Function And Regulatory Mechanisms Of Hmgb During Uterine Decidualization In Mice

To support blastocyst implantation and further development of the embryo,the endometrium of mice and humans undergoes a set of processes known as decidualization.Primed by estrogen and progesterone,endometrial fibroblasts transdifferentiate into decidual cells and acquire an epithelial-like phenotype.The high mobility group box(HMGB)family molecules that I studied are the main members of the non-histone family and are the main members of the high mobility group(HMG)superfamily.However,HMGB mediate a number of DNA-dependent biological activities,such as gene replication,transcription,DNA damage and other physiological processes.Once the expression of HMGB are disturbed,the expression of many genes will be up-regulated or down-regulated.The high-mobility group box(HMGB)family includes four members: HMGB1,2,3 and 4.HMGB is a type of dynamic protein that help transcription factors and other nuclear proteins to bind their binding sites by bending DNA molecules.The HMGB protein family is the most studied group in the HMG superfamily,and is mainly involved in embryonic development,cell proliferation and differentiation,and gene expression regulation.However,its role in the decidualization of mouse uterus and its regulatory mechanism have not been reported.In this study,in situ hybridization,overexpression vector construction,RNA interference and fluorescence quantitative PCR were used to study the implantation and decidualization in mouse uterus from the gene expression,function and related regulatory mechanisms of the Hmgb family.The results found that Hmgb1 m RNA was obviously observed in uterine epithelium on day 2 and 3 of pregnancy,but its expression was scarcely detected on day 4 of pregnancy.With the onset of embryo implantation,abundant Hmgb1 expression was noted in the subluminal stromal cells around the implanting blastocyst at implantation sites.Meanwhile,the accumulation of Hmgb1 m RNA was visualized in the decidual cells.Hmgb1 advanced the proliferation of uterine stromal cells and induced the expression of prolactin family 8,subfamily a,member 2(Prl8a2),a reliable differentiation marker for decidualization.In uterine stromal cells,c AMP analogue 8-Br-c AMP up-regulated the expression of Hmgb1,but the upregulation was abrogated by protein kinase A(PKA)inhibitor H89.Silencing of Hmgb1 by specific si RNA impeded the induction of 8-Br-c AMP on Prl8a2.Further analysis evidenced that Hmgb1 was a critical mediator of Kruppel-like factor 5(Klf5)function in stromal differentiation.Knockdown of bone morphogenetic protein 2(Bmp2)prevented the up-regulation of Prl8a2 elicited by Hmgb1 overexpression,whereas addition of exogenous recombinant BMP2 protein(r BMP2)reversed the repression of Hmgb1 si RNA on Prl8a2 expression.Conclusion: Hmgb1 may play an important role during mouse uterine decidualization.Hmgb2 m RNA had not been expressed in the uterus on days 1-2 of pregnancy.It was strongly expressed in the uterine epithelium(uterine cavity and glands)and was weak in the stromal cells around the uterine cavity on day 4 of pregnancy.Hmgb2 was highly expressed in the blastocysts and stromal cells around the uterine cavity on day 5 of pregnancy,and was widely expressed in the decidual region of the uteri on days 6-8 of pregnancy and artificial induced decidualization models.It has also been found that the expression of Hmgb2 m RNA was dependent on the activation of blastocysts.Studies using hormone models had found that Hmgb2 was an estrogen-dependent molecule.In vitro cultured uterine stromal cells,Hmgb2 could promote uterine stromal cell proliferation and differentiation;in cultured decidual cells,Hmgb2 had no effect on the proliferation of decidualized cells,but could inhibit the differentiation of decidualized cells.c AMP and steroid hormones significantly could inhibit the expression of Hmgb2 in stromal cells.In situ hybridization result exhibited a dynamic expression pattern of Hmgb3 m RNA during early gestation,and it was mainly localized to the decidua on days 6 to 8 of gestation.Consistently,elevated Hmgb3 expression was noted in the decidualizing stromal cells after intraluminal oil infusion.In uterine luminal epithelium of ovariectomized mice,estrogen induced the accumulation of Hmgb3 m RNA,which was dependent on the existence of implanting blastocyst.Simultaneously,Hmgb3 could stimulate the proliferation of uterine stromal cells and promote the expression of Prl8a2,a reliable marker for stromal cell differentiation.Further analysis evidenced that Hmgb3 might modulate the expression of Ptn in uterine stromal cells.Moreover,silencing of Ptn could impede the upregulation of Prl8a2 elicited by Hmgb3 overexpression,while overexpression of Ptn reversed the repressive effects of Hmgb3 si RNA on Prl8a2 expression.In conclusion,there may be a correlation between the Hmgb(Hmgb1,Hmgb2 and Hmgb3)family molecules during mouse embryo implantation and decidualization,and Hmgb may play an important role during mouse decidualization.