SMAD1/5 signaling in the early equine placenta regulates trophoblast differentiation and chorionic gonadotropin secretion.

Douglas F Antczak,Jordan E Read,Stephanie Richardson,Abir Mukherjee,Judith E Cartwright,Amanda M De Mestre,Victoria Cabrera-Sharp,Alycia A Kowalski

doi:10.1210/en.2013-2116

Douglas F Antczak, Jordan E Read + Show 6 more

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https://doi.org/10.1210/en.2013-2116

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Abstract

TGFβ superfamily proteins, acting via SMAD (Sma- and Mad-related protein)2/3 pathways, regulate placental function; however, the role of SMAD1/5/8 pathway in the placenta is unknown. This study investigated the functional role of bone morphogenetic protein (BMP)4 signaling through SMAD1/5 in terminal differentiation of primary chorionic gonadotropin (CG)-secreting trophoblast. Primary equine trophoblast cells or placental tissues were isolated from day 27–34 equine conceptuses. Detected by microarray, RT-PCR, and quantitative RT-PCR, equine chorionic girdle trophoblast showed increased gene expression of receptors that bind BMP4. BMP4 mRNA expression was 20- to 60-fold higher in placental tissues adjacent to the chorionic girdle compared with chorionic girdle itself, suggesting BMP4 acts primarily in a paracrine manner on the chorionic girdle. Stimulation of chorionic girdle-trophoblast cells with BMP4 resulted in a dose-dependent and developmental stage-dependent increase in total number and proportion of terminally differentiated binucleate cells. Furthermore, BMP4 treatment induced non-CG-secreting day 31 chorionic girdle trophoblast cells to secrete CG, confirming a specific functional response to BMP4 stimulation. Inhibition of SMAD2/3 signaling combined with BMP4 treatment further enhanced differentiation of trophoblast cells. Phospho-SMAD1/5, but not phospho-SMAD2, expression as determined by Western blotting was tightly regulated during chorionic girdle trophoblast differentiation in vivo, with peak expression of phospho-SMAD1/5 in vivo noted at day 31 corresponding to maximal differentiation response of trophoblast in vitro. Collectively, these experiments demonstrate the involvement of BMP4-dependent pathways in the regulation of equine trophoblast differentiation in vivo and primary trophoblast differentiation in vitro via activation of SMAD1/5 pathway, a previously unreported mechanism of TGFβ signaling in the mammalian placenta.

Highlights

TGF␤ superfamily proteins, acting via SMAD (Sma- and Mad-related protein)2/3 pathways, regulate placental function; the role of SMAD1/5/8 pathway in the placenta is unknown
We found no evidence of up-regulated expression of either TGF␤1-specific type I receptor (TGF␤R1) or its type II receptor (TGFBR2); neither did we find evidence for the expression of its SMAD1/5/8-specific pathway type I receptor activin receptor type-II-like I (ACVRL1)
BMPR1B was not detected in any of the chorionic girdle tissues tested. These results suggest that in these chorionic girdle trophoblast cells, bone morphogenetic protein (BMP) may signal through the BMPR1A and BMPR2 dimer

Summary

Introduction

TGF␤ superfamily proteins, acting via SMAD (Sma- and Mad-related protein)2/3 pathways, regulate placental function; the role of SMAD1/5/8 pathway in the placenta is unknown. Phospho-SMAD1/5, but not phospho-SMAD2, expression as determined by Western blotting was tightly regulated during chorionic girdle trophoblast differentiation in vivo, with peak expression of phospho-SMAD1/5 in vivo noted at day 31 corresponding to maximal differentiation response of trophoblast in vitro These experiments demonstrate the involvement of BMP4-dependent pathways in the regulation of equine trophoblast differentiation in vivo and primary trophoblast differentiation in vitro via activation of SMAD1/5 pathway, a previously unreported mechanism of TGF␤ signaling in the mammalian placenta. We have previously shown that it is possible to exploit the late implantation of the equine placenta to isolate pure populations of trophoblast cells at multiple stages of binucleate trophoblast development from the same stallion/mare combination [3] These features of equine pregnancy provide us with a unique resource to dissect molecular events that regulate differentiation of CG-secreting trophoblast cells in vivo, and so integrate molecular data with physiological function

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