TGF-beta enhances the production of hyaluronan in human lung but not in skin fibroblasts

Transforming growth factor-beta (TGF-beta) enhances the production of extracellular matrix components, such as type I and type III collagen, fibronectin, proteoglycans, in various cell types. The effect on hyaluronan synthesis in relation to proteoglycan synthesis has not been investigated. Human lung or skin fibroblast cultures were treated with TGF-beta in serum-free medium for various periods of time. 35SO4 or [3H]glucosamine was then added to the cultures in the absence of TGF-beta for up to 48 h. Hyaluronan and proteoglycans were isolated by ion-exchange chromatography and quantitated. TGF-beta induced a three- to fourfold increase in hyaluronan production by lung cells but had no effect on skin fibroblasts. In contrast, proteoglycan synthesis was enhanced in both cell types, although skin fibroblasts responded at lower concentrations of TGF-beta. Increased accumulation of hyaluronan was noted only in the cell medium, whereas proteoglycan accumulation was observed both in the medium and in the cell layer. The ED50 for TGF-beta on hyaluronan accumulation in lung cells was the same as that for proteoglycan accumulation, i.e., 40 pM. In skin fibroblasts the ED50 was considerably lower (4 pM). The induction time needed to attain full effect of TGF-beta was 6 h for both hyaluronan and proteoglycan synthesis. These results indicate that TGF-beta has tissue-specific effects on matrix production which may be of importance for control of cell proliferation in various disease states.     

Bifunctional effects of transforming growth factor-beta (TGF-beta) on endothelial cell growth correlate with phenotypes of TGF-beta binding sites.

Transforming growth factor-beta (TGF-beta) is a bifunctional, dose-dependent regulator of endothelial cell proliferation induced in vitro by heparin-binding growth factor 1 (HBGF-1, acidic FGF). Here we have examined the relationship between endothelial cell growth and the expression of cell surface binding sites for TGF-beta and HBGF-1. Fetal bovine heart endothelial cell (FBHEC) growth was stimulated by low concentrations of TGF-beta and inhibited by high concentrations of TGF-beta while expressing two distinct classes of TGF-beta binding sites with binding constants of 24 pM (6300 sites/cell) and 900 pM (12,000 sites/cell). In contrast, human umbilical vein endothelial cells (HUVEC), whose growth was slightly promoted by TGF-beta, exhibited a single class of high-affinity TGF-beta binding sites (Kd = 45 pM, 4500 sites/cell). Affinity crosslinking using [125I]TGF-beta showed that FBHEC expressed two distinct low molecular weight TGF-beta binding sites (Mr 85,000 and 58,000), while HUVEC expressed a single type of low molecular weight TGF-beta binding site (Mr 85,000). As detected by binding of [125I]HBGF-1, preincubation of FBHEC with high concentrations of TGF-beta transmodulated the expression of high-affinity HBGF-1 receptors. In contrast, no transmodulation of HBGF-1 receptors occurred in FBHEC during preincubation with low concentrations of TGF-beta. Furthermore, preincubation of HUVEC with TGF-beta did not transmodulate the expression of HBGF-1 receptors. The data suggest that the ability of TGF-beta to stimulate or inhibit endothelial cell proliferation in a dose-dependent manner correlated with the expression of specific TGF-beta binding site subtypes and involved the transmodulation of HBGF-1 receptors.     

Transforming growth factor-beta receptor profiles of human and murine embryonic palate mesenchymal cells.

Cell signalling in the developing mammalian palate appears to involve various growth factors and hormones. An important developmental role for the transforming growth factor-beta (TGF-beta) class of growth factors is suggested by the immunolocalization of TGF-beta 1 in the palate during its ontogeny. This study examined the effects of TGF-beta stimulation of, as well as TGF-beta receptor profiles in, murine embryonic palate mesenchymal (MEPM) and human embryonic palate mesenchymal (HEPM) cells. Results showed that TGF-beta 1 (1 ng/ml) stimulated proliferation of HEPM cells and inhibited proliferation of MEPM cells in a dose-dependent manner. The time course of 125I-TGF-beta 1 binding to specific receptors was determined by incubating cells in the presence of 170 pM 125I-TGF-beta 1 for up to 4 h. In both cell types, at 37 degrees C, the binding of 125I-TGF-beta decreased linearly over 4 h, while at 4 degrees C, binding increased with time of incubation. Incubation of both cell types at 4 degrees C for 4 h, with increasing concentrations of 125I-TGF-beta 1, resulted in binding which demonstrated saturation kinetics. Scatchard analyses revealed one class of receptors for HEPM (K 32.3 pM) and MEPM (K 26.3 pM). However, SDS-PAGE analyses of 125I-TGF-beta chemically crosslinked to specific receptor sites revealed that both cell types contained the types I (65,000 Mr) and III (230,000 Mr) TGF-beta receptors while MEPM also contained the type II (86,000 Mr) receptor. Binding studies further demonstrated the ability of platelet-derived growth factor to transmodulate TGF-beta binding. These results indicate that the HEPM cell line and primary cultures of MEPM cells, although obtained from palates at similar developmental stages, are dramatically different in their responsiveness to TGF-beta and have disparate TGF-beta receptor profiles.     

Transforming growth factor beta regulates the inhibitory actions of epidermal growth factor during granulosa cell differentiation

The effects of transforming growth factor beta (TGF-beta) on epidermal growth factor (EGF) receptor content and EGF action were studied in cultured granulosa cells from immature diethylstilbestrol-implanted rats. During follicle-stimulating hormone (FSH)-induced differentiation in vitro, EGF receptors increased by 20-fold as measured by the binding of 125I-EGF to the intact cells. Addition of TGF-beta during the 48-h culture period amplified the stimulatory effects of FSH on EGF receptors up to 2-fold, with ED50 and maximal concentrations of 2.5 and 8 pM, respectively. Also TGF-beta alone in amounts from 1.6 to 16 pM increased EGF receptor content 4-fold. The stimulatory effects of TGF-beta were due to increased numbers of EGF receptors/cell, since the growth factor had no effect on the Kd (3-5 X 10(-11) M) of the high-affinity EGF binding site. TGF-beta action was observed within 20 h of granulosa cell culture and was maximal by 48 h of a 96-h culture. The stimulatory actions of TGF-beta in gonadotropin-induced cells were exerted through the cAMP effector system of the granulosa cell, since the growth factor also amplified the induction of EGF receptors by cholera toxin, forskolin, and 8-bromo-cAMP. The augmentation of EGF receptors by TGF-beta resulted in a parallel 2-fold increase in the inhibitory effects of EGF on FSH-induced cAMP production and luteinizing hormone receptor expression during granulosa cell development. TGF-beta did not increase granulosa cell numbers during culture although it elevated [3H]thymidine incorporation into DNA by 2-fold over that of FSH-treated cells. These results indicate that TGF-beta regulates the effects of both FSH and EGF during granulosa cell differentiation and provides evidence that ovarian function may be controlled by the combined actions of gonadotropins and multiple growth factors.     

Vascular smooth muscle cells express distinct transforming growth factor-beta receptor phenotypes as a function of cell density in culture

Transforming growth factor-beta (TGF-beta) is a bifunctional, density-dependent regulator of vascular smooth muscle cell (SMC) proliferation in vitro (at sparse densities SMC are growth-inhibited by the peptide, whereas at confluent densities TGF-beta potentiates their growth). We have used affinity labeling and ligand binding techniques to characterize cell surface receptors for TGF-beta under sparse and confluent culture conditions. Confluent SMC, whose growth are promoted by TGF-beta, exhibited a single class of high affinity TGF-beta binding sites (Kd = 6 pM, 3,000 sites/cell). In contrast, sparse SMC (whose growth are inhibited by TGF-beta) expressed two distinct classes of high affinity binding sites with binding constants of 6 pM (3,000 sites/cell) and 88 pM (11,000 sites/cell). By affinity labeling using 125I-TGF-beta and disuccinimidyl suberate cross-linking, confluent cells were found to express a major Mr = 280,000 TGF-beta receptor as well as trace amounts of low molecular weight (Mr = 85,000 and 65,000) receptor subtypes. All three of these receptors were determined, by ligand competition, to show similar affinity for TGF-beta. The predominant receptor subtypes expressed by sparse SMC exhibited apparent Mr = 75,000 and 65,000. In ligand competition experiments, the Mr = 75,000 receptor subtype (never present in confluent cultures) exhibited lower relative affinity for TGF-beta than did the Mr = 65,000 form. The ability of TGF-beta to inhibit SMC proliferation, therefore, correlates with the expression of a unique TGF-beta-binding protein on the SMC surface. The data suggest that TGF-beta may exert opposite biological effects on the same cell type via an interaction with distinct, selectively expressed receptor subtypes.     

Continuous interferon-gamma or tumor necrosis factor-alpha exposure of enterocytes attenuates cell death responses

Short-term stimulation (i.e. <2 days) with tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) cause growth arrest and sensitize epithelial cells to CD95 (Fas/Apo-1)-mediated cell death. The effect of long-term cytokine exposure on viability, proliferation, and apoptosis response of colonic epithelial cells is unknown and addressed in this study. In the present study HT29 and DLD-1 colonic cells were stimulated with either TNF-alpha or IFN-gamma at varying concentrations for 2-9 days. Viability and proliferation was assessed. CD95-mediated cell death response was determined. IFN-gamma caused decreased viability at high concentrations (1 nM), whereas lower concentrations (10-100 pM) only caused a transient growth arrest. TNF-alpha (100 pM) did not affect cell growth. Cells stimulated for 8 days with IFN-gamma (10 pM) or TNF-alpha (100 pM) had higher proliferation rates than controls or cells stimulated for 2 days (p < 0.05). Whereas the spontaneous cell death increased slightly during continuous cytokine exposure the CD95L response decreased (P < 0.01). Colonic cells continuously exposed to IFN-gamma or TNF-alpha had cell turnover characteristics that resemble findings in patients with UC. Increased proliferation and decreased cell death response may act as a counter regulatory mechanism that limits the damaging effects of cytokines.     

Differential sensitivity of subclasses of human colon carcinoma cell lines to the growth inhibitory effects of transforming growth factor-beta 1

In this study we have employed a model system comprising three groups of colon carcinoma cell lines to examine the growth-inhibitory effects of two molecular forms of transforming growth factor-beta (TGF-beta), TGF-beta 1 and TGF-beta 2. Aggressive, poorly differentiated colon carcinoma cells of group I did not respond to growth inhibitory effects of TGF-beta 1 or TGF-beta 2, while less aggressive, well-differentiated cells of group III displayed marked sensitivity to both TGF-beta 1 and TGF-beta 2 in monolayer culture as well as in soft agarose. One moderately well-differentiated cell line from group II which has intermediate growth characteristics failed to respond to TGF-beta 1 or TGF-beta 2, but the growth of two other cell lines in this group was inhibited. TGF-beta 1 and TGF-beta 2 were equally potent, 50% growth inhibition for responsive cell lines being observed at a concentration of 1 ng/ml (40 pM). Antiproliferative effects of TGF-beta 1 and TGF-beta 2 in responsive cell lines of groups II and III were associated with morphological alterations and enhanced, concentration-dependent secretion of carcinoembryonic antigen. Radiolabeled TGF-beta 1 bound to all three groups of colon carcinoma cells with high affinity (Kd between 42 and 64 pM). These data indicate for the first time a strong correlation between the degree of differentiation of colon carcinoma cell lines and sensitivity to the antiproliferative and differentiation-promoting effects of TGF-beta 1 and TGF-beta 2.     

Characterization of a hepatic proliferation inhibitor (HPI): effect of HPI on the growth of normal liver cells--comparison with transforming growth factor beta

Improvements in the purification of a hepatic proliferation inhibitor (HPI) from adult rat liver have yielded a product that has an inhibitory activity 1,000-fold greater than previously reported. The growth inhibitory activity, which could be eluted from SDS-PAGE at 17-19 kilodaltons (kD), was compared to that of transforming growth factor beta (TGF-beta). The ID50 of the HPI preparation in Fischer rat liver epithelial cells was 50 pg/ml (2.5 pM) compared to a value of 260 pg/ml (10.4 pM) obtained for pure human TGF-beta. Both inhibitors also modulated the stimulation of DNA synthesis in primary hepatocytes by either epidermal growth factor or a growth stimulatory activity prepared from serum of hepatectomized rats. The ID50s of HPI and TGF-beta in these cells were 250 pg/ml and 40 pg/ml, respectively. In contrast to TGF-beta the growth inhibitory activity of HPI was unaltered in the presence of an antibody raised against TGF-beta. The possible mechanism of action of HPI is discussed.     

Transforming growth factor-beta s are equipotent growth inhibitors of interleukin-1-induced thymocyte proliferation

The effects of two forms of transforming growth factor-beta, TGF-beta 1 and TGF-beta 2, upon the proliferative response of murine thymocytes were investigated in this study. TGF-beta 1 and TGF-beta 2 were found to be equipotent growth inhibitors of interleukin-1 (IL-1)- and phytohemagglutinin (PHA)-stimulated thymocytes when added at the initiation of the cultures. These factors suppressed the proliferative response in a dose-dependent fashion between 0.4 and 100 pM. The proliferative response was maximally inhibited (90% inhibition) at 100 pM. The half-maximal inhibitory dose (ID50) was 6 and 4 pM for TGF-beta 1 and TGF-beta 2, respectively. These factors were less effective or ineffective at suppressing the proliferation of thymocytes which had been prestimulated for 24 to 48 hr by IL-1 and PHA. Neither factor inhibited interleukin-2 (IL-2)-dependent thymocyte proliferation or the proliferation of an IL-2-dependent cytotoxic T cell line (CTL-L), suggesting that the anti-proliferative actions of these factors was by inhibition of cellular events triggered by IL-1. Furthermore, anti-TGF-beta 1 antibodies did neutralize the biological actions of TGF-beta 1 and these antibodies did block the binding of 125I-labeled TGF-beta 1 to cell surface receptors showing that the inhibitory action is mediated through specific receptors for TGF-beta 1 on thymocytes. These antibodies, however, did not neutralize the anti-proliferative action of TGF-beta 2. Although TGF-beta 1 and TGF-beta 2 exhibit very similar biological activities, these molecules are antigenically different and, therefore, have different tertiary structures.     

Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells

We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-beta (TGF-beta), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-beta inhibited serum-induced proliferation of rat aortic smooth muscle cells (ED50 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-beta. TGF-beta concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47% increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-beta-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in G1 were hypertrophied with respect to the corresponding cells in vehicle-treated controls. Chronic treatment with TGF-beta (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-beta from these cultures was associated with the appearance of a significant fraction of cycling cells with greater than 4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-beta in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-beta.     

Regulation of colony-stimulating factor 1-dependent macrophage precursor proliferation by type beta transforming growth factor

Transforming growth factor (TGF) type beta, a potent growth modulator, has recently been shown to inhibit the proliferation and function of several types of immune cells. This report investigates the effect of human platelet purified TGF-beta on CSF-1-induced proliferation in liquid cultures. We used two cell types to study TGF-beta effects, bone marrow precursors and a c-myc partially transformed CSF-1-dependent macrophage cell line designated BMM-8. We found that CSF-1-dependent proliferation of both cell types was strongly inhibited by TGF-beta in a dose-dependent manner. Approximately 1.6 and 8 pM TGF-beta inhibited 50% of CSF-1 proliferation of the bone marrow precursors and BMM-8, respectively. Inhibition appeared to be reversible, as bone marrow and BMM-8 cells proliferated in response to CSF-1 after preincubation of the cells in TGF-beta. Interestingly, inhibition of hematopoietic cells was observed only after a lag period of 24 to 48 h after onset of cultures. TGF-beta inhibition was partially diminished when increasing amounts of CSF-1 were added to the cultures. TGF-beta inhibition did not involve secondary inhibitory factors such as IFN or PG, both of which have been previously shown to suppress CSF responsiveness. Finally, flow cytometric analysis of the cell cycle indicated that within 48 h, TGF-beta-treated BMM-8 cells were prevented from entering S phase. These results suggest that TGF-beta may play an important role in the negative regulation of macrophage production.     

Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes

This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis.     

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.     

Effects of transforming growth factors and regulation of their mRNA levels in two human endometrial adenocarcinoma cell lines.

The effects of the transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the growth of cells from 2 endometrial cancer lines, Ishikawa and HEC-50 were evaluated by measuring rates of DNA synthesis and changes in cell numbers during culture. EGF at 17 and 1.7 nM concentrations consistently enhanced HEC-50 cell proliferation. TGF-beta 1 inhibited Ishikawa cell proliferation but, unexpectedly for epithelium-derived cells, stimulated HEC-50 cell growth. This effect is of interest as it indicates that endometrial cells can acquire an altered responsiveness to a growth inhibitor during the process of malignant transformation. Northern blot analyses showed expression of TGF-alpha, TGF-beta 1 and EGF receptors mRNA in both cell lines. Neither estradiol (E2) nor 4-hydroxytamoxifen (OHTam) affected mRNA levels for either TGF-alpha or TGF-beta in HEC-50 cells, a line unresponsive to E2 for proliferation. In Ishikawa cells, previously shown to respond to both E2 and OHTam by increasing proliferation rates, E2 increased TGF-alpha mRNA and reduced TGF-beta mRNA levels. OHTam lowered the levels of both mRNA species, although the effect was greater on TGF-beta than TGF-alpha mRNA. These data are consistent with, but do not prove, the existence of a possible autocrine regulation by TGF-alpha and TGF-beta of human cancer cell proliferation, which might be under E2 influence in Ishikawa cells.     

Transforming growth factor-beta inhibits endothelial cell proliferation

Transforming growth factor-beta (TGF-beta) is an inhibitor of the proliferation of bovine aortic endothelial cells in culture. Basal cell growth in serum-containing medium and cell proliferation stimulated by fibroblast growth factor (FGF) are inhibited by TGF-beta in a dose-dependent manner. Half-maximal inhibition occurs at an inhibitor concentration of 0.5-1.0 ng/ml. TGF-beta does not appear to be cytotoxic and cells treated with the inhibitor grow normally after removal of TGF-beta. High concentrations of FGF are ineffective in overcoming TGF-beta-induced inhibition of cell proliferation, suggesting that antagonism of growth factor-induced cell proliferation by TGF-beta is of a noncompetitive nature.     

Transforming growth factor-beta modulates the high-affinity receptors for epidermal growth factor and transforming growth factor-alpha

The epidermal growth factor (EGF) receptor mediates the induction of a transformed phenotype in normal rat kidney (NRK) cells by transforming growth factors (TGFs). The ability of EGF and its analogue TGF-alpha to induce the transformed phenotype in NRK cells is greatly potentiated by TGF-beta, a polypeptide that does not interact directly with binding sites for EGF or TGF-alpha. Our evidence indicates that TGF-beta purified from retrovirally transformed rat embryo cells and human platelets induces a rapid (t 1/2 = 0.3 h) decrease in the binding of EGF and TGF-alpha to high-affinity cell surface receptors in NRK cells. No change due to TGF-beta was observed in the binding of EGF or TGF-alpha to lower affinity sites also present in NRK cells. The effect of TGF-beta on EGF/TGF-alpha receptors was observed at concentrations (0.5-20 pM) similar to those at which TGF-beta is active in promoting proliferation of NRK cells in monolayer culture and semisolid medium. Affinity labeling of NRK cells and membranes by cross-linking with receptor-bound 125I-TGF-alpha and 125I-EGF indicated that both factors interact with a common 170-kD receptor structure. Treatment of cells with TGF-beta decreased the intensity of affinity-labeling of this receptor structure. These data suggest that the 170 kD high-affinity receptors for EGF and TGF-alpha in NRK cells are a target for rapid modulation by TGF-beta.     

Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines

At least one member of the TGF-beta family, TGF-beta 1, has been previously shown to inhibit the anchorage-independent growth of some human breast cancer cell lines (Knabbe et al., 1987; Arteaga et al., 1988). Members of the TGF-beta family might, therefore, provide new strategies for breast cancer therapy. We have studied the inhibitory effects of TGF-beta 1 and TGF-beta 2 on the anchorage-independent growth of the oestrogen receptor-negative cell lines MDA-MB-231, SK-BR-3, Hs578T, MDA-MB-468, and MDA-MB-468-S4 (an MDA-MB-468 clone not growth inhibited by EGF) and the estrogen receptor-positive cell lines MCF7, ZR-75-1, T-47D. TGF-beta 1 and TGF-beta 2 caused a 75-90% growth inhibition of MDA-MB-231, SK-BR-3, Hs578T, and MDA-MB-468 cells and a 50% growth inhibition of ZR-75-1 and early passage (less than 100) MCF7 cells. T-47D cells responded to TGF-beta only in serum-free conditions in the presence of IGF-1 or EGF. The growth of MDA-MB-468-S4 cells and late passage (greater than 500) MCF7 cells was not inhibited by TGF-beta 1 or TGF-beta 2. TGF-beta-sensitive MCF7 and MDA-MB-231 cells did not respond to Muellerian inhibiting substance (MIS), a TGF-beta-related polypeptide. TGF-beta 1 or TGF-beta 2 were mutually competitive for receptor binding with a similar affinity (Kd 25-130 pM, 1,000-13,000 sites per cell). To determine the time course of the TGF-beta effect, an anchorage-dependent growth assay was carried out using MDA-MB-231 cells. Growth inhibition occurred at 6 days, and cell-cycle changes were seen 12 hr after the addition of TGF-beta. Cells accumulated in the G1 phase and were thus inhibited from entering the S-phase. These data indicate that TGF-beta is a potent growth inhibitor in most breast cancer cell lines and provide a basis for studying TGF-beta effects in vivo.     

Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide.

Recently, the simian type 1 transforming growth factor beta (TGF-beta 1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene amplification (L.E. Gentry, N.R. Webb, G.J. Lim, A.M. Brunner, J.E. Ranchalis, D.R. Twardzik, M.N. Lioubin, H. Marquardt, and A.F. Purchio, Mol. Cell. Biol. 7:3418-3427, 1987). We have now purified and characterized the recombinant proteins released by these cells. Analyses of the precursor proteins by amino acid sequencing identified potentially important proteolytic processing sites. Signal peptide cleavage occurs at the Gly-29-Leu-30 peptide bond of pre-pro-TGF-beta 1, yielding pro-TGF-beta 1 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-beta 1 occurs at the dibasic cleavage site immediately preceding Ala-279, indicating that CHO cells possess the appropriate processing enzyme. Greater than 95% of the biological activity detected in the conditioned medium of the CHO transfectant was due to mature, properly processed growth factor. Highly purified recombinant TGF-beta 1 had the same specific biological activity as natural TGF-beta 1. The concentration of TGF-beta 1 required for half-maximal inhibition of Mv1Lu mink lung epithelial cell growth was approximately 1 to 2 pM. Purified precursor inhibited mink lung cell proliferation at 50 to 60 pM concentrations. The purified precursor preparation was shown to consist of pro-TGF-beta 1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-beta 1 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-beta1 should prove useful for further structural and functional studies.     

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells

Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.     

Transforming growth factor beta is an important immunomodulatory protein for human B lymphocytes

The growth and differentiation of B cells to immunoglobulin (Ig)-secreting cells is regulated by a variety of soluble factors. This study presents data that support a role for transforming growth factor (TGF)-beta in this regulatory process. B lymphocytes were shown to have high-affinity receptors for TGF-beta that were increased fivefold to sixfold after in vitro activation. The addition of picogram quantities of TGF-beta to B cell cultures suppressed factor-dependent, interleukin 2 (IL 2) B cell proliferation and markedly suppressed factor-dependent (IL 2 or B cell differentiation factor) B cell Ig secretion. In contrast, the constitutive IgG production by an Epstein Barr virus-transformed B cell line was not modified by the presence of TGF-beta in culture. This cell line was found to lack high-affinity TGF-beta receptors. The degree of inhibition of B cell proliferation observed in in vitro cultures was found to be dependent not only on the concentration of TGF-beta added but also on the concentration of the growth stimulatory substance (IL 2) present. By increasing the IL 2 concentrations in culture, the inhibition of proliferation induced by TGF-beta could be partially overcome. In contrast, the inhibition of Ig secretion induced by TGF-beta could not be overcome by a higher concentration of stimulatory factor, demonstrating that the suppression of B cell differentiation by TGF-beta is not due solely to its effects on proliferation. Furthermore, it was demonstrated that B lymphocytes secrete TGF-beta. Unactivated tonsillar B cells had detectable amounts of TGF-beta mRNA on Northern blot analysis, and B cell activation with Staphylococcus aureus Cowan (SAC) resulted in a twofold to threefold increase in TGF-beta mRNA. Supernatants conditioned by unactivated B cells had small amounts of TGF-beta, SAC activation of the B cells resulted in a sixfold to sevenfold increase in the amount of TGF-beta present in the supernatants. Thus, B lymphocytes synthesize and secrete TGF-beta and express receptors for TGF-beta. The addition of exogenous TGF-beta to cultures of stimulated B cells inhibits subsequent proliferation and Ig secretion. TGF-beta may function as an autocrine growth inhibitor that limits B lymphocyte proliferation and ultimate differentiation.