TGF-beta Receptor Type Research Articles

Synergy between truncated c-Met (cyto-Met) and c-Myc in liver oncogenesis: importance of TGF-beta signalling in the control of liver homeostasis and transformation.

The c-Met tyrosine kinase receptor and its ligand, Hepatocyte Growth Factor/ Scatter Factor, have been implicated in human cancer. We have previously described that the transgenic expression of a truncated form of human c-Met (cyto-Met) in the liver confers resistance to several apoptotic stimuli. Here we show the impact of cyto-Met expression on liver proliferation and transformation. Despite a sixfold increase of hepatocyte proliferation, adult transgenic livers displayed normal size and architecture. We present evidence showing that activation of TGF-beta1 signalling controls the liver mass in cyto-Met mice. The oncogenic potential of cyto-Met was further assessed in the context of c-Myc-induced hepatocarcinogenesis, using WHV/c-Myc transgenic mice. Co-expression of cyto-Met and c-Myc further enhanced hepatocyte proliferation and caused a dramatic acceleration of the Myc-induced tumorigenesis, leading to the emergence of hepatocarcinomas in 3-4-month-old animals. Importantly, the TGF-beta receptor type II expression was strongly downregulated in most tumours, indicating that impairment of TGF-beta1-mediated growth inhibition plays a major role in accelerated neoplastic development. The strong potential of cyto-Met for oncogenic cooperation without direct transforming activity designates cyto-Met mice as an ideal tool for studying the early steps of multistage hepatocarcinogenesis and for identification of prognostic markers of transformation.

Soluble Transforming Growth Factor- β Type III Receptor Gene Transfection Inhibits Fibrous Airway Obliteration in a Rat Model of Bronchiolitis Obliterans

Post-transplant bronchiolitis obliterans (BO) is characterized by fibroproliferation and fibrous obliteration of distal airways in chronically rejected lungs. In this study, using a rat heterotopic allogeneic tracheal transplant model of BO, we evaluated the expression of transforming growth factor-beta (TGFbeta) during the development of airway fibrous obliteration. Immunohistochemical analysis revealed TGFbeta staining in infiltrating mononuclear cells at Days 2 and 7, and in the fibrous tissues until Day 21. Soluble TGFbeta receptor type III (TGFBIIIR), by blocking TGFbeta binding to its membrane receptors, functions as a TGFbeta antagonist. To study the role of TGFbeta in the development of BO, adenoviral-mediated soluble TGFBIIIR gene transfection (5 x 10(9) particles) was performed topically at the site of transplant on Day 5 after transplantation, which leads to inhibition of fibrous airway obliteration. In contrast, empty vector gene delivered through intramuscular injection, or given locally at Days 0 or 10 after tracheal transplantation had no significant effect. These results suggest that TGFbeta expressed in the allografts plays a pivotal role in the pathogenesis of BO. Soluble TGFBIIIR may competitively inhibit TGFbeta activity locally. Adenoviral-mediated soluble TGFBIIIR gene transfection should be further explored as a potential therapeutic modality for BO and other conditions involving chronic fibrosis.

Effect of age and hypoxia on TGFbeta1 receptor expression and signal transduction in human dermal fibroblasts: impact on cell migration.

Wound healing is critically affected by age, ischemia, and growth factors such as TGFbeta1. The combined effect of these factors on fibroblast migration, an essential component of wound healing, is poorly understood. To address this deficiency, we examined expression of TGFbeta receptor type I and II (TGFbetaRI and RII) under normoxia or hypoxia (1% O(2)) in cultured human dermal fibroblasts (HDFs) from young (ages 24-33) and aged (ages 61-73) adults. TGFbetaRI and RII expression was similar in both groups under normoxia. Hypoxia did not alter receptor levels in young HDFs but significantly decreased TGFbetaRI in aged cells (12 and 43%, respectively). Additionally, young cells displayed a 50% increase in activation of p42/p44 mitogen-activated kinase by TGFbeta1 (2-200 pg/ml) under hypoxia while aged cell levels of active p42/p44 decreased up to 24%. To determine functional outcomes of these findings, we measured the migratory capacity of the cells on type I collagen using a gold salt migration assay. Hypoxia increased the migratory index (MI) of young HDFs over normoxia by 30% but had no effect on aged cells. Under normoxia, TGFbeta1 (1-1000 pg/ml) increased young HDF migration in a concentration-dependent manner up to 109% over controls but minimally increased aged HDF migration (37%). Under hypoxia, TGFbeta1 significantly increased young cell MI at all concentrations but was without effect on the aged HDF response. These data demonstrate that aged fibroblasts have an impaired migratory capacity with complete loss of responsiveness to hypoxia and deficits in the migratory and signal transduction responsiveness to TGFbeta1 that may partly explain diminished healing capabilities often observed in aged patients.

Cytokines and cell cycle regulation in the fibrous progression of crescent formation in antiglomerular basement membrane nephritis of WKY rats.

Cytokines may regulate cell proliferation by cell-cycle-regulatory proteins, in which cyclin-dependent kinase inhibitors (CDKI) inhibit cell proliferation. We investigated whether CDKI p21 or p27, both of which are potentially regulated by transforming growth factor (TGF)-beta, a key cytokine in fibrogenesis, are involved together with TGF-beta and/or platelet-derived growth factor (PDGF) in the fibrous progression of glomerular crescent formation and examined the sequential change in the cell type and the cellular background of myofibroblasts in crescent formation. Crescentic glomerulonephritis (GN) was induced by i.v. injection of rabbit antirat glomerular basement membrane antiserum in WKY rats. Animals were killed 1, 2, 3 and 4 weeks after the induction of GN, and their kidneys were processed for immunohistochemical examination. After 1 week more than 85% of glomeruli showed cellular crescents, which became fibrocellular with decreased cellularity by 4 weeks. ED 1-positive macrophages were components of crescent cells in about 44% at 1-2 weeks, and this proportion declined markedly afterwards. Alpha smooth muscle actin (alpha SMA, a marker for myofibroblasts)-positive cells were found in Bowman's epithelial cells (BEP) and in some crescent cells at 1 week, becoming major components of crescent cells by 4 weeks (about 40%). It was 2 weeks before invasion of alpha SMA-positive interstitial cells into glomeruli was evident. PDGF-B and PDGF receptor beta-positive cells, indicating possible targets for PDGF, were found in BEP adjoining crescent formation almost exclusively from 1 to 2 weeks. By contrast, both TGF-beta receptor types I- and II-positive cells, indicating possible effectors for TGF-beta, were found in BEP and crescent formation, and the percentage of these in the crescent formation did not change until 4 weeks (about 32%). Cells with positive immunostaining for proliferating cell nuclear antigen and cyclin A, markers for cell proliferation, in the crescent formation peaked in number and proportion at 1-2 weeks, then decreased. In contrast, cells with positive immunostaining for p21 and p27, CDKI, were sparse at 1 week, and then increased markedly in number and in proportion, peaking at 3 (39.6%) or 2-3 weeks (about 25-30%), respectively. The present study demonstrates that restrained expression or a transient increase in p21 and p27 may be associated with proliferation or with inhibited proliferation of crescent cells, most of which are macrophages and myofibroblasts. The action, of PDGF and TGF-beta may contribute to the recruitment of myofibroblasts into the crescent. The action of TGF-beta on crescent cells might be linked to the expression of p21 and/or p27.

Transforming Growth Factor (TGF)-β1 Internalization: MODULATION BY LIGAND INTERACTION WITH TGF-β RECEPTORS TYPES I AND II AND A MECHANISM THAT IS DISTINCT FROM CLATHRIN-MEDIATED ENDOCYTOSIS

Transforming growth factor-beta (TGF-beta) internalization was studied by monitoring the uptake of (125)I-TGF-beta1 in Mv1Lu cells, which endogenously express TGF-beta receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 degrees C internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII. Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with (125)I- TGF-beta1 at 37 degrees C indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-beta1 at 4 degrees C for 2 h prior to switching the cells to 37 degrees C. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-beta1 bound to the cell surface at 37 degrees C and by a reduction in the labeling intensities of RI and RII in (125)I-TGF-beta1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-beta1 at 4 degrees C and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 degrees C, suggesting that prebinding promotes formation of stable RI.RII complexes that can signal independently of ligand.

TGF-beta isoform and receptor expression in giant cell tumor and giant cell lesions of bone.

The authors examined the distribution of tumor growth factor-beta (TGF-beta) isoforms and receptors in 35 giant cell tumor (GCT) of bone in comparison with a group of benign giant cell-containing lesions of bone, including 5 aneurysmal bone cysts, 2 cases of brown tumor of hyperparathyroidism, 3 nonossifying fibromas, and 7 cases of giant cell reparative granuloma. The results of immunohistochemical analysis of GCT showed a complete absence of TGF-beta1 expression in both mononuclear tumor cells and giant cells. Only reactive bone present within the tumor showed an intense immunoreactivity. Transforming growth factor-beta2 and TGF-beta3 were detected in the majority of cases (97.1% and 82.8%, respectively), whereas TGF-beta receptor type I (TGF-beta RI) and type II (TGF-beta RII) were diffusely expressed in all cases. Reverse transcription-polymerase chain reaction (RT-PCR) analysis performed on 10 GCTs with specific oligonucleotide primers demonstrated the presence of mRNA transcripts for TGF-beta1, 2, 3, and for TGF-beta RI and RII. Quantitative measurements of TGF-beta1 in conditioned media from primary cultures of GCT showed undetectable or very low amounts of the cytokine (0-23 pg/mL). The results of immunohistochemical analysis showed that all giant cell-containing lesions of bone were at least focally positive for the 3 isoform of TGF-beta, with positivity present both in osteoclast-like giant cells and mononuclear cells, and diffusely positive for TGF-beta RI and RII. Reverse transcription-polymerase chain reaction analysis conducted on samples from 3 nonossifying fibromas and 1 giant cell reparative granuloma confirmed the expression of the corresponding mRNA. In conclusion, according to the current data, GCT of bone can be distinguished from other giant cell-containing lesions of bone on the basis of the absence of TGF-beta1 expression at the protein level, which appears to be the result of posttranslational regulation processes.

High tumor grade in salivary gland mucoepidermoid carcinomas and loss of expression of transforming growth factor beta receptor type II.

Mucoepidermoid carcinoma (MEC) of salivary glands is a malignant, locally aggressive neoplasm with metastatic potential. The clinical course is usually dependent on histology; however, low-grade carcinomas can result in metastases and tumor-related death. Transforming growth factor beta1 (TGF-beta1) is a potent cytokine that affects growth inhibition of various cells and stimulates extracellular matrix production and angiogenesis. Loss of TGF-beta receptor type II (TGF-beta RII) expression has been related to resistance of TGF-beta1-mediated growth control and tumor progression. In this study, we correlate MEC tumor grade with expression of TGF-beta1 and TGF-beta RII. Immunohistochemical staining was performed on 16 MEC specimens for activated forms of TGF-beta1 and TGF-beta RII. The percentage of cells in which staining yielded positive findings for activated TGF-beta1 and TGF-beta RII was correlated with tumor grade. Activated TGF-beta1 was detected in 16 specimens (100%) of MEC and showed strong positive and diffuse staining. Predominately cytoplasmic staining of TGF-beta1 was seen in salivary gland ducts, stroma, and endothelial cells. There was an inverse correlation between tumor grade and loss of expression of TGF-beta RII. All low-grade MEC tumors yielded positive staining results, whereas only one case of intermediate-grade MEC had TGF-beta RII expression. No high-grade MEC showed TGF-beta RII expression. Loss of expression of TGF-beta RII correlates with tumor grade. The localization of activated TGF-beta1 within neoplastic epithelium, tumor-associated stroma, and endothelium suggests that it might play a role in the stromal proliferation and/or angiogenesis associated with MEC.

Toward an understanding of nonsyndromic craniosynostosis: altered patterns of TGF-beta receptor and FGF receptor expression induced by intrauterine head constraint.

Although the etiology of nonsyndromic forms of craniosynostosis remains uncertain, recent experiments from our laboratory have demonstrated that fetal head constraint induces cranial suture fusion in mice through a process associated with altered patterns of transforming growth factor beta (TGF-beta) isoform expression. Other recent studies have highlighted the role of secreted signaling molecules, including members of the TGF-beta superfamily and the fibroblast growth factors (FGFs), as well as their receptors, in regulating suture development and fusion. The purpose of these experiments was to examine the potential role of TGF-beta receptors and FGF receptor 2 (FGFR2) in nonsyndromic craniosynostosis by determining their temporospatial patterns of expression during development complicated by intrauterine head constraint. This study consisted of two groups of C57BI/6J mice: an experimental group subjected to intrauterine constraint and a control unconstrained group. Fetal head constraint was induced by performing uterine cerclage on day 17.5 of gestation and allowing intrauterine fetal growth to continue 24 and 48 hours beyond the normal gestational period. Control animals underwent hysterotomy on day 17.5 and the nonconstrained pups were allowed to continue intra-abdominal fetal growth 48 hours beyond normal gestation. Expression of TGF-beta receptor types I and II, and FGFR2 in the calvarial tissue was determined by immunohistochemical analysis. In the unconstrained control animals, there was minimal immunoreactivity for both TGF-beta receptors and FGFR2 within the coronal suture. After 24 hours of constraint, however, there was a marked increase in immunoreactivity of TGF-beta receptors and FGFR2 in the osteoblasts along the osteogenic fronts and in the dural cells. After 48 hours, there was continued expression of both type I and type II receptors and FGFR2 within the midsutural mesenchyme of the coronal suture, in the osteoblasts, and in the dura. The authors demonstrated substantial upregulation of TGF-beta receptor types I and II and FGFR2 in coronal sutures subjected to in utero constraint. These results suggest an important role for TGF-beta/TGF-beta receptor, and FGF/FGFR signaling in the pathogenesis of constraint-induced craniosynostosis.

Responsiveness to transforming growth factor-beta (TGF-beta)-mediated growth inhibition is a function of membrane-bound TGF-beta type II receptor in human breast cancer cells.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth and proliferation of breast epithelial cells, and loss of sensitivity to its effects has been associated with malignant transformation and tumorigenesis. The biological effects of TGF-beta are mediated by the TGF-beta receptor complex, a multimer composed of TGF-beta receptor type I (TbetaR-I) and TGF-beta receptor type II (TbetaR-II) subunits. Evidence suggests that loss of expression of Tbeta3R-II is implicated in the loss of sensitivity of tumorigenic breast cell lines to TGF-beta-mediated growth inhibition. A panel of human breast cell lines, including the immortalized MCF-10F and tumorigenic MCF-7, ZR75-1, BT474, T47-D, MDA-MB231, BT20, and SKBR-3 cell lines, was characterized for responsiveness to TGF-beta-induced G1 growth arrest. Only the nontumorigenic MCF-10F and the tumorigenic MDA-MB231 cell lines demonstrated a significant inhibitory response to TGF-beta1 and a significant binding of 125I-labeled TGF-beta ligand. While expression of TbetaR-I mRNA was similar across the panel of cell lines, TbetaR-II mRNA expression was decreased significantly in all seven tumorigenic cell lines in comparison with the nontumorigenic MCF- 10F cell line. When total cellular protein was fractionated by centrifugation, TbetaR-I protein was observed in both the cytosolic and membrane fractions at similar levels in all cell lines; however, TbetaR-II protein was present in the cytosolic fraction in all cell lines, but was observed in the membrane fraction of only the TGF-beta-responsive MCF-10F and MDA-MB231 cells. Thus, lack of membrane-bound TbetaR-II protein appears to be an important determinant of resistance to TGF-beta-mediated growth inhibition in this group of breast cell lines.

Microsatellite Instability in Transforming Growth Factor- β 1 Type II Receptor Gene in Alveolar Lining Epithelial Cells of Idiopathic Pulmonary Fibrosis

It has been reported that transforming growth factor (TGF)-beta, which plays an integral role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), suppresses proliferation of alveolar epithelial cells in vitro. Although hyperplastic lesions of alveolar lining epithelial cells (ALECs) are characteristic pathologic features of IPF, the mechanism of their involvement in the pathogenesis has not yet been extensively studied. On the assumption that the hyperplastic ALECs have escaped from the growth-inhibitory effects of TGF-beta, we searched for mutations in the microsatellite of the TGF-beta receptor type II (T beta RII) gene. To detect a deletion in the polyadenine tract in exon 3 of the T beta RII gene, cells were isolated by microdissection from lung sections of IPF patients, and DNA was extracted from these cells and amplified by high-fidelity polymerase chain reaction. A total of 121 sites of hyperplastic ALECs from 11 IPF patients were analyzed, and a one-base-pair deletion was detected in nine sites from five patients. The mutation was also detected in smooth muscle-like cells of the thickened pulmonary artery. In some tissue areas where the deletion was detected, low T beta RII expression was confirmed by immunohistochemical staining. These data suggest that microsatellite instability in the T beta RII gene occurred in some lesions of hyperplastic ALECs in IPF, although at a low incidence, and that this genetic disorder might play a partial role in the pathologic changes of IPF.

Transforming growth factor beta receptor induction in herniated intervertebral disc tissue: an immunohistochemical study.

Transforming growth factor beta (TGF-beta) is a potent inducer of angiogenesis and fibrogenesis. There is presently little information about the pathophysiological function of TGF-beta in herniated disc tissue. In order to analyze the cellular role and activation of TGF-beta after disc herniation we immunostained frozen material from 38 disc herniation operations and from eight macroscopically normal discs from organ donors. Polyclonal TGF-beta-I, TGF-beta-II and TGF-beta receptor type II antibodies were used with the avidin biotin complex (ABC-) immunoperoxidase method. All the herniated discs were TGF-beta immunopositive. Such immunoreactivity was mainly associated with disc cells. In a few samples, capillaries were also TGF-beta immunopositive. Immunopositivity was similarly observed in the control discs. To analyze possible differences between the two groups, we calculated the ratio of immunopositive disc cells. For all three antibodies, a statistically significantly (Mann-Whitney test, P = 0.0001) higher number of disc cells showed immunopositivity in the herniated discs. The increase in TGF-beta receptor immunopositivity suggested induction of TGF-beta receptors in herniated discs. Our results support an active regulatory role for TGF-beta in disc cell metabolism.

The chromosome 9q genes TGFBR1, TSC1, and ZNF189 are rarely mutated in bladder cancer.

This study assessed a series of bladder tumours and bladder tumour cell lines for sequence variation in the Krüppel-like zinc finger gene ZNF189, the tuberous sclerosis complex gene 1 (TSC1), and the TGF beta receptor type I (TGFBR1). All three genes have been mapped to 9q regions commonly deleted in transitional cell carcinoma of the bladder. Mutation analysis of the coding sequence of these genes revealed several variant bands that were shown to represent polymorphisms. Mutation analysis of the ZNF189 gene in bladder cancer cell lines identified one amino acid substitution (lysine-->isoleucine) at position 323 in exon 4. For the TSC1 gene, two mutations were identified in two out of 27 independent cell lines. Both mutations result in a truncated protein. Furthermore, one out of 36 bladder tumours had a frameshift mutation in exon 7 of the TSC1 gene. No tumour-specific mutations were found in the TGFBR1 gene. The length of the polyalanine tract present in exon 1 of the TGFBR1 gene was also investigated. It has been suggested that the allele with six alanines (6A) is more frequent in patients with bladder and other cancers, so bladder cancer patients were compared with normal controls. In both groups, the percentage of heterozygotes was 17%. These data do not support a role for the 6A allele in bladder cancer susceptibility.

Morphologic and molecular analysis of estrogen-induced pituitary tumorigenesis in targeted disruption of transforming growth factor-beta receptor type II and/or p27 mice.

The critical genes and products involved in estrogen-induced tumorigenesis of the pituitary gland were investigated in heterozygous transforming growth factor-beta (TGF-beta) receptor type II and p27 knockout mouse models. Tgfbr2(+/-), p27(+/-); Tgfbr2(+/-), and p27(+/-) mice and C57BL/6J wild-type mice received sc implantation of estrogen or placebo pellets for 16 or 25 wk, after which the mice were sacrificed and their pituitary glands removed for examination. The bromodeoxyuridine labeling indexes in tissue from both the anterior and intermediate pituitary lobes from p27 (+/-) and Tgfbr2(+/-); p27(+/-) mice were significantly higher than those from wild-type and Tgfbr2(+/-) mice after treatment with estrogen for 16 wk. Pituitary tumorigenesis was significantly accelerated in Tgfbr2(+/-), p27(+/-), and Tgfbr2(+/-); p27(+/-) mice compared with wild-type mice after treatment with estrogen for 16 wk. Pituitary tumorigenesis was not accelerated in Tgfbr2(+/-); p27(+/-) mice compared with Tgfbr2(+/-) or p27(+/-) mice. Expression of TGF-beta receptor type II mRNA was lower in the pituitary gland of Tgfbr2(+/-) mice than in wild-type mice before estrogen treatment and was significantly reduced after treatment. Pituitary tumorigenesis is accelerated in mice with severe TGF-beta resistance, and greatly accelerated in mice with TGF-beta resistance combined with decreased p27 expression compared with wild-type mice. Both the TGF-beta receptor type II gene and p27 gene and their products are involved in estrogen-induced tumorigenesis.

Role of transforming growth factor-beta1 in prostate cancer.

TGF-beta1 is an important regulator of the normal and malignant prostate. In the non-malignant prostate, TGF-beta1 stimulates cell differentiation, inhibits epithelial cell proliferation, and induces epithelial cell death. TGF-beta1 is secreted into semen where it is an important immunosuppressive factor. Prostate cancer cells express high levels of TGF-beta1, which seems to enhance prostate cancer growth and metastasis by stimulating angiogenesis and by inhibiting immune responses directed against tumour cells. Prostate cancer cells frequently lose their TGF-beta receptors and acquire resistance to the anti-proliferative and pro-apoptotic effects of TGF-beta1. Accordingly, high expression of TGF-beta1 and loss of TGF-beta receptor expression have been associated with a particularly bad prognosis in human prostate cancer patients. TGF-beta1 also seems to be a mediator of castration-induced apoptosis in androgen dependent normal and malignant prostate epithelial cells. The ability of some prostate tumours to avoid castration-induced apoptosis may not, however, be simply due to loss of TGF-beta receptor type I or type II expression in the tumour cells. It may also be related to an inability of these cells to up-regulate TGF-beta receptor levels in response to castration or possibly due to defects downstream of the receptors. Short-term therapy-induced changes in the TGF-beta system in prostate tumours can probably be used to predict the long-term response to androgen ablation treatment. Further investigations into the TGF-beta system in the prostate are needed, however, to elucidate how alterations in this system affect the behaviour of prostate tumours, and whether this system can be manipulated for therapeutical purposes.

Activin A and activin receptors in the human thyroid: a link to the female predominance of goiter?

Activin A belongs to the TGF-beta family of growth factors involved in control of tissue formation by growth inhibition and to a family of hormones involved in human reproduction. Serum levels of dimeric activin A display a sexual dimorphism with significantly higher circulating hormone concentrations in men than in women after menopause. Since goiter is far more frequent in women than in men, we investigated the role of this sex-related growth factor in the human thyroid. Primary cultures were obtained from 3 patients with goiter, and 4 with recurrent goiter. Activin A significantly inhibited the proliferation of human thyroid follicular epithelial cells in vitro in concentrations between 0.5 and 50 ng/ml and was almost as potent as TGFbeta-1. Analysis in native tissues from 12 normal thyroids, 16 goiters, and 5 Graves thyroids demonstrated mRNA expression of the activin subunit betaA, TGFbeta-receptor type I and II, activin receptors type I receptors alk2 and alk4, and activin type II receptors actRII and actRIIb. Isoform analysis of the major functional type I activin receptor alk4 revealed the full-length transcript SKR2-1 and transcripts for the partially truncated proteins SKR2-2 and SKR2-3 in normal thyroids and goiters. Semi-quantitative RT-PCR revealed a significant 2.5-fold decrease of mRNA for alk4-1 type I activin receptors in goiter as compared to normal thyroid (p < 0.05), whereas expression of the type II receptor actRII was unchanged. These data identify a novel growth inhibitory pathway by activin in the human thyroid. Down-regulation of activin receptor type lb in goiter hints towards end-organ changes which could contribute to deficient growth inhibition. These data provide a mechanistic model how the sexually dimorphic hormone activin A may protect males from goitrogenesis.

Repression of transforming growth factor-beta receptor type I promoter expression by Sp1 deficiency.

In this report, we describe the mechanism of TGF-beta receptor type I (RI) repression in the GEO human colon carcinoma cells. Treatment of GEO cells with the DNA methyltransferase inhibitor, 5 azacytidine induced RI expression and restored TGF-beta response. A stably transfected RI promoter-reporter construct (RI-Luc) expressed higher activity in the 5 aza C treated GEO cells, suggesting the activation of a transactivator for RI transcription. Gel shift analysis indicated enhanced binding of proteins from the 5 aza C treated nuclear extracts to radiolabeled Sp1 oligonucleotides specifically contained in the RI promoter. Protein stability studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5 aza C treated GEO cells. Further, transfection of Sp1 cDNA into untreated GEO control cells increased RI promoter activity and thus induced RI expression. 5 aza C mediated Sp1 expression in Sp1 deficient GEO colon and MCF-7 breast cancer cells also enhanced the activity of several other Sp1 dependent promoters such as TGF-beta receptor type II (RII), Cyclin A and p21/waf1/cip1. These results indicate that restoration of Sp1 in several different types of Sp1 deficient cells leads to enhanced activation of a wide range of Sp1 dependent promoters.

Modulation of collagenase 3 in human osteoarthritic cartilage by activation of extracellular transforming growth factor beta: role of furin convertase.

Treatment of normal cartilage with transforming growth factor beta (TGFbeta) can increase the synthesis of collagenase 3 by chondrocytes and mimic the in situ distribution of this enzyme in osteoarthritic (OA) cartilage, which occurs predominantly in the deep zone. In this study, we examined the elements of the TGFbeta system that are potentially relevant to this effect. TGFbeta1 and TGFbeta2 levels in cultured cartilage explants were determined by enzyme-linked immunosorbent assay (ELISA). OA cartilage explants were treated with small latent TGFbeta1 complex in the presence of various inhibitors, and collagenase 3 levels were determined by ELISA. The inhibitors were against serine proteases, plasmin, cathepsins, furin, and a neutralizing antibody against the mannose-6 phosphate/ insulin-like growth factor 2 receptor (M6P/IGF-2R). Small latent TGFbeta1, TGFbeta receptor types I, II, and III (TGFbetaRI, RII, and RIII), M6P/IGF-2R, and furin were immunolocalized in cartilage. Our data showed that latent TGFbeta1 is the major isoform that is synthesized; levels of 17.2 +/-1.7 pg/mg and 1.1 +/- 0.3 pg/mg tissue wet weight (mean +/- SEM) were found for total TGFbeta1 and TGFbeta2, respectively, in OA cartilage. A general serine protease inhibitor abrogated activation of both endogenous and exogenous small latent TGFbeta1. Plasmin and furin inhibitors and anti-M6P/IGF-2R reduced the levels of exogenous small latent TGFbeta1 complex-induced collagenase 3 by 33%, 95%, and 76%, respectively, but the cathepsin inhibitor had no effect. Immunolocalization of the small latent TGFbeta1 complex as well as of TGFbetaRI and RII revealed a statistically significant increase in the chondrocyte score in only the deep zone of OA cartilage. The M6P/IGF-2R level was significantly higher in OA cartilage in both the superficial and deep zones. Furin was found in normal cartilage exclusively in the superficial zone, whereas in OA cartilage, a level similar to that in normal cartilage was found in the superficial zone, but a significantly higher cell score (mean +/- SEM 23.6 +/- 4.7%) was registered in the deep zone. The mechanisms of TGFbeta activation/ activity with regard to collagenase 3 modulation in cartilage appear to be controlled by furin convertase with or without M6P/IGF-2R. These factors and the small latent TGFbeta complex are increased in the deep zone of OA cartilage, corresponding to the preferential site of collagenase 3 production.

Modulation of collagenase 3 in human osteoarthritic cartilage by activation of extracellular transforming growth factor β: Role of furin convertase

Treatment of normal cartilage with transforming growth factor beta (TGFbeta) can increase the synthesis of collagenase 3 by chondrocytes and mimic the in situ distribution of this enzyme in osteoarthritic (OA) cartilage, which occurs predominantly in the deep zone. In this study, we examined the elements of the TGFbeta system that are potentially relevant to this effect.TGFbeta1 and TGFbeta2 levels in cultured cartilage explants were determined by enzyme-linked immunosorbent assay (ELISA). OA cartilage explants were treated with small latent TGFbeta1 complex in the presence of various inhibitors, and collagenase 3 levels were determined by ELISA. The inhibitors were against serine proteases, plasmin, cathepsins, furin, and a neutralizing antibody against the mannose-6 phosphate/ insulin-like growth factor 2 receptor (M6P/IGF-2R). Small latent TGFbeta1, TGFbeta receptor types I, II, and III (TGFbetaRI, RII, and RIII), M6P/IGF-2R, and furin were immunolocalized in cartilage.Our data showed that latent TGFbeta1 is the major isoform that is synthesized; levels of 17.2 +/-1.7 pg/mg and 1.1 +/- 0.3 pg/mg tissue wet weight (mean +/- SEM) were found for total TGFbeta1 and TGFbeta2, respectively, in OA cartilage. A general serine protease inhibitor abrogated activation of both endogenous and exogenous small latent TGFbeta1. Plasmin and furin inhibitors and anti-M6P/IGF-2R reduced the levels of exogenous small latent TGFbeta1 complex-induced collagenase 3 by 33%, 95%, and 76%, respectively, but the cathepsin inhibitor had no effect. Immunolocalization of the small latent TGFbeta1 complex as well as of TGFbetaRI and RII revealed a statistically significant increase in the chondrocyte score in only the deep zone of OA cartilage. The M6P/IGF-2R level was significantly higher in OA cartilage in both the superficial and deep zones. Furin was found in normal cartilage exclusively in the superficial zone, whereas in OA cartilage, a level similar to that in normal cartilage was found in the superficial zone, but a significantly higher cell score (mean +/- SEM 23.6 +/- 4.7%) was registered in the deep zone.The mechanisms of TGFbeta activation/ activity with regard to collagenase 3 modulation in cartilage appear to be controlled by furin convertase with or without M6P/IGF-2R. These factors and the small latent TGFbeta complex are increased in the deep zone of OA cartilage, corresponding to the preferential site of collagenase 3 production.

Deletion of one adenine base within the polyadenine tract of transforming growth factor-beta receptor type II in human MDA-MB-231 breast cancer cell line.

Microsatellite mutation of the polyadenine tract (10 adenine repeat) within the TbetaR-II [transforming growth factor-beta (TGF-beta) receptor type II] coding region have been found in a variety of human cancers, particularly in association with microsatellite instability (MSI). Since breast cancers have been reported to carry MSI, although its frequencies were quite variable, we examined whether microsatellite mutation of the polyadenine tract occurs in the human breast cancer MDA-MB-231 cell line. TbetaR-II expression in MDA-MB-231 was found to be low on our Northern and Western analyses. Sequencing analysis for the polyadenine tract of TbetaR-II cDNA obtained from MDA-MB-231 cells indicated heterozygous deletion of one adenine base. Subsequently, sensitivity to TGF-beta induced growth-inhibitory effects of control and TbetaR-II transfected MDA-MB-231 cells was compared. The sensitivity of TbetaR-II transfectants to exogenous as well as endogenous TGF-beta1 was increased distinctly compared with control transfectant. These results suggest that heterozygous deletion of one adenine base within the polyadenine tract in MDA-MB-231 cells might lead to reduced TbetaR-II expression and sensitivity to TGF-beta.

The association of transforming growth factor-beta 1 with myometrial invasion of endometrial carcinomas through effects on matrix metalloproteinase.

The association of transforming growth factor-beta 1 (TGF-beta 1) with a matrix metalloproteinase (MMP) and a tissue inhibitor of metalloproteinase (TIMP), as well as myometrial invasion of endometrial cancer was studied. The effects of TGF-beta 1 on cellular invasiveness, gelatinase activity, and expression of TIMP-1 were examined in 2 endometrial adenocarcinoma cell lines, KLE and Ishikawa. Plasma was obtained from 8 endometrial cancer patients with Stage-Ia disease, from 6 with Stage-Ib disease, and from 4 with Stage-Ic disease, and the levels of TGF-beta 1 were measured by enzyme immunoassays. The immunohistochemical expression of MMP-9, TIMP-1, TGF-beta 1, and TGF-beta receptor Type I in tumor tissue from the same patients also was detected. Invasiveness, gelatinase activity, and the expression of TIMP-1 were higher in KLE cells than in Ishikawa cells, and they were increased by treatment with rTGF-beta 1. The expression of TGF-beta receptor Type I was higher in KLE cells than in Ishikawa cells, which were unresponsive to exogenous TGF-beta 1. The plasma levels of TGF-beta 1 were greater in Stage-Ib and Stage-Ic patients than in Stage-Ia patients. MMP-9 and TGF-beta receptor Type I were expressed mainly in tumor cells, while TIMP-1 and TGF-beta 1 were localized in both tumor epithelial cells and stromal cells. MMP-9 and TIMP-1 were expressed only in Stage-Ib and Stage-Ic patients, although TGF-beta 1 and TGF-beta receptor Type I were ubiquitous. Myometrial invasion of endometrial cancers involves an increase in gelatinase activity, regulated to some extent by TGF-beta 1 in an autocrine or paracrine fashion.