Bone morphogenetic proteins (BMPs) require major posttranslational modifications to become biologically active. One such key modification is endoproteolytic cleavage of the initially synthesized nonactive precursor protein to release the mature ligand. Here we show in a physiological context of uterine stromal decidualization that BMP2 cleavage is mediated by proprotein convertase 5/6 (PC6). Decidualization is a uterine remodeling event critical for embryo implantation. Deletion or knockdown of either BMP2 or PC6 inhibits decidualization causing implantation failure and female infertility. In this study we provide biochemical and physiological evidence that PC6 proteolytically activates BMP2. We used freshly isolated primary human endometrial stromal cells and demonstrated that PC6 was the sole member of the PC family significantly up-regulated during decidualization. The precursor form of BMP2 was reduced, whereas its active form was increased during decidualization. Inhibition of PC6 activity inhibited decidualization, and this was accompanied by a total blockade of BMP2 activation. Addition of recombinant active BMP2 partially rescued the decidualization arrest caused by PC6 inhibition. PC6 processed BMP2 at the KREKR(282) downward arrow cleavage site, and mutating this site prevented the cleavage. This study thus demonstrates for the first time that the proteolytic activation and thus bioavailability of BMP2 is controlled by PC6.
Embryo implantation requires a healthy embryo and a receptive endometrium (inner lining of the uterus); endometrial receptivity acquisition involves considerable epithelial surface remodeling. Dystroglycan (DG), a large cell surface glycoprotein, consists of a-and b-subunits; b-DG anchors within the plasma membrane whereas a-DG attaches extracellularly to b-DG. The glycosylated central a-DG mediates adhesion, but it is obstructed by its large N terminus (a-DG-N); a-DG-N removal enables DG's adhesive function. We demonstrate here that full-length a-DG in the human endometrial epithelium is a barrier for embryo attachment and that removal of a-DG-N by proprotein convertase 5/6 (PC6; a protease critical for implantation) regulates receptivity. This was evidenced by: 1) a-DG contains a PC6-cleavage site near a-DG-N, and PC6 cleaves a peptide harboring such a site; 2) PC6 knockdown reduces a-DG-N removal from endometrial epithelial cell surface and blastocyst adhesion; 3) mutating the PC6-cleavage site prevents a-DG-N removal, causing cell surface retention of full-length a-DG and loss of adhesiveness; 4) a-DG-N is removed from endometrial tissue in vivo for receptivity and uterine fluid a-DG-N reflects tissue removal and receptivity. We thus identified a-DG-N removal as an important posttranslational control of endometrial receptivity and uterine fluid a-DG-N as a potential biomarker for receptivity in women.-Heng, S., Paule, S. G., Li, Y., Rombauts, L. J., Vollenhoven, B., Salamonsen, L. A., Nie, G. Posttranslational removal of a-dystroglycan N terminus by PC5/6 cleavage is important for uterine preparation for embryo implantation in women. FASEB J. 29, 4011-4022 (2015). www.fasebj.org
Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.
Decidualization is a tissue remodelling process within the uterus in preparation for embryo implantation and pregnancy. In this study we isolated primary human endometrial stromal cells and stimulated decidualization with cAMP. We then used 2D- differential in-gel electrophoresis (DIGE) to identify proteins induced by decidualization. Eighty-eight out of 2714 spots were differentially regulated, 18 of which were assigned clear identities by mass spectrometry. Many of these are proteins known to be associated with cell structure and cytoskeletal remodelling. We validated five of these proteins by Western blot and immunohistochemistry on human endometrial tissue. The validated proteins are caldesmon 1, src substrate contactin 8, tropomyosin alpha-4 chain, protein disulfide isomerase 1A, and LIM and SH3 domain protein. With the exception of caldesmon 1, none of the identified proteins have previously been associated with decidualization. This study provides insight into our understanding of decidualization, which is important for successful embryo implantation and establishment of pregnancy.
STUDY QUESTION How is endometrial epithelial receptivity, particularly adhesiveness, regulated at the luminal epithelial surface for embryo implantation in the human? SUMMARY ANSWER Podocalyxin (PCX), a transmembrane protein, was identified as a key negative regulator of endometrial epithelial receptivity; specific downregulation of PCX in the luminal epithelium in the mid-secretory phase, likely mediated by progesterone, may act as a critical step in converting endometrial surface from a non-receptive to an implantation-permitting state. WHAT IS KNOWN ALREADY The human endometrium must undergo major molecular and cellular changes to transform from a non-receptive to a receptive state to accommodate embryo implantation. However, the fundamental mechanisms governing receptivity, particularly at the luminal surface where the embryo first interacts with, are not well understood. A widely held view is that upregulation of adhesion-promoting molecules is important, but the details are not well characterized. STUDY DESIGN, SIZE, DURATION This study first aimed to identify novel adhesion-related membrane proteins with potential roles in receptivity in primary human endometrial epithelial cells (HEECs). Further experiments were then conducted to determine candidates’ in vivo expression pattern in the human endometrium across the menstrual cycle, regulation by progesterone using cell culture, and functional importance in receptivity using in vitro human embryo attachment and invasion models. PARTICIPANTS/MATERIALS, SETTING, METHODS Primary HEECs (n = 9) were isolated from the proliferative phase endometrial tissue, combined into three pools, subjected to plasma membrane protein enrichment by ultracentrifugation followed by proteomics analysis, which led to the discovery of PCX as a novel candidate of interest. Immunohistochemical analysis determined the in vivo expression pattern and cellular localization of PCX in the human endometrium across the menstrual cycle (n = 23). To investigate whether PCX is regulated by progesterone, the master driver of endometrial differentiation, primary HEECs were treated in culture with estradiol and progesterone and analyzed by RT-PCR (n = 5) and western blot (n = 4). To demonstrate that PCX acts as a negative regulator of receptivity, PCX was overexpressed in Ishikawa cells (a receptive line) and the impact on receptivity was determined using in vitro attachment (n = 3–5) and invasion models (n = 4–6), in which an Ishikawa monolayer mimicked the endometrial surface and primary human trophoblast spheroids mimicked embryos. Mann–Whitney U-test and ANOVA analyses established statistical significance at *P ≤ 0.05 and **P ≤ 0.01. MAIN RESULTS AND THE ROLE OF CHANCE PCX was expressed on the apical surface of all epithelial and endothelial cells in the non-receptive endometrium, but selectively downregulated in the luminal epithelium from the mid-secretory phase coinciding with the establishment of receptivity. Progesterone was confirmed to be able to suppress PCX in primary HEECs, suggesting this hormone likely mediates the downregulation of luminal PCX in vivo for receptivity. Overexpression of PCX in Ishikawa monolayer inhibited not only the attachment but also the penetration of human embryo surrogates, demonstrating that PCX acts as an important negative regulator of epithelial receptivity for implantation. LIMITATIONS, REASONS FOR CAUTION Primary HEECs isolated from the human endometrial tissue contained a mixture of luminal and glandular epithelial cells, as further purification into subtypes was not possible due to the lack of specific markers. Future study would need to investigate how progesterone differentially regulates PCX in endometrial epithelial subtypes. In addition, this study used primary human trophoblast spheroids as human embryo mimics and Ishikawa as endometrial epithelial cells in functional models, future studies with human blastocysts and primary epithelial cells would further validate the findings. WIDER IMPLICATIONS OF THE FINDINGS The findings of this study add important new knowledge to the understanding of human endometrial remodeling for receptivity. The identification of PCX as a negative regulator of epithelial receptivity and the knowledge that its specific downregulation in the luminal epithelium coincides with receptivity development may provide new avenues to assess endometrial receptivity and individualize endometrial preparation protocols in assisted reproductive technology (ART). The study also discovered PCX as progesterone target in HEECs, identifying a potentially useful functional biomarker to monitor progesterone action, such as in the optimization of progesterone type/dose/route of administration for luteal support. STUDY FUNDING/COMPETING INTEREST(S) Study funding was obtained from ESHRE, Monash IVF and NHMRC. LR reports potential conflict of interests (received grants from Ferring Australia; personal fees from Monash IVF Group and Ferring Australia; and non-financial support from Merck Serono, MSD, and Guerbet outside the submitted work. LR is also a minority shareholder and the Group Medical Director for Monash IVF Group, a provider of fertility preservation services). The remaining authors have no potential conflict of interest to declare. TRIAL REGISTRATION NUMBER NA.
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