TGF-beta RI Research Articles

TGF-Beta Signaling Pathways in the Study of Expression in Patients with Atrial Fibrillation Atrial Tissue

Objective: To explore the relationship between TGF-beta signaling pathway and atrial fibrillation in patients with mitral valve disease, and reveal the mechanism of TGF-beta signaling pathway in development of atrial fibrillation. Methods: Firstly, the fibrosis extent of right atrial appendage tissues were tested by Masson’s trichrome staining. Secondly, the protein expression of TGF-beta1, TGF-beta RI, RII, RIII, P-Smad2/3, Smad2/3, Smad4, Smad7, TAK1, p38, ATF-2 were tested by Western blot. Results: There were significant difference between atrial fibrillation group and control group in fibrosis extent and the diameter of left atria (p0.05). The patients with atrial fibrillation are associated with the more serious fibrosis and larger diameter of left atria. For further study, the Western bolt results reveal that protein expression of TGF-beta1 and Smad4 were significant higher in atrial fibrillation and sever fibrosis group, other factors were no difference between trial and control group. Conclusion: There are severing atrial fibrosis in atrial fibrillation patients with mitral valve diseases. The over expression of TGF-beta1 and Smad4 maybe is an important reason that induces atrial fibrosis in atrial fibrillation, and the atrial fibrosis appears to play a role in the development of atrial fibrillation. Fibrosis response by TGF-beta1 initiating maybe only depends on mutations of the TGF-beta Smad signaling pathway.

Abstract 1876: Grape seed extract targets TGFβ/Smad signaling in its potential efficacy against human head and neck cancer cells

Abstract Head and neck squamous cell carcinoma(HNSCC) is the sixth most common malignancy and the major cause of morbidity and mortality worldwide. In HNSCC, various etiological factors produce mutations or epigenetic inactivation of numerous oncogenes and/or tumor suppressor genes, suggesting that all these factors synergistically enhance the progression of normal cells to carcinoma. Transforming growth factor (TGF-beta)/activin-Smad signaling acts as a potent tumor suppressor prior to tumor initiation; however, at later stages, resistance to TGF-beta mediated growth suppression develops and an increase in cell proliferation are observed in a variety of cancers including HNSCC, making this pathway a potential target for both prevention and therapy of various malignancies including HNSCC. Several animal studies in past have shown that consumption of fruits and vegetables and/or the supplements derived from them is associated with reduced risk of cancer development including HNSCC. Grape seed extract (GSE) is one such supplement that has received enormous attention in recent years due to its strong anti-cancer and chemopreventive potential in several cancer models. Accordingly, here we investigated the potential ability of GSE in targeting TGF-beta/Smad4 signaling in two human HNSCC cells, FaDu and Detroit 562 which differ in the status of TGF-beta and Smad4. GSE treatment inhibited growth and increased death in both HNSCC cell lines in a dose- and a time-dependent manner. Specifically, GSE treatment (40 µg/ml for12-72 h) resulted in 20-80% (p<0.001) and 13-60% (p<0.001) decrease in total cell number, increased cell death by 8-49% and 10-53% (p<0.001) in FaDu and Detroit 562 cells, respectively. To elucidate the molecular mechanism underlying cell growth inhibitory and increased cell death effects of GSE, we focused on the possible effect of GSE on TGF-beta/Smad-dependent and -independent signaling pathways in both FaDu and Detroit 562 HNSCC cells. We found that GSE treatment at 40 µg/ml conc. in DMSO decreases the expression of TGF-beta RI/ RII in both FaDu and Detroit 562 cells at 24, 48 and 72 h. GSE-treated cells also showed the reduced expression of Smad4, phosphorylation of Smad2 (at ser 465/467) without any effect on the phosphorylation of Smad3 (at ser 423/425) at 24, 48 and 72 h in Detroit 562 cells. Conversely, in FaDu cells which lack Smad4, similar GSE treatments decreased the phosphorylation of both Smad2 (at ser 465/467) and Smad3 (at ser 423/425). Taken together, our results show that GSE effectively inhibits tumor promoting function of dysfunctional TGF-beta/Smad signaling pathway by blocking the phosphorylation of Smad2/3, which in turn are surrogate marker for active TGF-beta signaling pathway. These effects of GSE seem to have a potential role in its overall anti-cancer activity against human HNSCC cells, suggesting future efficacy studies in pre-clinical animal models of HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1876.

Hyperosmolarity enhanced susceptibility to renal tubular fibrosis by modulating catabolism of type I transforming growth factor‐β receptors

Hyperosmolarity plays an essential role in the pathogenesis of diabetic tubular fibrosis. However, the mechanism of the involvement of hyperosmolarity remains unclear. In this study, mannitol was used to evaluate the effects of hyperosmolarity on a renal distal tubule cell line (MDCK). We investigated transforming growth factor-beta receptors and their downstream fibrogenic signal proteins. We show that hyperosmolarity significantly enhances the susceptibility to exogenous transforming growth factor (TGF)-beta1, as mannitol (27.5 mM) significantly enhanced the TGF-beta1-induced increase in fibronectin levels compared with control experiments (5.5 mM). Specifically, hyperosmolarity induced tyrosine phosphorylation on TGF-beta RII at 336 residues in a time (0-24 h) and dose (5.5-38.5 mM) dependent manner. In addition, hyperosmolarity increased the level of TGF-beta RI in a dose- and time-course dependent manner. These observations may be closely related to decreased catabolism of TGF-beta RI. Hyperosmolarity significantly downregulated the expression of an inhibitory Smad (Smad7), decreased the level of Smurf 1, and reduced ubiquitination of TGF-beta RI. In addition, through the use of cycloheximide and the proteasome inhibitor MG132, we showed that hyperosmolarity significantly increased the half-life and inhibited the protein level of TGF-beta RI by polyubiquitination and proteasomal degradation. Taken together, our data suggest that hyperosmolarity enhances cellular susceptibility to renal tubular fibrosis by activating the Smad7 pathway and increasing the stability of type I TGF-beta receptors by retarding proteasomal degradation of TGF-beta RI. This study clarifies the mechanism underlying hyperosmotic-induced renal fibrosis in renal distal tubule cells.

Attenuation of the Transforming Growth Factor β-Signaling Pathway in Chronic Venous Ulcers

Transforming growth factor beta (TGFbeta) is important in inflammation, angiogenesis, reepithelialization and connective tissue regeneration during wound healing. We analyzed components of TGFbeta signaling pathway in biopsies from 10 patients with nonhealing venous ulcers (VUs). Using comparative genomics of transcriptional profiles of VUs and TGFbeta-treated keratinocytes, we found deregulation of TGFbeta target genes in VUs. Using quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis, we found suppression of TGFbeta RI, TGFbeta RII and TGFbeta RIII, and complete absence of phosphorylated Smad2 (pSmad2) in VU epidermis. In contrast, pSmad2 was induced in the cells of the migrating epithelial tongue of acute wounds. TGFbeta-inducible transcription factors (GADD45beta , ATF3 and ZFP36L1) were suppressed in VUs. Likewise, genes suppressed by TGFbeta (FABP5, CSTA and S100A8) were induced in nonhealing VUs. An inhibitor of Smad signaling, Smad7 was also downregulated in VUs. We conclude that TGFbeta signaling is functionally blocked in VUs by downregulation of TGFbeta receptors and attenuation of Smad signaling resulting in deregulation of TGFbeta target genes and consequent hyperproliferation. These data suggest that application of exogenous TGFbeta may not be a beneficial treatment for VUs.

Changes in expression, and/or mutations in TGF-β receptors (TGF-β RI and TGF-β RII) and Smad 4 in human ovarian tumors

Loss of sensitivity to transforming growth factor beta (TGF-beta) signaling typically occurs in human ovarian cancer cells, but there is paucity of information regarding this in human ovarian tumors. Thus the association of inactivating mutations and/or variations in expression levels of TGF-beta signaling components with human ovarian tumors was evaluated. Forty human ovarian tissue samples were analyzed for mutations and/or variations in the expression of transforming growth factor beta signaling components. Mutation studies were done through reverse transcription (RT) PCR, single strand conformation polymorphism analysis and automated DNA sequencing. Expression studies were carried out by semi quantitative RT PCR and western blotting. DNA binding ability of Smad complexes and expression of downstream targets were also analyzed. The six alanine repeat containing variant of TGF-beta RI was seen in 27% of the tumor cases studied, in addition to the 45 bp nucleotide deletions in exon 1 of the receptor in two ovarian tumor samples. A deletion in the polyadenine tract of exon 3 of TGF-beta RII was seen in 22% of the tumor samples. We also report a loss or decrease in the expression of Smad 4 protein in tumor samples with a concurrent loss or reduced DNA binding ability of the Smad complex and deregulated expression of p21 and c-Myc. Our results suggest that mutations and/or alterations in expression of TGF-beta receptors and loss of Smad 4 are frequent in human ovarian cancers and may potentially explain the frequent loss of TGF-beta responsiveness that typically occurs in human ovarian cancer.

Antifibrogenic effects of tamoxifen in a rat model of periportal hepatic fibrosis

It has been reported that tamoxifen may affect hepatoma cell growth in vitro by suppressing transforming growth factor beta-1 (TGF-beta1) expression, suggesting that tamoxifen might also retard fibrogenesis. Thus, we examined whether tamoxifen might suppress TGF-beta1 expression and consequently inhibit the process of hepatic fibrosis in vivo. To induce periportal hepatic fibrosis, 50 male adult Sprague-Dawley rats were injected with 0.62 mmol/kg of allyl alcohol, intraperitoneally, twice a week for 8 weeks. Hepatic fibrosis scores, intrahepatic collagen levels and plasma TGF-beta1 expression levels were evaluated in three groups of 10 rats orally administered tamoxifen at 1, 5 and 10 mg/kg, respectively, and in 20 controls. Messenger RNAs (mRNAs) encoding TGF-beta1 and TGF-beta receptors in liver tissue were semiquantified using reverse transcriptase polymerase chain reaction. Hepatic fibrosis scores decreased progressively as the dose of tamoxifen increased, resulting in a significant change in rats treated with tamoxifen at 10 mg/kg compared with controls (P=0.018). Intrahepatic collagen content was significantly less in the group treated with tamoxifen at 10 mg/kg compared with the control (P=0.045). Plasma TGF-beta1 levels were also significantly lower in rats treated with tamoxifen at 10 mg/kg compared with controls (P=0.007). All three concentrations of tamoxifen tested decreased the expression levels of hepatic TGF-beta1 mRNA and type I TGF-beta receptor (TGF-beta RI) mRNA to similar extents. Tamoxifen seems to inhibit the process of hepatic fibrosis dose-dependently by suppressing the transcription of TGF-beta1 and TGF-beta RI in an experimental model of periportal hepatic fibrosis.

Transforming growth factor-? receptor II is preferentially expressed in the companion layer of the human anagen hair follicle

Transforming growth factor (TGF)-beta is a multifunctional growth factor with multiple roles in skin including hair follicle development and cycling, where it regulates cell proliferation, differentiation and apoptosis, as well as in wound healing. While TGF-beta receptor I (TGF-beta RI) and receptor II (TGF-beta RII) expression helps define early human hair follicle morphogenesis, expression in the adult human hair follicle remains to be established. To assess TGF-beta receptor expression in human scalp anagen hair follicles. Immunohistochemical and double immunofluorescence analysis of TGF-beta RI and RII was conducted on frozen sections of haired human scalp obtained from 10 healthy individuals. TGF-beta RI expression was detected in the outer root sheath of anagen hair follicles while TGF-beta RII was expressed almost exclusively in the companion layer of inner root sheath and less so in premedulla keratinocytes. Both receptors were colocalized in the companion layer of the proximal and mid follicle. The well-described role of TGF-beta in keratinocyte apoptosis during catagen is likely to involve anagen-specific hair follicle components including the companion layer, as this layer provides the slippage plane supporting the inner root sheath and hair shaft as they ascend to the skin surface. Results of this study suggest that the colocalization of TGF-beta RI/RII complexes at the companion layer would facilitate TGF-beta signalling at this site to regulate apoptosis of the companion layer keratinocytes, facilitating shrinkage/contraction of this cell layer during hair follicle regression/catagen.

Dihydropyrrolopyrazole Transforming Growth Factor-β Type I Receptor Kinase Domain Inhibitors: A Novel Benzimidazole Series with Selectivity versus Transforming Growth Factor-β Type II Receptor Kinase and Mixed Lineage Kinase-7

Novel dihydropyrrolopyrazole-substituted benzimidazoles were synthesized and evaluated in vitro as inhibitors of transforming growth factor-beta type I receptor (TGF-beta RI), TGF-beta RII, and mixed lineage kinase-7 (MLK-7). These compounds were found to be potent TGF-beta RI inhibitors and selective versus TGF-beta RII and MLK-7 kinases. Benzimidazole derivative 8b was active in an in vivo target (TGF-beta RI) inhibition assay.

Expression of bFGF receptor and TGF-β receptors in cultured human scleral fibroblasts

To examine the expression of basic fibroblast growth factor receptor (FGF R1) and transforming growth factor-beta receptors (TGF-beta RI and TGF-beta RII) in cultured human scleral fibroblasts. Scleral fibroblasts of passages 2-4 were used for the present studies. Polyclonal antibodies against FGF R1, TGF-beta RI and TGF-beta RII were used to detect the proteins of these receptors. Indirect immunofluorescence staining method (IIF) was used. Antibodies for FGF R1, TGF-beta RI and TGF-beta RII produced specific staining of the entire cell surface, including the cell membrane enveloping cytoplasmic projections; positive staining in some cells was most intensive in the perinuclear region. Immunostaining mainly originated from the cell membranes, indicating that the presence of the receptor proteins on the cell surface. The intensity of staining for TGF-beta RI and TGF-beta RII was relatively strong, while staining of FGF R1 was relatively weak. The cells treated with PBS instead of primary antibodies did not produce specific staining. This study shows that cultured human scleral fibroblasts express the receptor protein for FGF R1, TGF-beta RI and TGF-beta RII and also indicates that these growth factors may influence these cells. Exogenous bFGF and TGF-beta administration may elicit actions through the binding with these receptors in the scleral fibroblasts.

Ontogeny of expression of transforming growth factor‐β and its receptors and their possible relationship with scarless healing in human fetal skin

Fetal cutaneous wounds that occur in early gestation heal without scar formation. Although much work has been done to characterize the role of transforming growth factor-beta (TGF-beta) isoforms and their receptors in the wound healing process, their roles in scarless wound repair observed in early gestation and their functions in human fetal skin development, and structural and functional maintenance are still not well understood. In this study, we explore the expression and distribution characteristics of three TGF-beta isoforms and their receptors, TGF-betaRI (TBRI) and TGF-betaRII (TBRII), in fetal and postnatal skins to understand the relevance of these five proteins to skin development and elucidate the mechanism(s) underlying the phenotypic transition from scarless to scar-forming healing observed during fetal gestation. Fetal skin biopsies of human embryo were obtained from spontaneous abortions at different gestational ages from 13 to 32 weeks and postnatal skin specimens were collected from patients undergoing plastic surgery. Gene expression and positive immunohistochemical signals of TGF-beta(1), TGF-beta(2), TGF-beta(3), TBRI, and TBRII could all be detected in fetal and postnatal skins. In early gestation, gene expression of TGF-beta(1), TBRI, and TBRII was weaker and protein contents were less compared with postnatal skins (p < 0.05). In contrast, more TGF-beta(2) mRNA transcript was found in early gestation than in late gestation and in postnatal skins, whereas protein content of this growth factor increased during gestation. Lastly, mRNA transcript and protein contents of TGF-beta(3) were apparently higher in early gestation compared to postnatal skin (p < 0.05). In postnatal skin, granules containing the three TGF-beta isoforms were mainly distributed in the cytoplasm and extracellular matrix of epidermal cells, interfollicular keratinocytes, and some fibroblasts. TBRI and TBRII were chiefly located in the cellular membrane of epidermal keratinocytes and some fibroblasts. The endogenous three TGF-beta isoforms and their receptors may be involved in the development of embryonic skin and in the maintenance of cutaneous structure and function, and also in postnatal wound healing. The differential levels of TGF-beta isoforms may provide either a predominantly antiscarring or profibrotic signal upon wounding depending on the gestational period. Lower expression of their receptors in early gestational skins may be a reason for the reduced ability to perceive ligands, ultimately leading to scar-free healing.

A histological and immunohistochemical study of the subsynovial connective tissue in idiopathic carpal tunnel syndrome.

The most common histological finding in carpal tunnel syndrome is noninflammatory synovial fibrosis. The accumulated effect of minor injuries is believed to be an important etiologic factor in some cases of carpal tunnel syndrome. We sought evidence of such injuries in the synovial tissue of patients with carpal tunnel syndrome and in cadaver controls. We compared synovial specimens from thirty patients who had idiopathic carpal tunnel syndrome with specimens from a control group of ten fresh-frozen cadavers of individuals who had not had an antemortem diagnosis of carpal tunnel syndrome and who met the same exclusion criteria. Analysis included histological and immunohistochemical examination for the distribution of collagen types I, II, III, and VI and transforming growth factor-beta (TGF-beta) RI, RII, and RIII. Histological examination showed a marked increase in fibroblast density, collagen fiber size, and vascular proliferation in the specimens from the patients compared with the control specimens (p < 0.001). Collagen types I and II were not found in the synovium of either the patients or the controls, but collagen type VI was a major component of both. Collagen type-III fibers were more abundant in the patients than in the controls (p < 0.001). Expression of TGF-beta RI was found in the endothelial cells and fibroblasts in the patient and control specimens, with a marked increase in expression in the fibroblasts of the patients compared with that in the control tissue (p < 0.001). These findings are similar to those after injury to skin, tendon, and ligament and suggest that patients with idiopathic carpal tunnel syndrome may have sustained an injury to the subsynovial connective tissue.

Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion

Aryl hydrocarbon receptor (AHR)-null mice display a liver fibrosis phenotype that is associated with a concomitant increase in liver retinoid concentration, tissue transglutaminase type II (TGaseII) activity, transforming growth factor beta (TGF beta) overexpression, and accumulation of collagen. To test the hypothesis that this phenotype might be triggered by the observed increase in liver retinoid content, we induced the condition of retinoid depletion by feeding AHR-null mice a vitamin A- deficient diet with the purpose to reverse the phenotype. Liver retinoid content decreased sharply within the first few weeks on the retinoid-deficient diet. Analysis of TGF beta 1, TGF beta 2, and TGF beta 3 expression revealed a reduction to control levels in the AHR -/- mice accompanied by parallel changes in TGaseII protein levels. In addition, we observed an increase in the TGF beta receptors, TGF beta RI and TGF beta RII, as well as in Smad4, and their reduction to wild-type mouse liver levels in AHR -/- mice fed the retinoid-deficient diet. Reduction of peroxisomal proliferator-activated receptor gamma (PPAR gamma) messenger RNA (mRNA) and protein levels in AHR -/- mice was consistent with the presence of hepatic stellate cell (HSC) activation and liver fibrosis. Vitamin A deficiency normalized PPAR gamma expression in AHR -/- mice. In conclusion, livers from AHR -/- mice fed the vitamin A-deficient diet showed a decrease in collagen deposition, consistent with the absence of liver fibrosis.

Differential expression of transforming growth factors-β1, -β2, -β3 and the type I, II, III receptors in the lining epithelia of inflamed gingiva

To investigate the distribution of transforming growth factor-beta isoforms in chronically inflamed periodontal tissues. The present study determined, by immunohistochemistry, the expression patterns of TGF-betas and their receptors in the lining epithelia of inflamed gingiva. Frozen sections were obtained from 22 human gingival biopsies. TGF-beta 1 was not detected in gingival epithelial cells in examined sections. Detection of TGF-beta 2 indicated a progressive reduction of staining from the external oral epithelium through to gingival sulcus and the gingival attachment or pocket epithelium. TGF-beta 3 showed intense staining in all domains of both minimally inflamed gingiva and advanced periodontitis tissues. TGF-beta RI was visualised as focal staining of the spinous layer in the external oral epithelium of both periodontitis lesions and minimally inflamed tissues. TGF-beta RII was present throughout the strata, but with progressive reduction in intensity from the oral epithelium to gingival attachment or pocket epithelium respectively while, conversely, TGF-beta RIII showed an increase in diffuse staining intensity from external oral epithelium to pocket epithelium. A distinct expression profile was observed within different individuals for TGF-betas and the corresponding receptors. These findings provide a basis for evaluation of the role of these growth factors in the pathogenesis of periodontitis.

Decreased expression of human papillomavirus E2 protein and transforming growth factor‐β1 in human cervical neoplasia as an early marker in carcinogenesis

Human papillomavirus (HPV) is thought to be one of the possible causative factors in cervical carcinogenesis, and cervical carcinoma cells are refractory to tumor transforming growth factor (TGF)-beta1. The purpose of this study is to investigate the possible cause-effect association between HPV and TGF-beta1 during cervical tumorigenesis. We assessed the expression of HPV capsid proteins, HPV-16 E7, HPV-16 E2 (C and N terminals), TGF-beta1, and their receptors TGF-beta RI and RII by immunohistochemistry in 48 paraffin-embedded blocks of tumor tissue derived from patients of cervical neoplasia. Expression of TGF-beta1 decreased as tumor cells progressed from cervical intraepithelial neoplasia (CIN)1, CIN2, CIN3, to microinvasive carcinoma (P < 0.05). Levels of TGF-betaRI and TGFbeta-RII stayed the same in all cases. HPV was found in 89.6% of the studied sections, and cervical lesions without HPV infection expressed significantly less TGF-beta1 (P < 0.05). By comparing the expression pattern of TGF-beta1 and HPV in the neoplastic cells with that of normal cervical epithelium in each section, we found loss of HPV-16 E2 higher in CIN3 (15/24) than in CIN1 or CIN2 (3/7), and there is a significant trend that loss of HPV-16 E2 expression correlated with a >50% loss of TGF-beta1 at the lesion site (P < 0.05). Our result showed co-suppression of HPV and TGF-beta1 expression during progression of cervical squamous cell cancer. Using antibody against HPV-16 E2 may be an auxiliary tool for the investigation of cervical tumor progression.

Promoter methylation of TGFbeta receptor I and mutation of TGFbeta receptor II are frequent events in MSI sporadic gastric carcinomas.

Transforming growth factor beta (TGFbeta) is a potent inhibitor of cell growth, whose action is transduced through interaction between type I (RI) and type II (RII) receptors. Abnormal expression of these receptors has been identified in several human cancers and was found to be associated with resistance to TGFbeta. TGFbeta RII mutations occur in many types of malignancy. TGFbeta RI hypermethylation has been suggested as a cause of abnormal or absent expression of this receptor in cancer. This study has analysed the methylation status of the promoter region of the TGFbeta RI gene using a methylation-sensitive enzyme followed by polymerase chain reaction (PCR), and TGFbeta RII mutations (BAT-RII and a GT(3)) in order to determine the frequency of alteration of the TGFbeta receptors in a series of 40 sporadic gastric carcinomas (SGCs), 25 of which showed microsatellite instability (MSI) and 15 of which were microsatellite stable (MSS). Methylation in the promoter region of the TGFbeta RI gene was detected in 20 of the 40 (50%) SGCs (64% of the MSI cases and 26.7% of the MSS); 17 of the 40 (42.5%) cases had mutations in the BAT-RII region of the TGFbeta RII gene (68% in the MSI cases; 0% in the MSS). In total, 25 of the 40 (62.5%) SGCs had alterations in at least one of the TGFbeta receptors (84% of the cases in the MSI group, in contrast with 16% of the MSS cases) (p = 0.0003). The clinicopathological features of the cases were also studied and significant associations were found between the presence of alterations in TGFbeta receptors and the age of the patients (p = 0.046), size (p = 0.011), and proliferative rate of the tumours (p = 0.048). In conclusion, alterations in the receptors of TGFbeta (TGFbeta RI promoter hypermethylation and TGFbeta RII mutations) are frequent events in MSI SGC and are associated with large size and high proliferative activity of the tumours, in keeping with loss of the growth inhibitory effects of TGFbeta in this setting.

Increased transcriptional activities of transforming growth factor beta receptors in scleroderma fibroblasts.

To investigate the molecular mechanism of the overexpression of transforming growth factor beta receptors (TGF(beta)Rs) in dermal fibroblasts from patients with systemic sclerosis (SSc). Dermal fibroblasts from 7 patients with diffuse SSc of recent onset and from 7 healthy individuals were studied. The expression of TGF(beta)R type I (TGF(beta)RI), TGF(beta)RII, and type I collagen proteins in dermal fibroblasts was determined by immunoblotting. TGF(beta)RI, TGF(beta)RII, and alpha2(I) collagen messenger RNA (mRNA) were evaluated by Northern blot analysis. The transcriptional activities of the TGF(beta)RI and TGF(beta)RII genes were examined by luciferase assay. SSc fibroblasts expressed increased levels of TGF(beta)RI and TGF(beta)RII protein and mRNA, as well as increased levels of type I collagen protein and alpha2(I) collagen mRNA. Moreover, the half-lives of TGF(beta)RI and TGF(beta)RII mRNA in SSc fibroblasts did not change compared with those in control dermal fibroblasts. The promoter activities of the TGF(beta)RI and TGF(beta)RII genes were both significantly increased in SSc fibroblasts compared with those in control fibroblasts. Calphostin C, a specific inhibitor of protein kinase C (PKC), inhibited TGF(beta)RI promoter activity in SSc fibroblasts, and LY294002, an inhibitor of phosphoinositide 3-kinase (PI 3-kinase), inhibited TGF(beta)RII promoter activity in SSc fibroblasts. Moreover, calphostin C and LY294002 inhibited the up-regulation of TGF(beta)RI and TGF(beta)RII mRNA, respectively, in SSc fibroblasts. These results suggest that increased levels of TGF(beta)Rs in SSc fibroblasts play a role in excessive collagen production, and that up-regulation of TGF(beta)R expression might occur at the transcriptional levels. PKC and/or PI 3-kinase might contribute to the up-regulation of TGF(beta)R expression in SSc fibroblasts.

Decreased expression of TGF-beta type 2 receptor in primary B-cell lymphomas of the stomach.

TGF-beta insensitivity has been reported in some malignant lymphomas showing loss of TGF-beta receptor expression. This loss of TGF-beta sensitivity is thought to have removed the immunosuppressive properties of TGF-beta, thus enhancing cell proliferation and resulting in the development of malignant lymphoma. In this study, we performed immunohistochemical stains for TGF-beta1, TGF-beta RI and TGF-beta RII in primary gastric B-cell lymphomas in order to ascertain their possible roles in lymphomagenesis. A total of twenty cases of gastric lymphoma were included. All cases of low- and high-grade lymphomas were negative or weakly positive for TGF-beta1. Reactive lymphoid cells, including the germinal center, were also negative for TGF-beta1. In contrast, reactive germinal centers showed moderate to strong cytoplasmic or membranous staining for TGF-beta RI and TGF-beta RII. In malignant lymphomas, TGF-beta RI expression was maintained in all cases of low- and high-grade lymphomas. In contrast, TGF-beta RII expression was decreased in all low- and high-grade lymphoma cells. These findings suggest that the loss of TGF-beta RII expression may be involved in the lymphomagenesis of the stomach.

Differential expression of transforming growth factor-beta receptors in a rabbit zone II flexor tendon wound healing model.

Flexor tendon repair in zone II is complicated by adhesions that impair normal postoperative gliding. Transforming growth factor-beta (TGF-beta) is a family of growth factors that has been implicated in scar formation. The TGF-beta family of proteins binds to three distinct classes of membrane receptors, termed RI, RII, and RIII. In this study, we analyzed the temporal and spatial distribution of TGF-beta receptor isoforms (RI, RII, and RIII) in a rabbit zone II flexor tendon wound healing model.Twenty-eight adult New Zealand White rabbit forepaws underwent isolation of the middle digit flexor digitorum profundus tendon in zone II. The tendons underwent transection in zone II and immediate repair. The tendons were harvested at increasing time points: 1, 3, 7, 14, 28, and 56 days postoperatively (n = 4 at each time point). The control flexor tendons were harvested without transection and repair (n = 4). Immunohistochemical analysis was used to detect the expression patterns for TGF-beta receptors RI, RII, and RIII. Immunohistochemical staining of the transected and repaired tendons demonstrated up-regulation of TGF-beta RI, RII, and RIII protein levels. TGF-beta receptor production in the experimental group (transection and repair) was concentrated in the epitenon and along the repair site. Furthermore, the TGF-beta receptor expression levels peaked at day 14 and decreased by day 56 postoperatively. In contrast, minimal receptor expression was observed in the untransected and unrepaired control tendons. These data provide evidence that (1) TGF-beta receptors are up-regulated after injury and repair; (2) peak levels of TGF-beta receptor expression occurred at day 14 and decreased by day 56 after wounding and repair; and (3) both the tendon sheath and epitenon have the highest receptor expression, and both may play critical roles in flexor tendon wound healing. Understanding the up-regulation of TGF-beta isoforms and the up-regulation of their corresponding receptors during flexor tendon wound healing provides new targets for biomolecular modulation of postoperative scar formation.

Evidence that TGF beta may directly modulate POMC mRNA expression in the female rat arcuate nucleus.

The purpose of the present study was to determine whether TGF beta, a cytokine secreted by hypothalamic astrocytes, was able to regulate POMC neurons in the arcuate nucleus. In a first set of experiments, mediobasal hypothalamic fragments were exposed to TGF beta(1), and the relative POMC mRNA expression was assessed by in situ hybridization using a radiolabeled POMC riboprobe. The results showed that 4 x 10(-10) M TGF beta(1) was efficient in decreasing significantly the amounts of POMC mRNA (P < 0.01). Interestingly, the decrease of relative POMC mRNA levels was higher in the rostral than in the caudal parts of the arcuate nucleus. In a second set of experiments, we examined the occurrence of TGF beta receptors expression in arcuate POMC neurons. Dual labeling in situ hybridization and in situ hybridization, coupled to immunohistochemical labeling, were performed to examine mRNA expression of the type I serine-threonine kinase receptor for TGF beta and the presence of type II receptor for TGF beta, respectively, in POMC neurons. The results indicated that TGF beta receptor I mRNA and TGF beta receptor II protein were expressed in numerous POMC neurons. Regional analysis revealed that the highest proportion of POMC neurons expressing TGF beta receptors was located in the rostral part of the arcuate nucleus. Using dual labeling immunohistochemistry, we also found that Smad2/3 immunoreactivity, a TGF beta(1) downstream signaling molecule, was present in the cytoplasm and nucleus of some POMC (beta-endorphin) neurons. We next examined whether the number of POMC neurons expressing TGF beta-RI mRNA was affected by sex steroids. Quantification of the number of POMC neurons expressing TGF beta receptor I mRNA in ovariectomized, ovariectomized E2-treated, and ovariectomized E2 plus progesterone-treated animals revealed that estrogen treatment decreased the expression of TGF beta receptor I mRNA in POMC neurons located in the rostral half of the arcuate nucleus, an effect reversed by progesterone in a subset of the most rostral cells. Taken together, these data reveal that TGF beta(1) may directly modulate the activity of POMC neurons through the activation of TGF beta receptors. Therefore, the present study provides additional evidence for the involvement of TGF beta(1) in the regulation of neuroendocrine functions and supports the existence of a glial-to-neurons communication within the arcuate nucleus.

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.