NEDD8-mediated neddylation is required for human endometrial stromal proliferation and decidualization

Abstract

Study question: Does NEDD8-mediated neddylation regulate human endometrial stromal proliferation and decidualization?

Summary answer: Neddylation inhibition by a selective NEDD8-activating enzyme inhibitor, MLN4924, significantly impairs human endometrial stromal cell (HESC) proliferation and decidualization and facilitates cell senescence, via p21 accumulation.

What is known already: Neddylation regulates cell proliferation and tissue remodeling during embryogenesis and tumorigenesis, while human endometrial stroma undergoes sequential proliferation, differentiation, as well as dynamic tissue remodeling during each menstrual cycle.

Study design, size, duration: We first analyzed 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.

Participants/materials, setting, methods: We 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 by 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 (HESC) during proliferation and decidualization (N = 6). An 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 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 cullin1 and p21, and SA-β-Galactosidase staining were performed to reveal the potential molecular basis for the impaired HESC proliferation, decidualization and cellular senescence. The siRNA technique was applied to knockdown p21 expression to test whether a clearance of p21 accumulation would correct the HESC defects from neddylation inhibition.

Main results and the role of chance: We demonstrated that NEDD8 is ubiquitously expressed in human endometrium including luminal epithelium, glandular epithelium and the stromal cells during the menstrual cycle, as well as in the HESCs during proliferation and differentiation in culture. Employing multiple molecular, cellular and pharmacological approaches, we further observed that neddylation inhibition by MLN4924 significantly attenuates HESC proliferation (P-value < 0.05), impairs decidual transformation (P-value < 0.05), and facilitates cellular senescence. These abnormal HESC activities upon MLN4924 exposure were accompanied with reduced cullin1 neddylation and an aberrant accumulation of p21. While a clearance of p21 accumulation by siRNA knockdown could partially restore HESC proliferation and cellular viability, it failed to correct the decidualization defects.

Limitations, reasons for caution: Since NEDD8 was also intensely expressed in the endometrial epithelium, it is interesting to further study its potential role in stroma-epithelial interactions through isolating and culturing epithelial cells. p21 siRNA knockdown experiments revealed that there are differential molecular machineries, other than p21, that are subject to neddylation regulation during HESC proliferation compared with differentiation. This alternative mechanism warrants further investigation in future.

Wider implications of the findings: 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 of approaching the neddylation system for diagnosis and treatment of infertility in women.

Study funding/competing interests: This work was supported in parts by the National Basic Research Program of China (2011CB944400 to H.W.) and the National Natural Science Foundation (81130009, 81330017 to H.W., 81170575 to S.Q. and 31471106 to S.Z.). The author declares that there is no conflict of interest.

Keywords: MLN4924; NEDD8; decidualization; human endometrial stromal cells (HESCs).