Tripeptidyl peptidase I promotes human endometrial epithelial cell adhesive capacity implying a role in receptivity

Abstract

The endometrium undergoes cyclic remodelling throughout the menstrual cycle in preparation for embryo implantation which occurs in a short window during the mid-secretory phase. It is during this short ‘receptive window’ that the endometrial luminal epithelium acquires adhesive capacity permitting blastocysts firm adhesion to the endometrium to establish pregnancy. Dysregulation in any of these steps can compromise embryo implantation resulting in implantation failure and infertility. Many factors contribute to these processes including TGF-β, LIF, IL-11 and proteases. Tripeptidyl peptidase 1 (TPP1) is a is a lysosomal serine-type protease however the contribution of the TPP1 to receptivity is unknown. We aimed to investigate the role of TPP1 in receptivity in humans.In the current study, TPP1 was expressed in both epithelial and stromal compartments of the endometrium across the menstrual cycle. Expression was confined to the cytoplasm of luminal and glandular epithelial cells and stromal cells. Staining of mid-secretory endometrial tissues of women with normal fertility and primary unexplained infertility showed reduced immunostaining intensity of TPP1 in luminal epithelial cells of infertile tissues compared to fertile tissues. By contrast, TPP1 levels in glandular epithelial and stromal cells were comparable in both groups in the mid-secretory phase. Inhibition of TPP1 using siRNA compromised HTR8/SVneo (trophoblast cell line) spheroid adhesion on siRNA-transfected Ishikawa cells (endometrial epithelial cell line) in vitro. This impairment was associated with decreased sirtuin 1 (SIRT1), BCL2 and p53 mRNA and unaltered, CD44, CDH1, CDH2, ITGB3, VEGF A, OSTEOPONTIN, MDM2, CASP4, MCL1, MMP2, ARF6, SGK1, HOXA-10, LIF, and LIF receptor gene expression between treatment groups. siRNA knockdown of TPP1 in primary human endometrial stromal cells did not affect decidualization nor the expression of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Taken together, our data strongly suggests a role for TPP1 in endometrial receptivity via its effects on epithelial cell adhesion and suggests reduced levels associated with unexplained infertility may contribute to implantation failure.

Tripeptidyl Peptidase 1 Regulates Human Trophoblast Cell Proliferation Implying a Role in Placentation

“…We have previously shown that in human endometrial epithelial cells, knockdown of TPP1 impairs cell adhesion by targeting BCL2 and P53 [ 10 ]. Although TPP1 knockdown similarly affected cell adhesion in the HTR8/SVneo cells, no significant changes were recorded on the expression of BCL2 and P53, suggesting cell type-specific regulation of targeted genes to control cell adhesion.…”

“…Although TPP1 knockdown similarly affected cell adhesion in the HTR8/SVneo cells, no significant changes were recorded on the expression of BCL2 and P53, suggesting cell type-specific regulation of targeted genes to control cell adhesion. We have also reported that TPP1 knockdown in primary human endometrial stromal cells does not impact decidualization or expression of decidualization markers [ 10 ]. In this study, TPP1 was detected at appreciable levels in the decidual cells across all trimesters of pregnancy, with a lower level which has been observed in the second trimester.…”

“…Human placental villous and decidua tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at 4 μ m for immunohistochemistry staining. Sections were dewaxed, rehydrated, and subjected to antigen retrieval as previously optimized (10 mM sodium citrate for 5 min) [ 10 ]. After antigen retrieval, sections were treated with 3% hydrogen peroxide diluted in methanol to quench endogenous peroxidase.…”

“…Sections were then rinsed with Tris-buffered saline (TBS) and blocked with 10% goat serum and 2% human serum in TBS (v/v) for 30 min at room temperature. Sections were then incubated with TPP1 antibody (0.14 μ g/mL) or isotype negative control (0.14 μ g/mL) at 4°C overnight [ 10 ]. To determine if TPP1 localizes to the EVTs, HLA-G was used as an EVT marker (1 μ g/mL) and serial sections at 2 μ m were used to localize TPP1 and HLA-G in the first trimester human decidua/implantation sites.…”

“…Our recent work has demonstrated that TPP1 is expressed at significantly lower levels in human luminal epithelial cells of infertile endometrium compared to fertile. Functional analysis reveals that knockdown of TPP1 in human endometrial epithelial cells impairs cell adhesive capacity [ 10 ]. Since placentation requires highly regulated cell adhesion, proliferation, migration, and invasion, the involvement of TPP1 in mediating these activities support its potential roles in regulating normal placentation.…”