Transforming growth factor-beta1, transforming growth factor-beta2, and transforming growth factor-beta3 enhance ovarian cancer metastatic potential by inducing a Smad3-dependent epithelial-to-mesenchymal transition

Transforming growth factor-beta (TGF-beta) is thought to play a role in the pathobiological progression of ovarian cancer because this peptide hormone is overexpressed in cancer tissue, plasma, and peritoneal fluid. In the current study, we investigated the role of the TGF-beta/Smad3 pathway in ovarian cancer metastasis by regulation of an epithelial-to-mesenchymal transition. When cancer cells were cultured on plastic, TGF-beta1, TGF-beta2, and TGF-beta3 induced pro-matrix metalloproteinase (MMP) secretion, loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and acquisition of a fibroblastoid phenotype, consistent with an epithelial-to-mesenchymal transition. Furthermore, Smad3 small interfering RNA transfection inhibited TGF-beta-mediated changes to a fibroblastic morphology, but not MMP secretion. When cancer cells were cultured on a three-dimensional collagen matrix, TGF-beta1, TGF-beta2, and TGF-beta3 stimulated both pro-MMP and active MMP secretion and invasion. Smad3 small interfering RNA transfection of cells cultured on a collagen matrix abrogated TGF-beta-stimulated invasion and MMP secretion. Analysis of Smad3 nuclear expression in microarrays of serous benign tumors, borderline tumors, and cystadenocarcinoma revealed that Smad3 expression could be used to distinguish benign and borderline tumors from carcinoma (P = 0.006). Higher Smad3 expression also correlated with poor survival (P = 0.031). Furthermore, a direct relationship exists between Smad3 nuclear expression and expression of the mesenchymal marker N-cadherin in cancer patients (P = 0.0057). Collectively, these results implicate an important role for the TGF-beta/Smad3 pathway in mediating ovarian oncogenesis by enhancing metastatic potential.     

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.     

Adenosine mediates transforming growth factor-beta 1 release in kidney glomeruli of diabetic rats

Up regulation of the transforming growth factor-beta 1 (TGF-β1) axis has been recognized as a pathogenic event for progression of glomerulosclerosis in diabetic nephropathy. We demonstrate that glomeruli isolated from diabetic rats accumulate up to sixfold more extracellular adenosine than normal rats. Both decreased nucleoside uptake activity by the equilibrative nucleoside transporter 1 and increased AMP hydrolysis contribute to raise extracellular adenosine. Ex vivo assays indicate that activation of the low affinity adenosine A_2B receptor subtype (A_2BAR) mediates TGF-β1 release from glomeruli of diabetic rats, a pathogenic event that could support progression of glomerulopathy when the bioavailability of adenosine is increased.     

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.     

A new type of transforming growth factor-beta, TGF-beta 3.

A new type of TGF-beta, TGF-beta 3, has been identified by cDNA characterization. The amino acid sequence of mature TGF-beta 3 and its precursor has been derived from porcine and human cDNA sequences. The human TGF-beta 3 gene is spread over seven exons as in the case of the TGF-beta 1 gene. Comparison with TGF-beta 1 and -beta 2 indicates a strong conservation of the mature sequences, but a relaxed homology in the precursor segments. TGF-beta 3 mRNA is mainly expressed in cell lines from mesenchymal origin, suggesting a biological role different from the other TGFs-beta.     

The role of transforming growth factor-beta in atherosclerosis

Transforming growth factor-β (TGF-β) plays a pivotal role in a range of biological processes, including the control of cellular proliferation and differentiation, regulation of tissue repair and extracellular matrix accumulation, and modulation of the immune and inflammatory responses. The role of TGF-β in the pathogenesis of atherosclerosis, which is widely perceived as a form of chronic inflammation, has been the subject of debate for a number of years. A pro-atherogenic role was suspected because of its ability to promote fibrosis and to inhibit endothelial regeneration. However, several recent studies have shown that TGF-β limits atherosclerosis by modulating a number of processes, including the accumulation of lipids in the vessel wall and the inflammatory response. This review will discuss the role of TGF-β in atherosclerosis along with the molecular mechanisms underlying its action during the pathogenesis of the disease.     

The transforming growth factor-beta superfamily of receptors

The transforming growth factor-beta (TGF-beta) superfamily of receptors comprises two groups of transmembrane serine-threonine kinase receptors, so called type I, and type II receptors, that are activated following engagement by members of the TGF-beta superfamily of ligands. These events specify diverse downstream responses that are differentially regulated by controlling access and activation of the ligands, their receptors and downstream substrates in different cell types. The purpose of this review is to describe the biochemical properties of these receptors, focusing specifically on the mechanisms regulating receptor/ligand interactions and activation in mammalian cells.     

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.     

Conformation and self-association of human recombinant transforming growth factor-beta3 in aqueous solutions

The transforming growth factors-beta (TGF-beta) are important regulatory peptides for cell growth and differentiation with therapeutic potential for wound healing. Among the several TGF-beta isoforms TGF-beta3 has a particularly low solubility at physiological pH and easily forms aggregates. A spectroscopic structural analysis of TGF-beta3 in solution has thus been difficult. In this study, circular dichroism spectroscopy was used to determine the secondary structural elements of TGF-beta3. In addition, the aggregation of TGF-beta3 was investigated systematically as a function of pH and salt concentration using a rapid screening method. Sedimentation equilibrium and sedimentation velocity analysis revealed that TGF-beta3 exists predominantly in two major forms: (i) monomers in solution at low pH and (ii) large precipitating aggregates at physiological pH. Under acidic conditions (pH < 3.8) the protein was not aggregated. At pH approximately 3.9, a monomer right arrow over left arrow dimer equilibrium could be detected that transformed into larger aggregates at pH > 4.1. Aggregation was pronounced in the pH range of 4.3 < pH < 9.8 with the aggregation maximum between pH 6.5 and 8. 5. The aggregation process was accompanied by a structural change of the protein. The CD spectra were characterized by an isodichroic point at 209.5 nm indicating a two-state equilibrium between TGF-beta3 dissolved in solution and aggregated TGF-beta3. Aggregated TGF-beta3 showed a higher beta-sheet content and lower beta-turn and random coil contributions compared with monomeric TGF-beta3. Both the solution structure and the aggregate structure of TGF-beta3 were different from the crystal structure. This was in contrast to TGF-beta2, which showed very similar crystal and solution structures. Under alkaline conditions (pH > 9.8) the turbidity disappeared and a further conformational change was induced. The pH dependence of the TGF-beta3 conformation in solution in the range of 2.3 < pH < 11. 0 was reversible. Aggregation of TGF-beta3 was, furthermore, influenced by the presence of salt. For pH > 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under physiological conditions (pH 7.4, cNaCl = 164 mM) TGF-beta3 has almost the highest tendency to aggregate and will remain in solution only at nanomolar concentrations.     

Oxidative stress mediates cardiac fibrosis by enhancing transforming growth factor-beta1 in hypertensive rats

The methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism has been shown to be associated with cardiovascular disease and in patients with end-stage renal disease (ESRD). However, the relationship between MTHFR polymorphisms and cardiovascular disease (CVD) in patients on hemodialysis has not been examined. The aim of this study was to assess the association of polymorphisms of MTHFR gene with homocysteine (Hcy) and intimal medial thickness (IMT) in patients on hemodialysis. We performed case-control study involving107 patients with ESRD and 103 healthy controls. Plasma Hcy was measured in all the subjects and these subjects were genotyped for three MTHFR polymorphisms (C677T, A1298C, and G1793A). We observed significantly higher Hcy levels in patients as compared to controls. The frequency of MTHFR 1298CC genotype was significantly higher in ESRD patients than in controls (21.4% vs. 2.9%); the frequency of the MTHFR C677T genotypes did not differ between groups (26.1% vs. 17.4%). Compound heterozygous MTHFR 677CT/1298AC genotypes showed maximum association with the risk of ESRD (OR: 12.8; 5%CI: 1.64–10.01, P < 0.05). Concurrent occurrence of MTHFR 677CC wild genotype with either 1298CC or 1793GA significantly increased the risk of disease (OR: 7.20; 95%CI: 2.06–2.51, P < 0.001 and OR: 7.60; 95%CI: 1.68–34.35; P < 0.05, respectively). MTHFR 1298CC genotype was associated with higher Hcy levels. IMT was also significantly higher in patients with the 1298CC genotype (P < 0.05). Thus, A1298C polymorphism of MTHFR gene appears to be associated with the severity of carotid atherosclerosis and co-occurrence of MTHFR polymorphisms may be a risk factor for CVD in patients on hemodialysis.     

Actions of transforming growth factor-beta on muscle cells

It has recently been reported by three laboratories that transforming growth factor-beta (TGF-beta) is a potent and reversible inhibitor of differentiation in myogenic cells. To improve our understanding of this inhibition, we investigated the effects of TGF-beta on several other processes in L6 myoblasts, with emphasis on actions of the insulin-like hormones (which stimulate myoblast differentiation). We found that TGF-beta had no effect on the binding of insulin-like growth factors (IGFs) to their receptors on the cell surface, and it had little or no effect on some actions of the IGFs. There was essentially no change in the suppression of proteolysis or the stimulation of cell proliferation by IGFs when TGF-beta was also added to the medium. However, there was an effect of TGF-beta on another process stimulated by the IGFs; TGF-beta was an equally active and more potent stimulator of amino acid uptake than was IGF-I, and the stimulation was additive beyond the maximal response attained with IGF-I, suggesting that the two act by different mechanisms. TGF-beta had significant effects on myoblast morphology, causing the formation of abundant stress fibers containing cytoplasmic (but not myofibrillar) actin. Addition of TGF-beta at various times after initiation of differentiation demonstrated that TGF-beta inhibits an early process in differentiation. Thus it appears that the interactions of TGF-beta and the IGFs in myoblasts are complex; in some instances the effects of IGFs are inhibited and in others they are mimicked or are unaffected. It is clear that TGF-beta does not act by simply interfering with IGF binding or blocking early steps in its action on myoblasts.     

The role of proteases in transforming growth factor-beta activation

Transforming growth factor-beta (TGFbeta) plays a central role in a number of developmental and pathological processes. There are 3 isoforms of TGFbeta (1-3) and all are sequestered in the extracellular matrix as latent complexes. Activation of this complex is the key biological checkpoint controlling TGF-beta bioavailability. This process is tightly regulated in a temporal, spatial and isoform specific manner highlighting its importance. There are many different mechanisms by which TGF-beta can be activated. Both serine and metalloproteinases play an important role in TGF-beta activation, at least in vitro, and many of these proteases have been implicated in pathological conditions. The mechanism of activation is distinct between the different proteases, but is not conserved between the two groups. Both serine proteases, such as plasmin, and metalloproteases, such as MMP2, can directly cleave latent TGFbeta, whereas others, such as thrombin and MMP14, interact with integrin mediated TGFbeta activation pathways. However, further studies are still required to fully understand the relevance of all of these pathways in vivo. Currently, the best described mechanism of TGF-beta1 activation in vivo is by integrins, although this process can be modulated by proteases. The primary mechanism of TGF-beta2 and TGF-beta3 activation has yet to be defined in vivo, although it is likely that TGF-beta3 is activated in a similar manner to TGF-beta1. This review describes the mechanism of protease driven TGF-beta activation, and discusses the physiological and pathological relevance of this process.     

Lysosomotropic drugs inhibit maturation of transforming growth factor-beta

Transforming growth factor-beta (TGFbeta) is synthesized as a precursor protein, pro-TGFbeta, that must be cleaved by a furin-like proteinase before it becomes biologically active. We hypothesized that alkalinization of the trans-Golgi network (TGN)/endosome system may suppress pro-TGFbeta processing and decrease TGFbeta secretion. This hypothesis was tested in human A549 alveolar epithelial and T98G glioblastoma cell lines and in C57BL/6 mice. Inhibition of furin-like activity with decanoyl-RVKR chloromethylketone suppressed pro-TGFbeta processing, thereby significantly reducing the levels of secreted TGFbeta. Brefeldin A, bafilomycin A1, ammonium chloride, and monensin also prevented pro-TGFbeta processing. The alkalinizing lysosomotropic drugs chloroquine, hydroxychloroquine, amodiaquine, and azithromycin had a similar effect on the overall production of mature bioactive TGFbeta. Reduced levels of secreted TGFbeta were also associated with a decrease in Smad2 signaling. Mice treated with chloroquine showed a decrease in bronchoalveolar lavage fluid TGFbeta. We conclude that alkalinizing lysosomotropic drugs inhibit pro-TGFbeta processing.