Heightened Crescentic Glomerulonephritis in Immune Challenged 129sv Mice Is TGF-β/Smad3 Dependent

The 129sv mouse strain is particularly sensitive to experimental immune-mediated nephritis. Previous studies have indicated that transforming growth factor-β (TGF-β) plays a critical role in both immune modulation and tissue fibrogenesis in various diseases and that its biological activities are exerted via the SMAD family. In this study, we aimed to determine whether TGF-β/SMAD signaling is essential for the development of immune-mediated nephritis in 129sv mice. Relative to C57BL/6J control mice with anti-glomeruli basement membrane (GBM) nephritis, 129sv mice with anti-GBM nephritis exhibited increased renal collagen deposition. Additionally, higher mRNA levels of pro-collagen and collagen IV, higher serum levels of active and total TGF-β1, and increased TGF-β1, TGF-βIIR, and phosphorylated SMAD expression were detected in these mice. Deletion of Smad3 in 129sv mice ameliorated anti-GBM induced nephritis, including crescentic glomerulonephritis. Collectively, these findings indicate that the heightened experimental nephritis and fibrotic disease in the 129sv strain of mice are regulated by SMAD3, which could be a potential therapeutic target for immune-mediated nephritis.     

Smad2/3 Activation Regulates Smad1/5/8 Signaling via a Negative Feedback Loop to Inhibit 3T3-L1 Adipogenesis

Adipose tissues (AT) expand in response to energy surplus through adipocyte hypertrophy and hyperplasia. The latter, also known as adipogenesis, is a process by which multipotent precursors differentiate to form mature adipocytes. This process is directed by developmental cues that include members of the TGF-β family. Our goal here was to elucidate, using the 3T3-L1 adipogenesis model, how TGF-β family growth factors and inhibitors regulate adipocyte development. We show that ligands of the Activin and TGF-β families, several ligand traps, and the SMAD1/5/8 signaling inhibitor LDN-193189 profoundly suppressed 3T3-L1 adipogenesis. Strikingly, anti-adipogenic traps and ligands engaged the same mechanism of action involving the simultaneous activation of SMAD2/3 and inhibition of SMAD1/5/8 signaling. This effect was rescued by the SMAD2/3 signaling inhibitor SB-431542. By contrast, although LDN-193189 also suppressed SMAD1/5/8 signaling and adipogenesis, its effect could not be rescued by SB-431542. Collectively, these findings reveal the fundamental role of SMAD1/5/8 for 3T3-L1 adipogenesis, and potentially identify a negative feedback loop that links SMAD2/3 activation with SMAD1/5/8 inhibition in adipogenic precursors.     

Nuclear Respiratory Factor-1, a Novel SMAD4 Binding Protein,Represses TGF-β/SMAD4 Signaling by Functioning as a Transcriptional Cofactor

SMAD4, a key regulator of transforming growth factor-β (TGF-β) signaling, plays a major role in cell growth, migration, and apoptosis. In particular, TGF-β/SMAD induces growth arrest, and SMAD4 induces the expression of target genes such as p21WAF1 and p15INK4b through its interaction with several cofactors. Thus, inactivating mutations or the homozygous deletion of SMAD4 could be related to tumorigenesis or malignancy progression. However, in some cancer types, SMAD4 is neither mutated nor deleted. In the current study, we demonstrate that TGF-β signaling with a preserved SMAD4 function can contribute to cancer through associations with negative pathway regulators. We found that nuclear respiratory factor-1 (NRF1) is a novel interaction SMAD4 partner that inhibits TGF-β/SMAD4-induced p15INK4b mRNA expression by binding to SMAD4. Furthermore, we confirmed that NRF1 directly binds to the core region of the SMAD4 promoter, thereby decreasing SMAD4 mRNA expression. On the whole, our data suggest that NRF1 is a negative regulator of SMAD4 and can interfere with TGF-β/SMAD-induced tumor suppression. Our findings provide a novel perception into the molecular basis of TGF-β/SMAD4-signaling suppression in tumorigenesis.     

TGF-β/IL-7 Chimeric Switch Receptor-Expressing CAR-T Cells Inhibit Recurrence of CD19-Positive B Cell Lymphoma

Chimeric antigen receptor (CAR)-T cells are effective in the treatment of hematologic malignancies but have shown limited efficacy against solid tumors. Here, we demonstrated an approach to inhibit recurrence of B cell lymphoma by co-expressing both a human anti-CD19-specific single-chain variable fragment (scFv) CAR (CD19 CAR) and a TGF-β/IL-7 chimeric switch receptor (tTRII-I7R) in T cells (CD19 CAR-tTRII-I7R-T cells). The tTRII-I7R was designed to convert immunosuppressive TGF-β signaling into immune-activating IL-7 signaling. The effect of TGF-β on CD19 CAR-tTRII-I7R-T cells was assessed by western blotting. Target-specific killing by CD19 CAR-tTRII-I7R-T cells was evaluated by Eu-TDA assay. Daudi tumor-bearing NSG (NOD/SCID/IL2Rγ^-/-) mice were treated with CD19 CAR-tTRII-I7R-T cells to analyze the in vivo anti-tumor effect. In vitro, CD19 CAR-tTRII-I7R-T cells had a lower level of phosphorylated SMAD2 and a higher level of target-specific cytotoxicity than controls in the presence of rhTGF-β1. In the animal model, the overall survival and recurrence-free survival of mice that received CD19 CAR-tTRII-I7R-T cells were significantly longer than in control mice. These findings strongly suggest that CD19 CAR-tTRII-I7R-T cell therapy provides a new strategy for long-lasting, TGF-β-resistant anti-tumor effects against B cell lymphoma, which may lead ultimately to increased clinical efficacy.     

Protective Effect of Resveratrol against IL-1β-Induced Inflammatory Response on Human Osteoarthritic Chondrocytes Partly via the TLR4/MyD88/NF-κB Signaling Pathway: An “in Vitro Study”

Resveratrol is a natural polyphenolic compound that prevents inflammation in chondrocytes and animal models of osteoarthritis (OA) via yet to be defined mechanisms. The purpose of this study was to determine whether the protective effect of resveratrol on IL-1β-induced human articular chondrocytes was associated with the TLR4/MyD88/NF-κB signaling pathway by incubating human articular chondrocytes (harvested from osteoarthritis patients) with IL-1β before treatment with resveratrol. Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and TNFα levels in culture supernatants were measured by ELISA(Enzymelinked immunosorbent assay). The levels of TLR4 and its downstream signaling targets (MyD88 and TRAF6) and IL-1β were assessed by measuring the levels of mRNA and protein expression by real-time RT-PCR and western blot analysis, respectively, in addition to assessing NF-κB activation. In addition, TLR4 siRNA was used to block TLR4 expression in chondrocytes further demonstrating that resveratrol prevented IL-1β-mediated inflammation by TLR4 inhibition. We found that resveratrol prevented IL-1β-induced reduction in cell viability. Stimulation of chondrocytes with IL-1β caused a significant up-regulation of TLR4 and its downstream targets MyD88 and TRAF6 resulting in NF-κB activation associated with the synthesis of IL-1β and TNFα. These IL-1β-induced inflammatory responses were all effectively reversed by resveratrol. Furthermore, activation of NF-κB in chondrocytes treated with TLR4 siRNA was significantly attenuated, but not abolished, and exposure to resveratrol further reduced NF-κB translocation. These data suggested that resveratrol prevented IL-1β-induced inflammation in human articular chondrocytes at least in part by inhibiting the TLR4/MyD88/NF-κB signaling pathway suggesting that resveratrol has the potential to be used as a nutritional supplement to counteract OA symptoms.     

CCN2 Increases TGF-β Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-β Pathway Regulation

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.     

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1β-Induced Mitochondrial Dysfunction in Human Chondrocytes

Interleukin (IL)-1β is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1β-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1β in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE₂, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1β significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1β-induced NO release and mitochondrial dysfunction but not IL-1β-induced release of IL-6, PGE₂, and MMP3. Enhancement of cAMP by forskolin reduced IL-1β-induced NO release and prevented IL-1β-induced mitochondrial impairment. Activation of AMPK increased IL-1β-induced NO production and the negative impact of IL-1β on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1β-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1β-induced NO release and mitochondrial dysfunction.     

Effects of Visfatin on Intracellular Mechanics and Catabolism in Human Primary Chondrocytes through Glycogen Synthase Kinase 3β Inactivation

Osteoarthritis (OA) is still a recalcitrant musculoskeletal disease on account of its complex biochemistry and mechanical stimulations. Apart from stimulation by external mechanical forces, the regulation of intracellular mechanics in chondrocytes has also been linked to OA development. Recently, visfatin has received significant attention because of the clinical finding of the positive correlation between its serum/synovial level and OA progression. However, the precise mechanism involved is still unclear. This study determined the effect of visfatin on intracellular mechanics and catabolism in human primary chondrocytes isolated from patients. The intracellular stiffness of chondrocytes was analyzed by the particle-tracking microrheology method. It was shown that visfatin damages the microtubule and microfilament networks to influence intracellular mechanics to decrease the intracellular elasticity and viscosity via glycogen synthase kinase 3β (GSK3β) inactivation induced by p38 signaling. Further, microtubule network destruction in human primary chondrocytes is predominantly responsible for the catabolic effect of visfatin on the cyclooxygenase 2 upregulation. The present study shows a more comprehensive interpretation of OA development induced by visfatin through biochemical and biophysical perspectives. Finally, the role of GSK3β inactivation, and subsequent regulation of intracellular mechanics, might be considered as theranostic targets for future drug development for OA.     

Early Gβγ-GRK2 Inhibition Ameliorates Osteoarthritis Development by Simultaneous Anti-Inflammatory and Chondroprotective Effects

The G-protein-coupled receptor kinase 2 (GRK2) is an important regulator of inflammation and pathological macrophage phenotype in a variety of diseases. We hypothesize that Gβγ-GRK2 signaling promotes the early inflammatory response and chondrocyte loss in osteoarthritis (OA). Using the destabilization of the medial meniscus (DMM) model in 12-week-old male C57BL/6 mice, we determined the role of Gβγ-GRK2 signaling in synovitis, macrophage activation, and OA development. We achieved Gβγ-GRK2 inhibition at the time of DMM by administering the Gβγ inhibitor “gallein” and the GRK2 inhibitor “paroxetine” daily, starting from 2 days before DMM surgery, for a duration of 1 or 12 weeks. Synovial and cartilage structural changes were evaluated by histomorphometry, and molecular events and macrophage activation were examined. We studied the direct role of Gβγ-GRK2 in synovitis and macrophage activation in vitro using SW982 and THP1 cells. Continuous Gβγ-GRK2 inhibition initiated at the time of DMM attenuated OA development and decreased chondrocyte loss more effectively than delayed treatment. GRK2 expression and the M1 macrophage phenotype were elevated in the inflamed synovium, while early gallein and paroxetine treatment for 1 and 12 weeks following DMM resulted in their reduction and an upregulated M2 macrophage phenotype. In vitro experiments showed that Gβγ-GRK2 inhibition attenuated synoviocyte inflammation and the M1 phenotype. We show that early Gβγ-GRK2 inhibition is of higher therapeutic efficacy in OA than delayed inhibition, as it prevents OA development by inhibiting the early inflammatory response.     

Chondrocytes from Osteoarthritis Patients Adopt Distinct Phenotypes in Response to Central TH1/TH2/TH17 Cytokines

Chronic low-grade inflammation plays a central role in the pathogenesis of osteoarthritis (OA), and several pro- and anti-inflammatory cytokines have been implicated to mediate and regulate this process. Out of these cytokines, particularly IFNγ, IL-1β, IL-4 and IL-17 are associated with different phenotypes of T helper (T_H) cells and macrophages, both examples of cells known for great phenotypic and functional heterogeneity. Chondrocytes also display various phenotypic changes during the course of arthritis. We set out to study the hypothesis of whether chondrocytes might adopt polarized phenotypes analogous to T_H cells and macrophages. We studied the effects of IFNγ, IL-1β, IL-4 and IL-17 on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were harvested from the cartilage of OA patients undergoing knee replacement surgery and then cultured with or without the cytokines for 24 h. Total RNA was isolated and sequenced, and GO (Gene Ontology) functional analysis was performed. We also separately investigated genes linked to OA in recent genome wide expression analysis (GWEA) studies. The expression of more than 2800 genes was significantly altered in chondrocytes treated with IL-1β [in the C(IL-1β) phenotype] with a fold change (FC) > 2.5 in either direction. These included a large number of genes associated with inflammation, cartilage degradation and attenuation of metabolic signaling. The profile of genes differentially affected by IFNγ (the C(IFNγ) phenotype) was relatively distinct from that of the C(IL-1β) phenotype and included several genes associated with antigen processing and presentation. The IL-17-induced C(IL-17) phenotype was characterized by the induction of a more limited set of proinflammatory factors compared to C(IL-1β) cells. The C(IL-4) phenotype induced by IL-4 displayed a differential expression of a rather small set of genes compared with control, primarily those associated with TGFβ signaling and the regulation of inflammation. In conclusion, our results show that OA chondrocytes can adopt diverse phenotypes partly analogously to T_H cells and macrophages. This phenotypic plasticity may play a role in the pathogenesis of arthritis and open new therapeutic avenues for the development of disease-modifying treatments for (osteo)arthritis.     

Identification of Growth Factors,Cytokines and Mediators Regulated by Artemisia annua L. Polyphenols (pKAL) in HCT116 Colorectal Cancer Cells: TGF-β1 and NGF-β Attenuate pKAL-Induced Anticancer Effects via NF-κB p65 Upregulation

The anticancer effects of natural phytochemicals are relevant to the modulation of cytokine signaling pathways in various cancer cells with stem-like properties as well as immune cells. The aim of this study was to elucidate a novel anticancer mechanism of Artemisia annua L. polyphenols (pKAL) involved in the regulation of growth factors, cytokines and mediators in stem-like HCT116 colorectal cancer cells. Through RayBiotech human L-1000 antibody array and bioinformatics analysis, we show here that pKAL-induced anticancer effects are associated with downregulation of growth factor and cytokine signaling proteins including TGFA, FGF16, PDGFC, CCL28, CXCR3, IRF6 and SMAD1. Notably, we found that TGF-β signaling proteins such as GDF10, ENG and TGFBR2 and well-known survival proteins such as NGF-β, VEGFD and insulin were significantly upregulated by pKAL. Moreover, the results of hematoxylin staining, cell viability assay and Western blot analysis demonstrated that TGF-β1 and NGF-β attenuated pKAL-induced anticancer effects by inhibiting pKAL-induced downregulation of caspase-8, NF-κB p65 and cyclin D1. These results suggest that certain survival mediators may be activated by pKAL through the TGF-β1 and NGF-β signaling pathways during pKAL-induced cell death and thus, strategies to inhibit the survival signaling are inevitably required for more effective anticancer effects of pKAL.     

Novel Inhibitor for Downstream Targeting of Transforming Growth Factor-β Signaling to Suppress Epithelial to Mesenchymal Transition and Cell Migration

Cancer metastasis accounts for most of the mortality associated with solid tumors. However, antimetastatic drugs are not available on the market. One of the important biological events leading to metastasis is the epithelial to mesenchymal transition (EMT) induced by cytokines, namely transforming growth-factor-β (TGF-β). Although several classes of inhibitors targeting TGF-β and its receptor have been developed, they have shown profound clinical side effects. We focused on our synthetic compound, HPH-15, which has shown anti-fibrotic activity via the blockade of the TGF-β Smad-dependent signaling. In this study, 10 μM of HPH-15 was found to exhibit anti-cell migration and anti-EMT activities in non-small-cell lung cancer (NSCLC) cells. Although higher concentrations are required, the anti-EMT activity of HPH-15 has also been observed in 3D-cultured NSCLC cells. A mechanistic study showed that HPH-15 inhibits downstream TGF-β signaling. This downstream inhibition blocks the expression of cytokines such as TGF-β, leading to the next cycle of Smad-dependent and -independent signaling. HPH-15 has AMPK-activation activity, but a relationship between AMPK activation and anti-EMT/cell migration was not observed. Taken together, HPH-15 may lead to the development of antimetastatic drugs with a new mechanism of action.     

Ghrelin Attenuates Liver Fibrosis through Regulation of TGF-β1 Expression and Autophagy

Ghrelin is a stomach-derived growth hormone secretagogue that promotes various physiological effects, including energy metabolism and amelioration of inflammation. The purpose of this study was to investigate the protective mechanism of ghrelin against liver fibrosis. Liver fibrosis was induced in C57BL/6 mice by intraperitoneal injection of CCl₄ (2.0 mL/kg of 10% CCl₄ v/v solution in peanut oil) two times per week for eight weeks. Ghrelin (10 μg/kg) was intraperitoneally injected two times per week for eight weeks. A second murine liver fibrosis model was induced by bile duct ligation (BDL) and concurrent ghrelin administration for four weeks. Hematoxylin eosin (H&E), and Masson’s trichrome were used to detect pathological changes to liver tissue. Western blotting was used to detect protein levels of transforming growth factor (TGF)-β1, phosphorylated Smad3 (p-Smad3), I-collage, α-smooth muscle actin (α-SMA), matrix metalloproteinases (MMPs) 2, tissue inhibitor of matrix metalloproteinases (TIMPs) 1, phosphorylated NF-κB (p-NF-κB), and microtubule-associated protein light chain 3 (LC3). In addition, qRT-PCR was used to detect mRNA levels of TGF-β1, I-collage, α-SMA, MMP2, TIMP1 and LC3, while levels of TGF-β1, p-Smad3, I-collage, α-SMA, and LC3 were detected immunohistochemically. Levels of aspartate aminotransferase and alanine aminotransferase were significantly decreased by ghrelin treatment. Ghrelin administration also significantly reduced the extent of pathological changes in both murine liver fibrosis models. Expression levels of I-collage and α-SMA in both models were clearly reduced by ghrelin administration. Furthermore, ghrelin treatment decreased protein expression of TGF-β1 and p-Smad3. The protein levels of NF-κB and LC3 were increased in the CCl₄- and BDL-treatment groups but were significantly reduced following ghrelin treatment. In addition, ghrelin inhibited extracellular matrix formation by decreasing NF-κB expression and maintaining the balance between MMP2 and TIMP1. Our results demonstrated that ghrelin attenuates liver fibrosis via inhibition of the TGF-β1/Smad3 and NF-κB signaling pathways, as well as autophagy suppression.     

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.     

Smad3 Phospho-Isoform Signaling in Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis with insulin resistance, oxidative stress, lipotoxicity, adipokine secretion by fat cells, endotoxins (lipopolysaccharides) released by gut microbiota, and endoplasmic reticulum stress. Together, these factors promote NAFLD progression from steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and eventually end-stage liver diseases in a proportion of cases. Hepatic fibrosis and carcinogenesis often progress together, sharing inflammatory pathways. However, NASH can lead to hepatocarcinogenesis with minimal inflammation or fibrosis. In such instances, insulin resistance, oxidative stress, and lipotoxicity can directly lead to liver carcinogenesis through genetic and epigenetic alterations. Transforming growth factor (TGF)-β signaling is implicated in hepatic fibrogenesis and carcinogenesis. TGF-β type I receptor (TβRI) and activated-Ras/c-Jun-N-terminal kinase (JNK) differentially phosphorylate the mediator Smad3 to create two phospho-isoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). TβRI/pSmad3C signaling terminates cell proliferation, while constitutive Ras activation and JNK-mediated pSmad3L promote hepatocyte proliferation and carcinogenesis. The pSmad3L signaling pathway also antagonizes cytostatic pSmad3C signaling. This review addresses TGF-β/Smad signaling in hepatic carcinogenesis complicating NASH. We also discuss Smad phospho-isoforms as biomarkers predicting HCC in NASH patients with or without cirrhosis.     

The Involvement of Mutual Inhibition of ERK and mTOR in PLCγ1-Mediated MMP-13 Expression in Human Osteoarthritis Chondrocytes

The issue of whether ERK activation determines matrix synthesis or degradation in osteoarthritis (OA) pathogenesis currently remains controversial. Our previous study shows that PLCγ1 and mTOR are involved in the matrix metabolism of OA cartilage. Investigating the interplays of PLCγ1, mTOR and ERK in matrix degradation of OA will facilitate future attempts to manipulate ERK in OA prevention and therapy. Here, cultured human normal chondrocytes and OA chondrocytes were treated with different inhibitors or transfected with expression vectors, respectively. The levels of ERK, p-ERK, PLCγ1, p-PLCγ1, mTOR, p-mTOR and MMP-13 were then evaluated by Western blotting analysis. The results manifested that the expression level of ERK in human OA chondrocytes was lower than that in human normal articular chondrocytes, and the up-regulation of ERK could promote matrix synthesis, including the decrease in MMP-13 level and the increase in Aggrecan level in human OA chondrocytes. Furthermore, the PLCγ1/ERK axis and a mutual inhibition of mTOR and ERK were observed in human OA chondrocytes. Interestingly, activated ERK had no inhibitory effect on MMP-13 expression in PLCγ1-transformed OA chondrocytes. Combined with our previous study, the non-effective state of ERK activation by PLCγ1 on MMP-13 may be partly attributed to the inhibition of the PLCγ1/mTOR axis on the PLCγ1/ERK axis. Therefore, the study indicates that the mutual inhibition of ERK and mTOR is involved in PLCγ1-mediated MMP-13 expression in human OA chondrocytes, with important implication for the understanding of OA pathogenesis as well as for its prevention and therapy.     

Chrysin Ameliorates Cyclosporine-A-Induced Renal Fibrosis by Inhibiting TGF-β1-Induced Epithelial–Mesenchymal Transition

Cyclosporine A (CsA) is a nephrotoxicant that causes fibrosis via induction of epithelial–mesenchymal transition (EMT). The flavonoid chrysin has been reported to have anti-fibrotic activity and inhibit signaling pathways that are activated during EMT. This study investigated the nephroprotective role of chrysin in the prevention of CsA-induced renal fibrosis and elucidated a mechanism of inhibition against CsA-induced EMT in proximal tubule cells. Treatment with chrysin prevented CsA-induced renal dysfunction in Sprague Dawley rats measured by blood urea nitrogen (BUN), serum creatinine and creatinine clearance. Chrysin inhibited CsA-induced tubulointerstitial fibrosis, characterized by reduced tubular damage and collagen deposition. In vitro, chrysin significantly inhibited EMT in LLC-PK₁ cells, evidenced by inhibition of cell migration, decreased collagen expression, reduced presence of mesenchymal markers and elevated epithelial junction proteins. Furthermore, chrysin co-treatment diminished CsA-induced TGF-β₁ signaling pathways, decreasing Smad 3 phosphorylation which lead to a subsequent reduction in Snail expression. Chrysin also inhibited activation of the Akt/ GSK-3β pathway. Inhibition of both pathways diminished the cytosolic accumulation of β-catenin, a known trigger for EMT. In conclusion, flavonoids such as chrysin offer protection against CsA-induced renal dysfunction and interstitial fibrosis. Chrysin was shown to inhibit CsA-induced TGF-β₁-dependent EMT in proximal tubule cells by modulation of Smad-dependent and independent signaling pathways.