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Michael T Longaker - One of the best experts on this subject based on the ideXlab platform.

  • ontogeny of expression of transforming growth factor Beta 1 TGF Beta 1 TGF Beta 3 and TGF Beta Receptors i and ii in fetal rat fibroblasts and skin
    Plastic and Reconstructive Surgery, 2001
    Co-Authors: Meier Hsu, Ziv M Peled, Gyu S Chin, Wei Liu, Michael T Longaker
    Abstract:

    Fetal cutaneous wounds that occur in early gestation heal without scar formation. Although much work has been done to characterize the role of transforming growth factor-Beta (TGF-Beta) isoforms in the adult wound repair process, their function in fetal scarless wound repair is not well understood. The authors hypothesized that the pattern of expression for TGF-Beta isoforms and their Receptors may influence the phenotypic transition from scarless to scar-forming repair observed during fetal gestation. Using time-dated fetal Sprague-Dawley rat fibroblasts and unwounded skin at gestational ages 14, 16, 18, and 21 days postcoitum of the scarless ( 16 days) periods of gestation (term = 21.5 days), the authors analyzed the endogenous messenger RNA (mRNA) levels of TGF-Beta 1 and TGF-Beta 3 and their signaling Receptors TGF-Beta-RI and TGF-Beta-RII. Northern blot analyses in both fibroblasts and unwounded skin revealed that levels of TGF-Beta 1 were not differentially expressed, whereas more TGF-Beta 3 mRNA transcript was found in early than in late gestation. Fibroblast expression of TGF-Beta-RI showed no substantial differences, whereas expression of TGF-Beta-RII increased during gestation. In contrast, expression of both TGF-Beta-RI and TGF-Beta-RII in unwounded skin showed decreasing levels as a function of gestational age. The differential levels of TGF-Beta 1 and TGF-Beta 3 suggest that the ratio of these cytokines may provide a predominantly antiscarring or profibrotic signal upon wounding during the scar-free or scar-forming periods of gestation, respectively. Furthermore, lower amounts of the ligand-binding TGF-Beta-RII seen in early gestation fibroblasts suggest a decreased ability to perceive ligand during the period of scarless repair.

  • type i ri and type ii rii Receptors for transforming growth factor Beta isoforms are expressed subsequent to transforming growth factor Beta ligands during excisional wound repair
    American Journal of Pathology, 1997
    Co-Authors: Leslie I Gold, J J Sung, John W Siebert, Michael T Longaker
    Abstract:

    Transforming growth factor (TGF)-Beta isoforms (TGF-Beta 1, -Beta 2, and -Beta 3) regulate cell growth and differentiation and have critical regulatory roles in the process of tissue repair and remodeling. Signal transduction for TGF-Beta function is transmitted by a heteromeric complex of Receptors consisting of two serine/threonine kinase transmembrane proteins (RI and RII). We have previously shown that each TGF-Beta isoform is widely expressed in a distinct spatial and temporal pattern throughout the processes of excisional and incisional wound repair. As the presence of TGF-Beta Receptors determines cellular responsiveness, we have currently examined, by immunohistochemistry, the localization of RI (ALK-1, ALK-5) and RII throughout repair of full-thickness excisional wounds up to 21 days after wounding. The expression of RI (ALK-5) and RII co-localized in both the unwounded and wounded skin and was present in the same cell types as TGF-Beta ligands. However, immunoreactivity for TGF-Beta Receptors, throughout repair, occurred 1 to 5 days later than TGF-Beta isoform immunostaining. This implies that the presence of TGF-Beta ligands may up-regulate TGF-Beta Receptors for function and/or may reflect a lag due to local processing of latent TGF-Beta. As observed for the immunohistochemical localization of TGF-Beta isoforms in unwounded skin, RI and RII were expressed throughout the four layers of the epidermis, showing a wavy pattern of slight to moderate immunostaining, and hair follicles, sweat glands, and sebaceous glands were moderately immunoreactive. The extracellular matrix, fibroblasts, and blood vessels in the dermis were not immunoreactive. After injury, as observed for TGF-Beta ligands, RI and RII expression was increased in the epidermis adjacent to the wound and the epithelium migrating over the wound was completely devoid of TGF-Beta receptor immunoreactivity until re-epithelialization was completed by day 7 after wounding. The dermis was only slightly immunoreactive for RI and RII until day 5 when, immediately under the wound, immunostaining for fibroblasts, connective tissue cells, and newly forming vasculature began to increase and remained intense until day 14. Consistent with the role for TGF-Beta in scarring, numerous fibroblasts, ostensibly active in the production of extracellular matrix components, continued to be slightly immunoreactive for RI and RII at 21 days. The ALK-1 (TSR-1) type I receptor, which binds both activin and TGF-Beta, showed slight immunostaining early in repair (days 1 to 7) that progressively became more intense later in repair after day 10 and through day 21. This suggests that there may be a switch to a different type I receptor, implying different functions for the ALK-1 and ALK-5 Receptors. The concomitant expression of TGF-Beta isoforms and their signal-transducing Receptors denote potential spatial and temporal activity of TGF-Beta. Thus, although TGF-Beta ligand is present, TGF-Beta would not function in wound repair until a later time when RI and RII appear. This information should aid in the development of receptor antagonists as a therapeutic approach to scarring and fibrosis. In addition, these studies underscore the importance of defining the expression of proteins in vivo to establish a basis for the analysis of mechanisms in vitro.

Pontus Aspenstrom - One of the best experts on this subject based on the ideXlab platform.

  • smad7 is required for TGF Beta induced activation of the small gtpase cdc42
    Journal of Cell Science, 2004
    Co-Authors: Sofia Edlund, Marene Landstrom, Carlhenrik Heldin, Pontus Aspenstrom
    Abstract:

    Transforming growth factor Beta (TGF-Beta) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-Beta Receptors. The inhibitory Smads, Smad6 and Smad7, are considered to function as negative regulators of the TGF-Beta/Smad signaling cascade. In a previous study, we found that TGF-Beta induces rearrangements of the actin filament system in human prostate carcinoma cells and that this response requires the small GTPases Cdc42 and RhoA. On the basis of the current view on the function of Smad7 in TGF-Beta signaling, we hypothesized that Smad7 would function as a negative regulator of the TGF-Beta-induced activation of Cdc42 and RhoA, but instead we found that the reverse is the case; Smad7 is required for the TGF-Beta-induced activation of Cdc42 and the concomitant reorganization of the actin filament system. These observations propose a novel role for Smad7 in TGF-Beta-dependent activation of Rho GTPases.

  • transforming growth factor β induced mobilization of actin cytoskeleton requires signaling by small gtpases cdc42 and rhoa
    Molecular Biology of the Cell, 2002
    Co-Authors: Sofia Edlund, Marene Landstrom, Carlhenrik Heldin, Pontus Aspenstrom
    Abstract:

    Transforming growth factor-Beta (TGF-Beta) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-Beta Receptors. We studied TGF-Beta-induced rearrangements of the actin filament system and found that TGF-Beta 1 treatment of PC-3U human prostate carcinoma cells resulted in a rapid formation of lamellipodia. Interestingly, this response was shown to be independent of the Smad signaling pathway; instead, it required the activity of the Rho GTPases Cdc42 and RhoA, because ectopic expression of dominant negative mutant Cdc42 and RhoA abrogated the response. Long-term stimulation with TGF-Beta 1 resulted in an assembly of stress fibers; this response required both signaling via Cdc42 and RhoA, and Smad proteins. A known downstream effector of Cdc42 is p38(MAPK); treatment of the cells with the p38(MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(pyridyl)1H-imidazole (SB203580), as well as ectopic expression of a kinase-inactive p38(MAPK), abrogated the TGF-Beta-induced actin reorganization. Moreover, treatment of cells with the inhibitors of the RhoA target-protein Rho-associated coiled-coil kinase (+)-R-trans-4-(aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide (Y-27632) and 1-5(-isoquinolinesulfonyl)homopiperazine (HA-1077), as well as ectopic expression of kinase-inactive Rho coiled-coil kinase-1, abrogated the TGF-Beta 1-induced formation of stress fibers. Collectively, these data indicate that TGF-Beta-induced membrane ruffles occur via Rho GTPase-dependent pathways, whereas long-term effects require cooperation between Smad and Rho GTPase signaling pathways.

Rik Derynck - One of the best experts on this subject based on the ideXlab platform.

  • ligand independent activation of transforming growth factor TGF Beta signaling pathways by heteromeric cytoplasmic domains of TGF Beta Receptors
    Journal of Biological Chemistry, 1996
    Co-Authors: Xinhua Feng, Rik Derynck
    Abstract:

    Transforming growth factor Beta (TGF-Beta) transduces signals through two related serine/threonine kinase Receptors, the type I and type II Receptors, which have the ability to interact with each other. In the heteromeric complex, the type II receptor is the primary determinant of ligand binding and phosphorylates the cytoplasmic domain of the type I receptor. Using a chimeric receptor strategy, we and others have shown previously that a functional TGF-Beta receptor complex requires heteromerization of both extracellular and intracellular domains of type I and type II Receptors. In the current study, we show that overexpression of two Receptors carrying a heteromeric combination of cytoplasmic domains resulted in ligand-independent responses, further supporting the functional requirement of the two heterologous cytoplasmic domains in TGF-Beta signaling. Furthermore, coexpression of only the cytoplasmic domains of both the type I and II Receptors or tethering the type II to the type I cytoplasmic domain activated TGF-Beta responses in a ligand-independent manner. In cotransfected COS-1 cells, both cytoplasmic domains are associated with each other. Our results indicate that the cytoplasmic domains of the type I and type II TGF-Beta Receptors physically and functionally interact with each other in the heteromeric complex.

  • ligand independent activation of transforming growth factor TGF Beta signaling pathways by heteromeric cytoplasmic domains of TGF Beta Receptors
    Journal of Biological Chemistry, 1996
    Co-Authors: Xinhua Feng, Rik Derynck
    Abstract:

    Transforming growth factor β (TGF-β) transduces signals through two related serine/threonine kinase Receptors, the type I and type II Receptors, which have the ability to interact with each other. In the heteromeric complex, the type II receptor is the primary determinant of ligand binding and phosphorylates the cytoplasmic domain of the type I receptor. Using a chimeric receptor strategy, we and others have shown previously that a functional TGF-β receptor complex requires heteromerization of both extracellular and intracellular domains of type I and type II Receptors. In the current study, we show that overexpression of two Receptors carrying a heteromeric combination of cytoplasmic domains resulted in ligand-independent responses, further supporting the functional requirement of the two heterologous cytoplasmic domains in TGF-β signaling. Furthermore, coexpression of only the cytoplasmic domains of both the type I and II Receptors or tethering the type II to the type I cytoplasmic domain activated TGF-β responses in a ligand-independent manner. In cotransfected COS-1 cells, both cytoplasmic domains are associated with each other. Our results indicate that the cytoplasmic domains of the type I and type II TGF-β Receptors physically and functionally interact with each other in the heteromeric complex.

Harvey F Lodish - One of the best experts on this subject based on the ideXlab platform.

  • biosynthesis of the type i and type ii TGF Beta Receptors implications for complex formation
    Journal of Biological Chemistry, 1997
    Co-Authors: Rebecca G Wells, Herbert Y Lin, Haya Yankelev, Harvey F Lodish
    Abstract:

    The TGF-Beta type I and type II Receptors (TBetaRI and TBetaRII) are signaling Receptors that form heteromeric cell surface complexes with the TGF-Betas as one of the earliest events in the cellular response to these multifunctional growth factors. Using TGF-Beta-responsive mink lung epithelial cells (Mv1Lu), we have determined the half-lives of the endoplasmic reticulum (ER) and mature forms of these Receptors. In metabolically labeled cells, approximately 90% of newly synthesized type II receptor undergoes modification of N-linked sugars in the Golgi, with a half-life of 30-35 min; the Golgi-processed form of the receptor has a relatively short metabolic half-life of 2.5 h. In contrast, only 50% of pulse-labeled type I receptor is converted to the Golgi-processed and therefore endoglycosidase H-resistant form, and the endoglycosidase H-sensitive ER form has a half-life of 2.8-3 h. Addition of 100 pM TGF-Beta1 causes the Golgi-processed type II receptor to become less stable, with a half-life of 1.7 h, and also destabilizes the Golgi-processed type I receptor. TGF-Beta1 binding and cross-linking experiments on cells treated with tunicamycin for various times confirm different ER to cell surface processing times for TBetaRI and TBetaRII. Our results, which suggest that stable complexes between type I and II TGF-Beta Receptors do not form until the proteins reach a post-ER compartment (presumably the cell surface), have important implications for our understanding of complex formation and receptor regulation.

  • Signaling by chimeric erythropoietin-TGF-Beta Receptors: homodimerization of the cytoplasmic domain of the type I TGF-Beta receptor and heterodimerization with the type II receptor are both required for intracellular signal transduction.
    The EMBO Journal, 1996
    Co-Authors: Harvey F Lodish
    Abstract:

    Abstract Transforming growth factor-Beta (TGF-Beta) affects multiple cellular functions through the type I and type II receptor Ser/Thr kinases (TBetaRI and TBetaRII). Analysis of TGF-Beta signaling pathways has been hampered by the lack of cell lines in which both TBetaRI and TBetaRII are deleted, and by the inability to study signal transduction by TBetaRI independently of TBetaRII since TBetaRI does not bind TGF-Beta directly. To overcome these problems, we constructed and expressed chimeric Receptors with the extracellular domain of the erythropoietin receptor (EpoR) and the cytoplasmic domains of TBetaRI or TBetaRII. When expressed in Ba/F3 cells, which do not express EpoR, Epo induces the formation of a heteromeric complex between cell surface EpoR-TBetaRI and EpoR-TBetaRII chimeras. Neither the EpoR-TBetaRI nor the EpoR-TBetaRII chimera interacts with endogenous TGF-Beta Receptors. Ba/F3 cells expressing both EpoR-TBetaRI and EpoR-TBetaRII chimeras, but not EpoR-TBetaRI or EpoR-TBetaRII alone, undergo Epo-induced growth arrest. When expressed in Ba/F3 cells in the absence of the EpoR-TBetaRII chimera, EpoR-TBetaRI(T204D), a chimeric receptor with a point mutation in the GS domain of TBetaRI that is autophosphorylated constitutively, triggers growth inhibition in response to Epo. Thus, both homo- and heterodimerization of the cytoplasmic domain of the type I TGF-Beta receptor are required for intracellular signal transduction leading to inhibition of cell proliferation. These chimeric Receptors provide a unique system to study the function and signal transduction of individual TGF-Beta receptor subunits independently of endogenous TGF-Beta Receptors.

  • a dominant inhibitory mutant of the type ii transforming growth factor Beta receptor in the malignant progression of a cutaneous t cell lymphoma
    Molecular and Cellular Biology, 1996
    Co-Authors: Petra Knaus, Haya Yankelev, Dirk Lindemann, John F Decoteau, R Perlman, M Hille, M E Kadin, Harvey F Lodish
    Abstract:

    In many cancers, inactivating mutations in both alleles of the transforming growth factor Beta (TGF-Beta) type 11 receptor (TBetaRII) gene occur and correlate with loss of sensitivity to TGF-Beta. Here we describe a novel mechanism for loss of sensitivity to growth inhibition by TGF-Beta in tumor development. Mac-1 cells, isolated from the blood of a patient with an indolent form of cutaneous T-cell lymphoma, express wild-type TBetaRII and are sensitive to TGF-Beta. Mac-2A cells, clonally related to Mac-1 and isolated from a skin nodule of the same patient at a later, clinically aggressive stage of lymphoma, are resistant to TGF-Beta. They express both the wild-type TBetaRII and a receptor with a single point mutation (Asp-404-Gly [D404G]) in the kinase domain (D404G-->TBetaRII); no TBetaRI or TBetaRII is found on the plasma membrane, suggesting that D404G-TBetaRII dominantly inhibits the function of the wild-type receptor by inhibiting its appearance on the plasma membrane. Indeed, inducible expression, under control of a tetracycline-regulated promoter, of D404G-TBetaRII in TGF-Beta- sensitive Mac-1 cells as well as in Hep3B hepatoma cells results in resistance to TGF-Beta and disappearance of cell surface TBetaRI and TBetaRII. Overexpression of wild-type TBetaRII in Mac-2A cells restores cell surface TBetaRI and TBetaRH and sensitivity to TGF-Beta. The ability of the D404G-TBetaRH to dominantly inhibit function of wild-type TGF-Beta Receptors represents a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-Beta in tumor development.

  • expression cloning and characterization of the TGF β type iii receptor
    Cell, 1991
    Co-Authors: Xiaofan Wang, Harvey F Lodish, Herbert Y Lin, Elinor Ngeaton, Julian Downward, Robert A Weinberg
    Abstract:

    The rat TGF-Beta type III receptor cDNA has been cloned by overexpression in COS cells. The encoded receptor is an 853 amino acid protein with a large N-terminal extracellular domain containing at least one site for glycosaminoglycan addition, a single hydrophobic transmembrane domain, and a 41 amino acid cytoplasmic tail with no obvious signaling motif. Introduction of the cDNA into COS cells and L6 myoblasts induces expression of a heterogenously glycosylated 280-330 kd protein characteristic of the type III receptor that binds TGF-Beta 1 specifically. In L6 myoblasts lacking the endogenous type III receptor, expression of the recombinant receptor leads to an increase in the amount of ligand bound and cross-linked to surface type II TGF-Beta Receptors. This indicates that the type III receptor may regulate the ligand-binding ability or surface expression of the type II receptor.

Joan Massagué - One of the best experts on this subject based on the ideXlab platform.

  • the l3 loop a structural motif determining specific interactions between smad proteins and TGF β Receptors
    The EMBO Journal, 1998
    Co-Authors: Yeguang Chen, Yigong Shi, Nikola P Pavletich, Joan Massagué
    Abstract:

    Signal transduction specificity in the transforming growth factor-Beta (TGF-Beta) system is determined by ligand activation of a receptor complex which then recruits and phosphorylates a subset of SMAD proteins including Smads 1 and 2. These then associate with Smad4 and move into the nucleus where they regulate transcription. We have identified a discrete surface structure in Smads 1 and 2 that mediates and specifies their receptor interactions. This structure is the L3 loop, a 17 amino acid region that protrudes from the core of the conserved SMAD C-terminal domain. The L3 loop sequence is invariant among TGF-Beta- and bone morphogenetic protein (BMP)-activated SMADS, but differs at two positions between these two groups. Swapping these two amino acids in Smads 1 and 2 induces a gain or loss, respectively, in their ability to associate with the TGF-Beta receptor complex and causes a switch in the phosphorylation of Smads 1 and 2 by the BMP and TGF-Beta Receptors, respectively. A full switch in phosphorylation and activation of Smads 1 and 2 is obtained by swapping both these two amino acids and four amino acids near the C-terminal receptor phosphorylation sites. These studies identify the L3 loop as a determinant of specific SMAD-receptor interactions, and indicate that the L3 loop, together with the C-terminal tail, specifies SMAD activation.

  • Complementation between kinase-defective and activation-defective TGF-Beta Receptors reveals a novel form of receptor cooperativity essential for signaling.
    The EMBO Journal, 1996
    Co-Authors: Frances Weis-garcia, Joan Massagué
    Abstract:

    Transforming growth factor-Beta (TGF-Beta) signals through two transmembrane serine/threonine kinases, T Beta R-I and T Beta R-II. TGF-Beta binds to T Beta R-II, allowing this receptor to associate with and phosphorylate T Beta R-I which then propagates the signal. T Beta R-I is phosphorylated within its GS domain, a region immediately preceding the kinase domain. To further understand the function of T Beta R-I in this complex, we analyzed T Beta R-I-inactivating mutations identified in cell lines that are defective in TGF-Beta signaling yet retain ligand binding ability. The three mutations identified here all fall in the kinase domain of T Beta R-I. One mutation disrupts the kinase activity of T Beta R-I, whereas the other two mutations prevent ligand-induced T Beta R-I phosphorylation, and thus activation, by T Beta R-II. Unexpectedly, a kinase-defective T Beta R-I mutant can functionally complement an activation- defective T Beta R-I mutant, by rescuing its T Beta R-II- dependent phosphorylation. Together with evidence that the ligand-induced receptor complex contains two or more T Beta R-I molecules, these results support a model in which the kinase domain of one T Beta R-I molecule interacts with the GS domain of another, enabling its phosphorylation and activation by T Beta R-II. This cooperative interaction between T Beta R-I molecules appears essential for TGF-Beta signal transduction.

  • endoglin is a component of the transforming growth factor Beta receptor system in human endothelial cells
    Journal of Biological Chemistry, 1992
    Co-Authors: Sela Cheifetz, Carmela Cales, Sonia Vera, Joan Massagué, Carmelo Bernabeu, Teresa Bellon, Michelle Letarte
    Abstract:

    Abstract Endoglin, a dimeric membrane glycoprotein expressed at high levels on human vascular endothelial cells, shares regions of sequence identity with Betaglycan, a major binding protein for transforming growth factor-Beta (TGF-Beta) that co-exists with TGF-Beta Receptors I and II in a variety of cell lines but is low or absent in endothelial cells. We have examined whether endoglin also binds TGF-Beta and demonstrate here that the major TGF-Beta 1-binding protein co-existing with TGF-Beta Receptors I and II on human umbilical vein endothelial cells is endoglin, as determined by specific immunoprecipitation of endoglin affinity-labeled with 125I-TGF-Beta. Furthermore, endoglin ectopically expressed in COS cells binds TGF-Beta 1. Competition affinity-labeling experiments showed that endoglin binds TGF-Beta 1 (KD approximately 50 pM) and TGF-Beta 3 with high affinity but fails to bind TGF-Beta 2. This difference in affinity of endoglin for the TGF-Beta isoforms is in contrast to Beta-glycan which recognizes all three isoforms. TGF-Beta however is binding with high affinity to only a small fraction of the available endoglin molecules, suggesting that some rate-limiting event is required to sustain TGF-Beta binding to endoglin.

  • responsiveness to transforming growth factor Beta TGF Beta restored by genetic complementation between cells defective in TGF Beta Receptors i and ii
    Journal of Biological Chemistry, 1991
    Co-Authors: Marikki Laiho, Frances M B Weis, Frederick T Boyd, Ronald A Ignotz, Joan Massagué
    Abstract:

    Selection of mutant Mv1Lu mink lung epithelial cells resistant to growth inhibition by transforming growth factor-Beta (TGF-Beta) has led to the isolation of cell clones with distinct alterations in type I and II TGF-Beta Receptors. Certain mutant clones present a decreased number or complete loss of detectable type I receptor. Other clones show a loss and/or altered electrophoretic mobility of the type II receptor, with concomitant loss of the type I receptor. Using somatic cell hybridization analysis we demonstrate the recessive nature of these mutants with respect to the wild-type phenotype and define various mutant complementation groups. Among these, hybrids between cells that express only type II receptor (R mutants) and cells that express neither receptor type (DRa mutants) rescue wild-type expression of type I Receptors. Moreover, these hybrids regain full responsiveness to TGF-Beta 1, as measured by inhibition of DNA synthesis as well as stimulation of fibronectin and plasminogen activator inhibitor-1 production. These results provide evidence for an interaction between TGF-Beta receptor components I and II and show that, in Mv1Lu cells, expression of both receptor types is required for mediation of biological responses to TGF-Beta 1.