Trophoblast uptake of DBP regulates intracellular actin and promotes matrix invasion

in Journal of Endocrinology
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  • 1 Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, UK
  • 2 Birmingham Women’s & Children’s Foundation Hospital Trust, Mindelsohn Way, Edgbaston, Birmingham, UK
  • 3 CEDAM, Birmingham Health Partners, University of Birmingham, Birmingham, UK
  • 4 School of Life and Health Sciences, Aston University, Birmingham, UK

Correspondence should be addressed to M Hewison: m.hewison@bham.ac.uk
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Early pregnancy is characterised by elevated circulating levels of vitamin D binding protein (DBP). The impact of this on maternal and fetal health is unclear but DBP is present in the placenta, and DBP gene variants have been linked to malplacentation disorders such as preeclampsia. The functional role of DBP in the placenta was investigated using trophoblastic JEG3, BeWo and HTR8 cells. All three cell lines showed intracellular DBP with increased expression and nuclear localisation of DBP in cells treated with the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D). When cultured in the serum of mice lacking DBP (DBP−/−), JEG3 cells showed no intracellular DBP indicating uptake of exogenous DBP. Inhibition of the membrane receptor for DBP, megalin, also suppressed intracellular DBP. Elimination of intracellular DBP with DBP−/− serum or megalin inhibitor suppressed matrix invasion by trophoblast cells and was associated with increased nuclear accumulation of G-actin. Conversely, treatment with 1,25D enhanced matrix invasion. This was independent of the nuclear vitamin D receptor but was associated with enhanced ERK phosphorylation, and inhibition of ERK kinase suppressed trophoblast matrix invasion. When cultured with serum from pregnant women, trophoblast matrix invasion correlated with DBP concentration, and DBP was lower in first-trimester serum from women who later developed preeclampsia. These data show that the trophoblast matrix invasion involves uptake of serum DBP and associated intracellular actin-binding and homeostasis. DBP is a potential marker of placentation disorders such as preeclampsia and may also provide a therapeutic option for improved placenta and pregnancy health.

Supplementary Materials

    • Supplemental Figure 1. Effect of ERK kinase inhibition on expression of VDR and DBP in Matrigel cultures of BeWo and HTR8 cells. Immunofluorescence analysis of expression of protein for DBP (red) and VDR (pink) in BeWo and HTR8 cells cultured the presence or absence of 1,25D (100 nM, 48 hrs) without or with the pERK inhibitor U0126. Nuclear (Hoechst, blue) and membrane (NaKATPase, green) are also shown. Scale bar shows 20µm. Images were taken with 40x magnification.
    • Supplemental Figure 2. Expression of the vitamin D system and proliferation response in trophoblastic cells cultured on plastic. S2A. Expression of mRNA (1/δCt) for VDR, CYP24A1, and DBP and S2B. Cell proliferation (BrdU incorporation, absorbance units) in JEG3, BeWo, and TPC cells cultured on platic in the presence or absence of 1,25D (100 nM, 48 h). S2C. Expression of protein for the vitamin D receptor (VDR, pink) and vitamin D binding protein (DBP, red) in JEG3 and TPC cells cultured on plastic in the presence or absence of 1,25D (100 nM, 48 h). Immunofluorescence for each protein is shown in combination with nuclear (Hoechst, blue) and membrane (NaK ATPase, green) markers. Data (mean ± 95% CI) are shown for n= 3-4 separate experiments. Statistically different from vehicle-treated control, ** p < 0.01.
    • Supplemental Table 1. Antibodies used in immunofluorescence analysis and Western blot analysis

 

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