Trophinin-mediated cell adhesion induces apoptosis of human endometrial epithelial cells through PKC-δ

Abstract

Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-d protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-d associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-d became tyrosine phosphorylated, dissociated from trophinin, and entered the nucleus. In PKC-d knockdown endometrial cells, GWRQ did not induce apoptosis.

Acknowledgements

The authors thank Drs. Guy Salvesen and Ze'ev Ronai for their critical reading the manuscript, Dr. Elise Lamar for her excellent editing and Merryl Mathew for technical assistance. This study has been supported by the grant DoD prostate cancer research grant W81XWH040917.

Figures and Tables

Figure 1 Trophinin-mediated apoptosis of human endometrial epithelial cells. (A) Either trophinin-positive human trophoblastic embryonic carcinoma HT-H cells (a) or control trophinin-negative A431 cells (b) were added to monolayers of human endometrial adenocarcinoma SNG-M cells. Thirty minutes later, cells were removed from the monolayer, and an apoptag TUNEL assay was performed after 24 hours. Arrows in (a) indicate apoptotic nuclei. (B) Human endometrial adenocarcinoma SNG-M cells were subjected to a TUNEL assay 24 hours after adding none (a), control-MAPS (b) or GWRQ-MAPS (c). In all parts, cells were counterstained by hematoxylin. (C) Caspase 3/7 activities were measured using the lysates of SNG-M cells treated with 10 µg/mL each control-MAPS or with GWRQ-MAPS for the indicated times. Each bar and error bar represents mean ± SEM of triplicate measurements. (D) Human endometrial epithelial primary cell cultures were treated with hCG and IL-1β for 6 hours (5) to induce trophinin expression. Cells were subjected to a TUNEL assay 24 hours after addition of control-MAPS (a) or GWRQ-MAPS (b). Scale bars indicate 100 µm.

Figure 2 Summary of quantitative western blot analysis of phosphorylated proteins. Relative levels of phosphorylated proteins in SNG-M and HT-H cells after treatment with GWRQ-MAPS peptide for 30 minutes. Each point represents levels of phosphorylated protein, which either increased or decreased relative to levels seen in the absence of GWRQ-MAPS treatment. Phospho site-specific antibodies were used to detect each protein. The raw data are presented by Figure S5 and S6, Table S1 and S2.

Figure 3 Relationship of Fas/FasL- and trophinin-mediated apoptosis of SNG-M cells. (A) Nagative association of trophinin with Fas in SNG-M cells treated with or without GWRQ. Immunoprecipitation of SNG-M cell lysates with anti-trophinin antibody followed by western blot for FAS shows a lack of association between trophinin and FAS. (B) Western blot of immunoprecipitates with anti-fd phage antibody for FAS (upper parts) or trophinin (lower parts). Lane 1, total cell lysate from untreated SNG-M cells; lanes 2–4, immunoprecipitates of SNG-M cell lysates prepared from GWRQ (lanes 2 and 4) or control (lane 3) phage-bound SNG-M cells using control rabbit IgG (lane 2) or rabbit anti-phage antibody (lanes 3 and 4). (C) Immunocytochemistry of SNG-M cells using an anti-FAS antibody. SNG-M cells were transfected by negative control (a) or FAS (b) siRNA. Scale bars indicate 50 mm. (D) Western blot for Fas of SNG-M cell lysates 24 hours and 48 hours after being transfected with negative control siRNA or FAS siRNA. (E) TUNEL assay of SNG-M cells treated with control (a and b) or GWRQ (c and d) peptide after transfection with control (a and c) or FAS (b and d) siRNA. Scale bars indicate 50 mm.

Figure 4 Summary of quantitative western blot analysis of protein kinases in SNG-M cells before and after treatment with GWRQ-MAPS peptide for 30 minutes. The raw data are presented by Figure S7 and Table S3.

Figure 5 Involvement of PKC-δ in trophinin-mediated apoptosis in human endometrial epithelial SNG-M cells. (A) Immunocytochemistry of PKC-δ in SNG-M cells treated with (b) or without (a) GWRQ-MAPS for 30 minutes. Scale bars indicate 25 µm. (B) Western blot analysis of full-length (FL) PKC-δ and the caspase-3 cleaved form (CF) of PKC-δ distributed to either nuclear (N) or cytoplasmic (C) fractions prepared from SNG-M cells treated with or without GWRQ-MAPS peptide. Lamin and tubulin were included as a control for fractionation. (C) Time-dependency of nuclear translocation of PKC-δ in SNG-M cells cultured in media with or without GWRQ-MAPS. (D) Tyrosine phosphorylation of PKC-δ in SNG-M cells treated with or without GWRQ-MAPS for 30 min. (E) Immunocytochemistry of SNG-M cells by an anti-PKC-δ antibody. SNG-M cells transfected by control (a and b) or PKC-δ (c and d) siRNA were treated with GWRQ-MAPS (b and d) or with control peptide (a and c). Scale bars indicate 50 µm. (F) TUNEL assay of control (a) or PKC-δ (b) siRNA-transfected SNG-M cells treated with GWRQ peptide. A scale bar indicates 50 µm.

Figure 6 Association of trophinin with PKC-δ in SNG-M cells. (A) Immunoprecipitation by anti-trophinin antibody followed by western blot for PKC-δ. Cell lysates of SNG-M cells and HT-H cells were immunoprecipitated using an anti-trophinin antibody, followed by western blot analysis for PKC-δ. (B) Double immunofluorescence microscopy of SNG-M cells treated with or without GWRQ-MAPS for trophinin or PKC-δ. A scale bar presents 25 µm. (C) Dissociation of PKC-δ from trophinin in GWRQ-treated SNG-M cells. Western blot analysis of immunoprecipitates using an anti-trophinin antibody from lysates of SNG-M cells treated with or without GWRQ-MAPS.