Mechanical forces and TGFbeta1 reduce podocyte adhesion through alpha3beta1 integrin downregulation.

BACKGROUND: Podocyturia is a marker of diabetic nephr- opathy, a possible determinant of its progression and a powerful risk factor for cardiovascular disease. A reduction in podocyte adhesion to the glomerular basement membrane (GBM) via downregulation of alpha3beta1 integrin expression, the main podocyte anchoring dimer to the GBM, may represent one of the mechanisms of podocyturia in glomerular disease. This study investigated the role of mechanical forces and transforming growth factor beta1 (TGFbeta1) in podocyte adhesion and integrin expression. METHODS: Conditionally immortalized murine podocytes were exposed to mechanical stretch and/or TGFbeta1 for 48 h. Podocyte adhesion, apoptosis and alpha3beta1 integrin expression were assessed. RESULTS: Stretch and TGFbeta1 significantly reduced podocyte adhesion and alpha3beta1 integrin expression, events paralleled by increased apoptosis. Blockade of beta1 integrin, with a specific antibody, demonstrated a reduced podocyte adhesion indicating that beta1 integrin downregulation was required for the loss of podocyte adhesion. This was linked to an increase in podocyte apoptosis. The role of apoptosis in podocyte adhesion was further investigated using caspase-3 inhibitors. Podocyte apoptosis inhibition did not affect stretch- and TGFbeta1-mediated integrin downregulation and the loss of podocyte adhesion, suggesting that alpha3beta1 integrin downregulation is sufficient to alter cell adhesion. Although stretch significantly increased podocyte TGFbeta type I, II and III receptors but not podocyte TGFbeta1 secretion, the combination of stretch and TGFbeta1 did not show any additive or synergistic effects on podocyte adhesion and alpha3beta1 integrin expression. CONCLUSIONS: These results suggest that downregulation of alpha3beta1 integrin expression, by mechanical forces or TGFbeta1, is per se sufficient to reduce podocyte adhesion. Apoptosis may represent a parallel important determinant of the podocyte loss from the GBM.

[Identification of the isoform in type II receptor of transforming growth factor-beta in patients with acute leukemia and its clinical significance].

Recent research indicates that TGF-beta and type II receptor (TbetaR-II) play an important role in the pathogenesis of tumor. A high frequency of abnormalities in TbetaR-II has been demonstrated in various cancers. To identify the mutation of TbetaR-II in patients with acute leukemia, the bone marrow samples from 6 patients with acute leukemia and 11 normal individuals as control were detected by long-range RT-PCR. To detect a deletion in sequence of the TbetaR-IIgene, the PCR products were cloned to T vector and then sequenced. The results showed that there was existance of the isoform of TbetaR-II in 2 cases out of 6 patients with acute leukemia. These two patients had more poor prognosis than others. In conclusion, there was the isoform of TbetaR-II in partial patients with acute leukemia, and the isoform may be related with prognosis.

Transforming growth factor-beta-related proteins: an ancestral and widespread superfamily of cytokines in metazoans.

Members of the transforming growth factor beta (TGF-beta) superfamily of cell signalling polypeptides have attracted much attention because of their ability, from nematodes to mammals, to control cellular functions that in turn, regulate embryo development and tissue homeostasis. On the basis of structure similarities, the TGF-beta members (ligands, receptors and Smads) are subdivided into TGF-beta sensu stricto, bone morphogenetic proteins (BMP) and activins. Although BMP is the best characterized pathway in metazoans, recent findings in molluscs and non-bilateria as well as the analysis of nematode and arthropod genomes, demonstrate the early origin of these distinct subfamilies of ligands, receptors and Smads. This report analyses the large diversity of ligands, receptors and Smads in metazoans from cnidarians and molluscs to mammals. The contribution of new data, mainly from the lophochotrozoan Crassostrea gigas and other organisms on the fringe of the 'branded model organisms', will help us to demonstrate that TGF-betas are probably the most ancestral active cytokines characterized at the molecular level in both Protostome and Deuterostome lineages.

Targets of transcriptional regulation by two distinct type I receptors for transforming growth factor-beta in human umbilical vein endothelial cells.

Transforming growth factor-beta (TGF-beta) plays a crucial role in vascular development and homeostasis by regulating many transcriptional targets. Activin receptor-like kinase 5 (ALK-5) is a TGF-beta type I receptor expressed in various TGF-beta-responsive cells. In contrast, ALK-1 functions as a TGF-beta type I receptor in endothelial cells, and is responsible for human hereditary hemorrhagic telangiectasia (HHT) type II. ALK-5 and ALK-1 mediate TGF-beta signals through distinct Smad proteins, i.e., Smad2/Smad3 and Smad1/Smad5, respectively. To identify target genes of ALK-1 and ALK-5 in endothelial cells, we conducted oligonucleotide microarray analysis. Human umbilical vein endothelial cells (HUVEC) were infected with recombinant adenoviruses carrying a constitutively active form of ALK-1 or ALK-5. ALK-5 inhibited the proliferation, network formation, and tube formation of HUVEC and induced their apoptosis, whereas ALK-1 did not exhibit significant effects on HUVEC in vitro. mRNAs were extracted from HUVEC and used for hybridization of oligonucleotide arrays representing approximately 7,000 human genes. Northern blot and quantitative real-time polymerase chain reaction (PCR) analyses were also performed for some of these genes, confirming the validity of this microarray analysis. We found that ALK-1 specifically upregulated Smad6, Smad7, Id1, Id2, endoglin, STAT1, and interleukin 1 receptor-like 1. ALK-5, in contrast, upregulated PlGF, SM22alpha, connexin 37, betaIG-H3, and LTBP1. ALK-1 downregulated Smad1, CXCR4, Ephrin-A1, and plakoglobin, whereas ALK-5 downregulated claudin 5 and integrin beta(5). These results revealed some new targets of TGF-beta in endothelial cells, and differences in transcriptional regulation patterns between ALK-1 and ALK-5.

The TGF-beta family: signaling pathways, developmental roles, and tumor suppressor activities.

Intercellular communication is a critical process for all multicellular organisms, and communication among cells is required for proper embryonic development and adult physiology. Members of the Transforming Growth Factor-beta (TGF-beta) family of secreted proteins communicate information between cells via a complex signaling pathway, and family members are capable of inducing a wide range of cellular responses. The purpose of this review is to provide the reader with a broad introduction to our current understanding of three aspects of the TGF-beta family. These are the molecular mechanisms utilized by TGF-beta signaling pathways, the developmental roles played by TGF-beta family members in a variety of species, and the growing list of cancers in which various TGF-beta signaling pathways display tumor suppressor activity.

TGF-beta signaling by Smad proteins.

Members of the transforming growth factor-beta (TGF-beta) family bind to type II and type I serine/threonine kinase receptors, which initiate intracellular signals through activation of Smad proteins. Receptor-regulated Smads (R-Smads) are anchored to the cell membrane by interaction with membrane-bound proteins, including Smad anchor for receptor activation (SARA). Upon ligand stimulation, R-Smads are phosphorylated by the receptors and form oligomeric complexes with common-partner Smads (Co-Smads). The oligomeric Smad complexes then translocate into the nucleus, where they regulate the transcription of target genes by direct binding to DNA, interaction with various DNA-binding proteins, and recruitment of transcriptional coactivators or corepressors. A third class of Smads, inhibitory Smads (I-Smads), inhibits the signals from the serine/threonine kinase receptors. Since the expression of I-Smads is induced by the TGF-beta superfamily proteins, Smads constitute an autoinhibitory signaling pathway. The functions of Smads are regulated by other signaling pathways, such as the MAP kinase pathway. Moreover, Smads interact with and modulate the functions of various transcription factors which are downstream targets of other signaling pathways. Loss of function of certain Smads is involved in tumorigenesis, e.g., pancreatic and colorectal cancers. Analyses by gene targeting revealed pivotal roles of Smads in early embryogenesis, angiogenesis, and immune functions in vivo.

[The expression of TGF-beta receptors on cultured human dental papilla cells].

OBJECTIVE: To observe the expression of TGF-beta receptors on cultured human dental papilla cells and the effects of TGF-beta on TGF-beta receptors of the cells. METHODS: The expression of TGF-beta receptors on cultured human dental papilla cells and the effects of TGF-beta on TGF-beta receptors of the cells were examined by cell culture and immunohistochemical technique and image analysis. RESULTS: Type I, type II receptors for TGF-beta in human dental papilla cells were stained strongly and type III was weakly positive. Image analysis indicated that all experimental groups had no obvious difference with control groups (P > 0.05). CONCLUSIONS: Cultured human dental papilla cells express TGF-beta type I, type II and type III receptors. TGF-beta has no obvious effects on the cell receptor expression.

Multiple TGF-beta receptor related genes in sponge and ancient gene duplications before the parazoan-eumetazoan split.

Members of the transforming growth factor beta (TGF-beta) family mediate key events in cell growth and development. Various receptors for diverse members of the TGF-beta family have recently been isolated and sequenced. These receptors form a family (TbetaR family) with a Ser/Thr kinase domain in common. To understand the divergence pattern of the TbetaR family during animal evolution, we have conducted cloning of cDNAs encoding the TbetaR family members from Ephydatia fluviatilis, a freshwater sponge. We obtained seven cDNAs (sALK-1-sALK-7) which are closely related in structure to known family members. Including these sponge sequences, a phylogenetic tree of the family members was inferred by a maximum likelihood method. The phylogenetic tree suggests that the sponge receptors sALK-1-sALK-3, which are closely related to each other, are sponge homologs of vertebrate activin type I receptor (ActR-I). sALK-5 is likely to be a homolog of TGF-beta type II receptor. sALK-4 and sALK-6 might be ancestral precursors of type I and type II receptors, respectively, and sALK-7 is possibly an ancestral precursor of both types. The tree revealed that most, if not all, of the gene duplications that gave rise to known subtypes with distinct ligand specificities antedate the divergence of parazoans and eumetazoans, the earliest divergence of extant animal phyla.

Review article: Cellular markers in the gastric precancerous process.

Gastric cancer is the end result of a chronic process, which usually starts as Helicobacter pylori-associated chronic gastritis. Although some differences exist in the histological intermediary stages and in the frequency and timing of certain molecular alterations, both diffuse and intestinal cancer are accompanied by some important common cellular changes. These include an increase in cell proliferation, and an alteration in apoptosis, which may be secondary to loss of function of p53 and loss of growth inhibition by growth factor (TGF)-beta, due to mutation of the TGF-beta receptor type II. This review examines the potential role of H. pylori in the aetiology of the molecular changes during the progression to gastric cancer, and explores the usefulness of these changes as biomarkers of increased risk of neoplasia in the intermediate steps of gastric carcinogenesis.

Transforming growth factor-beta1 (TGF-beta1) in dentine matrix. Ligand activation and receptor expression.

Transforming Growth Factor-beta (TGF-beta) and other members of this family of growth factors have been implicated in tooth development and dental tissue repair. This study aimed to investigate regulatory factors for TGF-beta1 in rabbit incisor dentine matrix and the expression of receptors for TGF-beta in dental tissues better to understand the control of its biological activity. Approximately half of the TGF-beta1 in dentine matrix was present in active form. TGF-beta1 was found in association with latency associated peptide (LAP), betaglycan and decorin in an isolated dentine matrix preparation. Immunohistochemistry showed strong staining for TGF-beta type I and II receptors in odontoblasts with more variable and weaker staining of other pulpal cells. Association of TGF-beta1 with betaglycan, decorin and LAP may regulate the availability and biological activity of this growth factor and influence its presentation to the TGF-beta type I and II receptors on odontoblasts. During dental tissue repair, such control processes will be important in regulating the biological effects of TGF-beta1 on cells of the dentine-pulp complex.

Transforming growth factor-beta response and expression in junctional and oral gingival epithelial cells.

The junctional (JE) and oral gingival (OGE) epithelium show distinct morphological phenotypes and express different cell surface and keratin markers. Transforming growth factor-beta (TGF-beta) has been shown to stimulate extracellular matrix formation and inhibit proteolytic matrix degradation in periodontal wound healing. To elucidate potential roles of TGF-beta in gingival epithelial regeneration and reattachment, the present study examined the effects of TGF-beta on JE and OGE cell growth and determined the patterns of expression of mRNAs for the TGF-beta isotypes beta 1, beta 2 and beta 3 and TGF-beta receptor types I, II and III. Primary cell cultures were initiated from JE and OGE and the cell phenotypes confirmed using monoclonal antibodies to specific keratins. TGF-beta induced a significant growth inhibition in OGE cells derived from 6 different patients with a mean inhibition of 46% and a range of 16-70% (p = 0.031). Although responses varied between patients, in general maximum inhibition occurred at 10 ng/ml TGF-beta. JE cells from 5 patients showed no significant growth inhibition by TGF-beta (p = 0.125). Greater expression of TGF-beta 2 and receptor type I mRNA was found in OGE than JE cells and thus appeared to be associated with differentiating epithelial cells. JE cells expressed more TGF-beta type II receptor specific mRNA than did OGE cells, but TGF-beta 1 mRNA expression was similar in JE and OGE cells. JE or OGE cultures derived from 2 of 3 patients showed expression of mRNA for the TGF-beta type III receptor. TGF-beta 3 mRNA was not detected in any of the JE or OGE samples examined. The greater sensitivity of OGE than JE to the growth inhibiting effects of TGF-beta correlated with higher expression of receptor type I mRNA which, together with the type II receptor, is required for sensitivity to growth inhibition by TGF-beta. The results suggest that, in addition to structural differences, the development of functional differences in the responses of JE and OGE to TGF-beta may be associated with the formation of JE from OGE cells and the reformation of attachment after periodontal surgery.

Transforming growth factor-beta: the breaking open of a black box.

Transforming growth factor-beta (TGF-beta) and its related proteins regulate broad aspects of body development, including cell proliferation, differentiation, apoptosis and gene expression, in various organisms. Deregulated TGF-beta function has been causally implicated in the generation of human fibrotic disorders and in tumor progression. Nevertheless, the molecular mechanisms of TGF-beta action remained essentially unknown until recently. Here, we discuss recent progress in our understanding of the mechanism of TGF-beta signal transduction with respect to the regulation of gene expression, the control of cell phenotype and the potential usage of TGF-beta for the treatment of human diseases.

Transforming growth factor-beta 1 in hypertensive renal injury in Dahl salt-sensitive rats.

The expression of transforming growth factor-beta 1 (TGF-beta 1) for hypertensive renal injury was investigated in Dahl salt-sensitive (Dahl-S) rats fed a high-salt (HS; 8% NaCl) diet or a low-salt (LS; 0.3% NaCl) diet for 4 wk. The HS rats developed severe hypertension and renal damage, including glomerulosclerosis and arteriosclerosis. TGF-beta biosynthesis by isolated glomeruli, the TGF-beta localization, and the gene expression of TGF-beta 1, latent TGF-beta binding protein (LTBP), and TGF-beta receptors (Types I, II, and III) were compared between the HS rats and LS rats. A TGF-beta bioassay revealed that the isolated glomeruli from the HS rats secreted a larger amount of latent TGF-beta than those from the LS rats. Northern blotting analysis demonstrated that the HS diet led to the increases in cortical gene expression of TGF-beta 1, LTBP, and TGF-beta receptors, compared with the LS diet. The glomerular biosynthesis of fibronectin and plasminogen activator inhibitor-1 (PAI-1), and cortical mRNA expression for fibronectin, collagen I, and PAI-1 (which may be affected by TGF-beta) in the HS rats were elevated, compared with the LS rats. The latent TGF-beta immunostained by anti-LTBP antibody was localized on the sclerosing glomeruli and vascular walls. Furthermore, fibronectin, collagen I, and PAI-1 were also localized in the sclerotic area. The TGF-beta 1-positive cells, immunostained by antibody for latency-associated peptide of TGF-beta 1, increased in the glomeruli and vascular walls in the HS rats. These results thus suggested that TGF-beta 1 may be related to hypertensive renal injury in this model.

Signal transduction by members of the transforming growth factor-beta superfamily.

Transforming growth factor-beta (TGF beta) superfamily members exert their diverse biological effects through their interaction with heteromeric receptor complexes of transmembrane serine/threonine kinases. Both components of the receptor complex, known as receptor I and receptor II are essential for signal transduction. The composition of these complexes can vary significantly due to the promiscuous nature of the ligands and the receptors, and this diversity of interactions can yield a variety of biological responses. Several receptor interacting proteins and potential mediators of signal transduction have now been identified. Recent advances, particularly in our understanding of the function of Mothers against dpp-related (MADR) proteins, are providing new insights into how the TGF beta superfamily signals its diverse biological activities.

Lack of expression of transforming growth factor-beta type II receptor associated with malignant progression in human salivary gland cell clones.

To understand the molecular mechanisms whereby normal human salivary gland cells become malignant and escape growth-inhibitory control by transforming growth factor (TGF)-betaI, we examined the effect of TGF-betaI on the proliferation and expression of TGF-beta receptors in cells and the expression of TGF-beta type II receptor (TbetaR-II) mRNA. An SV40-immortalized normal human salivary gland duct cell clone (NS-SV-DC) with no tumorigenic ability, originally obtained via s.c. implantation into nude mice, was partially resistant to the growth-inhibitory effect of TGF-betaI, while a neoplastic human salivary gland duct cell clone (HSGc) with tumorigenic, but not metastatic, potential in nude mice was more resistant to the growth-suppressive effect of TGF-betaI than NS-SV-DC. Metastatic cell clones derived from carcinogen-treated HSGc were completely refractory to the anti-proliferative effect of TGF-betaI. Affinity cross-linking revealed that NS-SV-DC possesses the types I, II (TbetaR-II) and III receptors. However, HSGc and metastatic cell clones lacked expression of detectable levels of the TbetaR-II protein. Moreover, we evaluated TbetaR-II mRNA expression in these cell clones by Northern blot analysis and observed that, although NS-SV-DC expressed a large amount of TbetaR-II mRNA, a small amount of TbetaR-II mRNA was detectable in HSGc. In contrast, no significant bands were detected in metastatic cell clones. Our results, therefore, suggest that one of the possible mechanisms of escape from autocrine or paracrine growth inhibition by TGF-betaI during human salivary gland carcinogenesis involves reduced expression or lack of TbetaR-II.

Human thymic epithelial cells produce TGF-beta 3 and express TGF-beta receptors.

TGF-beta affects proliferation, differentiation and maturation of T cells; however, the effect of TGF-beta on thymic stromal cells has not been characterized. To better understand the role of TGF-beta in T cell development, we determined whether TGF-beta is present in the human thymus, and identified stromal cells that express TGF-beta receptors and respond to TGF-beta. We demonstrate that primary cultured human thymic epithelial cells (TEC) express TGF-beta 1, TGF-beta 2 and TGF-beta 3, as well as TGF-beta type I receptor (T beta RI) (ALK-5) and TGF-beta type II receptor (T beta RII) transcripts. In vitro, epidermal growth factor (EGF) increases transcript levels of TGF-beta 1, TGF-beta 3 and T beta RII, suggesting that EGF may modulate TGF-beta responses in TEC; however, TGF-beta 2 and T beta RI transcript levels were not affected. We also detect TGF-beta 3 and T beta RII protein in association with keratin-positive TEC in vitro and in vivo. TEC culture supernatants contain TGF-beta 3 as detected by Western blots and, upon heat and acid activation, display growth inhibitory activity on the CCL-64 cells that is neutralized by anti-TGF-beta mAb treatment. We further demonstrate that TGF-beta 1 increases leukemia inhibitory factor transcript levels in TEC, indicating that TEC express functional TGF-beta receptors. Thus, we have shown in the human thymus that TEC produce TGF-beta 3 and express T beta RI and T beta RII. The data suggest that TGF-beta is present in the human thymus and may indirectly affect T cell development by regulating TEC cytokine production.