Progranulin-derived granulin E and lysosome membrane protein CD68 interact to reciprocally regulate their protein homeostasis.

Progranulin (PGRN) is a glycoprotein implicated in several neurodegenerative diseases. It is highly expressed in microglia and macrophages and can be secreted or delivered to the lysosome compartment. PGRN comprises 7.5 granulin repeats and is processed into individual granulin peptides within the lysosome, but the functions of these peptides are largely unknown. Here, we identify CD68, a lysosome membrane protein mainly expressed in hematopoietic cells, as a binding partner of PGRN and PGRN-derived granulin E. Deletion analysis of CD68 showed that this interaction is mediated by the mucin-proline-rich domain of CD68. While CD68 deficiency does not affect the lysosomal localization of PGRN, it results in a specific decrease in the levels of granulin E but no other granulin peptides. On the other hand, the deficiency of PGRN, and its derivative granulin peptides, leads to a significant shift in the molecular weight of CD68, without altering CD68 localization within the cell. Our results support that granulin E and CD68 reciprocally regulate each other's protein homeostasis.

[Effect of Bushen Huoxue Recipe on Paracrine Gene Expression Profiling of Uterine Natural Killer Cells and Uterine Stromal Cells].

Objective To observe the effect of Bushen Huoxue Recipe (BHR) on paracrine gene expression profiling of uterine natural killer cells (uNK cells) and uterine stromal cells. Methods Human stromal cells were extracted from proliferative phase endometrium of child-bearing age females, which were then divided into the blank group, the control group, and the BHR group. DMEM/F12 was added in cells of the BHR group to dilute into final concentration of 2 mg/mL herbal liquor. Equal volume of DMEM/ F12 was added to cells in the normal group and the control group. Cells in the control group and the BHR group were cultured for 24 h, with 20% serum-free DMEM plus 80% uNK cell secretion extracting solution added. Then they were cultured in 5% CΟ2 at 37 °C for 6 h. Total RNAs were extracted after culture. The gene expression profile of stromal cells was detected using gene chip technology. At the same time mR- NA and protein expressions of chemokine (C-X-C motif) ligand 1 (CXCL1), intercellular cell adhesion molecule-1 (ICAM-1) , IL-8, and leukocyte inhibitor factor (LIF) were screened and detected using qRT- PCR and ELISA. Results Compared with the blank group, profiles of differentiated genes with 4-fold in- crease (a total of 63 genes) were basically agreeable in the control group and the BHR group. Compared with the control group, IL-15 receptor alpha (IL-15RA) was up-regulated by 1. 27 times, vascular endotheli- ai growth factor (VEGF) up-regulated by 1. 55 times, LIF up-regulated by 1. 45 times, IL-8 up-regulated by 1. 10 times, IL-11 up-regulated by 1. 23 times, transforming growth factor-ß (TGF-ß) up-regulated by 1. 40 times, epidermal growth factor (EGF) up-regulated by 1. 10 times, chemokine (C-C motif) ligand 8 (CCL8) up-regulated by 1.13 times, transporter 1 (TAP1 ) up-regulated by 1. 02 times, chemokine (C-X-C motif) receptor 2 (CXCR2) up-regulated by 1. 22 times, ICAM-1 up-regulated by 1. 15 times (P <0. 05) in the BHR group. Conclusion uNK paracrine played an important role in elevating endometrial receptivity and embry- o implantation, and BHR could improve and elevate the function of this paracrine system.

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

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-ß-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.

Bu Shen Huo Xue decoction restores endometrial leukemia-inhibitory factor but not Angiopoietin-2 expression, and improves uterine receptivity in the controlled ovarian stimulation rat model.

Leukemia-inhibitory factor (LIF) and Angiopoietin-2 (Ang-2) are important factors in fertility. In the present study, it was investigated whether Bu Shen Huo Xue Decoction (BSHXD) prevents controlled ovarian hyperstimulation (COH) treatment-induced changes in endometrial LIF and Ang-2 expression and whether it has an effect on the number of implantation sites and live births in rats. Uteri were collected on day (D) 3, 4 and 5 of pregnancy, and LIF and Ang-2 protein and mRNA expression were detected using western blot analysis and quantitative polymerase chain reaction. On pregnancy D10, the average number of implantation sites was observed. The number of live births from each group was recorded. The results indicated that BSHXD treatment markedly increased the number of live births by restoring endometrial LIF expression and the implantation capacity in the COH rat model. In addition, no association was identified between LIF and Ang-2 expression. Therefore, this suggests that BSHXD may be useful for female reproduction.