Transforming growth factor type beta can act as a potent competence factor for AKR-2B cells

Transforming growth factor type beta (TGF beta) is a pleiotropic regulator of cell growth with specific high-affinity cell-surface receptors on a large number of cells; its mechanism of action, however, is poorly defined. In this report, we utilized the mouse fibroblast line AKR-2B to explore the question of the temporal requirements during the cell cycle in regard to both the growth inhibitory and the growth stimulatory action of TGF beta. The results indicate that AKR-2B cells are most sensitive to the inhibitory action of TGF beta during early to mid-G1. In addition, TGF beta need be present only briefly (as little as 1 min) in order to exert its inhibitory effect on EGF-induced DNA synthesis. Likewise, the stimulatory effect of TGF beta in the absence of EGF requires only an equally brief exposure to TGF beta. Use of homogeneous 125I-labeled TGF beta in a cell-binding assay demonstrates that TGF beta bound to cell-surface receptors can readily exchange into the culture medium T1/2 = 120 min), helping to rule out the possibility that persistent receptor-bound TGF beta is the source of a continuous stimulus. The data indicate that TGF beta exposure induces a stable state in the cell (T1/2 = 20 h) similar to but distinct from the state of "competence" induced by platelet-derived growth factor (PDGF).     

Transforming growth factor type β can act as a potent competence factor for AKR-2B cells

Transforming growth factor type β (TGFβ) is a pleiotropic regulator of cell growth with specific high-affinity cell-surface receptors on a large number of cells; its mechanism of action, however, is poorly defined. In this report, we utilized the mouse fibroblast line AKR-2B to explore the question of the temporal requirements during the cell cycle in regard to both the growth inhibitory and the growth stimulatory action of TGFβ. The results indicate that AKR-2B cells are most sensitive to the inhibitory action of TGFβ during early to mid-G₁. In addition, TGFβ need be present only briefly (as little as l min) in order to exert its inhibitory effect on EGF-induced DNA synthesis. Likewise, the stimulatory effect of TGFβ in the absence of EGF requires only an equally brief exposure to TGFβ. Use of homogeneous ¹²⁵I-labeled TGFβ in a cell-binding assay demonstrates that TGFβ bound to cell-surface receptors can readily exchange into the culture medium T_1/2 = 120 min), helping to rule out the possibility that persistent receptor-bound TGFβ is the source of a continuous stimulus. The data indicate that TGFβ exposure induces a stable state in the cell (T_1/2 = 20 h) similar to but distinct from the state of “competence” induced by platelet-derived growth factor (PDGF).     

Coupling of TGF-beta-induced mitogenesis to G-protein activation in AKR-2B cells

We have investigated the signal transduction mechanisms by which TGF-beta stimulates proliferation of AKR-2B murine fibroblasts. Enhanced incorporation of [3H]-thymidine into TGF-beta challenged cells was inhibited in a dose-dependent manner by pertussis toxin. EGF stimulated DNA synthesis was unaffected. Parallel biochemical analysis of pertussis toxin-challenged cells revealed that TGF-beta-induced inhibition of DNA synthesis was associated with ADP-ribosylation of a 41 kDa membrane component and a concomitant decrease in TGF-beta stimulated GTPase activity. These data, along with the observation that Gpp(NH)p decreases the affinity of the TGF-beta receptor for its ligand, strongly suggest that a GTP-binding protein is involved in TGF-beta-induced mitogenesis in AKR-2B cells.     

Transforming growth factor beta 1 is a powerful modulator of platelet-derived growth factor action in vascular smooth muscle cells.

We have studied the effect of transforming growth factor beta 1 (TGF-beta 1) on vascular smooth muscle cell (SMC) mitogenesis and expression of thrombospondin and other growth related genes. We found that TGF-beta 1 treatment of vascular SMC induced a prolonged increase in steady-state mRNA levels of thrombospondin as well as alpha 1 (IV) collagen. The increase began at approximately 2 h, peaked by 24 h, and remained considerably elevated 48 h after growth factor addition. There was a corresponding increase in thrombospondin protein as well as increased expression of several other secreted polypeptides. The increase in thrombospondin contrasted sharply with that observed for platelet-derived growth factor (PDGF) which induced a rapid and transient increase in thrombospondin mRNA level. Although TGF-beta 1 was able to directly enhance expression of thrombospondin as well as the growth-related genes c-fos and c-myc, and induced c-fos expression with identical kinetics as PDGF, it was unable to elicit [3H]thymidine incorporation into DNA in three independent smooth muscle cell strains. However, TGF-beta 1 was able to strongly increase the mitogenic response of SMC to PDGF. Addition of both TGF-beta 1 and PDGF to SMC also caused a synergistic increase in the expression of thrombospondin as well as c-myc. Interestingly, in one other smooth muscle cell strain, a weak and delayed mitogenic response to TGF-beta 1 alone was observed. Our results strongly suggest that induction of thrombospondin expression by TGF-beta 1 and by PDGF occurs by distinct mechanisms. In addition, that TGF-beta 1 can enhance PDGF-induced mitogenesis may be due to the ability of TGF-beta 1 to directly induce the expression of thrombospondin, c-fos, c-myc, and the PDGF beta-receptor.     

Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells

Suramin, a polyanionic compound, has previously been shown to dissociate platelet-derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR-2B cells in fetal bovine serum (FBS)-supplemented medium in a dose-dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor beta (TGF beta), heparin-binding growth factor type-2 (HBGF-2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density-arrested cultures of AKR-2B cells. The inhibition of growth factor-stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I-labeled TGF beta, HBGF-2, and EGF to their cell surface receptors. Suramin affected TGF beta and HBGF-2-related events at a 10-15-fold lower dose than that required for EGF-related events. It was also noted that suramin inhibited TGF beta-stimulated soft agar colony formation of AKR-2B (clone 84A) cells as well as the spontaneous colony formation of AKR-MCA cells, a chemically transformed derivative of AKR-2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGF beta, HBGF-2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR-MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.     

Transforming growth factor beta1 receptor II is downregulated by E1A in adenovirus-infected cells

Transforming growth factor beta1 (TGF-beta1) signaling is compromised in many tumors, thereby allowing the tumor to escape the growth-inhibitory and proapoptotic activities of the cytokine. Human adenoviruses interfere with a number of cellular pathways involved in cell cycle regulation and apoptosis, initially placing the cell in a "tumor-like" state by forcing quiescent cells into the cell cycle and also inhibiting apoptosis. We report that adenovirus-infected cells resemble tumor cells in that TGF-beta1 signaling is inhibited. The levels of TGF-beta1 receptor II (TbetaRII) in adenovirus-infected cells were decreased, and this decrease was mapped, by using virus mutants, to the E1A gene and to amino acids 2 to 36 and the C-terminal binding protein binding site in the E1A protein. The decrease in the TbetaRII protein was accompanied by a decrease in TbetaRII mRNA. The decrease in TbetaRII protein levels in adenovirus-infected cells was greater than the decrease in TbetaRII mRNA, suggesting that downregulation of the TbetaRII protein may occur through more than one mechanism. Surprisingly in this context, the half-lives of the TbetaRII protein in infected and uninfected cells were similar. TGF-beta1 signaling was compromised in cells infected with wild-type adenovirus, as measured with 3TP-lux, a TGF-beta-sensitive reporter plasmid expressing luciferase. Adenovirus mutants deficient in TbetaRII downregulation did not inhibit TGF-beta1 signaling. TGF-beta1 pretreatment reduced the relative abundance of adenovirus structural proteins in infected cells, an effect that was potentiated when cells were infected with mutants incapable of modulating the TGF-beta signaling pathway. These results raise the possibility that inhibition of TGF-beta signaling by E1A is a means by which adenovirus counters the antiviral defenses of the host.     

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.     

Transforming growth factor beta treatment decreases ACTH receptors on ovine adrenocortical cells

Transforming growth factor beta (TGF beta) is a potent inhibitor of adrenocortical cell differentiated functions, whereas corticotropin (ACTH) is the main physiological hormone which acts positively on these functions. We have studied the effects of both TGF beta and ACTH on ovine adrenocortical cell ACTH receptors. Ovine adrenocortical cells contained specific high affinity (Kd = 2.7 +/- 1.6 x 10(-10) M) and low capacity (1190 +/- 120 sites/cell) ACTH receptors. Pretreatment of cells with TGF beta resulted in a time- and dose-dependent (ED50 = 50 pg/ml) decrease of 125I-ACTH1-39 binding. The observed decrease in ACTH binding was due to a 2-3-fold decrease in the number of binding sites without modification of the binding affinity. On the contrary, pretreatment of cells with ACTH caused a 4-4.5-fold increase in the number of ACTH binding sites without an effect on the Kd. When cells were pretreated with both ACTH and TGF beta, TGF beta blocked completely the positive trophic effect of ACTH on its own receptors. The variations in ACTH receptor number were associated with parallel changes on acute ACTH-induced cyclic AMP production. Thus, the effects of TGF beta on ACTH receptor content are likely another important negative action of this peptide on adrenocortical cell differentiation. Moreover, these results suggest that regulation of ACTH receptor number may be one mechanism by which hormones and growth factors control adrenocortical differentiation.     

Transforming growth factor beta2 is a neuronal death-inducing ligand for amyloid-beta precursor protein

APP, amyloid beta precursor protein, is linked to the onset of Alzheimer's disease (AD). We have here found that transforming growth factor beta2 (TGFbeta2), but not TGFbeta1, binds to APP. The binding affinity of TGFbeta2 to APP is lower than the binding affinity of TGFbeta2 to the TGFbeta receptor. On binding to APP, TGFbeta2 activates an APP-mediated death pathway via heterotrimeric G protein G(o), c-Jun N-terminal kinase, NADPH oxidase, and caspase 3 and/or related caspases. Overall degrees of TGFbeta2-induced death are larger in cells expressing a familial AD-related mutant APP than in those expressing wild-type APP. Consequently, superphysiological concentrations of TGFbeta2 induce neuronal death in primary cortical neurons, whose one allele of the APP gene is knocked in with the V642I mutation. Combined with the finding indicated by several earlier reports that both neural and glial expression of TGFbeta2 was upregulated in AD brains, it is speculated that TGFbeta2 may contribute to the development of AD-related neuronal cell death.     

Inhibition of mink lung epithelial cell proliferation by transforming growth factor-beta is coupled through a pertussis-toxin-sensitive substrate.

Transforming growth factor beta 1 (TGF beta 1) inhibits the proliferative response of mink lung epithelial cells (CCL64) to serum and to epidermal growth factor (EGF). This response to TGF beta 1 can be inhibited by prior exposure of the cells to nanogram concentrations of pertussis toxin (PT), suggesting the involvement of a guanine-nucleotide-binding regulatory protein (G-protein) in mediating TGF beta 1-induced growth inhibition. To characterize further this G-protein dependence, we have isolated, by chemical mutagenesis, a CCL64 variant (CCL64-D1) that is resistant to TGF beta 1. Whereas in the parental CCL64 cells TGF beta 1 stimulates both GTP[35S] (guanosine 5'-[gamma-[35S]thio]triphosphate) binding and GTPase activity, in the CCL64-D1 variants TGF beta 1 is without effect. Quantitative immunoblotting with antisera for G-protein alpha- and beta-subunits, as well as PT-catalysed ADP-ribosylation analyses, revealed no appreciable changes in the level of G-protein expression in the CCL64-D1 variants compared with parental cells. In contrast with another TGF beta-resistant clone, MLE-M, which we show lacks detectable type I receptor protein, the CCL64-D1 cells retain all three TGF beta cell-surface binding proteins. On the basis of these studies, we propose that a necessary component of TGF beta 1-mediated growth inhibition in CCL64 epithelial cells is the coupling of TGF beta 1 receptor binding to G-protein activation.     

Transforming growth factor beta 1 (TGF-beta 1) receptor expression on resting and mitogen-activated T cells

Transforming growth factor beta 1 (TGF-beta 1) is a potent autocrine growth inhibitor of lymphocytes. In this study, the expression of TGF-beta 1 binding proteins was characterized on murine splenic T cells. With an affinity cross-linking method and by neutralizing antibodies to TGF-beta 1, [125I] TGF-beta 1 was found to bind to three cell surface-binding proteins (280-200 kD, 95-85 kD, 65 kD) that were differentially expressed on resting and mitogen-stimulated T cells. Freshly prepared (resting) T cells were found to constitutively express the 95-85-kD form of these binding proteins, whereas mitogenic stimulation by either concanavalin-A (Con-A), interleukin-1 (IL-1), interleukin-2 (IL-2), or 12-tetradecanoyl-phorbol-13-acetate (TPA) for 12-72 h induced the appearance of all forms of the TGF-beta 1 binding proteins (280-200 kD, 95-85 kD, and 65 kD). Furthermore, antibodies that neutralized the biologic action of TGF-beta 1 also blocked the binding of [125I] TGF-beta 1 to all three binding proteins, suggesting that these binding proteins are involved with signal transduction. These results suggest that the expression of the TGF-beta 1 receptor on T cells is regulated by T cell mitogenic signals and that a regulatory relationship may exist between T cell growth-promoting cytokines (IL-1 and IL-2) and the T cell growth inhibitor, TGF-beta 1.     

Syndecan-1 expression in epithelial cells is induced by transforming growth factor beta through a PKA-dependent pathway

Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans bind and modulate a wide variety of biological molecules through their heparan sulfate (HS) moiety. Although all syndecans contain the ligand binding HS chains, they likely perform specific functions in vivo because their temporal and spatial expression patterns are different. However, how syndecan expression is regulated has yet to be clearly defined. In this study, we examined how syndecan-1 expression is regulated in epithelial cells. Our results showed that among several bioactive agents tested, only forskolin and three isoforms of TGFbeta (TGFbeta1-TGFbeta3) significantly induced syndecan-1, but not syndecan-4, expression on various epithelial cells. Steady-state syndecan-1 mRNA was not increased by TGFbeta treatment and cycloheximide did not inhibit syndecan-1 induction by TGFbeta, indicating that TGFbeta induces syndecan-1 in a post-translational manner. However, TGFbeta induction of syndecan-1 was inhibited by transient expression of a dominant-negative construct of protein kinase A (PKA) and by specific inhibitors of PKA. Further (i) syndecan-1 cytoplasmic domains were Ser-phosphorylated when cells were treated with TGFbeta and this was inhibited by a PKA inhibitor, (ii) PKA was co-immunoprecipitated from cell lysates by anti-syndecan-1 antibodies, (iii) PKA phosphorylated recombinant syndecan-1 cytoplasmic domains in vitro, and (iv) expression of a syndecan-1 construct with its invariant Ser(286) replaced with a Gly was not induced by TGFbeta. Together, these findings define a regulatory mechanism where TGFbeta signals through PKA to phosphorylate the syndecan-1 cytoplasmic domain and increases syndecan-1 expression on epithelial cells.     

Transforming growth factor beta 1 induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells

Transforming growth factor beta (TGF-beta) induces apoptosis in a variety of cells. We have previously shown that TGF-beta 1 rapidly induces apoptosis in the FaO rat hepatoma cell line. We have now studied the effect of TGF-beta 1 on the expression of different members of the Bcl-2 family in these cells. We observed no detectable changes in the steady-state levels of Bcl-2, Bcl-X(L), and Bax. However, TGF-beta 1 induced caspase-dependent cleavage of BAD at its N terminus to generate a 15-kDa truncated protein. Overexpression of the 15-kDa truncated BAD protein enhanced TGF-beta 1-induced apoptosis, whereas a mutant BAD resistant to caspase 3 cleavage blocked TGF-beta 1-induced apoptosis. Overexpression of Smad3 dramatically enhanced TGF-beta 1-induced cleavage of BAD and apoptosis, whereas antisense Smad3 blocked TGF-beta 1-induced apoptosis and BAD cleavage. These results suggest that TGF-beta 1 induces apoptosis through the cleavage of BAD in a Smad3-dependent mechanism.     

Transforming growth factor beta is a critical regulator of adult human islet plasticity

Tissue plasticity is well documented in the context of pancreatic regeneration and carcinogenesis, with recent reports implicating dedifferentiated islet cells both as endocrine progenitors and as the cell(s) of origin in pancreatic adenocarcinoma. Accordingly, it is noteworthy that accumulating evidence suggests that TGFbeta signaling is essential to pancreatic endocrine development and maintenance, whereas its loss is associated with the progression to pancreatic adenocarcinoma. The aim of this study was to examine the role of TGFbeta in an in vitro model of islet morphogenetic plasticity. Human islets were embedded in a collagen gel and cultured under conditions that induced transformation into duct-like epithelial structures (DLS). Addition of TGFbeta caused a dose-dependent decrease in DLS formation. Although it was demonstrated that collagen-embedded islets secrete low levels of TGFbeta, antibody-mediated neutralization of this endogenously released TGFbeta improved DLS formation rates, suggesting local TGFbeta concentrations may in fact be higher. Time course studies indicated that TGFbeta signaling was associated with an increase in ERK and p38 MAPK phosphorylation, although inhibitor-based studies were consistent with an islet endocrine-stabilizing effect mediated by p38 alone. Localization of TGFbeta signaling molecules suggested that the action of TGFbeta is directly on the beta-cell to inhibit apoptosis and thus stabilize endocrine phenotype.     

Transforming growth factor beta 1-specific binding proteins on human vascular endothelial cells.

Transforming growth factor beta (TGF beta) regulates the growth of human umbilical vein endothelial cells (HUVEC) differently depending on the isoform of TGF beta and the culture conditions. The cells are resistant to growth inhibition by TGF beta when the cells are cultured on substratum coated with gelatin. However, the proliferation of HUVEC cultured on substratum without a gelatin coating is inhibited by TGF beta, depending on the isoform and concentration of TGF beta. Binding assays with 125I-TGF beta 1 reveal that HUVEC contain a single class of high-affinity (Kd = 4.4 pM) TGF beta 1 binding sites with 8500 sites per cell. Affinity cross-linking studies demonstrate that HUVEC express 180 and 80 kDa TGF beta 1 binding sites that do not bind TGF beta 2. The reduction and the removal of glycosaminoglycans does not affect the electrophoretic mobility of the 180-kDa binding protein cross-linked with 125I-TGF beta 1. Therefore, the 180-kDa TGF beta 1 binding protein is not related to the type III TGF beta receptor, but might be a novel TGF beta 1-specific receptor/binding protein expressed on vascular endothelial cells. The expression of TGF beta 1 binding sites is not affected by the presence or absence of the gelatin coating on the culture substratum. The data suggest that a gelatin coating does not regulate the susceptibility of HUVEC to TGF beta 1 at the level of the receptor/binding proteins, and that growth inhibition of HUVEC by TGF beta 1 is linked to the regulation of extracellular matrices required for the interaction between the cells and the substratum.     

Transforming growth factor beta1 stimulates urokinase plasminogen activator system on prostate cancer cells

The effect of TGFbeta1 on the proliferation and plasminogen activator system (PA) of two prostate carcinoma cell lines, PC3 and DU145, was investigated. PA, particularly urokinase plasminogen activator (uPA), has been implicated in extracellular proteolysis, local invasiveness, metastatic spread and angiogenesis. High levels of uPA and plasminogen activator inhibitor-1 (PAI-1) correlate with poor prognosis in several cancers. TGFbeta1 had no significant effect on the proliferation of either cell line. TGFbeta1 increased the production of uPA in PC3 and DU145 cells. Despite the very low PAI-1 protein levels in both cell lines, TGFbeta1 treatment resulted in a remarkable increase in PAI-1 secretion. PAI-2 protein was also increased by 59% in the PC3 cells. A divergent effect of TGFbeta1 on the uPA enzyme activity was observed (28% decrease in PC3 and 131% increase in DU145 cells). Overall, TGFbeta1 treatment did not affect the invasion of reconstituted basement membrane of PC3 cells. In addition to the uPA:PAI-1 ratio, the presence of PAI-2 may be an important factor in the determination of metastatic sites for prostate cancer cells. In conclusion, the potential contribution of TGFbeta1 to tumor invasion may be considered as positive, based on both loss of growth inhibition and stimulation of components of the invasive system of prostate carcinoma.