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1.
Oncogene ; 29(6): 831-44, 2010 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-19935703

RESUMEN

Transforming growth factor (TGF)-beta plays a dual role in tumorigenesis, switching from acting as a growth inhibitory tumor suppressor early in the process, to a tumor promoter in late-stage disease. Since TGF-beta's prometastatic role may be linked to its ability to induce tumor cell epithelial-to-mesenchymal transition (EMT), we explored TGF-beta's EMT-promoting pathways by analysing the transcriptome changes occurring in BRI-JM01 mammary tumor epithelial cells undergoing a TGF-beta-induced EMT. We found the clusterin gene to be the most highly upregulated throughout most of the TGF-beta time course, and showed that this results in an increase of the secreted form of clusterin. By monitoring several hallmark features of EMT, we demonstrated that antibodies targeting secreted clusterin inhibit the TGF-beta-induced EMT of BRI-JM01 cells, as well as the invasive phenotype of several other breast and prostate tumor cell lines (4T1, NMuMG, MDA-MB231LM2 and PC3), without affecting the proliferation of these cells. These results indicate that secreted clusterin is a functionally important EMT mediator that lies downstream within TGF-beta's EMT-promoting transcriptional cascade, but not within its growth-inhibitory pathways. To further investigate the role played by secreted clusterin in tumor metastasis, we assessed the effect of several anti-clusterin monoclonal antibodies in vivo using a 4T1 syngeneic mouse breast cancer model and found that these antibodies significantly reduce lung metastasis. Taken together, our results reveal a role for secreted clusterin as an important extracellular promoter of EMT, and suggest that antibodies targeting clusterin may inhibit tumor metastasis without reducing the beneficial growth inhibitory effects of TGF-beta.


Asunto(s)
Anticuerpos/uso terapéutico , Clusterina/antagonistas & inhibidores , Clusterina/genética , Células Epiteliales/patología , Espacio Extracelular/metabolismo , Perfilación de la Expresión Génica , Mesodermo/patología , Factor de Crecimiento Transformador beta/farmacología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Clusterina/inmunología , Clusterina/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Espacio Extracelular/efectos de los fármacos , Femenino , Humanos , Inmunoglobulina G/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Transcripción Genética
2.
Oncogene ; 28(7): 1005-15, 2009 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-19079339

RESUMEN

Transforming growth factor (TGF)-beta signaling is a potent modulator of the invasive and metastatic behavior of breast cancer cells. Indeed, breast tumor responsiveness to TGF-beta is important for the development of osteolytic bone metastases. However, the specific TGF-beta isoforms that promote breast cancer outgrowth in bone is unknown. We demonstrate that expression of a TGF-beta ligand trap, which neutralizes TGF-beta1 and TGF-beta3, in MDA-MB-231 breast cancer cells diminished their outgrowth in bone and reduced the severity of osteolytic lesion formation when compared with controls. We further show that a reduction or loss of TGF-beta1 expression within the bone microenvironment of TGF-beta1+/- and TGF-beta1-/- mice significantly reduced the incidence of breast tumor outgrowth compared with wild-type animals. Interestingly, those tumors capable of growing within the tibiae of TGF-beta1-deficient mice had upregulated expression of all three TGF-beta isoforms. Finally, breast cancer cells expressing the TGF-beta ligand trap showed a pronounced reduction in their ability to form osteolytic lesions when injected into the tibiae of TGF-beta1+/- mice. Thus, our studies show that both host- and tumor-derived TGF-beta expression plays a critical role during the establishment and outgrowth of breast cancer cells in bone.


Asunto(s)
Neoplasias Óseas/patología , Neoplasias de la Mama/patología , Osteólisis/prevención & control , Factor de Crecimiento Transformador beta1/fisiología , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/prevención & control , Proteínas de Unión al ADN/fisiología , Femenino , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Noqueados , Ratones Desnudos , Osteólisis/patología , Fosforilación , Isoformas de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta3/antagonistas & inhibidores , Factor de Crecimiento Transformador beta3/genética , Factor de Crecimiento Transformador beta3/metabolismo
3.
Cell Death Differ ; 10(11): 1234-9, 2003 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-14576775

RESUMEN

Smac/Diablo and HtrA2/Omi promote apoptosis by binding to and antagonizing IAP proteins, including the 'X chromosome-linked inhibitor of apoptosis' (XIAP). Here we show that caspase-mediated proteolysis of a limited subset of cell death substrates exposes functional Smac/Diablo-like N-termini after cleavage, which are able to bind to and antagonize XIAP. We propose that this mechanism may establish a feedforward sensitization of the apoptotic pathway and contribute to the functional redundancy of IAP antagonism. In addition, this may be particularly relevant in Alzheimer's disease since the caspase-generated C31 peptide, an established cytotoxin, acquires Smac/Diablo-like properties after apoptotic processing.


Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Fragmentos de Péptidos/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/farmacología , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/fisiología , Caspasa 3 , Línea Celular Tumoral , Citocromos c/metabolismo , Inhibidores Enzimáticos/farmacología , Retroalimentación Fisiológica/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mitocondrias/metabolismo , Proteínas Mitocondriales/fisiología , Fragmentos de Péptidos/farmacología , Estructura Terciaria de Proteína/fisiología , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Transducción de Señal/fisiología , Proteína Inhibidora de la Apoptosis Ligada a X
4.
J Cell Physiol ; 188(2): 211-22, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11424088

RESUMEN

One of the differences between fetal and adult skin healing is the unique ability of fetal wounds to heal without contracture and scar formation. Studies have shown that the ratio between the three isoforms of TGFbeta is different in adult and fetal wounds. Thus, we analyzed the capacity of adult and fetal human skin fibroblasts to contract collagen gels after stimulation with TGFbeta isoforms. In control medium, fetal fibroblasts had a contractile capacity similar to that of adult fibroblasts. However, the growth capacity of fetal fibroblasts was completely inhibited, in contrast to adult fibroblasts. When cells were treated with TGFbeta, fetal fibroblasts showed an inhibition of their contractile capacity whereas adult fibroblasts further contracted gels. The contractile response was similar for all isoforms of TGFbeta although TGFbeta3 always had the strongest effect. We considered that the regulation of cell contractile capacity by TGFbeta may be dependent on receptor expression for this cytokine, on myofibroblast differentiation of the cells, or in cell links with matrix. Since TGFbeta receptor analysis did not show differences in receptor affinity, we studied the expression of alpha-smooth muscle (SM) actin, a fibroblast contractile marker and of three integrins, the cell surface receptors specific of the attachment of the fibroblasts with collagen matrix. We observed that the expression of alpha-SM actin and alpha3 and beta1 integrin subunits was increased when TGFbeta was added to the medium of adult fibroblasts whereas the levels of the alpha1 and alpha2 subunits were unchanged. In contrast, fetal fibroblasts treated with TGFbeta showed a decrease of alpha1, alpha2, and beta1 integrin expression but no change in alpha3 integrin and in alpha-SM actin expression. These results indicate that intrinsic differences between fetal and adult fibroblasts might explain their opposite responses to TGFbeta stimuli. The variations in their alpha-SM actin and integrin expression patterns represent potentially important mechanisms used by fetal fibroblasts to regulate their response to cytokines, and likely contribute to the resultant differences in the quality of wound repair.


Asunto(s)
Feto/citología , Fibroblastos/citología , Piel/citología , Cicatrización de Heridas/fisiología , Actinas/análisis , Adulto , Factores de Edad , Antígenos CD/análisis , Western Blotting , Cicatriz/patología , Fibroblastos/química , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Humanos , Integrina alfa1 , Integrina alfa2 , Integrina alfa3 , Integrina beta1/análisis , Integrinas/análisis , Radioisótopos de Yodo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1 , Factor de Crecimiento Transformador beta2 , Factor de Crecimiento Transformador beta3
5.
J Biol Chem ; 276(29): 27237-45, 2001 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-11356827

RESUMEN

Transforming growth factor-beta (TGF-beta) internalization was studied by monitoring the uptake of (125)I-TGF-beta1 in Mv1Lu cells, which endogenously express TGF-beta receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 degrees C internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII. Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with (125)I- TGF-beta1 at 37 degrees C indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-beta1 at 4 degrees C for 2 h prior to switching the cells to 37 degrees C. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-beta1 bound to the cell surface at 37 degrees C and by a reduction in the labeling intensities of RI and RII in (125)I-TGF-beta1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-beta1 at 4 degrees C and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 degrees C, suggesting that prebinding promotes formation of stable RI.RII complexes that can signal independently of ligand.


Asunto(s)
Clatrina/metabolismo , Endocitosis , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular , Humanos , Ligandos , Microscopía Electrónica , Visón , Unión Proteica , Transducción de Señal
6.
J Biol Chem ; 276(32): 29632-43, 2001 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-11382746

RESUMEN

Mature transforming growth factor-beta (TGF-beta) is proteolytically derived from the C terminus of a precursor protein. Latency-associated protein (LAP), the N-terminal remnant of the TGF-beta precursor, is able to bind and neutralize TGF-beta. Mature TGF-beta exerts its activity by binding and complexing members of two subfamilies of receptors, the type I and II receptors. In addition to these signaling receptors, TGF-beta can also interact with an accessory receptor termed the type III receptor. Using a surface plasmon resonance-based biosensor (BIAcore), we determined the mechanisms of interaction of four binding proteins (LAP, the type II and III receptor ectodomains (EDs), and a type II receptor ED/Fc chimera) with three TGF-beta isoforms, and we quantified their related kinetic parameters. Using global fitting based on a numerical integration data analysis method, we demonstrated that LAP and the type II receptor/Fc chimera interacted with the TGF-beta isoforms with a 1:1 stoichiometry. In contrast, the type II ED interactions with TGF-beta were best fit by a kinetic model assuming the presence of two independent binding sites on the ligand molecule. We also showed that the type III ED bound two TGF-beta molecules. Further experiments revealed that LAP was able to block the interactions of TGF-beta with the two EDs, but that the two EDs did not compete or cooperate with each other. Together, these results strongly support the existence of a cell-surface complex consisting of one type III receptor, two TGF-beta molecules, and four type II receptors, prior to the recruitment of the type I receptor for signal transduction. Additionally, our results indicate that the apparent dissociation rate constants are more predictive of the neutralizing potency of these TGF-beta-binding proteins (LAP, the type II and III receptor EDs, and the type II receptor/Fc chimera) than the apparent equilibrium constants.


Asunto(s)
Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/metabolismo , Animales , Sitios de Unión , Células Epiteliales/metabolismo , Cinética , Pulmón/citología , Visón , Modelos Químicos , Modelos Teóricos , Unión Proteica , Isoformas de Proteínas , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína , Proteoglicanos/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Resonancia por Plasmón de Superficie , Termodinámica , Factores de Tiempo
7.
J Biol Chem ; 276(27): 24627-37, 2001 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-11323414

RESUMEN

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.


Asunto(s)
Receptores de Activinas Tipo I , Citoplasma/metabolismo , Proteoglicanos/fisiología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Transducción de Señal , Factor de Crecimiento Transformador beta/fisiología , Animales , Células COS , Modelos Moleculares , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Relación Estructura-Actividad
8.
Biochem Biophys Res Commun ; 275(2): 631-6, 2000 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-10964715

RESUMEN

EGFRvIII is the most common deletion variant of the epidermal growth factor receptor and is found in cancers of the brain, breast, ovary, and lung. The complete absence of the receptor in healthy tissues makes it an ideal tumor marker. We sought to design a peptide ligand against EGFRvIII for development as a diagnostic imaging agent. We used the concept of hydropathic complementarity to search for sequences whose amino acid sidechains display a reciprocal pattern of hydropathicity to those of the deletion junction of EGFRvIII. The resulting peptide (PEPHC1) was synthesized and tested for binding to EGFRvIII and EGFR. In in vitro assays, PEPHC1 bound the recombinant EGFRvIII extracellular domain or full-length EGFRvIII solubilized from cell membranes in preference to native EGFR. These results demonstrate the utility of hydropathic complementarity as a basis for the design of highly specific ligands that may prove useful as tumor-targeting agents.


Asunto(s)
Receptores ErbB/genética , Péptidos/química , Células 3T3 , Secuencia de Aminoácidos , Animales , ADN Complementario , Ratones , Datos de Secuencia Molecular , Péptidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
9.
Exp Cell Res ; 258(1): 121-34, 2000 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-10912794

RESUMEN

Transforming growth factor-beta (TGF-beta) signaling requires the functional interaction of two distinct receptors, type I (RI) and type II (RII), at the cell surface. Exposure of cells to TGF-beta results in receptor internalization and down-regulation (Zwaagstra et al., 1999, Exp. Cell Res. 252, 352362); however, little is known about the subsequent fate of RI or RII. In this study the cellular distribution of RI was examined in cells before and after treatment with ligand. RI was localized by immunocytochemistry and confocal microscopy using two polyclonal antisera directed against two different epitopes, one in the C-terminal region and one in the N-terminal region of the cytoplasmic domain. The majority of RI molecules in untreated MvlLu and A549 cells were found to be intracellular. Treatment of MvlLu and A549 cells with 100 pM TGF-beta1 for 24 h at 37 degrees C caused a redistribution of surface RI on MvlLu cells, as evidenced by surface RI aggregation. Unexpectedly, this TGF-beta1 treatment also caused redistribution and accumulation of intracellular RI in and around the nucleus for both MvlLu and A549 cells. Nuclear accumulation of RI was also promoted independently of ligand receptor activation by treatment of MvlLu cells with olomoucine, an agent that results in growth arrest. The capacity of RI to localize in the nucleus was confirmed by microscopic examination of 293 cells transiently expressing RI fused to green fluorescent protein (RI-GFP). Olomoucine treatment of these cells resulted in the movement of RI-GFP into the nucleus. Our results indicate that growth arrest alters intracellular transport/routing of RI and may indicate that RI functions not only at the cell surface but inside the cell as well.


Asunto(s)
Receptores de Activinas Tipo I , Ciclo Celular/fisiología , Núcleo Celular/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular , Membrana Celular/fisiología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Retículo Endoplásmico/fisiología , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Aparato de Golgi/fisiología , Proteínas Fluorescentes Verdes , Humanos , Cinetina , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Neoplasias Pulmonares , Visón , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/genética , Purinas/farmacología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/análisis , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes de Fusión/biosíntesis , Mucosa Respiratoria , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transfección , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales Cultivadas
10.
J Biol Chem ; 275(35): 26748-53, 2000 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-10840042

RESUMEN

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) are mitogenic hormones that exert their activity primarily by binding to the EGF receptor, also known as ErbB-1. We have recently characterized a set of EGF/TGFalpha chimeric molecules with similar high affinity for ErbB-1 as EGF and TGFalpha and shown that three of these chimeras induce mitogenic cell stimulation at already a 10-fold lower concentration than their wild-type counterparts (Lenferink, A. E., Kramer, R. H., van Vugt, M. J., Königswieser, M., DiFiore, P. P., van Zoelen, E. J., and van de Poll, M. L. (1997) Biochem. J. 327, 859-865). In the present study we show that these so-called superagonistic chimeras do not differ from EGF and TGFalpha in their ability to induce ErbB-1 tyrosine phosphorylation but are considerably more potent in activation of mitogen-activated protein kinase phosphorylation. Direct cell binding studies and analysis of ligand-receptor interaction by surface plasmon resonance measurements revealed that both the association rate constant (k(on)) and the dissociation rate constant (k(off)) of these superagonists is 3-5-fold higher in comparison with the wild-type ligands and nonsuperagonistic chimeras. These data indicate that the dynamic on and off rate constants for receptor binding may be more specific parameters for determining the mitogenic activity of peptide hormones than their constants for equilibrium receptor binding.


Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Células 3T3 , Animales , Factor de Crecimiento Epidérmico/metabolismo , Humanos , Cinética , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie
11.
Exp Cell Res ; 252(2): 352-62, 1999 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-10527625

RESUMEN

The types I, II, and III receptors (RI, RII, RIII) for transforming growth factor-beta (TGF-beta) become down-regulated in response to ligand, presumably via their internalization from the cell surface. This report examines the down-regulation of full-length RI, RII, and RIII in cells endogenously or transiently expressing these receptors. Down-regulation occurred rapidly (within 2 h after TGF-beta1 treatment at 37 degrees C) and showed a dose response, between 10 and 200 pM TGF-beta1, in cells expressing RI, RII, and RIII (Mv1lu and A549 cells). A comparison between Mv1Lu and mutant cell derivatives R-1B (lacking RI) or DR-26 (lacking RII) indicated that all three receptors were necessary for efficient down-regulation. Down-regulation experiments, utilizing TGF-beta-treated 293 cells transiently expressing different combinations of these receptors indicated that neither RII or RIII were down-regulated when expressed alone and that RI was required for maximal down-regulation of RII. RII and RIII were partially down-regulated when these receptors were coexpressed in the absence of RI (in R-1B and 293 cells). Surprisingly, TGF-beta receptors were partially down-regulated in Mv1Lu, A549, and 293 cells treated with TGF-beta1 at 4 degrees C. Microscopic examination of 293 cells coexpressing RI fused to green fluorescent protein (RI-GFP) and RII indicated that, after treatment with TGF-beta1 at 4 degrees C, RI-GFP formed aggregates at the cell surface at this temperature. RI-GFP was not detected at the surface of these cells after TGF-beta1 treatment at 37 degrees C. Our results suggest a two phase mechanism for TGF-beta1 receptor down-regulation involving receptor modulation (aggregation) at the cell surface and internalization.


Asunto(s)
Membrana Celular/fisiología , Regulación hacia Abajo , Agregación de Receptores/fisiología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Animales , Línea Celular
12.
Growth Factors ; 17(1): 63-73, 1999.
Artículo en Inglés | MEDLINE | ID: mdl-10495963

RESUMEN

Transforming growth factor-beta (TGF-beta) is a multi-functional regulator of cell growth and differentiation. Three distinct isoforms of TGF-beta exist having similar, but not identical actions. TGF-beta 1, but not TGF-beta 2, binds to T beta RII and also to endoglin, a cell surface protein abundant on endothelial cells. In contrast, the affinity constant of TGF-beta 2 for alpha 2-macroglobulin is 10-fold greater than that of TGF-beta 1. TGF-beta 2 also binds better than TGF-beta 1 to a glycosyl phosphatidylinositol (GPI)-linked binding protein expressed on vascular endothelial cells. Using chimeric TGF-beta molecules, in which selected regions of TGF-beta 1 have been exchanged for the corresponding region of TGF-beta 2, we demonstrate here that amino acids 92-95 or 94-98 of TGF-beta determine isoform specific binding to endoglin. In contrast, exchange of only amino acids 95 and 98 did not alter TGF-beta specificity. Isoform specific binding to a GPI-linked protein on EJG endothelial cells was modulated by a region containing amino acids 40-68, although exchange of only amino acids 40-47 did not confer isoform specific binding. Significantly, the 92-98 region also modulates binding of TGF-beta to the type II receptor whereas isoform specific binding to alpha 2-macroglobulin requires concerted exchange of amino acids 45 and 47. Taken together, these results show that at least three different functional domains are important modulators of TGF-beta interaction with binding proteins and receptors.


Asunto(s)
Endotelio Vascular/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , alfa-Macroglobulinas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Células Cultivadas , Cricetinae , Endotelio Vascular/citología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína , Receptor Tipo II de Factor de Crecimiento Transformador beta , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Factor de Crecimiento Transformador beta/química
13.
FEBS Lett ; 456(1): 79-84, 1999 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-10452534

RESUMEN

Binding surfaces of the type II transforming growth factor (TGF)-beta receptor extracellular domain (TbetaRII-ECD) are mapped by combining scanning-deletion mutagenesis results with knowledge-based modeling of the ectodomain structure. Of the 17 deletion mutants produced within the core binding domain of TbetaRII-ECD, only three retained binding to TGF-beta. Comparative modeling based on the crystal structure of the activin type II receptor extracellular domain (ActRII-ECD) indicates that the TbetaRII mutants which retain TGF-beta binding are deleted in some of the loops connecting the beta-strands in the TbetaRII-ECD model. Interpretation of the mutagenesis data within the structural framework of the ectodomain model allows for the prediction of potential binding sites at the surface of TbetaRII-ECD.


Asunto(s)
Simulación por Computador , Modelos Moleculares , Mutagénesis , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Secuencia de Aminoácidos , Inteligencia Artificial , Sitios de Unión , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Glicosilación , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Eliminación de Secuencia , Transfección
14.
Exp Cell Res ; 238(1): 283-93, 1998 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-9457082

RESUMEN

Myofibroblasts play an important role in normal wound healing. They are present transiently during tissue repair. Their differentiation from fibroblasts and their role in granulation tissues are most likely to be modulated by cytokines. As these cells are derived from normal fibroblasts, their responses to cytokines are assumed to be similar. Until now, however, the difficulties in obtaining and maintaining normal human wound healing myofibroblasts in vitro have hampered comparison. The present study was designed to determine the effect of TGF-beta 1 and IFN-gamma, two cytokines known to modulate fibroblast morphology, on wound healing myofibroblasts and to compare it to fibroblasts. Morphological and phenotypic changes were followed by light and electron microscopy (stress fibers) and immunofluorescence cytochemistry (alpha-SM actin). Functional parameters such as the capacity to synthesize collagen and collagen gel contraction were studied. Both cytokines induced a strong modification of growth rate and phenotypic and morphological parameters in fibroblasts whereas collagen synthesis was slightly changed. Furthermore, TGF-beta 1 increased contractile capacity of fibroblasts whereas IFN-gamma greatly decreased it. In myofibroblasts, TGF-beta 1 and IFN-gamma did not induce any variation of morphology or growth rate. Interestingly, a strong modulation of functional parameters was observed: collagen synthesis was highly modified and, as for fibroblasts, the contractile capacity was altered. However, inhibition of contraction by IFN-gamma was irreversible in myofibroblasts but not in fibroblasts. These results suggest that fibroblastic cells show modulated responses to cytokines according to their stage of differentiation during wound healing.


Asunto(s)
Interferón gamma/farmacología , Músculos/fisiología , Piel/citología , Factor de Crecimiento Transformador beta/farmacología , Cicatrización de Heridas/efectos de los fármacos , Adulto , Diferenciación Celular , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Colágeno/biosíntesis , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Fibroblastos/ultraestructura , Humanos , Músculos/citología , Músculos/efectos de los fármacos , Biosíntesis de Proteínas , Proteínas Recombinantes/farmacología , Piel/efectos de los fármacos , Fenómenos Fisiológicos de la Piel/efectos de los fármacos
15.
Biochem Biophys Res Commun ; 220(2): 289-93, 1996 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-8645298

RESUMEN

There are three main types of receptors for TGF-beta termed receptor type I, type II and type III. TGF-beta receptor type II has a crucial role in the cell's responsiveness to TGF-beta as it is mandatory for the TGF-beta binding to the signaling complex (receptor type I and type II). Here we have used a scanning-deletion mutagenesis approach to determine the core binding domain of the extracellular domain of receptor type II that is required for interaction with TGF-beta. Deletions of three amino acids were systematically introduced at intervals of five amino acids in order to scan the N- and C-terminus of the extracellular domain of the receptor. We find that the N-terminal region which is devoid of cysteine residues is not critical for ligand binding. Similarly, the C-terminal region, i.e., the amino acids flanking the transmembrane domain, are dispensable for binding. These results suggest that the central 100 amino acid span that is rich in cysteine residues is the core binding domain for TGF-beta.


Asunto(s)
Eliminación de Gen , Mutagénesis , Receptores de Factores de Crecimiento Transformadores beta/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular , Datos de Secuencia Molecular , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Relación Estructura-Actividad , Transfección
16.
FEBS Lett ; 377(3): 368-72, 1995 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-8549757

RESUMEN

There are two TGF-beta binding subdomains in the extracellular domain of receptor type III (proximal and distal in relation to the transmembrane domain). Here we present an extension of our analysis of the proximal binding site of receptor type III. Due to the original deletion mutagenesis strategy, our proximal binding site contained 19 amino acids from the N-terminal part of the receptor. By deleting these, we demonstrated that they did not contribute to the binding ability of the proximal binding site. We also produced a soluble, secreted form of the proximal binding site and demonstrated that it was able to bind TGF-beta. Finally, we analyzed the role of the three asparagine residues (580, 591, 595) that are located in the region of the receptor that is necessary for expression of a functional proximal binding site, and found that mutation of these residues individually to alanine did not affect ligand binding.


Asunto(s)
Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Membrana Celular/metabolismo , Células Cultivadas , Análisis Mutacional de ADN , Glicosilación , Ligandos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteoglicanos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Eliminación de Secuencia , Solubilidad , Relación Estructura-Actividad
17.
J Cell Physiol ; 165(1): 201-11, 1995 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-7559802

RESUMEN

Recent studies of whole animal responses have defined a role for circulating TGF-beta in the preservation and stabilization of microvascular endothelial function (Lefer et al. [1993] Proc. Natl. Acad. Sci. U.S.A., 90:1018-1022; Pfister et al. [1992] J. Exp. Med., 176:265-269). In order to determine which TGF-beta receptor types are responsible for this endothelial cell responsiveness, we used an affinity-labeling technique with 125I-TGF-beta 1 and -beta 2 to characterize TGF-beta receptors on five different endothelial cell cultures: early passage bovine lung and rat epididymal fat pad microvascular endothelial cells (BLMEC and REEC), established endothelial cell lines from bovine adrenal medulla capillaries (EJG), fetal bovine heart (FBHE), and bovine pulmonary artery (CPAE). Since it is known that endothelial cells from different parts of the vasculature vary with respect to cell surface antigen expression (McCarthy et al. [1991] Trends Pharmacol. Sci., 12:462-467; Augustin et al. [1994] Bioessays, 16:901-906), it is important to compare TGF-beta receptor expression on microvascular and macrovascular endothelial cells. We observed 85 kDa and 200-400 kDa labeled receptor bands and analyzed their relationship to the cloned Type II and III receptors using peptide antibodies. We used dithiothreitol and phosphoinositol-phospholipase C pretreatments to establish whether the 65 kDa labeled band which we observed corresponded to the Type I receptor or a glycophosphotidylinositol-linked binding protein. The results demonstrated that microvascular but not macrovascular endothelial cells express high levels of the Type III receptor. This differential expression of the Type III receptor indicates that distinct anatomical segments of the vasculature have distinct TGF-beta receptor profiles. The presence of the Type III receptor on micro- but not macrovascular endothelial cells may account for the reportedly different potency of TGF-beta 1 and TGF-beta 2 on these two endothelial cell types. Analysis of the 85 kDa and 65 kDa affinity-labeled bands revealed that all the endothelial cells express the Type II receptor and a band consistent with the presence of a dithiothreitol-sensitive Type I receptor. Two isoform-specific phosphoinositol-phospholipase C releasable TGF-beta binding proteins were also detected: a 60 kDa protein on one micro- (EJG) and one macro- (FBHE) vascular endothelial cell line and a 150/180 kDa protein on the macrovascular cell lines (FBHE and CPAE). These studies emphasize the heterogeneous nature of endothelial cells and underline the importance of using microvascular endothelial cells when examining TGF-beta responses related to microvascular function.


Asunto(s)
Endotelio Vascular/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Médula Suprarrenal/irrigación sanguínea , Animales , Bovinos , Epidídimo/irrigación sanguínea , Masculino
19.
Mol Cell Endocrinol ; 111(1): 57-66, 1995 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-7649353

RESUMEN

In the present study, we have characterized the cell surface receptors for transforming growth factor-beta (TGF-beta) on monolayer cultures of stromal cells prepared from human endometrial biopsies, and on a human endometrial epithelial cell line (RL95-2) using affinity cross-link labeling techniques. On the stromal cells, five TGF-beta binding proteins were identified. Analysis of the sensitivity of these proteins to dithiothreitol and phosphatidylinositol-specific phospholipase C, together with results from immunoprecipitations with antibodies against the type II and III TGF-beta receptors, confirmed that three of these binding proteins correspond to the cloned type I, II, and III TGF-beta receptors. The other two binding proteins observed exhibit the characteristics of isoform-specific GPI-anchored TGF-beta binding proteins. On RL95-2 cells, three TGF-beta binding proteins, corresponding to the type I, II, and III TGF-beta receptors, were identified. The receptors which we have characterized on endometrial cells are responsive to physiological concentrations of TGF-beta as demonstrated by the effect of TGF-beta on endometrial cell proliferation. Accordingly, these receptors have the potential to respond to the TGF-beta isoforms which have recently been detected in the endometrium in an autocrine and/or paracrine manner.


Asunto(s)
Endometrio/química , Glicosilfosfatidilinositoles/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/análisis , Marcadores de Afinidad , División Celular , Células Cultivadas , Reactivos de Enlaces Cruzados , Ditiotreitol/farmacología , Endometrio/citología , Endometrio/metabolismo , Epitelio/química , Epitelio/metabolismo , Femenino , Humanos , Técnicas de Inmunoadsorción , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Hidrolasas Diéster Fosfóricas/farmacología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Células del Estroma/química , Células del Estroma/metabolismo , Factor de Crecimiento Transformador beta/farmacología
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