We showed in this study that trophinin-mediated cell adhesion, mimicked by a trophinin-binding GWRQ peptide, promotes apoptosis of human endometrial epithelial cells in primary cultures and in an endometrial adenocarcinoma SNG-M cell line (). Previously, we showed that trophinin-mediated cell adhesion triggers a molecular switch for trophectoderm activation and invasion.2,9
This study also shows that trophinin functions as a switch, but by contrast to activation of trophectoderm cells, for apoptosis in endometrial epithelia. Given that the endometrial epithelial layer represents a barrier to the blastocyst to be implanted, it may require a mechanism for overcoming this barrier prior to embryo invasion. Indeed, the morphologies of human embryo implantation sites in vivo indicate a lack of endometrial epithelial layer at the boundary between invading trophectoderm cells and the maternal stroma,8,20,21
suggesting that epithelial layers are degenerated. Trophinin's apoptotic activity in endometrial epithelial cells is consistent with these observations and supports the hypothesis that trophinin functions in human embryo implantation.2–5,7,9
In the first trimester placenta, invasive extravillous cytotrophoblasts confront the endometrial glandular epithelia, initiating the steps required for blood vessel formation of the placenta.22
Trophinin expression by extravillous cytotrophoblasts (Sup. Fig. 9
) suggests that it may function in these processes. In testicular and colon cancer, trophinin expression is associated with invasive activity of tumor cells.23–25
In the invasive trophoblast, interactions of trophinin with cytoplasmic signaling molecules may differ from those in silent trophectoderm cells described our previous study in reference 9
, or from those occurring in endometrial epithelial cells described here.
Since human trophectoderm cells express Fas ligand, and endometrial epithelial cells express Fas, the Fas/FasL system has been proposed as mediating implantation-dependent apoptosis of endometrial cells.12,17
Fas/FasL activity is linked to activation of mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase (JNK) in endometrial epithelial cells.14,17
Since trophinin-mediated apoptosis is independent of Fas (), it is likely that two apoptotic pathways, mediated by Fas/FasL or by trophinin, operate during human embryo implantation to ensure removal of endometrial epithelial layer.
The present study identifies PKC-δ as the mediator of trophinin-stimulated apoptosis. PKC-δ may be sequestered by trophinin before cell adhesion, whereas upon cell adhesion, PKC-δ is released from trophinin, phosphorylated and translocated to nuclei ( and
). PKC-δ contains a nuclear localization signal near the C-terminus.26
It is known that in non-apoptotic cells, PKC-δ remains in the cytoplasm and binds to cytoplasmic proteins including the tyrosine kinase c-Abl,27
whereas in apoptotic cells PKC-δ enters the nucleus in response to various apoptotic agents.28–32
Nuclear PKC-δ undergoes caspase 3-mediated proteolysis and caspase-3-cleaved PKC-δ accumulates in the nucleus, binds DNA and inhibits DNA polymerase activity.33,34
Therefore, nuclear localization of PKC-δ is necessary and sufficient for apoptosis.18,28,34
When the human blastocyst approaches endometrial epithelial cells, trophinin is induced in response to high concentrations of hCG secreted by the blastocyst,5
which may trap PKC-δ near the plasma membrane, reducing cytoplasmic PKC-δ levels. Once trophinin-mediated cell adhesion occurs, PKC-δ is released from trophinin to the cytoplasm, allowing its translocation to the nucleus, where full-length PKC-δ may be cleaved to an active form by caspase 3 (). Present study also suggest that trophinin-mediated cell adhesion induces the expression of PKC-δ (), which further facilitates apoptosis of endometrial epithelial cells.
Humans reproduce a limited number of offspring, requiring “selection” of optimal endometrial epithelial sites for implantation. Numerous mechanisms and factors govern the apposition and initial adhesion of the human embryo to endometrial epithelium: L-selectin/selectin ligand,6
all ensure strict quality control during the initial adhesion between trophectoderm and endometrial epithelia.3
Dual entries of endometrial epithelia via either Fas or trophinin activity may represent a redundant form of quality control in embryo implantation.
The mechanisms underlying human embryo implantation cannot be investigated directly. Therefore, the in vitro model employed in this and previous studies in reference 7
, provides valuable information relevant to this intriguing method of reproduction at the molecular level. Further understanding of the mechanism of human embryo implantation will provide us with new diagnostic and therapeutic approaches to improve human reproductive health.