肿瘤坏死因子相关的凋亡诱导配体(TRAIL)基因抑制小鼠子宫基质细胞的蜕膜化

为了观察肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因对体外培养的小鼠蜕膜基质细胞增殖及凋亡的作用,探讨TRAIL对小鼠子宫蜕膜化进程的影响,构建TRAIL过表达及干扰质粒,转染小鼠基质细胞后诱导蜕膜化发生.转染72h后,应用半定量RT-PCR和Western blotting检测蜕膜基质细胞中TRAILmRNA和蛋白质的表达情况、MTT法观察蜕膜基质细胞的生长和增殖能力、流式细胞术检测蜕膜基质细胞的细胞周期分布情况和凋亡率.经酶切和核苷酸测序证实,TRAIL基因正确克隆入真核表达载体且能够上调TRAIL的表达,干扰质粒能有效地抑制TRAIL基因的表达.TRAIL过表达和RNA干扰的结果表明:TRAIL具有将蜕膜基质细胞阻滞在G0/G1期、抑制蜕膜基质细胞增殖并促使其凋亡的功效,提示TRAIL可能参与调节胚胎植入后基质细胞的有序蜕膜化进程.     

子宫内膜蜕膜化的特征及其调节因素

子宫内膜向蜕膜的转化是正常着床和妊娠的一个重要特征,对于胚泡着床是必不可少的。在蜕膜化过程中,子宫内膜基质细胞在形态和生理等方面都发生了很大的变化。蜕膜化过程受多种因素的调节,包括cAMP、胰岛素样生长因子结合蛋白-1(IGFBP-1)、自然杀伤细胞、同源盒基因-10(HOXA10)、激活素等。但对蜕膜化的机制及调节等仍不清楚。     

Lefty inhibits in vitro decidualization by regulating P57 and cyclin D1 expressions

Endometrial decidualization is highly important for successful construction and maintenance of embryo implantation and pregnancy. Lefty gene at different menstrual cycle phases has different expressions, indicating its regulatory significance. To study the mechanism of Lefty in decidualization, human endometrial stromal cells (hESCs) were cultured and induced with medroxyprogesterone acetate (MPA) and 8‐bromoadenosine‐cAMP (8‐Br‐cAMP) in vitro as a research model. Our results showed that Lefty1 overexpression inhibited MPA‐ and 8‐Br‐cAMP‐induced hESC decidualization and significantly reduced the secretion of prolactin (PRL) and insulin‐like growth factor‐binding protein 1 (IGFBP‐1). With the inhibition of Lefty1 expression, hESC decidualization induced by MPA and 8‐Br‐cAMP became more remarkable, and the secretions of PRL and IGFBP‐1 were higher too. Further tests indicated that during the process of decidualization, P57 expression increased, whereas cyclin D1 expression decreased. Although Lefty1 overexpression did not significantly change the expressions of P57 and cyclin D1, inhibition of Lefty1 expression resulted in more evident changes in P57 and cyclin D1 expressions. Meanwhile, cell cycle examination showed that Lefty1 overexpression reduced the cell cycle arrest at G1/S phase in the in vitro hESC decidualization model. Therefore, Lefty1 could regulate the cell cycle via modulating the expressions of P57 and cyclin D1 and then inhibit the decidualization in vitro. Copyright © 2014 John Wiley & Sons, Ltd.     

Hmgn5 functions downstream of Hoxa10 to regulate uterine decidualization in mice

Although Hmgn5 is involved in the regulation of cellular proliferation and differentiation, its physiological function during decidualization is still unknown. Here we showed that Hmgn5 was highly expressed in the decidual cells. Silencing of Hmgn5 expression by specific siRNA reduced the proliferation of uterine stromal cells and expression of Ccnd3 and Cdk4 in the absence or presence of estrogen and progesterone, whereas overexpression of Hmgn5 exhibited the opposite effects. Simultaneously, Hmgn5 might induce the expression of Prl8a2 and Prl3c1 which were 2 well-known differentiation markers for decidualization. In the uterine stromal cells, cAMP analog 8-Br-cAMP and progesterone could up-regulate the expression of Hmgn5, but the up-regulation was impeded by H89 and RU486, respectively. Attenuation of Hmgn5 expression could block the differentiation of uterine stromal cells in response to cAMP and progesterone. Further studies found that regulation of cAMP and progesterone on Hmgn5 expression was mediated by Hoxa10. During in vitro decidualization, knockdown of Hmgn5 could abrogate Hoxa10-induced upregulation of Prl8a2 and Prl3c1, while overexpression of Hmgn5 reversed the inhibitory effects of Hoxa10 siRNA on the expression of Prl8a2 and Prl3c1. In the stromal cells undergoing decidualization, Hmgn5 might act downstream of Hoxa10 to regulate the expression of Cox-2, Vegf and Mmp2. Collectively, Hmgn5 may play an important role during mouse decidualization.     

Hmgn1 acts downstream of C/EBPβ to regulate the decidualization of uterine stromal cells in mice

Although Hmgn1 is involved in the regulation of gene expression and cellular differentiation, its physiological roles on the differentiation of uterine stromal cells during decidualization still remain unknown. Here we showed that Hmgn1 mRNA was highly expressed in the decidua on days 6-8 of pregnancy. Simultaneously, increased expression of Hmgn1 was also observed in the artificial and in vitro induced decidualization models. Hmgn1 induced the proliferation of uterine stromal cells and expression of Ccna1, Ccnb1, Ccnb2 and Cdk1 in the absence of estrogen and progesterone. Overexpression of Hmgn1 could enhance the expression of Prl8a2 and Prl3c1 which were 2 well-known differentiation markers for decidualization, whereas inhibition of Hmgn1 with specific siRNA could reduce their expression. Further studies found that Hmgn1 could mediate the effects of C/EBPβ on the expression of Prl8a2 and Prl3c1 during in vitro decidualization. In the uterine stromal cells, cAMP analog 8-Br-cAMP could stimulate the expression of Hmgn1 via C/EBPβ. Moreover, siRNA-mediated down-regulation of Hmgn1 could attenuate the effects of cAMP on the differentiation of uterine stromal cells. During in vitro decidualization, Hmgn1 might act downstream of C/EBPβ to regulate the expression of Cox-2, mPGES-1 and Vegf. Progesterone could up-regulate the expression of Hmgn1 in the ovariectomized mouse uterus, uterine epithelial cells and stromal cells. Knockdown of C/EBPβ with siRNA alleviated the up-regulation of progesterone on Hmgn1 expression. Collectively, Hmgn1 may play an important role during mouse decidualization.     

Diarylcarbonates are a new class of deubiquitinating enzyme inhibitor

Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be similarly valuable. The currently available promiscuous DUB inhibitors are highly reactive electrophilic compounds that can crosslink proteins. Herein we introduce diarylcarbonate esters as a novel class of promiscuous DUB inhibitors that do not have the liabilities associated with the previously reported compounds. Diarylcarbonates stabilize the high molecular weight ubiquitin pools in cells and lysates. They also elicit cellular phenotypes associated with DUB inhibition, demonstrating their utility in ubiquitin discovery. Diarylcarbonates may also be a useful scaffold for the development of specific DUB inhibitors.     

Noncanonical Wnt signaling in stromal cells regulates B-lymphogenesis through interleukin-7 expression

The regulation of early B cell development and the interaction of hematopoietic precursors with stromal cells in the bone marrow (BM) are controlled by various secreted signaling molecules. Several recent studies showed Wnt signaling involved in B-lymphogenesis through stromal cells. However, the molecules modulated by Wnt signaling in stromal cells regulating B-lymphogenesis have not been identified yet. Interleukin (IL)-7 and CXC chemokine ligand (CXCL) 12 are known to be express in stromal cells, and both molecules are essential for B-lymphogenesis. In the present study, we examined the role of Wnt signaling in regulating IL-7 and CXCL12 expression and in affecting B-lymphogenesis. In mouse stromal ST2 cells, expression of IL-7 and CXCL12 mRNA was augmented by noncanonical Wnt5a. When mouse BM-derived cells were cultured on Wnt5a-overexpressing ST2 cells, an increased number of B220+/IgM- B-lymphoid precursor cells was observed. These results show that Wnt5a regulates IL-7 gene expression in stromal cells and suggest the possibility that noncanonical Wnt regulates B-lymphogenesis via IL-7 expression in stromal cells.     

Spongiacidin C,a pyrrole alkaloid from the marine sponge Stylissa massa,functions as a USP7 inhibitor

USP7, a deubiquitylating enzyme hydrolyzing the isopeptide bond at the C-terminus of ubiquitin, is an emerging cancer target. We isolated spongiacidin C from the marine sponge Stylissa massa as the first USP7 inhibitor from a natural source. This compound inhibited USP7 most strongly with an IC₅₀ of 3.8 μM among several USP family members tested.     

USP7 controls Chk1 protein stability by direct deubiquitination

Chk1, an essential checkpoint kinase in the DNA damage response pathway (DDR), is tightly regulated by both ATR-dependent phosphorylation and proteasome-mediated degradation. Here we identify ubiquitin hydrolase USP7 as a novel regulator of Chk1 protein stability. USP7 was shown before to regulate other DDR proteins such as p53, Hdm2 and Claspin, an adaptor protein in the ATR-Chk1 pathway required for Chk1 activation. Depletion or inhibition of USP7 leads to lower Chk1 levels. The decreased Chk1 protein after USP7 knock down cannot be rescued by simultaneously elevating Claspin levels, demonstrating that the effect of USP7 on Chk1 is independent of its known effect on Claspin. Conversely, overexpression of USP7 wild type, but not a catalytic mutant version, elevates Chk1 levels and increases the half-life of Chk1 protein. Importantly, wild type, but not catalytic mutant USP7 can deubiquitinate Chk1 in vivo and in vitro, confirming that USP7 directly regulates Chk1 protein levels. Finally we show that USP7 catalytic mutant is (mono-)ubiquitinated, which suggests auto-deubiquitination by this ubiquitin hydrolase, possibly important for its regulation.     

Synthesis and biological evaluation of thiazole derivatives as novel USP7 inhibitors

Herpesvirus-associated Ubiquitin-Specific Protease (HAUSP, also called USP7) interacts with and stabilizes Mdm2, and represents one of the first examples that deubiquitinases oncogenic proteins. USP7 has been regarded as a potential drug target for cancer therapy. Inhibitors of USP7 have been recently shown to suppress tumor cell growth in vitro and in vivo. Based on leading USP7 inhibitors P5091 and P22077, we designed and synthesized a series of thiazole derivatives. The results of in vitro assays showed that the thiazole compounds exhibited low micromolar inhibition activity against both USP7 enzyme and cancer cell lines. The compounds induced cell death in a p53-dependent and p53-independent manner. Taken together, this study may provide thiazole compounds as a new class of USP7 inhibitors.     

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010. © 2009 Wiley‐Liss, Inc.