G. Nie

Pro-protein convertases (PCs) other than PC6 are not tightly regulated for implantation in the human endometrium

Pro-protein convertases (PCs) are a family of serine proteases (furin, PC1/3, PC2, PACE4, PC4, PC5/6, PC7/8) responsible for post-translational processing and activation of inactive precursors of many regulatory proteins. Endometrial PC6 is critical for implantation in mice and for decidualization of human endometrial stromal cells (ESCs). This study investigated the endometrial expression of other PCs during the menstrual cycle and early pregnancy to elucidate potential redundancies. Furin, PC4, PACE4, and PC7 along with PC6 transcripts were detected in total endometrial RNA, whereas PC1 and PC2 transcription levels were negligible. Quantitative RT-PCR demonstrated highest levels of furin mRNA during menstruation and lowest levels during the proliferative phase. Furin protein was immunolocalized in endometrial luminal and glandular epithelia, stromal fibroblasts, endothelia, and leukocytes. PACE4 and PC7 proteins were also immunodetected in endometrial stroma and glands. Total furin, PC7, and PACE4 proteins were constitutive in both stromal and glandular compartments throughout the cycle and during first trimester pregnancy. Furthermore, Furin and PC7 transcription was unaltered during decidualization of ESCs in vitro in contrast to PC6 which is significantly up-regulated during decidualization. Thus, whereas PC6 is tightly regulated during endometrial preparation for implantation, furin, PACE4, and PC7 are constitutively expressed in human endometrium, but must be considered if PC6 is to be targeted for manipulation of fertility.

Inhibition of HTRA3 stimulates trophoblast invasion during human placental development

Controlled invasion of extravillous trophoblast (EVT) is necessary for implantation and placentation. The serine protease HTRA3 is highly expressed in decidual cells in the late secretory phase of the menstrual cycle and throughout pregnancy. During the first trimester it is expressed in most trophoblast cell types, but not in the invading interstitial trophoblast. HTRA3 and its family members are down-regulated in a number of cancers and are proposed as tumour-suppressors. The current study investigated whether inhibiting HTRA3 in a first trimester trophoblast cell line expressing high levels of HTRA3 would alter invasion. HTR-8/SVneo (HTR-8, derived from first trimester placenta) and a number of choriocarcinoma cells (JEG-3, AC-1M88 and AC-1M32) were screened for HTRA3 expression. Only HTR-8 cells expressed high levels of HTRA3 mRNA, consistent with HTRA3 being down-regulated in cancer. Western blotting and immunofluorescence confirmed HTRA3 protein expression and localisation in HTR-8 cells. HTRA3 was detected in conditioned medium of HTR-8 cells, confirming its secretory nature. For functional studies, both long and short forms of recombinant human HTRA3, wild type and protease-inactive mutant (S(305)A) were produced using wheat-germ cell-free technology. Both have a similar molecular size, but the mutants have negligible protease activity. In addition, the mutants significantly inhibited the wild type protease activity, supporting their dominant-negative inhibition and utility as specific inhibitors of the wild type protein. Inhibition of HTRA3 by exogenous addition of HTRA3 mutant resulted in a significant increase in HTR-8 cell invasion. These results strongly support the hypothesis that HTRA3 is an inhibitor of trophoblast invasion during placental development.

211. Proteomic identification of caldesmon as one of the physiological substrates of proprotein convertase 6 during decidualisation

We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is a critical endometrial factor for implantation. PC6 is upregulated in the endometrium specifically at implantation in association with epithelial differentiation (in human and monkey) and stromal cell decidualisation (in the mouse, human and monkey). Knockdown of endometrial PC6 during early pregnancy in mice in vivo led to complete failure of implantation, while blocking of PC6 production in human endometrial stromal cells in vitro inhibited decidualisation. PCs convert a range of precursor proteins of important functions into their bioactive forms; they are thus regarded as critical ‘master switch’ molecules. We hypothesise that PC6 exerts its roles in the endometrium by regulating proteins of diverse functions essential for implantation. In this study, we utilised proteomic technology and aimed to identify proteins that are specifically cleaved by PC6 in human endometrial stromal cells (HESC) during decidualisation. HESC were decidualised with cyclic AMP, the cell lysates were treated with and without recombinant human PC6-A (rPC6-A), and the 2D Differential in Gel Electrophoresis (2D DiGE) protein profiles were compared between the two treatments. We identified several proteins which were differentially cleaved following the addition of rPC6-A. Mass spectrometric analysis confirmed that the most abundant of these were caldesmon, tropomyosin-2, tropomyosin-4, hypoxia Inducible factor-1 and chloride intracellular channel-1. These proteins showed spot shifts in hPC6-A treated HESC lysates consistent with hPC6-A cleavage. western blot analysis confirmed the specific cleavage of caldesmon by PC6 in HESCs, and immunohistochemical analysis showed co-localisation of caldesmon and PC6 in decidual cells in human endometrial tissue. Given that caldesmon is a structural protein previously found to be involved in actin filament reorganisation, our results strongly suggest that PC6 is a mediator of structural remodelling of stromal cells during decidualisation in the endometrium.