Hypoxia Conditions Differentially Modulate Human Normal and

Apr 14, 2010 - Dr. Francisco J. Blanco, Unidad de Investigación del Envejecimiento Osteoarticular, INIBIC-Complejo Hospitalario Universitario A CoruÃ...
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Hypoxia Conditions Differentially Modulate Human Normal and Osteoarthritic Chondrocyte Proteomes Cristina Ruiz-Romero, Valentina Calamia, Beatriz Rocha, Jesu ´ s Mateos, Patricia Ferna´ndez-Puente, and Francisco J. Blanco* Unidad de Investigacio´n del Envejecimiento Osteoarticular, INIBIC-Complejo Hospitalario Universitario A Corun ˜ a, Spain Received December 29, 2009

Osteoarthritis (OA) is a degenerative disease characterized by the degradation of articular cartilage. This tissue is avascular, and it is characterized by the low oxygen tension and poor nutrient availability for its cells, the chondrocytes. Hypoxia conditions have been reported to stimulate chondrogenesis and synthesis of extracellular matrix components. Therefore, we aimed to analyze the effect of hypoxia on normal and osteoarthritic cartilage cell cultures by a proteomic approach based in Two-dimensional gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry protein identification. Twentyeight proteins were found to be modulated by hypoxia in normal chondrocytes and 11 in OA cells when compared to their normoxia controls. In both cases, a hypoxia-dependent decrease in metabolismrelated proteins was detected. We also identified 42 protein forms that were altered in OA chondrocytes under hypoxia when compared to normal cells. The upregulation of cyclophylin A (PPIA) and Tumor necrosis factor receptor associated protein 1 (TRAP1) was confirmed both in cultured chondrocytes and in cartilage tissue. Our work shows how hypoxia conditions induce diverse modifications in the proteomic profile of normal and OA human articular chondrocytes, which probably renders a different capacity of OA and normal cells to react under a hypoxic environment. Keywords: hypoxia • osteoarthritis • chondrocyte • proteomics

Introduction Osteoarthritis (OA) is the most common age-related joint disease,1 affecting about 15% of the world’s population.2 OA is characterized by degeneration of articular cartilage, intraarticular inflammation and changes in peri-articular and subchondral bone.3 Multiple factors are involved in the pathogenesis of OA, including mechanical influences, effects of aging on cartilage matrix composition and structure, and genetic factors.4 OA is already the primary cause of permanent work incapacity and is one of the most common motives for visiting primary care physicians. Moreover, this disease is becoming increasingly prevalent worldwide because of the combination of an aging population and growing levels of obesity. Therefore, interest in elucidating the complex OA pathology has greatly increased in recent years. The lack of information about its pathogenesis contributes to the difficulties in developing early diagnosis strategies and efficient treatments, which are currently limited to controlling pain and improving function and quality of life while limiting adverse events. The damaged tissue in OA, articular cartilage, is a connective tissue composed of a single type of cells called chondrocytes (2-5% from total tissue) and an extracellular matrix (ECM) mainly comprised of water, proteoglycans and type-II collagen * To whom correspondence should be addressed. Dr. Francisco J. Blanco, Unidad de Investigacio´n del Envejecimiento Osteoarticular, INIBIC-Complejo Hospitalario Universitario A Corun ˜ a, C/Xubias, 84, 15006-A Corun ˜ a, Spain. Tel: 34-981-178272. Fax: 34-981-178273. E-mail: [email protected]. 10.1021/pr901209s

 2010 American Chemical Society

fibers. The chondrocytes are essential in the control of matrix turnover through the production of structural proteins and enzymes for the metabolism of the tissue. Therefore, cultures of articular chondrocytes isolated from various animal and human sources have served as useful models for studying the mechanisms controlling responses to growth factors and cytokines, as well as to understand the pathogenesis of some rheumatic diseases such as OA.5 A specific characteristic of mature articular cartilage is its avascularity. As a result, oxygen and other nutrients must diffuse into the tissue from the synovial fluid surrounding the joint in order to reach the cells,6 thus creating oxygen and glucose gradients in cartilage that ultimately lead to significantly reduced levels of these compounds in cartilage deep layer than in other vascularised tissues. Microelectrode studies have determined an oxygen tension of around 6-10% in cartilage surface, but less than 1% in the deep zones.7,8 These chronic hypoxia conditions of cartilage do not compromise chondrocyte viability, as these cells appear to have developed specific mechanisms to promote tissue function under this environment.9 Therefore, conventional cell cultures under normoxia (with O2 tensions around 21%) expose chondrocytes to much higher oxygen levels than physiological conditions. One strategy that aims to mimic the in vivo cellular environment, which is gaining increasing interest in cartilage research, is culturing chondrocytes under hypoxia.10 Hypoxic culture of cartilage cells Journal of Proteome Research 2010, 9, 3035–3045 3035 Published on Web 04/14/2010

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has shown several advantages, such as increased redifferentiation of dedifferentiated chondrocytes11-13 and increased synthesis of ECM components.14 Moreover, this model would allow a better understanding of chondrocyte metabolism. Consistently with this, the aim of this paper was to investigate which proteins are modulated by hypoxia in normal and OA human chondrocyte cultures, and to explore the differential proteomic profile of OA and normal cells under hypoxic conditions.

5. Protein Sample Preparation. Chondrocytes were recovered from culture plates by trypsinization and washed twice with PBS. Cells were then transferred to microcentrifuge tubes and resedimented. The cell pellets were solubilized by vortexing followed by one-hour incubation with gentle agitation in 200 µL of an isolectric focusing-compatible lysis buffer containing 8.4 M urea, 2.4 M thiourea, 5% CHAPS, 1% carrier ampholytes (IPG buffer), 0.4% Triton X-100 and 2 mM dithiothreitol (DTT). Total chondrocytic proteins in each lysate were quantified using the Bradford protein assay.

Experimental Section

6. Two-Dimensional Gel Electrophoresis (2-DE) and Protein Staining. The 2-DE technique used in this study has been previously described.15 Briefly, protein extracts (100 µg of protein) were incubated with rehydration buffer for one hour with gentle agitation and then applied to 24 cm, pH 3-11 NL, IPG strips by passive overnight rehydration. Isoelectric focusing (IEF), was performed at 20 °C in an IPGphor instrument (GE Healthcare) for a total of 64000 Vhr. The second dimension was run on an Ettan DALT six system (GE Healthcare) after equilibration of the strips for 15 min in 6 M urea, 50 mM TrisHCl (pH 8.8), 30% glycerol, 2% SDS and 1% DTT (reduction step), and then 15 min in the same buffer with 4% iodoacetamide instead of DTT (alkylation step). The equilibrated strips were transferred onto 10% homogeneous polyacrylamide gels (2.6% C). Electrophoresis was run at 2 W/gel for about 17 h at 20 °C, using Laemmli buffers. Gels were fixed and stained overnight with SYPRORuby (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. After image acquisition and data analysis, 2-DE gels were stained either with Coomassie Brilliant Blue (CBB) or silver nitrate, following standard procedures.15 7. 2-DE Image Acquisition and Data Analysis. SYPROstained gels were digitized using a CCD camera (LAS 3000 imaging system, Fuji, Tokyo, Japan) equipped with a blue (470 nm) excitation source and a 605DF40 filter. CBB and silver stained gels were digitized with a densitometer (ImageScanner, GE Healthcare). Images from SYPRO-stained gels were analyzed with the PDQuest 7.3.1 computer software (Bio-Rad, Hercules, CA). Using PDQuest tools, the protein spots were enumerated, quantified and characterized by their molecular mass and isoelectric point by bilinear interpolation between landmark features on each image previously calibrated with internal 2-DE standards (Bio-Rad). Protein expression data from each gel were normalized for the total density present in the gel images. 8. Mass Spectrometry (MS) Analysis. The gel spots of interest were manually excised and transferred to microcentrifuge tubes. Samples selected for analysis were reduced ingel, alkylated and digested with trypsin according to the method of Sechi and Chait.16 Briefly, spots were washed twice with water, shrunk with 100% acetonitrile (ACN) and dried in a Savant SpeedVac. The samples were reduced with DTT, subsequently alkylated with iodoacetamide and then digested with 12.5 ng/µL sequencing-grade trypsin (Roche Molecular Biochemicals, IN) at 37 °C. After digestion, the supernatant was collected and 1 µL was spotted onto a Matrix Assisted Laser Desorption/Ionization (MALDI) target plate, and allowed to airdry at room temperature. When dried, 0.5 µL of a 3 mg/mL solution of R-cyano-4-hydroxy-trans-cinnamic acid matrix in 0.1% trifluoroacetic acid (TFA)-50% ACN was added to the dried peptide digest spots and again allowed to air-dry. The samples were analyzed using the MALDI-Time of Flight (TOF)/TOF mass spectrometer 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA) and 4000 Series Explorer Software

1. Reagents, Chemicals and Antibodies. Culture media and fetal calf serum (FCS) were from Gibco BRL (Paisley, U.K.). Culture flasks and plates were purchased from Costar (Cambridge, MA). Hypoxia experiments were carried out using GasPak anaerobic pouch systems (BD Biosciences, Erembodegem, Belgium). This system consists of a reagent sachet that, once activated and placed into a resealable pouch, reduces the oxygen concentration within the pouch down to less than 2% and produces carbon dioxide. An anaerobic indicator is also introduced into the pouch and reports the amount of oxygen present during the incubation. Two-dimensional electrophoresis (2-DE) materials (Immobilized pH Gradient (IPG) buffer, strips, etc.) were from GE Healthcare (Uppsala, Sweden). Unless indicated, all other chemicals and enzymes were obtained from Sigma-Aldrich (St. Louis, MO). 2. Cartilage Procurement and Processing. Macroscopically normal human knee cartilage from adult donors with no history of joint disease was provided by the Tissue Bank and the Autopsy Service at CHU A Corun ˜ a. Osteoarthritic cartilage was obtained from patients undergoing joint surgery. The study was approved by the institutional Ethics Committee. After the cartilage surfaces were rinsed with saline, a scalpel was used to cut parallel vertical sections 5 mm apart from the cartilage surface to the subchondral bone. These cartilage strips were cut from the bone and the tissue was incubated with typsinEDTA solution (0.5 mg/mL) for 10 min at 37 °C. After removing the trypsin solution, the cartilage slices were treated for 14-16 h with 2 mg/mL clostridial collagenase type IV in Dulbecco’s modified Eagle’s medium (DMEM) with 5% FCS to release cartilage cells. 3. Primary Culture of Chondrocytes. The chondrocytes were recovered and plated at high density (4 × 106 per 162cm2 flask) in DMEM supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, 1% glutamine and 10% FCS. The cells were then incubated at 37 °C in humidified 5% CO2 and air. At confluency, the cells were recovered from culture flasks by trypsinization and seeded onto 100 mm culture plates at 2 × 106 per plate for proteomic studies, or into 6-well plates at 5 × 105 per well for RNA/protein extraction. Chondrocytes were used during the second or third week of primary culture (P1) after rendering them quiescent by incubation in a medium containing 0.5% FCS for 24 h. These cells were then cultured in hypoxic bags (4% CO2) during 96 h, or under normoxia (20% O2 approx.) for the control experiments. Cell viability was assessed by trypan blue dye exclusion. 4. Biochemical Determinations in Chondrocyte Culture Media. Glucose and Lactate dehydrogenase (LDH) were measured in the culture media of chondrocytes using a Vitalab Flexor (Vital Scientific, Spankeren, The Netherlands) and Biochemistry Control Serum I and II as standards (BioSystems S.A., Barcelona, Spain). 3036

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Effect of Hypoxia on Human Articular Chondrocytes (Applied Biosystems). MALDI-TOF spectra were acquired in reflector positive-ion mode using 1000 laser shots per spectrum. Data Explorer version 4.2 (Applied Biosystems) was used for spectra analyses and generating peak-picking lists. All mass spectra were internally calibrated using autoproteolytic trypsin fragments and externally calibrated using a standard peptide mixture (Sigma-Aldrich). TOF/TOF fragmentation spectra were acquired by selecting the 10 most abundant ions of each MALDI-TOF peptide mass map (excluding trypsin autolytic peptides and other known background ions) using an average of 2000 laser shots per fragmentation spectrum. The parameters used to analyze the data were a signal-to-noise threshold of 20, a minimum area of 100 and a resolution higher than 10000 with a mass accuracy of 20 ppm. 9. Database Search. The monoisotopic peptide mass fingerprinting data obtained by MS and the amino acid sequence tag obtained from each peptide fragmentation in MS/MS analyses were used to search for protein candidates using Mascot version 1.9 from Matrix Science (http:// www.matrixscience.com). Peak intensity was used to select up to 50 peaks per spot for peptide mass fingerprinting, and 50 peaks per precursor for MS/MS identification. Tryptic autolytic fragments, keratin- and matrix-derived peaks were removed from the data set used for the database search. The searches for peptide mass fingerprints and tandem MS spectra were performed in the Swiss-Prot release 53.0 (http:// www.expasy.ch/sprot) and TrEMBL release 37.0 (http:// www.ebi.ac.uk/trembl) databases without taxonomy restriction. Fixed and variable modifications were considered (Cys as S-carbamidomethyl derivate and Met as oxidized methionine, respectively) allowing one trypsin-missed cleavage site and a mass tolerance of 50 ppm. For MS/MS identifications, a precursor tolerance of 50 ppm and MS/MS fragments tolerance of 0.3 Da were used. Identifications were accepted as positive when at least five peptides matched and at least 20% of the peptide coverage of the theoretical sequences matched within a mass accuracy of 50 or 25 ppm with internal calibration. Probability scores were significant at p < 0.01 for all matches. 10. Real-Time PCR Assays. Primers for TRAP1, HIF1R, HPRT1 and PBGD (housekeeping genes) were intron-spanning designed using the Universal Probe Library tool available at Roche Web site (http://www.roche-applied-science.com). Primer sequences were as follows: TRAP1 forward, 5′-agaccaatgccgagaaagg-3′; TRAP1 reverse, 5′-tcctgtgtcatcccgatacc-3′; HIF1R forward, 5′-tttttcaagcagtaggaattgga-3′; HIF1R reverse, 5′-gtgatgtagtagctgcatgatcg-3′; HPRT forward, 5′- tgaccttgatttattttgcatacc-3′; HPRT1 reverse, 5′- cgagcaagacgttcagtcct-3′; PBGD forward, 5′-agctatgaaggatgggcaac-3′; PBGD reverse, 5′-ttgtatgctatctgagccgtcta-3′. Total RNA was isolated from cartilage or chondrocytes using Trizol LS Reagent (Invitrogen, Carlsbad, CA, USA), following manufacturer’s instructions. Whole RNA was treated with DNase (Invitrogen), and its concentration was determined by spectrophotometry. One µg of RNA from each sample was reverse-transcribed in a final volume of 20 µL using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science). cDNA synthesis was performed at 55 °C for 30 min followed by a final step of 5 min at 85 °C for inactivating the reverse transcriptase. Tubes were finally stored at -20 °C until PCR analyses. Real-time PCR was performed in the LightCycler 480 instrument (Roche Applied Science), with 20 µL reactions containing 10 µL LightCycler 480 SYBR Green I Master, 7.4 µL bidistilled

research articles water, 0.3 µL (0.3 µM) each primer and 2 µL cDNA as PCR template. Cycling parameters were 95 °C for 10 min to activate DNA polymerase, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s and a final extension of 72 °C for 10 s. Detection of fluorescence was carried out at the end of each extension step. After amplification, a melting curve was acquired by heating to 95 °C for 5 s, cooling to 70 °C for 1 min and slowly heating to 95 °C with a continuous fluorescence data collection of 10 acquisitions per °C. PCR data were analyzed using REST (Relative Expression Software Tool) software, which provides statistical information suitable for comparing groups of treated versus untreated samples while taking into account issues of reaction efficiency and reference gene normalization. 11. Western Blot Tests. One-dimensional Western blot analyses for PPIA and HIF-1R were performed utilizing standard procedures. Protein samples for PPIA detection were obtained as described above from 22 individuals (11 controls and 11 OAs) from both sexes and a broad range of ages. For HIF-1R analysis, OA chondrocytes (n ) 8) were stimulated with 200 µM CoCl2 where indicated, and cultured under normoxia or hypoxia conditions up to 96 h before protein extraction. Total cellular proteins (30 µg for PPIA immunodetection, and 60 µg for HIF1R) were loaded and resolved using standard 10% SDSpolyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were then electro-blotted onto polyvinylidene fluoride (PVDF) membranes (Immobilon P, Millipore Co., Bedford, MA). Equivalent loadings were verified by Ponceau Red staining after transfer. Membranes were blocked and subsequently hybridized overnight at 4 °C with monoclonal antibodies against PPIA (Santa Cruz Biotechnology, CA, at 1:1000 dilution), HIF-1R (Santa Cruz, at 1:250 dilution) and the housekeeping control R-tubulin (Sigma, 1:2000). Immunoreactive bands were detected by chemiluminescence using corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence (ECL) detection reagents (GE Healthcare), then digitized using the LAS 3000 image analyzer. Quantitative changes in band intensities were evaluated using ImageQuant 5.2 software (GE Healthcare). The densitometric values of the Western blot bands containing the proteins of interest were normalized against those of R-tubulin obtained from the same membranes. The relative abundance of PPIA and HIF-1R was calculated by obtaining the ratio of the normalized densitometric values between normal and treated samples. Statistical p values of the densitometry data were generated by the Mann-Whitney U test using SPSS version 15.0 program. 12. Immunohistochemistry. Normal and OA cartilages were fixed in formalin for 16 h and embedded in paraffin. Cartilage sections were serially sectioned (4 µm). Antigen retrieval was carried out using 0.1% Triton X-100 for 5 min. Nonspecific background staining was blocked with goat serum for 20 min. Sections were incubated with primary antibodies against PPIA (1:50), or a control antibody for 1 h. A horseradish peroxidaseconjugated secondary antibody (1:500) was applied for 30 min. Immunostaining was developed by applying freshly prepared 3,3′-diamino-benzidine-tetrahydrochloride (DAB) solution during 3 to 5 min. The samples were analyzed on a Leica DMLS microscope (Leica Microsystems, Wetzlar, Germany) connected to a Leica DC100 digital camera. The images were analyzed with the Leica QWin program. 13. Indirect Immunofluorescence. Normal and OA frozen cartilages were serially sectioned (4 µm) on cryostat at -30 °C. Slices were washed 10 min with PBS on glass slides, and Journal of Proteome Research • Vol. 9, No. 6, 2010 3037

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Table 1. Biochemical Parameters Measured in the Culture Media of Chondrocytes after 96 h of Culture under Normoxia or Hypoxia (into Hypoxic Bags)

96 h Normoxia 96 h Hypoxia a

glucose (mg/dL)

LDHa (UI/L)

400.75 ( 14.67 399.13 ( 11.85

103.25 ( 8.47 97.06 ( 7.42

LDH: lactate dehydrogenase.

mitochondria staining was performed with 100 µL of 100 nM Mitotracker Green (Invitrogen) for 30 min. Then, samples were washed again and fixed 10 min with acetone at 4 °C. Sections were incubated with primary anti-TRAP1 antibodies (BD Biosciences, 1:50 dilution) or a control antibody for 1 h. After three 10-min washes, a goat antimouse-PE-conjugated antibody (1:20 dilution) was applied for 1 h and the samples were washed again. Finally, nuclei staining was performed with 4′, 6-dianidino-2-phenylindole dihydrochloride (DAPI, 2 µg/mL) for 30 min at 37 °C. In TRAP1 immunofluorescence experiments carried out on primary cultured chondrocytes, cells were seeded at 5 × 105 cells per chamber in an 8-chamber slide. TRAP1 protein and nuclei were sequentially stained as described for cartilage. The chambers were stored at 4 °C in the dark until observation by fluorescence microscopy. Quantification of the emitted fluorescence was performed with AnalySISD 5.0 software (Olympus Biosystems, Hamburg, Germany).

Results and Discussion We have carried out a proteomic analysis of the effect of hypoxia on chondrocytes. Despite their limitations, monolayer cultures of chondrocytes have served as useful models in osteoarthritis research.17 These cells are known to be adapted to hypoxic environments, and some studies have been performed to study specific outcomes of culturing them under low oxygen tensions. Nevertheless, most of these works have employed short hypoxia-incubation periods9,18 thus really reporting the consequences of a definite hypoxic stress on the molecular biology of the chondrocyte. In the present work, we decided to expose the cells to hypoxia during 96 h, based in previous experiences from our group. This medium-term exposure avoids the promt stress response artifacts, and has been shown to be a useful model for metabolism studies in chondrocytes under low oxygen concentrations (personal, unpublished results). For the oxygen deprivation, we have employed hypoxic bags (BD) that guarantee a concentration of oxygen below 2%. Under these conditions of study, we had previously evaluated the ATP production and levels of apoptosis in normal and OA chondrocytes cultured into the hypoxic bags.19 In that work, we reported how chondrocytes are viable in the presence of glucose, whereas its depletion diminishes ATP production about a 90% and caused apoptosis (22%). The conclusions of this work were that, in the presented hypoxic conditions (hypoxic bags), glucose protects human chondrocytes from apoptosis, and also that the metabolic strategy for obtaining energy under low oxygen tension depends more on decreasing metabolic rate than increasing anaerobic glycolysis. We have now measured the amount of remaining glucose and lactate dehydrogenase release in the culture media of chondrocytes after 96 h under normoxia or hypoxia. As shown in Table 1, 3038

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Figure 1. Representative two-dimensional electrophoresis (2-DE) map of human articular chondrocyte proteins obtained in this work. Proteins were resolved in the 3-11 (non linear) pH range on the first dimension, and on 12% T gels on the second dimension. The 55 mapped and identified spots are annotated by numbers according to Tables 2, 3 and 4.

there is a high remnant of glucose in the media and no cytotoxic effect (LDH increase) is detected under the hypoxic stress. Differential Proteomic Analysis of the Effect of Hypoxia on Normal and Osteoarthritic Chondrocytes. We first aimed to analyze the consequences that low oxygen tensions may have on normal and osteoarthritic (OA) cells. To attain this objective, we employed a 2-DE-based approach using whole human articular chondrocyte protein extracts cultured under hypoxia, and compared their proteomes to the same cells cultured in normoxia conditions. Based in our previous experience,20 we performed the proteomic analysis on two protein pools, each made of samples from 3 donors. This strategy has been shown to reduce interindividual variability, which is very high when working with human samples. In order to minimize also the technical variability inherent to the 2-DE procedure, we made technical replicates from each sample. A representative image (corresponding to normal chondrocytes in normoxia) of one of the 2-DE gels obtained in this work is shown in Figure 1. The digitalized images of the gels were analyzed using PDQuest analysis software, which was able to detect more than 650 protein spots on each gel. An average of 540 spots, with a 19.5% coefficient of variation (CV), were matched among the gels with an average match rate of 83%. This evidence the highly reproducible protein pattern that was found between the gels. The matched spots were analyzed for their differential abundance. A comparison of the quantities after normalization to the total density in each gel allowed us to select a set of spots in hypoxia-subjected samples that exhibited significant variation from their controls. Proteins Modulated by Hypoxia in Normal Chondrocytes. We were able to detect 39 protein spots whose abundance was altered more than 1.5-fold (both increased and decreased) under hypoxia in normal chondrocytes when compared to their normoxia controls, considering only those with a significance level above 95% by the Student’s t test (p < 0.05). These spots were excised from the gels and analyzed by MALDI-TOF and MALDI-TOF/TOF MS. The resulting protein identifications led

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Effect of Hypoxia on Human Articular Chondrocytes Table 2. Proteins Modulated by Hypoxia in Normal Chondrocytes predictede

#a

protein IDb

Swiss-Protb

1 2 3 4 5 6 7

ACTB ANXA1 ANXA2 ARP3 CAZA1 LMNA VINC

P60709 P04083 P07355 P61158 P52907 P02545 P18206

8

AL941

P49189

9 10 11

DPYL2 NNMT DDAH1

Q16555 P40261 O94760

12 13 14

ALDOA ATPB G3P

P04075 P06576 P04406

15 16 17 18

KPYM PGAM1 PGK1 TPIS

P14618 P18669 P00558 P60174

Fructose-biphosphate aldolase A ATP synthase subunit beta Glyceraldehyde-3-phosphate dehydrogenase Pyruvate kinase isozymes M1/M2 Phosphoglycerate mutase 1 Phosphoglycerate kinase 1 Triosephosphate isomerase

19 20 21 22 23

PDIA1 HSPB1 PRDX1 PRDX2 SODM

P30101 P04792 Q06830 P32119 P04179

Chaperones and Protein disulfide isomerize precursor 1 0.41 Heat shock protein beta-1 0.32 Peroxiredoxin-1 0.25 Peroxiredoxin-2 0.26 Superoxide dismutase 0.50

24 25

RLA0 TCPE

P05388 P48643

26 27 28

GDIB RAN CLIC1

P50395 P62826 O00299

av. ratioc

protein name

locd

Cellular organization Actin, cytoplasmic 1 2.07 Cyt Annexin A1 0.34 Nuc, Cyt Annexin A2 0.45 Cyt, Exc Actin-related protein 3 0.36 Cyt F-actin-capping protein subunit alpha 1 2.65 Cyt Lamin A/C 3.54 Nuc Vinculin 4.27 Cyt

4-Trimethylaminobutyraldehyde dehydrogenase Dihydropyrimidinase-related protein 2 Nicotinamide N-methyltransferase N(G),N(G)-dimethylarginine dimethylaminohydrolase 1

Metabolism 2.78 Cyt

Mw

pI

scoref

41.7 38.7 38.6 47.4 31.2 74.1 123.8

5.29 6.57 7.57 5.61 5.41 6.57 5.50

176 181 153 178 98 106 97

54.67

5.69

85

no. peptg

% covh

14 41 13 42 15 48 18 33 7 44 13 21 GILSGTSDLLLTFDEAEVRI NFTVEKMSAEINEIIR EAFQPQEPDFPPPPPDLEQLR ANDTTFGLAAGVFTR

2.22 0.38 0.32

Cyt Cyt Cyt

62.71 30.01 31.44

5.95 5.56 5.53

224 141 33

18 10 ALPESLGQHALR

43 40

Energy 0.39 0.23 0.44

Cyt Mit Cyt

39.85 56.52 36.20

8.30 5.26 8.57

152 79 172

11 9 15

42 34 58

Cyt Nuc, Cyt Cyt Cyt

58.47 28.9 44.99 26.94

7.96 6.67 8.3 6.45

114 118 163 270

10 8 13 20

24 26 61 82

stress ER Nuc, Cyt Cyt Cyt Mit

57.48 22.83 22.32 22.04 24.87

4.76 5.98 8.27 5.66 8.35

228 125 103 161 64

18 9 7 10 5

37 51 39 40 24

Transcription and synthesis 60S acı´dic ribosomal protein P0 0.47 Rib T-complex protein 1 subunit epsilon 2.11 Cyt

34.42 60.08

5.71 5.45

108 65

10 7

28 18

Signaling and transport Rab GDP dissociation inhibitor beta 0.40 Cyt GTP-binding nuclear protein Ran 0.50 Nuc, Cyt Chloride intracellular channel protein 1 0.13 Nuc, Cyt

50.12 24.32 27.25

6.11 7.01 5.09

202 92 138

16 9 9

46 47 46

2.03 0.38 0.47 0.42

a Number of protein spot, as represented in Figure 2. b Protein ID and accession number according to SwissProt and TrEMBL databases. c Average volume ratio Hypoxia:Normoxia, quantified by PDQuest 7.3.0 software. d Protein localization: Nuc, nuclear; Cyt, cytoplasmic; ER, endoplasmic reticulum; Mit, mitochondrial; Rib, ribosomal. e Predicted Mr and pI according to protein sequence and Swiss-2DPAGE database. f Mascot protein identification score. g Number of peptide masses matching the top hit from MS-Fit PMF. h Amino acid sequence coverage for the identified proteins. I Identified by tandem mass spectrometry using MALDI-TOF/TOF MS.

to the recognition of 28 different proteins that were modulated by the hypoxia treatment. These proteins are listed in Table 2 and depicted in Figure 1. Interestingly, most of these proteins are decreased (71%), thus suggesting a reduction of the cellular activity under our hypoxia conditions. Database search allowed us to classify these 28 proteins according to their subcellular localization and cellular function, which are shown in Table 2. Regarding their putative cellular role (Figure 2A), one-fourth of them was involved on cellular organization, and another 25% in the production of energy. A remarkable issue of these data is that all the proteins that were identified as related with energy production, except a subunit of the ATP synthase, are enzymes that participate in the glycolytic pathway. Moreover, all of them were found to be decreased except the pyruvate kinase. The increase of this protein, which is the last one of the pathway, might be due to a compensatory effect that pursues to increase the amount of pyruvate. Proteins Modulated by Hypoxia in OA Chondrocytes. The second analysis aimed to evaluate the outcomes of hypoxia in

osteoarthritic cells. In this case we observed a more limited effect caused by the low oxygen tension, as we detected only 17 protein spots that were modulated with statistical confidence. We were able to identify 11 of them, which are listed in Table 3 and depicted in Figure 1. Some of these proteins were also altered in normal cells, such as two glycolytic enzymes (glyceraldehyde 3 phosphate dehydrogenase, G3P, and pyruvate kinase, KPYM), one chaperone (heat shock protein beta 1, HSPB1) and two stress response proteins (peroxiredoxin 1, PRDX1 and manganese superoxide dismutase, SODM). In all these cases the sense of the modulation was equal in OA cells than in normal, being always decreased under hypoxia except for the KPYM. Figure 2B represents the functional distribution of the proteins modulated in these pathologic cells, which can be classified into four groups. Differential Proteomic Analysis of OA and Normal Cells Cultured under Hypoxia. We have previously reported the protein alterations that are distinctive of OA cells when compared to normal.20 At this point, we aimed to perform the Journal of Proteome Research • Vol. 9, No. 6, 2010 3039

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Table 3. Proteins Modulated by Hypoxia in OA Chondrocytes protein ID

#

4 ARP3 29 TPM3 30 TPM4

predicted Swiss-Prot

P61158 P06753 067936

14 G3P

P04406

15 KPYM

P14618

protein name

av. ratio

pI

vScore

no. pept

% cov

178 74 131

18 RIQLVEEELDRAQER 12

33 33

Energy 0.37 Cyt

36.20 8.57

126

12

47

2.86

Cyt

58.47 7.96

184

12

29

Signaling 0.21 Cyt 0.49 Cyt 0.39 Cyt

29.33 4.63 28.18 4.76 21.17 7.01

200 166 94

17 14 7

61 54 41

Chaperones and stress 0.23 Nuc, Cyt 22.83 5.98 0.11 Cyt 22.32 8.27 0.48 Mit 24.87 8.35

59 61 161

4 27 4 25 GELLEAIKR LTAASVGVWGSGWGWLGFNKER AIWNVINWENVTER

Glyceraldehyde-3-phosphate dehydrogenase Pyruvate kinase isozymes M1/M2

P18669 P00558 P60174

14-3-3 protein epsilon 14-3-3 protein beta/alpha Phosphatidylethanolamine-binding protein 1

20 HSPB1 21 PRDX1 23 SODM

P04792 Q06830 P04179

Heat shock proteı´n beta-1 Peroxiredoxin-1 Superoxide dismutase

All abbreviations, as in Table 2.

same comparison but with the cells being cultured under hypoxia, in order to establish if the low oxygen tension (which is a characteristic of the tissue where these cells are embedded) modifies this differential protein profile. Using the same gel images obtained in the previous experiments with normal and OA cells under hypoxia, we carried out an independent analysis to detect those protein spots that were significantly altered. In this analysis, we found out 64 protein spots that were altered in OA cells under hypoxia when compared to normal cells in the same conditions. This more extended modulation was expected, as in this case we were working with nonpaired samples (tissue donors from one condition are different from the other), contrary to the prior analyses in which we used cells from the same donors but subjected to diverse culture conditions. We excised the spots from the gels and we were able to identify 43 proteins, which are listed in Table 4 and depicted in the gel image of Figure 1. These 43 proteins were classified according to their predicted cellular role (Figure 2C), being their pattern of functional distribution very similar to that obtained in the first analysis (the study of the effect of hypoxia on normal cells, Figure 2A). Again, the most represented groups involved proteins related with production of energy, cellular organization and chaperones/stress response. All proteins related with cellular metabolism or energy were found to be decreased. We identified in this group seven glycolytic enzymes, suggesting a reduction in the activity of this pathway that remained to be examined. Interestingly, four of them (aldolase A, enolase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase) were also found to be decreased in OA chondrocytes cultured in normoxia when compared to normal controls.20 Our results also confirmed the OA-dependent changes of other proteins whose alterations in pathologic cells were previously reported by our group using normoxia-cultured cells.20,21 These include three proteins related with the cytoskeletonsbeta-actin (ACTB), vinculin (VINC) and gelsolin (GELS)sand two molecular chaperonessthe peptidyl-prolyl 3040

Mw

Cellular organization 0.45 Cyt 47.8 5.61 0.47 Cyt 32.86 4.68 0.16 Cyt 28.62 4.67

Actin-related protein 3 Tropomyosin alpha-3-chain Tropomyosin alpha-4-chain

31 1433E 32 1433B 33 PEBP1

a

loc

Journal of Proteome Research • Vol. 9, No. 6, 2010

cis-trans isomerase A (PPIA) and the Tumor Necrosis factor type 1 receptor-associated protein 1 (TRAP1). The remarkable increase in GELS in OA cells (higher under hypoxia than in normoxia) is an interesting finding, as this protein has been suggested as novel mediator of cartilage pathology and as putative biomarker of joint diseases.22 Gelsolin is an actin- capping protein with important roles in extracellular actin scavenging23 and in regulating cytoskeletal architecture and cell-matrix interactions in many cell types, including developing chondrocytes.24 GELS was found to be decreased in synovial tissue from rheumatoid arthritis patients,25 its down-regulation being associated with cytoskeletal remodelling in arthritis synovial cells.23,26 A recent proteomic analysis has shown that its secretion by cartilage is also decreased after treatment with the proinflammatory cytokine IL-1β or retinoic acid.27 The abundance of gelsolin along with many other cytoskeletal proteins in synovial fluid28 suggests that GELS may serve an additional protective role in the synovium. Moreover, a cytoskeletal rearrangement in arthritis cartilage and chondrocytes has been reported,29,30 but little is known about this mechanism. Therefore, our finding of the increase of GELS in OA chondrocytes under hypoxia compared to normal suggests a role in this rearrangement. Cyclophilin A is Increased in OA Chondrocytes under Hypoxia. Another protein whose abundance is significantly increased in OA cells under hypoxia is the Peptidyl-prolylisomerase A (PPIA), also known as Cyclophilin A, which is a ubiquitous protein of eukaryotic cells.31 Several references support the role of PPIA as inflammatory mediator, as it is produced by macrophages following lipopolysaccharide stimulation.32 Moreover, it has been recently shown that PPIA induces the production of inflammatory cytokines and the expression of matrix metalloproteinase 9 (MMP-9) by monocytes, being these stimulatory effects mediated through the nuclear factor kappa B (NFκB) transcription factor.33 All these data, together with our finding of increased PPIA in the diseased cells, suggest a role of this protein in OA pathogenesis. Therefore, in this work we also verified this augment by

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Effect of Hypoxia on Human Articular Chondrocytes Table 4. Proteins Differentially Expressed in OA vs Normal Chondrocytes Cultured under Hypoxia protein ID

#

1 2 3 34

ACTB ANXA1 ANXA2 CO6A1

Swiss-Prot

av. ratioa

protein name

predicted loc

Cellular organization 0.34 Cyt 0.22 Nuc, Cyt 0.21 Cyt, Exc 0.33 Exc

P60709 P04083 P07355 P12109

Actin, cytoplasmic 1 Annexin A1 Annexin A2 Collagen alpha-1(VI) chain

35 GELS

P06396

Gelsolin

6.06 Cyt

36 TERA 37 VIME 7 VINC

P55072 P08670 P18206

Transitional endoplasmic reticulum Vimentin Vinculin

0.21 ER 2.92 Cyt 3.37 Cyt

38 9 10 39 11

CAPG DPYL2 NNMT UGDH DDAH1

P40121 Q16555 P40261 P12378 O94760

Macrophage-capping protein Dihydropyrimidinase-related protein 2 Nicotinamide N-methyltransferase UDP-glucose-6-dehydrogenase N(G),N(G)-dimethylarginine dimethylaminohydrolase 1

12 13 40 14

ALDOA ATPB ENOA G3P

P04075 P06576 P06733 P04406

15 41 16 17 42

KPYM LDHA PGAM1 PGK1 SCOT

P14618 P00338 P18669 P00558 P55809

Fructose-biphosphate aldolase A ATP synthase subunit beta Alpha enolase Glyceraldehyde-3-phosphate dehydrogenase Pyruvate kinase isozymes M1/M2 L-lactate dehydrogenase A chain Phosphoglycerate mutase 1 Phosphoglycerate kinase 1 Succinyl-CoA:3ketoacid-coenzyme A transferase 1 D-3-phosphoglycerate dehydrogenase

Metabolism 0.29 Nuc, Cyt 0.20 Cyt 0.18 Cyt 0.13 Cyt 0.07 Cyt

5.90

168

89.32 5.14 53.65 5.06 123.8 5.50

148 227 100

39.85 56.52 47.16 36.20

8.30 5.26 7.01 8.57

177 79 143 172

13 9 14 15

54 34 46 58

0.18 0.03 0.10 0.34 0.28

58.47 36.69 28.9 44.99 56.16

7.96 8.44 6.67 8.3 7.13

138 210 148 103 111

10 14 13 9 14

25 43 53 33 38

56.65 6.29

69

Cyt Cyt Nuc, Cyt Cyt Mit

50 GSTP1

P09211

51 EFTU 52 PSB2 53 RLA0

P49411 P49721 P05388

Transcription Elongation factor Tu 0.33 Proteasome subunit beta type-2 2.36 60S acı´dic ribosomal protein P0 5.90

54 SYWC

P23381

Tryptophanyl-tRNA synthetase

a

85.7

17 42 9 29 16 42 FIDNLRDR VFSVAITPDHLEPR AGKEPGLJWR EVQGFESATFLGYFK HUVPNEVVVQR YIETDPANR VPVDPATYGQFYGGDSYIILYNYR 13 20 21 56 AIPDLTAPVAAVQAAVSNLVR GILSGTSDLLLTFDEAEVR SFLDSGYR

Energy 0.23 Mem 0.23 Mit 0.26 Cyt 0.27 Cyt

Chaperones and stress 60 kDa heat shock protein 2.32 Mit Heat shock cognate 71 kDa protein 0.40 Nuc, Cyt Heat shock protein beta-1 0.20 Nuc, Cyt Protein disulfide isomerase precursor 1 2.81 ER Peptidyl-prolyl cis-trans isomerase A 2.08 Cyt T-complex protein 1 subunit epsilon 2.20 Cyt Protein DJ-1 0.06 Nuc, Cyt Superoxide dismutase 0.12 Mit Tumor necrosis factor type 1 2.03 Mit receptor-associated protein Glutathione S-transferase P 0.10 Cyt

P62826

228 119 193 63

20 23 44 31

P61604 P11142 P04792 P30101 P62937 P48643 Q99497 P04179 Q12931

27 RAN

5.29 6.57 7.57 5.26

% cov

6 11 11 14 ALPESLGQHALR

CH60 HSP7C HSPB1 PDIA1 PPIA TCPE PARK7 SODM TRAP1

P50395 P60174

41.7 38.7 38.6 108.5

no. pept

87 158 122 186 35

44 45 20 19 46 25 47 48 49

26 GDIB 33 PEBP1

score

5.88 5.95 5.56 6.73 5.53

O43175

P63104 O00299

pI

38.5 62.71 30.01 55.02 31.44

43 SERA

55 1433Z 28 CLIC1

Mw

0.20 Cyt

5.70 5.37 5.98 4.76 7.68 5.45 6.33 8.35 8.30

118 189 90 137 127 74 94 70 124

12 15 7 14 9 7 8 6 19

30 30 48 32 56 17 55 31 35

23.36 5.43

111

8

48

and synthesis Mit 50.14 9.10 Nuc, Cyt 22.84 6.52 Rib 34.42 5.71

81 120 276

9 29 9 53 GTIEILSDVQLIK VLALSVETDYTFPLAEK VLALSVETDYTFPLAEKVK 6 19

0.21 Cyt

61.05 70.90 22.83 57.48 18.00 60.08 19.89 24.87 80.3

LVINGNPITIFQER LVINGNPITIFQERDPSK

53.17 5.83

80

Signaling and transport 3.93 Cyt 17.75 4.73 0.13 Nuc, Cyt 27.25 5.09

83 253

10 16

44 66

14-3-3 protein zeta/delta Chloride intracellular channel protein 1 Rab GDP dissociation inhibitor beta Phosphatidylethanolamine-binding protein 1 GTP-binding nuclear protein Ran

0.05 Cyt 0.29 Cyt

50.12 6.11 21.17 7.01

202 94

16 7

46 41

0.50 Nuc, Cyt

24.32 7.01

92

9

47

Average volume ratio OA:Normal, quantified by PDQuest 7.3.0 software. Other abbreviations as in Table 2.

Western blot using chondrocytes from 22 age-matched OA and control donors (11 from each group). As shown in Figure 3A, PPIA is significantly increased in OA cells, with a ratio of 1.35

(OA:N) and p ) 0.029 obtained by densitometric analysis of the blots. Immunohistochemistry assays were subsequently performed in order to determine if this high abundance can Journal of Proteome Research • Vol. 9, No. 6, 2010 3041

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Ruiz-Romero et al.

Figure 2. Functional distribution of the proteins identified as altered in this work in any of the three analyses that were performed. (A) Modulated by hypoxia in normal chondrocytes; (B) modulated by hypoxia in OA chondrocytes; (C) altered in OA versus normal chondrocytes under hypoxic conditions.

Figure 3. Increased abundance of PPIA in OA chondrocytes and cartilage compared to normal. (A) Representative Western blot analysis on chondrocyte protein extracts from N and OA samples. On the right is shown a graphical representation of the densitometric data obtained from the blots (* ) P < 0.03, n ) 22). (B) Representative images from immunohistochemistry analyses performed on normal and OA paraffin-embedded cartilage sections (n ) 6).

also be detected in OA cartilage. As shown in Figure 3B, the stain intensity of chondrocytes in the diseased tissue is much higher than in normal, despite of the cartilage layer observed. This increased PPIA amount points out a role of this protein in OA pathogenesis. In this sense, it has been recently described how the use of Cyclosporin A (CSA), a calcineurin inhibitor, decreases cartilage damage in an animal model of experimental OA and appears to open a novel therapeutic strategy.34 Taking into account that PPIA is the major receptor of CSA and is essential for its calcineurin inhibitory activity,35 its high abundance in OA cartilage might underlie the positive effects of CSA on the disease. Hypoxia Conditions Up-Regulate TRAP1 Expression and Increase Its Protein Abundance. The third protein whose increased abundance in OA cells was confirmed under hypoxia is the TNFR-receptor associated protein 1, TRAP1. This protein is the mitochondrial member of the Hsp90 family of protein chaperones, and was first identified by its ability to bind the 3042

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Figure 4. Hypoxia positively modulates TRAP1 expression in chondrocytes. (A) Real-time PCR results obtained after subjecting cultured normal chondrocytes to hypoxia conditions during 1 (1d) and 4 days (4d). *, p < 0.05, n ) 4. (B) Indirect immunofluorescence (IFI) images of cultured chondrocytes, showing the increase of TRAP1 protein under hypoxia (4 days). Nuclei (DAPI) display a blue color, whereas TRAP1 protein is labeled in red (PE-conjugated antibody). Detailed cellular distribution of TRAP1 expression can be seen on the enlarged inserts showed at the bottom of each image.

type 1 tumor necrosis factor receptor.36 Despite its high homology with Hsp90, TRAP1 has distinct functional properties.37 In a previous work from our group, TRAP1 was found to be increased in the mitochondria from pathologic cells when compared to healthy controls.21 In the present study, our proteomic analysis points up that this protein spot exhibits a 2.03-fold increase in OA cells when compared to normal, both cultured under hypoxic conditions. To investigate if this increase is not only OA-dependent, but has also any dependence on the oxygen tension, we evaluated the effect of oxygen deprivation on TRAP1 gene expression by real-time PCR analysis. As shown in Figure 4A, PCR analysis found a TRAP1 overexpression of 2.38-fold and 2.3-fold after 1 and 4 days of hypoxia, respectively. This increase was statistically significant, with p ) 0.047 and p ) 0.032, respectively (n ) 4), and was

Effect of Hypoxia on Human Articular Chondrocytes

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Figure 5. High abundance of TRAP1 in chondrocytes from OA cartilage deep layer. Top row, hematoxylin and eosin (H&E) stained articular cartilage, showing the characteristics of its different layers. The other rows show indirect immunofluorescence labeling of Trap1 (red) and colocalization with mitochondria (stained with Mitotracker Green) in normal and OA cartilage. Increased abundance of mitochondrial TRAP1 can be detected in the deep layer of OA tissue. (Left) Chondrocyte lacunae from superficial and deep layers of OA cartilage.

confirmed by immunofluorescence on primary cultured chondrocytes (Figure 4B). We quantified the emitted fluorescence and obtained a significant (p ) 0.011) increase of the percentage of positive cells when subjected to hypoxia. TRAP1 is Increased Specifically in the Deep Layer of OA Cartilage. Immunohistofluorescence assays were subsequently performed in order to evaluate TRAP1 presence in cartilage tissue, and also to seek its subcellular localization. As shown in Figure 5, low presence of TRAP1 (red) can be detected in normal cartilage, either in its superficial or deep layers, whereas cartilage mitochondria are easily recognized by Mitotracker green labeling. In OA cartilage, however, a high presence of TRAP1 can be perceived mainly in the deep layer of the tissue (Figure 5). On the right on the figure, magnification of chondrocyte lacunae from superficial and deep layers (each with its characteristic shape) of OA cartilage showed the differences in abundance. Overlapping images of fluorescence from Mitotracker (green) and TRAP1 (red) evidenced the mitochondrial localization of TRAP1 in chondrocytes. This is in contrast to a recent microarray analysis carried out to detect genes differentially expressed in both normal and OA chondrocytes that were cultured under normoxic compared with hypoxic conditions.38 In this work, TRAP1 was not detected as regulated by O2 tension, but our real-time PCR results and their confirmation by immunocytofluorescence evidence this link. Our results suggest that increased synthesis of TRAP1 is necessary for OA chondrocytes to subsist under a low oxygen environment. According to this finding, the protective effect of TRAP1 overexpression against ischemia has been recently described, using both in vitro (primary astrocytic cultures) and in vivo (rat brain) models.39,40 These authors have reported how TRAP1 overexpression preserves ATP levels and cell viability during ischemia-like conditions such as oxygen and glucose deprivation, and improves mitochondrial function after ischemic brain injury. To investigate if this differential TRAP1 protein profile in the tissue is only dependent to the low oxygen tension, we tried to partially mimic other specific environmental characteristic of the cartilage deep layer, such as glucose deprivation.41 Realtime PCR and IFI analyses on chondrocytes cultured without

glucose for 1 and 4 days were performed, and we could observe how this nutrient limitation did not significantly affect neither TRAP1 gene expression (p ) 0.434, n ) 4) nor its protein abundance in cultured chondrocytes (data not shown), whereas we had previously shown how hypoxia conditions remarkably increased them (Figure 4). Modulation of HIF-1a under the Conditions of This Study. Finally, we have found in this work a significant decrease of a number of glycolytic proteins in chondrocytes cultured under hypoxia when compared to normoxia conditions. Interestingly, a positive transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1 (HIF-1, which is known to be up-regulated in hypoxia) has been reported.42 Therefore, we sought to investigate the levels of HIF-1 in the conditions of our proteomic study. As shown in Figure 6A, we first evaluated the amount of HIF-1R protein in chondrocytes cultured during 96 h under normoxia or hypoxia. Densitometric analysis of the blots render a 2-fold decrease of HIF-1R (ratio Hyp:Norm ) 0.492 ( 0.06, p ) 0.0053, n ) 8) in cells cultured into the hypoxic bags. These data were confirmed at the transcript level by Real-time PCR analysis, which show a 5-fold reduction of HIF-1R gene expression at 96 h of hypoxia (ratio Hyp:Norm ) 0.2067). We then performed a time-course analysis of HIF-1R protein abundance using chondrocytes cultured under hypoxia during 6, 12, 24, 48, and 96 h. As shown in Figure 6B, there is an initial increase of HIF-1R that reaches its top at 12 h, but after 48 h of culture this amount decreases significantly. Moreover, addition of a known HIF-1R inductor such as CoCl2 is not capable of increasing it in chondrocytes when they are cultured in hypoxia for 96 h (Figure 6B). Altogether, these data show a decrease of HIF-1R in the conditions of the present proteomic study. It is essential to point out that the reported upregulation of glycolytic enzymes by HIF-1R has been described at 1-16 h of hypoxia42 or up to 48 h.43 Besides, as written above, most of the experiments carried out to evaluate the effect of hypoxia in chondrocytes have also employed short incubation periods.9,18 Our model shows how articular chondrocytes are capable of adapting to medium-term cultures under low oxygen tensions (around 1% O2) without losing viability. The decrease in glycolytic enzymes Journal of Proteome Research • Vol. 9, No. 6, 2010 3043

research articles

Ruiz-Romero et al. PXIB916357PR and PGIDIT06PXIB916358PR. C.R.-R. was supported by Programa Parga Pondal, Secretaria Xeral I+D+i, Xunta de Galicia. J.M. was supported by Fondo Investigacio´n Sanitaria-Spain (CA07/00243). V.C. was supported by Xunta de Galicia (PGEIDIT06PXIC916175PN).

References

Figure 6. Modulation of Hypoxia-inducible Factor 1R (HIF-1R) under the conditions of our study. (A) Western Blot analysis of HIF-1R in OA chondrocytes cultured under normoxia (Norm) or hypoxia (Hyp) conditions during 96 h. A graphical representation of the densitometric data obtained from the blots is shown on the right (** ) P < 0.006, n ) 8). (B) Representative image of a time-course analysis of HIF-1R abundance in chondrocytes cultured into the hypoxic bags, showing its decrease after 1 day of culture. Addition of CoCl2 has no effect on HIF-1R in chondrocytes when cultured 96 h under hypoxia.

that we found under hypoxia also points to a reduction in the metabolic rate as adaptation mechanism.

Conclusions Our work describes those protein modulations caused by oxygen deprivation in chondrocyte cell culture. This data provide information about the intracellular molecular pathways that are involved in the chondrocyte adaptation to a low oxygen tension environment, such as cartilage. A study of the biological consequences of these modulations on the ECM metabolism, probably by the analysis of those proteins secreted from chondrocytes cultured under hypoxia, would be very useful to complement the data presented here. Interestingly, in this work OA cells showed a less extended modulation than normal, which brings to light a lower capacity of diseased cells to react under a hypoxic milieu. We found alterations in a number of proteins related with the cellular organization, and also a global decrease of energy and metabolism-related proteins in cells under hypoxia. These data point to a decrease in the cellular activity that remains to be studied more in detail. On the other hand, our analysis of the differential protein profile of OA chondrocytes under hypoxia illustrates various proteins that might be related with the OA process. Increase of protein chaperones, such as PPIA and TRAP1, in diseased cells and tissue highlights their essential role in maintaining chondrocyte viability in the OA process. All the evidence presented will increase the knowledge about chondrocyte physiology and its role in OA pathogenesis, which might help for the development of new early diagnosis and therapeutic alternatives.

Acknowledgment. We thank Ms. P. Cal Purrin˜os for her expert secretarial assistance, and express appreciation to the Pathology Service and to Mrs. Lourdes Sanjurjo and Mrs. Dolores Velo from the Orthopaedics Department of the CHU A Corun ˜ a for providing cartilage samples. This study was supported by grants from Fondo Investigacio´n Sanitaria-Spain ((CIBER-CB06/01/0040); PI-08/2028) and Secretaria I+D+I Xunta de Galicia: PGIDIT06PXIC916175PN; 3044

Journal of Proteome Research • Vol. 9, No. 6, 2010

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PR901209S

Journal of Proteome Research • Vol. 9, No. 6, 2010 3045