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.     

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.     

Syndecan-2 regulates transforming growth factor-beta signaling

Transforming growth factor-beta (TGF-beta) has multiple functions including increasing extracellular matrix deposition in fibrosis. It functions through a complex family of cell surface receptors that mediate downstream signaling. We report here that a transmembrane heparan sulfate proteoglycan, syndecan-2 (S2), can regulate TGF-beta signaling. S2 protein increased in the renal interstitium in diabetes and regulated TGF-beta-mediated increased matrix deposition in vitro. Transfection of renal papillary fibroblasts with S2 or a S2 construct that has a truncated cytoplasmic domain (S2DeltaS) promoted TGF-beta binding and S2 core protein ectodomain directly bound TGF-beta. Transfection with S2 increased the amounts of type I and type II TGF-beta receptors (TbetaRI and TbetaRII), whereas S2DeltaS was much less effective. In contrast, S2DeltaS dramatically increased the level of type III TGF-beta receptor (TbetaRIII), betaglycan, whereas S2 resulted in a decrease. Syndecan-2 specifically co-immunoprecipitated with betaglycan but not with TbetaRI or TbetaRII. This is a novel mechanism of control of TGF-beta action that may be important in fibrosis.     

Inhibition of Nb2 T-lymphoma cell growth by transforming growth factor-beta.

1. Mitochondria isolated from the gut-dwelling nematodes Nippostrongylus brasiliensis and Ascaridia galli (muscle and gut + reproductive tissue) were examined for cytochromes, and it was observed that N. brasiliensis and A. galli muscle tissue mitochondria contained a-, b- and c-type cytochromes, but their stoichiometries were quite different (1:2:1.9 and 1:11.4:13.6 respectively); A. galli gut + reproductive-tissue mitochondria, however, only contained b and c cytochromes, in a ratio of 1:0.8. 2. CO difference spectra showed the presence of CO-reacting b-type cytochrome(s) in all three types of mitochondria; the fast-reacting species comprised 30, 44 and 39% of the total in N. brasiliensis, A. galli muscle and A. galli gut + reproductive-tissue mitochondria respectively. 3. Cytochrome aa3 was observed in N. brasiliensis mitochondria and in those from A. galli muscle, but was below the level of detectability (less than 0.005 nmol/mg of protein) for A. galli gut + reproductive-tissue mitochondria. 4. Photochemical action spectra for the reversal of CO inhibition of the endogenous respiration of whole worms (at 24 microM- and 40 microM-O2 respectively for N. brasiliensis and A. galli) gave maxima at 598 and 542-543 nm, corresponding to the alpha- and beta-absorption maxima of cytochrome aa3, and at 567 nm (b-type cytochrome) for both worms. These results suggest that cytochrome aa3 is the major functional oxidase in N. brasiliensis, whereas the CO-reacting b-type cytochrome dominates in A. galli.     

Monoclonal antibodies recognizing transforming growth factor-beta. Bioactivity neutralization and transforming growth factor beta 2 affinity purification

Four mAb able to recognize transforming growth factor-beta 2 (TGF-beta)2 were obtained. One of these mAb, 1D11.16, was able to neutralize the biological activity of both TGF-beta 1 and beta 2 in vitro. This was demonstrated in an Il-1, PHA-dependent thymocyte mitogenic assay that is inhibitable by TGF-beta in a dose-dependent manner. All four mAb recognized the dimeric form of TGF-beta 2 in Western blots. The mAb were also found to immunoprecipitate [125I]-TGF-beta 2. mAb 3C7.14 coupled to Sepharose could efficiently immunoaffinity purify TGF-beta 2 from a complex mixture of proteins. Affinity constants were determined for the four mAb and they ranged from 3.4 x 10(8) to 1.6 x 10(7) L/mol.     

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.     

Cell-type-specific localization of transforming growth factor-beta 2 and transforming growth factor-beta 1 in the hamster ovary: differential regulation by follicle-stimulating hormone and luteinizing hormone.

Cell-type-specific localization and gonadotropin regulation of transforming growth factor-beta 1 (TGF-beta 1) and transforming growth factor-beta 2 (TGF-beta 2) in the hamster ovary were evaluated immunohistochemically under three conditions: (1) during the estrous cycle (Day 1 = estrus; Day 4 = proestrus); (2) after the blockade of periovulatory gonadotropin surges by phenobarbital, and (3) after FSH and/or LH treatment of long-term hypophysectomized hamsters. Ovarian TGF-beta 1 activity was primarily localized in theca and interstitial cells. The activity increased moderately but significantly after the preovulatory LH surge and reached a peak at 0900 h, Day 2 h; oocytes showed considerable activity. TGF-beta 1 immunoreactivity subsequently fell to low levels in theca-interstitial cells through 0900 h, Day 4. Significant TGF-beta 2 immunoreactivity appeared after the surge, mainly in the granulosa cells of both preantral and antral follicles; a few interstitial cells surrounding preantral follicles showed discrete staining. TGF-beta 2 immunoreactivity in granulosa cells and in interstitial cells next to preantral follicles reached a peak at 0900 h, Day 1, and persisted up to 0900 h, Day 2; oocytes showed no staining. Phenobarbital treatment blocked the appearance of TGF-beta 1 and TGF-beta 2 immunoreactivities at 1600 h, Day 4; however, a rebound in immunoreactivities was observed with the onset of the surge after a 1-day delay. Replacement of LH to long-term hypophysectomized hamsters resulted in a marked increase in TGF-beta 1 immunoreactivity in the interstitial cells, but FSH, although it induced follicular development, did not influence ovarian TGF-beta 1 activity. Treatment with FSH, however, induced a massive increase in TGF-beta 2 immunoreactivity in the granulosa cells of newly developed antral and preantral follicles but not in the interstitial cells; LH, on the other hand, had no significant effect on TGF-beta 2 activity. Treatment with FSH and LH combined resulted in a dramatic increase in TGF-beta 2 immunoreactivity in granulosa and interstitial cells and in TGF-beta 1 in theca and interstitial cells comparable to their peak activity in intact animals. Western analyses substantiated the presence of TGF-beta 1 and TGF-beta 2 in the hamster ovary and the specificity of immunolocalization. These studies, therefore, provide critical evidence that TGF-beta 1 and TGF-beta 2 in the hamster ovary are expressed in specific cell types and that their expression is differentially regulated by LH and FSH, respectively.     

Endothelial cell regulation by transforming growth factor-beta.

Pronounced changes including growth inhibition, increased matrix deposition and suppression of cell-associated proteolytic activity, take place in endothelial cells (EC) upon the application of TGF-beta. Interrelationships between these effects have shed some light on the mechanism of action of TGF-beta and on its role in regulating EC function vis-a-vis angiogenesis. For instance, preliminary evidence has indicated that increased levels of certain matrix components may be partly responsible for the antiproliferative action of TGF-beta. In addition, TGF-beta and bFGF have opposing effects on cellular proteolytic balance which may contribute to the antagonistic effect that TGF-beta has on bFGF-induced EC growth and possibly to the anti-angiogenic effect exerted by TGF-beta under certain circumstances. Of particular interest in this regard is the fact that physical contact between EC and vascular mural cells in EC:mural cell cocultures has been found to generate active TGF-beta, thus further implicating TGF-beta in the maintenance of the quiescent, differentiated aggregation of EC as found in vascular structures in vivo. While more information is needed to define what, if any role TGF-beta plays in endothelial differentiation, it is to be noted that many of the cellular and biochemical processes affected by TGF-beta are linked to differentiation. It is therefore possible that the growth inhibition of EC by TGF-beta primes them for differentiation and/or is critical for the maintenance of a differentiated state.     

Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia.

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.     

Retinoic acid induces transforming growth factor-beta 2 in cultured keratinocytes and mouse epidermis.

We have studied the functional interaction between retinoic acid and transforming growth factor-beta (TGF-beta), using the mouse epidermis as a model system. Treatment with retinoic acid increases expression of TGF-beta 2 in cultured keratinocytes in vitro, as well as in the epidermis in vivo. This TGF-beta 2 is secreted in a biologically active form that can bind to surface receptors, in contrast to most other conditions in which TGF-beta is secreted in a latent form. Specific antibodies to TGF-beta 2 partially reverse the ability of retinoic acid to inhibit DNA synthesis in cultured keratinocytes. The regulation of TGF-beta 2 expression by retinoic acid may have important physiological and pharmacological roles in the maintenance of epidermal homeostasis.     

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.     

Stimulation of normal human fibroblast collagen production and processing by transforming growth factor-beta

Transforming growth factor-beta (TGF beta) a growth factor with diverse effects on cellular growth and metabolism, caused dramatic stimulation of total protein and collagen synthesis by confluent normal human dermal fibroblasts in culture in a dose-dependent manner. Gel electrophoresis of the newly synthesized macromolecules from the culture media of TGF beta treated cultures demonstrated accelerated procollagen processing. These results indicate that TGF beta is capable of qualitatively and quantitatively influencing the biosynthesis of matrix molecules by fibroblasts, and raise the possibility that TGF may play a role in the development of normal and pathologic fibrogenesis.     

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.     

Binding of transforming growth factor-beta 1 to immobilized human alpha 2-macroglobulin.

Native alpha 2-macroglobulin (alpha 2M) and alpha 2M-methylamine were immobilized in 96-well microtiter plates. 125I-labeled transforming growth factor-beta 1 (TGF-beta 1) bound to both alpha 2M variants; however, greater binding was observed with alpha 2M-methylamine. Binding of 125I-TGF-beta 1 (0.2 nM) to immobilized alpha 2M-methylamine was inhibited by nonradiolabeled TGF-beta 1 (up to 74% with 0.4 microM TGF-beta 1). Approximately 10% of the TGF-beta 1-alpha 2M-methylamine complex was covalent. Treatment of alpha 2M-methylamine with iodoacetamide prior to immobilization completely eliminated covalent TGF-beta 1 binding; the total amount of 125I-TGF-beta 1-alpha 2M-methylamine complex detected was unchanged. The binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was not significantly inhibited by increasing the ionic strength to 1.0 M. Binding and complex dissociation were also unaffected by changes in pH within the range 6.9-8.9. Acidic pH dramatically decreased binding and promoted complex dissociation; no binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was detected at pH 3.5. The interaction of TGF-beta 1 with immobilized alpha 2M-methylamine was not significantly changed by 1.0 mM EDTA or 1.0 mM CaCl2. ZnCl2 (1.0 mM) completely eliminated binding. This result was not due to TGF-beta 1 precipitation or aggregation. Inhibition of 125I-TGF-beta 1 binding to alpha 2M-methylamine was 50% complete (IC50) with 30 microM ZnCl2. Native alpha 2M, thrombospondin, and alpha 2M-methylamine (in solution) decreased binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine. The IC50 values for these three proteins were 520, 160, and 79 nM, respectively. The TGF-beta 1-binding activity of native alpha 2M may have reflected, at least in part, trace-contamination with alpha 2M-proteinase complex.