Cellular distribution of type I and type II receptors for transforming growth factor-beta

Affinity labeling of target cells for transforming growth factor-beta (TGF beta) by cross-linking with 125I-TGF beta via disuccinimidyl suberate or by the photoreactive analogue 4-azidobenzoyl-125I-TGF beta has revealed the presence of multiple TGF beta receptor forms. Two distinct types of TGF beta receptors can be distinguished based on structural analysis of the 125I-TGF beta-labeled species by peptide mapping. Type I TGF beta receptors include the 280-kilodalton labeled receptor form previously found to be the subunit of a disulfide-linked TGF beta receptor complex. (Massagué, J. (1985) J. Biol. Chem. 260, 7059-7066), as well as a 65-kDa labeled receptor form present in all cell lines examined, and a 130-140-kDa labeled receptor form detected only in 3T3-L1 cells. The 280-kDa form is the major TGF beta receptor species in most cell lines examined, but is apparently absent in rat skeletal muscle myoblasts. Type I TGF beta receptors bind TGF beta with an apparent Kd of 50-500 pM. Type II TGF beta receptors include an 85-kDa labeled receptor form present in all mammalian cells examined and a 110-kDa labeled receptor form present in chick embryo fibroblasts. Type II TGF beta receptors bind TGF beta with an apparent Kd of about 50 pM. Except for the 280-kDa type I TGF beta receptor form, none of the TGF beta receptor forms described here is found as part of a disulfide-linked receptor complex. All the TGF beta receptor forms described here behave as intrinsic membrane proteins exposed on the surface of intact cells.     

The role of transforming growth factor-beta on retarded osteoblastic differentiation in vitro

Various matrix growth factors play important roles in the development and growth of cartilage and bone. Among them transforming growth factor-beta superfamily and especially bone morphogenetic proteins are known to be important factors, since they induce bone and cartilage formation in ectopic sites in vivo. We have previously shown that the human osteosarcoma cell line Saos-2 expresses molecules that in vivo induce new bone formation with asymmetric bone maturation. In this study we examined the role of Saos-2-conditioned medium in prolonged cultures of mesenchymal C3H/10T1/2 cells. The C3H/10T1/2 cells were cultured with Saos-2-conditioned medium for 28 days. We show that Saos-2-treated C3H/10T1/2 cells performed retarded osteoblastic differentiation when compared to recombinant BMP-2 and -4 induced differentiation. We further show that this retardation is due to excessive amounts of transforming growth factor-beta in Saos-2-conditioned medium. Our results also suggest that this model can well be used to study additional cofactors involved in retarded osteogenesis.     

Type I transforming growth factor-beta receptors on neutrophils mediate chemotaxis to transforming growth factor-beta.

Participation of human polymorphonuclear neutrophils in the inflammatory response is mediated, in part, by soluble factors such as chemotactic peptides and cytokines. Although the cytokine, transforming growth factor beta (TGF-beta), has been shown to recruit monocytes and promote the inflammatory process, its effects on neutrophils are unknown. In this investigation, [125I]TGF-beta 1 affinity binding studies were employed to show that neutrophils express TGF-beta receptors (350 +/- 20 receptors/cell), which exhibit high affinity for the ligand (dissociation constant, 50 pM). Affinity cross-linking studies identified the receptors to be primarily of the type I class. In contrast to the receptors on monocytes, neutrophil TGF-beta receptors were not down-regulated by exposure to specific inflammatory mediators. Additional studies examined whether exposure of neutrophils to TGF-beta could enhance specific functions, as occurs with monocytes. TGF-beta was shown to cause directed migration of neutrophils at femtomolar concentrations, thus it is the most potent neutrophil chemotactic factor yet identified. Neutrophil production of reactive oxygen intermediates was not stimulated by TGF-beta, nor did TGF-beta enhance or depress subsequent PMA- or FMLP-stimulated superoxide production. However, the stable expression of neutrophil TGF-beta receptors, and the capacity of this cytokine to stimulate neutrophil chemotaxis, suggest that the pro-inflammatory effects of TGF-beta are mediated by neutrophils in addition to monocytes.     

Differential expression of transforming growth factor-beta receptors I and II and activation of Smad 3 in keloid fibroblasts

Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially collagen. However, the molecular mechanisms regulating this pathologic collagen deposition still remain to be elucidated. A previous study by this group demonstrated that transforming growth factor (TGF)-beta1 and -beta2 ligands were expressed at greater levels in keloid fibroblasts when compared with normal human dermal fibroblasts (NHDFs), suggesting that TGF-beta may play a fibrosis-promoting role in keloid pathogenesis.To explore the biomolecular mechanisms of TGF-beta in keloid formation, the authors first compared the expression levels of the type I and type II TGF-beta receptors in keloid fibroblasts and NHDFs. Next, they investigated the phosphorylation of Smad 3, an intracellular TGF-beta signaling molecule, in keloid fibroblasts and NHDFs. Finally, they examined the regulation of TGF-beta receptor II by TGF-beta1, TGF-beta2, and TGF-beta3 ligands.Our findings demonstrated an increased expression of TGF-beta receptors (types I and II) and increased phosphorylation of Smad 3 in keloid fibroblasts relative to NHDFs. These data support a possible role of TGF-beta and its receptors as fibrosis-inducing growth factors in keloids. In addition, all three isoforms of recombinant human TGF-beta proteins could further stimulate the expression of TGF-beta receptor II in both keloids and NHDFs. Taken together, these results substantiate the hypothesis that the elevated levels of TGF-beta ligands and receptors present in keloids may support increased signaling and a potential role for TGF-beta in keloid pathogenesis.     

Modulation of monocyte type I transforming growth factor-beta receptors by inflammatory stimuli.

The regulatory mechanisms which control the wide array of cellular responses to transforming growth factor beta (TGF beta) are not understood. This report presents evidence that down-regulation of TGF beta receptors on human monocytes may be one mechanism by which the effects of TGF beta are regulated. Treatment of monocytes with interferon gamma (IFN gamma) and lipopolysaccharide for 18 h reduced monocyte receptor number (approximately 400/cell) in a dose-dependent fashion by 89 and 78%, respectively, as determined by 125I-TGF beta binding. Incubation with other cytokines (granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor-1, interleukin-1, tumor necrosis factor alpha) did not alter the amount of TGF beta bound. The decrease in 125I-TGF beta binding could not be attributed to competition for receptor sites by secreted TGF beta. Instead, the decline in binding was due to a loss of type I TGF beta receptors, the subtype primarily expressed by monocytes, with no decrease in receptor affinity. Lipopolysaccharide-induced receptor loss was rapid (1-4 h), in contrast to the prolonged (12 h) decline induced by IFN gamma. Loss of receptors was accompanied by a diminished ability of the cells to respond to TGF beta with an induction of TNF alpha mRNA. Thus, this monocyte system is the first example of a heterologous agent causing the down-regulation of TGF beta receptors with a concomitant decline in a TGF beta-stimulated function.     

Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation

Smad7 is an inhibitory Smad that acts as a negative regulator of signaling by the transforming growth factor-beta (TGF-beta) superfamily proteins. Smad7 is induced by TGF-beta, stably interacts with activated TGF-beta type I receptor (TbetaR-I), and interferes with the phosphorylation of receptor-regulated Smads. Here we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic protein-specific Smads, also interacts with Smad7 and induces Smad7 ubiquitination and translocation into the cytoplasm. In addition, Smurf1 associates with TbetaR-I via Smad7, with subsequent enhancement of turnover of TbetaR-I and Smad7. These results thus reveal a novel function of Smad7, i.e. induction of degradation of TbetaR-I through recruitment of an E3 ligase to the receptor.     

Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.     

Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells

Background  

Tumors expressing a transforming growth factor-beta type I receptor (TβRI) mutant with sequence deletions in a nine-alanine (9A) stretch of the signal peptide are reported to be highly associated with disease progression. Expression of this mutant could interfere with endogenous TGFβ signaling in the cell. However, little is known about the importance of the remaining part of the signal peptide on the cellular function of TβRI.     

Lysosomotropic drugs inhibit maturation of transforming growth factor-beta

Transforming growth factor-beta (TGFbeta) is synthesized as a precursor protein, pro-TGFbeta, that must be cleaved by a furin-like proteinase before it becomes biologically active. We hypothesized that alkalinization of the trans-Golgi network (TGN)/endosome system may suppress pro-TGFbeta processing and decrease TGFbeta secretion. This hypothesis was tested in human A549 alveolar epithelial and T98G glioblastoma cell lines and in C57BL/6 mice. Inhibition of furin-like activity with decanoyl-RVKR chloromethylketone suppressed pro-TGFbeta processing, thereby significantly reducing the levels of secreted TGFbeta. Brefeldin A, bafilomycin A1, ammonium chloride, and monensin also prevented pro-TGFbeta processing. The alkalinizing lysosomotropic drugs chloroquine, hydroxychloroquine, amodiaquine, and azithromycin had a similar effect on the overall production of mature bioactive TGFbeta. Reduced levels of secreted TGFbeta were also associated with a decrease in Smad2 signaling. Mice treated with chloroquine showed a decrease in bronchoalveolar lavage fluid TGFbeta. We conclude that alkalinizing lysosomotropic drugs inhibit pro-TGFbeta processing.     

Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.     

Smad7 regulates terminal maturation of chondrocytes in the growth plate

Members of the bone morphogenetic protein (BMP) superfamily, including transforming growth factor-betas (TGFβ), regulate multiple aspects of chondrogenesis. Smad7 is an intracellular inhibitor of BMP and TGFβ signaling. Studies in which Smad7 was overexpressed in chondrocytes demonstrated that Smad7 can impact chondrogenesis by inhibiting BMP signaling. However, whether Smad7 is actually required for endochondral ossification in vivo is unclear. Moreover, whether Smad7 regulates TGFβ in addition to BMP signaling in developing cartilage is unknown. In this study, we found that Smad7 is required for both axial and appendicular skeletal development. Loss of Smad7 led to impairment of the cell cycle in chondrocytes and to defects in terminal maturation. This phenotype was attributed to upregulation of both BMP and TGFβ signaling in Smad7 mutant growth plates. Moreover, Smad7−/− mice develop hypocellular cores in the medial growth plates, associated with elevated HIF1α levels, cell death, and intracellular retention of types II and X collagen. Thus, Smad7 may be required to mediate cell stress responses in the growth plate during development.     

The transforming growth factor-beta superfamily of receptors

The transforming growth factor-beta (TGF-beta) superfamily of receptors comprises two groups of transmembrane serine-threonine kinase receptors, so called type I, and type II receptors, that are activated following engagement by members of the TGF-beta superfamily of ligands. These events specify diverse downstream responses that are differentially regulated by controlling access and activation of the ligands, their receptors and downstream substrates in different cell types. The purpose of this review is to describe the biochemical properties of these receptors, focusing specifically on the mechanisms regulating receptor/ligand interactions and activation in mammalian cells.     

Bone morphogenetic proteins signal through the transforming growth factor-beta type III receptor

The bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-beta (TGF-beta) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-beta type III receptor (TbetaRIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-beta and inhibin receptor. Here we demonstrate that TbetaRIII functions as a BMP cell surface receptor. TbetaRIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-beta. TbetaRIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous TbetaRIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for TbetaRIII function, we demonstrate that BMP-2 specifically stimulates TbetaRIII-mediated epithelial to mesenchymal cell transformation. The ability of TbetaRIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-beta signaling suggests a broader role for TbetaRIII in orchestrating TGF-beta superfamily signaling.     

Molecular evolution of a developmental pathway: phylogenetic analyses of transforming growth factor-beta family ligands, receptors and Smad signal transducers.

Intercellular signaling by transforming growth factor-beta (TGF-beta) proteins coordinates developmental decisions in many organisms. A receptor complex and Smad signal transducers are required for proper responses to TGF-beta signals. We have taken a phylogenetic approach to understanding the developmental evolutionary history of TGF-beta signaling pathways. We were interested in detecting evolutionary influences among the physically interacting multigene families encoding TGF-beta ligands, receptors, and Smads. Our analyses included new ligands and Smads identified from genomic sequence as well as the newest published family members. From an evolutionary perspective we find that (1) TGF-beta pathways do not predate the divergence of animals, plants, and fungi; (2) ligands of the TGF-beta/activin subfamily likely originated after the divergence of nematodes and arthropods; (3) type I receptors from Caenorhabditis elegans are distinct from other receptors and may reflect an ancestral transitional state between type I and type II receptors; and (4) the Smad family appears to be evolving faster than, and independently of, ligands and receptors. From a developmental perspective we find (1) numerous phylogenetic associations not previously detected in each multigene family; (2) that there are unidentified pathway components that discriminate between type I and type II receptors; (3) that there are more Smads to be discovered in Drosophila and mammals; and (4) that the number of C-terminal serines is the best predictor of a Smad's role in TGF-beta signal transduction. We discuss these findings with respect to the coevolution of physically interacting genes.