Transforming Growth Factor-β Receptor Research Articles

Berberine reversed the epithelial-mesenchymal transition of normal colonic epithelial cells induced by SW480 cells through regulating the important components in the TGF-β pathway.

Stroma-tumor interactions within microenvironment play a crucial role in tumor development and growth. Cellular transdifferentiation in the stroma is a prerequisite for tumor formation. Targeting the interactions maybe a promising anticancer strategy. Berberine (BBR) has been confirmed to have anticancer and anti-inflammatory effects. We found for the first time that colon cancer cells SW480 induced spindle-like morphological changes and downregulation of E-cadherin and upregulation of vimentin and alpha-smooth muscle actin in colon epithelial cells HCoEpiCs by using transwell coculture system and conditioned medium from SW480. The conditioned medium also promoted the migration of HCoEpiCs. This transition was inhibited by a transforming growth factor-β receptor inhibitor LY364947. BBR (50 and 100 µg/ml) reversed the EMT-like transition and repressed the migration in HCoEpiCs. Further results demonstrated that downregulation of TβRII, Smad2, p-Smad3, and overexpression of Smad3 participated in the SW480-induced phenotypic transition of HCoEpiCs. In addition, BBR upregulated the expressions of TβRII, Smad2, and p-Smad3. In conclusion, our findings suggest that BBR exerts the anti-EMT and antimigration effect by mediating the expression of TβRII, Smad2, and p-Smad3.

Expression, purification, and evaluation of in vivo anti-fibrotic activity for soluble truncated TGF-β receptor II as a cleavable His-SUMO fusion protein.

Excessive production of transforming growth factor-β1 (TGF-β1) and its binding to transforming growth factor-β receptor type II (TGF-βRII) promotes fibrosis by activation of the TGF-β1-mediated signaling pathway. Thus, the truncated extracellular domain of TGF-βRII (tTβRII) is a promising anti-fibrotic candidate, as it lacks the signal transduction domain. In this work, the native N-terminal tTβRII was prepared as a His-SUMO fusion protein (termed His-SUMO-tTβRII) in Escherichia coli strain BL21 (DE3). His-SUMO-tTβRII was expressed as a soluble protein under optimal conditions (6h of induction with 0.5mM IPTG at 37°C). His-SUMO-tTβRII was purified by Ni-NTA resin chromatography, and then cleaved with SUMO protease to release native tTβRII, which was re-purified using a Ni-NTA column. Approximately 12mg of native tTβRII was obtained from a one liter fermentation culture with no less than 95% purity. In vivo studies demonstrated that tTβRII prevented CCl4-induced liver fibrosis, as evidenced by the inhibition of fibrosis-related Col I and α-SMA protein expression in C57BL/6 mice. In addition, tTβRII downregulated phosphorylation of SMAD2/3, which partly repressed TGF-β1-mediated signaling. These data indicate that the His-SUMO expression system is an efficient approach for preparing native tTβRII that possesses anti-liver fibrotic activity, allowing for the large-scale production of tTβRII, which potentially could serve as an anti-fibrotic candidate for treatment of TGF-β1-related diseases.

Influence of nourishing yin and tonifying yang sequential therapy combined with Western medicine on TGF-β1/Smads signaling pathway in anovulatory infertility rats with diminished ovarian reserve

To explore the influence for combination of nourishing yin and tonifying yang sequential therapy (NYTYST) with Western medicine in treating anovulatory infertility rats with diminished ovarian reserve (DOR) based on TGF-β1/Smads signaling pathway. Methods: A total of 40 female rats were randomly divided into 5 groups, a normal control group, a model group, a Western medicine group, a NYTYST group and a combination group (n=8 in each group). The DOR model was established through orally taking tripterygium pill for continuous 2 weeks. The normal control group and the model group were treated with saline for 10 days. The Western medicine group was treated with hormone replacement therapy (HRT) and ovarian stimulation. The NYTYST group was treated with nourishing yin herbs in proestrus and tonifying yang herbs in late estrus and the combination group was treated with Chinese herb and Western drugs for 10 days. HE staining was used to observe histopathologic changes in ovary. Expression levels of transforming growth factor β1 receptor (TGF-β1R) in rats ovarian were detected by immunohistochemistry. Expression levels of Smad2, Smad3 and Smad7 protein in rat ovarian were detected by Western blot. Results: Compared with the control group, the numbers of developing follicles, mature follicles and corpus luteum were decreased , while atrefic follicles were increased significantly in the model group (P<0.01); the levels of TGF-β1R, Smad2 and Smad3 were decreased significantly, while Smad7 was increased significantly (P<0.01). Compared with the model group, the numbers of developing follicles, mature follicles and corpus luteum, Smad2 and Smad3 expression were increased, while atrefic follicles and Smad7 were decreased significantly in the treatment group (P<0.05 or P<0.01). The numbers of developing follicles and corpus luteum in the combination group was superior to the Western medicine group (P<0.05). Compared with the Western medicine group, the levels of TGF-β1R, Smad2 and Smad3 were increased significantly, while Smad7 was decreased significantly in the combination group (P<0.05 or P<0.01). Conclusion: NYTYST combined with Western medicine can improve the function of ovaries reserve by up-regulation of TGF-β1R, Smad2 and Smad3 while down-regulation of Smad7 in DOR rats.

Luteolin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Inhibiting TGFBR1 Signaling.

Vascular smooth muscle cell (VSMC) proliferation and migration play a critical role in the development of arterial remodeling during various vascular diseases including atherosclerosis, hypertension, and related diseases. Luteolin is a food-derived flavonoid that exerts protective effects on cardiovascular diseases. Here, we investigated whether transforming growth factor-β receptor 1 (TGFBR1) signaling underlies the inhibitory effects of luteolin on VSMC proliferation and migration. We found that luteolin reduced the proliferation and migration of VSMCs, specifically A7r5 and HASMC cells, in a dose-dependent manner, based on MTS and EdU, and Transwell and wound healing assays, respectively. We also demonstrated that it inhibited the expression of proliferation-related proteins including PCNA and Cyclin D1, as well as the migration-related proteins MMP2 and MMP9, in a dose-dependent manner by western blotting. In addition, luteolin dose-dependently inhibited the phosphorylation of TGFBR1, Smad2, and Smad3. Notably, adenovirus-mediated overexpression of TGFBR1 enhanced TGFBR1, Smad2, and Smad3 activation in VSMCs and partially blocked the inhibitory effect of luteolin on TGFBR1, Smad2, and Smad3. Moreover, overexpression of TGFBR1 rescued the inhibitory effects of luteolin on the proliferation and migration of VSMCs. Additionally, molecular docking showed that this compound could dock onto an agonist binding site of TGFBR1, and that the binding energy between luteolin and TGFBR1 was -10.194 kcal/mol. Simulations of molecular dynamics showed that TGFBR1-luteolin binding was stable. Collectively, these data demonstrated that luteolin might inhibit VSMC proliferation and migration by suppressing TGFBR1 signaling.

Rhubarb Protect Against Tubulointerstitial Fibrosis by Inhibiting TGF-β/Smad Pathway and Improving Abnormal Metabolome in Chronic Kidney Disease.

Tubulointerstitial fibrosis is the final common pathway for all kidney diseases leading to chronic kidney disease (CKD). TGF-β/Smad signaling pathway plays a key role in renal fibrosis. Previous studies have revealed that rhubarb extracts attenuated the increase of transforming growth factor-β 1 (TGF-β1) in CKD rats. To gain an in-depth insight into the mechanism of the anti-fibrotic activities of the rhubarb extracts, we investigated the influence of rhubarb extracts on TGF-β/Smad signaling pathway and the influence on metabolome in a rat model of CKD with adenine-induced chronic tubulointerstitial nephropathy. Male Sprague-Dawley rats were divided into four groups, including control, CKD, CKD + petroleum ether extract, CKD + ethyl acetate extract, and CKD + n-butanol extract groups. Kidneys harvested on the week three were evaluated for renal fibrosis, the expression of proteins in TGF-β/Smad signaling pathway and metabolomic study. We found rhubarb extracts suppressed TGF-β/Smad3-mediated renal fibrosis by reducing the TGF-β1, transforming growth factor-β receptor I (TGF-β RI), transforming growth factor-β receptor II (TGF-β RII), Smad2, p-Smad2, Smad3, p-Smad3, and Smad4, meanwhile increased Smad7. In addition, rhubarb extracts mitigated renal injury and dysfunction, and either fully or partially reversed the abnormalities of tissue metabolites. Thus, rebalancing the disorder of TGF-β/Smad signaling and metabolic dysfunction by treatment with rhubarb extracts may represent as an effective therapy for CKD associated with fibrosis.

Peptidyl-prolyl cis-trans isomerase NIMA interacting 1 regulates skeletal muscle fusion through structural modification of Smad3 in the linker region.

Myoblast fusion is critical for muscle growth, regeneration, and repair. We previously reported that the enzyme peptidyl‐prolyl cis–trans isomerase NIMA interacting 1 (Pin1) is involved in osteoclast fusion. The objective of this study was to investigate the possibility that Pin1 also inhibits myoblast fusion. Here, we show the increased number of nuclei in the Pin1+/− mice muscle fiber compared to that in wild‐type mice. Moreover, we show that low dose of the Pin1 inhibitor dipentamethylene thiuram monosulfide treatment caused enhanced fusion in C2C12 cells. The R‐Smads are well‐known mediators of muscle hypertrophy and hyperplasia as well as being substrates of Pin1. We found that Pin1 is crucial for maintaining the stability of Smad3 (homologues of the Drosophila protein, mothers against decapentaplegic (Mad) and the Caenorhabditis elegans protein Sma). Our results show that serine 204 within Smad3 is the key Pin1‐binding site during inhibition of myoblast fusion and that both the transforming growth factor‐β receptor and extracellular signal‐regulated kinase (ERK)‐mediated phosphorylation are required for the interaction of Pin1 with Smad3. These findings suggest that a precise level of Pin1 activity is essential for regulating myoblast fusion during myogenesis and muscle regeneration.

The t(1;10)(p22;q24) TGFBR3/MGEA5 Translocation in Pleomorphic Hyalinizing Angiectatic Tumor, Myxoinflammatory Fibroblastic Sarcoma, and Hemosiderotic Fibrolipomatous Tumor.

Pleomorphic hyalinizing angiectatic tumor (PHAT) of soft parts, hemosiderotic fibrolipomatous tumor (HFLT), and myxoinflammatory fibroblastic sarcoma (MIFS) are 3 distinct entities of low-grade spindle cell mesenchymal neoplasm. These tumors have similar clinical presentations and partially overlapping but distinctive pathologic features. A recurrent translocation, t(1;10)(p22;q24), has been detected in a subset of PHAT, HFLT, MIFS, and HFLT/MIFS hybrid cases. Translocation t(1;10)(p22;q24) involves transforming growth factor β-receptor 3 ( TGFBR3) and meningioma-expressed antigen 5 ( MGEA5) genes on chromosomes 1p22 and 10q24, respectively. However, the percentage of translocation in PHAT, HFLT, and MIFS varies significantly among different studies. The relationship among these tumors has been a controversial topic among experts. To discuss the diagnostic and functional significance of translocation t(1;10)(p22;q24) TGFBR3/MGEA5 rearrangement in HFLT, PHAT, and MIFS. PubMed was used for this study. Diagnosis of HFLT, PHAT, and MIFS is challenging because of a lack of unique morphologic, immunophenotypic, molecular, and cytogenetic markers. The recurrent t(1;10)(p22;q24) translocation and/or TGFBR3/MGEA5 rearrangement was reported in 55 patients, with a relatively even distribution among HFLT, PHAT, and MIFS (17 HFLT, 15 MIFS, 13 MIFS/HFLT, and 10 PHAT). This indicates that current morphology-based diagnostic criteria do not identify reliably the subset of soft tissue tumor with t(1;10) translocation. Genetic heterogeneity of these tumors is supported by the recent detection of a mutually exclusive, second recurrent genetic change, t(7;17) TOM1L2-BRAF translocation or BRAF amplification, in a subset of MIFS.

Insulin Regulates Lipolysis and Fat Mass by Upregulating Growth/Differentiation Factor 3 in Adipose Tissue Macrophages.

Previous genetic studies in mice have shown that functional loss of activin receptor-like kinase 7 (ALK7), a type I transforming growth factor-β receptor, increases lipolysis to resist fat accumulation in adipocytes. Although growth/differentiation factor 3 (GDF3) has been suggested to function as a ligand of ALK7 under nutrient-excess conditions, it is unknown how GDF3 production is regulated. Here, we show that a physiologically low level of insulin converts CD11c- adipose tissue macrophages (ATMs) into GDF3-producing CD11c+ macrophages ex vivo and directs ALK7-dependent accumulation of fat in vivo. Depletion of ATMs by clodronate upregulates adipose lipases and reduces fat mass in ALK7-intact obese mice, but not in their ALK7-deficient counterparts. Furthermore, depletion of ATMs or transplantation of GDF3-deficient bone marrow negates the in vivo effects of insulin on both lipolysis and fat accumulation in ALK7-intact mice. The GDF3-ALK7 axis between ATMs and adipocytes represents a previously unrecognized mechanism by which insulin regulates both fat metabolism and mass.

Aortic carboxypeptidase-like protein enhances adipose tissue stromal progenitor differentiation into myofibroblasts and is upregulated in fibrotic white adipose tissue.

White adipose tissue expands through both adipocyte hypertrophy and hyperplasia and it is hypothesized that fibrosis or excess accumulation of extracellular matrix within adipose tissue may limit tissue expansion contributing to metabolic dysfunction. The pathways that control adipose tissue remodeling are only partially understood, however it is likely that adipose tissue stromal and perivascular progenitors participate in fibrotic remodeling and also serve as adipocyte progenitors. The goal of this study was to investigate the role of the secreted extracellular matrix protein aortic carboxypeptidase-like protein (ACLP) on adipose progenitor differentiation in the context of adipose tissue fibrosis. Treatment of 10T1/2 mouse cells with recombinant ACLP suppressed adipogenesis and enhanced myofibroblast differentiation, which was dependent on transforming growth factor-β receptor kinase activity. Mice fed a chronic high fat diet exhibited white adipose tissue fibrosis with elevated ACLP expression and cellular fractionation of these depots revealed that ACLP was co-expressed with collagens primarily in the inflammatory cell depleted stromal-vascular fraction (SVF). SVF cells isolated from mice fed a high fat diet secreted increased amounts of ACLP compared to low fat diet control SVF. These cells also exhibited reduced adipogenic differentiation capacity in vitro. Importantly, differentiation studies in primary human adipose stromal cells revealed that mature adipocytes do not express ACLP and exogenous ACLP administration blunted their differentiation potential while upregulating myofibroblastic markers. Collectively, these studies identify ACLP as a stromal derived mediator of adipose progenitor differentiation that may limit adipocyte expansion during white adipose tissue fibrosis.

Macrophages Promote Wound-Induced Hair Follicle Regeneration in a CX3CR1- and TGF-β1-Dependent Manner.

Hair follicle stem cells are regulated by intrafollicular and extrafollicular niche signals. Appropriate hair follicle regeneration relies on the coordinated release and integration of these signals. How immune cells, particularly cutaneous macrophages, influence the hair follicle stem cell niche and regeneration is not well understood. We took advantage of wound-induced hair growth (WIHG) to explore the relationship between wound macrophages and hair follicle regeneration. First, we showed that WIHG is dependent on CD11b+F4/80+ macrophages at 7-11 days after injury. Next, using CX3CR1gfp/+:CCR2rfp/+ mice to capture the dynamic spectrum of macrophage phenotypes during wound healing, we showed that wound macrophages transition from a CX3CR1lo/med to a CX3CR1hi phenotype at the onset of WIHG. Finally, WIHG is abolished in mice deficient for CX3CR1, delayed with pharmacological inhibition of transforming growth factor-β receptor type 1, and rescued with exogenous transforming growth factor-β1. Overall, we propose a model in which transforming growth factor-β1 and CX3CR1 are critical for recruiting and maintaining the CCR2+CX3CR1hiLy6CloTNFα+ macrophages critical for stimulating WIHG.

MiR-17-5p promotes cervical cancer cell proliferation and metastasis by targeting transforming growth factor-β receptor 2.

MicroRNAs (miRNAs) play critical roles in post-translational gene expression. The aim of the current study was to investigate the effects of miR-17-5p in cervical cancer. Fifteen clinical cervical cancer tissue samples, as well as their paired adjacent noncancerous tissues, were collected. The microarray was performed to identify differential miRNAs in cervical cancer. Luciferase reporter assay was conducted to identify the target gene of selected miRNA. SiHa was transfected with mimics, inhibitors as well as negative controls of miR-17-5p and Targeting Transforming Growth Factor-β Receptor 2 (TGFBR2) open reading frame or siRNA. Cell counting kit-8 (CCK-8) assay and transwell experiment were performed to detect the proliferation rate and metastasis, respectively. Western blotting and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis were used to analyze TGFBR2 expression. Balb/c nude mice were utilized to verify the effect of miR-17-5p in vivo. Microarray analysis identified miR-17-5p as our interesting miRNA, and luciferase reporter assay identified TGFBR2 as its target gene. MiR-17-5p overexpression significantly enhanced cervical cancer cell proliferation and metastasis. In-vivo study also verified that miR-17-5p overexpression stimulated cervical cancer growth. MiR-17-5p enhances cervical cancer proliferation and metastasis via targeting TGFBR2. It is proposed that targeting miR-17-5p may be a promising therapeutic approach for cervical cancer.

Downregulation of microRNA-193-3p inhibits the progression of intrahepatic cholangiocarcinoma cells by upregulating TGFBR3.

The aim of this study was to investigate the function of microRNA (miR)-193-3p in human intrahepatic cholangiocarcinoma (ICC) tissues and cells. To evaluate whether miR-193-3p was aberrantly upregulated, we used reverse transcription-quantitative polymerase chain reaction to detect the level of miR-193-3p in ICC tissues and ICC-9810 cells. The effects of miR-193-3p downregulation on ICC cell proliferation, migration and invasion were also measured by MTT, wound-healing and Transwell assays. Additionally, transforming growth factor-β receptor type 3 (TGFBR3) was investigated as a direct target of miR-193-3p by dual-luciferase reporter assays and western blot analyses. The results demonstrated that miR-193-3p was aberrantly upregulated in ICC tissues and cell lines. Furthermore, TGFBR3 was confirmed to be a target of miR-193-3p in ICC and was notably upregulated by miR-193-3p knockdown in the ICC-9810 cells. The knockdown of miR-193-3p also exerted direct inhibitory effects on the proliferation, migration and invasion of the ICC-9810 cells. Therefore, we present evidence that miR-193-3p plays a key role in promoting human ICC by regulating TGFBR3.

Prognostic Value of ACVRL1 Expression in Metastatic Colorectal Cancer Patients Receiving First-line Chemotherapy With Bevacizumab: Results From the Triplet Plus Bevacizumab (TRIBE) Study

BackgroundNo biomarkers exist to predict benefit from antiangiogenic therapy in metastatic colorectal cancer patients. ACVRL1 (activin receptor like-protein 1) encodes for ALK1, a member of the transforming growth factor-β receptor family, which directs pathologic angiogenesis. We examined the intratumoral expression of ACVRL1 and other angiogenesis pathway-related genes to identify molecular markers in the TRIBE study. Materials and MethodsOf 503 randomized patients, 228 had sufficient tissue for analysis. Formalin-fixed paraffin-embedded specimens were examined for expression of VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR2, ACVRL1, EphB4, and EGFL7 using reverse transcription polymerase chain reaction. A maximal χ2 approach was used to determine the messenger RNA levels associated with progression-free survival (PFS), overall survival (OS), response rate, early tumor shrinkage, and depth of response. Recursive partitioning trees were constructed to identify composite prognostic biomarker profiles. External validation was conducted in silico using the Oncomine database. ResultsHigh ACVRL1 expression was associated with superior OS in both treatment arms (FOLFOXIRI [5-fluorouracil, leucovorin, oxaliplatin, irinotecan]-bevacizumab, 32.7 vs. 13.5 months, hazard ratio [HR], 0.38, P = .023; FOLFIRI [5-fluorouracil, leucovorin, irinotecan]-bevacizumab, 35.1 vs. 22.0 months, HR, 0.36, P = .006) and prolonged PFS (11.7 vs. 5.9 months, multivariate HR, 0.17; P = .001) for patients receiving FOLFOXIRI-bevacizumab on univariate and multivariate analyses. In recursive partitioning analysis, ACVRL1 was the strongest discriminator of the response rate, PFS, and OS in patients receiving FOLFOXIRI-bevacizumab and of OS in patients receiving FOLFIRI-bevacizumab. In silico validation revealed significant associations between ACVRL1 expression, disease recurrence, and 1-year survival (P < .05) among all colorectal cancer stages. ConclusionACVRL1 expression could serve as a prognostic biomarker in metastatic colorectal cancer patients receiving chemotherapy and bevacizumab and warrants further evaluation in prospective studies.

Administration of metformin in clinical medicine; an updated mini-review on current findings

Metformin is in the biguanide class that has been considered as the treatment for insulin resistance diabetes and polycystic ovarian disease. Many mechanisms have been suggested for it such as inhibition of the mitochondrial respiratory chain and mitochondrial glycerophosphate dehydrogenase, activation of adenosine monophosphate-activated protein kinase, inhibition of glucagon-induced elevation of cyclic adenosine monophosphate with reduced activation of protein kinase A, an effect on gut microbiota and activation of adenosine monophosphateactivated protein kinase. Metformin is a suppressor for transforming growth factor-β1 via directly binding and interact with transforming growth factor-β1 receptor. Lactic acidosis is one of the adverse and noxious effects of metformin. Nowadays, metformin has an important role in inflammation pathways and antioxidant pathways that can prevent or decrease kidney fibrosis, cardiac remodeling in hypertensive heart disease, and cell death in cerebral ischemia, kidney crystal formation, immunological diseases and cancer. Although there has been strong evidence for the potential harm caused by metformin, several studies have shown beneficial effects for it. Hence, it is necessary to revision and modification in contraindications for prescription of this drug

TGF-β1 protection against Aβ1-42-induced hippocampal neuronal inflammation and apoptosis by TβR-I.

Alzheimer's disease (AD), the most common chronic neurodegenerative disease, is pathologically characterized by the formation of neurofibrillary tangles because of hyperphosphorylation of tau protein and extracellular deposits of amyloid-β (Aβ) protein termed senile plaques. Recent studies indicate that neuronal apoptosis caused by chronic neuroinflammation is one of the important pathogenesis of AD. Transforming growth factor (TGF)-β1 is a pleiotropic cytokine with immunosuppressive and anti-inflammatory properties. However, it is poorly known whether the anti-inflammatory property of TGF-β1 is involved in a neuroprotection in AD. Here, an AD cell model of hippocampal neurons induced by Aβ1-42 was used to show an anti-inflammatory and neuroprotective effect of TGF-β1 through its receptor transforming growth factor-β receptor type I (TβR-I). As expected, Aβ1-42-induced an upregulation in neuronal expression of amyloid precursor protein (APP), tumor necrosis factor-α, cyclooxygenase-2, Bax, cleaved caspase-3, and cleaved caspase-9, and a downregulation in the expression of Bcl-2, as well as an increase in the number of NeuN/terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) double-positive cells. TGF-β1 pretreatment reduced the Aβ1-42-induced effects of upregulating APP, tumor necrosis factor-α, Bax, cleaved caspase-3 and cleaved caspase-9, and downregulating Bcl-2, in addition to increasing NeuNTUNEL cell number. TβR-I expression in hippocampal neurons was downregulated by Aβ1-42 exposure, but upregulated by TGF-β1 pretreatment. Silencing of the TβR-I gene in the neurons abolished the anti-inflammatory and antiapoptotic effects of TGF-β1 in the Aβ1-42-induced AD cell model. These findings suggest that TGF-β1 protects neurons against Aβ1-42-induced neuronal inflammation and apoptosis by activation of TβR-I.

TGF-β-mediated NADPH oxidase 4-dependent oxidative stress promotes colistin-induced acute kidney injury.

Colistin (polymyxin E) is an important constituent of the polymyxin class of cationic polypeptide antibiotics. Intrarenal oxidative stress can contribute to colistin-induced nephrotoxicity. Nicotinamide adenine dinucleotide 3-phosphate oxidases (Noxs) are important sources of reactive oxygen species. Among the various types of Noxs, Nox4 is predominantly expressed in the kidney. We investigated the role of Nox4 and benefit of Nox4 inhibition in colistin-induced acute kidney injury using in vivo and in vitro models. Human proximal tubular epithelial (HK-2) cells were treated with colistin with or without NOX4 knockdown, or GKT137831 (most specific Nox1/4 inhibitor). Effects of Nox4 inhibition on colistin-induced acute kidney injury model in Sprague-Dawley rats were examined. Nox4 expression in HK-2 cells significantly increased following colistin exposure. SB4315432 (transforming growth factor-β1 receptor I inhibitor) significantly inhibited Nox4 expression in HK-2 cells. Knockdown of NOX4 transcription reduced reactive oxygen species production, lowered the levels of pro-inflammatory markers (notably mitogen-activated protein kinases) implicated in colistin-induced nephrotoxicity and attenuated apoptosis by altering Bax and caspase 3/7 activity. Pretreatment with GKT137831 replicated these effects mediated by downregulation of mitogen-activated protein kinase activities. In a rat colistin-induced acute kidney injury model, administration of GKT137831 resulted in attenuated colistin-induced acute kidney injury as indicated by attenuated impairment of glomerulus function, preserved renal structures, reduced expression of 8-hydroxyguanosine and fewer apoptotic cells. Collectively, these findings identify Nox4 as a key source of reactive oxygen species responsible for kidney injury in colistin-induced nephrotoxicity and highlight a novel potential way to treat drug-related nephrotoxicity.

Gene Silencing of TGFβRII Can Inhibit Glioblastoma Cell Growth

Objective:Glioblastoma (GBM) is the most malignant and aggressive type of glioma, associated with a high rate of mortality. The transforming growth factor-β receptor II (TGFβ RII) is involved in glioma initiation and progression. On the other hand, TGFβ RII silencing is critical to the inhibition of GBM. Therefore, we aimed to determine the effects of specific TGFβ RII siRNA on the survival of U-373MG cells.Methods:TGFβ RII siRNA was transfected, and qRT-PCR was performed to examine TGFβ RII mRNA expression. Cell survival was determined using colorimetric MTT assay, and platelet-derived growth factor-BB (PDGF-BB) level was measured in the culture supernatant using ELISA assay.Result:Our findings indicated that specific siRNAs could dose-dependently suppress TGFβ RII mRNA expression after 48 hours. In addition, treatment with TGFβ RII siRNA significantly reduced tumor cell survival and decreased the amount of PDGF-BB protein in the cell culture supernatant.Conclusion:Our results suggest that TGFβ RII silencing can be a promising complementary treatment for glioma.

Preclinical assessment of galunisertib (LY2157299 monohydrate), a first-in-class transforming growth factor-β receptor type I inhibitor.

Transforming growth factor-β (TGFβ) is an important driver of tumor growth via intrinsic and extrinsic mechanisms, and is therefore an attractive target for developing cancer therapeutics. Using preclinical models, we characterized the anti-tumor activity of a small molecule inhibitor of TGFβ receptor I (TGFβRI), galunisertib (LY2157299 monohydrate). Galunisertib demonstrated potent and selective inhibition of TGFβRI with corresponding inhibition of downstream signaling via inhibition of SMAD phosphorylation (pSMAD). Galunisertib also inhibited TGFβ-induced pSMAD in vivo, which enabled a pharmacokinetic/pharmacodynamic profile in Calu6 and EMT6-LM2 tumors. Galunisertib demonstrated anti-tumor activity including inhibition of tumor cell migration and mesenchymal phenotype, reversal of TGFβ-mediated immune-suppression, and tumor growth delay. A concentration-effect relationship was established with a dosing schedule to achieve the optimal level of target modulation. Finally, a rat model demonstrated a correlation between galunisertib-dependent inhibition of pSMAD in tumor tissues and in PBMCs, supporting the use of PBMCs for assessing pharmacodynamic effects.Galunisertib has been tested in several clinical studies with evidence of anti-tumor activity observed in subsets of patients. Here, we demonstrate that galunisertib inhibits a number of TGFβ-dependent functions leading to anti-tumor activity. The enhanced understanding of galunisertib provides rationale for further informed clinical development of TGFβ pathway inhibitors.

MiR-33a hinders the differentiation of adipose mesenchymal stem cells towards urothelial cells in an inductive condition by targeting β‑catenin and TGFR.

Tissue engineering technology offers an appealing approach for tissue reconstruction of the urothelium. Adipose-derived mesenchymal stem cells (ADSCs) represent an abundant source for tissue engineering applications. However, ASCs primarily possess mesoderm lineage differentiation potential. It is difficult to induce differentiation of ASCs towards urothelial cells that are derived from the endoderm, although a recent findings have reported that a conditioned medium may drive ADSCs towards differentiation into the urothelium phenotype. In the present study, human ADSCs were isolated from abdominal adipose tissues and incubated in this conditioned medium for indicated time periods. Western blotting showed that protein expression levels of urothelial specific marks, including CK7, CK20 and UPIII, were increased after seven days' incubation, but immunofluorescence microscopy determined that cells with CK7 and UPIII staining were scarce, which suggested a low-efficiency for the differentiation. Prolonging the incubation time did not further increase CK20 and UPIII expression. Furthermore, miR-33a expression was increased with ADSC differentiation. Using synthetic miRNAs to mimic or inhibit the action of miR-33a revealed that miR-33a hinders the differentiation of ADSCs towards urothelial cells. Furthermore, luciferase reporter assay confirmed that β-catenin and transforming growth factor-β receptor (TGFR) are targets of miR-33a. Inhibition of miR-33a expression increased β-catenin and TGFR expression and improved the efficiency of ADSCs towards differentiation into the urothelium phenotype. The present novel finding suggests that miR-33 may be an important target in tissue engineering and regenerative medicine for urothelium repair.

The Cytokine TGF-β Promotes the Development and Homeostasis of Alveolar Macrophages

Alveolar macrophages (AMs) derive from fetal liver monocytes, which colonize the lung during embryonic development and give rise to fully mature AMs perinatally. AM differentiation requires granulocyte macrophage colony-stimulating factor (GM-CSF), but whether additional factors are involved in AM regulation is not known. Here we report that AMs, in contrast to most other tissue macrophages, were also dependent on transforming growth factor-β receptor (TGF-βR) signaling. Conditional deletion of TGF-βR in mice at different time points halted the development and differentiation of AMs. In adult mice, TGF-β was also critical for AM homeostasis. The source of TGF-β was AMs themselves, indicative of an autocrine loop that promotes AM self-maintenance. Mechanistically, TGF-βR signaling resulted in upregulation of PPAR-γ, a signature transcription factor essential for the development of AMs. These findings reveal an additional layer of complexity regarding the guidance cues, which govern the genesis, maturation, and survival of AMs.