Characterisation of novel mechanisms for the regulation of trophoblast function by vitamin D

Ganguly, Ankana ORCID: 0000-0002-5225-0461 (2020). Characterisation of novel mechanisms for the regulation of trophoblast function by vitamin D. University of Birmingham. Ph.D.

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Abstract

Introduction: Vitamin D deficiency is common during pregnancy but its impact on maternal and foetal health is still unclear. The placenta is a key target for vitamin D: the vitamin D activating enzyme 1α-hydroxylase (CYP27B1), vitamin D receptor (VDR) and vitamin D binding protein (DBP) are all expressed by trophoblast cells early in pregnancy. This PhD aimed to identify new effects of the vitamin D system on trophoblast cell function.

Hypothesis: We hypothesised that vitamin D plays a key role in trophoblast behaviour and that this may be important for healthy placental development.

Methods: In vitro studies were carried out using trophoblast cell lines JEG3, BeWo and HTR8, with thyroid cancer cells (TPC) as a non-trophoblast comparator. Expression of mRNA and protein was compared for cells grown on plastic and extra-cellular matrix. Cells were assessed for motility (wound closure assay), proliferation (BrdU assay) and matrix invasion (Boyden chambers). Protein and mRNA expression were assessed following treatments using an ERK1/2 blocker (U0126), a megalin blocker (RAP), VDR siRNA, and DBP free serum. Pregnancy serum samples from 1st-trimester healthy pregnancy patients and 1st-trimester pre-eclampsia patients were assessed for DBP concentration and in vitro matrix invasion of trophoblast cells.

Results: In plasticware cultures, JEG3, BeWo and HTR8 cells showed weak expression of VDR and no induction of the vitamin D-response gene CYP24A1 by 1,25-dihydroxyvitamin D (1,25D). TPC cells showed strong nuclear VDR expression and potent 1,25D-mediated induction of CYP24A1. Conversely, for matrix-grown trophoblast cells there was abundant intracellular VDR and DBP, with apparent nuclear localisation of both, in the presence of 1,25D, but no induction of CYP24A1. Treatment with 1,25D significantly enhanced the invasion of MatrigelTM by trophoblast cells. Cells cultured on matrix with serum from DBP knockout mice (DBP-/-) or in the presence of an inhibitor of megalin-mediated endocytosis (RAP) showed no intracellular DBP, indicating uptake of exogenous DBP by these cells. These treatments also resulted in low matrix invasion by trophoblast cells. Trophoblast and TPC cells showed non-genomic induction and nuclear localisation of phospho-ERK1/2 (pERK1/2) in response to 1,25D, and inhibition of pERK1/2 blocked DBP uptake and trophoblast matrix invasion. DBP is also an actin-binder and decreased cellular uptake of DBP resulting in impaired matrix invasion were associated with elevated intracellular levels of G-actin and concomitant lower F-actin. Levels of DBP and 1,25D from 1st-trimester pregnancy serum samples significantly correlated with JEG3 matrix invasion, suggesting that this mechanism may be important for normal healthy pregnancies. In 1st-trimester serum samples, the concentration of DBP was lower in women who subsequently went on to develop pre-eclampsia.

Discussion: These data suggest that 1,25D enhances cellular uptake of serum DBP by pERK-mediated signalling and that this is important for maintaining homeostasis of intracellular actin. We, therefore, propose that optimal trophoblast function and healthy placentation requires the actions of maternal 1,25D and DBP. Both factors should be measured in studies of vitamin D and pregnancy.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):