Silencing of TGF-beta signalling by the pseudoreceptor BAMBI.

Members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta, bone morphogenetic proteins (BMPs), activins and nodals, are vital for regulating growth and differentiation. These growth factors transduce their signals through pairs of transmembrane type I and type II receptor kinases. Here, we have cloned a transmembrane protein, BAMBI, which is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. We show that BAMBI is co-expressed with the ventralizing morphogen BMP4 (refs 5, 6) during Xenopus embryogenesis and that it requires BMP signalling for its expression. The protein stably associates with TGF-beta-family receptors and inhibits BMP and activin as well as TGF-beta signalling. Finally, we provide evidence that BAMBI's inhibitory effects are mediated by its intracellular domain, which resembles the homodimerization interface of a type I receptor and prevents the formation of receptor complexes. The results indicate that BAMBI negatively regulates TGF-beta-family signalling by a regulatory mechanism involving the interaction of signalling receptors with a pseudoreceptor.     

Betaglycan binds inhibin and can mediate functional antagonism of activin signalling

Activins and inhibins, structurally related members of the TGF-beta superfamily of growth and differentiation factors, are mutually antagonistic regulators of reproductive and other functions. Activins bind specific type II receptor serine kinases (ActRII or IIB) to promote the recruitment and phosphorylation of the type I receptor serine kinase, ALK4 (refs 7-9), which then regulates gene expression by activating Smad proteins. Inhibins also bind type II activin receptors but do not recruit ALK4, providing a competitive model for the antagonism of activin by inhibin. Inhibins fail to antagonize activin in some tissues and cells, however, suggesting that additional components are required for inhibin action. Here we show that the type III TGF-beta receptor, betaglycan, can function as an inhibin co-receptor with ActRII. Betaglycan binds inhibin with high affinity and enhances binding in cells co-expressing ActRII and betaglycan. Inhibin also forms crosslinked complexes with both recombinant and endogenously expressed betaglycan and ActRII. Finally, betaglycan confers inhibin sensitivity to cell lines that otherwise respond poorly to this hormone. The ability of betaglycan to facilitate inhibin antagonism of activin provides a variation on the emerging roles of proteoglycans as co-receptors modulating ligand-receptor sensitivity, selectivity and function.     

A truncated activin receptor inhibits mesoderm induction and formation of axial structures in Xenopus embryos.

Activins can induce mesoderm in embryonic explants and have been proposed as the natural inducer in Xenopus. A mutant activin receptor that inhibits activin signalling is used to show that activin is required for the induction of mesoderm in vivo and the patterning of the embryonic body plan. Blocking the activin signal transduction pathway also reveals autonomous induction of a neural marker and unmasks a relationship between activin and fibroblast growth factor.     

Activin-like factor from a Xenopus laevis cell line responsible for mesoderm induction

Induction of mesoderm during early amphibian embryogenesis can be mimicked in vitro by adding growth factors, including heparin-binding and type-beta transforming growth factors (TGF-beta), to isolated ectoderm explants from Xenopus laevis embryos. Although the mesoderm-inducing factor (MIF) from X. laevis XTC cells (XTC-MIF) has properties similar to TGF-beta, this factor is still unidentified. Recently, we obtained a number of homogeneous cell lines from the heterogeneous XTC population, which differ in their MIF production. Only one, XTC-GTX-11, produced MIF, although it was similar to the rest of the clones in its production of known growth factors, including TGF-beta activity. This observation, together with the identification of activin A as a potent MIF led us to study the parallel activities of MIF and activin. Here we report an analysis of activin-like activity from XTC cells and some of the XTC clones, including XTC-GTX-11. There is a clear consistent correlation between MIF activity and presence of activin activity, indicating that XTC-MIF is the Xenopus homologue of mammalian activin.     

Smurf2单克隆抗体的制备与鉴定

Smad泛素调节因子家族的两个E3泛素连接酶成员Smurf1和Smurf2 (Smad ubiquitin regulatory factor 1 and 2)具有至少80％的同源性.为了更好地研究它们的功能,需要获得一个能区分Smurf1和Smurf2的单克隆抗体.选取Smurf2与Smurf1蛋白序列中仅有的非同源区域序列作为抗原免疫Bal b/C小鼠,成功制备若干株能稳定分泌抗Smurf2抗体的杂交瘤细胞株.对所获得的单克隆抗体的效价和特异性等进行一系列检测,结果表明该抗体能特异性地识别过表达及细胞内源Smurf2蛋白.此外该抗体能被应用于Western blot和免疫荧光实验,从而为深入研究Smurf2的细胞生物学功能提供了良好的实验基础.     

BMP2，BMP4及BMP信号通路相关成员在发情周期小鼠子宫中的表达

研究目的：研究骨形态发生蛋白（BMP）2,4及BMP信号通路相关成员在发情周期小鼠子宫中的表达模式。创新要点：运用实时荧光定量聚合酶链式反应（PCR）系统地研究了Bmp2和Bmp4及其BMP信号通路相关成员在小鼠子宫中mRNA水平表达模式,同时运用免疫组化方法研究了BMP2蛋白在小鼠子宫中的定位模式。研究方法：重要结论：收集发情周期各个时期小鼠子宫,一侧子宫角贮存于液氮或-80℃冰箱用于实时荧光定量PCR,另一侧子宫角用40mg／ml多聚甲醛固定用于BMP2蛋白免疫组化定位。实时荧光定量PCR结果表明,Bmp2的表达水平在发情前期显著高于发情期和发情后期（P〈0．05）,Bmp4的表达水平呈现显著波动,但Bmp2与Bmp4差异不显著（P〉0．05）。Bmprla和Bmpr2的表达水平在整个发情周期无显著变化（P〉0．05）。然而,Bmprlb的mRNA水平在发情期显著下降（P〈0．05）,在发情后期上升。此外,Bmprlb的mRNA水平显著低于相应时期Bmprla和Bmpr2的mRNA水平（P〈0．05）。三种R—Smads差异地表达于小鼠子宫,并且Smad1和Smad5的表达水平显著高于Smad8（P〈0．05）。此外,Smad4的表达水平在整个发情周期无显著变化。免疫组化结果显示,BMP2蛋白在整个发情周期差异表达,并主要定位于子宫腔上皮细胞和腺上皮细胞。我们的结果提供了BMP2和BMP4及其BMP信号通路相关成员mRNA水平表达变化信息,这些数据为论证BMP在子宫内膜中的作用如子宫内膜的退化与重塑提供量化和有用的信息。     

DNA damage stress induces the dissociation of Smurf1/2 from MDM2 in a slow manner

The tumor suppressor p53 locates at the key point of cell growth or apoptosis balance, and the expression level of p53 is tightly controlled by ubiquitin ligases including MDM2. Upon DNA damage stresses, p53 was accumulated and activated, leading to cell cycle arrest or apoptosis. We previously showed that Smad ubiquitylation regulatory factor 1/2 (Smurf1/2) promotes p53 degradation by interacting with and stabilizing MDM2, and consequently enhancing MDM2-mediated ubiquitylation of p53. However, it is unclear how the Smurf1-MDM2 interaction is regulated in response to DNA damage stress. Here, we show that in response to etoposide treatment Smurf1 dissociates from MDM2, resulting in MDM2 destabilization and p53 accumulation. The negative regulation of Smurf1 on apoptosis is released. Notably, this dissociation is a slow process rather than a rapid response, implicating high expression of Smurf1 might confer the resistance against p53 activation. Consistent with this notion, we observed that Smurf1/2 ligases are highly expressed in colon cancer, esophageal squamous cell carcinoma and pancreatic cancer tissues, suggesting the oncogenic tendency of Smurf1/2.     

Smad4 signalling in T cells is required for suppression of gastrointestinal cancer

SMAD4 (MAD homologue 4 (Drosophila)), also known as DPC4 (deleted in pancreatic cancer), is a tumour suppressor gene that encodes a central mediator of transforming growth factor-beta signalling. Germline mutations in SMAD4 are found in over 50% of patients with familial juvenile polyposis, an autosomal dominant disorder characterized by predisposition to hamartomatous polyps and gastrointestinal cancer. Dense inflammatory cell infiltrates underlay grossly normal appearing, non-polypoid colonic and gastric mucosa of patients with familial juvenile polyposis. This prominent stromal component suggests that loss of SMAD4-dependent signalling in cells within the epithelial microenvironment has an important role in the evolution of intestinal tumorigenesis in this syndrome. Here we show that selective loss of Smad4-dependent signalling in T cells leads to spontaneous epithelial cancers throughout the gastrointestinal tract in mice, whereas epithelial-specific deletion of the Smad4 gene does not. Tumours arising within the colon, rectum, duodenum, stomach and oral cavity are stroma-rich with dense plasma cell infiltrates. Smad4(-/-) T cells produce abundant T(H)2-type cytokines including interleukin (IL)-5, IL-6 and IL-13, known mediators of plasma cell and stromal expansion. The results support the concept that cancer, as an outcome, reflects the loss of the normal communication between the cellular constituents of a given organ, and indicate that Smad4-deficient T cells ultimately send the wrong message to their stromal and epithelial neighbours.     

Cytoplasmic PML function in TGF-beta signalling

Transforming growth factor beta (TGF-beta) is a pluripotent cytokine that controls key tumour suppressive functions, but cancer cells are often unresponsive to it. The promyelocytic leukaemia (PML) tumour suppressor of acute promyelocytic leukaemia (APL) accumulates in the PML nuclear body, but cytoplasmic PML isoforms of unknown function have also been described. Here we show that cytoplasmic Pml is an essential modulator of TGF-beta signalling. Pml-null primary cells are resistant to TGF-beta-dependent growth arrest, induction of cellular senescence and apoptosis. These cells also have impaired phosphorylation and nuclear translocation of the TGF-beta signalling proteins Smad2 and Smad3, as well as impaired induction of TGF-beta target genes. Expression of cytoplasmic Pml is induced by TGF-beta. Furthermore, cytoplasmic PML physically interacts with Smad2/3 and SARA (Smad anchor for receptor activation) and is required for association of Smad2/3 with SARA and for the accumulation of SARA and TGF-beta receptor in the early endosome. The PML-RARalpha oncoprotein of APL can antagonize cytoplasmic PML function and APL cells have defects in TGF-beta signalling similar to those observed in Pml-null cells. Our findings identify cytoplasmic PML as a critical TGF-beta regulator, and further implicate deregulated TGF-beta signalling in cancer pathogenesis.     

The DIX domain targets dishevelled to actin stress fibres and vesicular membranes

Colorectal cancer results from mutations in components of the Wnt pathway that regulate beta-catenin levels. Dishevelled (Dvl or Dsh) signals downstream of Wnt receptors and stabilizes beta-catenin during cell proliferation and embryonic axis formation. Moreover, Dvl contributes to cytoskeletal reorganization during gastrulation and mitotic spindle orientation during asymmetric cell division. Dvl belongs to a family of eukaryotic signalling proteins that contain a conserved 85-residue module of unknown structure and biological function called the DIX domain. Here we show that the DIX domain mediates targeting to actin stress fibres and cytoplasmic vesicles in vivo. Neighbouring interaction sites for actin and phospholipid are identified between two helices by nuclear magnetic resonance spectroscopy (NMR). Mutation of the actin-binding motif abolishes the cytoskeletal localization of Dvl, but enhances Wnt/beta-catenin signalling and axis induction in Xenopus. By contrast, mutation of the phospholipid interaction site disrupts vesicular association of Dvl, Dvl phosphorylation, and Wnt/beta-catenin pathway activation. We propose that partitioning of Dvl into cytoskeletal and vesicular pools by the DIX domain represents a point of divergence in Wnt signalling.     

MiR-205 inhibits the invasion and migration of esophageal squamous cell carcinoma by modulating SMAD1 expression

Esophageal squamous cell carcinoma （ESCC） is one of the most lethal cancers worldwide. In this study, we aimed to investigate the underlying mechanisms of metastasis inhibition by miR-205 in ESCC. In microRNA （miRNA） array and quantitative RT-PCR analyses, we found that the expression level of miR-205 was significantly lower in patients with lymph node metastasis compared with that in patients without lymph node metastasis. After transfection of miR-205 mimics or inhibitors into ESCC cell lines, a significant negative correlation was observed between the expression level of miR-205 and Smad 1. In luciferase reporter assays, we revealed that miR- 205 inhibited the expression of SMAD1 by targeting the 3＇ untranslated region （3＇-UTR） of SMAD1 mRNA in ESCC cells. Furthermore, our results showed that miR-205 sup- pressed the invasion and migration of ESCC cells, whereas Smadl increased their invasion and migration. Taken together, our study demonstrates that miR-205 functions as a suppressor of tumor metastasis by regulating SMAD1 expression through targeting the 3＇-UTR of SMAD1 mRNAin ESCC. Therefore, miR-205 may be a potential therapeutic target for miRNA-based therapy of ESCC.     

Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the 'DNA damage' S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine gamma-aminobutyric acid (GABA) signalling by means of GABA(A) receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABA(A) receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABA(A) receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.     

Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin A

The first inductive interaction in amphibian development is mesoderm induction, when a signal from the vegetal hemisphere of the blastula induces mesoderm from overlying equatorial cells. Recently, several 'mesoderm-inducing factors' (MIFs) have been discovered. These cause isolated Xenopus animal caps to form mesodermal cell types such as muscle, instead of their normal fate of epidermis. The MIFs fall into two classes. One comprises members of the fibroblast growth factor (FGF) family, and the other members of the transforming growth factor type beta (TGF-beta) family. Of the latter group, the most potent is XTC-MIF, a protein produced by Xenopus XTC cells. Here we show that XTC-MIF is the homologue of mammalian activin A. Activins modulate the release of follicle-stimulating hormone from cultured anterior pituitary cells and cause the differentiation of two erythroleukaemia cell lines. Our results indicate that these molecules may also act in early development during formation of the mesoderm.