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.     

Role of transforming growth factor-beta superfamily signaling pathways in human disease

Transforming growth factor beta (TGF-beta) superfamily signaling pathways are ubiquitous and essential regulators of cellular processes including proliferation, differentiation, migration, and survival, as well as physiological processes, including embryonic development, angiogenesis, and wound healing. Alterations in these pathways, including either germ-line or somatic mutations or alterations in the expression of members of these signaling pathways often result in human disease. Appropriate regulation of these pathways is required at all levels, particularly at the ligand level, with either a deficiency or an excess of specific TGF-beta superfamily ligands resulting in human disease. TGF-beta superfamily ligands and members of these TGF-beta superfamily signaling pathways also have emerging roles as diagnostic, prognostic or predictive markers for human disease. Ongoing studies will enable targeting of TGF-beta superfamily signaling pathways for the chemoprevention and treatment of human disease.     

The types II and III transforming growth factor-beta receptors form homo-oligomers

Affinity-labeling experiments have detected hetero-oligomers of the types I, II, and III transforming growth factor beta (TGF-beta) receptors which mediate intracellular signaling by TGF-beta, but the oligomeric state of the individual receptor types remains unknown. Here we use two types of experiments to show that a major portion of the receptor types II and III forms homo-oligomers both in the absence and presence of TGF-beta. Both experiments used COS-7 cells co-transfected with combinations of these receptors carrying different epitope tags at their extracellular termini. In immunoprecipitation experiments, radiolabeled TGF-beta was bound and cross-linked to cells co-expressing two differently tagged type II receptors. Sequential immunoprecipitations using anti-epitope monoclonal antibodies showed that type II TGF-beta receptors form homo-oligomers. In cells co- expressing epitope-tagged types II and III receptors, a low level of co- precipitation of the ligand-labeled receptors was observed, indicating that some hetero-oligomers of the types II and III receptors exist in the presence of ligand. Antibody-mediated cross-linking studies based on double-labeling immunofluorescence explored co-patching of the receptors at the cell surface on live cells. In cells co-expressing two differently tagged type II receptors or two differently tagged type III receptors, forcing one receptor into micropatches by IgG induced co- patching of the receptor carrying the other tag, labeled by noncross- linking monovalent Fab'. These studies showed that homo-oligomers of the types II and III receptors exist on the cell surface in the absence or presence of TGF-beta 1 or -beta 2. In cells co-expressing types II and III receptors, the amount of heterocomplexes at the cell surface was too low to be detected in the immunofluorescence co-patching experiments, confirming that hetero-oligomers of the types II and III receptors are minor and probably transient species.     

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.     

Cell adhesion protein receptors as targets for transforming growth factor-beta action

Transforming growth factor-beta (TGF-beta) increases the incorporation of fibronectin and type I collagen into the extracellular matrix of fibroblasts and epithelial cells and enhances the attachment of thymocytes onto a fibronectin substratum. Investigation of the molecular basis for these effects showed that TGF-beta elevates specifically the expression of cell adhesion protein receptors. Treatment of cells with either form of TGF-beta, TGF-beta 1, or TGF-beta 2, increases the rate of receptor synthesis and the level of receptors on the cell surface. TGF-beta acts via two complementary mechanisms, elevation of receptor mRNA and faster kinetics of receptor beta subunit precursor to product conversion. The results show that the expression of cell adhesion receptors is susceptible to pretranslational and posttranslational regulation by factors that control cell morphology, proliferation, and differentiation such as TGF-beta.     

Ligand binding and functional properties of betaglycan, a co-receptor of the transforming growth factor-beta superfamily. Specialized binding regions for transforming growth factor-beta and inhibin A

Betaglycan, also known as the transforming growth factor-beta (TGF-beta) type III receptor, is a membrane-anchored proteoglycan that binds TGF-beta via its core protein. Deletion mutagenesis analysis has revealed two regions of betaglycan ectodomain capable of binding TGF-beta: one at the amino-terminal half, the endoglin-related region (López-Casillas, F., Payne, H., Andres, J. L., and Massagué, J. (1994) J. Cell Biol. 124, 557-568), and the other at the carboxyl-terminal half, the uromodulin-related region (Pepin, M.-C., Beauchemin, M., Plamondon, J., and O'Connor-McCourt, M. D. (1994) Proc. Natl. Acad. Sci. U. S. A 91, 6997-7001). In the present work we have functionally characterized these ligand binding regions. Similar to the wild type receptor, both regions bind TGF-beta2 with higher affinity than TGF-beta1. However, only the endoglin-related region increases the TGF-beta2 labeling of the TGF-beta type II receptor, the so-called "TGF-beta -presentation" function of the wild type receptor. Despite this preference, both regions as well as the wild type receptor mediate the TGF-beta2-dependent Smad2 phosphorylation, indicating that they can function indistinguishably as TGF-beta-enhancing co-receptors. On the other hand, we found that the recently described ability of the wild type betaglycan to bind inhibin A is a property of the core protein that resides in the uromodulin-related region. Binding competition experiments indicate that this region binds inhibin and TGF-beta with the following relative affinities: TGF-beta2 > inhibin A > TGF-beta1. All together, the present results suggest that betaglycan ectodomain is endowed with two bona fide independent ligand binding domains that can perform specialized functions as co-receptors of distinct members of the TGF-beta superfamily.     

The transforming growth factor-beta system, a complex pattern of cross-reactive ligands and receptors

A new homodimer form of transforming growth factor-beta (TGF-beta), TGF-beta 2, has been identified in porcine blood platelets. TGF-beta 2 is homologous to ordinary TGF-beta (TGF-beta 1), which is also present in platelets. TGF-beta 1.2, a heterodimer containing one TGF-beta 1 chain and one TGF-beta 2 chain, has also been isolated. TGF-beta 1 and TGF-beta 2 interact differently with a family of receptors in target cells. A 280 kd receptor displays high affinity for both TGF-beta 1 and TGF-beta 2. Occupancy of this receptor by TGF-beta 1 or TGF-beta 2 correlates with the ability of these TGF-beta s to inhibit cell proliferation. In contrast, 65 kd and 85 kd receptors have high affinity for TGF-beta 1 but lower affinity for TGF-beta 2. The existence of distinct forms of TGF-beta that interact differently with a family of TGF-beta receptors could provide flexibility to the regulation of tissue growth and differentiation by the TGF-beta system.     

Visualisation of transforming growth factor-beta 1, tissue kallikrein, and kinin and transforming growth factor-beta receptors on human clear-cell renal carcinoma cells

Transforming growth factor-beta1 (TGF-beta1) has a biphasic effect on the growth of renal epithelial cells. In transformed cells, TGF-beta1 appears to accelerate the proliferation of malignant cells. The diverse cellular functions of TGF-beta1 are regulated by three high-affinity serine/threonine kinase receptors, namely TbetaRI, TbetaRII and TbetaRIII. The renal serine protease tissue kallikrein acts on its endogenous protein substrate kininogen to form kinin peptides. The cellular actions of kinins are mediated through B1 and B2 G protein-coupled rhodopsin receptors. Both kinin peptides and TGF-beta1 are mitogenic, and therefore may play an important role in carcinogenesis. Experiments were designed to immunolabel tissue kallikrein, TGF-beta1, TbetaRII, TbetaRIII and kinin receptors using specific antibodies on serial sections of normal kidney and clear-cell renal carcinoma (CCRC) tissue, which included both the tumour and the adjacent renal parenchyma. The essential result was the localisation of tissue kallikrein, kinin B 1 and B 2 receptors and TGF-beta1 primarily on the cell membranes of CCRC cells. In the distal and proximal tubules of the renal parenchyma adjacent to the carcinoma (RPTAC), immunolabelling for tissue kallikrein was reduced, but the expression of kinin B1 and B2 receptors was enhanced. Immunolabelling for TbetaRII and TbetaRIII was more pronounced in the proximal tubules of the tissue adjacent to the carcinoma when compared to the normal kidney. The expression of tissue kallikrein, kinin receptors, and TbetaRII and TbetaRIII may be relevant to the parenchymal invasion and metastasis of clear-cell renal carcinoma.     

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.     

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.     

Identification of a novel member (GDF-1) of the transforming growth factor-beta superfamily.

A cDNA clone encoding a new member (designated GDF-1) of the transforming growth factor-beta (TGF beta) superfamily was isolated from a library prepared from day 8.5 mouse embryos. The nucleotide sequence of GDF-1 predicts a protein of 357 amino acids with a mol wt of 38,600. The sequence contains a pair of arginine residues at positions 236-237, which is likely to represent a site for proteolytic processing. The C-terminus following the presumed dibasic cleavage site shows significant homology with the known members of the TGF beta superfamily, matching the other family members at all of the invariant positions, including the seven cysteine residues with their characteristic spacing. GDF-1 is most homologous to Xenopus Vg-1 (52%), but is not likely to be the murine homolog of Vg-1. In vitro translation experiments were consistent with GDF-1 being a secreted glycoprotein. Genomic Southern analysis indicated that GDF-1 may be highly conserved across species. These results suggest that GDF-1 is most likely an extracellular factor mediating cell differentiation events during embryonic development.     

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

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

Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.     

Modulation of monocyte type I transforming growth factor-beta receptors by inflammatory stimuli.

The regulatory mechanisms which control the wide array of cellular responses to transforming growth factor beta (TGF beta) are not understood. This report presents evidence that down-regulation of TGF beta receptors on human monocytes may be one mechanism by which the effects of TGF beta are regulated. Treatment of monocytes with interferon gamma (IFN gamma) and lipopolysaccharide for 18 h reduced monocyte receptor number (approximately 400/cell) in a dose-dependent fashion by 89 and 78%, respectively, as determined by 125I-TGF beta binding. Incubation with other cytokines (granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor-1, interleukin-1, tumor necrosis factor alpha) did not alter the amount of TGF beta bound. The decrease in 125I-TGF beta binding could not be attributed to competition for receptor sites by secreted TGF beta. Instead, the decline in binding was due to a loss of type I TGF beta receptors, the subtype primarily expressed by monocytes, with no decrease in receptor affinity. Lipopolysaccharide-induced receptor loss was rapid (1-4 h), in contrast to the prolonged (12 h) decline induced by IFN gamma. Loss of receptors was accompanied by a diminished ability of the cells to respond to TGF beta with an induction of TNF alpha mRNA. Thus, this monocyte system is the first example of a heterologous agent causing the down-regulation of TGF beta receptors with a concomitant decline in a TGF beta-stimulated function.     

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.     

Recent development in pleiotropic effects of statins on cardiovascular disease through regulation of transforming growth factor-beta superfamily

Background

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, also known as statins, are a drug class that reduce the level of cholesterol in the blood. As a result, statins are used to suppress the progression of cardiovascular disease. Evidence points to another component of statins involving the non-lipid effects of the drug class in preventing cardiovascular disease. One specific mediator of this action is the transforming growth factor β (TGF-β) superfamily. The TGF-β superfamily consists of proteins that include TGF-β and bone morphogenetic proteins (BMPs). These proteins regulate cellular pathways to mediate effects including immunomodulation, cell cycling, and angiogenesis. One pathway that mediates these effects is Ras. Moreover, within this pathway, different functions are possible depending on the activation of the specific receptor subtype. This review discusses the recent development of the non-lipid effects of statins in preventing cardiovascular disease progression by regulating Ras pathway of the TGF-β superfamily, especially RhoA/ROCK pathway.

Methods

A systematic PubMed database search of all English-language articles up to 2011 was conducted using the following terms: statin, TGF-β, Ras, ROCK, GGPP, inducible nitric oxide synthase, endothelial nitric oxide synthase, actin filament formation, PPARγ, MMP-2, and human trials.

Conclusion

With better understanding of the pathway, various mediators were identified; some of these mediators are important biomarkers producing more specific and accurate assessment of the pleiotropic effects of statins. The review of human trials also highlights that more specific biomarkers are employed in recent studies, and the non-lipid effects on human subjects are more accurately documented. Confirmation of the accuracy of these biomarkers by further large-scale studies and further development of new biomarkers may prove an important path leading to better patient selection for treatment, and thus better cost-effectiveness may be achieved.