156. IDENTIFICATION OF SCAFFOLDING PROTEINS AS PC6 SUBSTRATES IN THE HUMAN ENDOMETRIAL EPITHELIAL CELLS FOR EMBRYO IMPLANTATION

Abstract

Successful embryo implantation is an important step in establishing pregnancy, which requires a healthy embryo and a receptive endometrium. Establishment of endometrial receptivity involves morphological and physiological changes initially in the endometrial epithelium, however the underlying molecular mechanisms are not fully understood. We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is up-regulated in the endometrium specifically at implantation in association with epithelial differentiation in the human and monkey. PCs convert a range of precursor proteins of important functions into their bioactive forms, they are thus regarded as critical ‘master switch’ molecules. The aim of this study was to identify target proteins of PC6 in the endometrial epithelial cells important for implantation. We used a HEC1A cell line in which PC6 was stably knocked down by siRNA approach (HEC1A-PC6). HEC1A cells that were similarly transfected with a scrambled siRNA sequence (HEC1A-control) were used as the control. Previous study confirmed that HEC1A-PC6 cells had much reduced capacity to adhere to blastocyst. A proteomic comparison between HEC1A-PC6 treated with or without human recombinant PC6 identified ezrin as a potential PC6 substrate. Ezrin is a cytoplasmic protein which is known to bind to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) thereby translocating to the plasma membrane. This complex has been associated with cytoskeletal re-organisation and changes in cell polarity. Co-immunoprecipitation of ezrin and EBP50 showed that knockdown of PC6 allowed the binding of ezrin to the C-terminus of EBP50 in HEC1A-PC6, whereas PC6 cleavage of EBP50 in HEC1A-control prevented the binding. This was also confirmed by immunofluorescence showing that ezrin and EBP50 were co-localized to the plasma membrane in HEC1A-PC6. This study thus identified that PC6 regulates scaffolding proteins such as EBP50 and ezrin in the endometrium for embryo implantation.