TGF-ß1-induced expression of collagen type II and ACAN is regulated by 4E-BP1, a repressor of translation.

Eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) binds eIF4E and represses protein translation by displacing it from the mRNA. In this study, we investigated the influence of 4E-BP1 translational apparatus on the regulation of transforming growth factor-beta 1 (TGF-ß1)-induced anabolic signaling in chondrocytes. The level of 4E-BP1 expression was significantly higher in human OA cartilage than normal cartilage. TGF-ß1 increased total protein synthesis, including aggrecan (ACAN) and collagen type II (Col II), together with activation of Akt/mTOR signaling pathway. mTOR silencing significantly suppressed ACAN and Col II expressions through decreasing TGF-ß1-induced phosphorylation of 4E-BP1. On the contrary, 4E-BP1 knockdown promoted total protein synthesis but suppressed Col II and ACAN expressions with decreased expression of Smad2/3 and Smad4 and increased expression of inhibitory Smad6 and Smad7. TGF-ß1 suppressed the interaction of 4E-BP1 and eIF4E and subsequently enhanced protein synthesis. Furthermore, 4E-BP1 regulated translation levels of inhibitory Smads, which decreased the accumulation of nuclear Smad2/3 complexes on the promoter of ACAN and Col II genes, subsequently affecting transcription of ACAN and Col II. These results demonstrated that TGF-ß1-modulated phosphorylation of 4EBP1 plays a role in the expression of Col II and ACAN through differential alteration of Smad signaling pathway.

Norepinephrine modulates IL-1ß-induced catabolic response of human chondrocytes.

BACKGROUND: The influence of the sympathetic nervous system (SNS) on metabolism of bone and cartilage expressing ß-adrenergic receptors (AR) was suggested. Here, we investigated whether the SNS functions as a modulator of cartilage metabolism induced by interleukin-1beta (IL-1ß). METHODS: Human articular chondrocytes and articular cartilage were collected from patients with osteoarthritis (OA). Chondrocyte monolayer and cartilage explant culture were stimulated with IL-1ß. The activity of ß-ARs was modulated by an agonist, norepinephrine (NE), and antagonists, including propranolol, atenolol, nebivolol, and nadolol. RESULTS: The levels of ß1-, ß2-, and ß3-AR in OA cartilage and IL-1ß-treated chondrocytes were lower than normal cartilage and untreated cells. Treatment of chondrocytes with IL-1ß and ß-blockers, including propranolol, atenolol, nebivolol, and nadolol, for 6 h significantly upregulated IL-1ß-induced expression of MMP-1, -3, and - 13, compared to chondrocytes treated with IL-1ß alone, indicating that antagonism of ß-AR confers catabolic signals. On the other hand, NE antagonized IL-1ß-induced catabolic response. In addition, NE significantly inhibited IL-1ß-induced release of glycosaminoglycan (GAG) from cartilage explant culture. In addition, ß-AR activity significantly affected IL-1ß-stimulated phosphorylation of JNK and ERK. These results indicate that ß-AR signal is associated with cartilage metabolism. CONCLUSIONS: Our findings showed that ß-ARs is a regulator of cartilage catabolism induced with IL-1ß.

PEP-1-GRX-1 Modulates Matrix Metalloproteinase-13 and Nitric Oxide Expression of Human Articular Chondrocytes.

BACKGROUND: The protein transduction domain (PTD) enables therapeutic proteins to directly penetrate the membranes of cells and tissues, and has been increasingly utilized. Glutaredoxin-1 (GRX-1) is an endogenous antioxidant enzyme involved in the cellular redox homeostasis system. In this study, we investigated whether PEP-1-GRX-1, a fusion protein of GRX-1 and PEP-1 peptide, a PTD, could suppress catabolic responses in primary human articular chondrocytes and a mouse carrageenan-induced paw edema model. METHODS: Human articular chondrocytes were isolated enzymatically from articular cartilage and cultured in a monolayer. The transduction efficiency of PEP-1-GRX-1 into articular chondrocytes was measured by western blot and immunohistochemistry. The effects of PEP-1-GRX-1 on matrix metalloproteinases (MMPs) and catabolic factor expression in interleukin (IL)-1ß- and lipopolysaccharide (LPS)-treated chondrocytes were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and western blot. The effect of PEP-1-GRX1 on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) signaling pathway were also analyzed by western blot. Finally, the inhibitory effect of PEP-1-GRX-1 on MMP-13 production was measured in vivo in a mouse carrageenan-induced paw edema model. RESULTS: PEP-1-GRX-1 significantly penetrated into human chondrocytes and mouse cartilage, whereas GRX-1 did not. PEP-1-GRX-1 significantly suppressed MMP-13 expression and nitric oxide (NO) production in LPS-stimulated chondrocytes, and NO production in IL-1ß-stimulated chondrocytes, compared with GRX-1. In addition, PEP-1-GRX-1 decreased IL-1ß- and LPS-induced activation of MAPK and NF-κB. In the mouse model of carrageenan-induced paw edema, PEP-1-GRX-1 significantly suppressed carrageenan-induced MMP-13 production as well as paw edema. CONCLUSION: These results demonstrate that PEP-1-GRX-1 can be transduced efficiently in vitro and in vivo into human chondrocytes and mouse cartilage tissue and downregulate catabolic responses in chondrocytes by inhibiting the MAPK and NF-κB pathway. PEP-1-GRX-1 thus has the potential to reduce catabolic responses in chondrocytes and cartilage.

Neuroprotective effect of PEP-1-peroxiredoxin2 on CA1 regions in the hippocampus against ischemic insult.

BACKGROUND: Oxidative stress is a leading cause of various diseases, including ischemia and inflammation. Peroxiredoxin2 (PRX2) is one of six mammalian isoenzymes (PRX1-6) that can reduce hydrogen peroxide (H2O2) and organic hydroperoxides to water and alcohols. METHODS: We produced PEP-1-PRX2 transduction domain (PTD)-fused protein and investigated the effect of PEP-1-PRX2 on oxidative stress-induced neuronal cell death by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western blot, immunofluorescence microscopy, and immunohistochemical analysis. RESULTS: Our data showed that PEP-1-PRX2, which can effectively transduce into various types of cells and brain tissues, could be implicated in suppressing generation of reactive oxygen species, preventing depolarization of the mitochondrial membrane, and inhibiting the apoptosis pathway in H2O2-stimulated HT22, murine hippocampal neuronal cells, likely resulting in protection of HT22 cells against H2O2-induced toxicity. In addition, we found that in a transient forebrain ischemia model, PEP-1-PRX2 inhibited the activation of astrocytes and microglia in the CA1 region of the hippocampus and lipid peroxidation and also prevented neuronal cell death against ischemic damage. CONCLUSIONS: These findings suggest that the transduced PEP-1-PRX2 has neuroprotective functions against oxidative stress-induced cell death in vitro and in vivo. GENERAL SIGNIFICANCE: PEP-1-PRX2 could be a potential therapeutic agent for oxidative stress-induced brain diseases such as ischemia.

Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis.

Apoptosis is a highly-regulated, active process of cell death involved in development, homeostasis and aging. Dysregulation of apoptosis leads to pathological states, such as cancer, developmental anomalies and degenerative diseases. Osteoarthritis (OA), the most common chronic joint disease in the elderly population, is characterized by progressive destruction of articular cartilage, resulting in significant disability. Because articular cartilage depends solely on its resident cells, the chondrocytes, for the maintenance of extracellular matrix, the compromising of chondrocyte function and survival would lead to the failure of the articular cartilage. The role of subchondral bone in the maintenance of proper cartilage matrix has been suggested as well, and it has been proposed that both articular cartilage and subchondral bone interact with each other in the maintenance of articular integrity and physiology. Some investigators include both articular cartilage and subchondral bone as targets for repairing joint degeneration. In late-stage OA, the cartilage becomes hypocellular, often accompanied by lacunar emptying, which has been considered as evidence that chondrocyte death is a central feature in OA progression. Apoptosis clearly occurs in osteoarthritic cartilage; however, the relative contribution of chondrocyte apoptosis in the pathogenesis of OA is difficult to evaluate, and contradictory reports exist on the rate of apoptotic chondrocytes in osteoarthritic cartilage. It is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a byproduct of cartilage destruction. Chondrocyte death and matrix loss may form a vicious cycle, with the progression of one aggravating the other, and the literature reveals that there is a definite correlation between the degree of cartilage damage and chondrocyte apoptosis. Because current treatments for OA act only on symptoms and do not prevent or cure OA, chondrocyte apoptosis would be a valid target to modulate cartilage degeneration.

Monosodium Urate Crystal-Induced Chondrocyte Death via Autophagic Process.

Monosodium urate (MSU) crystals, which are highly precipitated in the joint cartilage, increase the production of cartilage-degrading enzymes and pro-inflammatory mediators in cartilage, thereby leading to gouty inflammation and joint damage. In this study, we investigated the effect of MSU crystals on the viability of human articular chondrocytes and the mechanism of MSU crystal-induced chondrocyte death. MSU crystals significantly decreased the viability of primary chondrocytes in a time- and dose-dependent manner. DNA fragmentation was observed in a culture medium of MSU crystal-treated chondrocytes, but not in cell lysates. MSU crystals did not activate caspase-3, a marker of apoptosis, compared with actinomycin D and TNF-α-treated cells. MSU crystals did not directly affect the expression of endoplasmic reticulum (ER) stress markers at the mRNA and protein levels. However, MSU crystals significantly increased the LC3-II level in a time-dependent manner, indicating autophagy activation. Moreover, MSU crystal-induced autophagy and subsequent chondrocyte death were significantly inhibited by 3-methyladenine, a blocker of autophagosomes formation. MSU crystals activated autophagy via inhibition of phosporylation of the Akt/mTOR signaling pathway. These results demonstrate that MSU crystals may cause the death of chondrocytes through the activation of the autophagic process rather than apoptosis or ER stress.

Antitumor activity of methyl gallate by inhibition of focal adhesion formation and Akt phosphorylation in glioma cells.

BACKGROUND: Methyl gallate (MG) possesses a wide range of biological properties that include anti-oxidant, anti-inflammatory, and anti-microbial activities. However, its anti-tumor activity has not been extensively examined in cancer cells. Thus, we examined the effect of MG in both glutamate-induced rat C6 and human U373 glioma cell proliferation and migration. METHODS: MG was isolated from the stem bark of Acer barbinerve. Cell viability and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch wound-healing assay, respectively. Focal adhesion formation was detected with immunofluorescence. RESULTS: Treatment of C6 and U373 glioma cells with MG significantly reduced cell viability, migration, and Akt phosphorylation level. Glutamate stimulation markedly increased the level of ERK1/2 phosphorylation. However, cells treated with MG displayed decreased ERK1/2 phosphorylation. Inhibition of ERK1/2 by MG or MEK1/2 inhibitor significantly inhibited paxillin phosphorylation at Ser(83) and focal adhesion turn-over produced inefficient glioma cell migration. In addition, activation of Akt and ERK1/2 upon glutamate stimulation was independently regulated by Ca(2+) and protein kinase C activity, respectively, via the α-amino-3-hydroxy-5-methy-4-isoxazolepropionate acid glutamate receptor and metabotropic glutamate receptor. GENERAL SIGNIFICANCE: Our results clearly indicate that MG has a strong anti-tumor effect through the down-regulation of the Akt and ERK1/2 signaling pathways. Thus, methyl gallate is a potent anti-tumor and novel therapeutic agent for glioma.

The family-wide structure and function of human dual-specificity protein phosphatases.

Dual-specificity protein phosphatases (DUSPs), which dephosphorylate both phosphoserine/threonine and phosphotyrosine, play vital roles in immune activation, brain function and cell-growth signalling. A family-wide structural library of human DUSPs was constructed based on experimental structure determination supplemented with homology modelling. The catalytic domain of each individual DUSP has characteristic features in the active site and in surface-charge distribution, indicating substrate-interaction specificity. The active-site loop-to-strand switch occurs in a subtype-specific manner, indicating that the switch process is necessary for characteristic substrate interactions in the corresponding DUSPs. A comprehensive analysis of the activity-inhibition profile and active-site geometry of DUSPs revealed a novel role of the active-pocket structure in the substrate specificity of DUSPs. A structure-based analysis of redox responses indicated that the additional cysteine residues are important for the protection of enzyme activity. The family-wide structures of DUSPs form a basis for the understanding of phosphorylation-mediated signal transduction and the development of therapeutics.

PEP-1-metallothionein-III protein ameliorates the oxidative stress-induced neuronal cell death and brain ischemic insults.

BACKGROUND: Oxidative stress is considered to be involved in a number of human diseases including ischemia. Metallothioneins (MT)-III can protect neuronal cells from the cytotoxicity of reactive oxygen species (ROS). However, MT-III proteins biological function is unclear in ischemia. Thus, we examined the protective effects of MT-III proteins on oxidative stress-induced neuronal cell death and brain ischemic insult. METHODS: A human MT-III gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1-MT-III protein. PEP-1-MT-III protein was purified using affinity chromatograph. Transduced PEP-1-MT-III proteins were detected by Western blotting and immunoflourescence. Cell viability and DNA fragmentation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay and terminal dexoynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Brain ischemic injury was detected with immunohistochemistry. RESULTS: Purified PEP-1-MT-III proteins transduced into astrocytes in a time- and dose-dependent manner and protected against oxidative stress-induced cell death. Also, transduced PEP-1-MT-III proteins efficiently protected cells against DNA fragmentation. Furthermore, immunohistochemical analysis revealed that PEP-1-MT-III prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. We demonstrated that transduced PEP-1-MT-III protein protects against oxidative stress induced cell death in vitro and in vivo. GENERAL SIGNIFICANCE: Transduced PEP-1-MT-III protein has neuroprotective roles as an antioxidant in vitro and in vivo. PEP-1-MT-III protein is a potential therapeutic agent for various human brain diseases such as stroke, Alzheimer's disease, and Parkinson's disease.

Transduced Tat-glyoxalase protein attenuates streptozotocin-induced diabetes in a mouse model.

Diabetes mellitus (DM) is characterized by hyperglycemia. Glyoxalase 1 (GLO) has considerable potential as a possible therapeutic agent for DM. However, the precise action of GLO remains unclear in DM. In this study, we examined the protective effects of GLO protein in a streptozotocin (STZ)-induced diabetes animal model using cell-permeable Tat-GLO protein. Purified Tat-GLO protein was efficiently transduced into RINm5F cells in a time- and dose-dependent manner and protected cells against sodium nitroprusside (SNP)-induced cell death and DNA fragmentation. Furthermore, Tat-GLO protein significantly inhibited blood glucose levels and altered the serum biochemical parameters in STZ-induced diabetic mice. These results demonstrate that transduced Tat-GLO protein protects pancreatic cells by the inhibition of STZ-mediated toxicity. Therefore, Tat-GLO protein could be useful as a therapeutic agent against DM.

Metabolic obesity and the risk of knee osteoarthritis progression in elderly community residents: A 3-year longitudinal cohort study.

OBJECTIVE: Metabolic syndrome is a major health problem worldwide associated with obesity, thus drawing attention to its relation to osteoarthritis (OA). However, it is still uncertain whether metabolic syndrome or body fat distribution is associated with knee OA. The aim of this longitudinal study was to elucidate the association between metabolic obesity and adverse structural changes of knee OA assessed by magnetic resonance imaging (MRI). METHODS: Participants were recruited from the Hallym Aging Study cohort in Korea. Knee MRI scans, along with dual-energy X-ray absorptiometry, were assessed in 226 participants at baseline and after 3 years. The structural progression in the tibiofemoral joint was evaluated using the semi-quantitative Whole-Organ MRI Score (WORMS) for cartilage morphology and bone marrow lesions (BML). Logistic regression with generalized estimating equation was performed for associations of metabolic risk factors with worsening of WORMS scores at the subregional level. RESULTS: In the medial compartment, fat mass in women was associated with cartilage loss, but the statistical significance disappeared after adjusting for body mass index. Metabolic syndrome and each of its components were not associated with cartilage loss or increase of BML. On the other hand, the interaction effects of metabolic syndrome on the association between obesity and knee OA progression were not significant. CONCLUSION: In this cohort, metabolic effects of obesity on knee cartilage damage and BML were not demonstrated. Further large-scale studies are required to prove the causal relationship between metabolic obesity and knee OA.

Effects of pergolide mesylate on transduction efficiency of PEP-1-catalase protein.

The low transduction efficiency of various proteins is an obstacle to their therapeutic application. However, protein transduction domains (PTDs) are well-known for a highly effective tool for exogenous protein delivery to cells. We examined the effects of pergolide mesylate (PM) on the transduction of PEP-1-catalase into HaCaT human keratinocytes and mice skin and on the anti-inflammatory activity of PEP-1-catatase against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation using Western blot and histological analysis. PM enhanced the time- and dose-dependent transduction of PEP-1-catalase into HaCaT cells without affecting the cellular toxicity. In a mouse edema model, PEP-1-catalase inhibited the increased expressions of inflammatory mediators and cytokines such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1ß, and tumor necrosis factor-α induced by TPA. On the other hand, PM alone failed to exert any significant anti-inflammatory effects. However, the anti-inflammatory effect of co-treatment with PEP-1-catalase and PM was more potent than that of PEP-1-catalase alone. Our results indicate that PM may enhance the delivery of PTDs fusion therapeutic proteins to target cells and tissues and has potential to increase their therapeutic effects of such drugs against various diseases.

Anti-inflammatory effect of transduced PEP-1-heme oxygenase-1 in Raw 264.7 cells and a mouse edema model.

Heme oxygenase-1 (HO-1), which catalyzes the degradation of free heme to biliverdin, carbon monoxide (CO), and free iron (Fe(2+)), is up-regulated by several cellular stress and cell injuries, including inflammation, ischemia and hypoxia. In this study, we examined whether fusion of HO-1 with PEP-1, a protein transduction domain that is able to deliver exogenous molecules to living cells or tissues, would facilitate HO-1 delivery to target cells and tissues, and thereby effectively exert a therapeutically useful response against inflammation. Western blot analysis demonstrated that PEP-1-HO-1 fusion proteins were transduced into Raw 264.7 cells in time- and dose-dependent manners, and were stably maintained in the cells for about 60h. In addition, fluorescence analysis revealed that only PEP-1-HO-1 fusion proteins were significantly transduced into the cytoplasm of cells, while HO-1 proteins failed to be transduced. In lipopolysaccharide (LPS)-stimulated Raw 264.7 cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse edema model, transduced PEP-1-HO-1 fusion proteins effectively inhibited the overexpression of pro-inflammatory mediators and cytokines. Also, histological analysis demonstrated that PEP-1-HO-1 remarkably suppressed ear edema. The results suggest that the PEP-1-HO-1 fusion protein can be used as a therapeutic molecule against reactive oxygen species-related inflammatory diseases.

The TLR-2/TonEBP signaling pathway regulates 29-kDa fibronectin fragment-dependent expression of matrix metalloproteinases.

Tonicity-responsive enhancer-binding protein (TonEBP; nuclear factor of activated T cells 5) is a transcription factor that responds to changes in osmolality. However, recent studies have shown that it also modulates immune responses under inflammatory conditions independently of hyperosmolality. Fibronectin fragments (FN-fs), which are abundant in the synovial fluid of patients with osteoarthritis (OA), induce expression of matrix metalloproteinases (MMPs) via the toll-like receptor-2 (TLR-2) signaling pathway. In this study we examined whether TonEBP is involved in 29-kDa FN-f-induced expression of MMPs. The expression of TonEBP was significantly higher in human osteoarthritis compared with normal cartilage samples. 29-kDa FN-f affected the expression of MMPs 1, 3, and 13 via TonEBP, and expression and nuclear accumulation of TonEBP were induced by activation of the phospholipase C/NF-κB/MAPK signaling pathway and, in particular, modulated by TLR-2. In addition, 29-kDa FN-f induced the expression of osmoregulatory genes, including Tau-T, SMIT, and AR, as well as voltage-dependent calcium channels via the TonEBP/TLR-2 signaling pathway. These results show that 29-kDa FN-f upregulates MMPs in chondrocytes via the TLR-2/TonEBP signaling pathway.

NOD2 signaling pathway is involved in fibronectin fragment-induced pro-catabolic factor expressions in human articular chondrocytes.

The nucleotide-binding and oligomerization domain (NOD) is an innate pattern recognition receptor that recognizes pathogen- and damage-associated molecular patterns. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) is a matrix degradation product found in the synovial fluids of patients with osteoarthritis (OA). We investigated whether NOD2 was involved in 29-kDa FN-f-induced pro-catabolic gene expression in human chondrocytes. The expression of mRNA and protein was measured using quantitative real-time polymerase chain reaction (qrt-PCR) and Western blot analysis. Small interfering RNAs were used for knockdown of NOD2 and toll-like receptor 2 (TLR-2). An immunoprecipitation assay was performed to examine protein interactions. The NOD2 levels in human OA cartilage were much higher than in normal cartilage. NOD1 and NOD2 expression, as well as pro-inflammatory cytokines, including interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α), were upregulated by 29-kDa FN-f in human chondrocytes. NOD2 silencing showed that NOD2 was involved in the 29-kDa FN-f-induced expression of TLR-2. Expressions of IL-6, IL-8, matrix metalloproteinase (MMP)-1, -3, and -13 were also suppressed by TLR-2 knockdown. Furthermore, NOD2 and TLR-2 knockdown data demonstrated that both NOD2 and TLR-2 modulated the expressions of their adaptors, receptorinteracting protein 2 (RIP2) and myeloid differentiation 88, in 29-kDa FN-f-treated chondrocytes. 29-kDa FN-f enhanced the interaction of NOD2, RIP2 and transforming growth factor beta-activated kinase 1 (TAK1), an indispensable signaling intermediate in the TLR-2 signaling pathway, and activated nuclear factor-κB (NF-κB), subsequently leading to increased expressions of pro-inflammatory cytokines and cartilagedegrading enzymes. These results demonstrate that 29-kDa FN-f modulated pro-catabolic responses via cross-regulation of NOD2 and TLR-2 signaling pathways. [BMB Reports 2019; 52(6): 373-378].

Induction of pro-inflammatory cytokines by 29-kDa FN-f via cGAS/STING pathway.

The cGAS-STING pathway plays an important role in pathogen-induced activation of the innate immune response. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) found predominantly in the synovial fluid of osteoarthritis (OA) patients increases the expression of catabolic factors via the toll-like receptor-2 (TLR-2) signaling pathway. In this study, we investigated whether 29-kDa FN-f induces inflammatory responses via the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) pathway in human primary chondrocytes. The levels of cGAS and STING were elevated in OA cartilage compared with normal cartilage. Long-term treatment of chondrocytes with 29-kDa FN-f activated the cGAS/STING pathway together with the increased level of gamma-H2AX, a marker of DNA breaks. In addition, the expression of pro-inflammatory cytokines, including granulocytemacrophage colony-stimulating factor (GM-CSF/CSF-2), granulocyte colony-stimulating factor (G-CSF/CSF-3), and type I interferon (IFN-α), was increased more than 100-fold in 29-kDa FN-f-treated chondrocytes. However, knockdown of cGAS and STING suppressed 29-kDa FN-f-induced expression of GM-CSF, G-CSF, and IFN-α together with the decreased activation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and inhibitor protein κBα (IκBα). Furthermore, NOD2 or TLR-2 knockdown suppressed the expression of GM-CSF, G-CSF, and IFN-α as well as decreased the activation of the cGAS/STING pathway in 29-kDa FN-f-treated chondrocytes. These data demonstrate that the cGAS/STING/TBK1/IRF3 pathway plays a critical role in 29-kDa FN-f-induced expression of pro-inflammatory cytokines. [BMB Reports 2019; 52(5): 336-341].

29-kDa FN-f inhibited autophagy through modulating localization of HMGB1 in human articular chondrocytes.

Fibronectin fragments found in the synovial fluid of patients with osteoarthritis (OA) induce the catabolic responses in cartilage. Nuclear high-mobility group protein Box 1 (HMGB1), a damage-associated molecular pattern, is responsible for the regulation of signaling pathways related to cell death and survival in response to various stimuli. In this study, we investigated whether changes induced by 29-kDa aminoterminal fibronectin fragment (29-kDa FN-f) in HMGB1 expression influences the pathogenesis of OA via an HMGB1- modulated autophagy signaling pathway. Human articular chondrocytes were enzymatically isolated from articular cartilage. The level of mRNA was measured by quantitative real-time PCR. The expression of proteins was examined by western blot analysis, immnunofluorescence assay, and enzyme-linked immunosorbent assay. Interaction of proteins was evaluated by immunoprecipitation. The HMGB1 level was significantly lower in human OA cartilage than in normal cartilage. Although 29-kDa FN-f significantly reduced the HMGB1 expression at the mRNA and protein levels 6 h after treatment, the cytoplasmic level of HMGB1 was increased in chondrocytes treated with 29-kDa FN-f, which significantly inhibited the interaction of HMGB1 with Beclin-1, increased the interaction of Bcl-2 with Beclin-1, and decreased the levels of Beclin-1 and phosphorylated Bcl-2. In addition, the level of microtubule-associated protein 1 light chain 3-II, an autophagy marker, was down-regulated in chondrocytes treated with 29-kDa FN-f, whereas the effect was antagonized by mTOR knockdown. Furthermore, prolonged treatment with 29-kDa FN-f significantly increased the release of HMGB1 into the culture medium. These results demonstrated that 29-kDa FN-f inhibits chondrocyte autophagy by modulating the HMGB1 signaling pathway. [BMB Reports 2018; 51(10): 509-514].

Structure-activity relationships of the human prothrombin kringle-2 peptide derivative NSA9: anti-proliferative activity and cellular internalization.

The human prothrombin kringle-2 protein inhibits angiogenesis and LLC (Lewis lung carcinoma) growth and metastasis in mice. Additionally, the NSA9 peptide (NSAVQLVEN) derived from human prothrombin kringle-2 has been reported to inhibit the proliferation of BCE (bovine capillary endothelial) cells and CAM (chorioallantoic membrane) angiogenesis. In the present study, we examined the structure-activity relationships of the NSA9 peptide in inhibiting the proliferation of endothelial cells lines e.g. BCE and HUVE (human umbilical vein endothelial). N- or C-terminal truncated derivatives and reverse sequence analogues of NSA9 were prepared and their anti-proliferative activities were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. This cell proliferation assay demonstrated that both the N-terminal region and sequence orientation of NSA9 are important for inhibiting the proliferation of endothelial cells. In particular 2 C-terminal truncation derivatives of NSA9 [NSA7 (NSAVQLV) and NSA8 (NSAVQLVE)] inhibited cellular proliferation to a greater extent than did NSA9. The heptapeptide NSA7, was found to be more potent than NSA9 in inhibiting CAM angiogenesis, and tubular formation and migration of HUVE cells. In addition NSA9, NSA8 and NSA7 peptides exhibited considerable inhibitory effects on the proliferation of tumour cells such as B16F10 (murine melanoma), LLC and L929 (murine fibroblast). Also, cellular internalization studies demonstrated that NSA7 was internalized into both endothelial and tumour cells more easily than was NSA9. In conclusion, these results suggest that NSA7, residing within the full sequence of NSA9, contains the required sequence for anti-proliferative activity and cellular internalization.

MicroRNA-365 regulates IL-1ß-induced catabolic factor expression by targeting HIF-2α in primary chondrocytes.

Endothelial Per-Arnt-Sim domain protein-1/hypoxia-inducible factor-2α (EPAS-1/ HIF-2α) is a catabolic transcription factor that regulates osteoarthritis (OA)-related cartilage destruction. Here, we examined whether microRNA-365 (miR-365) affects interleukin (IL)-1ß-induced expression of catabolic factors in chondrocytes via regulation of HIF-2α. MiR-365 levels were significantly decreased in human OA cartilage relative to normal cartilage. Overexpression of miR-365 significantly suppressed IL-1ß-induced expression of HIF-2α in human articular chondrocytes. Pharmacological inhibition of various IL-1ß-associated signaling pathways revealed mitogen-activated protein kinase and nuclear factor-κB as the primary pathways driving IL-1ß-mediated decreases in miR-365 and subsequent increase in HIF-2α expression. Using a luciferase reporter assay encoding the 3' untranslated region (UTR) of human HIF-2α mRNA, we showed that overexpression of miR-365 significantly suppressed IL-1ß-induced up-regulation of HIF-2α. AGO2 RNA-immunoprecipitation (IP) assay demonstrated that miR-365 and HIF-2α mRNA were enriched in the AGO2-IP fraction in miR-365-transfected primary chondrocytes compared to miR-con-transfected cells, indicating that HIF-2α is a target of miR-365. Furthermore, miR-365 overexpression significantly suppressed IL-1ß-induced expression of catabolic factors, including cyclooxygenase-2 and matrix metalloproteinase-1, -3 and -13, in chondrocytes. In pellet culture of primary chondrocytes miR-365 prevented IL-1ß-stimulated extracellular matrix loss and matrix metalloproteinase-13 expression. MiR-365 regulates IL-1ß-stimulated catabolic effects in human chondrocytes by modulating HIF-2α expression.

Fibronectin fragment-induced expression of matrix metalloproteinases is mediated by MyD88-dependent TLR-2 signaling pathway in human chondrocytes.

INTRODUCTION: Fibronectin fragments (FN-fs) are increased in the cartilage of patients with osteoarthritis (OA) and have a potent chondrolytic effect. However, little is known about the cellular receptors and signaling mechanisms that are mediated by FN-fs. We investigated whether the 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) regulates cartilage catabolism via the Toll-like receptor (TLR)-2 signaling pathway in human chondrocytes. METHODS: Small interfering RNA was used to knock down TLR-2 and myeloid differentiation factor 88 (MyD88). TLR-2 was overexpressed in chondrocytes transfected with a TLR-2 expression plasmid. The expression levels of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 were analyzed using quantitative real-time reverse transcription polymerase chain reactions, immunoblotting, or enzyme-linked immunosorbent assay. The effect of TLR-2 on 29-kDa FN-f-mediated signaling pathways was investigated by immunoblotting. RESULTS: TLR-2, TLR-3, TLR-4, and TLR-5 mRNA were significantly overexpressed in OA cartilage compared with normal cartilage, whereas no significant difference of TLR-1 mRNA expression was found. 29-kDa FN-f significantly increased TLR-2 expression in human chondrocytes in a dose- and time-dependent manner. Knockdown of TLR-2 or MyD88, the latter a downstream adaptor of TLR-2, significantly inhibited 29-kDa FN-f-induced MMP production at the mRNA and protein levels. Conversely, TLR-2 overexpression led to enhanced MMP production by 29-kDa FN-f. In addition, TLR-2 knockdown apparently inhibited 29-kDa FN-f-mediated activation of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, and p38, but not of c-Jun N-terminal kinase or extracellular signal-regulated kinase. Exposure to synovial fluid (SF) from affected joints of patients with OA elevated MMP-1, MMP-3, and MMP-13 expression markedly in primary chondrocytes without reducing cell viability. However, TLR-2 knockdown in chondrocytes significantly suppressed SF-induced MMP induction. CONCLUSIONS: Our data demonstrate that the MyD88-dependent TLR-2 signaling pathway may be responsible for 29-kDa FN-f-mediated cartilage catabolic responses. Our results will enhance understanding of cartilage catabolic mechanisms driven by cartilage degradation products, including FN-f. The modulation of TLR-2 signaling activated by damage-associated molecular patterns, including 29-kDa FN-f, is a potential therapeutic strategy for the prevention of cartilage degradation in OA.