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.     

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.     

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.     

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.     

Activating mutations in ALK provide a therapeutic target in neuroblastoma

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.     

Pituitary FSH is released by a heterodimer of the beta-subunits from the two forms of inhibin

Inhibin is a gonadal protein that specifically inhibits the secretion of pituitary follicle-stimulating hormone (FSH). Two forms of inhibin (A and B) have been purified from porcine follicular fluid and characterized as heterodimers of relative molecular mass (Mr) 32,000 (ref. 2). Each inhibin is comprised of an identical alpha-subunit of Mr 18,000 and a distinct but related beta-subunit of Mr 13,800-14,700 linked by interchain disulphide bond(s). Throughout the purification of inhibins, we consistently observed two fractions which stimulated the secretion of pituitary FSH. We report here the isolation of one of the FSH-releasing proteins; it has a Mr of 24,000 and its N-terminal sequences up to residue 32 are identical to those of each beta-subunit of inhibins A and B. In the presence of reducing agents, SDS-polyacrylamide gel electrophoresis resolves the FSH-releasing substance into two subunits which are identical in their migration behaviour to the reduced beta-subunits of inhibins A and B. Based on the N-terminal sequence data and Mr of the intact and reduced molecules, we propose that the FSH-releasing substance, which is active in picomolar concentrations, is a heterodimeric protein composed of the two beta-subunits of inhibins A and B linked by interchain disulphide bond(s). The structural organization of the FSH-releasing substance is homologous to that of transforming growth factor-beta (TGF-beta), which also possesses FSH-releasing activity in the same bioassay. We suggest that the substance be called activin to signify the fact that it has opposite biological effects to inhibin.     

An unusual feature revealed by the crystal structure at 2.2 A resolution of human transforming growth factor-beta 2.

Transforming growth factor type beta 2 (TGF-beta 2) is a member of an expanding family of growth factors that regulate proliferation and differentiation of many different cell types. TGF-beta 2 binds to various receptors, one of which was shown to be a serine/threonine kinase. TGF-beta 2 is involved in wound healing, bone formation and modulation of immune functions. We report here the crystal structure of TGF-beta 2 at 2.2 A resolution, which reveals a novel monomer fold and dimer association. The monomer consists of two antiparallel pairs of beta-strands forming a flat curved surface and a separate, long alpha-helix. The disulphide-rich core has one disulphide bone pointing through a ring formed by the sequence motifs Cys-Ala-Gly-Ala-Cys and Cys-Lys-Cys, which are themselves connected through the cysteines. Two monomers are connected through a single disulphide bridge and associate such that the helix of one subunit interacts with the concave beta-sheet surface of the other. Four exposed loop regions might determine receptor specificity. The structure provides a suitable model for the TGF-beta s and other members of the super-family and is the basis for the analysis of the TGF-beta 2 interactions with the receptor.     

Impaired type II glucocorticoid-receptor function in mice bearing antisense RNA transgene.

Glucocorticoids, in conjunction with their cognate receptors, exert negative-feedback effects on the hypothalamus-pituitary-adrenal axis, suppressing adrenal steroid secretions. Two types of corticosteroid receptor, distinguishable by their ability to bind corticosterone, have been identified as classical mineralocorticoid (type I) and glucocorticoid (type II) receptors by cloning their complementary DNAs. The type I receptor controls the basal circadian rhythm of corticosteroid secretion. Both receptor types are involved in negative feedback, but the type II receptor may be more important for terminating the stress response as it is the only one to be increased in animals rendered more sensitive to corticosteroid negative-feedback effects. Here we create a transgenic mouse with impaired corticosteroid-receptor function by partially knocking out gene expression with type II glucocorticoid receptor antisense RNA. We use this animal to study the glucocorticoid feedback effect on the hypothalamus-pituitary-adrenal axis.     

Cloning of the full-length gene of activin receptor-interacting protein2a and characterization of its interaction with ActRⅡA

To investigate the mechanism of intracellular signal transduction mediated by activin receptors, the full-length gene encoding a novel activin receptor-interacting protein2a (ARIP2a) was identified from a mouse brain cDNA library. The sequences of ARIP2a and ARIP2, distribution of ARIP2a and ARIP2 mRNA in mouse tissues, and expression of ARIP2a and ARIP2 in activin-induced RAW264.7 cell were compared, and the interaction between ARIP2a and ActRIIA was confirmed. The sequence analysis revealed that the full-length gene of ARIP2a, which composed of 1008 bp and encoded 153 amino acid residues, shared high sequence identity with ARIP2 except the position of the 99th amino acid. RT-PCR assay showed that ARIP2a mRNA was highly expressed in brain, pituitary and testis, and moderately in pancreas and ovary, but undetectable in other tissues. Whereas, ARIP2 mRNA was widely distributed in all mouse tissues that we tested. Moreover, expression of ARIP2a mRNA was significantly decreased in activin-stimulated RAW264.7 cells; however, the expression of ARIP2 mRNA was increased. Additionally, the interaction between ARIP2a and activin type IIA receptor (ActRIIA) was further demonstrated by mammalian two-hybrid assays and pull-down assays. Taken together, those results indicate that although ARIP2a is homologous to ARIP2, they are different in tissue distribution and responses to activin. ARIP2a could also interact with activin type II receptor as a novel member of ARIP family.     

Cloning of the full-length gene of activin receptor-interacting protein2a and characterization of its interaction with ActR Ⅱ A

To investigate the mechanism of intracellular signal transduction mediated by activin receptors, the full-length gene encoding a novel activin receptor-interacting protein2a (ARIP2a) was identified from a mouse brain cDNA library. The sequences of ARIP2a and ARIP2, distribution of ARIP2a and ARIP2 mRNA in mouse tissues, and expression of ARIP2a and ARIP2 in activin-induced RAW264.7 cell were compared, and the interaction between ARIP2a and ActRIIA was confirmed. The sequence analysis revealed that the full-length gene of ARIP2a, which composed of 1008 bp and encoded 153 amino acid residues, shared high sequence identity with ARIP2 except the position of the 99th amino acid. RT-PCR assay showed that ARIP2a mRNA was highly expressed in brain, pituitary and testis, and moderately in pancreas and ovary, but undetectable in other tissues. Whereas, ARIP2 mRNA was widely distributed in all mouse tissues that we tested. Moreover, expression of ARIP2a mRNA was significantly decreased in activin-stimulated RAW264. 7 cells; however, the expression of ARIP2 mRNA was increased. Additionally, the interaction between ARIP2a and activin type IIA receptor (ActRIIA) was further demonstrated by mammalian two-hybrid assays and pull-down assays. Taken together, those results indicate that although ARIP2a is homologous to ARIP2, they are different in tissue distribution and responses to activin. ARIP2a could also interact with activin type II receptor as a novel member of ARIP family.     

Importance of FSH-releasing protein and inhibin in erythrodifferentiation

Inhibin is a hypophysiotropic hormone which selectively suppresses the secretion of pituitary follicle-stimulating hormone. It has been isolated from gonadal fluids and characterized as a protein heterodimer consisting of an alpha subunit and one of two beta subunits (beta A or beta B). FSH-releasing protein (FRP), also named activin, is a dimer consisting of two inhibin beta-chains. A factor from conditioned medium of a leukaemia cell line has been isolated which can induce mouse Friend cells to become benzidine-positive, and which shares a similar N-terminal sequence with porcine FRP. In this report, we find that FRP and inhibin modulate both the induction of haemoglobin accumulation in a human erythroleukaemic cell line, K562, and the proliferation of erythroid progenitor cells in human bone marrow culture. These two proteins could constitute a novel humoral regulatory control of erythropoiesis which would involve two types of related protein dimers with functionally opposite effects.     

卵泡分化排卵和黄体发生的分子基础

本文综述了近二十年来在雌性生殖研究领域中五个方面的研究进展。（1）原始卵泡的启动生长和分化：原始卵泡生长启动可能受卵巢内在因子,像生长因子的调控,而不是人所想象的FSH;启动生长的卵泡受生长因子,FSH,激活素,孤儿受体和其他卵巢旁分泌因子调控,并发展成“优势卵泡”;（2）优势卵泡和闭锁卵泡：FSH,激活素／抑制素,卵泡抑素和雌激素调节卵泡分化的趋向,它们的相互作用和平衡决定“优热卵泡”和“闭锁卵泡”的形成,其中雌激素在此起关键性作用;它一方面促进FSH对LH受体的诱导作用,增加GC上的LH受体数目;另一方面可促进GC芳香水酶的产生,使此卵泡进入良性循环,发展为优势卵泡;（3）两种细胞两种促性尕激素学说：在FSH和LH作用下,GC产生的孕酮可被膜细胞利用转化为雄激素;因此GC和TC相互作用是卵巢雌激素产生的前提;（4）排卵:GC主要产生tPA,而TC主要表达tPA的抑制因子PAI－1,在激素作用下,GC的tPA和TC的PAI－1相互作用,形成一个局部蛋白水解窗口,它对卵泡破裂起决定性作用;（5）黄体形成和萎缩：uPA和MMP／TIMP系统对黄体发生起重要作用;而tPA对黄体萎缩起关键作用。     

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.     

Recognition and plasma clearance of endotoxin by scavenger receptors.

Lipid A is the active moiety of lipopolysaccharide (LPS, also referred to as endotoxin), a surface component of Gram-negative bacteria that stimulates macrophage activation and causes endotoxic shock. Macrophages can bind, internalize and partially degrade LPS, lipid A and its bioactive precursor, lipid IVA. We report here that lipid IVA binding and subsequent metabolism to a less active form by macrophage-like RAW 264.7 cells is mediated by the macrophage scavenger receptor. Scavenger-receptor ligands inhibit lipid IVA binding to, and metabolism by, RAW cells, and lipid IVA binds to type I and type II bovine scavenger receptors on transfected Chinese hamster ovary cells. Although in vitro competition studies with RAW cells indicate that scavenger receptor binding is not involved in LPS or lipid IVA-induced stimulation of macrophages, in vivo studies show that scavenger-receptor ligands greatly inhibit hepatic uptake of lipid IVA in mice. Thus, scavenger receptors expressed on macrophages may have an important role in the clearance and detoxification of endotoxin in animals.     

Interaction between CD4 and class II MHC molecules mediates cell adhesion

The CD4 glycoprotein is expressed on T-helper and cytotoxic lymphocytes which are restricted to class II major histocompatibility complex (MHC) antigens on target cells. Antibody inhibition studies imply that CD4 acts to increase the avidity of effector-target cell interactions. These observations have led to the speculation that CD4 binds to a monomorphic class II antigen determinant, thereby augmenting low affinity T-cell receptor-antigen interactions. However, no direct evidence has been presented indicating that CD4 and class II molecules interact. To address this issue, we have used a vector derived from simian virus 40 (SV40) to express a complementary DNA (cDNA) encoding the human CD4 glycoprotein. When CV1 cells expressing large amounts of the CD4 protein at the cell surface are incubated with human B cells bearing MHC-encoded class II molecules, they are bound tightly to the infected monolayer, whereas mutant B cells which lack class II molecules fail to bind. Furthermore, the binding reaction is specifically inhibited by anti-class II and anti-CD4 antibodies. Thus, the CD4 protein, even in the absence of T-cell receptor-antigen interactions, can interact directly with class II antigens to function as a cell surface adhesion molecule.     

The alpha 1 domain of the HLA-DR molecule is essential for high-affinity binding of the toxic shock syndrome toxin-1

Several exoproteins from the bacterium Staphylococcus aureus are highly potent polyclonal activators of T cells in the presence of cells bearing class II antigens of the major histocompatibility complex (MHC). These toxins, including the toxic shock syndrome toxin (TSST-1), act at nanomolar concentrations, bind directly to class II molecules, and do not require the processing typical of nominal antigen. Each toxin is capable of stimulating a subpopulation of peripheral T lymphocytes bearing particular V beta sequences as part of their alpha beta T-cell receptors. It is not known how these so-called 'superantigens' bind to class II and how this binding stimulates T cells. In this study, the different affinities of TSST-1 for human class II molecules DR and DP were exploited to define the region of a class II molecule necessary for high-affinity binding. Using chimaeric alpha- and beta-chains of DR and DP expressed at the surface of transfected murine fibroblasts and a binding assay with TSST-1, it was shown that the alpha 1 domain of DR is essential for high-affinity binding, and further that TSST-1 binding did not prevent subsequent binding of a DR-restricted antigenic peptide. This is compatible with a model of superantigen making external contacts with both class II and T cell receptor, and suggests that the V beta portion of the T-cell receptor interacts with the nonpolymorphic alpha-chain of DR.