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

1992 ◽  
Vol 12 (4) ◽  
pp. 1674-1679
Author(s):  
A R Lopez ◽  
J Cook ◽  
P L Deininger ◽  
R Derynck
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Dominant Negative ◽  
Tgf Beta ◽  
Growth Factor Beta ◽  
Beta 2

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.

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

1992 ◽  
Vol 12 (4) ◽  
pp. 1674-1679 ◽  
Author(s):  
A R Lopez ◽  
J Cook ◽  
P L Deininger ◽  
R Derynck
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Dominant Negative ◽  
Tgf Beta ◽  
Growth Factor Beta ◽  
Beta 2

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.

scholarly journals Transforming growth factor beta 1 (TGF-beta 1) induced neutrophil recruitment to synovial tissues: implications for TGF-beta-driven synovial inflammation and hyperplasia.

1991 ◽  
Vol 173 (5) ◽  
pp. 1121-1132 ◽  
Author(s):  
R A Fava ◽  
N J Olsen ◽  
A E Postlethwaite ◽  
K N Broadley ◽  
J M Davidson ◽  
...  
Keyword(s):  
Growth Factor ◽  
Synovial Tissue ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Biologically Active ◽  
Tgf Beta ◽  
Histological Techniques ◽  
Growth Factor Beta

We have studied the consequences of introducing human recombinant transforming growth factor beta 1 (hrTGF-beta 1) into synovial tissue of the rat, to begin to better understand the significance of the fact that biologically active TGF-beta is found in human arthritic synovial effusions. Within 4-6 h after the intra-articular injection of 1 microgram of hrTGF-beta 1 into rat knee joints, extensive recruitment of polymorphonuclear leukocytes (PMNs) was observed. Cytochemistry and high resolution histological techniques were used to quantitate the influx of PMNs, which peaked 6 h post-injection. In a Boyden chamber assay, hrTGF-beta 1 at 1-10 fg/ml elicited a chemotactic response from PMNs greater in magnitude than that evoked by FMLP, establishing that TGF-beta 1 is an effective chemotactic agent for PMNs in vitro as well as in vivo. That PMNs may represent an important source of TGF-beta in inflammatory infiltrates was strongly suggested by a demonstration that stored TGF-beta 1 was secreted during phorbol myristate acetate-stimulated degranulation in vitro. Acid/ethanol extracts of human PMNs assayed by ELISA contained an average of 355 ng of TGF/beta 1 per 10(9) cells potentially available for secretion during degranulation of PMNs. [3H]Thymidine incorporation in vivo and autoradiography of tissue sections revealed that widespread cell proliferation was triggered by TGF-beta 1 injection. Synovial lining cells and cells located deep within the subsynovial connective tissue were identified as sources of at least some of the new cells that contribute to TGF-beta 1-induced hyperplasia. Our results demonstrate that TGF-beta is capable of exerting pathogenic effects on synovial tissue and that PMNs may represent a significant source of the TGF-beta present in synovial effusions.

scholarly journals Regulation of Trypanosoma cruzi infections in vitro and in vivo by transforming growth factor beta (TGF-beta).

1991 ◽  
Vol 174 (3) ◽  
pp. 539-545 ◽  
Author(s):  
J S Silva ◽  
D R Twardzik ◽  
S G Reed
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Tgf Beta ◽  
Ifn Gamma ◽  
Human Macrophages ◽  
Growth Factor Beta ◽  
Cruzi Infection

The effects of transforming growth factor beta (TGF-beta) on interferon gamma-mediated killing of the intracellular protozoan parasite Trypanosoma cruzi and on the course of T. cruzi infection in mice were investigated. Spleen cells from mice with acute T. cruzi infections were found to produce elevated levels of biologically active TGF-beta in vitro, and the possibility that TGF-beta may mediate certain aspects of T. cruzi infection was then addressed. When mouse peritoneal macrophages were treated with TGF-beta in vitro, the ability of IFN-gamma to activate intracellular inhibition of the parasite was blocked. This occurred whether cells were treated with TGF-beta either before or after IFN-gamma treatment. TGF-beta treatment also blocked the T. cruzi-inhibiting effects of IGN-gamma on human macrophages. Additionally, treatment of human macrophages with TGF-beta alone led to increased parasite replication in these cells. The effects of TGF-beta on T. cruzi infection in vivo were then investigated. Susceptible C57BL/6 mice developed higher parasitemias and died earlier when treated with TGF-beta during the course of infection. Resistant C57BL/6 x DBA/2 F1 mice treated with TGF-beta also had increased parasitemias, and 50% mortality, compared with no mortality in infected, saline-treated controls. A single dose of TGF-beta, given at the time of infection, was sufficient to significantly decrease resistance to infection in F1 mice and to exacerbate infection in susceptible C57BL/6 mice. Furthermore, a single injection of TGF-beta was sufficient to counter the in vivo protective effects of IFN-gamma. We conclude that TGF-beta, produced during acute T. cruzi infection in mice, is a potent inhibitor of the effects of macrophage activating cytokines in vivo and in vitro and may play a role in regulating infection.

scholarly journals Immunoregulatory role of transforming growth factor beta (TGF-beta) in development of killer cells: comparison of active and latent TGF-beta 1.

1990 ◽  
Vol 172 (6) ◽  
pp. 1777-1784 ◽  
Author(s):  
S C Wallick ◽  
I S Figari ◽  
R E Morris ◽  
A D Levinson ◽  
M A Palladino
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Recombinant Dna ◽  
Chinese Hamster ◽  
Tgf Beta ◽  
Recombinant Dna Technology ◽  
Growth Factor Beta

Using recombinant DNA technology, we have generated Chinese hamster ovary (CHO) cell lines that synthesize latent transforming growth factor beta 1 (TGF-beta 1) to study immune regulation by TGF-beta 1. In vitro, latent TGF-beta 1 synthesized by transfectants or added exogenously as a purified complex after activation inhibited CTL generation to a similar extent as seen with acid-activated recombinant human (rHu) TGF-beta 1. In vivo, serum from nu/nu mice bearing CHO/TGF-beta 1 tumors contained significant levels of latent TGF-beta 1 in addition to depressed natural killer (NK) activity in spleens which paralleled that seen in C3H/HeJ mice treated with acid-activated rHuTGF-beta 1. rHuTGF-beta 1 treatment of mice receiving heart allografts resulted in significant enhancement of organ graft survival. Because of possible regulated tissue-specific activation, administration of latent rather than active TGF-beta may provide a better route to deliver this powerful immunosuppressive agent in vivo.

scholarly journals Transforming growth factor-beta stimulates collagen VII expression by cutaneous cells in vitro.

1992 ◽  
Vol 117 (3) ◽  
pp. 679-685 ◽  
Author(s):  
A König ◽  
L Bruckner-Tuderman
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Human Keratinocytes ◽  
Steady Increase ◽  
Tgf Beta ◽  
Growth Factor Beta ◽  
Beta 2 ◽  
Collagen Vii

Collagen VII, the major component of cutaneous anchoring fibrils is expressed at a low level by normal human keratinocytes and fibroblasts in vitro. In cocultures of these two cell types, signals from fibroblasts enhance expression of collagen VII by keratinocytes and vice versa. In this study, the effects of a possible mediator of such a stimulation, transforming growth factor-beta (TGF-beta), were investigated. Its effect on the expression and deposition of the highly insoluble collagen VII was assessed in a semiquantitative manner by a newly developed enzyme-linked immunoassay which is based on immunoblotting. In keratinocyte monocultures, 0.5-20 ng/ml of TGF-beta 2 induced a dose-dependent stimulation of collagen VII expression as measured per microgram of DNA. The maximal enhancement was about sevenfold compared to controls. The effect of TGF-beta 2 was observed already after 12 h, with a steady increase at least up to 3 d. As previous studies have implicated, untreated cocultures of keratinocytes and fibroblasts exhibited a higher basic level of collagen VII expression, which could be further stimulated about twofold by TGF-beta 2. Fibroblasts alone synthesized very minor quantities of collagen VII and could be only weakly stimulated by TGF-beta 2. This growth factor seems a specific enhancer of collagen VII since the expression of laminin, collagen IV, as well as total protein was increased to a much lesser extent. Our data suggest that TGF-beta may be an important mediator of epithelial-mesenchymal interactions and may regulate the synthesis of the anchoring fibrils at the skin basement membrane zone.

A GC-rich domain with bifunctional effects on mRNA and protein levels: implications for control of transforming growth factor beta 1 expression

1993 ◽  
Vol 13 (6) ◽  
pp. 3588-3597
Author(s):  
L Scotto ◽  
R K Assoian
Keyword(s):  
Growth Hormone ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Mrna Levels ◽  
Tgf Beta ◽  
Rich Domain ◽  
Growth Factor Beta

Chimeric plasmids containing selected reporter coding domains and portions of the transforming growth factor beta 1 (TGF-beta 1) 3' untranslated region (UTR) were prepared and used to identify potential mechanisms involved in regulating the biosynthesis of TGF-beta 1. Transient transfections with core and chimeric constructs containing the chloramphenicol acetyltransferase (CAT) reporter showed that steady-state CAT mRNA levels were decreased two- to threefold in response to the TGF-beta 1 3' UTR. Interestingly, CAT activity was somewhat increased in the same transfectants. Thus, production of CAT protein per unit of mRNA was stimulated by the TGF-beta 1 3' UTR (approximately fourfold in three cell lines of distinct lineage). The translation-stimulatory effect of the TGF-beta 1 3' UTR suggested by these studies in vivo was confirmed in vitro by cell-free translation of core and chimeric transcripts containing the growth hormone coding domain. These studies showed that production of growth hormone was stimulated threefold by the TGF-beta 1 3' UTR. A deletion analysis in vivo indicated that the GC-rich domain in the TGF-beta 1 3' UTR was responsible for both the decrease in mRNA levels and stimulation of CAT activity-mRNA. We conclude that this GC-rich domain can have a bifunctional effect on overall protein expression. Moreover, the notable absence of this GC-rich domain in TGF-beta 2, TGF-beta 3, TGF-beta 4, and TGF-beta 5 indicates that expression of distinct TGF-beta family members can be differentially controlled in cells.

scholarly journals A GC-rich domain with bifunctional effects on mRNA and protein levels: implications for control of transforming growth factor beta 1 expression.

1993 ◽  
Vol 13 (6) ◽  
pp. 3588-3597 ◽  
Author(s):  
L Scotto ◽  
R K Assoian
Keyword(s):  
Growth Hormone ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Mrna Levels ◽  
Tgf Beta ◽  
Rich Domain ◽  
Growth Factor Beta

Chimeric plasmids containing selected reporter coding domains and portions of the transforming growth factor beta 1 (TGF-beta 1) 3' untranslated region (UTR) were prepared and used to identify potential mechanisms involved in regulating the biosynthesis of TGF-beta 1. Transient transfections with core and chimeric constructs containing the chloramphenicol acetyltransferase (CAT) reporter showed that steady-state CAT mRNA levels were decreased two- to threefold in response to the TGF-beta 1 3' UTR. Interestingly, CAT activity was somewhat increased in the same transfectants. Thus, production of CAT protein per unit of mRNA was stimulated by the TGF-beta 1 3' UTR (approximately fourfold in three cell lines of distinct lineage). The translation-stimulatory effect of the TGF-beta 1 3' UTR suggested by these studies in vivo was confirmed in vitro by cell-free translation of core and chimeric transcripts containing the growth hormone coding domain. These studies showed that production of growth hormone was stimulated threefold by the TGF-beta 1 3' UTR. A deletion analysis in vivo indicated that the GC-rich domain in the TGF-beta 1 3' UTR was responsible for both the decrease in mRNA levels and stimulation of CAT activity-mRNA. We conclude that this GC-rich domain can have a bifunctional effect on overall protein expression. Moreover, the notable absence of this GC-rich domain in TGF-beta 2, TGF-beta 3, TGF-beta 4, and TGF-beta 5 indicates that expression of distinct TGF-beta family members can be differentially controlled in cells.

scholarly journals Autoantibodies produced spontaneously by young 1pr mice carry transforming growth factor beta and suppress cytotoxic T lymphocyte responses.

1995 ◽  
Vol 181 (5) ◽  
pp. 1875-1880 ◽  
Author(s):  
D A Rowley ◽  
E T Becken ◽  
R M Stach
Keyword(s):  
Growth Factor ◽  
T Lymphocyte ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Cytotoxic T Lymphocyte ◽  
Tgf Beta ◽  
Growth Factor Beta ◽  
Ctl Responses

Young MRL/MPJ-lpr (lpr) mice 8-12 wk old challenged with alloantigen had significantly lower specific cytolytic T lymphocyte (CTL) responses than control MRL/MPJ +/+ mice. Serum from lpr mice compared with serum from ++ or normal C3H mice powerfully suppressed CTL responses in mixed lymphocyte cultures (MLC); absorbing lpr serum on protein G, adding antibody against transforming growth factor beta (TGF-beta) to cultures or dissociating immunoglobulin G (IgG) and TGF-beta before additions to cultures prevented suppression. Apparently autoantibody, similar to IgG produced by normal mice in response to immunization, carries TGF-beta which suppresses CTL responses in vivo and in vitro.

Growth-factor-related proteins that are inducers in early amphibian development may mediate similar steps in amniote (bird) embryogenesis

Development ◽  
1991 ◽  
Vol 111 (1) ◽  
pp. 197-212
Author(s):  
J. Cooke ◽  
A. Wong
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Normal Development ◽  
Tgf Beta ◽  
A Cell ◽  
Growth Factor Beta ◽  
Beta 2 ◽  
Related Proteins

Xenopus and murine activin A homologues (XTC-MIF and WEHI-MIF) and Xenopus and bovine basic fibroblast growth factor (bFGFs) are potent inducers of mesodermal and endodermal pathways of development in amphibian blastular animal cap cells. Porcine transforming growth factor beta 2 (TGF beta 2) is a weaker inducer in the same assay but human platelet-derived growth factor (PDGF) is inactive. We have assayed these factors for evidence of homologous effects in bird development. Unlike amphibians, bird embryos never exhibit a clean segregation of a cell layer that has a uniform specification when uninduced, and can be cultured in isolation as an assay after exposure to soluble factors. We have therefore performed less direct experiments, of three types. We have briefly cultured early chick epiblast cells with and without factors and then assayed their capacity to attach and spread upon fibronectin, in comparison with young streak and substreak hypoblast cells. We have asked whether similar microculture with factors alters the ability of quail epiblast cells to disrupt morphogenesis, and to integrate into the structure, of host chick blastoderms into which they are seeded. Finally, whole early chick blastoderms have been preincubated with or without factors for a brief period before setting them up to develop in vitro under circumstances usually permitting successful formation of axial pattern. Strong effects of the activin-like factors, of bFGF and of TGF beta 2 were seen in all three procedures, while PDGF was essentially inactive. In epiblast cells, effective factors at picomolar concentrations induced stable spreading upon fibronectin, and a capacity to adhere and spread upon basal epiblast surface and prevent morphogenesis in host blastoderms. Preincubation of whole early blastoderms with these factors led to characteristic deviation from normal development over the subsequent 24 h. We therefore suggest that peptides from the particular families that are active as inducers in amphibian blastula ectoderm may mediate homologous or closely related steps in respecification throughout vertebrates.

scholarly journals Bacterial cell wall-induced immunosuppression. Role of transforming growth factor beta.

1988 ◽  
Vol 168 (4) ◽  
pp. 1403-1417 ◽  
Author(s):  
S M Wahl ◽  
D A Hunt ◽  
G Bansal ◽  
N McCartney-Francis ◽  
L Ellingsworth ◽  
...  
Keyword(s):  
Cell Wall ◽  
T Cell ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Tgf Beta ◽  
Con A ◽  
Growth Factor Beta

Group A streptococcal cell wall (SCW)-injected rats exhibit a profound immunosuppression that persists for months after the initial intraperitoneal injection of SCW. The goal of this study was to determine the mechanisms for the suppressed T lymphocyte proliferative responses in this experimental model of chronic inflammation. When spleen cell preparations were depleted of adherent cells, restoration of T cell proliferative responses to Con A and PHA occurred, implicating adherent macrophages in the regulation of immunosuppression. Furthermore, macrophages from SCW-treated animals, when cocultured with normal spleen cells in the presence of Con A or PHA, effectively inhibited the proliferative response. Supernatants from suppressed spleen cell cultures were found to inhibit normal T cell mitogenesis. Taken together, these results implicated a soluble macrophage-derived suppressor factor in the down regulation of T cell proliferation after exposure to SCW in vivo. Subsequent in vitro studies to identify this suppressor molecule(s) revealed the activity to be indistinguishable from the polypeptide transforming growth factor beta (TGF-beta). Furthermore, TGF-beta was identified by immunolocalization within the spleens of SCW-injected animals. The cells within the spleen that stained positively for TGF-beta were phagocytic cells that had ingested, and were presumably activated by, the SCW. These studies document that TGF-beta, previously shown to be a potent immunosuppressive agent in vitro, also effectively inhibits immune function in chronic inflammatory lesions in vivo.

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