TGF-β1、TβRⅡ、c-myc在急性白血病中的表达及其意义

为了探讨转化生长因子β（transforming growth factor—beta，TGF-β）信号转导途径失活与急性白血病的关系，用S—P免疫细胞化学法检测急性白血病患者骨髓单个核细胞中TGF—β1及其Ⅱ型受体（TβRⅡ）和信号下游靶基因蛋白C-myc的表达。结果发现，白血病患者骨髓单个核细胞中TGF—β1的表达水平与正常对照无显著性差异（P〉0．05），TβRⅡ的表达水平明显低于正常对照（P〈0．05），c-myc的表达水平明显高于正常对照（P〈0．05），但以上各指标在急性淋巴细胞白血病与急性非淋巴细胞白血病之间无显著性差异（P〉0．05），TβRⅡ和c-myc表达率有负相关关系（r=-0．474，P〈0．01）。结论：TGF-β信号转导途径失活使细胞逃逸了TGF-β的生长抑制，TGF-β信号转导途径失活的一个重要机制就是通过TGF-βⅡ型受体的下调使c-myc失去抑制而高表达，促进白血病发生。     

Dysfunction of TGF-beta signaling in Alzheimer's disease

Accumulation of beta-amyloid peptide (Abeta) in the brain is believed to trigger a complex and poorly understood pathologic reaction that results in the development of Alzheimer's disease (AD). Despite intensive study, there is no consensus as to how Abeta accumulation causes neurodegeneration in AD. In this issue of the JCI, Tesseur et al. report that the expression of TGF-beta type II receptor (TbetaRII) by neurons is reduced very early in the course of AD and that reduced TGF-beta signaling increased Abeta deposition and neurodegeneration in a mouse model of AD (see the related article beginning on page 3060). Intriguingly, reduced TGF-beta signaling in neuroblastoma cells resulted in neuritic dystrophy and increased levels of secreted Abeta. Collectively, these data suggest that dysfunction of the TGF-beta/TbetaRII signaling axis in the AD brain may accelerate Abeta deposition and neurodegeneration.     

Blockade of TGF-beta inhibits mammary tumor cell viability,migration, and metastases

TGF-betas are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-beta interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-beta type II receptor fusion protein (Fc:TbetaRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TbetaRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TbetaRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TbetaRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TbetaRII. Therefore, blockade of TGF-beta signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.     

Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease

TGF-beta1 functions as a negative regulator of T cell immune responses, signaling to target cells using the Smad family of proteins. We show here that Smad7, an inhibitor of TGF-beta1 signaling, is overexpressed in inflammatory bowel disease (IBD) mucosa and purified mucosal T cells. Both whole tissue and isolated cells exhibit defective signaling through this pathway, as measured by phospho-Smad3 immunoreactivity. Specific antisense oligonucleotides for Smad7 reduce Smad7 protein expression in cells isolated from patients with IBD, permitting the cells to respond to exogenous TGF-beta1. TGF-beta1 cannot inhibit proinflammatory cytokine production in isolated lamina propria mononuclear cells from patients with Crohn disease (CD), but inhibition of Smad7 restores TGF-beta1 signaling and enables TGF-beta1 to inhibit cytokine production. In inflamed mucosal tissue explants from patients with CD, inhibition of Smad7 also restores p-Smad3 and decreases proinflammatory cytokine production, an effect that is partially blocked by anti-TGF-beta1. These results show that Smad7 blockade of TGF-beta1 signaling helps maintain the chronic production of proinflammatory cytokines that drives the inflammatory process in IBD and that inhibition of Smad7 enables endogenous TGF-beta to downregulate this response.     

The type III TGF-beta receptor suppresses breast cancer progression

The TGF-beta signaling pathway has a complex role in regulating mammary carcinogenesis. Here we demonstrate that the type III TGF-beta receptor (TbetaRIII, or betaglycan), a ubiquitously expressed TGF-beta coreceptor, regulated breast cancer progression and metastasis. Most human breast cancers lost TbetaRIII expression, with loss of heterozygosity of the TGFBR3 gene locus correlating with decreased TbetaRIII expression. TbetaRIII expression decreased during breast cancer progression, and low TbetaRIII levels predicted decreased recurrence-free survival in breast cancer patients. Restoring TbetaRIII expression in breast cancer cells dramatically inhibited tumor invasiveness in vitro and tumor invasion, angiogenesis, and metastasis in vivo. TbetaRIII appeared to inhibit tumor invasion by undergoing ectodomain shedding and producing soluble TbetaRIII, which binds and sequesters TGF-beta to decrease TGF-beta signaling and reduce breast cancer cell invasion and tumor-induced angiogenesis. Our results indicate that loss of TbetaRIII through allelic imbalance is a frequent genetic event during human breast cancer development that increases metastatic potential.     

SB-431542, a transforming growth factor beta inhibitor, impairs Trypanosoma cruzi infection in cardiomyocytes and parasite cycle completion

The antiinflammatory cytokine transforming growth factor beta (TGF-beta) plays an important role in Chagas disease, a parasitic infection caused by the protozoan Trypanosoma cruzi. In the present study, we show that SB-431542, an inhibitor of the TGF-beta type I receptor (ALK5), inhibits T. cruzi-induced activation of the TGF-beta pathway in epithelial cells and in cardiomyocytes. Further, we demonstrate that addition of SB-431542 greatly reduces cardiomyocyte invasion by T. cruzi. Finally, SB-431542 treatment significantly reduces the number of parasites per infected cell and trypomastigote differentiation and release. Taken together, these data further confirm the major role of the TGF-beta signaling pathway in both T. cruzi infection and T. cruzi cell cycle completion. Our present data demonstrate that small inhibitors of the TGF-beta signaling pathway might be potential pharmacological tools for the treatment of Chagas disease.     

Requirement for transforming growth factor beta1 in controlling T cell apoptosis

Transforming growth factor (TGF)-beta1, a potent immunoregulatory molecule, was found to control the life and death decisions of T lymphocytes. Both thymic and peripheral T cell apoptosis was increased in mice lacking TGF-beta1 (TGF-beta1(-/-)) compared with wild-type littermates. Engagement of the T cell receptor enhanced this aberrant T cell apoptosis, as did signaling through either the death receptor Fas or the tumor necrosis factor alpha receptor in peripheral T cells. Strikingly, TGF-beta was localized within the mitochondria of normal T cells, and the absence of TGF-beta1 resulted in disruption of mitochondrial membrane potential (Deltapsi(m)), which marks the point of no return in a cell condemned to die. This TGF-beta-dependent regulation of viability appears dissociable from the TGF-beta1 membrane receptor-Smad3 signaling pathway, but associated with a mitochondrial antiapoptotic protein Bcl-XL. Thus, TGF-beta1 may protect T cells at multiple sites in the death pathway, particularly by maintaining the essential integrity of mitochondria. These findings may have broad implications not only for T cell selection and death in immune responses and in the generation of tolerance, but also for defining the mechanisms of programmed cell death in general.     

Inhibition of intracellular transport of B cell antigen receptor complexes by Kaposi's sarcoma-associated herpesvirus K1

The B cell antigen receptor (BCR) is a large complex that consists of a disulfide-linked tetramer of two transmembrane heavy (mu) chains and two light (lambda or kappa) chains in association with a heterodimer of Igalpha and Igbeta. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a transforming protein called K1, which has structural and functional similarity to Igalpha and Igbeta. We demonstrate that K1 downregulates the expression of BCR complexes on the surface. The NH(2)-terminal region of K1 specifically interacts with the mu chains of BCR complexes, and this interaction retains BCR complexes in the endoplasmic reticulum, preventing their intracellular transport to the cell surface. Thus, KSHV K1 resembles Igalpha and Igbeta in its ability to induce signaling and to interact with mu chains of the BCR. However, unlike Igalpha and Igbeta, which interact with mu chains to direct BCR complexes to the cell surface, K1 interacts with mu chains to block the intracellular transport of BCR complexes to the cell surface. These results demonstrate a unique feature of the K1 transforming protein, which may confer virus-infected cells with a long-term survival advantage.     

A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells

Caspase-3 is a critical enzyme for apoptosis and cell survival. Here we report delayed ossification and decreased bone mineral density in caspase-3-deficient (Casp3(-/-) and Casp3(+/-)) mice due to an attenuated osteogenic differentiation of bone marrow stromal stem cells (BMSSCs). The mechanism involved in the impaired differentiation of BMSSCs is due, at least partially, to the overactivated TGF-beta/Smad2 signaling pathway and the upregulated expressions of p53 and p21 along with the downregulated expressions of Cdk2 and Cdc2, and ultimately increased replicative senescence. In addition, the overactivated TGF-beta/Smad2 signaling may result in the compromised Runx2/Cbfa1 expression in preosteoblasts. Furthermore, we demonstrate that caspase-3 inhibitor, a potential agent for clinical treatment of human diseases, caused accelerated bone loss in ovariectomized mice, which is also associated with the overactivated TGF-beta/Smad2 signaling in BMSSCs. This study demonstrates that caspase-3 is crucial for the differentiation of BMSSCs by influencing TGF-beta/Smad2 pathway and cell cycle progression.     

TGF-beta switches from tumor suppressor to prometastatic factor in a model of breast cancer progression

The TGF-beta signaling network plays a complex role in carcinogenesis because it has the potential to act as either a tumor suppressor or a pro-oncogenic pathway. Currently, it is not known whether TGF-beta can switch from tumor suppressor to pro-oncogenic factor during the course of carcinogenic progression in a single cell lineage with a defined initiating oncogenic event or whether the specific nature of the response is determined by cell type and molecular etiology. To address this question, we have introduced a dominant negative type II TGF-beta receptor into a series of genetically related human breast-derived cell lines representing different stages in the progression process. We show that decreased TGF-beta responsiveness alone cannot initiate tumorigenesis but that it can cooperate with an initiating oncogenic lesion to make a premalignant breast cell tumorigenic and a low-grade tumorigenic cell line histologically and proliferatively more aggressive. In a high-grade tumorigenic cell line, however, reduced TGF-beta responsiveness has no effect on primary tumorigenesis but significantly decreases metastasis. Our results demonstrate a causal role for loss of TGF-beta responsiveness in promoting breast cancer progression up to the stage of advanced, histologically aggressive, but nonmetastatic disease and suggest that at that point TGF-beta switches from tumor suppressor to prometastatic factor.     

Developmentally regulated association of a 56-kD member of the surface immunoglobulin M receptor complex

Immature and mature B cells differ in the signals generated and transduced through their antigen receptor, surface immunoglobulin M (sIgM). Whereas signals generated through sIgM on mature B cells initiate a program leading to the positive activation of these cells, signaling through this receptor at the immature stage of development leads to a state of induced unresponsiveness or tolerance. Our previous studies have described developmental differences in sIgM transmembrane signaling that are independent of ligand-receptor affinity. In an attempt to understand the molecular basis for signaling differences between immature and mature B cells, we have analyzed the sIgM receptor complex in neonatal and adult mouse splenic B cells. While previously described components of this complex do not exhibit marked developmentally regulated differences in their association with sIgM, we have identified a 56-kD protein that associates with sIgM in mature (antigen-responsive), but not immature (tolerance-sensitive) B cells. This protein (p56) associates with sIgM as a homodimer, is constitutively phosphorylated on tyrosine, and is coimmunoprecipitated with IgM but not IgD. The observed inability to iodinate p56 suggests it is an intracellular component of the receptor complex. Based upon its migration in one- and two-dimensional gel electrophoresis we show, however, that p56 is distinct from the blk, lyn, or fyn src family kinases that have been shown to be associated with sIgM in mature B cells. The developmentally regulated participation of p56 in the B cell antigen receptor complex suggests a role in the differential signaling mediated via sIgM on immature and mature B cells.     

Small intestinal CD8+TCRgammadelta+NKG2A+ intraepithelial lymphocytes have attributes of regulatory cells in patients with celiac disease

Intraepithelial lymphocytes (IELs) bearing the gammadelta TCR are more abundant in the small intestinal mucosa of patients with celiac disease (CD) compared with healthy individuals. However, their role in disease pathogenesis is not well understood. Here, we investigated the functional attributes of TCRgammadelta+ IELs isolated from intestinal biopsies of patients with either active celiac disease (ACD) or those on a gluten-free diet (GFD). We found that compared with individuals with ACD, individuals on GFD have a higher frequency of CD8+TCRgammadelta+ IELs that express the inhibitory NK receptor NKG2A and intracellular TGF-beta1. TCR triggering as well as cross-linking of NKG2A increased both TGF-beta1 intracellular expression and secretion in vitro. Coculture of sorted TCRgammadelta+NKG2A+ IELs, IL-15-stimulated TCRalphabeta+ IELs, and HLA-E+ enterocytes resulted in a decreased percentage of cytotoxic CD8+TCRalphabeta+ IELs expressing intracellular IFN-gamma and granzyme-B and surface NKG2D. This inhibition was partially abrogated by blocking either TGF-beta alone or both NKG2A and HLA-E. Thus, our data indicate that suppression was at least partially mediated by TGF-beta secretion as a result of engagement of NKG2A with its ligand, HLA-E, on enterocytes and/or TCRalphabeta+ IELs. These findings demonstrate that human small intestinal CD8+TCRgammadelta+ IELs may have regulatory potential in celiac disease.     

Selective upregulation of platelet-derived growth factor alpha receptors by transforming growth factor beta in scleroderma fibroblasts

Transforming growth factor beta (TGF-beta), a multifunctional cytokine, is an indirect mitogen for human fibroblasts through platelet-derived growth factor (PDGF), particularly the A ligand-alpha receptor arm of that system. TGF-beta effects on PDGF alpha receptor expression were studied in vitro using ligand binding techniques in three human dermal fibroblast strains: newborn foreskin, adult skin, and scleroderma (systemic sclerosis, SSc). Each cell strain responded differently to TGF-beta. In newborn foreskin fibroblasts, PDGF alpha receptor number decreased in a dose-dependent manner after exposure to low concentrations of TGF-beta (0.1-1 ng/ml). Responses of normal skin fibroblasts were varied, and mean net receptor number was unchanged. Increases in PDGF alpha receptor number by TGF-beta occurred consistently with SSc fibroblasts and low concentrations of TGF-beta (0.1-1 ng/ml) were particularly stimulatory. Increased surface expression of alpha receptor subunit by TGF-beta in SSc fibroblasts correlated with increased new PDGF alpha receptor synthesis as demonstrated by radioimmunoprecipitation analysis of metabolically labeled cells and with increased steady-state levels of corresponding mRNAs. In normal adult skin fibroblasts, TGF-beta had no effect on either synthesis or mRNA expression of alpha receptor subunits. Proliferative responses to PDGF-AA after pretreatment with TGF-beta correlated positively with effects of TGF-beta on expression of alpha receptor subunit. Decreased mitogenic responses to PDGF-AA were observed in foreskin fibroblasts, small changes in responses in adult fibroblasts, and significant increases in SSc fibroblasts. Thus, costimulation with PDGF-AA and TGF-beta selectively enhanced proliferation of fibroblasts with the SSc phenotype. Immunohistochemical examination of SSc and control skin biopsies revealed the presence of PDGF-AA in SSc skin. Data obtained by ligand binding, immunoprecipitation, mRNA, and mitogenic techniques are consistent with the hypothesis that activation of the PDGF-AA ligand/alpha receptor pathway is a characteristic of the SSc fibroblast and may contribute to the expansion of fibroblasts in SSc.     

Tumor cell adhesion and migration supported by interaction of a receptor-protease complex with its inhibitor

Tissue factor (TF), the cell-surface receptor for coagulation factor VIIa, supports metastasis. Equally important for this process are (a) interactions of the TF cytoplasmic domain, which binds the mobility-enhancing actin-binding protein 280, and (b) the formation of a proteolytically active TF-VIIa complex on the tumor cell surface. In primary bladder carcinoma cells, we find that this complex localizes to the invasive edge, in proximity to tumor-infiltrating vessels that stain intensely for TF pathway inhibitor (TFPI-1), the major inhibitor of the protease activity of the complex. In culture, binding of VIIa to TF-expressing tumor cells is sufficient to allow cell adhesion, migration, and intracellular signaling on immobilized TFPI-1. Immobilized heparin, a mimic for extracellular matrix-associated proteoglycans, binds physiological concentrations of TFPI-1 in a conformation that supports TF-VIIa-dependent cell adhesion. Consistent with a functional role of TFPI-1 in complex extracellular matrices, we show that TF cooperates with integrin-mediated adhesion and migration on composite matrices that contain ligands for both integrins and the TF-VIIa complex. This study thus provides evidence for a novel mechanism of protease-supported migration that is independent of proteolytic matrix degradation but rather involves protease-dependent bridging of TF's extracellular domain to an ECM-associated inhibitor.     

Constitutive expression of the B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be rescued by CD40 signaling

The recently described ligand-receptor pair, B7h-inducible costimulator (ICOS), is critical for germinal center formation and antibody responses. In contrast to the induced expression of the related costimulatory ligands B7.1 and B7.2, B7h is constitutively expressed on naive B cells and is surprisingly extinguished after antigen engagement and interleukin (IL)-4 cytokine signaling. Although signaling through both B cell receptor (BCR) and IL-4 receptor (R) converge on the extinction of B7h mRNA levels, BCR down-regulation occurs through Ca2+ mobilization, whereas IL-4R down-regulation occurs through a distinct Stat6-dependent pathway. During antigen-specific B cell activation, costimulation through CD40 signaling can reverse both BCR- and IL-4R-mediated B7h down-regulation. These data suggest that the CD40-CD40 ligand signaling pathway regulates B7h expression on activated B cells and may control whether antigen-activated B cells can express B7h and costimulate cognate antigen-activated T cells through ICOS.     

Transforming growth factor-beta activation in irradiated murine mammary gland.

The biological activity of TGF-beta, an important modulator of cell proliferation and extracellular matrix formation, is governed by dissociation of mature TGF-beta from an inactive, latent TGF-beta complex in a process that is critical to its role in vivo. So far, it has not been possible to monitor activation in vivo since conventional immunohistochemical detection does not accurately discriminate latent versus active TGF-beta, nor have events associated with activation been defined well enough to serve as in situ markers of this process. We describe here a modified immunodetection method using differential antibody staining that allows the specific detection of active versus latent TGF-beta. Under these conditions, we report that an antibody raised to latency-associated peptide detects latent TGF-beta, and we demonstrate that LC(1-30) antibodies specifically recognize active TGF-beta 1 in tumor xenografts overproducing active TGF-beta 1, without cross-reactivity in tumors expressing similar levels of latent TGF-beta 1. We previously reported that TGF-beta immunoreactivity increases in murine mammary gland after whole-body 60Co-gamma radiation exposure. Using differential antibody staining we now show that radiation exposure specifically generates active TGF-beta 1. While latent TGF-beta 1 was widely distributed in unirradiated tissue, active TGF-beta 1 distribution was restricted. Active TGF-beta 1 increased significantly within 1 h of irradiation concomitant with decreased latent TGF-beta immunoreactivity. This rapid shift in immunoreactivity provides the first evidence for activation of TGF-beta in situ. This reciprocal pattern of expression persisted for 3 d and was accompanied by decreased recovery of latent TGF-beta 1 from irradiated tissue. Radiation-induced activation of TGF-beta may have profound implications for understanding tissue effects caused by radiation therapy.