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PubMed1995

Progressive loss of sensitivity to growth control by retinoic acid and transforming growth factor-beta at late stages of human papillomavirus type 16-initiated transformation of human keratinocytes.

Retinoids (vitamin A and its natural and synthetic derivatives) have shown potential as chemopreventive agents, and diets poor in vitamin A and/or its precursor beta-carotene have been linked to an increased risk of cancer at several sites including the cervix. Human papillomavirus (HPV) plays an important role in the etiology of cervical cancer. We have developed an in vitro model of cancer progression using human keratinocytes (HKc) immortalized by HPV16 DNA (HKc/HPV16). Although immortal, early passage HKc/HPV16, like normal HKc, require epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation and undergo terminal differentiation in response to serum and calcium. However, following prolonged culture, growth factor independent HKc/HPV16 lines that no longer require EGF and BPE can be selected (HKc/GFI). Further selection of HKc/GFI produces lines that are resistant to serum- and calcium- induced terminal differentiation (HKc/DR). HKc/DR, but not early passage HKc/HPV16, are susceptible to malignant conversion following transfection with viral Harvey ras or Herpes simplex virus type II DNA. We have investigated the sensitivity of low to high passage HKc/HPV16 and HKc/GFI to growth control by all-trans-retinoic acid (RA, an active metabolite of vitamin A). Early passage HKc/HPV16 are very sensitive to growth inhibition by RA, and in these cells RA decreases the expression of the HPV16 oncogenes E6 and E7. However, as the cells progress in culture they lose their sensitivity to RA. Growth inhibition by RA may be mediated through the cytokine transforming growth factor-beta (TGF-beta), a potent inhibitor of epithelial cell proliferation. RA treatment of HKc/HPV16 and HKc/GFI results in a dose-and time-dependent induction (maximal of 3-fold) in secreted levels of TGF-beta. Also, Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced TGF-beta 1 and TGF-beta 2 expression about 3- and 50-fold, respectively. We next studied the effect of TGF-beta 1 and TGF-beta 2 on the proliferation of early to late passage HKc/HPVa6, HKc/GFI and HKc/DR. While early passage HKc/HPV16 were as sensitive as normal HKc to growth inhibition by TGF-beta 1 and TGF-beta 2, the cells became increasingly resistant to TGF-beta during in vitro progression, with the proliferation of HKc/DR being virtually unaffected by TGF-beta 1 or TGF-beta 2 treatment. Overall, loss of growth inhibition by RA parallels loss of TGF-beta sensitivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Division↗

PubMed2002

Dynamic expression patterns of transforming growth factor-beta(2) and transforming growth factor-beta receptors in experimental glomerulonephritis.

Numerous studies have demonstrated the involvement of the transforming growth factor (TGF) isoform beta(1) in the pathogenesis of renal fibroproliferative diseases. Although in vitro studies suggest that TGF-beta(2) is equally potent to TGF-beta(1) in terms of its antimitogenic and fibrogenic effects, much less is known about the regulation of TGF-beta(2) in renal diseases associated with glomerular cell hyperplasia and matrix expansion. Here we investigated the glomerular expression patterns of TGF-beta(2) and of the TGF-beta receptors I, II, and III during the course of rat anti-Thy1.1 nephritis (days 2, 6, 12, and 56), a model characterized by transient mesangial hypercellularity and extracellular matrix accumulation. TGF-beta(2) exhibited dynamic changes in expression. Immunohistochemical double-staining of renal sections revealed that most TGF-beta(2)-positive cells in control glomeruli were podocytes with few TGF-beta(2)-positive mesangial cells. This staining pattern could also be observed in human kidney. On day 6 of anti-Thy1.1 nephritis both TGF-beta(2) positive podocytes and mesangial cells were more abundant. By western blot analysis of isolated glomeruli from nephritic rats, protein expression of TGF-beta(2) was upregulated tenfold over control glomeruli, peaking on day 6 of the disease. In cultured rat mesangial cells we found that the TGF-beta(2) and TGF-beta(1) isoforms were equally potent in terms of nuclear accumulation of phosphorylated Smad 2/3, inhibition of DNA synthesis, and induction of beta(1)-integrin and type I collagen protein synthesis. Protein expression of the TGF-beta receptor I was not detected by immunohistochemistry in control glomeruli but was markedly induced in the mesangium on day 6 of nephritis. Mesangial staining for TGF-beta receptors II and III was detected in normal kidneys. Expression of TGF-beta receptor II was strongly enhanced on days 6 and 12 of disease, while TGF-beta receptor III was upregulated only on day 6. In summary, we report marked yet transient upregulation of TGF-beta(2) protein and of TGF-beta receptors I, II, and III in glomerular cells during anti-Thy1.1 nephritis. These results are in keeping with the notion that TGF-beta(2) and its receptors participate in the pathogenesis and/or resolution of this transient form of glomerulonephritis.

Animals↗

PubMed2002

Polymorphisms of transforming growth factor-beta1 and transforming growth factor-beta1 type II receptor genes are associated with acute graft-versus-host disease in children with HLA-matched sibling bone marrow transplantation.

The aim of this study was to determine whether the gene polymorphisms of Th1/Th2 and immunoregulatory cytokines were associated with aGVHD in Japanese children receiving allogeneic bone marrow transplantation (allo BMT). We investigated polymorphisms of genes encoding interleukin (IL)-4, IL-4 receptor (IL-4 R), IL-10, transforming growth factor (TGF)-beta1, TGF-beta1 type II receptor (TGF-beta1 RII), interferon (IFN)-gamma, IFN-gamma type 2 receptor (IFN-gamma R2), and IFN regulatory factor (IRF)-1. Sixty-seven patients were treated with allo BMT from HLA-identical siblings, and aGVHD was observed in 38. TGF-beta1 codon 10 leucine (Leu) /proline (Pro) polymorphism in donors was associated with the development of aGVHD. Patients having donors with the Pro allele had aGVHD more frequently than those without Pro allele (30/45 vs 8/20, odds ratio = 3.00; P = 0.04). TGF-beta1 RII 1167 C/T polymorphism in recipients was also associated with the development of aGVHD. The incidence was significantly higher in recipients with T allele than in those without T allele (21/27 vs 16/35, odds ratio = 4.16; P = 0.01). In conclusion, genetic backgrounds of TGF-beta1 and TGF-beta1 RII may be involved in the development of aGVHD in HLA-matched sibling BMT in Japanese children.

Acute Disease↗

PubMed2002

Expression of transforming growth factor beta1, transforming growth factor type I and II receptors, and TNF-alpha in the mucosa of the small intestine in infants with food protein-induced enterocolitis syndrome.

BACKGROUND: TNF-alpha secreted by activated T cells is known to increase intestinal permeability, whereas transforming growth factor (TGF) beta has the ability to protect the epithelial barrier. OBJECTIVE: We determined the expression of TGF-beta1, its receptors, and TNF-alpha on the mucosa of small intestine to investigate their roles in the pathogenesis of food protein-induced enterocolitis syndrome (FPIES). METHODS: Twenty-eight infants diagnosed with FPIES by means of clinical criteria and challenge test results were included. Immunohistochemical stains for TGF-beta1, type 1 and 2 TGF-beta receptors, and TNF-alpha on duodenal biopsy specimens were performed. RESULTS: TGF-beta1 expression was generally depressed in patients. Expression of type 1 TGF-beta receptor was significantly lower in the patients who had villous atrophy compared with expression in those patients who did not (P <.001) and negatively correlated with the severity of atrophy (r = -0.59, P <.001). Expression of type 2 TGF-beta receptor showed no significant difference between the patients with or without villous atrophy. The immunoreactivity for both TGF-beta receptors on lamina proprial cells was slight or negative. TNF-alpha expression was detected on both epithelial and lamina proprial cells and was significantly greater in the patients who had villous atrophy compared with that in the patients who did not (P <.01). CONCLUSION: Our results suggest that decreased countering activity of TGF-beta1 against T-cell cytokines is implicated in the pathogenesis of FPIES. The significantly lower expression of type 1 TGF-beta receptor compared with type 2 receptor suggests the differential contribution of each receptor to the diverse biologic activities of TGF-beta in the intestinal epithelium.

Activin Receptors, Type I↗

PubMed1997

Function of the type V transforming growth factor beta receptor in transforming growth factor beta-induced growth inhibition of mink lung epithelial cells.

The type V transforming growth factor beta (TGF-beta) is a 400-kDa nonproteoglycan membrane protein that co-expresses with the type I, type II, and type III TGF-beta receptors in most cell types. The type V TGF-beta receptor exhibits a Ser/Thr-specific protein kinase activity with distinct substrate specificity (Liu, Q., Huang, S. S., and Huang, J. (1994) J. Biol. Chem. 269, 9221-9226). In mink lung epithelial cells, the type V TGF-beta receptor was found to form heterocomplexes with the type I TGF-beta receptor by immunoprecipitation with antiserum to the type V TGF-beta receptor after 125I-TGF-beta affinity labeling or Trans35S-label metabolic labeling of the cells. The kinase activity of the type V TGF-beta receptor was stimulated after treatment of mink lung epithelial cells with TGF-beta. TGF-beta stimulation resulted in the growth inhibition of wild-type mink lung epithelial cells and to a lesser extent of the type I and type II TGF-beta receptor-defective mutants, although higher concentrations of TGF-beta were required for the growth inhibition of these mutants. TGF-beta was unable to induce growth inhibition in human colorectal carcinoma cells lacking the type V TGF-beta receptor but expressing the type I and type II TGF-beta receptors. These results suggest that the type V TGF-beta receptor can mediate the TGF-beta-induced growth inhibitory response in the absence of the type I or type II TGF-beta receptor. These results also support the hypothesis that loss of the type V TGF-beta receptor may contribute to the malignancy of certain carcinoma cells.

Activin Receptors, Type I↗

PubMed2001

Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling.

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.

Activin Receptors, Type I↗

PubMed2000

In situ expression of transforming growth factor-beta1-3, latent transforming growth factor-beta binding protein and tumor necrosis factor-alpha in liver tissue from patients with chronic hepatitis C.

BACKGROUND: The mechanisms determining liver damage in chronic hepatitis C remain unclear. The aim was to evaluate the in situ expression of transforming growth factor beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha), two key cytokines implicated as important pathogenic mediators in the development of liver fibrosis. METHODS: In situ expression of TNF-alpha and of TGF-beta isoforms 1-3, and its transport protein latent TGF-beta binding protein (LTBP), was determined by immunohistochemistry in 9 untreated patients with chronic hepatitis C infection and in 6 controls without liver disease. In addition, TGF-beta1 expression was analyzed in 10 HCV patients before and after treatment with interferon-alpha alone, or in combination with ribavirin. RESULTS: Liver biopsies from HCV patients showed positive staining for TGF-beta1-3 isoforms and LTBP, and to a lesser degree for TNF-alpha, in areas with inflammation and fibrosis. Normal control liver showed no positive staining. TGF-beta1 expression before treatment, quantified by morphometric analysis, did not differ between non-responders and sustained responders. In patients responding to therapy, TGF-beta1 expression decreased in parallel with histological improvement, while no difference in TGF-beta1 expression was seen before and after treatment in non-responders. CONCLUSION: These results suggest that TNF-alpha and all three isoforms of TGF-beta are involved in the pathogenesis of HCV related liver disease, and that treatment leading to eradication of the virus affects the expression of TGF-beta1.

Adult↗

PubMed1992

Transforming growth factor beta stimulates mitogenically mouse NIH3T3 fibroblasts and those cells transformed by the EJ-H ras oncogene.

TGF-beta 1 stimulates thymidine incorporation and the growth rate of mouse NIH3T3 fibroblasts and of those cells transformed by the EJ-H-ras oncogene (TR15 cells), in the presence and the absence of serum. Thymidine incorporation, in serum-deprived cells, is stimulated to a higher degree by 0.1-1 ng/ml of TGF-beta in NIH3T3 than in TR15 cells, which have a 10-fold higher basal level of incorporation. In both cell types TGF-beta 1 is as active, or more active than other mitogens (TGF-alpha, PDGF-AB, bFGF) at the same concentration. The growth rate of NIH3T3 cells, in low serum or serum-free (S-) medium, is stimulated by only 10 picograms/ml of TGF-beta 1, and that of TR15 cells, in S- medium, by only 1 picogram/ml. In contrast, TGF-beta 1 inhibits mitogenically unestablished mouse embryo fibroblasts and these fibroblasts immortalized spontaneously and able to grow in S- medium. It also inhibits the anchorage-independent growth of TR15 cells. NIH3T3 and TR15 cells respond, similarly, to TGF-beta activated by acification of their culture medium. The kinetics of thymidine incorporation and of activation of the c-myc proto-oncogene, observed already after 1 hr, in treated NIH3T3 and TR15 cells, suggests a direct mitogenic stimulation. The level of activated c-myc RNA is 2-fold higher at 2 hr, and subsequently decreases relatively less in the TR15 cells.

3T3 Cells↗

PubMed2003

Development and characterization of DP-153, a nontumorigenic prostatic cell line that undergoes malignant transformation by expression of dominant-negative transforming growth factor beta receptor type II.

We have developed a nontumorigenic epithelial cell line, DP-153, from the dorsal prostate of a Lobund/Wistar rat treated with N-methyl-N-nitrosourea and testosterone propionate. DP-153 cells express cytokeratins 5 and 14, but not cytokeratin 18, consistent with a basal epithelial cell phenotype. Similar to the nontumorigenic NRP-152 prostatic cell line, DP-153 cells do not form tumors in athymic mice and retain many of the properties of normal prostatic cells. They express prostatic acid phosphatase and androgen receptors and require several mitogens (epidermal growth factor, insulin, dexamethasone, and cholera toxin) for sustained growth in culture under serum-containing conditions. DP-153 cells are also growth-stimulated by keratinocyte growth factor and basic fibroblast growth factor and growth-inhibited by all-trans-retinoic acid, 1,25-dihydroxyvitamin D(3), and transforming growth factor (TGF)-beta1. We demonstrate that expression of dominant-negative TGF-beta receptor type II by retroviral transduction of DP-153 cells leads to complete loss of TGF-beta1-induced growth inhibition. When transplanted s.c. in athymic mice, DP-153 cells expressing dominant-negative TGF-beta receptor type II form tumors as early as 4 weeks, in contrast to the vector control and parental cell line, which do not form tumors even 8 months after transplantation, supporting the observation that TGF-beta functions as a tumor suppressor in these cells. Our data further support that DP-153 is a suitable cell line for analysis of normal prostatic growth and carcinogenesis.

Animals↗

PubMed2004

The expression of matrix metalloproteinases-9, transforming growth factor-beta1 and transforming growth factor-beta receptor I in human atherosclerotic plaque and their relationship with plaque stability.

BACKGROUND: Transforming growth factor-beta (TGF-beta) and matrix metalloproteinases-9 (MMP-9) have been implicated in the pathogenesis of human atherosclerosis but their relationship during lesion progression are poorly understood. The objective of this study was to investigate the expression of MMP-9, TGF-beta1 and TGF-beta receptor I (TbetaR-I) in human atherosclerotic plaque and their relationship and plaque stability. METHODS: Specimens of human coronary artery atherosclerotic plaques were obtained from 41 patients undergoing coronary endarterectomy, and were paraffin embedded, sectioned at 4 microm intervals then stained with haematoxylin and eosin. They were divided into stable (with no or only little lipid core) and unstable plaque groups (with lipid core size > 40%): the immunohistochemical staining were performed for MMP-9, TGF-beta1 and TbetaR-I. RESULTS: The expression of MMP-9 in the unstable plaques was much higher than in the stable ones, but the expression of TGF-beta1 was higher in the stable plaques. There was no similar significant difference for TbetaR-I. Correlation analysis showed that there was a negative correlation between the expression of MMP-9 and TGF-beta1 (r = -0.332, P = 0.034 for average areal density; r = -0.373, P = 0.016 for average optical density). CONCLUSIONS: There were close relationships between MMP-9, TGF-beta1 and plaque stability. Enhanced production of MMP-9 may participate in the formation of unstable plaque, while TGF-beta1 maybe an important stabilizing factor in preventing transition into an unstable plaque phenotype.

Activin Receptors, Type I↗

PubMed2004

An increased transforming growth factor beta receptor type I:type II ratio contributes to elevated collagen protein synthesis that is resistant to inhibition via a kinase-deficient transforming growth factor beta receptor type II in scleroderma.

OBJECTIVE: Aberrant transforming growth factor beta (TGFbeta) signaling has been implicated in the pathogenesis of scleroderma (systemic sclerosis [SSc]), but the contribution of specific components in this pathway to SSc fibroblast phenotype remains unclear. This study was undertaken to delineate the role of TGFbeta receptor type I (TGFbetaRI) and TGFbetaRII in collagen overexpression by SSc fibroblasts. METHODS: Primary dermal fibroblasts from SSc patients and healthy adults were studied (n = 10 matched pairs). Adenoviral vectors were generated for TGFbetaRI (AdTGFbetaRI), TGFbetaRII (AdTGFbetaRII), and kinase-deficient TGFbetaRII (AdDeltakRII). TGFbetaRI basal protein levels were analyzed by (35)S-methionine labeling/immunoprecipitation and immunohistochemistry. Type I collagen and TGFbetaRII basal protein levels were analyzed by Western blot and newly secreted collagen by (3)H-proline incorporation assay. RESULTS: Analysis of endogenous TGFbetaRI and TGFbetaRII protein levels revealed that SSc TGFbetaRI levels were increased 1.7-fold (P = 0.008; n = 7) compared with levels in healthy controls, while TGFbetaRII levels were decreased by 30% (P = 0.03; n = 7). This increased TGFbetaRI:TGFbetaRII ratio correlated with SSc collagen overexpression. To determine the consequences of altered TGFbetaRI:TGFbetaRII ratio on collagen expression, healthy fibroblasts were transduced with AdTGFbetaRI or AdTGFbetaRII. Forced expression of TGFbetaRI in the range corresponding to elevated SSc TGFbetaRI levels increased basal collagen expression in a dose-dependent manner, while similar TGFbetaRII overexpression had no effect, although transduction of fibroblasts at higher multiplicities of infection led to a marked reduction of basal collagen levels. Blockade of TGFbeta signaling via AdDeltakRII resulted in approximately 50% inhibition of basal collagen levels in healthy fibroblasts and in 5 of 9 SSc cell lines. A subset of SSc fibroblasts (4 of 9 cell lines) was resistant to this treatment. SSc fibroblasts with the highest levels of TGFbetaRI were the least responsive to collagen inhibition via DeltakRII. CONCLUSION: This study indicates that an increased TGFbetaRI:TGFbetaRII ratio may underlie aberrant TGFbeta signaling in SSc and contribute to elevated basal collagen production, which is insensitive to TGFbeta signaling blockade via DeltakRII.

Activin Receptors, Type I↗

PubMed1998

Expression of recombinant human soluble type II transforming growth factor-beta receptor in Pichia pastoris and Escherichia coli: two powerful systems to express a potent inhibitor of transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell metabolism, proliferation, and differentiation. To study the role of endogenous TGF-beta in processes such as tissue repair and inflammation, potent and specific inhibitors are required. Because the type II TGF-beta receptor (TGF beta RII) has a high affinity for TGF-beta, the extracellular domain of TGF beta RII (TGF-beta sRII) was expressed in Pichia pastoris and Escherichia coli. Expression of the soluble TGF beta sRII using P. pastoris resulted in a soluble, heterogeneously glycosylated protein which was secreted into the medium. Although expression of TGF beta sRII in E. coli resulted in the formation of insoluble inclusion bodies, solubilization and refolding resulted in a biologically active protein. Because in both systems a C-terminal 6x His coding sequence was inserted behind the coding sequence for the extracellular domain of TGF beta RII the recombinant proteins could be purified by a powerful, single-step procedure using a Ni-NTA agarose. The purified proteins appeared to be potent inhibitors of TGF-beta 1 and TGF-beta 3. In contrast, TGF beta sRII was less effective in neutralization of TGF-beta 2. In conclusion, biologically active TGF beta sRII can be produced using P. pastoris and E. coli expression systems. The ease of these expression systems, the powerful single step purification and low costs makes it possible to produce TGF beta s RII in large amounts to further elucidate the role of TGF-beta 1 and TGF-beta 3 in physiological processes like tissue repair and inflammation.

Base Sequence↗

PubMed2001

The effect of pasteurization on transforming growth factor alpha and transforming growth factor beta 2 concentrations in human milk.

Transforming growth factor alpha (TGF-alpha) and beta 2 (TGF-beta2) are present in human milk and are involved in growth differentiation and repair of neonatal intestinal epithelia. Heat treatment at 56 degrees C has been shown effective for providing safe banked donor milk, with good retention of other biologically active factors. The purpose of our study was to determine the effect of heat sterilization on TGF-alpha and TGF-beta2 concentrations in human milk. Twenty milk samples were collected from 20 lactating mothers in polypropylene containers and frozen at -20 degrees C for transport or storage. Before heat treatment by holder pasteurization, the frozen milk was thawed and divided into 1-mL aliquots. All samples were heated in an accurately regulated water bath until a holding temperature was achieved, then held for 30 minutes using constant agitation. Holding temperature ranged from 56.5 degrees C to 56.9 degrees C. The milk was then stored at 4 degrees C overnight for analysis the following day. The concentration of TGF-alpha was measured by radioimmunoassay. Mean concentration +/- SD of TGF-alpha in raw milk samples was 119+/-50 pg/mL, range 57 to 234. The mean concentration +/- SD of TGF-alpha in heat treated samples was 113+/-50 pg/mL, range 51 to 227. TGF-alpha concentration was minimally affected by pasteurization, with an overall loss of 6.1%. Of 19 samples, 4 had increased and 15 had decreased concentrations after pasteurization (mean percent SEM: 94%+/-7% of raw milk, range 72%+/-107%). The concentration of acid-activated TGF-beta2 was measured by enzyme-linked immunosorbent assay. Mean concentration +/- SD of TGF-beta2 in raw milk samples was 5624+/-5038 pg/mL, range 195 to 15480. The mean concentration +/- SD of TGF-beta2 in heat-treated samples was 5073+/-4646 pg/mL, range 181 to 15140. TGF-beta2 survived with relatively little loss (0.6%): of 18 samples, 11 had increased and 7 had decreased concentrations after pasteurization (mean percent +/- SEM: 99.4+/-6.7% of raw milk, range 79%-120%). In conclusion, both TGF-alpha and TGF-beta2 were well-preserved in whole milk after holder pasteurization at 56.5 degrees C. The relative increase in growth factor concentration in some of the samples may be attributable to the release of that factor from the cellular and/or fat compartments into the aqueous fraction of human milk. These findings have implications regarding use of donor milk as an alternate source of growth factors and cytokines for the newborn gut when mother's milk is unavailable.

Epithelial Cells↗

PubMed1997

Mutation analysis of the transforming growth factor-beta type II receptor in human cell lines resistant to growth inhibition by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta) binds the type II TGF-beta growth factor receptor (RII) to inhibit the growth of most epithelial tissues. Most human colon and gastric cancers with microsatellite instability (MI) have frameshift mutations in polynucleotide repeats within the RII coding region; these mutations truncate the receptor protein and disable the serine/threonine kinase to produce TGF-beta resistance. To further investigate the type, frequency and tissue distribution of RII mutations, we selected 24 human cancer cell lines from various tissues which were previously reported to be resistant to the inhibitory effects of TGF-beta. We developed protocols for non-isotopic SSCP analysis of PCR products from genomic DNA samples, and we tested them for microsatellite instability. PCR-SSCP analysis followed by DNA sequencing identified deletion mutations in the exon 3 poly-adenine tract in three colon tumor cell lines: LS174T and SW48 had a single base deletion and LS411 had a two base deletion. Among the 24 previously unreported cell lines, only these three demonstrated microsatellite instability. These and other recent data indicate that RII mutations are essentially confined to colon and gastric cancers with microsatellite instability. The narrow spectrum of tissues containing RII mutations illustrates the complexity of genetic checkpoints in human carcinogenesis.

Cell Division↗

PubMed1998

Expression of transforming growth factor-beta1 and transforming growth factor-beta Type-II receptor mRNA in papillary thyroid carcinoma.

Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of epithelial cell proliferation. Signal transduction by TGF-beta1 involves direct binding to the TGF-beta Type-II receptor and then the formation of a heterodimeric complex of TGF-beta Type-I and Type-II receptor. To explore the role of TGF-beta1 in thyroid carcinoma, we examined the expression of TGF-beta1 and TGF-beta Type-II receptor mRNA by northern blotting analysis in both 14 papillary thyroid carcinomas and surrounding normal thyroid tissues. Relative mRNA level was determined by scanning densitometry of the autoradiogram and corrected for loading differences using a human beta-actin cDNA probe. The relative mRNA levels of TGF-beta1 in 12 out of 14 papillary thyroid carcinomas were higher than those in surrounding normal thyroid tissues. In contrast, the relative mRNA levels of TGF-beta Type-II receptor were reduced to 60.1+/-18.3% of those of normal thyroid tissues in 10 papillary thyroid carcinomas. There were no clear relationships between the relative mRNA levels for TGF-beta1 and TGF-beta Type-II receptor and the histological characteristics of papillary thyroid carcinomas. The relative mRNA levels for TGF-beta1 and TGF-beta Type-II receptor did not show significant differences in thyroid carcinomas with or without lymph node metastases. There was a negative correlation between the TGF-beta Type-II receptor mRNA level and tumor size, while no significant correlation was observed between the TGF-beta1 mRNA level and tumor size. In conclusion, most papillary thyroid carcinomas overexpress TGF-beta1 mRNA but exhibit a reduction in TGF-beta Type-II receptor mRNA. The reduction of TGF-beta Type-II receptor mRNA may play a role in the pathogenesis of papillary thyroid carcinoma.

Adult↗

PubMed2000

Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway.

Our previous results have shown that transforming growth factor beta (TGFbeta) rapidly activates Ras, as well as both ERKs and SAPKs. In order to address the biological significance of the activation of these pathways by TGFbeta, here we examined the role of the Ras/MAPK pathways and the Smads in TGFbeta(3) induction of TGFbeta(1) expression in untransformed lung and intestinal epithelial cells. Expression of either a dominant-negative mutant of Ras (RasN17) or a dominant-negative mutant of MKK4 (DN MKK4), or addition of the MEK1 inhibitor PD98059, inhibited the ability of TGFbeta(3) to induce AP-1 complex formation at the TGFbeta(1) promoter, and the subsequent induction of TGFbeta(1) mRNA. The primary components present in this TGFbeta(3)-inducible AP-1 complex at the TGFbeta(1) promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGFbeta(1) promoter or addition of PD98059 inhibited the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Collectively, our data demonstrate that TGFbeta(3) induction of TGFbeta(1) is mediated through a signaling cascade consisting of Ras, the MAPKKs MKK4 and MEK1, the MAPKs SAPKs and ERKs, and the specific AP-1 proteins Fra-2 and JunD. Although Smad3 and Smad4 were not detectable in TGFbeta(3)-inducible AP-1 complexes at the TGFbeta(1) promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGFbeta(3) to transactivate the TGFbeta(1) promoter. Thus, although the Ras/MAPK pathways are essential for TGFbeta(3) induction of TGFbeta(1), Smads may only contribute to this biological response in an indirect manner.

Animals↗

PubMed2003

Adenovirus-mediated gene transfer of transforming growth factor-beta3, but not transforming growth factor-beta1, inhibits constrictive remodeling and reduces luminal loss after coronary angioplasty.

BACKGROUND: Extracellular matrix (ECM) remodeling is central to the development of restenosis after PTCA. Substantial evidence implicates transforming growth factor-beta1 (TGF-beta1), a regulator of ECM deposition by vascular cells, in its pathogenesis. TGF-beta3 reduces TGF-beta1-induced ECM deposition in cutaneous wounds. We therefore investigated the effects of intracoronary expression of TGF-beta3 and TGF-beta1 on luminal loss after angioplasty. METHODS AND RESULTS: Porcine coronary arteries received an adenovirus expressing TGF-beta3, TGF-beta1, or lacZ (beta-galactosidase), or PBS only, at the site of angioplasty. Morphometric analysis 28 days after angioplasty confirmed reduced luminal loss in TGF-beta3 vessels (-0.65+/-0.10 mm2) compared with lacZ (-1.18+/-0.19 mm2) or PBS only (-1.19+/-0.17 mm2; P=0.003). Luminal loss was not reduced in TGF-beta1 vessels (-1.02+/-0.19 mm2; P=0.48). An increase in the external elastic lamina area in TGF-beta3-treated vessels (+0.73+/-0.32 mm2) contrasted with decreases in control vessels (mean, -0.53+/-0.17 mm2; P=0.001) and TGF-beta1 vessels (-0.87+/-0.34 mm2; P=0.003). Collagen content increased at the site of injury in TGF-beta3-treated vessels (26.1+/-14.2%) but decreased in the lacZ (-22.8+/-6.6%) and PBS-only (-23.4+/-7.0%; P=0.002) groups and was not significantly changed in TGF-beta1-treated vessels. CONCLUSIONS: Expression of TGF-beta3 inhibits constrictive remodeling after PTCA and reduces luminal loss. This is accompanied by increased adventitial collagen, which may act as an external "scaffold" preventing vessel constriction. These findings confirm the potential of gene therapies that modify ECM remodeling for prophylaxis of restenosis.

Adenoviridae↗

PubMed1997

Induction of apoptosis in rat thecal/interstitial cells by transforming growth factor alpha plus transforming growth factor beta in vitro.

In each estrous cycle dominant follicles are selected from a growing pool to develop to the preovulatory stage and to ovulate. Those follicles that do not ovulate must be eliminated in order to maintain the constant mass and homeostasis of the ovary. Granulosa cells are lost by apoptosis at the onset of follicular atresia, whereas apoptotic thecal cells are identified at later stages of atresia. Since transforming growth factor (TGF) alpha and TGF beta 1 have been implicated in the regulation of thecal cell physiology we have localized these growth factors by immunohistochemistry in sections of ovaries from 25-day-old rats, an age at which the ovary exhibits a wave of atresia of preantral follicles. Thecal cells contained TGF alpha and TGF beta 1 throughout the entire process of follicular atresia. To determine if these growth factors could influence thecal cell death, thecal/interstitial cells were isolated from 25-day-old rats, and maintained in culture with growth factors. Subconfluent cultures treated with TGF alpha or TGF beta 1 alone remained healthy whereas in the presence of both TGF alpha and TGF beta 1 there was light microscopical evidence of rounding up of cells and detachment from the monolayer. Chromatin condensation and internucleosomal fragmentation, characteristic of apoptosis, were observed by nucleic acid staining and fluorescence microscopy of thecal/interstitial cells treated with TGF alpha plus TGF beta 1. Further evidence that these cells were undergoing apoptosis came from DNA analysis and the demonstration of DNA laddering. This response of thecal/interstitial cells to TGF alpha plus TGF beta 1 was density dependent; confluent cultures were protected from the induction of apoptosis under these conditions. We conclude that thecal cells are eliminated from atretic follicles by the active and strictly regulated process of involving the combined actions of TGF alpha and TGF beta 1.

Animals↗