TGF-βRII Protein Research Articles

Involucrasin B Inhibits the Proliferation of Caco-2 Cells by Regulating the TGFβ/SMAD2-3-4 Pathway.

(1) Background: Colorectal cancer (CRC) is the third most common malignant tumor worldwide and the second most common cause of cancer death. However, effective anti-CRC drugs are still lacking in clinical settings. This article investigated the anti-proliferative effect of involucrasin B on CRC Caco-2 cells. (2) Methods: This study employed a sulforhodamine B (SRB) method, colony formation experiments, flow cytometry, FastFUCCI assay, dual luciferase assay, and Western blot analysis for the investigation. (3) Results: The SRB method and colony formation experiments showed that involucrasin B exhibited an inhibitory effect on the Caco-2 cells cultured in vitro. Subsequently, the flow cytometry, FastFUCCI assay, and Western blotting results showed that involucrasin B induced cell cycle arrest in the G1 phase dose-dependently. Involucrasin B significantly enhanced the TGFβ RII protein level and SMAD3 phosphorylation, thus inhibiting the expression of CDK4 and cyclin D1 and causing G1 cell cycle arrest. (4) Conclusion: This study shows that involucrasin B exerts its anti-proliferative effect by regulating the TGFβ/SMAD2-3-4 pathway to cause G1 cycle arrest in Caco-2 cells.

486P AK130, a first-in-class Fc-mutant anti-TIGIT antibody fused with TGF-βRII protein, elicits potent anti-tumor efficacy in pre-clinical studies

TIGIT (T-cell immunoglobulin and ITIM domain), which is expressed on T and NK cells, can interact with its ligands (i.e., CD155 and CD122), leading to inhibitory signaling in T cells and promoting exhaustion of lymphocytes. Although TIGIT is considered as a promising immune checkpoint molecule, the single-agent efficacy of anti-TIGIT therapy is limited. TGF-β (Transforming growth factor-beta), serves as an immune regulator in the tumor microenvironment (TME), is elevated in various tumor types and contributes to the resistance to checkpoint inhibitors.

AAV-mediated expression of galactose-1-phosphate uridyltransferase corrects defects of galactose metabolism in classic galactosemia patient fibroblasts.

Classic galactosemia (CG) is a rare disorder of autosomal recessive inheritance. It is caused predominantly by point mutations as well as deletions in the gene encoding the enzyme galactose-1-phosphate uridyltransferase (GALT). The majority of the more than 350 mutations identified in the GALT gene cause a significant reduction in GALT enzyme activity resulting in the toxic buildup of galactose metabolites that in turn is associated with cellular stress and injury. Consequently, developing a therapeutic strategy that reverses both the oxidative and ER stress in CG cells may be helpful in combating this disease. Recombinant adeno-associated virus (AAV)-mediated gene therapy to restore GALT activity offers the potential to address the unmet medical needs of galactosemia patients. Here, utilizing fibroblasts derived from CG patients we demonstrated that AAV-mediated augmentation of GALT protein and activity resulted in the prevention of ER and oxidative stress. We also demonstrate that these CG patient fibroblasts exhibit reduced CD109 and TGFβRII protein levels and that these effectors of cellular homeostasis could be restored following AAV-mediated expression of GALT. Finally, we show initial in vivo proof-of-concept restoration of galactose metabolism in a GALT knockout mouse model following treatment with AAV-GALT.

Inhibition of TGF-β1 on Gli2 expression was promoted by TNF-α in primary leukemia cells

Purpose Hedgehog (Hh) signaling pathway regulates a variety of tumors-related diseases including leukemia. Whether inhibition of TGF-β1 on Gli2 expression is promoted by TNF-α in primary leukemia cells remains to be determined. Methods Primary leukemia cells were treated with TGF-β1, TNF-α or SIS3 at different concentrations. Gli2 expression was detected by quantitative real-time PCR and western blot analyses. Results We found that TGF-β significantly decreased Gli2 expression, and co-treatment with TNF-αfurther decreased Gli2 expression in primary leukemia cells. TNF-α can increased TGF-βRI and TGF-βRII protein expression in primary leukemia cells, while SIS3 inhibited the effect of TGF-β. Conclusion Our results suggest that Gli2 expression in primary leukemia cells is induced by TGF-β in a Smad3-dependent manner, and independent of Hh receptor signaling.

Abstract 2661: AUTO6NG overcomes immune suppressive mechanisms in the TME and demonstrate preclinical anti-tumor activity in GD2-expressing solid tumors

Abstract Immune-checkpoint blockade therapies has had impressive results in the clinic and are approved in multiple cancer indications. However, meaningful responses remain elusive in several solid tumor types and durable responses in some of these indications, or subtypes, are rarely observed. It has been postulated that this is in part due to elaborate poorly understood immunosuppressive mechanisms that the therapeutic approach must overcome to elicit the desired durable responses. Chimeric Antigen Receptor T (CAR-T) cell therapies had been therapeutically highly successful in haematological malignancies but such successes have not been replicated yet in solid tumors, primarily due to immune suppressive mechanisms in the tumor microenvironment (TME) of these settings. To address these issues in developing our CAR-T therapeutics, we deployed tissue cytometry to conduct a multiplex analysis of 30 markers on the same tissue sections from each patient from a set of patients from various solid tumor indications expressing the antigen GD2 (e.g. SCLC, Melanoma, Neuroblastoma), a disialoganglioside to which we have developed a selective CAR. The panel aimed to characterize and phenotype key players within the TME (i.e. tumour cells, T cells, DCs, macrophages, MDSCs, CAFs, as well as endothelial cells). We then deployed machine learning analytics to assist in inferring about potential interactions the different cell types have in the 3D context of the tumor tissue which allowed the identification of therapeutically relevant elements in the TMEs of these indications. Next, we generated CAR-T cells from healthy donors targeting GD2, an antigen we validated its expression in multiple solid tumor types. A first generation GD2 CAR is currently being developed in the clinic for the treatment of Neuroblastoma. We proceeded to incorporate a CCR module (IL7R chimeric protein designed to improve cell persistence), a dnTGFβ (dominant negative TGF-βRII protein designed to block inhibitor signals from TGFβ), and other function enhancing modules into the GD2 CAR. The multi module CAR-T cells showed potent in vitro activity against human patient derived cancer cell lines as measured by both tumor lysis and cytokine secretion. These CAR-T cells also demonstrated enhanced tumor infiltration and anti-tumor activity in vivo in xenografts mouse models. Our findings suggest that GD2 targeted CAR-T cell therapy is a viable novel approach for the treatment of multiple solid tumors indications. Citation Format: Margarida Neves, Lukas Stanczuk, Alireza Alavi, Meggan Czapiga, Rebecca Moore, Frederick Vargas, Muhammad A. Al-Hajj. AUTO6NG overcomes immune suppressive mechanisms in the TME and demonstrate preclinical anti-tumor activity in GD2-expressing solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2661.

Expression of Transforming Growth Factor β Type I and II Receptor in Prostate Cancer

This study was carried out to establish the correlation between expression of Transforming growth factor beta receptor one (TGF-βRI) and Transforming growth factor beta receptor two (TGF-βRII) and prostate cancer progression, and to establish the role of prostate cancer development. Immunohistochemistry (IHC) technique was used to detect the level of expression of TGF-βRI and TGF-βRII protein in tissues of patients and healthy control groups. TGF-βR1 protein was expressed in 3 (18.7%) and 14 (56%) of poorly and moderately differentiated malignancy respectively. There was significant difference in mean level of TGF-βRI protein expression among all studied groups. TGF-βRII protein was expressed in 6 (37.5%) and 22 (88%) of poorly and moderately differentiated malignancy respectively, There was significant difference in mean level of TGF-βRII protein expression among all studied groups. We concluded that there was statistically significant association between the loss of expression of TGF-β1 signaling receptors, especially TGF-βRI, and increasing grades of malignancy in prostate cancer, leading to a more malignant phenotype

Down-regulation of TGF-β RII expression is correlated with tumor growth and invasion in non-functioning pituitary adenomas

The two types of TGF-β receptors play a crucial role in TGF-β signaling. It has been reported that TGF-β signaling activity may be associated with the development and invasion of NFPAs. However, the role of TGF-β receptor signaling in NFPAs has not been fully explored. In this study, the expression of TGF-β RI and TGF-β RII in normal anterior pituitaries, invasive NFPAs and non-invasive NFPAs was analyzed by qRT-PCR, western blot, and immunohistochemistry. We found that TGF-β RII protein and mRNA levels gradually decreased from normal anterior pituitaries, noninvasive NFPAs to invasive NFPAs. There was no significant difference in the expression of TGF-β RI mRNA and protein level between normal anterior pituitaries and NFPAs. PCNA mRNA level was significantly higher in the invasive NFPAs than noninvasive NFPAs and PCNA mRNA had a negative correlation with TGF-β RII mRNA. We may draw the conclusion that low expression of TGF-β RII may contribute to the development and invasion of NFPAs and TGF-β RII promises to be a useful biomarker for the diagnosis of invasive NFPAs.

The role of miR‑370 in fibrosis after myocardial infarction.

In the present study, we investigated the expression of miR-370 in the border area of infarction after myocardial infarction and its role in the process of post-infarction fibrosis. A myocardial infarction model in Sprague-Dawley rats was established. After two weeks, the mRNA levels of transforming growth factor-β1 (TGFβ1), TGFβRII, ColIa1, ColIIIa1 and miR-370 and the expression of TGFβ1, TGFβRII and α-smooth muscle actin (α-SMA) proteins in the border area of infarction were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot analysis. Cardiac fibroblasts in neonatal rat were isolated and cultured, and the changes in the above indicators were detected after AngII and miR-370 intervention. Luciferase reporter gene assay was conducted to verify whether TGFβRII was a target gene of miR-370. In the border area after myocardial infarction, the expression of miR-370 decreased, while mRNA levels of TGFβ1, TGFβRII, ColIa1 and ColIIIa1 and levels of TGFβ1, TGFβRII and α-SMA proteins were all increased. Luciferase reporter gene assay confirmed that TGFβRII was the target gene of miR-370. miR-370 reduced the expression of TGFβRII and inhibited the increased expression of TGFβRII and collagen protein caused by AngII. As well, its inhibited the differentiation effect of muscle fibroblasts while it did not inhibit the expression of TGFβ1. miR-370 inhibited the expression of TGFβRII protein by combining with TGFβRII mRNA. miR-370 also partially blocked TGFβ1-TGFβRII and induced the downstream signal transduction pathways, thus exerting anti-fibrotic effects.

Angiotensin II Type 2 Receptor Decreases Transforming Growth Factor-β Type II Receptor Expression and Function in Human Renal Proximal Tubule Cells.

Transforming growth factor-β (TGF-β), via its receptors, induces epithelial-mesenchymal transition (EMT) and plays an important role in the development of renal tubulointersitial fibrosis. Angiotensin II type 2 receptor (AT2R), which mediates beneficial renal physiological functions, has received attention as a prospective therapeutic target for renoprotection. In this study, we investigated the effect and underlying mechanism of AT2R on the TGF-β receptor II (TGF-βRII) expression and function in human proximal tubular cells (HK-2). Here, we show that the AT2R agonist CGP42112A decreased TGF-βRII protein expression in a concentration- and time-dependent manner in HK-2 cells. The inhibitory effect of the AT2R on TGF-βRII expression was blocked by the AT2R antagonists PD123319 or PD123177. Stimulation with TGF-β1 enhanced EMT in HK-2 cells, which was prevented by pre-treatment with CGP42112A. One of mechanisms in this regulation is associated with the increased TGF-βRII degradation after activation of AT2R. Furthermore, laser confocal immunofluorescence microscopy showed that AT2R and TGF-βRII colocalized in HK-2 cells. AT2R and TGF-βRII coimmunoprecipitated and this interaction was increased after AT2R agonist stimulation for 30 min. The inhibitory effect of the AT2R on TGF-βRII expression was also blocked by the nitric oxide synthase inhibitor L-NAME, indicating that nitric oxide is involved in the signaling pathway. Taken together, our study indicates that the renal AT2R regulates TGF-βRII expression and function via the nitric oxide pathway, which may be important in the control of renal tubulointerstitial fibrosis.

STAT3-mediated activation of microRNA cluster 17 92 promotes proliferation and survival of ALK-positive anaplastic large cell lymphoma

Systemic anaplastic large cell lymphoma is a category of T-cell non-Hodgkin's lymphoma which can be further subdivided into two distinct entities (ALK(+) and ALK(-)) based on the presence or absence of ALK gene rearrangements. Among several pathways triggered by ALK signaling, constitutive activation of STAT3 is strictly required for ALK-mediated transformation and survival. Here we performed genome-wide microRNA profiling and identified 48 microRNA concordantly modulated by the inducible knock-down of ALK and STAT3. To evaluate the functional role of differentially expressed miRNA, we forced their expression in ALK(+) anaplastic large cell lymphoma cells, and monitored their influence after STAT3 depletion. We found that the expression of the microRNA-17~92 cluster partially rescues STAT3 knock-down by sustaining proliferation and survival of ALK(+) cells. Experiments in a xenograft mouse model indicated that forced expression of microRNA-17~92 interferes with STAT3 knock-down in vivo. High expression levels of the microRNA-17~92 cluster resulted in down-regulation of BIM and TGFβRII proteins, suggesting that their targeting might mediate resistance to STAT3 knock-down in anaplastic large cell lymphoma cells. We speculate that the microRNA-17~92 cluster is involved in lymphomagenesis of STAT3(+) ALCL and that its inhibition might represent an alternative avenue to interfere with ALK signaling in anaplastic large cell lymphomas.

Abstract 2569: Evaluation of TGFBR2 gene polymorphisms with TGF-βRII protein expression in breast cancer tissues: results from the Shanghai Breast Cancer Study.

Abstract The transforming growth factor (TGF)-β signaling pathway plays a significant role in the carcinogenic process of breast cancer. We have previously shown that the minor G allele of the single nucleotide polymorphism (SNP) rs1078985 in the TGFBR2 gene was associated with reduced risk of breast cancer. To further investigate the role of genetic variants of TGFBR2 in breast cancer etiology, we systematically evaluated common genetic variants (minor allele frequency > 5%) in the TGFBR2 gene (80 SNPs) for associations with TGF-βRII protein expression in breast tissue from 970 women with invasive breast cancer who participated in the Shanghai Breast Cancer Study. Expression of TGF-βRII was detected by immunofluorescence staining. The immunofluorescence signals were designated as negative, positive, or strong-positive based on a modified Allred scoring system. Associations of genetic polymorphisms with TGF-βRII protein expression were estimated using linear regression models adjusted for age at diagnosis. Variant alleles of SNPs rs1078985 and rs3773634 were associated with increased TGF-βRII expression intensity in breast cancer tissue (P = 0.0004 for rs1078985 and 0.0022 for rs3773634). However, these two SNPs were not associated with TGF-βRII protein expression intensity in adjacent normal tissue or in in situ breast cancer tissue. The sample sizes for the adjacent normal tissue and in situ breast cancer groups were small. SNPs rs1078985 (located in intron 3) and rs3773634 (located in intron 4) are approximately 7 Kb apart with strong linkage disequilibrium (r2=0.962 in Asians). We then performed electrophoretic mobility shift assays to examine whether the DNA sequence containing SNPs rs1078985 or rs3773634 interacts with nuclear proteins and whether the single nucleotide change in these polymorphic sites alters protein-DNA interactions. The variant G allele of SNP rs1078985 altered both DNA-protein complex intensity and patterns in breast cancer MCF7 and MDA231 cells, but not in MCF10A cells, whereas SNP rs3773634 did not change DNA-protein intensity or patterns in any of the three types of cells. Our data suggest that common variants in the TGFBR2 gene are associated with TGF-βRII protein expression in breast cancer tissue. The variant G allele of SNP rs1078985 may affect transcription factor binding, and in turn, increase TGF-βRII protein expression in breast tumor tissue. Characterization of genetic variants and their associations with protein expression in tumor tissue is valuable for elucidating the role of TGF-β signaling in the development and prognosis of breast cancer. Citation Format: Qingchao Qiu, Yinghao Su, Wei Lu, Ben Zhang, Ying Zheng, Xiao Ou Shu, Wei Zheng, Qiuyin Cai. Evaluation of TGFBR2 gene polymorphisms with TGF-βRII protein expression in breast cancer tissues: results from the Shanghai Breast Cancer Study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2569. doi:10.1158/1538-7445.AM2013-2569

Absence of TGF-βRII predicts bone and lung metastasis and is associated with poor prognosis in stage III breast tumors

In the case of operated breast cancer (BC), prognostic markers help to determine if the patient needs additional treatment and predictive markers help the clinician to decide which treatment to use. Thus, a better knowledge of known predictive and prognostic markers and the identification of new markers, may improve the treatment of BC patients. The transforming growth factor-beta type II receptor (TGF-βRII), a main receptor of transforming growth factor beta pathway, is a potential new prognostic marker. The aims of the present study were to investigate both the predictive and prognostic impact of TGF-βRII in BC samples. TGF-βRII protein expression was evaluated using immunohistochemistry on a tissue microarray containing 110 TNM stage III BC samples obtained prior to doxorubicin-based neoadjuvant chemotherapy (NAC). Our results demonstrate that TGF-βRII did not predict the response to NAC. On the other hand, an association between TGF-βRII-negative tumor and higher risk of metastasis to lungs and bones was verified. TGF-βRII negativity was an independent prognostic factor for decreased disease-free and overall survival.

Loss of Transforming Growth Factor Beta Type II Receptor Increases Aggressive Tumor Behavior and Reduces Survival in Lung Adenocarcinoma and Squamous Cell Carcinoma

Lung adenocarcinoma and lung squamous cell carcinoma (SCC) are the most common non-small cell lung cancer (NSCLC) subtypes. This study was designed to determine whether reduced expression of TGFβ type II receptor (TGFβRII) promotes lung adenocarcinoma and SCC carcinogenesis. We examined TGFβRII expression at the protein and mRNA levels in human NSCLC samples and assessed the relationship between TGFβRII expression and clinicopathologic parameters. To determine whether sporadic TGFβRII deletion in airway epithelial cells induces NSCLC formation, we targeted TGFβRII deletion alone and in combination with oncogenic Kras(G12D) to murine airways using a keratin 5 (K5) promoter and inducible Cre recombinase. Reduced TGFβRII expression in human NSCLC is associated with male gender, smoking, SCC histology, reduced differentiation, increased tumor stage, increased nodal metastasis, and reduced survival. Homozygous or heterozygous TGFβRII deletion in mouse airway epithelia increases the size and number of Kras(G12D)-initiated adenocarcinoma and SCC. TGFβRII deletion increases proliferation, local inflammation, and TGFβ ligand elaboration; TGFβRII knockdown in airway epithelial cells increases migration and invasion. Reduced TGFβRII expression in human NSCLC is associated with more aggressive tumor behavior and inflammation that is, at least partially, mediated by increased TGFβ1 expression. TGFβRII deletion in mouse airway epithelial cells promotes adenocarcinoma and SCC formation, indicating that TGFβRII loss plays a causal role in lung carcinogenesis. That TGFβRII shows haploid insufficiency suggests that a 50% TGFβRII protein reduction would negatively impact lung cancer prognosis.

Absence of transforming growth factor-β type II receptor is associated with poorer prognosis in HER2-negative breast tumours

BackgroundThe clinical relevance of transforming growth factor-beta (TGF-β)-signalling pathway in breast carcinomas (BCs) remained elusive. This study aimed to evaluate the prognostic value of TGF-β1 and transforming growth factor-beta type II receptor (TGF-βRII) expression levels in tumour cells and their association with the established biomarkers in BC. Patients and methodsIn 324 BC from patients with long-term follow-up, the TGF-β1 and TGF-βRII transcript and protein expression levels were assessed. ResultsTGF-β1 and TGF-βRII down-expression was significantly associated with BC. Negative TGF-β1 and TGF-βRII protein status was associated with the development of distant metastasis (P=0.003 and P=0.029, respectively). In multivariate analysis, TGF-β1-positive tumours were associated with increased disease-free survival (DFS) [hazard ratio (HR)=0.489, P=0.003]. TGF-βRII positivity was an independent prognostic factor for DFS (HR=0.439, P=0.001) and overall survival (OS) (HR=0.409, P=0.003) in human epidermal growth factor receptor-2 (HER2)-negative patients. Absence of TGF-β1 and TGF-βRII proteins in breast tumour cells was significantly associated with metastasis development. ConclusionsTo the best of our knowledge, this is the first report indicating the relevance of HER2 status in discriminating TGF-βRII as a prognostic marker for DFS and OS in human BC. These data indicate that TGF-βRII protein analysis in tumour cells could be introduced in clinical practice as additional prognostic biomarker in HER2-negative BC.

Evaluation of Transforming Growth Factor-β Signaling Pathway in the Wall of Normal and Varicose Veins

Objective(s): Extracellular matrix remodeling in the vein wall is involved in varicose vein pathogenesis, with transforming growth factor β₁ (TGF-β₁) playing a potential role. The aim of the study was to assess the TGF-β signaling pathway including its receptor (TGF-β RII) and phosphorylated receptor-regulated Smads (p-Smad2/3) in varicose veins. Methods: Varicose veins from patients undergoing varicose vein surgery were the studied material, whereas normal greater saphenous veins from patients undergoing infrainguinal arterial bypass surgery were the control material. Expression of TGF-β RII mRNA was assessed with RT-PCR, whereas expression of TGF-β RII and p-Smad2/3 proteins was assessed with Western blot. Results: A significantly increased TGF-β RII mRNA level was found in varicose veins (287 ± 24%), when compared with normal veins (100 ± 26%). The receptor protein expression reflected a changed mRNA level with significantly increased TGF-β RII protein in varicose veins (290 ± 21%), when compared with controls (100 ± 16%). Enhanced TGF-β RII expression was accompanied by increased p-Smad2/3 protein expression in varicose veins (257 ± 19%) in comparison with normal veins (100 ± 9%). Conclusion(s): Increased TGF-β RII expression and activation in the wall of varicose veins may be involved in extracellular matrix remodeling related to TGF-β₁ and supports its role in the disease pathogenesis.

267. A role for transforming growth factor-β 1 (TGF-β1) during the establishment of folliculogenesis

A group of structurally related proteins, known as the transforming growth factor-β (TGF- β) superfamily, have been implicated in the local regulation of ovarian function. It is unclear what role TGF-β1–3 plays in folliculogenesis during the period after birth in the rat. We investigated whether the TGF-β ligands and their receptors were present during this period of development and the effects of TGF-β1 on granulosa cell function (proliferation, apoptosis, steroidogenesis). Ovaries from rats 4, 8 and 12 days of age were isolated and RNA extracted and reverse transcribed for real-time PCR. The expression of the TGF-β ligands and TGFβRI and TGFβRII were measured. Granulosa cells isolated from DES treated immature rats were treated with FSH (100ng/mL) and TGF-β1 (1 or 10ng/mL) for 2hr, n = 4 replicates. The RNA was extracted and prepared for RT–PCR. The expression of cyclin D2, FKHR, SCC, 3βHSD and StAR were measured. TGFβRI and TGFβRII proteins were localised to postnatal rat ovary by immunohistochemistry. TGF-β1–3, TGFβRI and TGFβRII were present in rat ovaries as early as 4 days after birth. Expression of TGF-β1 mRNA increased 2-fold between day 4 and 12. TGF-β2 and TGF-β3 mRNAs declined between day 4 and 8 and remained low at day 12. The type I and II TGF-β receptors were differentially regulated with TGFβRI expression high at day 4, declining at day 8. In contrast, TGFβRII appeared to be ubiquitously expressed. Cyclin D2 mRNA expression was enhanced in the presence of both TGF-β1 and FSH, whereas FKHR mRNA expression declined. TGF-β1 had no impact on the steroidogenic mRNAs. TGFβRI and TGFβRII proteins were localised to the cytoplasm of oocytes, granulosa cells and theca cells. These studies indicate that TGF-β1 can exert effects on ovarian folliculogenesis as it is established during the postnatal period. Proliferation and apoptosis appear to be targets of TGF-β1 action. Supported by the NHMRC of Australia (Regkeys 241000, 198705, 441101 & 465415)

Adenovirus-Delivered Antisense RNA and shRNA Exhibit Different Silencing Efficiencies for the Endogenous Transforming Growth Factor-β(TGF-β) Type II Receptor

Gene silencing is an essential tool in gene discovery and gene therapy. Traditionally, viral delivery of antisense RNA and, more recently, small interfering RNA (siRNA) molecules in the form of small hairpin RNAs (shRNA) has been used as a strategy to achieve gene silencing. Nevertheless, the enduring challenge is to identify molecules that specifically and optimally silence a given target gene. In this study, we tested a set of adenovirus-delivered antisense RNA fragments and adenovirus-delivered shRNA molecules for their ability to target human transforming growth factor-beta type II receptor (TGFbetaRII). We used a dicistronic reporter, consisting of the coding sequences for TGFbetaRII and green fluorescent protein (GFP) to screen for optimal silencing agents targeting TGFbetaRII. Our results show, for both antisense RNA and shRNA molecules, that their effectiveness in the GFP screen correlated directly with their ability to reduce exogenously expressed TGFbetaRII. Unexpectedly, the antisense RNAs were unable to silence endogenous TGFbetaRII. In contrast, the shRNAs were able to silence endogenous TGFbetaRII. The shRNA that demonstrated the most pronounced effect on the dicistronic TGFbetaRII/GFP reporter reduced endogenous TGFbetaRII protein expression by 70% in A549 cells and reduced TGFbeta signaling by >80% in HeLa cells.

Altered Expression of TGF-β Receptors in Hepatocellular Carcinoma – Effects of a Constitutively Active TGF-β Type I Receptor Mutant

Background/Aims: Hepatocellular carcinomas (HCC) often show resistance to the effects of transforming growth factor-β (TGF-β). This study focuses on molecular mechanisms of this resistance to explore ways to overcome it. Methods: Transcription and protein expression of TGF-β type I and type II receptors (TGF-βRI/RII) were analyzed in clinical HCCs and the human hepatoma cell lines HuH-7 and HepG2. HuH-7 cells were transiently and stably transfected with a constitutively active TGF-βRI mutant (CA TGF-βRI). Resulting growth kinetics, integrin expression, invasiveness, TGF-β-mediated activation of human plasminogen activator inhibitor type-1 (PAI-1) promoter and Smad expression were determined. Results: In clinical HCCs, there was less TGF-βRII (6/10 cases) and more TGF-βRI (8/10 cases) protein expression detectable in tumor compared to adjacent liver tissue. In HuH-7 cells, TGF-βRII expression was likewise decreased. Cells transiently transfected with CA TGF-βRI exhibited strong TGF-β-related PAI-1 promoter activation. Stably transfected cells showed an attenuated response of the PAI-1 promoter, but increased Smad7 expression. Proliferation of stable clones was decreased. There was no change in integrin expression or invasiveness. Conclusions: Decreased TGF-βRII protein expression might cause TGF-β resistance in a subset of clinical HCCs. Stable transfection with CA TGF-βRI reverses this in HuH-7 cells without increasing invasiveness.

Mechanisms through which bradykinin promotes glomerular injury in diabetes

In diabetes, mesangial cell proliferation and extracellular matrix expansion are critical components in the development of glomerulosclerosis. We reported that diabetes alters the activity of the kallikrein-kinin system and that these alterations contribute to the development of diabetic nephropathy. The present study examined the influence of streptozotocin-induced diabetes on the renal expression of bradykinin (BK) B2 receptors (B2KR), connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta), and TGF-beta type II receptor (TGF-betaRII) and assessed the signaling mechanisms through which B2KR activation may promote glomerular injury. Eight weeks after the induction of diabetes, renal mRNA levels of B2KR, CTGF, and TGF-beta as well as protein levels of CTGF and TGF-betaRII were measured in control (C), diabetic (D), and insulin-treated diabetic (D+I) rats. Renal B2KR and TGF-beta mRNA levels expressed relative to beta-actin mRNA levels and CTGF and TGF-betaRII protein levels were significantly increased in D and D+I rats compared with C rats (P < 0.03, n = 5). To assess the contribution of B2KR activation on modulating the expression of CTGF, TGF-betaRII, and collagen I, mesangial cells (MC) were treated with BK (10(-8) M) for 24 h and CTGF and TGF-betaRII protein levels were measured by Western blots and collagen I mRNA levels were measured by RT-PCR. A two- to threefold increase in CTGF and TGF-betaRII protein levels was observed in response to BK stimulation (P < 0.001, n = 6). In addition, a marked increase in collagen I mRNA levels was observed in response to BK stimulation. Treatment of MC with BK (10(-8) M) for 5 min significantly increased the tyrosine phosphorylation of p60src kinase and of p42/p44 MAPK (P < 0.05, n = 4). Inhibition of src kinase by PP1 (10 microM) inhibited the increase in p42/p44 MAPK activation in response to BK. Finally, to determine whether BK stimulates CTGF, TGF-betaRII, and collagen I expression via activation of MAPK pathways, MC were pretreated with an inhibitor of p42/p44 MAPK (PD-98059) for 45 min, followed by BK (10(-8) M) stimulation for 24 h. Selective inhibition of p42/p44 MAPK significantly inhibited the BK-induced increase in CTGF, TGF-betaRII, and collagen I levels. These findings are the first to demonstrate that BK regulates the expression of CTGF, TGF-betaRII, and collagen I in MC and provide a mechanistic pathway through which B2KR activation may contribute to the development of diabetic nephropathy.

TGF-β signaling is disrupted in endometrioid-type endometrial carcinomas

ObjectivePrevious studies have demonstrated deregulation of the expression and changes in the intracellular distribution of TGF-β pathway components in human endometrial cancer (EC). The aim of this study was to assess the relationship between the expression of TGF-β cascade components, including TGF-β receptor type I (TGFβRI) and type II (TGFβRII), SMAD2, SMAD3, SMAD4, and clinicopathological features—tumor grade, FIGO classification, and depth of myometrial invasion—of type I (endometrioid-type) ECs to give some insight into the role of TGF-β cascade components in endometrial tumorigenesis. MethodsThe expression of TGFβRI, TGFβRII, SMAD2, SMAD3, and SMAD4 was evaluated both at the mRNA and protein level using reverse transcription polymerase chain reaction (RT-PCR) and ELISA, respectively. ResultsInfiltrating endometrial carcinomas (less and more than half of the myometrial wall thickness) express significantly higher TGFβRII protein level compared with non-infiltrating tumors (P = 0.04 and P = 0.01, respectively). Decreased level of SMAD2 and SMAD4 mRNAs was observed in the uterine tumors infiltrating less and more than half of the myometrial wall (P = 0.03 and P = 0.02, respectively) compared with noninfiltrating ECs. Significantly higher SMAD4 protein level in the cytoplasmic fraction of ECs was found when tumor grade and depth of myometrial invasion were considered (P < 0.05). Generally, tumor progression was associated with a decreased number of cases characterized by the presence of SMADs in the nuclear fraction only. ConclusionOur data suggest that disturbances of the TGFβRII and SMAD4 expression as well as localization of SMADs may be important to the infiltration of the myometrial wall by the type I endometrial carcinomas.