TGF-βRII Expression Research Articles

MiR-18a-5p Inhibits Sub-pleural Pulmonary Fibrosis by Targeting TGF-β Receptor II

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease that typically leads to respiratory failure and death within 3-5 years of diagnosis. Sub-pleural pulmonary fibrosis is a pathological hallmark of IPF. Bleomycin treatment of mice is a an established pulmonary fibrosis model. We recently showed that bleomycin-induced epithelial-mesenchymal transition (EMT) contributes to pleural mesothelial cell (PMC) migration and sub-pleural pulmonary fibrosis. MicroRNA (miRNA) expression has recently been implicated in the pathogenesis of IPF. However, changes in miRNA expression in PMCs and sub-pleural fibrosis have not been reported. Using cultured PMCs and a pulmonary fibrosis animal model, we found that miR-18a-5p was reduced in PMCs treated with bleomycin and that downregulation of miR-18a-5p contributed to EMT of PMCs. Furthermore, we determined that miR-18a-5p binds to the 3' UTR region of transforming growth factor β receptor II (TGF-βRII) mRNA, and this is associated with reduced TGF-βRII expression and suppression of TGF-β-Smad2/3 signaling. Overexpression of miR-18a-5p prevented bleomycin-induced EMT of PMC and inhibited bleomycin-induced sub-pleural fibrosis in mice. Taken together, our data indicate that downregulated miR-18a-5p mediates sub-pleural pulmonary fibrosis through upregulation of its target, TGF-βRII, and that overexpression of miR-18a-5p might therefore provide a novel approach to the treatment of IPF.

Cancer-associated fibroblasts enact field cancerization by promoting extratumoral oxidative stress

Histological inspection of visually normal tissue adjacent to neoplastic lesions often reveals multiple foci of cellular abnormalities. This suggests the presence of a regional carcinogenic signal that spreads oncogenic transformation and field cancerization. We observed an abundance of mutagenic reactive oxygen species in the stroma of cryosectioned patient tumor biopsies, indicative of extratumoral oxidative stress. Diffusible hydrogen peroxide (H2O2) was elevated in the conditioned medium of cultured skin epithelia at various stages of oncogenic transformation, and H2O2 production increased with greater tumor-forming and metastatic capacity of the studied cell lines. Explanted cancer-associated fibroblasts (CAFs) also had higher levels of H2O2 secretion compared with normal fibroblasts (FIBs). These results suggest that extracellular H2O2 acts as a field effect carcinogen. Indeed, H2O2-treated keratinocytes displayed decreased phosphatase and tensin homolog (PTEN) and increased Src activities because of oxidative modification. Furthermore, treating FIBs with CAF-conditioned medium or exogenous H2O2 resulted in the acquisition of an oxidative, CAF-like state. In vivo, the proliferative potential and invasiveness of composite tumor xenografts comprising cancerous or non-tumor-forming epithelia with CAFs and FIBs could be attenuated by the presence of catalase. Importantly, we showed that oxidatively transformed FIBs isolated from composite tumor xenografts retained their ability to promote tumor growth and aggressiveness when adoptively transferred into new xenografts. Higher H2O2 production by CAFs was contingent on impaired TGFβ signaling leading to the suppression of the antioxidant enzyme glutathione peroxidase 1 (GPX1). Finally, we detected a reduction in Smad3, TAK1 and TGFβRII expression in a cohort of 197 clinical squamous cell carcinoma (SCC) CAFs, suggesting that impaired stromal TGFβ signaling may be a clinical feature of SCC. Our study indicated that CAFs and cancer cells engage redox signaling circuitries and mitogenic signaling to reinforce their reciprocal relationship, suggesting that future anticancer approaches should simultaneously target ligand receptor and redox-mediated pathways.

Role of the TGFβ/PDCD4/AP-1 Signaling Pathway in Nasopharyngeal Carcinoma and Its Relationship to Prognosis

The objective of the present study was to evaluate the role of the TGFβ/PDCD4/AP-1 pathway in nasopharyngeal carcinoma (NPC) and its relationship to NPC prognosis. NPC tissues collected from 66 NPC patients were compared to 17 nasopharyngeal mucosa biopsy specimens collected as normal tissues. Immunohistochemical staining was performed to assess expression of transforming growth factor-β receptor I (TGFβRI), programmed cell death 4 (PDCD4) and activator protein-1 (AP-1). The Kaplan-Meier method was applied to evaluate NPC patient overall survival (OS) and progression-free-survival (PFS). Cox regression analysis was used to estimate independent prognostic factors for NPC. The human NPC cell line CNE2 was selected and treated with SB431542, an inhibitor of TGFβRI; expression of TGFβRI and PDCD4 in CNE2 cells was determined by western blotting. NPC tissues showed higher expression of TGFβRI and AP-1 but lower expression of PDCD4 than normal tissues (all P < 0.05). The results of Kaplan-Meier analysis showed that TGFβRI-positive patients and AP-1-positive patients had shorter OS and PFS than TGFβRI-negative patients and AP-1-negative patients; additionally, PDCD4-positive patients had higher OS and PFS than PDCD4-negative patients. Cox regression analysis revealed that advanced tumor stage, overexpression of TGFβRI and AP-1, and low expression of PDCD4 were unfavorable factors influencing OS and PFS in NPC patients. Compared with the control group, expression of TGFβRI decreased and that of PDCD4 increased significantly in CNE2 cells treated with the inhibitor (all P < 0.05). These findings indicate that the TGFβ/PDCD4/AP-1 pathway may be associated with NPC development and progression. High expression of TGFβRI and AP-1 and low expression of PDCD4 may be unfavorable prognostic factors for NPC.

ROLE OF TRANSFORMING GROWTH FACTOR RECEPTOR B I TYPE (TGF-BRI) IN THE PROGRESSION OF THE LUMINAL BREAST CANCER SUBTYPE

Introduction. The crosstalk between the estrogen and growth factor receptors signaling may play an important role in the resistance to endocrine therapy. The aim of the study was to examine the relationship between the protein and receptor gene expression of transforming growth factor β type I (TGF-βRI) and its polymorphism with progression of luminal breast cancer patients treated by adjuvant tamoxifen. Material and methods . The study included 105 patients with luminal breast cancer (T 1-3 N 0–3 M 0 ), who had received adjuvant tamoxifen at a dose of 20 mg/day for at least 5 years. Patients who developed distant metastasis or recurrence after tamoxifen therapy were defined as tamoxifen resistance (TR) group, while distant metastasis-free patients were analyzed as tamoxifen sensitive (TS) group. TGF-βRI expression level was evaluated using immunohistochemistry. TGF-BRI gene expression and genotypes for rs334354 SNP were detected by a Real-time PCR. Results. We found high TGF-βRI gene expression in patients with luminal A subtype compared with luminal B breast cancer (p=0.050). The Int7G24AA and Int 7G24A mutant carriers were more prevalent in luminal A breast cancer patients (p=0.019 and p=0.007, respectively). TGF-βRI protein expression level was significantly higher in the tamoxifen sensitive group compared to tamoxifen resistance breast cancer patients regardless of molecular subtypes (p=0.043). There was a trend for a tamoxifen sensitivity among luminal B breast cancer patients with a high TGF-βRI protein expression (p=0.090). Conclusion. TGF-βRI protein expression level can be a potential molecular marker of tamoxifen resistance in luminal breast cancer patients.

MiR-17∼92 family clusters control iNKT cell ontogenesis via modulation of TGF-β signaling

Invariant natural killer T cells (iNKT) cells are T lymphocytes displaying innate effector functions, acquired through a distinct thymic developmental program regulated by microRNAs (miRNAs). Deleting miRNAs by Dicer ablation (Dicer KO) in thymocytes selectively impairs iNKT cell survival and functional differentiation. To unravel this miRNA-dependent program, we systemically identified transcripts that were differentially expressed between WT and Dicer KO iNKT cells at different differentiation stages and predicted to be targeted by the iNKT cell-specific miRNAs. TGF-β receptor II (TGF-βRII), critically implicated in iNKT cell differentiation, was found up-regulated in iNKT Dicer KO cells together with enhanced TGF-β signaling. miRNA members of the miR-17∼92 family clusters were predicted to target Tgfbr2 mRNA upon iNKT cell development. iNKT cells lacking all three miR-17∼92 family clusters (miR-17∼92, miR-106a∼363, miR-106b∼25) phenocopied both increased TGF-βRII expression and signaling, and defective effector differentiation, displayed by iNKT Dicer KO cells. Consistently, genetic ablation of TGF-β signaling in the absence of miRNAs rescued iNKT cell differentiation. These results elucidate the global impact of miRNAs on the iNKT cell developmental program and uncover the targeting of a lineage-specific cytokine signaling by miRNAs as a mechanism regulating innate-like T-cell development and effector differentiation.

Lin28b Regulates Fetal Regulatory T Cell Differentiation through Modulation of TGF-β Signaling.

Immune tolerance between the fetus and mother represents an active process by which the developing fetus must not mount immune responses to noninherited Ags on chimeric maternal cells that reside in fetal tissue. This is, in part, mediated by the suppressive influence of CD4+FOXP3+CD25+ regulatory T cells (Tregs). Fetal secondary lymphoid organs have an increased frequency of Tregs and, as compared with adult T cells, fetal naive CD4+ T cells exhibit a strong predisposition to differentiate into Tregs when stimulated. This effect is mediated by the TCR and TGF-β pathways, and fetal T cells show significantly increased Treg differentiation in response to anti-CD3 and TGF-β stimulation. Naive fetal T cells also exhibit increased signaling through the TGF-β pathway, with these cells demonstrating increased expression of the signaling mediators TGF-βRI, TGF-βRIII, and SMAD2, and higher levels of SMAD2/SMAD3 phosphorylation. Increased fetal Treg differentiation is mediated by the RNA-binding protein Lin28b, which is overexpressed in fetal T cells as compared with adult cells. When Lin28b expression is decreased in naive fetal T cells, they exhibit decreased Treg differentiation that is associated with decreased TGF-β signaling and lowered expression of TGF-βRI, TGF-βRIII, and SMAD2. Lin28b regulates the maturation of let-7 microRNAs, and these TGF-β signaling mediators are let-7 targets. We hypothesize that loss of Lin28b expression in fetal T cells leads to increased mature let-7, which causes decreased expression of TGF-βRI, TGF-βRIII, and SMAD2 proteins. A reduction in TGF-β signaling leads to reduced Treg numbers.

Tolerogenic dendritic cells generated with dexamethasone and vitamin D3 regulate rheumatoid arthritis CD4+ T cells partly via transforming growth factor-β1.

Tolerogenic dendritic cells (tolDC) are a new immunotherapeutic tool for the treatment of rheumatoid arthritis (RA) and other autoimmune disorders. We have established a method to generate stable tolDC by pharmacological modulation of human monocyte-derived DC. These tolDC exert potent pro-tolerogenic actions on CD4+ T cells. Lack of interleukin (IL)-12p70 production is a key immunoregulatory attribute of tolDC but does not explain their action fully. Here we show that tolDC express transforming growth factor (TGF)-β1 at both mRNA and protein levels, and that expression of this immunoregulatory cytokine is significantly higher in tolDC than in mature monocyte-derived DC. By inhibiting TGF-β1 signalling we demonstrate that tolDC regulate CD4+ T cell responses in a manner that is at least partly dependent upon this cytokine. Crucially, we also show that while there is no significant difference in expression of TGF-βRII on CD4+ T cells from RA patients and healthy controls, RA patient CD4+ T cells are measurably less responsive to TGF-β1 than healthy control CD4+ T cells [reduced TGF-β-induced mothers against decapentaplegic homologue (Smad)2/3 phosphorylation, forkhead box protein 3 (FoxP3) expression and suppression of (IFN)-γ secretion]. However, CD4+ T cells from RA patients can, nonetheless, be regulated efficiently by tolDC in a TGF-β1-dependent manner. This work is important for the design and development of future studies investigating the potential use of tolDC as a novel immunotherapy for the treatment of RA.

Effects of somatostatin and its analogues on progenitor mesenchymal cells isolated from human pituitary adenomas.

Progenitor mesenchymal cells (PMCs) have been found also in epithelial tumors and may derive from cancer stem cells (CSCs) by EMT mechanism. In this scenario, the effects of traditionally drugs on PMCs become of primary concern for therapeutic approaches. Previously, we isolated PMCs from acromegalic (GHomas) and not-functioning pituitary adenomas (NFPAs). Here we evaluate: (1) the role of EMT on their origin; (2) the presence of the somatostatin receptors (SSTR1-5); (3) the effects of somatostatin (SST) and its analogues (SSAs) on PMCs proliferation, apoptosis and SSTR1-5 expression. PMCs were isolated from GHomas and NFPAs; the expression of E-CADHERIN and TGFβRII (referred to EMT), the expression of the SSTR1-5 as well as the proliferation and apoptosis were tested before and after drugs administration. Results show a decrease of E-CADHERIN and an increase of TGFβRII, confirming an EMT involvement; SSTR1-5 are more expressed by PMCs from GHomas than from NFPAs. SST and SSAs administration does not affect cell proliferation and SSTR1-5 expression on PMCs from NFPAs while in PMCs from GHomas, cell proliferation showed a marked decrease and a corresponding increase in the expression of SSTR1-2. Apoptosis rate and EMT were not affected by drugs administration. Results indicate as EMT may be related to the presence of PMCs on pituitary tumors; SSAs, currently used in the management of human GHomas, exert anti-proliferative effect also in PMCs that, because of their derivation from CSCs, may be a new meaningful target for drugs treatment.

Collagen regulates transforming growth factor-β receptors of HL-1 cardiomyocytes through activation of stretch and integrin signaling.

The extracellular matrix (ECM) and transforming growth factor-β (TGF)-β are important in cardiac fibrosis, however, the effects of the ECM on TGF‑β signaling remain to be fully elucidated. The aims of the present study were to evaluate the role of collagen in TGF‑β signaling and examine the underlying mechanisms. In the present study, western blot analysis was used to examine TGF‑β signaling in HL‑1cells treated with and without (control) typeI collagen (10µg/ml), which was co‑administered with either an anti‑β1 integrin antibody (10µg/ml) or a stretch‑activated channel inhibitor (gadolinium; 50µM). Cell proliferation and adhesion assays were used to investigate the roles of integrin, mechanical stretch and mitogen‑activated protein kinases (MAPKs) on cell proliferation and adhesion. The typeI collagen (10µg/ml)‑treated HL‑1 cells were incubated with or without anti‑β1 integrin antibody (10µg/ml), gadolinium (50µM) or inhibitors of p38 (SB203580; 3µM), extracellular signal‑regulated kinase (ERK; PD98059; 50µM) and c‑Jun N‑terminal kinase (JNK; SP600125; 50µM). Compared with the control cells, the collagen‑treated HL‑1 cells had lower expression levels of typeI and type II TGF‑β receptors (TGFβRI and TGFβRII), with an increase in phosphorylated focal adhesion kinase (FAK), p38 and ERK1/2, and a decrease in JNK. Incubation with the anti‑β1 integrin antibody reversed the collagen‑induced downregulation of the expression of TGFβRII and phosphorylated FAK. Gadolinium downregulated the expression levels of TGFβRI and small mothers against decapentaplegic (Smad)2/3, and decreased the levels of phosphorylated p38, ERK1/2 and JNK. In addition, gadolinium reversed the collagen‑induced activation of p38 and ERK1/2. In the presence of gadolinium and anti‑β1 integrin antibody, collagen regulated the expression levels of TGFβRI, TGFβRII and Smad2/3, but did not alter the phosphorylation of p38, ERK1/2 or JNK. In addition, collagen increased cell proliferation and adhesion, and this collagen‑induced cell proliferation was inhibited by the anti‑β1 integrin antibody and ERK inhibitor. Taken together, the data obtained suggested that collagen differentially regulated the expression levels of TGFβRI and TGFβRII, and modulated the phosphorylation of MAPKs through integrin‑ or stretch‑dependent and ‑independent signaling pathways.

Thymoquinone subdues tumor growth and potentiates the chemopreventive effect of 5-fluorouracil on the early stages of colorectal carcinogenesis in rats.

Colorectal cancer (CRC) is one of the most prevalent cancers and has a high mortality rate. Insensitivity and the limited therapeutic efficacy of its standard chemotherapeutic drug, 5-fluorouracil (5-FU), represents an important challenge in CRC treatment. The robust antitumor properties of thymoquinone (TQ), the main bioactive constituent of Nigella sativa, have recently been demonstrated on different cancers. We investigated whether TQ could potentiate the chemopreventive effect of 5-FU to eradicate the early stages of CRC and elucidated its underlying mechanisms. An intermediate-term (15 weeks) model of colorectal tumorigenesis was induced in male Wistar rats by azoxymethane (AOM), and the animals were randomly and equally divided into five groups: control, AOM, AOM/5-FU, AOM/TQ, and AOM/5-FU/TQ. TQ (35 mg/kg/d; 3 d/wk) was given during the seventh and 15th weeks post-AOM injection, while 5-FU was given during the ninth and tenth weeks (12 mg/kg/d for 4 days; then 6 mg/kg every other day for another four doses). At week 15, the resected colons were subjected to macroscopic, histopathological, molecular, and immunohistochemical examinations. Interestingly, 5-FU/TQ combination therapy resulted in a more significant reduction on AOM-induced colorectal tumors and large aberrant crypts foci than treatment with the individual drugs. Mechanistically, 5-FU and TQ remarkably cooperated to repress the expression of procancerous Wnt, β-catenin, NF-κB, COX-2, iNOS, VEGF, and TBRAS and upregulate the expression of anti-tumorigenesis DKK-1, CDNK-1A, TGF-β1, TGF-βRII, Smad4, and GPx. Overall, our findings present the first report describing the in vivo enhancement effect of combined TQ and 5-FU against early stages of CRC; however, further studies are required to determine the value of this combination therapy in an advanced long-term model of CRC and also to realize its clinical potential.

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.

Elevated TGF-β2 level in aqueous humor of cataract patients with high myopia: Potential risk factor for capsule contraction syndrome

To evaluate the level of transforming growth factor-β2 (TGF-β2) in the aqueous humor of highly myopic cataract patients and its correlation with capsule contraction syndrome. Eye and ENT Hospital of Fudan University, Shanghai, China. Prospective comparative case series. The highly myopic cataract patients were divided into the following 2 groups according to the Lens Opacity Classification System III: nuclear color (NC) 2 to 3 and NC 5 to 6. Aqueous humor TGF-β2 concentrations were assayed in the highly myopic cataract and age-related cataract groups. The TGF-β2, TGF-βRII (the type II receptor for TGF-β2), and α-smooth muscle actin (α-SMA) expressions in lens epithelial cells (LECs) were detected by real-time polymerase chain reaction and immunofluorescent staining. The study comprised 40 highly myopic cataract patients (40 eyes) and 20 patients (20 eyes) with age-related cataract as the control group. Compared with the control group, the highly myopic cataract group had significantly higher TGF-β2 concentration in the aqueous humor and increased TGF-βRII expression in LECs, especially in NC 5 to 6 cases. Expression of α-SMA was barely detectable in both groups. In highly myopic cataract patients, especially those with dark nuclei, elevated aqueous humor TGF-β2 levels and the upregulated TGF-βRII expression in LECs might contribute to the pathogenesis of capsule contraction syndrome through transdifferentiation of LECs into myofibroblasts. None of the authors has a financial or proprietary interest in any material or method mentioned.

Association of down-regulation of CD109 expression with up-expression of Smad7 in pathogenesis of psoriasis.

Transforming growth factor (TGF)-β signaling plays an important role in the pathogenesis of psoriasis. CD109, a novel TGF-β co-receptor, which inhibits TGF-β signaling by enhancing Smad7-dependent degradation of TGF-β type I receptor (TGF-β RI), is abnormally expressed in psoriasis. To date, the expression of Smad7 and the correlation between CD109 and Smad7 expression in psoriasis have not been fully elucidated. This study was designed to investigate the expression and the correlation of CD109 and TGF-β signaling associated proteins in psoriasis and their roles in the pathogenesis of psoriasis. Thirty-two psoriasis specimens were subjected to immunohistochemical staining for CD109, Smad7, TGF-β RI and Ki67. Ten normal skin (NS) specimens served as controls. The positive expression rate (% positive cells) of Smad7 and Ki67 in psoriasis was significantly higher than in NS (62.6%±19.9% vs. 17.2%±4.4%, and 50.7%±14.3% vs. 19.5%±3.2%, respectively, P<0.001), and the expression levels of CD109 and TGF-β RI were reduced significantly in psoriasis as compared with NS (8.1%±6.7% vs. 35.8%±6.7% and 27.3%±3.4% vs. 3.0%±3.4%, respectively, P<0.001). There were significantly negative correlations between CD109 and Smad7 (r=-0.831, P<0.01). These findings indicated that CD109 might play a certain role in the pathogenesis of psoriasis. Lower expression of CD109 and TGF-β RI was highly correlated with higher expression of Smad7 and Ki67, suggesting that CD109 may induce the pathogenesis of psoriasis through Smad7-mediated degradation of TGF-β RI, and lead to the termination of TGF-β signaling.

Exosomes-Derived MiR-302b Suppresses Lung Cancer Cell Proliferation and Migration via TGFβRII Inhibition

Several studies have reported that tumor-derived exosomes contain kinds of miRNAs, including oncogenic miRNAs and tumor suppressor miRNAs. It has been reported that miR-302b could inhibit cancer progression by targeting oncogenes post-transcriptionally. Whether miR-302b is involved in the regulation of tumor-derived exosomes on lung cancer cells proliferation has not yet been demonstrated. This study aimed to examine the effect of exosomes-derived miR-302b on lung cancer cells proliferation and explain the potential mechanism.The effect of exosomes derived from 95C cells and 95D cells with different metastatic ability on lung cancer cell proliferation and migration were analyzed by MTT assay and Transwell assays, respectively. Exosomes of 95C and 95D cells derived miR-302b was quantified by qRT-PCR, the effect of miR-302b on proliferation and migration of 95D cells was analyzed by MTT assays and Transwell assays respectively after transfection with miR-302b, while the expression of phosphorylated ERK1/2, MMP9 and TGFβRx2161; was analyzed by Western blot. The target gene of miRNA-302b was analyzed by Luciferase reporter assays.95C-derived exosomes significantly decreased the migration ability of 95D cells. miRNA-302b was significantly overexpressed in 95C cells and 95C-derived exosomes compared with 95D cells and 95D-derived exosomes. Transfection of miR-302b into 95D cells significantly suppressed cells proliferation and migration capabilities along with the down-regulated expression of TGFβRII, phosphorylated ERK1/2 and MMP9 induced by TGF-β1.Exosomes-derived miR-302b could suppress lung cancer cell proliferation and migration via the TGFβRII/ERK pathway and thus provides a potential target for human lung cancer therapy.

Effect of Kuijie Granule on the Expression of TGF-β/Smads Signaling Pathway in Patients with Ulcerative Colitis.

The dysregulation of TGF-β/Smads signaling pathway has been postulated to contribute to the development of ulcerative colitis (UC) and the manifestation of clinical symptoms. Kuijie Granule is a prescription medicine used clinically in China to alleviate the symptoms associated with UC. To evaluate whether the clinical benefit of Kuijie Granule is associated with TGF-β/Smads signaling, we measured the expression levels of TGF-β/Smads signaling proteins (TGF-β1, TGF-βRII, Smad2, Smad4, Smad6, and Smad7) in the intestinal mucosa of 72 patients with UC treated with Kuijie Granule for 60 days. Colonic tissues were obtained by a virtual colonoscopy guided biopsy before and after Kuijie Granule treatment followed by pathological analysis and quantitative analysis of TGF-β/Smads using immunohistochemistry. Kuijie Granule treatment significantly improved symptoms associated with UC, which include diarrhea, mucus production, pus and blood in stool, abdominal pain and distention, and tenesmus. The clinical benefit of Kuijie Granule treatment correlated with decreased expression of TGF-β1 and Smad7 and increased expression of TGF-βRII and Smad4. These clinical results indicate that Kuijie Granule can alleviate the symptoms associated with UC and modulate TGF-β/Smads signaling.

MiR-22 may Suppress Fibrogenesis by Targeting TGFβR I in Cardiac Fibroblasts

Background/Aims: Cardiac fibrosis after myocardial infarction (MI) has been identified as a key factor in the development of heart failure, but the mechanisms undelying cardiac fibrosis remained unknown. microRNAs (miRNAs) are novel mechanisms leading to fibrotic diseases, including cardiac fibrosis. Previous studies revealed that miR-22 might be a potential target. However, the roles and mechanisms of miR-22 in cardiac fibrosis remained ill defined. The present study thus addressed the impact of miR-22 in cardiac fibrosis. Methods: After seven days following coronary artery occlusion in mice, tissues used for histology were collected and processed for Masson's Trichrome staining. In addition, cardiac fibroblasts were transfected with mimics and inhibitors of miR-22 using Lipofectamin 2000, and luciferase activity was measured in cell lysates using a luciferase assay kit. Western blotting was used to detect the expression of collagen1, α-SMA and TGFβRI proteins levels, and real time-PCR was employed to measure the Col1α1, Col3α1, miR-22 and TGFβRI mRNA levels. Results: In this study, we found that miR-22 was dynamically downregulated following MI induced by permanent ligation of the left anterior descending coronary artery for 7 days, an effect paralleled by significant collagen deposition. Inhibition of miR-22 with AMO-22 resulted in increased expression of Col1α1, Col3α1 and fibrogenesis in cultured cardiac fibroblasts. Conversely, overexpression of miR-22 in cultured cardiac fibroblasts significantly abrogated angiotensin II-induced collagen formation and fibrogenesis. Furthermore, we found that TGFβRI is a direct target for miR-22, and downregulation of TGFβR may have mediated the antifibrotic effect of miR-22. Conclusion: Our data clearly demonstrate that miR-22 acts as a novel negative regulator of angiotensin II-induced cardiac fibrosis by suppressing the expression of TGFβRI in the heart and may represent a new potential therapeutic target for treating cardiac fibrosis.

Krüppel-like factor KLF10 deficiency predisposes to colitis through colonic macrophage dysregulation.

Krüppel-like factor (KLF)-10 is an important transcriptional regulator of TGF-β1 signaling in both CD8(+) and CD4(+) T lymphocytes. In the present study, we demonstrate a novel role for KLF10 in the regulation of TGFβRII expression with functional relevance in macrophage differentiation and activation. We first show that transfer of KLF10(-/-) bone marrow-derived macrophages into wild-type (WT) mice leads to exacerbation of experimental colitis. At the cell biological level, using two phenotypic strategies, we show that KLF10-deficient mice have an altered colonic macrophage phenotype with higher frequency of proinflammatory LyC6(+)MHCII(+) cells and a reciprocal decrease of the anti-inflammatory LyC6(-)MHCII(+) subset. Additionally, the anti-inflammatory CD11b(+)CX3CR1(hi) subset of colonic macrophages is significantly decreased in KLF10(-/-) compared with WT mice under inflammatory conditions. Molecularly, CD11b(+) colonic macrophages from KLF10(-/-) mice exhibit a proinflammatory cytokine profile with increased production of TNF-α and lower production of IL-10 in response to LPS stimulation. Because KLF10 is a transcription factor, we explored how this protein may regulate macrophage function. Consequently, we analyzed the expression of TGFβRII expression in colonic macrophages and found that, in the absence of KLF10, macrophages express lower levels of TGFβRII and display an attenuated Smad-2 phosphorylation following TGF-β1 stimulation. We further show that KLF10 directly binds to the TGFβRII promoter in macrophages, leading to enhanced gene expression through histone H3 acetylation. Collectively, our data reveal a critical role for KLF10 in the epigenetic regulation of TGFβRII expression in macrophages and the acquisition of a "regulatory" phenotype that contributes to intestinal mucosal homeostasis.

Abstract 2054: In pancreatic cancer, MUC1 regulates function of TGF-β and thus enhances metastasis

Abstract Background Pancreatic Cancer (PC) is the 4th leading cause of cancer-related deaths in the United States with 94% of PC patients dying within 5 years of diagnosis. Pancreatic Ductal Adenocarcinoma (PDA) characterizes 90% of PC. Over 80% of PDA overexpresses tumor associated Mucin-1 (MUC1), a membrane bound glycoprotein that is hypoglycosylated compared to normally expressed MUC1. Overexpression of MUC1 is associated with increased metastasis and poor prognosis. However the mechanism remains elusive. Transforming growth factor-β (TGF-β) is similarly overexpressed in most PDA. TGF-β is a cytokine with dual functionality. In normal cells, TGF-β functions as a tumor suppressor and induces apoptosis. This effect is mediated by activation of the canonical Smad pathway via engagement of TGF-β Receptor 1 (TGF-βRI). However, during cancer development, TGF-β becomes a tumor promoter and stimulates epithelial to mesenchymal transition, migration, and invasion of tumor cells thus enhancing metastasis. This effect of TGF-β is mediated by activation of the noncanonical Erk1/2 pathway through the engagement of TGF-β Receptor 2 (TGF-βRII). Therefore, we hypothesize that overexpression of MUC1 in PDA transforms the function of TGF-β from a tumor suppressor to a tumor promoter. Further, we postulate that signaling through the MUC1 cytoplasmic tail (CT) is necessary to activate the noncanonical Erk1/2 pathway via phosphorylation of the TGF-βRII, thus leading to enhanced metastasis. Methods We first assessed the ability of TGF-β to induce apoptosis versus invasiveness in PDA cell lines that express variable levels of MUC1 with gain of function (forced expression) and loss of function (knock down using specific MUC1 siRNA) studies. We determined the levels of TGF-βRI, RII, MUC1, and Smad 4 proteins in these PDA cell lines. We evaluated the activation status of the Erk1/2 and Smad pathways. Since c-Src is known to phosphorylate the 6th tyrosine residue of MUC1 CT and is essential for oncogenic signaling, we determined if c-Src is associated with MUC1 and is phosphorylated upon treatment with exogenous TGF-β in PDA cells. Results In MUC1-high expressing PDA cells, TGF-β acts as a tumor promoter while in MUC1-low expressing PDA cells, TGF-β induces apoptosis and acts as a tumor suppressor. We show a clear association between the expression of MUC1 and TGF-βRI and RII activation. In MUC1-high expressing cells, TGF-βRII is over expressed while in MUC1-low expressing cells, TGF-βRI is over expressed and this correlates with the activation status of Erk1/2 and Smad 4. Finally, we show that MUC1 specifically associates with c-Src and without this association, downstream oncogenic signaling is impeded. Conclusion MUC1 expression is directly correlated with TGF-β function and expression of TGF-βRII, while being negatively correlated with TGF-βRI expression. This has high clinical significance for patients with PDA. Citation Format: Priyanka Grover, Sritama Nath, Mohammad Ahmad, Pinku Mukherjee. In pancreatic cancer, MUC1 regulates function of TGF-β and thus enhances metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2054. doi:10.1158/1538-7445.AM2015-2054

Transforming growth factor β type II receptor as a marker in diffuse large B cell lymphoma.

The objective of this study was to investigate the expression and significance of the transforming growth factor β type II receptor (TGFβRII) in diffuse large B cell lymphoma. All patients were enrolled at the First Affiliated Hospital of Liaoning Medical University between 2001 and 2007. The median follow-up period was 53.3 months. Of the 338 patients studied, 131 (38.76 %) had TGFβRII positive expression on immunohistochemistry. The 5 year survival rate was significantly higher in patients with TGFβRII expression than in those without TGFβRII expression (40.3 vs. 31.6 %, P = 0.041). Multivariate analysis identified TGFβRII expression as an independent predictive parameter for survival, in addition to lactate dehydrogenase, clinical stage, and histologic subtype. TGFβRII expression may be considered a new prognostic factor of diffuse large B cell lymphoma.

Vitamin D levels in multiple sclerosis patients: Association with TGF-β2, TGF-βRI, and TGF-βRII expression.

A variety of evidence suggests that vitamin D can prevent the development of multiple sclerosis (MS). TGF-β pathway genes also play important roles in MS. Here, we aim to study whether vitamin D affects TGF-β pathway gene expression and Expanded Disability Status Scale (EDSS) scores in MS patients. A randomized clinical trial was conducted on 31 relapsing-remitting (RR) MS patients. Using real-time RT-PCR, we tested the levels of TGF-β2, TGF-βRI and TGF-βRII mRNAs in the RRMS patients before and after 8 weeks of supplementation with vitamin D. Expression of TGF-β2 mRNA increased 2.84-fold, while TGF-βRI and TGF-βRII mRNA levels did not change after vitamin D treatment. In addition, these results revealed no correlation between the normalized expression of TGF-β2, TGF-βRI, or TGF-βRII and EDSS scores. Here, we demonstrate new evidence for the complex role of vitamin D in the pathogenesis, activity and progression of MS through the TGF-β signaling pathway.