The murine transforming growth factor-beta precursor

Transforming growth factor-beta (TGF-beta) is a homodimeric polypeptide which can act, often in cooperation with other growth factors, as a mitogenic factor for a variety of cells. TGF-beta can also exert growth inhibitory activity on many other cell lines. We have isolated cDNAs coding for the murine TGF-beta cDNA precursor. The deduced amino acid sequence localizes the 112-amino acid long TGF-beta monomer to the C terminus of the precursor. Two areas of the precursor exhibit a marked degree of homology to the human counterpart. One of these regions comprises the mature TGF-beta monomer, while the other corresponds to the NH2 terminus of the precursor and suggests an important biological function for this area. Northern hybridization results identify a major 2.5-kilobase TGF-beta mRNA and several minor TGF-beta mRNA species.     

Interaction of transforming growth factor-beta 1 with alpha 2-macroglobulin. Role in transforming growth factor-beta 1 clearance.

It has been widely assumed that the interaction of transforming growth factor-beta 1 (TGF-beta 1) with its serum-binding protein, alpha 2-macroglobulin (alpha 2M), mediates the rapid clearance of TGF-beta 1 from the circulation. To test this, we have analyzed the effect of TGF-beta 1 binding on the conformational state of alpha 2M. Our results demonstrate that the binding of TGF-beta 1 to alpha 2M does not lead to the conformational change in the alpha 2M molecule that is required for the clearance of the alpha 2M.TGF-beta 1 complex via the alpha 2M receptor. Furthermore, endogenous TGF-beta 1 is associated with the conformationally unaltered slow clearance form of alpha 2M. Clearance studies in mice show that the half-life of 125I-TGF-beta 1 in the circulation (1.6 +/- 0.71 min) is not affected by blocking the alpha 2M receptor with excess conformationally altered alpha 2M. These results suggest that TGF-beta 1 is rapidly cleared from the circulation after injection by a pathway not involving alpha 2M.     

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.     

Localization and actions of transforming growth factor-beta s in the embryonic nervous system.

We present evidence for unique localization and specific biological activities for transforming growth factor-beta s (TGF-beta s) 2 and 3, as compared to TGF-beta 1, in the nervous system of the 12-18 day mouse embryo. Each TGF-beta isoform was localized immunohistochemically by specific antibodies raised to peptides corresponding to unique sequences in the respective TGF-beta proteins. Staining for TGF-beta 1 was principally in the meninges, while TGF-beta s 2 and 3 co-localized in neuronal perikarya and axons, as well as in radial glial cells. In the central nervous system, staining was most prominent in zones where neuronal differentiation occurs and less intense in zones of active proliferation, while in the peripheral nervous system, many nerve fibers as well as their cell bodies were strongly immunoreactive for TGF-beta s 2 and 3. Functionally, we have also found that in the presence of an extract of chick eye tissue, TGF-beta s 2 and 3 inhibit survival of cultured embryonic chick ciliary ganglionic neurons in a dose-dependent fashion; TGF-beta 1 shows no inhibitory effects. Our data suggest that TGF-beta s 2 and 3 may play a role in regulation of neuronal migration and differentiation, as well as in glial cell proliferation and differentiation.     

Adenoviral supply of active transforming growth factor-beta1 (TGF-beta1) did not prevent lethality in transforming growth factor-beta1-knockout embryos.

In TGF-beta1-knockout mice, TGF-beta1-null conceptuses die during embryonic development with a penetrance of lethality that depends on the mouse genetic background. Studies have suggested that transplacental passage of maternal TGF-beta1 could account for the rescue of some TGF-beta1-null embryos. Herein, we have used an adenovirus-based gene delivery system and a strain of mice where most TGF-beta1-null conceptuses die prior to parturition, to investigate whether an increase in maternal TGF-beta1 during pregnancy would rescue TGF-beta1-null embryos. A single intravenous injection of an adenovirus containing a modified version of TGF-beta1 cDNA (Ad-TGF-beta1S223/S225), coding for a biologically active form of the cytokine, induced a 20-fold increase in plasma TGF-beta1 (active and latent forms) levels for up to 3 months in adult mice. Similar levels of TGF-beta1 were detected in 13-day post co?tum (dpc) embryos from Ad-TGF-beta1-treated mothers, demonstrating an efficient maternal/fetal transfer of the cytokine. However, no increase in the frequencies of TGF-beta1-null neonates nor in day 11.5 dpc TGF-beta1-null conceptuses was observed despite elevated levels of TGF-beta1 delivered throughout gestation. In addition, we show that the high levels of TGF-beta1-titrated in the plasma from Ad-TGF-beta1S223/S225-treated mice were partly the consequence of a stimulation of an autocrine production by exogenous bioactive TGF-beta1. These results indicate that transplacental passage of TGF-beta1 was not effective in rescuing TGF-beta1-null conceptuses from embryonic lethality.     

Inhibition of basal and tumor necrosis factor-enhanced binding of murine tumor cells to murine endothelium by transforming growth factor-beta 1.

The adherence of cells to microvascular endothelium is important in a number of processes, including inflammatory responses and metastasis. It has been demonstrated that in human models, cytokines such as TNF, IL-1, IFN-gamma increase the adhesiveness of endothelium for cells of the immune and inflammatory system by stimulating the expression of cell adhesion molecules on endothelial cell surfaces. We and others have shown similar cytokine-induced endothelial adhesiveness for tumor cells in murine and human models. In contrast to the effect of those modulators, transforming growth factor-beta (TGF-beta) has been shown to inhibit the binding of human neutrophils and T lymphocytes to human endothelium, although the mechanism of TGF-beta action remains unknown. Little is known about the effect of TGF-beta on tumor cell-endothelial interaction. In the present study, we demonstrate that TGF-beta inhibits basal and TNF-enhanced binding of murine P815 mastocytoma cells to murine microvascular endothelium (MME). The alterations in MME mediated by TGF-beta, also lead to the inhibition of adherence of murine splenocytes, thymocytes, and human lymphoblastoid cells but do not inhibit adherence of murine B16 melanoma cells. The effect of TGF-beta is transient and inhibition of the endothelial adhesive phenotype is strongest 12 to 24 h after addition of the factor to MME. The TGF-beta-mediated inhibition of P815 basal binding to endothelium is dependent on protein synthesis because cycloheximide reverses the TGF-beta effect. TGF-beta does not appear to activate classical signal transduction pathways. Inhibitors of G proteins do not abolish TGF-beta action, protein kinase C and protein kinase A activators elicit an effect opposite to that of the factor, TGF-beta does not increase intracellular cAMP levels, and finally calcium-mobilizing agents do not mimic, but rather inhibit the effect of TGF-beta. However, TGF-beta-mediated inhibition of both basal binding and TNF-enhanced P815 binding to MME is completely abolished in the presence of the protein phosphatase inhibitor okadaic acid which suggests that TGF-beta may elicit its effect by stimulating protein phosphatase activity.     

Type I transforming growth factor-beta receptors on neutrophils mediate chemotaxis to transforming growth factor-beta.

Participation of human polymorphonuclear neutrophils in the inflammatory response is mediated, in part, by soluble factors such as chemotactic peptides and cytokines. Although the cytokine, transforming growth factor beta (TGF-beta), has been shown to recruit monocytes and promote the inflammatory process, its effects on neutrophils are unknown. In this investigation, [125I]TGF-beta 1 affinity binding studies were employed to show that neutrophils express TGF-beta receptors (350 +/- 20 receptors/cell), which exhibit high affinity for the ligand (dissociation constant, 50 pM). Affinity cross-linking studies identified the receptors to be primarily of the type I class. In contrast to the receptors on monocytes, neutrophil TGF-beta receptors were not down-regulated by exposure to specific inflammatory mediators. Additional studies examined whether exposure of neutrophils to TGF-beta could enhance specific functions, as occurs with monocytes. TGF-beta was shown to cause directed migration of neutrophils at femtomolar concentrations, thus it is the most potent neutrophil chemotactic factor yet identified. Neutrophil production of reactive oxygen intermediates was not stimulated by TGF-beta, nor did TGF-beta enhance or depress subsequent PMA- or FMLP-stimulated superoxide production. However, the stable expression of neutrophil TGF-beta receptors, and the capacity of this cytokine to stimulate neutrophil chemotaxis, suggest that the pro-inflammatory effects of TGF-beta are mediated by neutrophils in addition to monocytes.     

Increased toxicity by transforming growth factor-beta 1 in liver cells overexpressing CYP2E1

Ethanol treatment causes an increase in expression of TGF-beta1 and CYP2E1 in the centrilobular area. Alcoholic liver disease is usually initiated in the centrilobular region of the liver. We hypothesized that the combination of TGF-beta1 and CYP2E1 produces increased oxidative stress and liver cell toxicity. To test this possibility, we studied the effects of TGF-beta1 on the viability of HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells, which do not express CYP2E1. E47 cells underwent greater growth inhibition and enhanced apoptosis after TGF-beta1 treatment, as compared to the C34 cells. There was an enhanced production of reactive oxygen species (ROS) and a decline in reduced glutathione (GSH) levels in the TGF-beta1-treated E47 cells and the enhanced cell death could be prevented by antioxidants. The CYP2E1 inhibitor diallyl sulfide prevented the potentiated cell death in E47 cells validating the role of CYP2E1. Mitochondrial membrane potential declined in the TGF-beta1-treated E47 cells, prior to developing toxicity, and cell death could be prevented by trifluoperazine, an inhibitor of the mitochondrial membrane permeability transition. TGF-beta1 also produced a loss of cell viability in hepatocytes from pyrazole-treated rats with elevated levels of CYP2E1, compared to control hepatocytes. In conclusion, increased toxic interactions by TGF-beta1 plus CYP2E1 can occur by a mechanism involving increased production of intracellular ROS and depletion of GSH, resulting in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of TGF-beta1-induced cell death by CYP2E1 may contribute to mechanisms of alcohol-induced liver disease.     

Localization of transforming growth factor-beta at the human fetal-maternal interface: role in trophoblast growth and differentiation.

We examined the localization of transforming growth factor (TGF)-beta in first-trimester and term human decidua and chorionic villi and explored the role of this factor on the proliferation and differentiation of cultured trophoblast cells. Two antibodies, 1D11.16.8, a mouse monoclonal neutralizing antibody capable of recognizing both TGF-beta 1 and TGF-beta 2 and CL-B1/29, a rabbit polyclonal antibody capable of recognizing TGF-beta 2, were used to immunolocalize TGF-beta in fixed, paraffin-embedded, or fixed, frozen sections of placenta and decidua, providing similar results. Intense labeling was observed in the extracellular matrix (ECM) of the first-trimester decidua and cytoplasm of term decidual cells. Syncytiotrophoblast cell cytoplasm as well as the ECM in the core of the chorionic villi of both first-trimester and term placentas exhibited a moderate degree of labeling. Strong cytoplasmic labeling was observed in the cytotrophoblastic shell of the term placenta. To examine the role of TGF-beta on trophoblast proliferation and differentiation, early passage cultures of first-trimester and primary cultures of term trophoblast cells were established and characterized on the basis of numerous immunocytochemical and functional markers. These cells expressed cytokeratin, placental alkaline phosphatase, urokinase-type plasminogen activator, and pregnancy-specific beta glycoprotein, but not factor VIII or 63D3; they also produced hCG and collagenase type IV. Exposure of first-trimester trophoblast cultures to TGF-beta 1 significantly inhibited proliferation in a dose-dependent manner. An antiproliferative effect was also noted in the presence of TGF-beta 2. These effects were abrogated in the presence of the neutralizing anti-TGF-beta antibody (1D11.16.8) in a concentration-dependent manner. In a 3-day culture, exogenous TGF-beta 1 stimulated formation of multinucleated cells by the first trimester as well as term trophoblast cells. Addition of neutralizing anti-TGF-beta antibody to first-trimester trophoblast cells stimulated proliferation beyond control levels in a 24-h culture and reduced formation of multinucleated cells in a 3-day culture, indicating the presence of endogenous TGF-beta activity. These results indicate that TGF-beta produced at the human fetal-maternal interface plays a major regulatory role in the proliferation and differentiation of the trophoblast.     

Possible involvement of transforming growth factor-beta 1 and transforming growth factor-beta receptor type II during luteinization in the marmoset ovary

The expression of transforming growth factor-beta 1 (TGF-beta 1), and transforming growth factor-beta receptor type II (T beta R-II), were evaluated in periovulatory marmoset ovaries. Histochemical methods were used, in particular double-labelling techniques, in order to correlate growth factor/receptor expression with proliferation (Ki 67), apoptosis (TUNEL method) and luteinization (3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)). The latter was used as a luteinization marker. Periovulatory ovaries are especially suited for studying all aspects since they typically consist of small non-luteinized follicles, large luteinizing follicles and corpora lutea accessoria (Clas), which have developed from large luteinizing follicles. TGF-beta 1 and T beta R-II expression was found in luteinizing theca cells of large periovulatory follicles and in all luteal cells of Clas. Non-luteinized theca cells, including those of small follicles were always devoid of any immunostaining. Granulosa cells of small follicles were immunopositive for T beta R-II. Large follicles with granulosa cell immunoreactivity of both antibodies coexisted with non-reactive follicles of comparable size. The highest activity of the luteal marker enzyme 3 beta-HSD was co-localized in the same cells that expressed TGF-beta 1 and T beta R-II. The double-labelling experiments revealed that TGF-beta 1 and T beta R-II expression is not correlated with proliferation or apoptosis of follicular cells. Our results indicate that TGF-beta 1 and T beta R-II participate in differentiation processes, i.e. luteinization, rather than proliferation. In particular, the dynamics of T beta R-II expression appear highly related to the process of luteinization.     

Modulation of insulin-like growth factor-II/mannose 6-phosphate receptors and transforming growth factor-beta 1 during liver regeneration.

Transforming growth factor-beta 1 (TGF-beta 1) is a potent mito-inhibiting substance that is thought to play an important function in regulating hepatocyte proliferation during liver regeneration. In this investigation, we have shown by immunohistochemistry that hepatocytes containing significant intracellular concentrations of TGF-beta 1 12 h after a two-thirds partial hepatectomy. This increase in hepatocyte TGF-beta 1 concentration was initially confined to those cells that resided in the periportal region of the liver. The elevation of intracellular TGF-beta 1 was, however, transient, and within 36 h, the hepatocytes positive for TGF-beta 1 had changed in a wavelike fashion from the periportal to the pericentral region of the liver lobules. By 48 h, most hepatocytes no longer contained TGF-beta 1. Interestingly, this temporary increase in TGF-beta 1 always preceded the onset of hepatocyte replication by approximately 3-6 h. Since TGF-beta 1 mRNA has been shown to be absent from hepatocytes normally and throughout liver regeneration, these results imply that the increase in intracellular TGF-beta 1 resulted from an augmented uptake. We have further shown that the insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man-6-P) receptors were up-regulated during liver regeneration and that the increased expression of this receptor co-localized in those hepatocytes containing elevated concentrations of TGF-beta 1. The latent TGF-beta 1 phosphomannosyl glycoprotein complex has been shown to bind to the IGF-II/Man-6-P receptor. Therefore, our data are consistent with the hypothesis that this latent complex is internalized through the IGF-II/Man-6-P receptor to the intracellular acidic prelysosomal/endosomal compartments where the mature TGF-beta 1 molecule could be activated by dissociation from the latent complex.     

Co-stimulation of T cell proliferation by transforming growth factor-beta 1.

Transforming growth factor-beta (TGF-beta) exhibits diverse regulatory roles in the immune system and has been described as a potent inhibitor of lymphocyte and hemopoietic progenitor cell growth. The present studies investigated the effects of TGF-beta 1 on murine T cell proliferation triggered through the T cell receptor/CD3 complex. In contrast to previously reported T cell growth inhibition, TGF-beta 1 costimulated splenic T cell proliferation in the presence of immobilized anti-CD3 antibody 2C11, with maximal effect at anti-CD3 concentration of 50 micrograms/ml. Although TGF-beta 1 induced a modest increase in IL-2R display, TGF-beta 1 co-stimulated proliferation was largely independent of IL-2 and/or IL-4. Anti-IL-2 and/or anti-IL-4 antibody did not significantly block the TGF-beta 1 co-stimulated T cell growth, and no IL-2 or IL-4 bioactivity was detected in TGF-beta 1 co-stimulated cultures. TGF-beta 1 did not enhance IL-2 mRNA expression beyond control levels. However, TGF-beta 1 co-stimulation caused an accelerated evolution of a memory or mature T cell population phenotype. Both CD4+ and CD8+ T cell subsets were significantly enriched for cells of the mature CD45RBloPgp-1hi phenotype, in comparison with T cells stimulated by anti-CD3 alone or with PMA, and CD8+ T cells predominated. These results thus provide initial evidence that TGF-beta 1 is capable of bifunctional T cell growth regulation, which occurs largely via an IL-2- and IL-4-independent pathway.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Hyaluronan regulates transforming growth factor-beta1 receptor compartmentalization

Transforming growth factor-beta1 (TGF-beta1) is a key cytokine involved in the pathogenesis of fibrosis in many organs. We previously demonstrated in renal proximal tubular cells that the engagement of the extracellular polysaccharide hyaluronan with its receptor CD44 attenuated TGF-beta1 signaling. In the current study we examined the potential mechanism by which the interaction between hyaluronan (HA) and CD44 regulates TGF-beta receptor function. Affinity labeling of TGF-beta receptors demonstrated that in the unstimulated cells the majority of the receptor partitioned into EEA-1-associated non-lipid raft-associated membrane pools. In the presence of exogenous HA, the majority of the receptors partitioned into caveolin-1 lipid raft-associated pools. TGF-beta1 increased the association of activated/phosphorylated Smad proteins with EEA-1, consistent with activation of TGF-beta1 signaling following endosomal internalization. Following addition of HA, caveolin-1 associated with the inhibitory Smad protein Smad7, consistent with the raft pools mediating receptor turnover, which was facilitated by HA. Antagonism of TGF-beta1-dependent Smad signaling and the effect of HA on TGF-beta receptor associations were inhibited by depletion of membrane cholesterol using nystatin and augmented by inhibition of endocytosis. The effect of HA on TGF-beta receptor trafficking was inhibited by inhibition of HA-CD44 interactions, using blocking antibody to CD44 or inhibition of MAP kinase activation. In conclusion, we have proposed a model by which HA engagement of CD44 leads to MAP kinase-dependent increased trafficking of TGF-beta receptors to lipid raft-associated pools, which facilitates increased receptor turnover and attenuation of TGF-beta1-dependent alteration in proximal tubular cell function.