TRPV4 Channel Inhibits TGF-β1-Induced Proliferation of Hepatic Stellate Cells

TRPV4, one of the TRP channels, is implicated in diverse physiological and pathological processes including cell proliferation. However, the role of TRPV4 in liver fibrosis is largely unknown. Here, we characterized the role of TRPV4 in regulating HSC-T6 cell proliferation. TRPV4 mRNA and protein were measured by RT-PCR and Western blot in patients and rat model of liver fibrosis in vivo and TGF-β1-activated HSC-T6 cells in vitro. Both mRNA and protein of TRPV4 were dramatically increased in liver fibrotic tissues of both patients and CCl₄-treated rats. Stimulation of HSC-T6 cells with TGF-β1 resulted in increase of TRPV4 mRNA and protein. However, TGF-β1-induced HSC-T6 cell proliferation was inhibited by Ruthenium Red (Ru) or synthetic siRNA targeting TRPV4, and this was accompanied by downregulation of myofibroblast markers including α-SMA and Col1α1. Moreover, our study revealed that miR-203 was downregulated in liver fibrotic tissues and TGF-β1-treated HSC-T6 cell. Bioinformatics analyses predict that TRPV4 is the potential target of miR-203. In addition, overexpression of miR-203 in TGF-β1-induced HSC significantly reduced TRPV4 expression, indicating TRPV4, which was regulated by miR-203, may function as a novel regulator to modulate TGF-β1-induced HSC-T6 proliferation.     

Functional Implications of MicroRNA-215 in TGF-β1-Induced Phenotypic Transition of Mesangial Cells by Targeting CTNNBIP1

Mesangial cell (MC) phenotypic transition is crucial for the progression of diabetic nephropathy. A major stimulus mediating high glucose-induced MC phenotypic transition is TGF-β1. Our current study focuses on microRNA-215 (miR-215) and investigates its role in TGF-β1-mediated MC phenotypic transition. Using real-time quantitative PCR (qRT-PCR) and northern blotting, we determined that the miR-192/215 family is dramatically upregulated under diabetic conditions both in vitro and in vivo. Gain- and loss-of-function approaches demonstrated that miR-215 inhibition significantly inhibited TGF-β1-induced mouse mesangial cell (MMC) phenotypic transition, whereas miR-215 upregulation promoted MMC phenotypic transition. Interestingly, these changes were not detected in cells that were treated with TGF-β1 and miR-192 mimics or inhibitors. These results suggest that miR-215 participates in TGF-β1-induced MMC phenotypic transition. Luciferase reporter assays were used to identify whether catenin-beta interacting protein 1 (CTNNBIP1) is a direct target of miR-215, which was predicted by bioinformatic analysis. Mechanistic studies revealed that CTNNBIP1 suppresses Wnt/β-catenin signaling and that miR-215 promotes β-catenin activation and upregulates α-SMA and fibronectin expression in TGF-β1-treated MMCs by targeting CTNNBIP1. In addition, in vivo miR-215 silencing with a specific antagomir significantly increased CTNNBIP1 protein expression, resulting in reduced β-catenin activity and decreased α-SMA and fibronectin expression in db/db mouse kidney glomeruli. Taken together, our findings indicate that miR-215 plays an essential role in MC phenotypic transition by regulating the CTNNBIP1/β-catenin pathway, which is related to the pathogenesis of diabetic nephropathy.     

Sorafenib Ameliorates Renal Fibrosis through Inhibition of TGF-β-Induced Epithelial-Mesenchymal Transition

ObjectiveThis study was to investigate whether sorafenib can inhibit the progression of renal fibrosis and to study the possible mechanisms of this effect.MethodsEight-week-old rats were subjected to unilateral ureteral obstruction (UUO) and were intragastrically administered sorafenib, while control and sham groups were administered vehicle for 14 or 21 days. NRK-52E cells were treated with TGF-β1 and sorafenib for 24 or 48 hours. HE and Masson staining were used to visualize fibrosis of the renal tissue in each group. The expression of α-SMA and E-cadherin in kidney tissue and NRK-52E cells were performed using immunohistochemistry and immunofluorescence. The apoptosis rate of NRK-52E cells was determined by flow cytometry analysis. The protein levels of Smad3 and p-Smad3 in kidney tissue and NRK-52E cells were detected by western blot analysis.ResultsHE staining demonstrated that kidney interstitial fibrosis, tubular atrophy, and inflammatory cell infiltration in the sorafenib-treated-UUO groups were significantly decreased compared with the vehicle-treated-UUO group (p<0.05). Masson staining showed that the area of fibrosis was significantly decreased in the sorafenib-treated-UUO groups compared with vehicle-treated-UUO group (p<0.01). The size of the kidney did not significantly increase; the cortex of the kidney was thicker and had a richer blood supply in the middle-dose sorafenib group compared with the vehicle-treated-UUO group (p<0.05). Compared with the vehicle-treated-UUO and TGF-β-stimulated NRK-52E groups, the expression of a-SMA and E-cadherin decreased and increased, respectively, in the UUO kidneys and NRK-52E cells of the sorafenib-treated groups (p<0.05). The apoptotic rate of NRK-52E cells treated with sorafenib decreased for 24 hours in a dose-dependent manner (p<0.05). Compared with the vehicle-treated UUO and TGF-β-stimulated NRK-52E groups, the ratio of p-Smad3 to Smad3 decreased in the sorafenib-treated groups (p<0.05).ConclusionOur results suggest that sorafenib may useful for the treatment of renal fibrosis through the suppression of TGF-β/Smad3-induced EMT signaling.     

Col1A1 Production and Apoptotic Resistance in TGF-β1-Induced Epithelial-to-Mesenchymal Transition-Like Phenotype of 603B Cells

Recent studies have suggested that proliferating cholangiocytes have an important role in the induction of fibrosis, either directly via epithelial-to-mesenchymal transition (EMT), or indirectly via activation of other liver cell types. Transforming growth factor beta 1 (TGF-β1), a critical fibrotic cytokine for hepatic fibrosis, is a potent EMT inducer. This study aimed to clarify the potential contributions of TGF-β1-induced EMT-like cholangiocyte phenotype to collagen production and cell survival of cholangiocytes in vitro. Mouse cholangiocytes (603B cells) were treated with TGF-β1 and EMT-like phenotype alterations were monitored by morphological changes and expression of EMT-associated genes. Alterations in Col1A1 gene, Col1A1-associated miR-29s, and pro-apoptotic genes were measured in TGF-β1-treated 603B cells. Snail1 knockdown was achieved using shRNA to evaluate the contribution of EMT-associated changes to Col1A1 production and cell survival. We found TGF-β1 treatment induced partial EMT-like phenotype transition in 603B cells in a Snail1-dependent manner. TGF-β1 also stimulated collagen α1(I) expression in 603B cells. However, this induction was not parallel to the EMT-like alterations and independent of Snail1 or miR-29 expression. Cells undergoing EMT-like changes showed a modest down-regulation of multiple pro-apoptotic genes and displayed resistance to TNF-α-induced apoptosis. TGF-β1-induced apoptosis resistance was attenuated in Snail1 knockdown 603B cells. TGF-β1-induced Col1A1 production seems to be independent of EMT-like transition and miR-29 expression. Nevertheless, TGF-β1-induced EMT may contribute to the increased survival capacity of cholangiocytes via modulating the expression of pro-apoptotic genes.     

Inhibition of Glomerular Mesangial Cell Proliferation by siPDGF-B- and siPDGFR-β-Containing Chitosan Nanoplexes

Mesangioproliferative glomerulonephritis is a disease that has a high incidence in humans. In this disease, the proliferation of glomerular mesangial cells and the production of extracellular matrix are important. In recent years, the RNAi technology has been widely used in the treatment of various diseases due to its capability to inhibit the gene expression with high specificity and targeting. The objective of this study was to decrease mesangial cell proliferation by knocking down PDGF-B and its receptor, PDGFR-β. To be able to use small interfering RNAs (siRNAs) in the treatment of this disease successfully, it is necessary to develop appropriate delivery systems. Chitosan, which is a biopolymer, is used as a siRNA delivery system in kidney drug targeting. In order to deliver siRNA molecules targeted at PDGF-B and PDGFR-β, chitosan/siRNA nanoplexes were prepared. The in vitro characterization, transfection studies, and knockdown efficiencies were studied in immortalized and primary rat mesangial cells. In addition, the effects of chitosan nanoplexes on mesangial cell proliferation and migration were investigated. After in vitro transfection, the PDGF-B and PDGFR-β gene silencing efficiencies of PDGF-B and PDGFR-β targeting siRNA-containing chitosan nanoplexes were 74 and 71% in immortalized rat mesangial cells and 66 and 62% in primary rat mesangial cells, respectively. siPDGF-B- and siPDGFR-β-containing nanoplexes indicated a significant decrease in mesangial cell migration and proliferation. These results suggested that mesangial cell proliferation may be inhibited by silencing of the PDGF-B signaling pathway. Gene silencing approaches with chitosan-based gene delivery systems have promise for the efficient treatment of renal disease.     

The Polyphenols (?)-Epigallocatechin-3-Gallate and Luteolin Synergistically Inhibit TGF-β-Induced Myofibroblast Phenotypes through RhoA and ERK Inhibition

The presence of reactive stroma, predominantly composed of myofibroblasts, is directly associated with and drives prostate cancer progression. We have previously shown that (−)-Epigallocatechin-3-gallate (EGCG), in the form of Polyphenon E, significantly decreases serum levels of HGF and VEGF in prostate cancer patients. Given that HGF and VEGF are secreted from surrounding tumor myofibroblasts, these observations suggested that EGCG may inhibit prostate cancer-associated myofibroblast differentiation. Herein, we demonstrate that micromolar combinations of EGCG and a second polyphenol, luteolin, synergistically inhibit TGF-β-induced myofibroblast phenotypes in prostate fibroblast cell lines, as observed primarily by potentiation of fibronectin expression. Functionally, EGCG and luteolin inhibited TGF-β-induced extracellular matrix contraction, an enhancer of tumor cell invasion. EGCG and luteolin inhibited downstream TGF-β-induced signaling, including activation of ERK and AKT, respectively, but mechanistically, only ERK appeared to be necessary for TGF-β-induced fibronectin expression. Furthermore, neither EGCG nor luteolin affected Smad signaling or nuclear translocation. Rho signaling was found to be necessary for TGF-β-induced fibronectin expression and EGCG and luteolin each reduced RhoA activation. Finally, EGCG and luteolin were shown to reverse TGF-β-induced fibronectin expression, implicating that these natural compounds may be useful not only in preventing but also in treating already activated myofibroblasts and the diseases they cause, including cancer. The ability of EGCG and luteolin to synergistically target myofibroblasts suggests that combined clinical use of these compounds could prevent or reverse cancer progression through targeting the tumor microenvironment, in addition to the tumor itself.     

ShcA Protects against Epithelial–Mesenchymal Transition through Compartmentalized Inhibition of TGF-β-Induced Smad Activation

Epithelial–mesenchymal transition (EMT) is a normal cell differentiation event during development and contributes pathologically to carcinoma and fibrosis progression. EMT often associates with increased transforming growth factor-β (TGF-β) signaling, and TGF-β drives EMT, in part through Smad-mediated reprogramming of gene expression. TGF-β also activates the Erk MAPK pathway through recruitment and Tyr phosphorylation of the adaptor protein ShcA by the activated TGF-β type I receptor. We found that ShcA protects the epithelial integrity of nontransformed cells against EMT by repressing TGF-β-induced, Smad-mediated gene expression. p52ShcA competed with Smad3 for TGF-β receptor binding, and down-regulation of ShcA expression enhanced autocrine TGF-β/Smad signaling and target gene expression, whereas increased p52ShcA expression resulted in decreased Smad3 binding to the TGF-β receptor, decreased Smad3 activation, and increased Erk MAPK and Akt signaling. Furthermore, p52ShcA sequestered TGF-β receptor complexes to caveolin-associated membrane compartments, and reducing ShcA expression enhanced the receptor localization in clathrin-associated membrane compartments that enable Smad activation. Consequently, silencing ShcA expression induced EMT, with increased cell migration, invasion, and dissemination, and increased stem cell generation and mammosphere formation, dependent upon autocrine TGF-β signaling. These findings position ShcA as a determinant of the epithelial phenotype by repressing TGF-β-induced Smad activation through differential partitioning of receptor complexes at the cell surface.     

Inhibition of Survivin Reduces HIF-1α, TGF-β1 and TFE3 in Salivary Adenoid Cystic Carcinoma

In the present study, we explored the expression and correlation of survivin with HIF-1α, TGF-β1 and TFE3 in adenoid cystic carcinoma (AdCC). The expression of survivin, HIF-1α, TGF-β1 and TFE3 was assessed by immunohistochemical staining of a tissue microarray containing tissue samples of normal salivary gland (NSG), pleomorphic adenoma (PA) and AdCC. Correlation analysis of these proteins revealed that increased survivin expression was associated with the overexpression of HIF-1α (P<0.001, r = 0.5599), TGF-β1 (P<0.001, r = 0.6616) and TFE3 (P<0.001, r = 0.7747). The expression of survivin, HIF-1α, TGF-β1 and TFE3 was not correlated with the pathological type of human AdCC (P>0.05). Selective inhibition of survivin by YM155 and siRNA significantly reduced human SACC-83 cell proliferation, with the corresponding decrease in expression of HIF-1α, TGF-β1 and TFE3. The data indicate that the overexpression of survivin in AdCC is related to HIF-1α, TGF-β1 and TFE3. We hypothesize from these findings that the inhibition of survivin may be a novel strategy for neoadjuvant chemotherapeutic and radiosensitive treatment of AdCC.     

Redox Control of the Senescence Regulator Interleukin-1α and the Secretory Phenotype

Senescent cells accumulate in aged tissue and are causally linked to age-associated tissue degeneration. These non-dividing, metabolically active cells are highly secretory and alter tissue homeostasis, creating an environment conducive to metastatic disease progression. IL-1α is a key senescence-associated (SA) proinflammatory cytokine that acts as a critical upstream regulator of the SA secretory phenotype (SASP). We established that SA shifts in steady-state H₂O₂ and intracellular Ca²⁺ levels caused an increase in IL-1α expression and processing. The increase in intracellular Ca²⁺ promoted calpain activation and increased the proteolytic cleavage of IL-1α. Antioxidants and low oxygen tension prevented SA IL-1α expression and restricted expression of SASP components IL-6 and IL-8. Ca²⁺ chelation or calpain inhibition prevented SA processing of IL-1α and its ability to induce downstream cytokine expression. Conditioned medium from senescent cells treated with antioxidants or Ca²⁺ chelators or cultured in low oxygen markedly reduced the invasive capacity of proximal metastatic cancer cells. In this paracrine fashion, senescent cells promoted invasion by inducing an epithelial-mesenchymal transition, actin reorganization, and cellular polarization of neighboring cancer cells. Collectively, these findings demonstrate how SA alterations in the redox state and Ca²⁺ homeostasis modulate the inflammatory phenotype through the regulation of the SASP initiator IL-1α, creating a microenvironment permissive to tumor invasion.     

The Class II Phosphatidylinositol 3 kinase C2β Is Required for the Activation of the K+ Channel KCa3.1 and CD4 T-Cells

The Ca²⁺-activated K⁺ channel KCa3.1 is required for Ca²⁺ influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2β (PI3K-C2β) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2β by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2β siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2β in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca²⁺ influx, whereas silencing of PI3K-C2β inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2β colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.     

Negligible Effect of Sodium Chloride on the Development and Function of TGF-β-Induced CD4+ Foxp3+ Regulatory T Cells

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Interleukin-1β-Induced Autophagy-Related Gene 5 Regulates Proliferation of Embryonic Stem Cell-Derived Odontoblastic Cells

We previously established a method for the differentiation of induced pluripotent stem cells and embryonic stem cells into α2 integrin-positive odontoblast-like cells. We also reported that Wnt5 in response to interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in these cells. Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state. Here, we examined whether up-regulation of autophagy-related gene (Atg) 5 by IL-1β was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8) and Atg12. Treatment with siRNAs against Atg5, but not LC3 and Atg12, suppressed the IL-1β-induced increase in MMP-3 expression and cell proliferation. Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation. These results demonstrate the unique involvement of Atg5 in IL-1β-induced proliferation of embryonic stem cell-derived odontoblast-like cells.     

SeMet Inhibits IL-1β-Induced Rheumatoid Fibroblast-Like Synoviocytes Proliferation and the Production of Inflammatory Mediators

Previous studies demonstrated that Se has anti-inflammatory activities and that it plays an important role in maintaining normal cartilage metabolism. Nevertheless, little is known about the effects of Se on the production of inflammatory mediators in rheumatoid fibroblast-like synoviocytes (FLSs). The objective of this study was to determine the effects of Se on the interleukin-1β (IL-1β)-induced proliferation of FLSs and production of matrix metalloproteinases (MMPs) and inflammatory mediators by FLSs. In this study, the proliferation of FLSs was assessed using the MTT assay after cultured with/without the presence of IL-1β and SeMet. Human FLSs were pretreated with SeMet (0.5 μM) and subsequently stimulated with IL-1β (5 ng/ml) for 24 h. Production of NO and PGE2 were evaluated by the Griess reaction and ELISA. Gene expression of MMP-3, MMP-13, iNOS, and COX-2 was measured by real-time PCR. MMP-3 and MMP-13 proteins in culture medium were determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the iNOS and COX-2 protein production in the culture medium and the activity of phosphorylation of P38 MAPK pathways. We found that SeMet significantly inhibits IL-1β-induced proliferation of FLSs. SeMet also inhibited the production of PGE2 and NO induced by IL-1β. SeMet significantly decreased IL-1β-stimulated gene expression and production of MMP-3, MMP-13, iNOS, and COX-2 in human FLSs. In addition, we found SeMet partly inhibited the IL-1β-induced activation of p38 MAPK pathways. The present report is first to demonstrate that SeMet inhibits IL-1β-induced expression of MMPs and production of inflammatory factors in cultured FLSs, indicating that SeMet may be a potential agent in the treatment of rheumatoid arthritis.     

Naringenin Decreases Invasiveness and Metastasis by Inhibiting TGF-β-Induced Epithelial to Mesenchymal Transition in Pancreatic Cancer Cells

Epithelial to mesenchymal transition (EMT) promotes cellular motility, invasiveness and metastasis during embryonic development and tumorigenesis. Transforming growth factor-β (TGF-β) signaling pathway is a key regulator of EMT. A lot of evidences suggest that this process is Smad3-dependent. Herein we showed that exposure of aspc-1 and panc-1 pancreatic cancer cells to TGF-β1 resulted in characteristic morphological alterations of EMT, and enhancement of cell motility and gemcitabine (Gem) resistance along with an up-regulation of EMT markers genes such as vimentin, N-cadherin, MMP2 and MMP9. Naringenin (Nar) down-regulated EMT markers expression in both mRNA and protein levels by inhibiting TGF-β1/Smad3 signal pathway in the pancreatic cancer cells. Consequently, Nar suppressed the cells migration and invasion and reversed their resistance to Gem.     

Mouse Hepatic Oval Cells Require Met-Dependent PI3K to Impair TGF-β-Induced Oxidative Stress and Apoptosis

We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met^−/− oval cells) are more sensitive to TGF-β-induced apoptosis than cells expressing a functional Met (Met^flx/flx), demonstrating that the HGF/Met axis plays a pivotal role in oval cell survival. Here, we have examined the mechanism behind this effect and have found that TGF-β induced a mitochondria-dependent apoptotic cell death in Met^flx/flx and Met^−/− oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-β-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met^−/− oval cells. TGF-β also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4) mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-β-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-β. Notably, oxidative stress-related events were strongly amplified in Met^−/− oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-β-induced apoptosis and increased sensitivity of Met^flx/flx oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met^−/− oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Met^flx/flx oval cells, whereas no effect was observed in Met^−/− oval cells. Altogether, results presented here provide solid evidences that both paracrine and autocrine HGF/Met signaling requires PI3K to promote mouse hepatic oval cell survival against TGF-β-induced oxidative stress and apoptosis.