Revisiting the role of hCG: new regulation of the angiogenic factor EG-VEGF and its receptors

Abstract

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is an angiogenic factor reported to be specific for endocrine tissues, including the placenta. Its biological activity is mediated via two G protein-coupled receptors, prokineticin receptor 1 (PROKR1) and prokineticin receptor 2 (PROKR2). We have recently shown that (i) EG-VEGF expression peaks between the 8th and 11th weeks of gestation, (ii) its mRNA and protein levels are up-regulated by hypoxia, (iii) EG-VEGF is a negative regulator of trophoblast invasion and (iv) its circulating levels are increased in preeclampsia (PE), the most threatening pathology of pregnancy. Here, we investigated the regulation of the expression of EG-VEGF and its receptors by hCG, a key pregnancy hormone that is also deregulated in PE. During the first trimester of pregnancy, hCG and EG-VEGF exhibit the same pattern of expression, suggesting that EG-VEGF is potentially regulated by hCG. Both placental explants (PEX) and primary cultures of trophoblasts from the first trimester of pregnancy were used to investigate this hypothesis. Our results show that (i) LHCGR, the hCG receptor, is expressed both in cyto- and syncytiotrophoblasts, (ii) hCG increases EG-VEGF, PROKR1 and PROKR2 mRNA and protein expression in a dose- and time-dependent manner, (iii) hCG increases the release of EG-VEGF from PEX conditioned media, (iv) hCG effects are transcriptional and post-transcriptional and (v) the hCG effects are mediated by cAMP via cAMP response elements present in the EG-VEGF promoter region. Altogether, these results demonstrate a new role for hCG in the regulation of EG-VEGF and its receptors, an emerging regulatory system in placental development.

Fig. 1

LHCGR protein expression in placental tissue at 8 and 10 weeks of gestation. A LHCGR expression in human placental sections. The photographs in a and b show chorionic villi sections immunostained with anti-LHCGR antibody at 8 wg and 10 wg, respectively. The photographs in c and d show tissue sections incubated with pre-immune sera at 8 and 10 wg, respectively. Cytotrophoblast (Ct), Hofbauer cells (Ho), syncytiotrophoblast (St). Scale bar 50 μm. B Representative Western-blot analysis of LHCGR expression in human placental extracts collected at 6–8 wg, 9–11 wg and at term. Two major bands with molecular masses of 55 and 70 kDa were observed at all gestational periods

Fig. 2

Effect of hCG on EG-VEGF expression. The photographs in this figure show EG-VEGF staining in sections of placental explants that have been cultured in the absence or presence of hCG (10 and 100 IU/ml) at 8 and 10 wg. a and g show non-treated explant sections at 8 and 10 wg, respectively. b and h show sections of explants that have been treated with 10 IU/ml hCG for 48 h at 8 and 10 wg, respectively. c and i show sections of explants that have been treated with 100 IU/ml hCG for 48 h at 8 and 10 wg, respectively. Undersized photographs in d–f and in j–l show tissue sections incubated with pre-immune serum to EG-VEGF at 8 wg and 10 wg, respectively. Cytotrophoblast (Ct), Hofbauer cells (Ho), syncytiotrophoblast (St). Scale bar 50 μm, (d–f) and (j–l)

Fig. 3

Effect of hCG on EG-VEGF secretion. A, B Temporal (0–48 h) and dose-dependence analysis of the effect of hCG (10–100 IU/ml) on EG-VEGF secretion in placental conditioned media collected from placental explants at 6–8 wg and 9–11 wg, respectively. C Time course (0–48 h) of the effect of forskolin (10 μM) on EG-VEGF secretion. D Effects of the PKA inhibitors Rp-cAMP (100 μM) and PKI (2 μM) on the effects of hCG (25 μM) and forskolin (10 μM) on EG-VEGF secretion. The treatments lasted for 10 h. Data represent the mean ± SEM from three independent experiments (*p < 0.05, **p < 0.001)

Fig. 4

Effect of hCG on EG-VEGF mRNA expression in placental explants and in syncytiotrophoblasts. A Temporal effect of hCG (10 IU/ml) on EG-VEGF mRNA expression measured by qRT-PCR in placental explants. Data represent the means ± SEM from 3 independent experiments (*p < 0.05). B Representative analysis of the effect of hCG (10, 50 and 100 IU/ml) on EG-VEGF mRNA expression in syncytiotrophoblast cultures (12 h). C Dose–response effect of hCG (10, 50 and 100 IU/ml) on EG-VEGF mRNA expression in syncytiotrophoblast cultures (12 h) monitored by q-PCR. D Effects of the PKA inhibitors Rp-cAMP (100 μM) and PKI (2 μM) on the effects of hCG (25 μM) and forskolin (10 μM) on EG-VEGF mRNA expression. The treatments lasted for 10 h. E Effects of DRB (50 μg/ml) and CHX (10 μg/ml) on basal and hCG-induced EG-VEGF mRNA expression in primary trophoblast cells. Data represent the mean ± SEM from three independent experiments (*p < 0.05, **p < 0.001)

Fig. 5

Mechanism by which hCG up-regulates EG-VEGF expression. A Effect of 8-bromo-cAMP on EG-VEGF promoter activation in Cos7 cells transfected with pGL3b pEG-VEGF or pGL3b (used as control). Luciferase activity was measured after 4 h and 6 h of cAMP stimulation. Data represent the mean ± SEM from three independent experiments. (*p < 0.05). B Effect of 8-bromo-cAMP on EG-VEGF promoter activation in Cos7 cells transfected with the following plasmids: pGL3b, pGL3b pEG-VEGF, pGL3b pEG-VEGF mutated at the CRE1 site, pGL3b pEG-VEGF mutated at the CRE2 site, and pGL3b pEG-VEGF mutated at the CRE1 and CRE2 sites. Luciferase activity was measured after 6 h of cAMP stimulation. Data represent the mean ± SEM from three independent experiments

Fig. 6

Effect of hCG on PROKR1 mRNA and protein expression. A Dose–response effect of hCG (12 h) on PROKR1 mRNA expression measured by qRT-PCR in cytotrophoblast cells. B PROKR1 staining in sections of placental explants that have been cultured in the absence or presence of hCG (10 and 100 IU/ml) at 10 wg. Non-treated explant sections are shown in a, and treated explant sections with hCG at 10 IU/ml and 100 IU/ml are shown in b and c, respectively. Undersized photographs in d, e, and f show tissue sections incubated with pre-immune serum to PROKR1. Cytotrophoblast (Ct), Hofbauer cells (Ho), syncytiotrophoblast (St). Scale bar 50 μm. C Representative Western-blot analysis of PROKR1 expression in cytotrophoblasts incubated in the absence or presence of hCG (100 IU/ml). D Histograms of the mean relative OD of PROKR1 protein signals normalized to β-actin. Data are mean ± SEM (**p < 0.001)

Fig. 7

Effect of hCG on PROKR2 mRNA and protein expression. A Dose–response effect of hCG (12 h) on PROKR2 mRNA expression, measured by qRT-PCR, in syncytiotrophoblast cells. B PROKR2 staining in sections of placental explants that have been cultured in the absence or presence of hCG (10 and 100 IU/ml) at 10 wg. Non-treated explants sections are shown in a and treated explant sections with hCG at 10 IU/ml and (100 IU/ml) are shown in b and c, respectively. Undersized photographs in d, e, and f show tissue sections incubated with pre-immune serum to PROKR2. Cytotrophoblast (Ct), Hofbauer cells (Ho), syncytiotrophoblast (St). Scale bar 50 μm. C Representative Western-blot analysis of PROKR2 expression in syncytiotrophoblast incubated in the absence or presence of hCG (10 IU/ml). D Histograms of the mean relative OD of PROKR2 protein signals normalized to β-actin protein. Data are mean ± SEM (**p < 0.001)

Fig. 8

Proposed model of hCG stimulation of the EG-VEGF/PROKR1 and PROKR2 system in human placenta during the first trimester of pregnancy. A Illustration of placental villi with the new physiological regulation between hCG and EG-VEGF. hCG released from the syncytiotrophoblast layer will activate EG-VEGF and PROKR2 expression in the ST layer in an autocrine manner and PROKR1 expression in the cytotrophoblast layer in a paracrine manner. B Summary of the proposed model according to which PROKR1, expressed by cytotrophoblasts, is only increased in response to high hCG levels. In contrast, EG-VEGF and PROKR2, which are mainly expressed by differentiated syncytiotrophoblasts, are increased in response to low hCG levels