| BackgroundHuman endometrium is a steroid-responsive tissue that undergoes cyclic regeneration involving sequential proliferation, differentiation, breakdown and repair.During each menstrual cycle, this dynamic tissue remodeling ensures the endometrium exhibiting a short period of receptivity for successful implantation known as the’implantation window’. It refers to the function of the endometrium that will allow the blastocyst to attach, penetrate, and induce localized changes in the stroma resulting in decidualization. If implantation fails, the endometrium degenerates and manifests as menstruation with subsequent regeneration and repair in preparation for the next implantation window. When the blastocyst implants into the womb, the endometrial stromal bed embedding the implanting blastocyst undergo extensive differentiation, vascular reconstruction and immune cell recruitment, a process termed decidualization crucial for successful pregnancy. The decidual tissue provides important secretory factors for nourishing the developing embryo before the maturation of placenta and forms an immune tolerance environment for the allograft embryo. It has been well established that an impaired endometrial stromal proliferation and differentiation is often associated with recurrent miscarriage, preeclampsia, intrauterine growth restriction and unexplained infertility in the clinical setting. Despite that a range of signaling molecules have been shown to contribute to the regulatory network of endometrial remodeling and transformation for implantation, the molecular machinery governing normal endometrial functions during menstrual cycle and early pregnancy remained largely unknown.Ubiquitylation is a form of protein modification mediated by ubiquitin that involves in degrading modified proteins. Increasing evidences suggest that ubiquitylation participates in various developmental events and diseases. In this respect, previous studies have demonstrated that ubiquitin and ubiquitin-related proteins are potentially important players during endometrial transformation and placental development. For example, while endometrial stromal cells contain apparently low levels of ubiquitin in the non-pregnant uterus, its abundance increases dramatically during early pregnancy, with decidualized stromal cells containing high levels of both nuclear and cytoplasmic ubiquitin. Moreover, human endometrial decidual cells can secrete ubiquitin in culture. However, it remains to be determined how ubiquitylation modification via ubiquitin or ubiquitin-like proteins ensures normal human endometrial functions during early pregnancy.NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) is a 76 amino acid polypeptide that was originally identified as a highly expressed gene from embryonic mouse brains and conjugated to its target proteins as an ubiquitin-like modification through the covalent linkage with the lysine residue. Among the several identified ubiquitin-like proteins, NEDD8 is the closest relative to ubiquitin and undergo the active neddylation processes analogous to the ubiquitylation pathway. Neddylation is a process of substrate protein modification by NEDD8, which is catalyzed by cascaded enzymes similar with the ubiquitin system. These enzymes consisted of the NEDD8-activating enzyme E1 (NAE) with two subunits, APP-BP1 and UBA3, NEDD8-conjugating enzyme E2 (Ubc12), and NEDD8-E3 ligases targeted different substrates. Similar to classic ubiquitination, the process of neddylation also involves a chronological modification process in which small proteins need to be conjugated, activated, transferred to the substrates and E3 ligases determine the specific targets. However, tagging NEDD8 onto a substrate protein during neddylation is not for targeting protein degradation, but for modulation of protein activity and function. The most well characterized substrates for the neddylation are cullins, a highly conserved family of proteins. Cullin proteins function as a core scaffold for assembling ubiquitin E3 ligases, the Cullin-RING ubiquitin ligases (CRLs), with its founding member of SKP1-Cullins-F-box proteins (SCF) E3 ubiquitin ligase. This ubiquitin ligase assembling requires cullin neddylation for its activation. Thus, through modulating CRLs, neddylation regulates many biological processes, including cell cycle progression and signal transduction during embryogenesis and tumorigenesis. Since Cullins have been shown to be expressed in human fetal-maternal interface during early pregnancy, it would be interesting to further explore the physiological function of NEDD8 mediated neddylation during human endometrial proliferation and differentiation.In the present study, we demonstrated that NEDD8 is ubiquitously expressed in human endometrium during the menstrual cycle. Using human endometrial stromal cell line (HESC) and a selective NEDD8 activating enzyme inhibitor, we further revealed that neddylation inhibition impairs HESC proliferation and decidualization and facilitates cellular senescence accompanied with an aberrant p21 accumulation. These findings added a new line of evidence supporting the fact that NEDD8 mediated neddylation is required for human endometrial stromal proliferation and decidualization.ObjectiveThe central issue in all these studies is to analyze the expression of NEDD8 in human endometrial tissues from 50 subjects, and then explored the consequence of neddylation inhibition by MLN4924 on HESCs proliferation, decidualization and cellular senescence. Our findings add novel evidence showing for what we believe the first time that NEDD8 mediated neddylation is required for normal human endometrial functions, which raises the possibility for exploring the mechanism of neddylation system in endometrial receptivity for embryo implantation.MethodsWe collected 50 dated human endometrial tissues from early proliferative stage to late secretory phase of the menstrual cycle and analyzed the NEDD8 expression and cellular location in human endometrium employing quantitative real-time PCR (qRT-PCR) and immunohistochemistry staining. Similar approaches were also used to explore the mRNA and protein expression of NEDD8 in an immortalized human endometrial stromal cell line during proliferation and decidualization (N=6). MTS assay was performed to evaluate the effects of neddylation inhibition by MLN4924 on HESC proliferation. Flow cytometry and BrdU incorporation assay were conducted to determine the HESC cell cycle progression in response to MLN4924 exposure during proliferation. We also analyzed the F-actin distribution by phalloidin staining and decidual marker gene expression by qRT-PCR to accesses the consequence of neddylation inhibition on HESC decidualization. Immunoblotting analysis of cullinl and p21, and SA-β-Galactosidase staining were performed to reveal the potential molecular basis for impaired HESC proliferation, decidualization and cellular senescence. siRNA technique was applied to knockdown p21 expression to test whether a clearance of p21 accumulation would correct the HESC defects from neddylation inhibition.Statistical analysisAll values were shown as means ± SEM of at least three independent experiments. A parametric test was performed. We examined an overall effect using one-way analysis of variance (ANOVA), then multiple comparisons between control and specific dose or time groups were made using the method of least significant difference (LSD) if the variance among the groups were equal or the method of Dunnett’s T3 in the case of heterogeneity of variance. Statistical analysis was performed using SPSS 19.0 program. The difference was regarded statistically significant if the two tailed P value was less than 0.05.Results1. NEDD8 was widely expressed in the luminal epithelium, gland epithelium as well as the stromal cells at different phases of menstrual cycle. This intensely persisted expression and nuclear localization of NEDD8 in human endometrium indicated that NEDD8-mediated neddylation might actively participate in the dynamic regulation of human stromal proliferation and decidualization.2. Using quantitative real-time PCR and immunocytochemistry staining, we examined the NEDD8 expression in HESCs undergoing either proliferation or decidualization. Both NEDD8 mRNA and protein can be abundantly detected in the either proliferating or differentiating HESCs in culture, similar to observations of persisted expression of NEDD8 in different phases of endometrium in vivo.3.0.3 μM of MLN4924 can efficiently block cullinl neddylation. Neddylation inhibition attenuates HESC proliferation and a significantly slow cell cycle progression in MLN4924-treated HESCs. A time course analysis of bromodeoxyuridine (BrdU) incorporation assay revealed massively reduced HESCs in the S phase during neddylation inhibition and a unique mitotic phase marker p-Histone H3 (p-H3, ser10) was drastically reduced in MLN4924-treated stromal cells.4. Neddylation inhibition impairs HESC decidualization. Neddylation inhibition by MLN4924 disrupted the normal actin network in HESCs and failed to go through decidualization. The findings collectively ascribed an indispensable role to NEDD8-mediated neddylation during HESCs decidualization.5. An obvious inhibition of cullinl neddylation and a significantly increased accumulation of p21 proteins by MLN4924 treatment were obversed in proliferating and differentiating HESCs. The results reinforced the notion that MLN4924-induced p21 accumulation reduces cell viability via facilitating cellular senescence.6. MLN4924-exerted neddylation inhibition fails to trigger the apoptotic cell death pathway in human endometrial stromal cells in neither proliferation nor differentiation culture conditions.7. Cdknla-targeting siRNA can largely reduce p21 expression at both mRNA and protein levels in either the control or MLN4924-treated HESCs during proliferation. This reduced p21 accumulation upon MLN4924 exposure can partially restore the HESC proliferation activities as assessed by Ki67 staining and knockdown of p21 expression also largely corrected the cellular senescence status. Despite an efficient clearance of p21 accumulation by Cdknla siRNA in MLN4924-treated HESCs, the expression of decidual markers such as PRL and IGFBP1 remained persistently lower in HESCs under neddylation inhibition. These findings suggested there are differential molecular machineries rather than p21 only subjected to neddylation regulation during HESC proliferation versus differentiation.Conclusion1. NEDD8 is ubiquitously expressed in human endometrium during the menstrual cycle.2. Both NEDD8 mRNA and protein can be abundantly detected in the either proliferating or differentiating HESCs in culture, similar to observations of persisted expression of NEDD8 in different phases of endometrium in vivo.3. MLN4924 can efficiently block cullinl neddylation. NEDD8-mediated neddylation is required for normal HESC proliferation, whereas its inhibition strikingly inhibits the consecutive progression of cell cycle through the S-phase and entering the G2-M phase.4. Neddylation inhibition by MLN4924 disrupted the normal actin network in HESCs and failed to go through decidualization.5. Neddylation inhibition induces p21 accumulation, facilitating cell senescence.6. Neddylation inhibition fails to activate the apoptotic cell death pathway in HESCs in culture.7. p21 knockdown partially restores HESC proliferation and cellular viability, but not decidualization. |
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