Prokineticin-1: a novel mediator of the inflammatory response in third-trimester human placenta

Abstract

Prokineticin-1 (PK1) is a recently described protein with a wide range of functions, including tissue-specific angiogenesis, modulation of inflammatory responses, and regulation of hemopoiesis. The aim of this study was to investigate the localization and expression of PK1 and PK receptor-1 (PKR1), their signaling pathways, and the effect of PK1 on expression of the inflammatory mediators cyclooxygenase (COX)-2 and IL-8 in third-trimester placenta. PK1 and PKR1 were highly expressed in term placenta and immunolocalized to syncytiotrophoblasts, cytotrophoblasts, fetal endothelium, and macrophages. PK1 induced a time-dependent increase in expression of IL-8 and COX-2, which was significantly reduced by inhibitors of Gq, cSrc, epidermal growth factor receptor (EGFR), and MAPK kinase. Treatment of third-trimester placenta with 40 nm PK1 induced a rapid phosphorylation of cSrc, EGFR, and ERK1/2. Phosphorylation of ERK1/2 in response to PK1 was dependent on sequential phosphorylation of cSrc and EGFR. Using double-immunofluorescent immunohistochemistry, PKR1 colocalized with IL-8 and COX-2 in placenta. These data suggest that PK1 may have a novel role as a mediator of the inflammatory response in placenta.

Fig. 1

Relative expression of PK1 and PKR1 in third-trimester human placenta and pregnant (preg) and nonpregnant (nonpreg) myometrium (myo). Relative expression of PK1 and PKR1 mRNA were determined by real-time quantitative RT-PCR analysis in third-trimester placenta (n = 16), third-trimester myometrium (n = 9), and nonpregnant myometrium (n = 6). Relative expression of PK1 mRNA was significantly higher in placenta than pregnant or nonpregnant myometrium. There was no significant difference in relative PKR1 mRNA expression between third-trimester placenta and pregnant or nonpregnant myometrium. Data are expressed as mean ± SEM; b is significantly different from a (P < 0.01).

Fig. 2

Immunohistochemical localization of PK1 and PKR1 in third-trimester human placenta (n = 8). PK1 was immunolocalized to the syncytiotrophoblast (st) layer of placental villi, cytotrophoblasts (ct), and endothelium (e) of fetal blood vessels (bv) (A and B). PKR1 was immunolocalized to macrophages (m) in the villous core, syncytiotrophoblast (st), cytotrophoblasts (ct), and endothelium (e) (C and D). Immunohistochemistry negatives (Neg, insets) were incubated with isotype-matched IgG in place of primary antibody and displayed no immunoreactivity. Scale bars, 50 μm).

Fig. 3

Colocalization of PKR1 with cytokeratin, CD31, and CD68 in third-trimester human placenta (n = 4). PKR1 was colocalized with cytokeratin 18 (trophoblast marker), CD31 (blood vessel marker), CD68 (macrophage marker), CD3 (T-cell marker), and CD 20 (B-cell marker) by dual-immunofluorescence immunohistochemistry in third-trimester placenta. PKR1 (red, A and D) and cytokeratin 18 (green, B and E) were colocalized (yellow channel, merged, C and F) in syncytiotrophoblasts (st) and in some cytotrophoblasts (ct). PKR1 (red, G) and CD31 (green, H) were colocalized in blood vessel (bv) endothelium within villi (yellow channel, merged, I). PKR1 (red, J) colocalized with CD68 (green, K) to macrophages (m, yellow channel, merged, L). PKR1 did not colocalize with CD3 (M, merged) or CD20 (N, merged). Control sections were incubated with normal IgG from the same host species and displayed no immunoreactivity (O). Original magnification, ×40.

Fig. 4

Relative expression of COX-2 and IL-8 in response to PK1 in third-trimester human placenta. Third-trimester placental explants were treated for 2, 4, 6, 8, and 24 h in the presence of vehicle or 40 nm PK1, and COX-2 and IL-8 expression was measured by real-time RT-PCR (n = 4). To investigate the signaling pathway, placental explants were treated for 4 h in the presence of vehicle or 40 nm PK1 and chemical inhibitors of Gq (YM254890, 1 μm), EGFR kinase (AG1478, 200 nm), cSrc (PP2, 10 μm), or MEK (PD98059, 50 μm), with COX-2 and IL-8 expression being measured as before (n = 3). A, There was a significant fold increase in COX-2 expression at 4 h in placenta treated with 40 nm PK1 compared with vehicle-treated control (P < 0.05); B, there was a significant fold decrease in COX-2 in explants treated with PK1 in the presence of chemical inhibitors relative to placenta treated with PK1 alone (P < 0.05); C and D, similarly, there was a significant fold increase in IL-8 expression at 2 and 4 h in placenta treated with PK1 compared with vehicle-treated control (P < 0.05; n = 4) (C) with expression being significantly reduced in the presence of chemical inhibitors (P < 0.05; n = 3) (D). Results are expressed as mean ± SEM; b is significantly different from a (P < 0.05), and c is not significantly different from a or b.

Fig. 5

Phosphorylation of ERK, EGF, and cSrc in response to PK1 in third-trimester human placenta. Placental explants were incubated with 40 nm PK1 for 5, 10, 20, and 30 min and subsequently analyzed for phosphorylation of ERK1/2, cSrc, and EGFR (n = 3). A, There was a time-dependent increase in phosphorylation of ERK1/2 that peaked at 30 min (c is significantly different from a but not b; P < 0.05). Explants treated for 30 min with vehicle or 40 nm PK1 in the presence of chemical inhibitors of Gq (YM254890), EGFR kinase (AG1478), cSrc (PP2), or MEK (PD98059) were then subsequently analyzed for ERK phosphorylation (n = 7). B, There was a significant decrease in ERK 1/2 phosphorylation in explants incubated with inhibitors compared with explants treated with PK1 alone (b is significantly different from a; P < 0.05). Placental explants were stimulated with 40 nm PK1 for 0, 10, 20, and 30 min, and lysates were immunoprecipitated with anti-phosphotyrosine antibody and then subjected to Western immunoblot analysis using antibodies against cSrc and EGFR (n = 4). Treatment with 40 nm PK1 resulted in a 2-fold increase in phosphorylation of Src and EGFR within 20 min of treatment. C, IgG was used to demonstrate equal sample loading (representative blot). Data are shown as mean ± SEM.

Fig. 6

Colocalization of PKR1 with COX-2 and IL-8 in third-trimester human placenta (n = 4). PKR1 was colocalized with COX-2 and IL-8 by dual immunofluorescence in third-trimester placenta. PKR1 (red, A, D, and G) and COX-2 (green, B, E, and H) were colocalized in syncytiotrophoblasts (st), cytotrophoblasts (ct), and macrophages (m) in placenta (yellow, merged, C, F, and I, respectively). PKR1 (red, J) and IL-8 (green, K) were colocalized to fetal blood vessels (bv) within villi (yellow channel, L). Inset in B shows negative (Neg) control (original magnification ×40).