Danièle Evain-Brion

Hypoxia impairs cell fusion and differentiation process in human cytotrophoblast, in vitro

During human pregnancy, the trophoblast develops from differentiation of cytotrophoblast cells into an endocrine active syncytiotrophoblast. In culture, isolated mononuclear cytotrophoblasts aggregate and then fuse to form a syncytium, reproducing the in vivo process. In this study, we examined the effect of low oxygen tension (approximately 9%, hypoxia) compared to standard conditions (approximately 19% oxygen, normoxia) on these cellular events. Under hypoxia, syncytial formation was less frequently observed, cell staining and electron microscopy revealed that cytotrophoblasts remain aggregated, with a positive proliferative cell nuclear antigen (PCNA) immunostaining. Desmoplakin and E‐cadherin, both known to disappear with cytotrophoblast fusion, showed persistent expression in hypoxic cells after 3 days of culture. In contrast, the expression of actin and ezrin, two cytoskeletal proteins, was unchanged. hCG secretion and hPL expression were both decreased in hypoxic cells, reflecting a reduced syncytial formation. Thus, on day 3, the mean values for hCG secretion were 1,100 ± 155 and 289 ± 26 mlU/mL in normoxic and hypoxic conditions, respectively. The reduced cell fusion process as well as hCG secretion and hPL expression under hypoxia were reversed by reoxygenation of the cells. We conclude that under hypoxia, the formation of functional syncytiotrophoblast is impaired due to a defect in the cytotrophoblast fusion process. This may explain the observation of a higher number of cytotrophoblast cells and a reduced syncytial layer in placentas of some pathological pregnancies.

Physiological role of human placental growth hormone

Placental growth hormone (PGH) is the product of the GH-V gene specifically expressed in the syncytiotrophoblast layer of the human placenta. PGH differs from pituitary growth hormone by 13 amino acids. It has high somatogenic and low lactogenic activities. Assays of PGH by specific monoclonal antibodies reveal that in the maternal circulation from 15-20 weeks up to term, PGH gradually replaces pituitary growth hormone which becomes undetectable. It is secreted by the placenta in a non-pulsatile manner. This continuous secretion appears to have important implications for physiological adjustment to gestation and especially in the control of maternal IGF1 levels. PGH secretion is inhibited by glucose in vitro and in vivo, and is significantly decreased in the maternal circulation in cases of pregnancies with intrauterine growth retardation. PGH does not appear to have a direct effect on fetal growth, as this hormone is not detectable in the fetal circulation. However the physiological role of PGH might also include a direct influence on placental development via an autocrine or paracrine mechanism as suggested by the presence of specific GH receptors in this tissue.

Biochemical characterization and modulation of LH/CG—receptor during human trophoblast differentiation

Due to the key role of the human chorionic gonadotropin hormone (hCG) in placental development, the aim of this study was to characterize the human trophoblastic luteinizing hormone/chorionic gonadotropin receptor (LH/CG‐R) and to investigate its expression using the in vitro model of human cytotrophoblast differentiation into syncytiotrophoblast. We confirmed by in situ immunochemistry and in cultured cells, that LH/CG‐R is expressed in both villous cytotrophoblasts and syncytiotrophoblasts. However, LH/CG‐R expression decreased during trophoblast fusion and differentiation, while the expression of hCG and hPL (specific markers of syncytiotrophoblast formation) increased. A decrease in LH/CG‐R mRNA during trophoblast differentiation was observed by means of semi‐quantitative RT‐PCR with two sets of primers. A corresponding decrease (∼60%) in LH/CG‐R protein content was shown by Western‐blot and immunoprecipitation experiments. The amount of the mature form of LH/CG‐R, detected as a 90‐kDa band specifically binding 125I‐hCG, was lower in syncytiotrophoblasts than in cytotrophoblasts. This was confirmed by Scatchard analysis of binding data on cultured cells. Maximum binding at the cell surface decreased from 3,511 to about 929 molecules/seeded cells with a kDa of 0.4–0.5 nM. Moreover, on stimulation by recombinant hCG, the syncytiotrophoblast produced less cyclic AMP than cytotrophoblasts, indicating that LH/CG‐R expression is regulated during human villous trophoblast differentiation. J. Cell. Physiol. 212: 26–35, 2007.

OXIDATIVE MODULATION OF CYCLIC AMP-DEPENDENT PROTEIN KINASE IN HUMAN FIBROBLASTS : POSSIBLE ROLE IN PSORIASIS

Previous studies have established that cyclic AMP-dependent protein kinase (PKA) activity, as well as 8-azido-[32P]-cAMP binding to the RI and RII regulatory subunits, are decreased in cells from psoriatic patients compared to cells from normal patients. Here we show that the exposure of normal human dermal fibroblasts in culture to hydrogen peroxide and to oxygen free-radical generating systems decreased PKA activity, as well as cyclic AMP binding to the RI and RII regulatory subunits, to levels similar to those observed with psoriatic fibroblasts. Likewise, treatment of normal cytosolic preparations of PKA, as well as purified bovine PKA II, in vitro with free radical generating systems also resulted in decreased PKA activity and 8-azido [32P]-cAMP binding to the RI and RII regulatory subunits. Further, treatment of psoriatic fibroblasts with free radical scavenging agents such as vitamins E and C, and mannitol, and also with superoxide dismutase, restored the ability of RI and RII to bind 8-azido-[32P]-cAMP toward normal levels. Western blot analysis showed that the protein levels of the RI and RII subunits are similar in normal and psoriatic fibroblasts, and that the amounts of RI and RII are not altered by treatment of the cells with free radical-generating systems. These results suggest that oxidative modification may serve as a mechanism to alter PKA activity in human cells, and that an altered oxidative state may be involved in mediating the decrease in PKA activity and cyclic AMP binding noted in cells from psoriatic patients.

High retinoid X receptor expression in JEG-3 choriocarcinoma cells: Involvement in cell function modulation by retinoids

Retinoic acid (RA) is an important mediator of cell differentiation. It stimulates hCG secretion by JEG‐3 choriocarcinoma cells in vitro after a time lag. The first aim of this study was to characterize which types of retinoid receptors (RARs and RXRs) are present in JEG‐3 cells. Using Western blot analysis and immunocytochemistry with specific antibodies as well as Northern blot analysis, we found that JEG‐3 cells expressed RARα and RXRα, the latter being the predominant receptor. We then analyzed the action on cell proliferation and hCG secretion of the physiological retinoids all‐trans RA (RA) and 9 cis RA as well as synthetic retinoids with specific affinity for RARα and RXRα. All these retinoids were potent inhibitors of cell growth, maximal inhibition (72 ± 2%) being observed after 4 days of treatment with Ro 25, a RXRα specific ligand. Within 24 h, 9 cis RA and Ro 25 stimulated hCG secretion, and maximal stimulation (1,472 ± 10%) occurred at 48 h with the RXRα‐specific ligand. The RARα‐specific ligand also stimulated hCG secretion but to a lower extend and after a delay of 48 h. These results suggest a predominant role of RXRα in mediating the biological effects of retinoids on JEG‐3 cells and the possible induction by RA itself of the metabolic pathway leading to 9 cis RA. J. Cell. Physiol. 176:595–601, 1998.

EGF increases retinoid X receptor-α expression in human trophoblastic cells in culture: relationship with retinoic acid induced human chorionic gonadotropin secretion

In the present study, the effect of retinoic acid (RA) and epidermal growth factor (EGF) on the functions of human trophoblastic cells in culture were analysed. In these cells, RA potentiated the hCG secretion increase induced by EGF. To gain a better understanding of such a synergistic effect, the expression of retinoic acid receptors (RAR alpha and beta) and retinoid X receptor (RXR alpha) was studied by immunoblotting in RA- and EGF-treated cells. EGF treatment specifically increased the level of RXR alpha protein and RXR alpha transcripts. In parallel, we demonstrated that the choriocarcinoma cells JEG 3, which respond to RA by an increase in hCG secretion, express constitutively high levels of RXR alpha protein. Furthermore, RXR alpha-transfected trophoblastic cells also become RA responsive for hCG secretion. All these data suggest that RXR alpha expression is modulated by EGF, and may be involved in the effect of RA on hCG secretion.

Post-translational modifications of the regulatory subunits of cAMP-dependent protein kinases during G1/S progression.

During the G1/S transition of the cell cycle variations in the labelling by 8-N3-[32P]cAMP of the protein kinase A regulatory subunits RI and RII, used as a probe to monitor post-translational modifications that may regulate cAMP binding, were observed in synchronized HeLa cells. A decrease in 8-N3-[32P]cAMP labelling of RI, RII and RII phosphorylated by the catalytic subunit of PKA was correlated with the increased percentage of cells in phases G1. An increase in 8-N3-[32P]cAMP incorporated into the 54-kDa RII subunit during progression from G1 to S was correlated with an increase in intracellular cAMP. A transient increase in Mn-SOD activity was detected in cells arrested at the G1/S transition using two different techniques, suggesting that oxidative modulation of regulatory subunits by free radicals may modify cAMP binding sites during the cell cycle. Decreased photoaffinity labelling by 8-N3-[32P]cAMP of RI, RII and autophosphorylated RII subunits was found to be an inherent characteristic of PKA in the G1/S transition.