Bats as instructive animal models for studying longevity and aging.

Bats (order Chiroptera) are emerging as instructive animal models for aging studies. Unlike some common laboratory species, they meet a central criterion for aging studies: they live for a long time in the wild or in captivity, for 20, 30, and even >40 years. Healthy aging (i.e., healthspan) in bats has drawn attention to their potential to improve the lives of aging humans due to bat imperviousness to viral infections, apparent low rate of tumorigenesis, and unique ability to repair DNA. At the same time, bat longevity also permits the accumulation of age-associated systemic pathologies that can be examined in detail and manipulated, especially in captive animals. Research has uncovered additional and critical advantages of bats. In multiple ways, bats are better analogs to humans than are rodents. In this review, we highlight eight diverse areas of bat research with relevance to aging: genome sequencing, telomeres, and DNA repair; immunity and inflammation; hearing; menstruation and menopause; skeletal system and fragility; neurobiology and neurodegeneration; stem cells; and senescence and mortality. These examples demonstrate the broad relevance of the bat as an animal model and point to directions that are particularly important for human aging studies.

A retrotransposon gag-like-3 gene RTL3 and SOX-9 co-regulate the expression of COL2A1 in chondrocytes.

PURPOSE: Transposable elements are known to remodel gene structure and provide a known source of genetic variation. Retrotransposon gag-like-3 (RTL3) is a mammalian retrotransposon-derived transcript (MART) whose function in the skeletal tissue is unknown. This study aimed to elucidate the biological significance of RTL3 in chondrogenesis and type-II collagen (COL2A1) gene expression in chondrocytes. MATERIALS AND METHODS: Expression of RTL3, SOX-9 and COL2A1 mRNAs was determined by TaqMan assays and the protein expression by immunoblotting. RTL3 and Sox-9 depletion in human chondrocytes was achieved using validated siRNAs. An RTL3 mutant (∆RTL3) lacking the zinc finger domain was created using in vitro mutagenesis. Forced expression of RTL3, ∆RTL3, and SOX-9 was achieved using CMV promoter containing expression plasmids. CRISPR-Cas9 was utilized to delete Rtl3 and create a stable ATDC5Rlt3-/- cell line. Matrix deposition and Col2a1 quantification during chondrogenesis were determined by Alcian blue staining and Sircol™ Soluble Collagen Assay, respectively. RESULTS: RTL3 is not ubiquitously expressed but showed strong expression in cartilage, chondrocytes and synoviocytes but not in muscle, brain, or other tissues analyzed. Loss-of-function and gain-of-function studies demonstrated a critical role of RTL3 in the regulation of SOX-9 and COL2A1 expression and matrix synthesis during chondrogenesis. Both RTL3 and SOX-9 displayed co-regulated expression in chondrocytes. Gene regulatory activity of RTL3 requires the c-terminal CCHC zinc-finger binding domain. CONCLUSIONS: Our results identify a novel regulatory mechanism of COL2A1 expression in chondrocytes that may help to further understand the skeletal development and the pathogenesis of diseases with altered COL2A1 expression.

Assessing Chondrocyte Status by Immunofluorescence-Mediated Localization of Parkin Relative to Mitochondria.

Immunofluorescence staining is a widely used and powerful tool for the visualization and colocalization of two or more proteins and/or cellular organelles. For colocalization studies in fixed cells, one target protein/organelle is immunostained and visualized by one fluorophore and the other target protein/organelle is immunostained and visualized by a different fluorophore whose excitation emission spectra does not overlap with the first fluorophore. Parkin (PARK2) is an E3 ubiquitin ligase which performs ubiquitination of surface proteins of dysfunctional mitochondria to mark them for autolysosomal degradation. Here we describe the immunofluorescence staining of parkin protein and immunofluorescence or dye-based methods to visualize mitochondria and study the colocalization of parkin and mitochondria in primary human or mouse chondrocytes or cell lines.

Mitochondrial dysfunction triggers a catabolic response in chondrocytes via ROS-mediated activation of the JNK/AP1 pathway.

Mitochondrial function is impaired in osteoarthritis (OA) but its impact on cartilage catabolism is not fully understood. Here, we investigated the molecular mechanism of mitochondrial dysfunction-induced activation of the catabolic response in chondrocytes. Using cartilage slices from normal and OA cartilage, we showed that mitochondrial membrane potential was lower in OA cartilage, and that this was associated with increased production of mitochondrial superoxide and catabolic genes [interleukin 6 (IL-6), COX-2 (also known as PTGS2), MMP-3, -9, -13 and ADAMTS5]. Pharmacological induction of mitochondrial dysfunction in chondrocytes and cartilage explants using carbonyl cyanide 3-chlorophenylhydrazone increased mitochondrial superoxide production and the expression of IL-6, COX-2, MMP-3, -9, -13 and ADAMTS5, and cartilage matrix degradation. Mitochondrial dysfunction-induced expression of catabolic genes was dependent on the JNK (herein referring to the JNK family)/activator protein 1 (AP1) pathway but not the NFκB pathway. Scavenging of mitochondrial superoxide with MitoTEMPO, or pharmacological inhibition of JNK or cFos and cJun, blocked the mitochondrial dysfunction-induced expression of the catabolic genes in chondrocytes. We demonstrate here that mitochondrial dysfunction contributes to OA pathogenesis via JNK/AP1-mediated expression of catabolic genes. Our data shows that AP1 could be used as a therapeutic target for OA management.This article has an associated First Person interview with the first author of the paper.

Imperatorin suppresses IL-1ß-induced iNOS expression via inhibiting ERK-MAPK/AP1 signaling in primary human OA chondrocytes.

Joint inflammation is a key player in the pathogenesis of osteoarthritis (OA). Imperatorin, a plant-derived small molecule has been reported to have anti-inflammatory properties; however, its effect on chondrocytes is not known. Here, we investigated the effects of Imperatorin on interleukin-1ß (IL-1ß) induced expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in primary human OA chondrocytes and cartilage explants culture under pathological conditions and explored the associated signaling pathways. We pretreated chondrocytes or explants with Imperatorin (50 µM) followed by IL-1ß (1 ng/ml), and the culture supernatant was used to determine the levels of nitrite production by Griess assay and chondrocytes were harvested to prepare cell lysate or RNA for gene expression analysis of iNOS by Western blot or qPCR and in explants by immunohistochemistry (IHC). Pretreatment of primary chondrocytes and cartilage explants with Imperatorin suppressed IL-1ß induced expression of iNOS and NO production. Imperatorin blocked the IL-1ß-induced phosphorylation of ERK-MAPK/AP1 signaling pathway to suppress iNOS expression. The role of ERK in the regulation of iNOS expression was verified by using ERK inhibitor. Interestingly, we also found that Imperatorin binds to iNOS protein and inhibits its activity in vitro. Our data demonstrated that Imperatorin possess strong anti-inflammatory activity and may be developed as a therapeutic agent for the management of OA.

Role of iNOS in osteoarthritis: Pathological and therapeutic aspects.

Accumulating evidence suggests that inflammation has a key role in the pathogenesis of osteoarthritis (OA). Nitric oxide (NO) has been established as one of the major inflammatory mediators in OA and drives many pathological changes during the development and progression of OA. Excessive production of NO in chondrocytes promotes cartilage destruction and cellular injury. The synthesis of NO in chondrocytes is catalyzed by inducible NO synthase (iNOS), which is thereby an attractive therapeutic target for the treatment of OA. A number of direct and indirect iNOS inhibitors, bioactive compounds, and plant-derived small molecules have been shown to exhibit chondroprotective effects by suppressing the expression of iNOS. Many of these iNOS inhibitors hold promise for the development of new, disease-modifying therapies for OA; however, attempts to demonstrate their success in clinical trials are not yet successful. Many plant extracts and plant-derived small molecules have also shown promise in animal models of OA, though further studies are needed in human clinical trials to confirm their therapeutic potential. In this review, we discuss the role of iNOS in OA pathology and the effects of various iNOS inhibitors in OA.

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation.

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit ß (IKKß) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.

Autophagy plays an essential role in bone homeostasis.

Autophagy is very critical for multiple cellular processes. Autophagy plays a critical role in bone cell differentiation and function.

Genetic Inactivation of ZCCHC6 Suppresses Interleukin-6 Expression and Reduces the Severity of Experimental Osteoarthritis in Mice.

OBJECTIVE: Cytokine expression is tightly regulated posttranscriptionally, but high levels of interleukin-6 (IL-6) in patients with osteoarthritis (OA) indicate that regulatory mechanisms are disrupted in this disorder. The enzyme ZCCHC6 (zinc-finger CCHC domain-containing protein 6; TUT-7) has been implicated in posttranscriptional regulation of inflammatory cytokine expression, but its role in OA pathogenesis is unknown. The present study was undertaken to investigate whether ZCCHC6 directs the expression of IL-6 and influences OA pathogenesis in vivo. METHODS: Human and mouse chondrocytes were stimulated with recombinant IL-1ß. Expression of ZCCHC6 in human chondrocytes was knocked down using small interfering RNAs. IL-6 transcript stability was determined by actinomycin D chase, and 3'-uridylation of microRNAs was determined by deep sequencing. Zcchc6-/- mice were produced by gene targeting. OA was surgically induced in the knee joints of mice, and disease severity was scored using a semiquantitative grading system. RESULTS: ZCCHC6 was markedly up-regulated in damaged cartilage from human OA patients and from wild-type mice with surgically induced OA. Overexpression of ZCCHC6 induced the expression of IL-6, and its knockdown reduced IL-6 transcript stability and IL-1ß-induced IL-6 expression in chondrocytes. Reintroduction of Zcchc6 in Zcchc6-/- mouse chondrocytes rescued the IL-1ß-induced IL-6 expression. Knockdown of ZCCHC6 reduced the population of micro-RNA 26b (miR-26b) with 3'-uridylation by 60%. Zcchc6-/- mice with surgically induced OA produced low levels of IL-6 and exhibited reduced cartilage damage and synovitis in the joints. CONCLUSION: These findings indicate that ZCCHC6 enhances IL-6 expression in chondrocytes through transcript stabilization and by uridylating miR-26b, which abrogates repression of IL-6. Inhibition of IL-6 expression and significantly reduced OA severity in Zcchc6-/- mice identify ZCCHC6 as a novel therapeutic target to inhibit disease pathogenesis.

Butein Activates Autophagy Through AMPK/TSC2/ULK1/mTOR Pathway to Inhibit IL-6 Expression in IL-1ß Stimulated Human Chondrocytes.

BACKGROUND/AIMS: Butein (2',3,4,4'-Tetrahydroxychalcone), a polyphenol produced by several plants including Butea monoserpma, has been reported to exert potent anti-inflammatory activity but the mechanism remains unknown. In the present work we investigated the mechanism of Butein-mediated suppression of IL-6 expression in normal and human osteoarthritis (OA) chondrocytes under pathological conditions. METHODS: Expression level of interleukin-6 (IL-6) protein in OA cartilage was analyzed by immunohistochemistry using a validated antibody. Chondrocytes derived from normal or OA cartilage by enzymatic digestion were pretreated with Butein followed by stimulation with interleukin-1ß (IL-1ß) and the levels of IL-6 mRNA were quantified by TaqMan assay and the protein levels were measured by Western immunoblotting. Autophagy activation was determined by Western blotting and confocal microscopy. Autophagy was inhibited by siRNA mediated knockdown of ATG5. RESULTS: Expression of IL-6 protein was high in the OA cartilage compared to smooth cartilage from the same patient. OA chondrocytes and cartilage explants stimulated with IL-1ß showed high level expression of IL-6 mRNA and protein. Butein increased the phosphorylation of AMPKαThr-172, TSC2Ser-1387 and ULK1Ser-317 and inhibited the phosphorylation of mTORSer-2448 and its downstream target p70S6K and increased autophagy flux that correlated with the suppression of the IL-1ß mediated expression of IL-6 in normal and OA chondrocytes. In OA chondrocytes with siRNA-mediated knockdown of ATG5 expression, treatment with Butein failed to activate autophagy and abrogated the suppression of IL-1ß induced IL-6 expression. CONCLUSION: Our findings demonstrate for the first time that Butein activate autophagy in OA chondrocytes via AMPK/TSC2/ULK1/mTOR pathway. Additionally, activation of autophagy was essential to block the IL-1ß-induced expression of IL-6 in OA chondrocytes. These data support further studies to evaluate the use of Butein or compounds derived from it for the management of OA.

A standardized extract of Butea monosperma (Lam.) flowers suppresses the IL-1ß-induced expression of IL-6 and matrix-metalloproteases by activating autophagy in human osteoarthritis chondrocytes.

BACKGROUND/OBJECTIVE: Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1ß. METHODS: The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. RESULTS: BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1ß induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that BME activated autophagy in chondrocytes via inhibition of mammalian target of rapamycin (mTOR) pathway. Of importance is our finding that BME-mediated suppression of IL-1ß induced expression of IL-6, MMP-3, -9, and -13 was autophagy dependent and was abrogated by inhibition of autophagy. CONCLUSION: The above results show that the Butea monosperma (Lam.) extract has strong potential to activate autophagy and suppress IL-1ß induced expression of IL-6 and MMP-3, -9 and -13 in human OA chondrocytes. This study shows that BME or compounds derived from BME can be developed as safe and effective chondroprotective agent(s) that function by activating autophagy to suppress the expression of inflammatory and catabolic factors associated with OA pathogenesis.

Wogonin, a natural flavonoid, intercalates with genomic DNA and exhibits protective effects in IL-1ß stimulated osteoarthritis chondrocytes.

Wogonin has recently been shown to possess anti-inflammatory and chondroprotective properties and is of considerable interest due to its broad pharmacological activities. The present study highlights that Wogonin binds DNA and exerts chondroprotective effects in vitro. Wogonin showed strong binding with chondrocytes genomic DNA in vitro. The mode of binding of Wogonin to genomic-DNA was assessed by competing Wogonin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. EtBr fluorescence reduced significantly with increase in Wogonin concentration suggesting possible DNA intercalation of Wogonin. Further, in silico molecular docking of Wogonin on mammalian DNA also indicated possible intercalation of Wogonin with DNA. The denaturation and FRET studies revealed that Wogonin prevents denaturation of DNA strands and provide stability to genomic DNA against a variety of chemical denaturants. The cellular uptake study showed that Wogonin enters osteoarthritis chondrocytes and was mainly localized in the nucleus. Wogonin treatment to OA chondrocytes protects the fragmentation of genomic DNA in response to IL-1ß as evaluated by DNA ladder and TUNEL assay. Treatment of chondrocytes with Wogonin resulted in significant suppression of IL-1ß-mediated induction of ROS. Further, Wogonin exhibited protective potential through potent suppression of extrinsic and intrinsic apoptotic pathways and induction of anti-apoptotic proteins in IL-1ß-stimulated osteoarthritis chondrocytes. Our data thus suggest that DNA intercalation by Wogonin may result in the stabilization of genomic DNA leading to protective activity.

Dataset of effect of Wogonin, a natural flavonoid, on the viability and activation of NF-κB and MAPKs in IL-1ß-stimulated human OA chondrocytes.

This article contains data related to the article "Wogonin, a plant derived small molecule exerts potent anti-inflammatory and chondroprotective effects through activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes" (Khan et al. 2017) [1]. The data are related to effects of Wogonin on the viability and IL-1ß-stimulated activation of NF-κB and ERK1/2, JNK1/2 and p38 MAPKs in human OA chondrocytes. Gene expression data representing the chondrogenic phenotype and the efficiency of Nrf2 knockdown in monolayer culture of human OA chondrocytes were shown. Moreover, mass spectrometric calibration curve of Wogonin used to quantify the intracellular uptake were also presented. The data are presented in the form of figures and significance of these has been given in the research article (Khan et al. 2017) [1].

A wogonin-rich-fraction of Scutellaria baicalensis root extract exerts chondroprotective effects by suppressing IL-1ß-induced activation of AP-1 in human OA chondrocytes.

Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation and sustained oxidative stress. The root extract of Scutellaria baicalensis (SBE) has been used for the treatment of inflammatory and other diseases. Here, we performed activity-guided HPLC-fractionation of SBE, identified the active ingredient(s) and investigated its chondroprotective potential. We found that the Wogonin containing fraction-4 (F4) was the most potent fraction based on its ability to inhibit ROS production and the suppression of catabolic markers including IL-6, COX-2, iNOS, MMP-3, MMP-9, MMP-13 and ADAMTS-4 in IL-1ß-treated OA chondrocytes. OA chondrocytes treated with F4 in the presence of IL-1ß showed significantly enhanced expression of anabolic genes ACAN and COL2A1. In an in vitro model of cartilage degradation treatment with F4 inhibited s-GAG release from IL-1ß-treated human cartilage explants. The inhibitory effect of F4 was not mediated through the inhibition of MAPKs and NF-κB activation but was mediated through the suppression of c-Fos/AP-1 activity at transcriptional and post transcriptional levels in OA chondrocytes. Purified Wogonin mimicked the effects of F4 in IL-1ß-stimulated OA chondrocytes. Our data demonstrates that a Wogonin-rich fraction of SBE exert chondroprotective effects through the suppression of c-Fos/AP-1 expression and activity in OA chondrocytes under pathological conditions.

Wogonin, a plant derived small molecule, exerts potent anti-inflammatory and chondroprotective effects through the activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes.

Osteoarthritis (OA), characterized by progressive destruction of articular cartilage, is the most common form of human arthritis. Here, we evaluated the potential chondroprotective and anti-inflammatory effects of Wogonin, a naturally occurring flavonoid, in IL-1ß-stimulated human OA chondrocytes and cartilage explants. Wogonin completely suppressed the expression and production of inflammatory mediators including IL-6, COX-2, PGE2, iNOS and NO in IL-1ß-stimulated OA chondrocytes. Further, Wogonin exhibits potent chondroprotective potential by switching the signaling axis of matrix degradation from catabolic towards anabolic ends and inhibited the expression, production and activities of matrix degrading proteases including MMP-13, MMP-3, MMP-9, and ADAMTS-4 in OA chondrocytes, and blocked the release of s-GAG and COL2A1 in IL-1ß-stimulated OA cartilage explants. Wogonin also elevated the expression of cartilage anabolic factors COL2A1 and ACAN in chondrocytes and inhibited the IL-1ß-mediated depletion of COL2A1 and proteoglycan content in the matrix of cartilage explants. The suppressive effect of Wogonin was not mediated through the inhibition of MAPKs or NF-κB activation. Instead, Wogonin induced mild oxidative stress through the generation of ROS and depletion of cellular GSH, thereby modulating the cellular redox leading to the induction of Nrf2/ARE pathways through activation of ROS/ERK/Nrf2/HO-1-SOD2-NQO1-GCLC signaling axis in OA chondrocytes. Molecular docking studies revealed that Wogonin can disrupt KEAP-1/Nrf-2 interaction by directly blocking the binding site of Nrf-2 in the KEAP-1 protein. Genetic ablation of Nrf2 using specific siRNA, significantly abrogated the anti-inflammatory and chondroprotective potential of Wogonin in IL-1ß-stimulated OA chondrocytes. Our data indicates that Wogonin exerts chondroprotective effects through the suppression of molecular events involved in oxidative stress, inflammation and matrix degradation in OA chondrocytes and cartilage explants. The study provides novel insights into the development of Nrf2 as a promising candidate and Wogonin as a therapeutic agent for the management of OA.

Sucrose, But Not Glucose, Blocks IL1-ß-Induced Inflammatory Response in Human Chondrocytes by Inducing Autophagy via AKT/mTOR Pathway.

Pathogenesis of osteoarthritis (OA) is multifactorial but interleukin-1ß (IL-1ß) is known to be an important mediator of cartilage degradation. Autophagy is an essential cellular homeostasis mechanism and has been proposed to protect against cartilage degradation and chondrocyte death under pathological conditions. We investigated the role of autophagy activated by sucrose, a natural disaccharide, in suppressing inflammatory mediator's expression and cell death under pathological conditions in human chondrocytes. Autophagy activation was investigated by Western blotting for LC3 and Beclin-1, immunofluorescence staining for LC3 puncta, and measuring autophagic flux. Activation of mTOR, AKT, and P70S6K was evaluated by Western blotting. Chondrocyte apoptosis was evaluated by propidium iodide (PI) staining using flowcytometry, expression of Bax by Western blotting, gene expression by TaqMan assays and caspase 3/7 activity was measured using a luminescence-based assay. We found that sucrose-induced active autophagy in OA chondrocytes in vitro was dependent on the activation of AKT/mTOR/P70S6K signaling pathways but was independent of reactive oxygen species (ROS) production. Sucrose activated autophagy blocked IL-1ß-induced apoptosis and mRNA expression of MMP-13, COX-2, and IL-6 in human OA chondrocytes. Glucose or fructose, the two metabolites of sucrose, failed to induce autophagy indicating that autophagy was specifically mediated by sucrose. In conclusion, sucrose attenuated IL-1ß induced apoptosis and the expression of catabolic mediators by inducing autophagy, and the autophagy in part was mediated through the activation of AKT/mTOR/P70S6K signaling pathway in human OA chondrocytes. J. Cell. Biochem. 118: 629-639, 2017. © 2016 Wiley Periodicals, Inc.

Interleukin-1ß induced Stress Granules Sequester COX-2 mRNA and Regulates its Stability and Translation in Human OA Chondrocytes.

Enhanced and immediate expression of cyclooxygenase-2 (COX-2) mRNA is observed in IL-1ß-stimulated OA chondrocytes but the synthesis of protein found significantly delayed. Here we investigated the role of stress granules (SGs), ribonucleoprotein complexes that regulate mRNA translation, in the delayed translation of COX-2 mRNAs in IL-1ß-stimulated OA chondrocytes. Stimulation of human chondrocytes with IL-1ß activated the stress response genes and the phosphorylation of eIF2α that triggered the assembly of SGs. Using combined immunofluorescence staining of SGs markers and COX-2 protein, RNA fluorescence in situ hybridization and RNA immunoprecipitation, the COX-2 mRNAs were found sequestered in SGs in IL-1ß-stimulated OA chondrocytes. No increase in COX-2 protein expression was observed during the persistence of SGs but enhanced expression of COX-2 protein was noted upon clearance of the SGs. Inhibition of SGs clearance blocked COX-2 mRNA translation whereas blocking the assembly of SGs by TIA-1 depletion resulted in rapid and increased production of COX-2 and PGE2. Our findings show for the first time assembly of SGs and sequestration of COX-2 mRNAs in human OA chondrocytes under pathological conditions. Post-transcriptional regulation of COX-2 mRNAs translation by SGs indicates a role in IL-1ß-mediated catabolic response that could be therapeutically targeted in OA.