The role of GPR55 in human trophoblast differentiation

In the human placenta two trophoblast subpopulations arise and differentiate from trophoblast progenitor cells. One subpopulation forms the villous trophoblast, which covers placental villi as a two-layered epithelium and thereby functions as the placental barrier between maternal blood and fetal tissues. The villous trophoblast consists of mononucleated cytotrophoblasts and an overlying multinucleated syncytium, the so-called syncytiotrophoblast. Cellular turnover within the villous trophoblast is unique as a subset of progenitor cells amongst the cytotrophoblast population undergoes differentiation and subsequently fuses with the overlying syncytiotrophoblast. Beside the villous trophoblast population, a second subpopulation detaches from anchoring placental villi and differentiates into invasive extravillous trophoblasts. These extravillous trophoblasts invade the interstitium of the maternal endometrium and are thus also referred to as interstitial trophoblasts. On their way through the endometrium a subset of interstitial trophoblasts encounters maternal spiral arteries and differentiates into endovascular trophoblasts, with the mission to transform spiral arteries to deliver large quantities of blood into the intervillous space. When interstitial trophoblasts reach and invade uterine glands, they differentiate into endoglandular trophoblasts, which enable histiotrophic nutrition of the embryo during the first trimester of pregnancy. Defects in both the villous and/or the extravillous trophoblast differentiation routes have been suggested to result in severe pregnancy disorders and thus differentiation routes have to be tightly regulated. Amongst a plethora of factors described to drive trophoblast differentiation endocannabinoids have recently attracted increasing attention. Endocannabinoids not only bind the classical cannabinoid receptors CB1 and CB2, but were also shown to bind the G-protein coupled receptor (GPR) 55. GPR55 has a widespread expression pattern throughout human tissues, including the central nervous system, adrenal glands, spleen and gut. Yet to date, there have been no reports on GPR55 expression in human placenta and its putative role in trophoblast differentiation. Thus, this project aims to determine GPR55 gene expression in human placenta and primary placental cells. Once expression is determined GPR55 downstream signalling and its role in human trophoblast differentiation will be explored in detail. Moreover, the effect of different oxygen levels on GPR55 expression and signalling will be analysed in human trophoblasts. To determine a potential role of GPR55 in pregnancy pathologies, the expression of this receptor will be compared between healthy and pathologic placentas.

Endocannabinoids and their receptors play key roles in human pregnancy, by regulating important aspects such as implantation, decidualization, placentation and labour. G protein- coupled receptor 55 (GPR55) has recently been suggested as another cannabinoid receptor, even though it lacks the typical cannabinoid binding pocket and shares only low sequence homology with classical cannabinoid receptors. While in silico screenings suggested potential interactions of GPR55 with some cannabinoid receptor ligands, several pharmacological studies showed that the non-cannabinoid bioactive lipid, L-a- lysophosphatidylinositol (LPI), is the specific and functional ligand for GPR55. In healthy individuals, GPR55 is expressed in various regions of the human brain, but is also detected in a wide range of peripheral tissues including adrenals, jejunum, ileum, spleen, and bone. Very little is known about GPR55 expression and function in human placenta. Thus one aim of the project was to determine expression and localization of GPR55 in human placental tissues at early and late stages of pregnancy. Moreover, GPR55 expression was analyzed in different cell types, isolated from placenta tissues. Additionally, a functional role of GPR55 in human placenta was tested by treating isolated placenta cells with the receptor ligand LPI. Gene expression analysis showed that placental GPR55 levels were low when compared to other tissues, such as spleen and lung. For human placenta, expression analysis suggested a clear increase in GPR55 levels over the period of gestation. In first trimester of pregnancy, i.e. between gestational weeks 7 and 12, GPR55 was abundantly detected in the endothelium lining fetal blood vessels of placental villi. Interestingly, GPR55 was not only detected in well established, large vessels in the center of villi, but was also expressed in newly emerging capillaries just underneath the villous surface. Like in early pregnancy, GPR55 was predominantly detected at the endothelium of fetal vessels in placenta villi at term, i.e. right after delivery. This observation was confirmed in primary endothelial cells isolated from human placenta tissues, whereas GPR55 expression seems to be higher in venous endothelium than in arterial. Functional assays revealed that GPR55 ligand LPI increased the migratory activity of venous, but not arterial placental endothelial cells, suggesting a role of the LPI-GPR55 axis in placental venous endothelium function. Recently, GPR55 expression and LPI serum levels have been associated with obesity, a condition assumed as low-grade systemic inflammation with a number of elevated inflammatory cytokines. Results of the current project showed that inflammatory cytokines tumor necrosis factor (TNF)-alpha and Interleukin (IL)-6 considerably induced up-regulation of GPR55 in endothelial cells isolated from human placenta. Obesity is rising worldwide and the associated low-grade systemic inflammation promotes occurrence of gestational diabetes mellitus (GDM). Thus, placental GPR55 and LPI levels may be altered in pregnancies complicated by obesity and/or GDM, which could influence venous endothelium functions in human placenta development and in turn could affect fetal growth.