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R Derynck

Publications and source records attributed to R Derynck.

At least 91 records · Page 5Linked to original sources
PubMed1993

Extracellular matrix regulates expression of the TGF-beta 1 gene.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell proliferation and modulates the interactions of cells with their extracellular matrix (ECM), in part by inducing the synthesis of various ECM proteins. Three different isoforms of TGF-beta are synthesized in a defined pattern in specific cell populations in vivo. In the specific case of TGF-beta 1, this well-defined and limited expression stands in sharp contrast to its synthesis by virtually all cells in culture. Using mammary epithelial cells as a model system, we evaluated the substratum dependence of the expression of TGF-beta 1. The level of TGF-beta 1 expression is high in cells on plastic, but is strongly downregulated when cells are cultured on a reconstituted basement membrane matrix. In contrast, TGF-beta 2 mRNA levels in cells on either substratum remain unchanged. Using the chloramphenicol acetyl transferase gene as reporter gene under the control of the TGF-beta 1 promoter, we show that transcription from this promoter is suppressed when the cells are in contact with either endogenously synthesized or exogenously administered basement membrane. TGF-beta 1 promoter activity is strongly induced by the absence of basement membrane, i.e., by direct contact of the cells with plastic. This modulation of transcription from the TGF-beta 1 promoter occurs in the absence of lactogenic hormones which allow full differentiation. Our results thus indicate that basement membrane is an important regulator of TGF-beta 1 synthesis, and explain why most cells in culture on plastic express TGF-beta 1 in contrast with the more restricted TGF-beta 1 synthesis in vivo. We propose that there is a feedback loop whereby TGF-beta 1-induced synthesis of basement membrane components is repressed once a functional basement membrane is present. Finally, these results together with our current knowledge of regulation of TGF-beta 1 and TGF-beta 2 synthesis, suggest that, in vivo, TGF-beta 1 may play a major role in regulating the ECM synthesis and the cell-ECM interactions, whereas TGF-beta 2 may be more important in morphogenetic processes.

Animals↗
PubMed1993

Modulation of expression and cell surface binding of members of the transforming growth factor-beta superfamily during retinoic acid-induced osteoblastic differentiation of multipotential mesenchymal cells.

We have evaluated the effects of retinoic acid as a differentiating agent on two pluripotential mesenchymal stem cell lines, the mouse cell line C3H-10T1/2 (10T1/2), which has the capacity to differentiate in vitro into myoblasts, adipocytes, chondrocytes, and osteoblasts, and the rat cell line ROB-C26 (C26), which can, in culture, give rise to adipocytes, myoblasts, and osteoblasts. Retinoic acid (10(-6) M) reduces the incidence of myoblast and adipocyte formation and induces or increases alkaline phosphatase expression and responsiveness to PTH, two indicators of the osteoblastic phenotype. Because transforming growth factor-beta (TGF beta) superfamily members, including the different TGF beta isoforms and the bone morphogenetic proteins (BMPs), are thought to play a role in regulating bone and cartilage formation, and because exogenous TGF beta and BMP-2 have already been found to modulate osteoblastic differentiation of C26 and 10T1/2 cells, we evaluated the endogenous expression of these factors in both cell lines cultured in the presence or absence of retinoic acid. Our data show that C26 and 10T1/2 cells constitutively express a broad spectrum of TGF beta superfamily members. However, this pattern of expression is dramatically altered in response to retinoic acid. Specifically, expression of TGF beta 1 and especially TGF beta 2 is strongly increased, whereas TGF beta 3 expression is down-regulated. These changes are accompanied by a striking decline in TGF beta receptor expression levels at the cell surface. Furthermore, BMP-2 and -4 expression are decreased after treatment with retinoic acid, whereas vgr-1/BMP-6 expression is induced in C26 cells, but decreased in 10T1/2 cells. These results clearly show a dynamic changing pattern of TGF beta superfamily expression consequent to the induction of osteogenic differentiation and provide the first indication that TGF beta receptor down-regulation may be an essential part of this differentiation process. These data also establish the C26 and 10T1/2 cell lines as convenient in vitro model systems for exploring the autoregulation of osteogenic differentiation by members of the TGF beta superfamily.

Animals↗
PubMed1993

Autonomous growth of human keratinocytes requires epidermal growth factor receptor occupancy.

In the studies reported here, we demonstrate that transforming growth factor alpha (TGF-alpha) or epidermal growth factor (EGF) is required for the establishment of small colonies of human keratinocytes at clonal densities, but once small (10-15 cells) colonies have formed, the continued growth of these colonies can proceed in the absence of exogenous TGF-alpha or EGF. Equivalent receptor-binding concentrations of TGF-alpha and EGF were equipotent in stimulating colony formation. We also demonstrate that the growth of keratinocytes at high densities proceeds in the absence of exogenous peptide growth factors or hormones. The expression of TGF-alpha mRNA and protein is regulated by both cell density and the presence of exogenous growth factors. The addition of an antibody which blocks the mitogenic effect of mature TGF-alpha had no effect on the autocrine/paracrine growth of these cells at either density. However, monoclonal antibodies which antagonize ligand activation of the EGF receptor inhibit the autonomous proliferation of keratinocytes at high density and abrogate the exogenous TGF-alpha/EGF-independent expansion of colonies at clonal density. The results of these experiments are among the first evidence to demonstrate that normal human epithelial cells in culture exhibit autocrine/paracrine-mediated proliferation. Exogenous growth factors initiate colonies of human keratinocytes that become self-perpetuating in culture. Keratinocytes regulate production of the mitogenic ligand, TGF-alpha, through a density-dependent mechanism, and cell density stringently controls proliferation.

Amphiregulin↗
PubMed1992

Altered metabolic and adhesive properties and increased tumorigenesis associated with increased expression of transforming growth factor beta 1.

Transforming growth factor-beta (TGF-beta) is a potent mediator of cell proliferation and extracellular matrix formation, depending on the cell type and the physiological conditions. TGF-beta is usually secreted in a "latent" complex that needs activation before it can exert its effects. Several observations correlate increased expression of TGF-beta 1 with tumorigenesis. To evaluate the physiological relevance of increased TGF-beta 1 synthesis in tumor cells we established cell clones overexpressing TGF-beta 1 and observed the resulting physiological changes in TGF-beta overproducing cells in vitro and in vivo. As a model system we used the human E1A-transformed 293 tumor cells, which are insensitive to the direct growth modulatory effects of TGF-beta. The selection of this cell line allows an assessment of physiological alterations independent of TGF-beta induced proliferative changes. The use of two TGF-beta 1 expression vectors containing either the natural or a modified TGF-beta 1 precursor cDNA permitted the establishment of separate 293 cell lines overexpressing latent or active TGF-beta. Comparison of the resulting changes in glycolytic rate, adhesiveness and integrin and plasminogen activator expression established that, in vitro, both types of clones behaved similarly, indicating that expression of latent TGF-beta induces autocrine changes in the tumor cells and thus suggesting that some level of cell-associated activation occurs. TGF-beta overexpression resulted in an increased metabolic rate due to enhanced glycolysis, a property long associated with tumor cells. This increased glycolysis was not associated with altered proliferation. Cells overexpressing TGF-beta also displayed enhanced fibronectin mRNA and plasminogen activator synthesis and increased adhesiveness in vitro. They showed enhanced survival when plated sparsely on plastic in the absence of serum, and attached more readily to laminin. In addition, synthesis of several beta 1 integrins, in particular the alpha 1/beta 1, alpha 2/beta 1, and alpha 3/beta 1, all of which recognize laminin, were enhanced. Finally, cells overexpressing active TGF-beta, but not latent TGF-beta, also showed increased tumorigenicity in nude mice. Thus, an increase in endogenous TGF-beta synthesis confers several proliferation-independent phenotypic changes which may be of significance for the survival of the tumor cell inoculum or its subsequent growth, and for tumor formation and development. In the case of cells expressing active TGF-beta, the release of active TGF-beta into the vicinity of the tumor cells may also result in a more hospitable environment for tumor growth.

Animals↗
PubMed1992

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

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

Biological Transport, Active↗
PubMed1991

Epidermal growth factor and transforming growth factor-alpha: differential intracellular routing and processing of ligand-receptor complexes.

Two structurally related but different polypeptide growth factors, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), exert their activities after interaction with a common cell-surface EGF/TGF-alpha-receptor. Comparative studies of the effects of both ligands have established that TGF-alpha is more potent than EGF in a variety of biological systems. This observation is not explained by differences in affinities of the ligands for the receptor, because the affinity-constants of both factors are very similar. We have compared the intracellular processing of ligand-receptor complexes using either EGF or TGF-alpha in two different cell systems. We found that TGF-alpha dissociates from the EGF/TGF-alpha-receptor at much higher pH than EGF, which may reflect the substantial difference in the calculated isoelectric points. After internalization, the intracellular TGF-alpha is more rapidly cleared than EGF, and a substantial portion of the released TGF-alpha represents undegraded TGF-alpha in contrast to the mostly degraded EGF. In addition, TGF-alpha did not induce a complete down-regulation of cell surface receptors, as observed with EGF, which is at least in part responsible for a much sooner recovery of the ligand-binding ability after down-regulation, in the case of TGF-alpha. These differences in processing of the ligand-receptor complexes may explain why TGF-alpha exerts quantitatively higher activities than EGF.

Affinity Labels↗
PubMed1991

Inhibition of translation of transforming growth factor-beta 3 mRNA by its 5' untranslated region.

We have cloned and sequenced the 5' untranslated region of the transforming growth factor-beta 3 (TGF-beta 3) mRNA as well as the adjacent genomic sequence. S1 nuclease analysis identified a single transcription start site. We have thus determined that the 5' untranslated region is about 1.1 kb long and contains 11 open reading frames. In vitro translation of the TGF-beta 3 precursor coding sequence was markedly inhibited by the presence of the 5' untranslated region. Similarly, when the 5' untranslated region of TGF-beta 3 was introduced upstream of the coding sequence of chloramphenicol acetyltransferase, in vitro translation was inhibited. Furthermore, upon transfection into 293 cells, chloramphenicol acetyltransferase expression was inhibited by the 5' untranslated region of TGF-beta 3. The degree of translational inhibition was inversely proportional to the amount of transfected DNA. Mutation analysis implicated multiple segments of the 5' untranslated region as contributing to the inhibitory effect. Deletion of much of the 5'-most 640 nucleotides, including 8 of the 11 upstream ATGs, relieved much but not all of the inhibitory influence of the 5' untranslated region of TGF-beta 3 mRNA. The two upstream open reading frames closest to the initiator codon for the TGF-beta 3 coding sequence also decreased translational efficiency, since mutation of either ATG resulted in increased translation. Transfection results with T47-D cells, a cell line which expresses TGF-beta 3 mRNA, were similar to those obtained with the 293 cell line. Thus, TGF-beta 3 mRNA is a recent example of an expanding group of growth-related mRNAs in which the 5' untranslated region contains upstream open reading frames and other sequences which inhibit translation.

Amino Acid Sequence↗
PubMed1991

Differential binding of transforming growth factor-beta 1, -beta 2, and -beta 3 by fibroblasts and epithelial cells measured by affinity cross-linking of cell surface receptors.

A murine fibroblast cell line (AKR-2B clone 84A) and an epithelial cell line (BALB/MK) were compared for their ability to bind different transforming growth factor-beta (TGF beta) species. The results of competitive binding assays indicated that the epithelial cells had a higher affinity for TGF beta than the fibroblasts. This difference may be the basis for the sensitivity of epithelial cells to much lower concentrations of TGF beta than fibroblasts. Affinity cross-linking studies showed that both cell types express the three cell surface TGF beta-binding molecules that have been previously described for a variety of cell types. The complexity of these cell surface binding proteins was further evaluated using all possible combinations of radiolabeled ligands in competition with each of the three unlabeled TGF beta species. Differences in the ability of specific TGF beta types to compete with radiolabeled TGF beta 2 for binding to the type I and II receptors were observed, with TGF beta 1 being more potent for epithelial cells, and TGF beta 2 being more potent for fibroblasts. In addition, a difference in the ability of different TGF beta species to compete the [125I]TGF beta 3 from epithelial cell surface receptors was apparent. TGF beta 2 was not able to compete with [125I]TGF beta 3 for binding to the type II receptor at any concentration tested, while TGF beta 1 and TGF beta 3 were about equally potent in competition for this receptor type. These differences in cell surface receptor binding of structurally and biologically similar molecules may reflect different functions for these molecules.

Animals↗
PubMed1991

Effects of MGSA/GRO alpha on melanocyte transformation.

In the work described here we demonstrate that the clonal cell line Mel-a-6, produced by transfection of mouse Melan-a cells with human MGSA, had an increased ability to form large colonies in soft agar and increased ability to form tumors when injected into nude mice as compared to cells transfected with the neomycin resistance gene alone. This effect appeared to be dependent on the levels of MGSA produced since another transfected clone, Mel-a-l, produced only a low level of MGSA transgene mRNA, formed only minimal large colonies in soft agar and had a tumorigenic rate equal to that of neomycin resistant controls. The histology of the Mel-a-6 tumors is compatible with features normally exhibited by melanoma tumors. The cells do not stain for melanin, and they are positive for the neural crest marker protein S-100 as well as the HMB 45 melanoma specific antigen. Immunohistochemical studies revealed expression of the human MGSA in tumor cells from tissues, excised from animals injected with cells from clone Mel-a-6. Whereas DNA ploidy analysis suggests that in vitro the Mel-a parent cell line, control Mel-a-neo, Mel-a-1 and Mel-a-6 cells show no evidence of aneuploidy, the nuclei isolated from the tumors from animals injected with Mel-a-6 do exhibit aneuploidy. These data suggest that over-expression of MGSA in immortalized melanocytes contributes to transformation.

Aneuploidy↗
PubMed1990

Macrophage deactivating factor and transforming growth factors-beta 1 -beta 2 and -beta 3 inhibit induction of macrophage nitrogen oxide synthesis by IFN-gamma.

Macrophage deactivating factor (MDF) and three members of the transforming growth factor-beta family (TGF-beta 1, -beta 2, and -beta 3) blocked the ability of IFN-gamma to induce release of reactive nitrogen intermediates from mouse peritoneal macrophages. Raising the concentration of IFN-gamma did not diminish the potency of the inhibitors (50% inhibition by approximately 7 nM MDF, 2 pM TGF-beta 1, 4 pM TGF-beta 2, and 8 pM TGF-beta 3). These inhibitors partially blocked induction of nitrite release in macrophages activated with the combination of IFN-gamma plus TNF-alpha, but were incapable of inhibiting when macrophages were activated by the combination of IFN-gamma plus LPS. MDF and TGF-beta 1, -beta 2, and -beta 3 inhibited IFN-gamma-induced nitrite release only if present during the induction phase; once IFN-gamma-nitrite release had commenced, addition of the same cytokines was no longer inhibitory. Maximum inhibition of synthesis over a 48-h period required that the inhibitors be present during the first 3 h of induction. Thus, cytokines can suppress as well as induce macrophage synthesis of reactive nitrogen intermediates, products with cytotoxic, antimicrobial, and vasodilatory properties.

Animals↗
PubMed1990

Differential regulation of expression of three transforming growth factor beta species in human breast cancer cell lines by estradiol.

Transforming growth factor (TGF)-beta is a potent regulator of many cell functions and a growth inhibitor for mammary epithelial cells. We now know of three highly homologous members of the human TGF-beta gene family. We have studied the expression of TGF-beta 1, -beta 2, and -beta 3 mRNA in four human breast cancer cell lines. Using the RNase protection assay, we have detected mRNA expression of TGF-beta 1, -beta 2, and -beta 3 by T-47D cells, TGF-beta 1 and -beta 3 by ZR-75-1 cells, and TGF-beta 1 by MCF-7 cells. Treatment of these estrogen receptor-positive cells with 10 nM estradiol for 48 h resulted in decreased mRNA levels of TGF-beta 2 and -beta 3 but did not affect mRNA levels of TGF-beta 1. Expression of TGF-beta 1 and -beta 2 mRNA by an estrogen receptor-negative cell line, MDA-MB-231, was not changed by estradiol treatment. Treatment of cells with the antiestrogen tamoxifen (1 microM) did not significantly alter mRNA levels for any of the three TGF-beta species. We have further determined that estradiol treatment of T-47D was associated with diminished secretion of TGF-beta into the medium. Both TGF-beta 1 and -beta 2 inhibited the proliferation of MCF-7 cells, and neither protein affected the growth of T-47D cells. TGF-beta 1 was at least 10-fold more potent than TGF-beta 2 at inhibiting the growth of MCF-7 cells.

Breast Neoplasms↗
PubMed1990

Transforming growth factor-alpha.

In summary, although TGF-alpha was initially found in tumors, a number of later studies, some of them from the author's laboratory, have shown that TGF-alpha should no longer be considered a tumor associated growth factor. Rather, TGF-alpha is a normal physiological ligand for the EGF receptor. Table 2 lists some of the normal cellular sources of TGF-alpha. Our list is incomplete, but we know that TGF-alpha is made in keratinocytes and a number of epithelial cells, including gut and breast epithelial cells. It seems very likely that TGF-alpha is a major growth factor secreted by cells of epithelial origin. Zena Werb's and Russell Ross's groups have shown that activated macrophages make TGF-alpha. We have shown that brain makes TGF-alpha and Jeff Kudlow has found TGF-alpha made in the pituitary. Data from several sources, including David Lee, the author's laboratory, and Zena Werb's laboratory has shown that TGF-alpha is made during embryonic development. Therefore, it is now important to look at TGF-alpha in its normal physiological context. Finally, it should be stressed that, as was mentioned above, TGF-alpha is not necessarily a secreted growth factor 50 amino acids long. There is quite a bit of processing of the larger precursor that may or may not take place. This processing, which determines the ultimate size and location of the molecule, is also likely to influence its physiological action.

Amino Acid Sequence↗
PubMed1990

Transforming growth factor-beta. A family of growth regulatory peptides.

TGF beta, initially described as a factor that stimulates rodent fibroblast cell lines to proliferate in soft agar, has been shown to be active in several biological processes. The in vitro biological activities of the closely related molecules, TGF beta 1, TGF beta 2, and TGF beta 3, are comparable. Northern blot analyses of adult and embryonic tissues have shown the TGF beta mRNAs to be expressed in vivo, yet their patterns of expression appear somewhat different. In addition, even when all the TGF beta s are expressed in a tissue at the same time, the expression observed has been shown to be localized to different cells within the organ in some cases. This suggests that perhaps these molecules may have activities or functions in mice that are not apparent in vitro. Several members of the TGF beta family of genes have been mapped to mouse chromosome locations near loci previously assigned morphogenetic mutant loci. Although the relationship between the TGF beta genes and these loci have not been proven to be allelic, they may reveal important clues to the true activities of these molecules in vivo.

Animals↗
PubMed1989

Induction of transforming growth factor-alpha expression in human keratinocytes by phorbol esters.

Exposure of cultured human epidermal keratinocytes to the protein kinase C (Ca2+- and phospholipid-dependent protein kinase)-activating phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) or 4-beta-phorbol-12,13-didecanoate markedly enhanced accumulation of transforming growth factor-alpha (TGF-alpha) mRNA and secretion of TGF-alpha protein. The nonactivating phorbol ester, 4-alpha-phorbol 12,13-didecanoate, had no effect. In the absence of exogenous growth factors, confluent cultures of keratinocytes express low or undetectable levels of TGF-alpha mRNA and protein. While TPA and epidermal growth factor treatment of keratinocyte cultures deprived of growth factors both induced TGF-alpha mRNA expression, maximum induction by TPA is 5-fold greater than epidermal growth factor. Furthermore, the addition of epidermal growth factor did not enhance TPA-mediated induction of TGF-alpha mRNA expression. Under these experimental conditions, TPA increased levels of secreted TGF-alpha protein by 20-fold at 24 h. Concentration dependence and kinetic studies of TGF-alpha expression showed that TPA (greater than or equal to 1 ng/ml) induced accumulation of TGF-alpha mRNA with an optimum concentration of 10 ng/ml. TGF-alpha mRNA expression increased within 1 h following TPA treatment (10 ng/ml) and peaked at 5 h. At 24 h, TPA-treated cultures still expressed elevated levels of TGF-alpha mRNA (1.7-fold). Protein secretion into the medium was enhanced 2-fold (5 h) to 3-fold (24 h) by TPA treatment of keratinocyte cultures containing growth factors. Prolonged pretreatment (24 h) of keratinocyte cultures with TPA caused marked desensitization of TGF-alpha mRNA expression to repeated stimulation by phorbol ester. The synthetic diacylglycerol, 1,2-sn-dioctanoylglycerol, enhanced levels of TGF-alpha transcription and secretion of TGF-alpha protein. The rate of TGF-alpha mRNA accumulation peaked and declined earlier for 1,2-sn-dioctanoylglycerol compared to TPA. 1,2-sn-Dioctanoylglycerol (50 micrograms/ml) increased production and secretion of TGF-alpha protein, but less than TPA treatment. An inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, also inhibited 1,2-sn-dioctanoylglycerol-mediated accumulation of TGF-alpha mRNA. Cycloheximide failed to inhibit TGF-alpha mRNA expression induced by TPA and, when added alone to keratinocyte cultures, significantly enhanced TGF-alpha mRNA accumulation. Actinomycin D abrogated transcriptional activation of TGF-alpha mRNA by TPA. These studies suggest that activation of protein kinase C by active phorbol esters or diacylglycerols is responsible, at least in part, for TGF-alpha gene expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗