikawa cells, compared with WT Ishikawa cells as assessed by real-time PCR analysis. Similarly, immunofluorescent histochemistry demonstrates expression of PROKR1 protein (red staining) in PROKR1-Ishikawa cells, which is absent in WT Ishikawa cells. B, Inositol phosphate mobilization is elevated in PROKR1, compared with WT Ishikawa cells after treatment with 40 nM PROK1. C, ERK1/2 phosphorylation was determined by Western blot analysis; phosphorylation is maximal in PROKR1-Ishikawa cells after treatment with 40 nM PROK1. D, ERK1/2 phosphorylation in PROKR1-Ishikawa cells incubated with 40 nM PROK1 is maximal at 5 min. E, ERK 1/2 phosphorylation was measured in PROKR1-Ishikawa cells in response to the administration of vehicle or 40 nM PROK1 for 5 min in the presence or absence of pretreatment with YM254890 (Gq inhibitor, lane 3), U73122 (PLC inhibitor, lane 4), PP2 (cSrc inhibitor, lane 5), AG1478 (EGFR inhibitor, lane 6), or PD98059 (MEK inhibitor, lane 7). The PROK1-induced ERK 1/2 response was significantly inhibited in the presence of these inhibitors. F, PROKR1-Ishikawa cells were transiently cotransfected with Myc-tagged ERK and either empty vector (pcDNA3) or cDNA constructs encoding DN isoforms of cSrc, EGFR, Ras, or MEK. Transfected cells were subjected to stimulation with vehicle or 40 nM PROK1 for 5 min. PROK1-induced ERK response was significantly inhibited by the DN cDNA constructs. G and H, cSrc and EGFR phosphorylation is increased in PROKR1-Ishikawa cells after treatment with 40 nM PROK1. The PROK1-induced cSrc and EGFR phosphorylation peaked at 5 min. ERK1/2, cSrc and EGFR phosphorylation was calculated as fold above vehicle-treated controls. For each, a representative Western immunoblot is shown with semiquantitative analysis determined as described in Materials and Methods. In all panels each bar represents the mean ± sem of at least three individual experiments (*, P < 0.05). −, Absence of agent; +, presence of agent.

FIG. 3

Validation of gene array analysis by real-time PCR. Expression of COX-2 (PTGS2), LIF, IL-6, IL-8, and IL-11 is significantly elevated in PROKR1-Ishikawa cells stimulated with 40 nM PROK1 for 8 h. No elevation was observed in WT Ishikawa cells. Each bar represents the mean ± sem of at least three individual experiments (*, P < 0.05; **, P < 0.01).

FIG. 4

PROK1-induced COX-2 mRNA expression is mediated via ERK 1/2 and results in de novo synthesis of prostanoids. A, COX-2 expression in WT and PROKR1-Ishikawa cells after treatment with 40 nM PROK1. No elevation in COX-2 mRNA expression was observed in WT Ishikawa cells, whereas COX-2 expression peaked at 6 h in PROKR1-Ishikawa cells. B, COX-2 protein expression, as determined by Western blot analysis, was maximal in PROKR1-Ishikawa cells after treatment with 40 nM PROK1 (p) for 8 h. No elevation in COX-2 protein expression was observed in vehicle (v)-treated cells. C, COX-2 protein expression was measured in PROKR1-Ishikawa cells in response to the administration of vehicle or 40 nM PROK1 for 8 h in the presence or absence of pretreatment with YM254890 (Gq inhibitor, lane 3), U73122 (PLC inhibitor, lane 4), PP2 (cSrc inhibitor, lane 5), AG1478 (EGFR inhibitor, lane 6), or PD98059 (MEK inhibitor, lane 7). The PROK1-induced COX-2 expression was significantly inhibited in the presence of these inhibitors. PROK1-induced de novo synthesis of PGF_2α (D) and PGE₂ (E) is COX-2 dependent, indicated by abrogation of prostaglandin production in presence of specific COX-2 inhibitor NS-398. For B and C, a representative Western immunoblot is shown with semiquantitative analysis determined as described in Materials and Methods. In all panels each bar represents the mean ± sem of at least three independent experiments (*, P < 0.05). −, Absence of agent; +, presence of agent.

Fig. 5

PROK1 induces ERK 1/2 and COX-2 expression in first-trimester decidua. A, PROKR1 (red) and COX-2 (green) colocalize (yellow) to the glandular epithelium and a subset of stromal cells in human endometrium (n = 5) and first-trimester decidua (n = 5; representative sections shown, inset, negative control incubated with control IgG). B, Treatment of first-trimester decidua explants with 40 nM PROK1 resulted in ERK1/2 phosphorylation (pERK), which was maximal at 30–45 min. C, ERK 1/2 phosphorylation was measured in first-trimester decidua in response to the administration of vehicle or 40 nM PROK1 for 30 min in the presence or absence of pretreatment with YM254890 (Gq inhibitor, lane 3), U73122 (PLC inhibitor, lane 4), PP2 (cSrc inhibitor, lane 5), AG1478 (EGFR inhibitor, lane 6), or PD98059 (MEK inhibitor, lane 7). PROK1-induced ERK 1/2 response was significantly inhibited in the presence of the above inhibitors. D, Treatment of first-trimester decidua explants with 40 nM PROK1 resulted in increased expression of COX-2, which was maximal at 6 h. E, PROK1-induced COX-2 mRNA expression was inhibited by preincubation of first-trimester decidua with YM254890 (Gq inhibitor, lane 3), U73122 (PLC inhibitor, lane 4), PP2 (cSrc inhibitor, lane 5), AG1478 (EGFR kinase inhibitor, lane 6), or PD98059 (MEK inhibitor, lane 7). For B and C, a representative Western immunoblot is shown, with semiquantitative analysis determined as described in Materials and Methods. In all panels each bar represents mean ± sem of at least five experiments (*, P < 0.05). −, Absence of agent; +, presence of agent.