Concomitant loss of transforming growth factor (TGF)-beta receptor types I and II in TGF-beta-resistant cell mutants implicates both receptor types in signal transduction

A panel of 71 chemically mutagenized Mv1Lu mink lung epithelial cell clones were selected based on their resistance to the growth inhibitory action of transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2. Characterization of TGF-beta receptors in these mutants indicates that the TGF-beta-binding membrane proteoglycan, betaglycan, is apparently normal in all of them. However, 14 of the mutant clones are defective in TGF-beta receptor type I, and 22 clones are simultaneously defective in receptor types I and II. The clones with type I receptor defects fall into two distinct phenotypes, called R and LR. The R phenotype is characterized by the lack of detectable type I receptors, and has been previously described (Boyd, F. T., and Massagué, J. (1989) J. Biol. Chem. 264, 2272-2278). LR mutants are characterized by expression of low levels of type I receptor and are, like the R mutants, completely resistant to growth inhibition by TGF-beta 1 or -beta 2. Mutant clones that are simultaneously defective in receptor types I and II fall into three distinct phenotypes. These included DRa mutants which are characterized by lack of detectable receptor types I and II, DRb mutants which are characterized by low expression of both receptor types and an anomalously fast electrophoretic mobility of the type II receptor protein. All mutants that have a low level of type II receptor are also defective in type I receptor. In addition to the loss of growth inhibitory response, the receptor-defective mutants described here have lost all other responses to TGF-beta 1 and -beta 2 known to occur in parental Mv1Lu cells. The defects present in these mutant clones are not encountered in clones isolated from nonmutagenized parental Mv1Lu cells or in mutagenized cells that had not been exposed to selection with TGF-beta. The results implicate TGF-beta receptor types I and II in the mediation of a common set of cellular responses to TGF-beta. Furthermore, the high relative frequency of isolation of DR mutants raises the possibility that receptor types I and II interact as part of a common signaling TGF-beta receptor complex.     

Combinatorial activation of FAK and AKT by transforming growth factor-beta1 confers an anoikis-resistant phenotype to myofibroblasts

Transforming growth factor-beta (TGF-beta) is a prototypical tumour-suppressor cytokine with cytostatic and pro-apoptotic effects on most target cells; however, mechanisms of its pro-survival/anti-apoptotic signalling in certain cell types and contexts remain unclear. In human lung fibroblasts, TGF-beta1 is known to induce myofibroblast differentiation in association with the delayed activation of focal adhesion kinase (FAK) and protein kinase B (PKB/AKT). Here, we demonstrate that FAK and AKT are independently regulated by early activation of SMAD3 and p38 MAPK, respectively. Pharmacologic or genetic approaches that disrupt SMAD3 signalling block TGF-beta1-induced activation of FAK, but not AKT; in contrast, disruption of early p38 MAPK signalling abrogates AKT activation, but does not alter FAK activation. TGF-beta1 is able to activate AKT in cells expressing mutant FAK or in cells treated with an RGD-containing peptide that interferes with integrin signalling, inhibits FAK activation and induces anoikis (apoptosis induced by loss of adhesion signalling). TGF-beta1 protects myofibroblasts from anoikis, in part, by activation of the PI3K-AKT pathway. Thus, TGF-beta1 co-ordinately and independently activates the FAK and AKT protein kinase pathways to confer an anoikis-resistant phenotype to myofibroblasts. Activation of these pro-survival/anti-anoikis pathways in myofibroblasts likely contributes to essential roles of TGF-beta1 in tissue fibrosis and tumour-promotion.     

Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause a different phenotype than do previously isolated missense mutations.

Missense mutations in the SNF3 gene of Saccharomyces cerevisiae were previously found to cause defects in both glucose repression and derepression of the SUC2 (invertase) gene. In addition, the growth properties of snf3 mutants suggested that they were defective in uptake of glucose and fructose. We have cloned the SNF3 gene by complementation and demonstrated linkage of the cloned DNA to the chromosomal SNF3 locus. The gene encodes a 3-kilobase poly(A)-containing RNA, which was fivefold more abundant in cells deprived of glucose. The SNF3 gene was disrupted at its chromosomal locus by several methods to create null mutations. Disruption resulted in growth phenotypes consistent with a defect in glucose uptake. Surprisingly, gene disruption did not cause aberrant regulation of SUC2 expression. We discuss possible mechanisms by which abnormal SNF3 gene products encoded by missense alleles could perturb regulatory functions.     

TGF-beta control of cell proliferation

This article focuses on recent findings that the type V TGF-beta receptor (TbetaR-V), which co-expresses with other TGF-beta receptors (TbetaR-I, TbetaR-II, and TbetaR-III) in all normal cell types studied, is involved in growth inhibition by IGFBP-3 and TGF-beta and that TGF-beta activity is regulated by two distinct endocytic pathways (clathrin- and caveolar/lipid-raft-mediated). TGF-beta is a potent growth inhibitor for most cell types, including epithelial and endothelial cells. The signaling by which TGF-beta controls cell proliferation is not well understood. Many lines of evidence indicate that other signaling pathways, in addition to the prominent TbetaR-I/TbetaR-II/Smad2/3/4 signaling cascade, are required for mediating TGF-beta-induced growth inhibition. Recent studies revealed that TbetaR-V, which is identical to LRP-1, mediates IGF-independent growth inhibition by IGFBP-3 and mediates TGF-beta-induced growth inhibition in concert with TbetaR-I and TbetaR-II. In addition, IRS proteins and a Ser/Thr-specific protein phosphatase(s) are involved in the TbetaR-V-mediated growth inhibitory signaling cascade. The TbetaR-V signaling cascade appears to cross-talk with the TbetaR-I/TbetaR-II, insulin receptor (IR), IGF-I receptor (IGF-IR), integrin and c-Met signaling cascades. Attenuation or loss of the TbetaR-V signaling cascade may enable carcinoma cells to escape from TGF-beta growth control and may contribute to the aggressiveness and invasiveness of these cells via promoting epithelial-to-mesenchymal transdifferentiation (EMT). Finally, the ratio of TGF-beta binding to TbetaR-II and TbetaR-I is a signal controlling TGF-beta partitioning between two distinct endocytosis pathways and resultant TGF-beta responsiveness. These recent studies have provided new insights into the molecular mechanisms underlying TGF-beta-induced cellular growth inhibition, cross-talk between the TbetaR-V and other signaling cascades, the signal that controls TGF-beta responsiveness and the role of TbetaR-V in tumorigenesis.     

GM-CSF increases airway smooth muscle cell connective tissue expression by inducing TGF-beta receptors

Fibrosis around the smooth muscle of asthmatic airway walls leads to irreversible airway obstruction. Bronchial epithelial cells release granulocyte/macrophage colony-stimulating factor (GM-CSF) in asthmatics and are in close proximity to airway smooth muscle cells (ASMC). The findings in this study demonstrate that GM-CSF induces confluent, prolonged, serum-deprived cultures of ASMC to increase expression of collagen I and fibronectin. GM-CSF also induced ASMC to increase the expression of transforming growth factor (TGF)-beta receptors type I, II, and III (TbetaR-I, TbetaR-II, TbetaR-III), but had no detectable effect on the release of TGF-beta1 by the same ASMC. The presence of GM-CSF also induced the association of TGF-beta1 with TbetaR-III, which enhances binding of TGF-beta1 to TbetaR-II. The induction of TbetaRs was parallel to the increased induction of phosphorylated Smad2 (pSmad2) and connective tissue growth factor (CTGF), indicative of TGF-beta-mediated connective tissue synthesis. Dexamethasone decreased GM-CSF-induced TbetaR-I, TbetaR-II, TbetaR-III, pSmad2, CTGF, collagen I, and fibronectin. In conclusion, GM-CSF increases the responsiveness of ASMC to TGF-beta1-mediated connective tissue expression by induction of TbetaRs, which is inhibited by corticosteroids.     

Growth differentiation factor 11 signals through the transforming growth factor-beta receptor ALK5 to regionalize the anterior-posterior axis

Growth differentiation factor 11 (GDF11) contributes to regionalize the mouse embryo along its anterior-posterior axis by regulating the expression of Hox genes. The identity of the receptors that mediate GDF11 signalling during embryogenesis remains unclear. Here, we show that GDF11 can interact with type I receptors ALK4, ALK5 and ALK7, but predominantly uses ALK4 and ALK5 to activate a Smad3-dependent reporter gene. Alk5 mutant embryos showed malformations in anterior-posterior patterning, including the lack of expression of the posterior determinant Hoxc10, that resemble defects found in Gdf11-null mutants. A heterozygous mutation in Alk5, but not in Alk4 or Alk7, potentiated Gdf11(-/-)-like phenotypes in vertebral, kidney and palate development in an Acvr2b(-/-) background, indicating a genetic interaction between the two receptor genes. Thus, the transforming growth factor-beta (TGF-beta) receptor ALK5, which until now has only been associated with the biological functions of TGF-beta1 to TGF-beta3 proteins, mediates GDF11 signalling during embryogenesis.     

Dominant negative mutants of transforming growth factor-beta 1 inhibit the secretion of different transforming growth factor-beta isoforms.

Transforming growth factor-beta (TGF-beta) is a secreted polypeptide factor that is thought to play a major role in the regulation of proliferation of many cell types and various differentiation processes. Several related isoforms have been structurally characterized, three of which, TGF-beta 1, -beta 2, and -beta 3, have been detected in mammalian cells and tissues. Each TGF-beta form is a homodimer of a 112-amino-acid polypeptide which is encoded as a larger polypeptide precursor. We have introduced several mutations in the TGF-beta 1 precursor domain, resulting in an inhibition of TGF-beta 1 secretion. Coexpression of these mutants with wild-type TGF-beta 1, -beta 2, and -beta 3 results in a competitive and specific inhibition of the secretion of different TFG-beta forms, indicating that these mutated versions act as dominant negative mutants for TGF-beta secretion. Overexpression of dominant negative mutants can thus be used to abolish endogenous secretion of TGF-beta and structurally related family members, both in vitro and in vivo, and to probe in this way the physiological functions of the members of the TGF-beta superfamily.     

Expression of TGF-beta signaling genes in the normal, premalignant, and malignant human trophoblast: loss of smad3 in choriocarcinoma cells

We had earlier shown that TGF-beta controls proliferation, migration, and invasiveness of normal human trophoblast cells, whereas premalignant and malignant trophoblast cells are resistant to TGF-beta. To identify signaling defects responsible for TGF-beta resistance in premalignant and malignant trophoblasts, we have compared the expression of TGF-beta signaling molecules in a normal trophoblast cell line (HTR-8), its premalignant derivative (RSVT2/C), and two choriocarcinoma cell lines (JAR and JEG-3). RT-PCR analysis revealed that all these cell lines expressed the mRNA of TGF-beta1, -beta2, and -beta3, TGF-beta receptors type I, II, and III, and post-receptor signaling genes smad2, smad3, smad4, smad6, and smad7 with the exception that TGF-beta2 and smad3 were undetectable in JAR and JEG-3 cells. Immunoblot analysis confirmed the absence of smad3 protein in choriocarcinoma cells. Treatment with TGF-beta1 induced smad3 phosphorylation and smad3 translocation to the nucleus in the normal and premalignant trophoblast cells. These results suggest that loss of smad3 may account for a functional disruption in the TGF-beta signaling pathway in choriocarcinomas, but not in the premalignant trophoblast.     

In the absence of type III receptor, the transforming growth factor (TGF)-beta type II-B receptor requires the type I receptor to bind TGF-beta2

Transforming growth factor beta (TGF-beta) ligands exert their biological effects through type II (TbetaRII) and type I receptors (TbetaRI). Unlike TGF-beta1 and -beta3, TGF-beta2 appears to require the co-receptor betaglycan (type III receptor, TbetaRIII) for high affinity binding and signaling. Recently, the TbetaRIII null mouse was generated and revealed significant non-overlapping phenotypes with the TGF-beta2 null mouse, implying the existence of TbetaRIII independent mechanisms for TGF-beta2 signaling. Because a variant of the type II receptor, the type II-B receptor (TbetaRII-B), has been suggested to mediate TGF-beta2 signaling in the absence of TbetaRIII, we directly tested the ability of TbetaRII-B to bind TGF-beta2. Here we show that the soluble extracellular domain of the type II-B receptor (sTbetaRII-B.Fc) bound TGF-beta1 and TGF-beta3 with high affinity (K(d) values = 31.7 +/- 22.8 and 74.6 +/- 15.8 pm, respectively), but TGF-beta2 binding was undetectable at corresponding doses. Similar results were obtained for the soluble type II receptor (sTbetaRII.Fc). However, sTbetaRII.Fc or sTbetaRII-B.Fc in combination with soluble type I receptor (sTbetaRI.Fc) formed a high affinity complex that bound TGF-beta2, and this complex inhibited TGF-beta2 in a biological inhibition assay. These results show that TGF-beta2 has the potential to signal in the absence of TbetaRIII when sufficient TGF-beta2, TbetaRI, and TbetaRII or TbetaRII-B are present. Our data also support a cooperative model for receptor-ligand interactions, as has been suggested by crystallization studies of TGF-beta receptors and ligands. Our cell-free binding assay system will allow for testing of models of receptor-ligand complexes prior to actual solution of crystal structures.     

Transforming growth factor-beta stimulates cyclin D1 expression through activation of beta-catenin signaling in chondrocytes

Transforming growth factor-beta (TGF-beta) plays an essential role in chondrocyte maturation. It stimulates chondrocyte proliferation but inhibits chondrocyte differentiation. In this study, we found that TGF-beta rapidly induced beta-catenin protein levels and signaling in murine neonatal sternal primary chondrocytes. TGF-beta-increased beta-catenin induction was reproduced by overexpression of SMAD3 and was absent in Smad3(-/-) chondrocytes treated with TGF-beta. SMAD3 inhibited beta-transducin repeat-containing protein-mediated degradation of beta-catenin and immunoprecipitated with beta-catenin following TGF-beta treatment. Both SMAD3 and beta-catenin co-localized to the nucleus after TGF-beta treatment. Although both TGF-beta and beta-catenin stimulated cyclin D(1) expression in chondrocytes, the effect of TGF-beta was inhibited with beta-catenin gene deletion or SMAD3 loss of function. These results demonstrate that TGF-beta stimulates cyclin D(1) expression at least in part through activation of beta-catenin signaling.     

Restoration of expression of transforming growth factor-beta type II receptor in murine renal cell carcinoma (renca) cells by 5-Aza-2'-deoxycytidine

The murine renal cell carcinoma (Renca) cells are insensitive to TGF-beta due to a lack of TGF-beta type II receptor (TbetaR-II). The objective of the present study is to determine the mechanism of this loss of sensitivity to TGF-beta in Renca cells. Renca cells were cultured and treated with 5-Aza-2'-Deoxycytidine (5-Aza), a specific inhibitor of methylation. Expression of TGF-beta type I receptor (TbetaRI) and TbetaRII was determined by RT-PCR and Western blot analysis before and after the treatment of Renca cells with 5-Aza. The expression of phosphorylated Smad2 (P-Smad2) was determined by Western blot analysis. TGF-beta levels in the conditioned medium were measured by ELISA. Renca cells did not express TbetaR-II prior to 5-Aza treatment. After 5-Aza treatment, these cells expressed TbetaR-II at both mRNA and protein levels, which corresponded to the restoration of sensitivity to TGF-beta by an increase in P-Smad2. Levels of TGF-beta1 were similar before and after 5-Aza treatment. Results of the present study indicated that, in Renca cells, the loss of sensitivity to TGF-beta is likely due to a promoter hypermethylation in the TbetaR-II gene.     

Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas

Four long-flagella (LF) genes are important for flagellar length control in Chlamydomonas reinhardtii. Here, we characterize two new null lf3 mutants whose phenotypes are different from previously identified lf3 mutants. These null mutants have unequal-length flagella that assemble more slowly than wild-type flagella, though their flagella can also reach abnormally long lengths. Prominent bulges are found at the distal ends of short, long, and regenerating flagella of these mutants. Analysis of the flagella by electron and immunofluorescence microscopy and by Western blots revealed that the bulges contain intraflagellar transport complexes, a defect reported previously (for review see Cole, D.G., 2003. Traffic. 4:435-442) in a subset of mutants defective in intraflagellar transport. We have cloned the wild-type LF3 gene and characterized a hypomorphic mutant allele of LF3. LF3p is a novel protein located predominantly in the cell body. It cosediments with the product of the LF1 gene in sucrose density gradients, indicating that these proteins may form a functional complex to regulate flagellar length and assembly.