Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.     

Alterations of expression and regulation of transforming growth factor beta in human cancer prostate cell lines

TGFβ can promote and/or suppress prostate tumor growth through multiple and opposing actions. Alterations of its expression, secretion, regulation or of the sensitivity of target cells can lead to a favorable environment for tumor development. To gain a better insight in TGFβ function during cancer progression, we have used different cultured human prostate cells: preneoplastic PNT2 cells, the androgen-dependent LNCaP and the androgen-independent PC3 and DU145 prostate cancer cell lines. We have studied by specific ELISA assays in conditioned media (CM), the secretion of TGFβ1 and TGFβ2 in basal conditions and after hormonal treatment (DHT or E2) and the expression of TGFβ1 mRNA by Northern blot. We have also compared the effect of fibroblast CM on TGFβ secretion by the different cell types. Compared to PNT2 cells, cancer cell lines secrete lower levels of active TGFβ which are not increased in the presence of fibroblast CM. LNCaP cells respond to androgen or estrogen treatment by a 10-fold increase of active TGFβ secretion while PC3 and DU145 are unresponsive. In conclusion, prostate cancer cell lines have lost part of their ability to secrete and activate TGFβ, and to regulate this secretion through stromal–epithelial interactions. Androgen-sensitive cancer cells may compensate this loss by hormonal regulation.     

Effects of phorbol ester on cell growth inhibition by transforming growth factor beta 1 in human hepatoma cell lines

The effects of phorbol ester on cell growth inhibition by transforming growth factor beta 1 (TGF-beta 1) in human hepatoma cell lines, Mahlavu and PLC/PRF/5, were investigated. TGF-beta 1 (2.5 to 10 pM) alone could not inhibit the growth of Mahlavu cells, whereas in the presence of 12-O-tetradecanoyl phorbol 13-acetate (TPA) at 1 ng/ml, TGF-beta 1 could suppress their growth in a dose-dependent manner. The growth of PLC/PRF/5 cells could be inhibited by addition of TGF-beta 1 (2.5 to 10 pM) alone in a dose-dependent manner, and this action was not affected by TPA (1 ng/ml). The TGF-beta 1 inhibition induced by TPA in Mahlavu cells could not be cancelled by addition of protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) (10 microM) or staurosporin (1 nM). Thus, TPA could induce TGF-beta 1 inhibition of cell growth in Mahlavu cells which did not respond to TGF-beta 1 alone, and activation of protein kinase C does not seem to be behind this TPA action.     

Preparation and binding of radioactively labeled porcine transforming growth factor type beta

This report describes the labeling of porcine transforming growth factor type beta (TGF-beta) with 125-iodine. Its binding to NRK cells and three other cell lines has been examined. The data indicate that NRK cells exhibit approximately 10,000 receptors for porcine TGF-beta per cell, with an apparent dissociation constant of 45 pM. The binding of porcine 125I-TGF-beta can be blocked by porcine, murine and human TGF-beta but not by several well characterized growth factors. In all respects examined, the binding observed with porcine 125I-TGF-beta appears to be the same as that observed with human TGF-beta. The findings reported here argue that porcine 125I-TGF-beta can be used to quantitate TGF-beta receptors on a wide range of mammalian cells.     

Monoclonal antibodies recognizing transforming growth factor-beta. Bioactivity neutralization and transforming growth factor beta 2 affinity purification

Four mAb able to recognize transforming growth factor-beta 2 (TGF-beta)2 were obtained. One of these mAb, 1D11.16, was able to neutralize the biological activity of both TGF-beta 1 and beta 2 in vitro. This was demonstrated in an Il-1, PHA-dependent thymocyte mitogenic assay that is inhibitable by TGF-beta in a dose-dependent manner. All four mAb recognized the dimeric form of TGF-beta 2 in Western blots. The mAb were also found to immunoprecipitate [125I]-TGF-beta 2. mAb 3C7.14 coupled to Sepharose could efficiently immunoaffinity purify TGF-beta 2 from a complex mixture of proteins. Affinity constants were determined for the four mAb and they ranged from 3.4 x 10(8) to 1.6 x 10(7) L/mol.     

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

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

Biphasic effects of type beta transforming growth factor on epidermal growth factor receptors in NRK fibroblasts. Functional consequences for epidermal growth factor-stimulated mitosis

Exposure of confluent NRK cells to transforming growth factor-beta (TGF-beta) results in distinct alterations in subpopulations of plasma membrane epidermal growth factor (EGF) receptors. The low affinity sites increase in number, whereas the high affinity sites undergo a transient decrease in affinity followed by a prolonged increase in number. Cycloheximide inhibits both of these effects. Functional assays measuring EGF-stimulated thymidine incorporation in the presence of TGF-beta show that the resulting long-term stimulation of EGF receptor binding is associated with an increased sensitivity to EGF. Similarly, the initial, transient decrease in EGF binding is associated with a temporary inhibition of EGF-stimulated thymidine incorporation. The results describe a bifunctional effect of TGF-beta at the biochemical level consistent with the action of this peptide on NRK cell growth.     

Complete amino acid sequence of human transforming growth factor type beta 2

The complete amino acid sequence of human type beta 2 transforming growth factor (hTGF-beta 2) was determined by automated Edman degradation of S-pyridylethylated hTGF-beta 2 and selected fragments. Cleavage of hTGF-beta 2 by enzymatic and chemical techniques established all the fragments in an unambiguous sequence. Human TGF-beta 2 consists of two disulfide-linked, identical subunits. Each hTGF-beta 2 subunit is a single-chain polypeptide of 112 residues, with a calculated molecular weight of 12,720. Human TGF-beta 2 displays 71.4% sequence homology with the functionally related human TGF-beta 1, and is distantly related (23-40% amino acid identity) to porcine inhibins and activins, the carboxyl-terminal regions of human Müllerian inhibiting substance, and the putative decapentaplegic gene complex protein of Drosophila.     

Heart and liver defects and reduced transforming growth factor beta2 sensitivity in transforming growth factor beta type III receptor-deficient embryos

The type III transforming growth factor beta (TGFbeta) receptor (TbetaRIII) binds both TGFbeta and inhibin with high affinity and modulates the association of these ligands with their signaling receptors. However, the significance of TbetaRIII signaling in vivo is not known. In this study, we have sought to determine the role of TbetaRIII during development. We identified the predominant expression sites of TbetaRIII mRNA as liver and heart during midgestation and have disrupted the murine TbetaRIII gene by homologous recombination. Beginning at embryonic day 13.5, mice with mutations in TbetaRIII developed lethal proliferative defects in heart and apoptosis in liver, indicating that TbetaRIII is required during murine somatic development. To assess the effects of the absence of TbetaRIII on the function of its ligands, primary fibroblasts were generated from TbetaRIII-null and wild-type embryos. Our results indicate that TbetaRIII deficiency differentially affects the activities of TGFbeta ligands. Notably, TbetaRIII-null cells exhibited significantly reduced sensitivity to TGFbeta2 in terms of growth inhibition, reporter gene activation, and Smad2 nuclear localization, effects not observed with other ligands. These data indicate that TbetaRIII is an important modulator of TGFbeta2 function in embryonic fibroblasts and that reduced sensitivity to TGFbeta2 may underlie aspects of the TbetaRIII mutant phenotype.     

Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling

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.     

The cell biology of transforming growth factor beta

The TGF beta family of polypeptide growth factors regulates a remarkable diversity of cellular functions, many of which are not directly associated with cell growth. The present review has summarized many of the recent studies that have just begun to conceptually integrate this expanding array of TGF beta functions into the context of a three-dimensional, multicellular organ or tissue, be it normal or diseased. This fascinating research strongly implicates TGF beta as a key modulator of a wide variety of important physiologic and pathophysiologic processes.     

Heterodimeric transforming growth factor beta. Biological properties and interaction with three types of cell surface receptors

Type beta transforming growth factors (TGF) are disulfide-linked homo- and heterodimers of two related polypeptide chains, beta 1 and beta 2. The homodimers TGF-beta 1 and TGF-beta 2 are widely distributed, but the heterodimer TGF-beta 1.2 has been found only in porcine platelets (Cheifetz, S., Weatherbee, J.A., Tsang, M.L.-S., Anderson, J.K., Mole, J.E., Lucas, R., and Massagué, J. (1987) Cell 48, 409-415). Here we characterize the receptor binding and biological properties of TGF-beta 1.2 and compare them with those of TGF-beta 1 and TGF-beta 2. Three types of cell surface receptors previously identified by affinity labeling with 125I-TGF-beta 1 are available for binding to TGF-beta 1.2. These three types of receptors are detected as 65-kDa (type I), 85-95-kDa (type II), and 250-350-kDa (type III) affinity-labeled receptor complexes on electrophoresis gels. They co-exist in many cell types, have high affinity for TGF-beta 1, and varying degrees of affinity for TGF-beta 2. Of the 11 cell lines screened in the present study none showed evidence for additional receptor types that would bind TGF-beta 2 but not TGF-beta 1. In receptor competition studies, TGF-beta 1, TGF-beta 1.2, and TGF-beta 2 competed for binding to type I and type II receptors with a relative order of potencies of 16:5:1 and 12:3:1, respectively, whereas all three forms of TGF-beta were equipotent as ligands for the type III receptors. The three forms of TGF-beta were equally potent at stimulating the biosynthesis of extracellular sulfated proteoglycan in BRL-3A rat liver epithelial cells, a response that presumably involves the type III receptor present in these cells. In contrast, the ability of the three ligands to inhibit the growth of B6SUt-A multipotential hematopoietic progenitor cells which display only type I receptors decreased in the order TGF-beta 1, TGF-beta 1.2, and TGF-beta 2 with a relative potency of 100:30:1. The results indicate that the presence of one beta 1 chain in TGF-beta 1.2 increases (with respect to TGF-beta 2) the biological potency and binding affinity toward receptor types I and II, but the presence of a second beta 1 chain in the dimer is required for full potency.     

A binding assay for the solubilized receptors of type beta transforming growth factor: adsorption and removal of free ligand by dextran-coated charcoal

A binding assay was developed for the measurement of solubilized receptors for transforming growth factor type beta (TGF-beta). Solubilized receptors were incubated with 125I-TGF-beta, then the unbound ligand was removed by adsorption to dextran-coated charcoal. The binding of TGF-beta to solubilized receptors was saturable and specific, and increased in a linear manner with respect to the amount of membrane protein present. Crosslinking of radioactive complexes after adsorptive removal of unbound TGF-beta yielded complexes similar to affinity-labeled TGF-beta receptors from whole cells. Treatment of a 20% charcoal suspension with 0.2-0.4% dextran was optimal for the protection of receptors from adsorption to charcoal while allowing free TGF-beta to be removed; Mr approximately 250,000 dextran was most effective. This method can assay receptors from purified membranes and crude extracts of cells and tissues, and was used to demonstrate that TGF-beta receptors are glycosylated and retain a high affinity (Kd approximately 530 pM) for ligand after solubilization.     

Differential expression of platelet-derived growth factor receptors in human malignant glioma cell lines.

Glioma cells in culture express platelet-derived growth factor (PDGF) A- and B-chains and secrete PDGF-like activity that is mainly PDGF-AA. In this work, we show that the PDGF alpha- and beta-receptors are independently expressed in human malignant glioma cells. We also define three different receptor phenotypes that are related to the morphology of glioma cells: cells with only alpha-receptors, only beta-receptors, or with both types of receptors. By the help of Northern blot analyses, 125I-PDGF-binding experiments, and immunoprecipitations the receptors are shown to be structurally normal PDGF receptors, except for minor variations in size that probably are due to differences in glycosylation. PDGF-BB induces DNA synthesis in cells of all three receptor phenotypes, whereas PDGF-AA or PDGF-AB has this effect only on cells with alpha- or with alpha- and beta-receptors. 125I-PDGF-AB binds with high affinity and down-regulates beta-receptors only in cells where alpha-receptors are present in addition to beta-receptors. Thus, the different functional capacities of PDGF isoforms on glioma cells fit with their known receptor-binding specificities and are compatible with the hypothesis that the isoforms act by inducing dimeric receptor complexes. When data on PDGF A- and B-chains, as well as alpha- and beta-receptor expression are compiled and the pattern of receptor binding specificity is taken into account, the majority of glioma cell lines are found to have a phenotype that makes autocrine stimulation possible.     

A novel glycosylation signal regulates transforming growth factor beta receptors as evidenced by endo-beta-galactosidase C expression in rodent cells

The αGal (Galα1-3Gal) epitope is a xenoantigen that is responsible for hyperacute rejection in xenotransplantation. This epitope is expressed on the cell surface in the cells of all mammals except humans and Old World monkeys. It can be digested by the enzyme endo-β-galactosidase C (EndoGalC), which is derived from Clostridium perfringens. Previously, we produced EndoGalC transgenic mice to identify the phenotypes that would be induced following EndoGalC overexpression. The mice lacked the αGal epitope in all tissues and exhibited abnormal phenotypes such as postnatal death, growth retardation, skin lesion and abnormal behavior. Interestingly, skin lesions caused by increased proliferation of keratinocytes suggest the role of a glycan structure [in which the αGal epitope has been removed or the N-acetylglucosamine (GlcNAc) residue is newly exposed] as a regulator of signal transduction. To verify this hypothesis, we introduced an EndoGalC expression vector into cultured mouse NIH3T3 cells and obtained several EndoGalC-expressing transfectants. These cells lacked αGal epitope expression and exhibited 1.8-fold higher proliferation than untransfected parental cells. We then used several cytokine receptor inhibitors to assess the signal transduction cascades that were affected. Only SB431542 and LY364947, both of which are transforming growth factor β (TGFβ) receptor type-I (TβR-I) inhibitors, were found to successfully reverse the enhanced cell proliferation rate of EndoGalC transfectants, indicating that the glycan structure is a regulator of TβRs. Biochemical analysis demonstrated that the glycan altered association between TβR-I and TβR-II in the absence of ligands.     

Loss of transforming growth factor beta 1 receptors and its effects on the growth of EBV-transformed human B cells.

Transforming growth factor-beta (TGF-beta) is a potent negative regulator of normal human B cell growth mediated by exogenous signals, including IL-2 and low m.w. B cell growth factor 12 kDa (BCGF-12 kDa). In the present study, we investigated the regulatory linkage between viral or nonviral transformation of human B cells and the growth inhibitory effects of TGF-beta 1. A panel of EBV+ and EBV- B cell lines, derived either by in vitro EBV B cell transformation, or from cases of lymphoma was used to quantitate the negative growth effects of TGF-beta 1. The proliferative response of three EBV- B cell lines to rBCGF-12 kDa or serum was inhibited by low concentrations of TGF-beta 1 (0.2-0.5 ng/ml for 50% maximal effect), as measured by tritiated thymidine uptake and viable cellular recovery. In contrast, rBCGF-12 kDa or serum mediated proliferation of three EBV+ B cell lines was refractory to the growth inhibitory effects of TGF-beta 1. In an attempt to understand the mechanism(s) for this differential growth control in EBV+ and EBV- B cells, we studied the expression of TGF-beta 1, c-myc, and TGF-beta 1 receptors. No correlation was observed between the expression of TGF-beta 1 or c-myc gene and growth inhibition by TGF-beta 1 in the cell lines studied. Our results indicate that sensitivity or resistance to TGF-beta 1 correlated with the presence or absence (loss) of high affinity receptors for TGF-beta 1. EBV- B cell lines expressed levels of high affinity receptors similar to those found on activated normal B or T cells. In contrast, EBV+ B cell lines showed no detectable high affinity receptors. Chemical cross-linking studies with a bifunctional reagent, dissuccinimidyl suberate revealed a normal expression of type I (65-70 kDa), type II (85-90 kDa), and type III (280-300 kDa) TGF-beta 1 high affinity receptors on EBV- B cell lines. In contrast, EBV+ B cell lines did not express type I and type II receptors, whereas type III receptors were expressed but could not be inhibited by unlabeled TGF-beta 1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two roles for transforming growth factor beta 1 in colon enterocytic cell differentiation.

The role of transforming growth factor beta 1 (TGF-beta 1) in enterocytic differentiation was examined by treating two undifferentiated HT29 colon carcinoma sublines, U4 and U9, with hexamethylene bisacetamide to up-regulate their level of TGF-beta 1 mRNA expression. Although both lines after treatment secreted approximately equal levels of biologically active TGF-beta 1, only U4H cells were found to undergo enterocytic differentiation when cultured postconfluence on collagen I-coated transwells, forming polarized monolayer cells with an apical brush border, whereas U9H cells remained multilayered and undifferentiated. Enterocytic U4H cells exhibited four times as much cell surface expression of the collagen I-binding protein alpha 2-integrin, twice as much of the accessory collagen-binding protein carcinoembryonic antigen, and almost twice as much binding to collagen I films as undifferentiated U9H cells. TGF-beta 1 treatment doubled U4 cell collagen I binding, increased expression of alpha 2-integrin 4-fold, but increased carcinoembryonic antigen expression only marginally. U4H cells displayed cell cycle regulation by arresting reversibly at a restriction point in G1 when placed in the postconfluent culture conditions which initiated enterocytic differentiation. In contrast, undifferentiated U9H cells exhibited no restriction point but arrested throughout G1. TGF-beta 1 blocked synchronized U4H cells in G1, whereas it stimulated the growth of U9H cells. Thus, TGF-beta 1 has two roles in enterocytic differentiation: to increase levels of collagen I adhesion proteins and to block enterocytic cells in G1 so that they can differentiate.