| Luman recruiting factor(LRF) also named CREB3 regulate factor(CREBRF), which is an endoplasmic reticulum stress-related leucine zipper protein transcription factor. LRF is a negative feedback regulating factor of Luman, which is a member of CREB family. LRF was found to recruit the nuclear form of Luman to discrete nuclear foci and repress the transactivation activity of Luman. LRF interacts with Luman through the leucine zipper region and promotes Luman degradation. LRF plays a critical role in the attenuation of the HPA axis through repression of glucocorticoid stress signaling during parturition and the postpartum period. The expression of glucocorticoid receptor was dramatically up-regulated in LRF gene knockout mice. The elevated glucocorticoid receptor expression can repress the synthesis of prolactin and further result to maternal behavioral defect. In our previous study, the expression of LRF was spatiotemporal in the uterus of mice during estrus cycle and early embryo implantation. The expression level of LRF in uterine primary decidual zone and secondary decidual zone of mice on day 6-7 of pregnancy was significantly increased. Collectively, the above studies indicated that LRF may be regulated by steroid hormone E2 and P4, and LRF may involve in embryo implantation and decidualization during pregnancy. In our present study, q RT-PCR, Western blotting, immunohistochemical staining and RNA interference were used to monitor(1) The regulated expression of LRF by steroid hormone E2 and P4 in mice uterus;(2) The effects of LRF silencing on embryo implantation and decidualization in vivo and in vitro.The full-length CDS region of LRF gene was amplified and three sh RNA fragments were synthetized and cloned into the lentiviral vector by T4 DNA ligase ligation. After transformation into competent E. coli DH5α cells, the candidate clones were identified by PCR and sequencing. The recombinant plasmid and the three packaging plasmids were co-transfected into HEK 293 T cells by Tubo Fect to produce the lentiviral particles, and the viral titer was determined. The HEK 293 T cells were infected by the lentiviral particles obtained and the transfection efficiency was assessed under fluorescent microscope. q RT-PCR and Western blotting was used to detect the expression and interference efficiency of LRF m RNA and protein in the transfected NIH 3T3 cells. The lentiviral vectors for LRF overexpression and RNA interferece were constructed successfully. The overexpression vector significantly increased the expression of exogenous LRF compared to controls. A effective sh RNA lentiviral vectors p CD513B-U6-sh LRF-2205 lead to significant knockdown of the LRF gene. The lentiviral vector for LRF has been successfully constructed with a high yield of lentivirus, which facilitate further investigation of the roles of LRF in the reproductive physiology function of mice.Using western blotting and immunohistochemical staining, we investigated LRF expression in primary endometrial stromal cells and ovariectomized mice uteri, which were administered with P4 and E2. The results showed that the expression of LRF was upregulated in the primary ESCs of mice and ovariectomized mice uteri in a time-dependent pattern following administration of progesterone. However, this stimulatory effect of P4 was eradicated by the progesterone receptor antagonist mifepristone(RU486). Although the expression of LRF did not obviously change in E2-treated primary stromal cells and ovariectomized mice uteri, LRF expression was down-regulated after the combinatorial treatment of E2 and P4 compared to treatment with P4 alone. In addition, LRF expression was increased compared to E2 and the control groups when stromal cells and ovariectomized mice uteri were treated with an estrogen receptor antagonist fulvestrant(ICI 182780). Our results indicated that LRF is expressed in mice uteri and that LRF is primarily upregulated by P4-PGR pathway. In addition, the upregulation of LRF expression by P4 can be partly mitigated with administration of E2. Using the sh LRF lentivirus to infected the mice uteri of day 3 of pregnancy to attenuated the expression of LRF, which significantly reduced the weight and size of implantation sites on days 7-8 of pregnancy. In a stromal cell culture model, LRF m RNA and protein levels were increased significantly during the process of stromal cell decidualization induced by E2 and P4. Utilizing LRF silencing, we show here that the decidual markers decidual prolactin-related protein, insulin-like growth factor-binding protein 1 and progesterone receptor were dramatically reduced and the decidual process was significantly inhibited. Cell cycle analysis and cell apoptosis analysis revealed that although no obvious apoptosis occurred in sh LRF lentivirus-infected stromal cells during decidualization, proliferation was inhibited via S-phase cell cycle arrest and the mitotic activity of uterine stromal cells was inhibited. An examination of cell cycle regulatory factors indicated that the m RNA expression levels of cyclin A and cyclin B1 were significantly down-regulated after treatment with the sh LRF lentivirus. However, there were no significant changes in the m RNA expression levels of cyclin D3 and cyclin E. Collectively, our study indicates that LRF is involved in the regulation of decidualization during pregnancy by modulating the expression of the key cell cycle regulatory factors cyclin A and cyclin B1. In summary, this study provides evidences that the expression of endoplasmic reticulum stress-related LRF is regulated by steroid hormone E2 and P4; and LRF regulates the cell cycle via cyclin A and cyclin B1 and plays a role in ESC proliferation during decidualization. This work reveals that LRF involves in physiological regulation in the reproductive system of female mice. In addition, this study provides a starting point for exploring the function of LRF in mammalian reproduction and adds a new dimension to our understanding of the relationship between endoplasmic reticulum stress and decidualization. |
PDF Full Text Request