Biochimica et Biophysica Acta 1823 (2012) 2179–2189

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Biochimica et Biophysica Acta

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Endoplasmic reticulum stress induces the expression of COX-2 through activation ofeIF2α, p38-MAPK and NF-κB in advanced glycation end products stimulatedhuman chondrocytes

Zafar Rasheed ⁎, Tariq M. HaqqiDepartment of Medicine, Division of Rheumatology, MetroHealth Medical Center, 2500 Metro Health Drive, Cleveland, OH 44109, United States

Abbreviations: ER stress, endoplasmic reticulum stadvanced glycation end products; RAGE, receptor for Aprotein‐78 (bip); Bag‐1, Bcl‐2 associated athanogenefactor α‐2; COX‐2, cyclooxygenase‐2; PGE2, prostaglandactivated protein kinase; NF‐κB, nuclear factor‐κb⁎ Corresponding author. Present address: College of M

Saudi Arabia. Tel.: +966 531269227.E-mail address: zafarrasheed@rocketmail.com (Z. Ra

0167-4889/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bbamcr.2012.08.021

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 21 May 2012Received in revised form 11 August 2012Accepted 29 August 2012Available online 6 September 2012

Keywords:ER stressChondrocyteOAeIF2-αp38-MAPKNF-κB

Introduction: During aging, advanced glycation end products (AGEs) accumulate in articular cartilage. In this studywe determined whether AGEs induce endoplasmic reticulum (ER) stress and studied the ER stress-activated path-ways that stimulate cyclooxygenase-2 (COX-2) expression in human chondrocytes.Methods: Chondrocytes were stimulated with AGE-BSA. Gene expression was determined by quantitative PCR andprotein expression was studied by immunoblotting. Studies to elucidate involved pathways were executed usingsiRNAs and specific inhibitors of eukaryotic initiation factor-2α (eIF2α), MAPKs and NF-κB.Results: AGE-BSA induced expression of GRP78 with concomitant increase in COX-2 expression was observed inhuman chondrocytes. In addition, expression of Bag-1, an ER stress marker was also increased by AGE-BSA.RAGE knockdown inhibited AGE-BSA-induced expression of GRP78 and COX-2. Treatment with eIF2α inhibitoror eIF2α knockdown inhibited AGE-BSA-induced expression of GRP78 and COX-2 with decreased PGE2 produc-tion. Treatment with SB202190 inhibited AGE-BSA-induced expression of GRP78 and COX-2, while treatmentwith PD98051 inhibited AGE-BSA-induced GRP78 protein expression but had no effect on COX-2 protein expres-

sion. SP600125 had no effect on either GRP78 or COX-2 protein expression. Bay 11-7082 suppressedAGE-BSA-induced GRP78 and COX-2 expression. AGE-BSA-induced activation of NF-κB was inhibited by treat-ment with SB202190 and by eIF2α knockdown, but was not inhibited when chondrocytes were treated withSP600125 or PD98059.Conclusion: This study demonstrates that AGEs induce ER stress and stimulate the expression of COX-2 througheIF2α, p38-MAPK and NF-κB pathways in human chondrocytes. Our results provide important insights into car-tilage degradation in osteoarthritis associated with latent ER stress.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

In the endoplasmic reticulum (ER), secretory and transmembraneproteins fold into their native conformation andundergo posttranslation-al modifications important for their activity and structure [1]. Interfer-ence with proper protein folding activity results in the accumulation ofmisfolded proteins in ER, a phenomenon commonly referred to as ERstress [1,2]. To help proteins fold properly, ER contains a master molecu-lar chaperone GRP78 (BiP). When protein folding is disturbed, GRP78synthesis is increased and GRP78 subsequently binds to misfolded pro-teins to prevent them from forming aggregates and assists them to refoldproperly [3]. Bag-1 has recently been shown to be a downstream

ress; OA, osteoarthritis; AGEs,GEs; GRP78, glucose regulated1;eIF2‐α, eukaryotic initiationin E2; p38‐MAPK, p38‐mitogen

edicine, Qassim University,

sheed).

rights reserved.

regulator of ER stress in chondrocytes [4]. It is now well establishedthat overexpression of Bag-1 protects chondrocytes from ER stress [4,5].Therefore elevated expressions of GRP78 and Bag-1 are considered tobe reliable markers of ER stress [3,4]. The ER stress response is suggestedto contribute in several human diseases, including osteoarthritis (OA)and rheumatoid arthritis (RA) [2].

OA, the most common forms of arthritis, is a progressive degener-ative joint disease that has a major impact on joint function and thepatient's quality of life [6]. The most important risk factor for OA be-sides female sex, obesity, and joint trauma is aging [6,7]. However,it is relatively unknown how aging contributes to the onset and pro-gression of OA. Advanced glycation end products (AGEs) are pro-duced by the non-enzymatic glycation of macromolecules andaccumulate in a number of different tissues [8]. In articular cartilage,accumulation of AGEs makes the collagen network brittle thus in-creasing the risk for the development of OA [8,9]. The biological activ-ities of AGEs are thought to be mediated by specific receptors for AGE(RAGE) and activation of RAGE engages critical signaling pathwayslinked to pro-inflammatory responses and activation of various in-flammatory genes [10]. Chondrocytes are secretory cells and are the

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only resident cell type found in cartilage which are responsible forsynthesis and turnover of extracellular matrix (ECM) [11]. In OA, ac-tivation of RAGE has been shown to stimulate chondrocytes, resultingin increased production of cartilage degrading enzymes through theactivation of intracellular signaling pathways comprising of MAPKS,and NF-κB [12,13]. Despite studies on AGEs induced cartilage destruc-tion in OA, ER stress related cartilage degradation remains nebulous.

Signal transduction from theER to the cell nucleus could bemediatedby a signaling cascade similar to theplasmamembrane-initiated cell sig-naling. ER stress, for example, is coupled to the activation of stress-activated protein kinases such as MAP kinases [15,16]. Members of theMAPK family phosphorylate a number of transcription factors includingNF-κB, with subsequent induction of inflammatory genes expres-sion [14]. p38-MAPK, in particular, is activated by a diverse array of ERstress inducing agents such as tunicamycin [15] and its activation is as-sociatedwith inflammation and cartilage and bone destruction [17] as itregulates cytokine production through a variety of transcriptional andtranslational mechanisms [18]. In a rodent model of inflammatory ar-thritis, p38-MAPK inhibition suppressed the inflammation and bone de-struction [19]. Phosphorylation of the subunit of eukaryotic initiationfactor-2 (eIF2) is another important mechanism involved in the regula-tion of protein synthesis during ER stress [extensively reviewed in [20]and is required for the activation of NF-κB [21]. Activation of NF-κB byER stress leads to induction of many cellular genes that are largelyanti-apoptotic in function [22]. Now, it is well established that underthe influence of inflammation, COX-2 is upregulated, which results in adisturbed cartilage matrix turnover [23–25]. It is also reported that se-lective COX-2 inhibition prevents proinflammatory cytokine-inducedcartilage damage [23,24]. Furthermore, over expression of COX-2 me-diated PGE2 production has been found in OA and was linked to OAprogression and drug resistance [25,26]. Chondrocytes are thoughtto be the major source of PGE2 production in OA joints and suppres-sion of COX-2 expression and PGE2 production was found to bechondroprotective [25].

Although AGEs have been shown to induce the inflammatory re-sponse via activation of NF-κB andMAPKs in various cell types includingchondrocytes, but whether they induce the ER stress in chondrocyteshas not yet been reported. In this report, we demonstrate that AGEs in-duce ER stress which leads to increased expression of COX-2 in humanchondrocytes. Our results also showed that in human chondrocyteswith ER stress induction of COX-2 was mediated through eIF2α,p38-MAPK and NF-κB pathways.

2. Patients, materials and methods

2.1. Cartilage selection and chondrocytes preparation

With Institutional Review Board (IRB) approval, discarded cartilagesamples were obtained from the knee or hip joints of 44 OA patientsaged 38–69 years (32 female and 12 male Caucasian; mean age, 48±26 years) who underwent joint replacement surgery at MetrohealthMedical Center, Cleveland, OH. The macroscopic cartilage degenerationwas determined by staining of femoral head samples with India ink[27], and the cartilage with smooth articular surface (unaffected carti-lage)was resected andused to prepare chondrocytes by enzymatic diges-tion as previously described [14,28,29]. Histological analysis of theunaffected cartilage samples was performed on 5-μm-thick sectionsstained with H & E and Safranin O and graded using the Mankin score[30]. Grading of the histology slides revealed that all of the cartilagepieces taken from the unaffected area had a Mankin score b2 for struc-ture and a Mankin score of 1 for cellularity. Normal cartilage sampleswere obtained from trauma patients (n=2) with no known history ofOA or RA. Isolated chondrocytes were plated at a density of 1.2×106/mlin 35-mm tissue culture dishes (0.125×106 cells/cm2) in completeDMEM/Ham's F-12medium as previously described [14,28]. Only prima-ry human OA chondrocytes were used in studies described here.

2.2. AGE-BSA preparation

AGE-BSA was prepared by reacting BSA (Sigma Chemical Co., StLouis, MO, USA) with glycoaldehyde (Sigma), according to the meth-od described by Valencia and colleagues [31] with slight modifica-tions. Briefly, endotoxin-free BSA (2 mg/ml) was incubated undernonreducing conditions with 70 mM fresh glycoaldehyde in PBS(pH 7.4) without calcium chloride and magnesium chloride for3 days at 37 °C. The reaction was terminated by removing nonreactedglycoaldehyde by dialyzing extensively against PBS.

Endotoxicity of glycoaldehyde was also tested on chondrocytes byincubating with freshly prepared glycoaldehyde (1–50 μM) and wasfound to be non-toxic for tested chondrocytes.

2.3. Treatment of chondrocytes with AGE-BSA and inhibitors for MAPKs,eIF2α and NF-κB

Human OA chondrocytes (1.2×106/ml) were plated in 35 mm cul-ture dishes (0.125×106 cells/cm2) in complete DMEM/Hams-F12medi-um and serum-starved for 12 h/overnight and then treated with5–100 μg/ml of AGE-BSA for 0–24 h. Primary chondrocyteswere treatedwith MAPKs, eIF2α or NF-κB inhibitors for 1 h prior to stimulation withAGE-BSA. Chondrocytes cultured without AGE-BSA or cultured with na-tive BSA served as controls. Inhibitors used were SB202190 (catalog#1264, R&D Systems Inc., MN), SP600125 (catalog #40119, Calbiochem,MA); PD98059 (catalog #513000, Calbiochem); 2 Aminopurine (catalog#A3509, Sigma, St. Louis, MO), Bay 11–7082 (catalog #B5556, Sigma).The concentration of SB202190, SP600125, PD98059, 2 Aminopurine(2AP), Bay 11–7082 used in the study was 100 μM, 10 μM, 50 μM,10 μM, 10 μM, respectively.

2.4. Knockdown of RAGE and eIF2α by transfection with small interferingRNA (siRNA)

Human OA chondrocytes were transfected with human RAGE-specific siRNA (On-Target SMARTPool, catalog#L00362500, DharmaconRNA Technologies, Lafayette, CO), eIF2α-specific siRNA (catalog #sc-35272, Santa Cruz Biotechnology, CA) or with a negative humanGAPDH control siRNA (catalog #D-0018301020, Dharmacon) usingAmaxa Human Chondrocytes Nucleofector Kit (catalogue #VPF-1001, Lonza, Walkersville, MD). Transfection efficiency was monitoredwith red fluorescent siRNA oligonucleotides (siGLO red indicator,catalogue #D0016300220, Dharmacon). Approximately 70–80% of thechondrocytes emitted red fluorescence signal when transfected withsiGLO.

2.5. Cell viability assays

Human OA chondrocytes viability after nucleofection was exam-ined using the CytoTox-Glo™ Luminescent Cell Viability Assay Kit(Promega, Madison, WI). The viability of the transfected cell was70% in comparison to non-transfected cells. Whereas AGE-BSA or in-hibitors used in the study have no cytotoxic effect on human OAchondrocytes.

2.6. Real time-PCR

Real time quantitative polymerase chain reaction (qRT-PCR) wasused to quantify the expression of mRNAs for GRP78 and COX-2with expression of GAPDH as endogenous control. Primers used forPCR assisted amplification were listed in Table 1. PCR amplificationwas carried out using the core kit for SYBR Green or Taqman (AppliedBiosystem, Foster City, CA) and the Step One Real Time PCR System(Applied Biosystems). Typical profile times used were initial step,95 °C for 10 minutes, followed by a second step at 95 °C for 15 secand 60 °C for 60 sec for 40 cycles with melting curve analysis. The

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level of target mRNA was normalized to the level of GAPDH and com-pared to control (untreated sample). Data was analyzed using com-parative ΔΔCT method [32].

2.7. Western immunoblotting

Stimulated and control human OA chondrocytes were washedwith cold PBS and lysed using the cell lysis RIPA buffer (50 mMTris:HCl, pH 7.5; 150 mM NaCl; 1% IGEPAL, 4 mM EDTA, 0.1% sodiumdeoxycholate; 10 mM Na4P2O7, 10 mM NaF, 2 mM Na3VO4, 1 mMPMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin). Cytoplasmic and nuclearfractions were prepared as previously described [33]. Blots were ana-lyzed as described previously [33,34] and images were captured usingAFP-Imaging System (Minimedical Series, Elms Ford, NY).

2.8. Enzyme-linked immunosorbent assay (ELISA)

Human OA chondrocytes were stimulated with or withoutAGE-BSA (100 μg/ml) for 0–24 h. PGE2 produced in the culture medi-um was quantified using commercially available ELISA kit (CaymanChemical Company, Ann Arbor, MI). Plates were read using SynergyHT microplate reader (Biotek Instrument, Winooski, VT).

2.9. NF-κB activity assays

Activation of NF-κBp65 in AGE-BSA stimulated human OAchondrocyteswas determined using a highly sensitive Transcription Fac-tor ELISAKit (AssayDesigns, AnnArbor,MI). Activation of NF-κBp50 andRel B in AGE-BSA stimulated chondrocytes was determined by Westernimmunoblotting using specific antibodies against NF-κBp50 and Rel B(Cell signaling technology, Beverly, MA). Upregulation of NF-κBp50 byAGE-BSA was further verified by Sandwich ELISA established in our lab-oratory. Briefly, flat bottomed 96-well microtiter plates (polystryrenepolysorp immunoplates, Nunc-Immuno™ MicroWell, Sigma) werecoated with anti-NF-κBp50 (N-19) polyclonal antibodies (catalog#sc-1191, Santa Cruz Biotechnology, CA; diluted 1:100 in coating buffer)overnight at 4 °C. The plates were washed with TBS-Tween 20 and thenon-specific binding sites were blocked with TBS containing 1% BSA(Sigma) at room temperature for 1 h. After washing extensively withTBS-Tween 20, 100 μl of nuclear cell extracts (10 μg/ml) were added toduplicate wells of the coated plate and incubated at room temperaturefor 2 h and overnight at 4 °C. The plates were washed five times withTBS-Tween 20 and 100 μl of anti-NF-κBp50 (D-6)monoclonal IgG (cata-log #sc-166588, Santa Cruz Biotechnology; diluted 1:100)was incubatedat room temperature for 2 h. The plates were washed extensively and100 μl of anti-human IgG-horseradish peroxidase (catalog #sc2769,Santa Cruz Biotechnology)was added and incubated for 2 h. Afterwash-ing, 100 μl of 3,3′,5,5′-Tetramethylbenzidine peroxidase substrate (TMB,

Table 1Sequences of primers used in gene expression studies.

Gene Accessionnumber

Forward primer Backward primer

GRP78 NM_005347 5′-TCC TGC GTC GGC GTGT-3′

5′-GTT GCC CTG ATC GTTGGC-3′

COX-2 NM_000963 5′-CAA ATC CTT GCT GTTCCC ACC CAT-3′

5′-GTG CAC TGT GTT TGGAGT GGG TTT-3′

GAPDH NM_002046 5′-TCG ACA GTC AGC CGCATC TTC TTT-3′

5′-ACC AAA TCC GTT GACTCC GAC CTT-3′

COL2A1 NM_001844 5′-ACG TGA AAG ACT GCCTCA GC-3′

5′-TTT CAT CAA ATC CTCCAG CC-3′

COL10A1 NM_000493 5′-TGA TCC TGG AGT TGGAGG AC-3′

5′-GAG ATC GAT GAT GGCACT CC-3′

ACAN NM_013227 5′-GAC CTG CAA GGA GACAGA GG-3′

5′-CCA CTG GTA GTC CTTGGG CAT-3′

SOX-9 NM_011448 5′-GAT TTT TCA CGC AGCCCT AA-3′

5′-ATA CAG TCC AGG CAGACC CA-3′

catalog #206697A, Santa Cruz Biotechnology) was added to each well.The reactionwas stopped after 10 minutes by adding 100 μl of stop solu-tion (2 M H2SO4) and OD was read at 450 nm on an automaticmicroplate reader (Anthos Zenyth 3100 Multimode Detectors, Salzburg,Austria).

2.10. Statistical analysis

All measurements were performed in triplicates and repeated atleast three times using chondrocytes prepared from different OA pa-tients. Data sets were analyzed by analysis of variance followed byTukey's post hoc multiple comparison tests using Graph Pad Prismversion 5.0 (Graph Pad Software Inc., San Diego, CA) or Origin soft-ware package version 5.0 (Origin Lab Corporation, Northampton,MA). A p value of b0.05 was considered statistically significant. Valuesshown are mean with 95% CI unless stated otherwise.

3. Results

3.1. Human chondrocytes in monolayers maintain their chondrogenicphenotype

We determined whether primary human chondrocytes used inthese studiesmaintained their phenotype - by analyzing the expressionof COL2A1, ACAN, and SOX-9 mRNA, which are considered to be signa-tures of the chondrogenic phenotype [35]. PCR primers are listed inTable 1. Our results show that primary human chondrocytes in mono-layer culture maintained their phenotype, when they were plated(1.2×106/ml) in 35-mm culture dishes (0.125×106 cells/cm2) in com-plete DMEM with 10% FBS and were allowed to grow at 37 °C and 5%CO2 in a tissue culture incubator, as judged by the continued expres-sion of COL2A1, ACAN, and SOX-9 mRNAs, whereas COL10A1 mRNAwas not expressed (data not given). Based on these data, humanchondrocytes were used within 72 h after plating to avoid possiblededifferentiation.

3.2. AGE-BSA induce ER stress and the expression of COX-2 in humanchondrocytes

This study comprised of 44 OA patients, out of which 24 OA pa-tients primary chondrocytes showed up-regulation of mRNA and pro-tein expression of ER stress marker GRP78, and COX-2 upon AGE-BSAstimulation (pb0.05, Fig. 1A). Treatment of OA chondrocytes with awell known ER stress inducer drug tunicamycin (TM), used as a pos-itive control for ER stress, also increased the gene/protein expressionof GRP78 and COX-2 (Fig. 1B). Similarly, enhanced mRNA and proteinexpression of COX-2 by AGE-BSA or by TM, subsequently increasedthe production of PGE2 in the culture medium (Fig. 1A, B). To furtherstrengthen that AGE-BSA induced ER stress, we estimated the proteinexpression of another ER stress marker protein Bag-1 [4,5] inAGE-BSA stimulated human OA chondrocytes. Our results showedthat AGE-BSA time dependently induced the protein expression ofBag-1 (p50/p36) in human OA chondrocytes (Fig. 1C). These findingwere further re-confirmed by well known ER stress inducertunicamycin. Tunicamycin treated human OA chondrocytes also in-creased the protein expression of Bag-1 (Fig. 1C). Similarly, humanOA chondrocytes were treated with native BSA (nBSA) and expres-sion of GRP78, Bag1, COX-2 was determined. Our data showed thatnBSA was not involved in up-regulation of GRP78, Bag1, COX-2 orPGE2 production in human OA chondrocytes (Fig. 1D). We have alsotested the role of AGE-BSA or nBSA on chondrocytes obtained fromnormal cartilage. Normal human chondrocytes were stimulatedwith either AGE-BSA or nBSA (100 μg/ml) for 0–24 h and levels ofGRP78, Bag1 and COX-2 was quantified by immunoblotting and com-pared with the levels in untreated normal chondrocytes. Our resultsshowed that AGE-BSA increased the protein expression of GRP78,

Fig. 1. Elevated expression of GRP-78, Bag-1 and COX-2 in AGE-BSA stimulated human chondrocytes. (A) Human OA chondrocytes (70–80% confluent) were treated with AGE-BSA(100 μg/ml) for 0, 1, 6, 12 and 24 h. Gene expression of GRP78 and COX-2 was determined by real time quantitative PCR and normalized to GAPDH and compared to the levelspresent in untreated OA chondrocytes using comparative ΔΔCT method. Cell lyastes were analyzed by immunoblotting with antibodies specific for GRP78 (cat. #3183, Cell SignalingTech), COX-2 (cat. #4842, Cell Signaling Tech) and β-actin (cat. #sc-47778, Santa Cruz Biotech). β-actin was used as a loading control. Production of PGE2 in the culture mediumwas quantified by ELISA. (B) Human OA chondrocytes were treated with tunicamycin, TM (2.5 μM) for 0, 1, 6, 12 and 24 h. Gene or protein expressions were determined asdescribed in section A. (C) Bag-1 expression in human OA chondrocytes stimulated by AGE-BSA or TM. Cell lyastes were analyzed by immunoblotting with antibodies specificfor Bag-1 (cat. #sc-135844, Santa Cruz Biotech). (D) Human OA chondrocytes were treated with native (n)BSA (100 μg/ml) for 0, 1, 6, 12 and 24 h. Gene or protein expressionswere determined as described in section A. Western blots are from single chondrocytes sample, but are representative of three chondrocytes samples. Bars show the mean with95% confidence interval (CI) results of three independent assays, each of which run in triplicate experiments. *pb0.05 versus control; #pb0.0001 versus control.

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Bag1, COX-2 or PGE2 production in a time dependent manner(Fig. 2A). Treatment of normal human chondrocytes with nBSA forthe same time points showed no increase in GRP78, Bag1 andCOX-2 expressions or PGE2 production (Fig. 2B).

3.3. Necessity of RAGE for AGE-BSA stimulation of COX-2 during ER stressin human chondrocytes

To determine whether AGE-BSA-induced expression of GRP78 andCOX-2 in primary human OA chondrocytes was mediated via bindingto RAGE, we used siRNA-mediated knockdown of RAGE expression inOA chondrocytes. Transfection of human OA chondrocytes withRAGE-specific siRNAs significantly knocked down the expression ofRAGE and inhibited the AGE-BSA-induced GRP78 and COX-2 expres-sion and PGE2 production (Fig. 3A and B). Taken together these

findings provide strong evidence of the involvement of RAGE inAGE-BSA-mediated induction of ER stress and the expression ofCOX-2 in human OA chondrocytes.

3.4. Activation of eIF2α is required for ER stress stimulated expression ofCOX-2 in human OA chondrocytes

Treatment of primary human OA chondrocytes with AGE-BSA, in-duced eIF2α phosphorylation in a time-dependent manner, and themaximum increase over the basal levels was observed 24 h after thetreatment (Fig. 4A). To determine whether AGE-BSA-induced phos-phorylation of eIF2α is required for the elevated expression ofCOX-2, human chondrocytes were transfected with eIF2α-specificsiRNAs and then stimulated with AGE-BSA for 24 h. Total RNA wasisolated and then subjected to real time PCR. Our results showed

Fig. 2. Elevated expression of GRP78, Bag1 and COX-2 in AGE-BSA stimulated normal human chondrocytes. Normal human chondrocytes (70–80% confluent) were treated with AGE-BSA(100 μg/ml) (A) orwith nBSA (100 μg/ml) (B) for 0, 1, 6, 12 and24 h. Cell lyasteswere analyzed by immunoblottingwith antibodies specific for GRP78, COX-2.β-actinwas usedas a loadingcontrol. Production of PGE2 in the culturemediumwas quantified by ELISA. Normal chondrocytes were obtained from trauma patients. Immunoblots are from single chondrocytes sample,but are representative of two chondrocytes samples. Bars show themeanwith 95% CI results of two independent assays, eachofwhich run in triplicate experiments. *pb0.05 versus control;#pb0.0001 versus control.

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that eIF2α knockdown significantly inhibited the AGE-BSA-inducedmRNA expression of GRP78 and COX-2 (pb0.05; Fig. 4B). To deter-mine whether mRNA inhibition of GRP78 and COX-2 by eIF2α knock-down also affects the protein levels and the production of PGE2,

Fig. 3. AGE-BSA induced ER stress is mediated through RAGE in human chondrocytes. (A) RAOA chondrocytes. OA chondrocytes were transfected with RAGE-siRNA or control siRNA anddetermined as described under Fig. 1. (B) Protein expression of RAGE, GRP78 and COX-2 wasof PGE2 was quantified by ELISA. Immunoblots are from single chondrocytes sample, but arefive independent assays, each of which run in triplicate experiments. #pb0.05 versus ctransfected with control siRNA alone.

chondrocyte lysates were analyzed by Western immunoblotting forGRP78 and COX-2 proteins and cell culture supernatants wereassayed for PGE2 production using ELISA. As shown in Fig. 4C, knock-down of eIF2α significantly inhibited the AGE-BSA-induced protein

GE-specific siRNA transfection on AGE-BSA-induced expression of GRP78 and COX-2 inthen stimulated with AGE-BSA for 24 h. (A) Expression of GRP78 and COX-2 mRNA wasdetermined by Immunoblot analysis. β-actin was used as a loading control. Productionrepresentative of five chondrocytes samples. Bars show the mean with 95% CI results ofhondrocytes treated with AGE-BSA+control siRNA; *pb0.001 versus chondrocytes

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expression of GRP78 and COX-2 or production of PGE2 (pb0.05) in OAchondrocytes. Furthermore, treatment of human OA chondrocyteswith 2-aminopurine (2-AP), an inhibitor of eIF2α phosphorylation at-tenuated the AGE-BSA-induced mRNA and protein expression ofGRP78, COX-2 and PGE2 production (Fig. 4D and E). Taken together,these results clearly indicate that activating eIF2α is essential forthe AGE-BSA-induced ER stress and expression of COX-2 in humanOA chondrocytes.

3.5. MAPKs activation in human OA chondrocytes is required for the AGE-BSA-induced COX-2 expression during ER stress

As the expression of COX-2 is regulated through the MAPKs path-ways [15], we investigated whether these pathways played a role inthe induction of COX-2 during ER stress in AGEs stimulated OAchondrocytes. Primary human OA chondrocytes were treated withAGE-BSA for 0–24 h and the phosphorylation of p38-MAPK, JNK andERK was examined by Western immunoblotting. All three MAPKswere phosphorylated in AGE-BSA-stimulated OA chondrocytes butthe duration of phosphorylation was different with phosphorylatedJNK being detectable for 1 h, p38-MAPK for 15 minutes while ERKwas found to be phosphorylated at all the time points analyzed(Fig. 5A). To determine the relation of MAPKs p38, JNK, ERK activationand GRP78 or COX-2 expressions in AGE-BSA-stimulated human OAchondrocytes, we used pharmacological agents that inhibit activationof p38-MAPK (SB202190), JNK (SP600125) and ERK (PD89059). Our

Fig. 4. AGE-BSA-induced stimulation of GRP78 and COX-2 ismediated throughα subunit of eIFindicated times, the phosphorylation of eIF2αwas determined by immunobloting. (B) eIF2α-spOA chondrocytes. (C) eIF2α-specific siRNA transfection andprotein expression ofGRP78 and COgene expression of GRP78 and COX-2. OA chondrocytes were pretreated with 2AP (10 μM) for 2and COX-2 or PGE2 production. OA chondrocytes were pretreated with 2AP for 2 h and stimuchondrocytes sample, but are representative of three chondrocytes samples. Bars show themeaperiments. *pb0.001 versus chondrocytes treatedwith AGE-BSA+control siRNA; #pb0.05 versuAGE-BSA alone; $pb0.0001 versus untreated chondrocytes.

results showed that pretreatment with SB202190 caused significantdecrease in mRNA and protein expression of GRP78 (pb0.05) but incontrast, treatment with the PD98059 and SP600125 had no signifi-cant effect on AGE-BSA induced GRP78 mRNA or protein expressionscompared to controls (Fig. 5A and B). Expression of COX-2 mRNA orprotein and PGE2 production in the culture medium was significantlyinhibited with SB202190 treatment (pb0.05), whereas treatmentwith SP600125 and PD98059 had no effect on AGE-BSA-inducedCOX-2 expression and PGE2 production in AGE-BSA-stimulated OAchondrocytes (pb0.05; Fig. 5 B&C). Taken together, our results dem-onstrated that p38-MAPK is the only MAPK involved in AGE-BSA-induced induction of ER stress characterized by the expression ofGRP78 and COX-2 in OA chondrocytes (Fig. 5B and C).

3.6. Induction of NF-κB is dependent on the activation of p38-MAPK andeIF2α in AGE-BSA-stimulated human OA chondrocytes

To evaluate the mechanism of AGE-BSA induced expression ofCOX-2 in OA chondrocytes with ER stress, NF-κB activation was stud-ied in detailed. Stimulation of OA chondrocytes with AGE-BSA in-duced the degradation of cytoplasmic IκBα and increased thenuclear level of NF-κB subunits p65 and Rel-B (Fig. 6A). Our resultsalso showed that after 5 h of AGE-BSA treatment, the level of nuclearNF-κBp50 was enhanced (Fig. 6A). These observations were furtherverified by NF-κBp50 specific ELISA and the results are shown inFig. 6D. The co-relation of NF-κB activation and the expression of

2 in human chondrocytes. (A) Human OA chondrocytes were treatedwith AGE-BSA for theecific siRNA transfection and gene expression of GRP78 and COX-2 in AGE-BSA-stimulatedX-2 in AGE-BSA-stimulated OA chondrocytes. (D) eIF2α-phosphorylation inhibitor 2AP andh and stimulatedwith AGE-BSA for 24 h. (E) 2AP and protein expression of eIF2α, GRP78

lated with AGE-BSA for 24 h. See Fig. 1 for details. Western immunoblots are from singlen with 95% CI results of three independent experiments, each of which run in triplicate ex-s chondrocytes transfectedwith control siRNA; **pb0.05 versus chondrocytes treatedwith

Fig. 5. AGE-BSA-induced activation of p38-MAPKs is essential for COX-2 induction during ER stress in human chondrocytes. (A) Human OA chondrocytes were treated withAGE-BSA for the times indicated and the activation of MAPKs was determined by Immunoblot analysis. GRP78 and COX-2 were indicators for ER stress. (B) Effect of specific inhib-itors for MAPKs on the gene expression of GRP78 and COX-2 in AGE-BSA-stimulated OA chondrocytes. (C) Effect of specific inhibitors for MAPKs on the protein expression of GRP78and COX-2, and production of PGE2 in AGE-BSA-stimulated OA chondrocytes. OA chondrocytes were pretreated with inhibitors of p38 (SB202190), JNK (SP600125) and ERK(PD98059) for 1 h and then stimulated with AGE-BSA for 24 h. The concentration of SB202190, SP600125 and PD98059 used in these studies was 100 μM, 10 μM and 50 μM, re-spectively. Western immunoblots are from single chondrocytes sample, but are representative of five chondrocytes samples. Bars show the mean with 95% CI results of five inde-pendent assays, each of which run in triplicate experiments. *pb0.001 versus chondrocytes treated with AGE-BSA alone; #pb0.0001 versus untreated chondrocytes; $p>0.05versus chondrocytes treated with AGE-BSA alone.

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GRP78 and COX-2 in our studies, were investigated by the use ofpharmacological agent, Bay 11‐7082, a known inhibitor of NF-κB(IKKα/β). Treatment of primary human OA chondrocytes with Bay11‐7082, significantly blocked the AGE-BSA-induced gene/protein ex-pression of GRP78 or COX-2 (pb0.05) in human OA chondrocytes(Fig. 6B and C).

Phosphorylation of the α subunit of eIF2α is required for the activa-tion of NF-κB in response to ER stress [21]; therefore we examinedwhether inhibition of eIF2α-activation by 2-AP treatment or eIF2α-siRNA knockdown affects the activation of NF-κB subunits p50 or p65in AGE-BSA-stimulated human OA chondrocytes. Treatment of humanOA chondrocytes with 2-AP or knockdown of eIF2α expression usingspecific siRNAs significantly inhibited the activation andDNAbinding ac-tivity of NF-κBp65 (pb0.05) (Fig. 6E and F) and also inhibited the trans-location of nuclear NF-κBp50 (data not shown). Similarly, treatmentwith p38-MAPK inhibitor SB202190 caused a significant decrease inthe DNA binding activity of NF-κBp65 (Fig. 6F) in AGE-BSA stimulatedOA chondrocytes (pb0.05). In contrast, treatment with JNK inhibitorSP600125 or ERK inhibitor PD98059 had no significant effect onAGE-BSA induced activation of NF-κBp65 (Fig. 6F, p>0.05). Togetherthese results indicate that activation of NF-κB is dependent on the activa-tion of p38-MAPK and eIF2α during ER stress in AGE-BSA-stimulatedhuman OA chondrocytes.

3.7. eIF2α is required to activate the stress-activated p38-MAPK in OAchondrocytes

Transfection with eIF2α-specific siRNA (30 nM) significantlyinhibited the AGE-BSA-induced phosphorylation of p38-MAPK in OAchondrocytes (Fig. 6G), indicating that activation eIF2α is upstreamof p38-MAPK and is required for its activation during ER stress inAGE-BSA-stimulated OA chondrocytes.

4. Discussion

This report shows AGEs induce ER stress in human chondrocytes, afinding in agreement with previous reports showing that AGEs medi-ated ER stress in other cell types [36,37]. Iwawaki et al. [38] used atransgenic mouse model to monitor ER stress in vivo, and demon-strated that ER stress occurred in a variety of organs such as liver,spleen, brain, and kidney. However, little is known about the role ofER stress in cartilage but ER stress likely plays an important role incartilage biology as the expression of GRP78 and Bag-1, ER stressmarker proteins, were found to be upregulated several folds inhuman as well as in mouse model of arthritis [4,5,39,40]. In addition,recent reports suggest that multiple outcomes exist following induc-tion of ER stress in chondrocytes [2,17,40,41]. Despite these advances,additional studies are needed to fully understand the action of variousinducers of ER stress on chondrocytes and the consequences of ERstress.

Accumulation of AGEs in articular cartilage has been proposed as amechanism for the age-related development of OA [8–10]. Theage-related accumulation of AGEs results in crosslinking of cartilagematrix components and suggests a putative molecular mechanismfor the development of OA [9]. Therefore, the levels of AGEs mightpredict susceptibility for developing OA. In contrast, it is recentlyreported that In end stage osteoarthritis, cartilage tissue pentosidinelevels are inversely related to parameters of cartilage damage [42].This is likely due to the ongoing (ineffective) increased turnover ofcartilage matrix protein even in end stage disease. Moreover, eleva-tion of cartilage AGEs does not accelerate initiation of canine experi-mental OA upon mild surgical damage [43]. These data furthersupport the diminished cartilage turnover in OA, but only a tendencytowards enhanced cartilage damage in AGEs accumulated articularcartilage. However, in OA chondrocytes, elevated levels of AGEs may

Fig. 6. AGE-BSA-induced activation of NF-κB ismediated through eIF2α or p38-MAPK in human chondrocytes during ER stress. (A) OA chondrocytes were incubatedwith AGE-BSA for thetime indicated and NF-κB subunits were analyzed by Immunoblotting. PARP-1 and Paxillin were used as internal markers for nuclear and cytoplasmic proteins. (B–C) NF-κB inhibitor (BAY11‐7082) inhibited theAGE-BSA-inducedGRP78 andCOX-2. Chondrocyteswere pretreatedwith Bay 11‐7082 (10 μM) for 1 h and then stimulatedwith AGE-BSA for 24 h. (D)Activation ofNF-κBp50 was further verified by NF-κBp50 specific ELISA using nuclear extracts at indicated time intervals. (E) eIF2α-knockdown and NF-κBp65 activation in AGE-BSA-stimulated OAchondrocytes. eIF2α-knockdown chondrocytes were incubated with AGE-BSA for 45 minutes, and activated NF-κBp65 was determined by ELISA. (F) p38-MAPK and eIF2α inhibitorsinhibited the AGE-BSA-induced activation of NF-κBp65 in chondrocytes. Chondrocytes were pretreated with SB202190, SP600125 and PD98059, 2AP for 1 h and then stimulated withAGE-BSA for 45 minutes and activated NF-κBp65 was determined ELISA. TNF-α treated HeLa cell extract and Bay 11-7082/AGE-BSA treated chondrocytes extract were used as positivecontrols. (G) AGE-BSA-induced phosphorylation of p38-MAPKwas inhibited by eIF2α-knockdown. eIF2α-knockdown chondrocyteswere incubatedwith AGE-BSA for 45 minutes. Immu-noblots are from single chondrocytes sample, but are representative of five chondrocytes samples. Bars show themeanwith 95% CI results of five independent assays, each ofwhich run intriplicate experiments. *pb0.0001 versus chondrocytes treatedwith AGE-BSA alone; #pb0.0001 versus untreated chondrocytes; ##pb0.001 versus chondrocytes transfected with controlsiRNA alone; $pb0.05 versus chondrocytes treated with AGE-BSA+control siRNA; **p>0.05 versus chondrocytes treated with AGE-BSA alone.

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also trigger the activation of catabolic pathways throughRAGE (receptorfor AGEs) [10,12–14,26]. The activation of RAGE stimulates critical sig-naling pathways linked to the activation and expression of various in-flammatory genes [12–14,26]. Studies have shown that abnormallyhigh levels of COX-2-PGE2 are present in the synovial fluid, synoviumand cartilage tissues of arthritic patients [24,25]. In this study, using pri-mary human OA chondrocytes, we have demonstrated that in-vitro gen-erated AGE-BSA activates a RAGE-mediated signaling cascade thatresults in the induction of ER stress which leads to increased expressionof COX-2 and PGE2 production. We used AGE-BSA as a model for AGEs,which is basically a complex that includes N3-carboxymethyllysine(CML), pentosidine, and other AGEs, produced by reacting BSA withglycoaldehyde under sterile conditions, followed by extensive dialysisas previously described [31]. Several lines of evidence support the no-tion of an interaction of AGE-BSA with RAGE. Knockdown of RAGE bysiRNAs significantly inhibited the AGE-BSA‐induced GRP78 and COX-2

expression (pb0.05), confirming that AGE-BSA-induced ER stress relat-ed stimulation of COX-2 expression requires signaling through RAGE. ERstress and its associated affects on COX-2 expression arewell establishedin other cell types [15]. Tunicamycin is a well known ER stress inducerdrug [15,17]. Studies have shown that tunicamycin in various celltypes enhanced COX-2 expression [15,26]. In addition, similar stimula-tion of COX-2 expression was observed by another ER stressinducer brefeldin A [15]. These studies clearly indicate that COX-2 is apart of ER stress. Furthermore, ER stress induced by expression of hepa-titis B virus surface protein also stimulates the expression of COX-2in-vitro [15,22] and in-vivo [26]. These studies further support thatCOX-2 is a part of ER stress. In the present study, we have establisheda relation between ER stress and COX-2 in human chondrocytes forthe first time. Treatment of human chondrocytes with tunicamycin, sig-nificantly increased the expression of COX-2. This again indicating thatCOX-2 is a part of ER stress.

Fig. 7. Signaling of endoplasmic stress to COX-2 in AGEs stimulated human chondrocytes. AGEs through RAGE activates eIF2α which relays ER stress signals to the nuclear tran-scription factor NF-κB via activation of p38-MARK. Different NF-κB complexes were demonstrated by NF-κBp50, NF-κBp65, Rel B and by the proteasomal degradation of IκBα. In-termediates indicated in blue were not demonstrated in this report. Abbreviations: ER stress: endoplasmic reticulum stress; AGEs: advanced glycation end products; RAGE; receptorfor AGEs; eIF2-α: eukaryotic initiation factor α-2; p38-MAPK: p38-mitogen activated protein kinase; NF-κB; nuclear factor-Κb; COX-2: cyclooxygenase-2.

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ER stress can activate multiple signal transduction pathways includ-ing the eIF2α, MAPK and NF-κB [2,15–17]. These pathways maycross-talk with each other or converge on common downstream effec-tors [15,16]. Here we demonstrated that AGE-BSA-induced expressionof COX-2 and PGE2 production required the phosphorylation of eIF2αduring ER stress in human OA chondrocytes. These are novel findingsand have not been reported previously. In addition, we also demonstrat-ed that treatment of OA chondrocytes with SB202190 (p38 inhibitor)significantly down regulated the AGE-BSA-stimulated GRP78 andCOX-2 expression and PGE2 production. Whereas, PD98059 (ERK inhib-itor) or SP600125 (JNK inhibitor) had no effect on AGE-BSA-inducedGRP78 and COX-2 expressions and PGE2 production. But this is in con-trast to the previous study that showed all MAPK inhibitors (SB202190,SP600125, PD98059) inhibited AGE-BSA induction of COX-2 expressionand PGE2 production in human OA chondrocytes [26]. Although wecould not find how much inhibitors concentrations authors were usedin this study [26], but we cannot rule out of SP600126 or PD98059 inAGE-BSA stimulated expression of COX-2 and PGE2 production. But ourfindings in the present study demonstrate that AGE-BSA-stimulated ex-pression of COX-2 and PGE2 productionwasmediated via p38-MAPK ac-tivation in OA chondrocytes during ER stress.

NF-κB is a transcription factor that participates in immunity andinflammation [44]. NF-κB proteins include p65 (Rel A), Rel B, c-Rel,p50/p105 and p52/p100, which normally bind to IκB in the cytoplasmusually in three different complexes [18,44]. It is well documentedthat NF-κB is involved in AGE-mediated effects of RAGE signaling[10,12–14,26] and during ER stress the expression of COX-2 is depen-dent on the activation of NF-κB [15]. In order to gain further insightinto the mechanism, AGE-BSA induced IκBα degradation and nuclear

translocation of NF-κB p65, p50 and Rel B have been studies in this re-port (Fig. 6 and 7). Direct effect of NF-κB on COX-2 expression duringER stress was investigated using the NF-κB (IKKα/β) inhibitor Bay 11‐7082. Treatment of OA chondrocytes with Bay 11‐7082, significantlyblocked the AGE-BSA-induced gene/protein expression of GRP78 orCOX-2 (pb0.05) which showed that AGE-BSA-induced ER stress stim-ulate the expression of COX-2 via activation of NF-κB.

It has been reported that phosphorylation of eIF2α is required for theactivation of NF-κB in response to ER stress [21]; thereforewe examinedwhether inhibition of eIF2α-activation affects the activation of NF-κB inAGE-BSA-stimulated OA chondrocytes. Treatment of OA chondrocyteswith 2-aminopurine (2AP, an eIF2α inhibitor) or eIF2α-knockdown bysiRNAs significantly inhibited the activation and DNA binding activityof NF-κB (pb0.05) in AGE-BSA-stimulated OA chondrocytes. On theother hand, treatment with p38-MAPK inhibitor caused significant de-crease in the DNA binding activity of NF-κB in AGE-BSA stimulated OAchondrocytes (pb0.05). In contrast, treatment with either JNK inhibitoror ERK inhibitor had no significant effect on AGE-BSA induced activationof NF-κB. These data indicate that activation of p38-MAPK appears to beessential for the activation of NF-κB in AGE-BSA-stimulated OAchondrocytes. We also investigated, whether eIF2α is required for theAGE-BSA-induced activation of p38-MAPK during ER stress. eIF2αknockdown by eIF2α-specific siRNAs inhibited the AGE-BSA-inducedphosphorylation of p38-MAPK in OA chondrocytes, indicating thateIF2α is required for the ER stress-mediated activation of p38-MAPKin OA chondrocytes. All together these results suggest that activationof NF-κB is dependent on the phosphorylation of eIF2α and activationof p38-MAPK during ER stress in AGE-BSA-stimulated humanchondrocytes (data summarized in Fig. 7). In short, our novel data

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strongly suggest that AGE-BSA-induced ER stress stimulates COX-2 ex-pression and PGE2 production via activation of eIF2α, p38-MAPK, andNF-κB pathways in human chondrocytes.

5. Conclusions

This study demonstrates that AGEs stimulate ER stress via RAGE inhuman chondrocytes. Activation of RAGE by AGEs induce ER stressand trigger a cascade of signaling events, which includes phosphory-lation of eIF2α and p38-MAP kinase which in turn activates NF-κBand increases the COX-2 expression and PGE2 production. Taken to-gether, our data suggest that the stress induced by AGEs in the endo-plasmic reticulum activates multiple signaling pathways that mayplay an important role in the degeneration of cartilage in OA. Addi-tional work is needed to further define the role of AGEs induced ERstress in cartilage matrix turnover and to determine if RAGEmediated-ER stress inhibition could reduce the progression of carti-lage damage in arthritis.

Authors' contributions

ZR carried out the experimental work, data collection, interpreta-tion and manuscript drafting. TMH conceived of the study, its design,coordinated, data interpretation and manuscript drafting.

Conflict of interest statement

The authors declare that they have no conflict of interests.

Acknowledgments

This work was supported by the USPHS/NIH grants RO1-AT-003267,RO1-AT-005520, R21-AT504615 to TMH and financial support fromMetrohealth, Cleveland, Ohio is also gratefully acknowledged. We alsothank Dr. Brendan Patterson (Department of Orthopedics, MHHC) forproviding human cartilage samples.

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