Miha Milek

Natasa Karas Kuzelicki

Alenka Smid

Irena Mlinaric-Rascan

S-adenosylmethionine regulates thiopurine methyltransferase activity and decreases 6mercaptopurine cytotoxicity in MOLT lymphoblasts

Keywords: S-adenosylmethionine, thiopurine methyltransferase, cytotoxicity, lymphoblasts, 6-mercaptopurine

Six-mercaptopurine (6-MP) is a pro-drug widely used in treatment of various diseases, including acute lymphoblastic leukaemia (ALL). Side-effects of thiopurine therapy have been correlated with thiopurine methyltransferase (TPMT) activity.

We propose a novel TPMT-mediated mechanism of S-adenosylmethionine (SAM)-specific effects on 6-mercaptopurine (6-MP) induced cytotoxicity in a model cell line for acute lymphoblastic leukemia (MOLT). Our results show that exogenous SAM (10-50 µM) rescues cells from the toxic effects of 6-MP (5 µM) by delaying the onset of apoptosis. We prove that the extent of methylthioinosine monophosphate (MeTIMP) induced inhibition of de novo purine synthesis (DNPS) determines the concentrations of intracellular ATP, and consequently SAM, which acts as a positive modulator of TPMT activity. This leads to a greater conversion of 6-MP to inactive 6-methylmercaptopurine, and thus lower availability of thioinosine monophosphate for the biotransformation to cytotoxic thioguanine nucleotides (TGNs) and MeTIMP. We further show that the addition of exogenous SAM to 6-MP treated cells maintains intracellular SAM levels, TPMT activity and protein levels, all of which are diminished in cells incubated with 6-MP. Since TPMT mRNA levels remained unaltered, the effect of SAM appears to be restricted to protein stabilisation rather than an increase of TPMT expression. We thus propose that SAM reverses the extent of 6-MP cytotoxicity, by acting as a TPMT-stabilizing factor.

This study provides new insights into the pharmacogenetics of thiopurine drugs. Identification of SAM as critical modulator of TPMT activity and consequently thiopurine toxicity may set novel grounds for the rationalization of thiopurine therapy.

A c c e p t e d M a n u s c r i p t

Introduction

Thiopurines (e.g. 6-mercaptopurine, 6-MP; 6-thioguanine, 6-TG) are pro-drugs widely used in treatment of various diseases, including acute lymphoblastic leukaemia (ALL) [START_REF] Lennard | The clinical pharmacology of 6-mercaptopurine[END_REF]. The cytotoxic effect of 6-MP depends on two distinct metabolic pathways (Fig. 1) [START_REF] Chrzanowska | Metabolites of mercaptopurine in red blood cells: a relationship between 6-thioguanine nucleotides and 6methylmercaptopurine metabolite concentrations in children with lymphoblastic leukaemia[END_REF]. The conversion of 6-MP by hypoxanthine phosphoribosyltransferase (HPRT) yields thioinosine monophosphate (TIMP), which can either be converted to active thioguanine nucleotides (TGNs), or methylated by thiopurine methyltransferase (TPMT; EC 2.1.1.67) to methylthioinosine monophosphate (MeTIMP) [START_REF] Dervieux | Differing contribution of thiopurine methyltransferase to mercaptopurine versus thioguanine effects in human leukaemic cells[END_REF]. Incorporation of TGNs into DNA and RNA results in S phase arrest [START_REF] Panetta | Mechanistic mathematical modelling of mercaptopurine effects on cell cycle of human acute lymphoblastic leukaemia cells[END_REF] and programmed cell death [START_REF] Hortelano | 6-mercaptopurine decreases the Bcl-2/Bax ratio and induces apoptosis in activated splenic B lymphocytes[END_REF], triggered via the mismatch repair pathway [START_REF] Yamane | Mismatch repair-mediated G2/M arrest by 6thioguanine involves the ATR-Chk1 pathway[END_REF]. On the other hand, MeTIMP is a potent inhibitor of de novo purine synthesis (DNPS), causing depletion of purine nucleotides, which results in cell growth arrest and cytotoxicity [START_REF] Tay | Inhibition of phosphoribosyl pyrophosphate amidotransferase from Ehrlich ascites-tumor cells by thiopurine nucleotides[END_REF]. Nevertheless, the incorporation of TGNs is considered to be the main mode of action of 6-MP [START_REF] Relling | Prognostic importance of 6mercaptopurine dose intensity in acute lymphoblastic leukemia[END_REF].

TPMT also catalyzes the direct methylation of 6-MP to produce the inactive 6methylmercaptopurine (6-MMP) leading to lower toxic potential of the drug and consequently to higher leukemic cell survival (Fig. 1) [START_REF] Otterness | Human thiopurine methyltransferase pharmacogenetics: gene sequence polymorphisms[END_REF]. Due to the presence of genetic polymorphisms, heterozygous and homozygous individuals for mutant alleles display intermediate and low TPMT activity, respectively [START_REF] Milek | Thiopurine Smethyltransferase pharmacogenetics: genotype to phenotype correlation in the Slovenian population[END_REF][START_REF] Salavaggione | Thiopurine Smethyltransferase pharmacogenetics: variant allele functional and comparative genomics[END_REF]. ALL patients with decreased TPMT activity require dosage adjustment protocols, since treatment with standard doses of 6-MP results in high cytotoxic TGN levels, causing severe life-threatening toxicity [START_REF] Lennard | Genetic variation in response to 6-mercaptopurine for childhood acute lymphoblastic leukemia[END_REF][START_REF] Evans | Altered mercaptopurine metabolism, toxic effects and dosage requirement in a thiopurine methyltransferase-deficient child with acute lymphoblastic leukemia[END_REF]. Molecular studies have shown that non-synonymous amino-acid substitutions in mutant TPMT isoforms cause the disruption of intra-molecular van der Waals contacts [START_REF] Wu | Structural basis of allele variation of human thiopurine-S-methyltransferase[END_REF]. Consequently, such variant proteins are readily degradable via proteasome-mediated proteolysis [START_REF] Tai | Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT*3A, A c c e p t e d M a n u s c r i p t 24 TPMT*2): mechanisms for the genetic polymorphism of TPMT activity[END_REF], a process which can be partially prevented by S-adenosylmethionine (SAM), the methyl donor in the S-A c c e p t e d M a n u s c r i p t 5 methylation reactions catalyzed by TPMT [START_REF] Tai | Enhanced proteasomal degradation of mutant human thiopurine S-methyltransferase (TPMT) in mammalian cells: mechanism for TPMT protein deficiency inherited by TPMT*2, TPMT*3A, TPMT*3B or TPMT*3C[END_REF]. The binding of SAM has been shown to stabilise the 3D structure of the enzyme [START_REF] Scheuermann | Consequences of binding an Sadenosylmethionine analogue on the structure and dynamics of the thiopurine methyltransferase protein backbone[END_REF].

With respect to the thiopurine toxicity, decreased SAM recycling appears to be a consequence of MeTIMP inhibition of DNPS [START_REF] Vogt | The importance of methylthio-IMP for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity in Molt F4 human malignant T-lymphoblasts[END_REF], since the ATP-dependent synthesis of SAM from methionine is limited [START_REF] Stet | Decrease in S-adenosylmethionine synthesis by 6-mercaptopurine and methylmercaptopurine ribonucleoside in Molt F4 human malignant lymphoblasts[END_REF]. In addition, the presence of low-activity mutations in 5,10methylenetetrahydrofolate reductase (MTHFR), the enzyme that catalyzes the formation of 5methyltetrahydrofolate, a rate-determining step for the conversion of methionine to homocysteine (Fig. 1) has been found to correlate with decreased TPMT activity [START_REF] Arenas | Genetic variation in the MTHFR gene influences thiopurine methyltransferase activity[END_REF] and toxic side-effects of 6-MP in patients with ALL [START_REF] Kuzelicki | Heterozygosity at the TPMT gene locus, augmented by mutated MTHFR gene, predisposes to 6-MP related toxicities in childhood ALL patients[END_REF]. It was postulated that low MTHFR activity results in limited SAM synthesis and consequently lower TPMT stability.

Since low TPMT activity is strongly correlated with unfavourable side effects during thiopurine treatment, availability of endogenous SAM could contribute to the extent of 6-MP induced cytotoxic effects. To test this hypothesis, we used a human ALL cell line MOLT, and found that SAM-specific effects include the regulation of thiopurine metabolism by acting as a TPMT-stabilizing factor, resulting in decreased amounts of cytotoxic 6-MP metabolites, and diminished cytotoxic effects in these cells.

Materials and methods

1. Chemicals

Allopurinol

Cell culture

Human T-lymphoblastic cell line MOLT was purchased from American Type Culture Collection (LGC Promochem, Wesel, Germany) and grown in RPMI 1640 medium (Sigma), supplemented with 10% fetal bovine serum (Gibco, Invitrogen, Carlsbad, CA, USA), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin and 50 µM 2-mercaptoethanol (all from Sigma). Cells were incubated in a sterile environment at 37°C with a humidified atmosphere of 5% CO 2 , and maintained in the density range of 0.4-2.0x10 6 /ml. In all experiments, the concentration of cells at the beginning of incubation with 6-MP, SAM, or vehicles (t = 0) was 0.5x10 6 /ml.

3. Trypan blue exclusion assay

Cells (5.0x10 5 /ml) were incubated in culture media containing 5 µM 6-MP, 25 µM SAM, or vehicles for different time periods. Afterwards, the cells were suspended in 0.2% Trypan Blue solution (Sigma), stained for 5 min, and counted on a haemocytometer as described previously [START_REF] Kristl | Surface active stabilizer Tyloxapol in colloidal dispersions exerts cytostatic effects and apoptotic dismissal of cells[END_REF]. Viability (%) was determined by dividing the number of viable cells (white) by the total cell count (white and blue).

A c c e p t e d M a n u s c r i p t 7

4. MTS assay

Metabolic activity of cells was determined by the CellTiter Aqueous One Proliferation

Assay™ (Promega, Madison, WI, USA) as described previously [START_REF] Egle | A novel gene delivery system for stable transfection of thiopurine-S-methyltransferase gene in versatile cell types[END_REF]. Briefly, cells (1.5x10 4 /well) were seeded into 96-well plates and incubated in culture media containing different concentrations of 6-MP, SAM, or vehicles for 24 or 48 h. Afterwards, the MTS reagent (1:10) was added and the plates were incubated for another 3 h at 37 °C. Absorbance (492 nm) readings were carried out by an automated plate reader (Tecan Safire2, Austria).

Assays performed without cells served as background controls.

5. Detection of apoptotic cells

The Annexin V-FITC Apoptosis Detection Kit™ (Sigma) was used to detect apoptotic cells, according to the manufacturer's instructions. Flow-cytometric analysis was carried out immediately after staining on a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany), utilizing the fluorescence detectors FL1 (530 nm) and FL3 (679 nm) for the detection of Annexin V-FITC and PI, respectively. Percentages of Annexin V + /PI -(early apoptotic) and Annexin V + /PI + (dead) cells were determined by CellQuest Pro software (BD Biosciences).

6. DEVDase activity

Caspase-3 activity was measured in total cell lysates using a labelled Ac-DEVD.AFC peptide substrate (Sigma) as described previously [START_REF] Murn | Internucleosomal DNA cleavage in apoptotic WEHI 231 cells is mediated by a chymotrypsin-like protease[END_REF]. Incubation of cells with 100 µM TPCK was

7. Cell cycle analysis

The DNA content of cells was determined by the flow cytometric analysis as previously described [START_REF] Kristl | Surface active stabilizer Tyloxapol in colloidal dispersions exerts cytostatic effects and apoptotic dismissal of cells[END_REF], with the exception that cells (2.0x10 6 ) were fixed in 80% ethanol at -20 °C overnight.

8. Fluorescence microscopy

The DNA intercalating Hoechst 33342 dye (Sigma) was used for live-cell fluorescence imaging of nuclei. Cells (1.2x10 6 ) were washed in PBS and stained with Hoechst (5.0 µg/ml) for 20 min at room temperature. Afterwards, the cells were washed twice, resuspended in PBS (1.5x10 6 /ml), placed into a cytospin chamber (Cytospin2, StatSpin Inc., Norwood, MA, USA), and centrifuged (1100 rpm, 10 min). Fluorescence imaging was carried out immediately on an Olympus IX 81 inverted microscope (Tokyo, Japan) with the 360nm/420nm excitation/emission filter to visualize the Hoechst-stained nuclei. Images were taken at a total magnification of 960x and processed by the Cell^R software (Olympus).

9. Cytosolic TGN levels

Intracellular TGNs were measured by the modified reversed-phase HPLC method described previously [START_REF] Dervieux | Identification of 6-methylmercaptopurine derivative formed during acid hydrolysis of thiopurine nucleotides in erythrocytes, using liquid chromatographymass spectrometry, infrared spectroscopy, and nuclear magnetic resonance assay[END_REF][START_REF] Stefan | Improved HPLC methodology for monitoring thiopurine metabolites in patients on thiopurine therapy[END_REF]. Cells (3.0x10 6 ) were washed twice, resuspended in PBS and frozen at -80 °C. The cells were lysed by multiple freezing and thawing cycles, followed by centrifugation A c c e p t e d M a n u s c r i p t 9 (14000g, 5 min, 4 °C). After the addition of 70 % HClO 4 (10 µl) and 0.65 M DTT (10 µl), the samples were incubated on ice (10 min) and centrifuged. The supernatants were hydrolyzed by heating (100°C, 75 min) to release 6-TG. Afterwards, they were immediately placed on ice and re-centrifuged to remove any remaining debris. Aliquots (40 µl) were separated on a Zorbax ODS column (25x0.46 cm, 5 µm; Agilent Technologies, Santa Clara, CA, USA) utilizing an automated Agilent Technologies HPLC 1100 system by isocratic elution (10 mM potassium phosphate buffer, pH 2.2, 4% acetonitrile) at a flow rate of 1.0 ml/min. Integration of peaks was carried out by the Chemstation for LC 3D Systems software (Agilent). The concentration of TGNs is reported as the amount (pmol) of 6-TG present after hydrolysis per 10 6 viable cells.

10. Cytosolic MeTIMP levels

Methylated metabolites of 6-MP were detected as MMPR after the digestion of cell lysate with alkaline phosphatase (AP), according to the previously described reversed-phase HPLC method [START_REF] Giverhaug | Increased concentrations of methylated 6mercaptopurine metabolites and 6-thioguanine nucleotides in human leukemic cells in vitro by methotrexate[END_REF], with minor modifications. Cells were lysed as for TGN determinations. The resulting supernatants (85 µl), with the addition (10 µl) of 0.5 M Tris buffer (pH 7.8), were treated with calf intestine AP (10 U, Calbiochem, Gibbstown, NJ, USA) for 2 h at 37 °C. 1.8 M HClO 4 (10 µl) was then added and the samples were incubated on ice (10 min). After centrifugation (14000g, 5 min, 4 °C), the supernatants (aliquots of 70 µl) were used for HPLC analysis. MMPR was eluted isocratically in 180 mM potassium phosphate buffer, pH 6.85, supplemented with 15% acetonitrile, at a flow rate of 1.0 ml/min, and was detected at 290 nm.

HPLC instrumentation was the same as above and the concentration of MeTIMP was reported as the amount (pmol) of MMPR present per 10 6 viable cells. (1:1) for 2 min on an orbital shaker to induce cell lysis and facilitate the ATP-dependent luciferase reaction, resulting in the production of oxyluciferin. The plates were then equilibrated at room temperature for 10 min, and luminescence was recorded on an automated plate reader (Tecan Safire2, Zürich, Switzerland). Standards solutions of ATP were prepared in culture media on the same plate and processed as described above. The concentration of ATP was reported as the amount (pmol) of ATP per 10 4 viable cells.

1Intracellular SAM levels

Cells (8.0x10 6 ) were washed twice in PBS and an aliquot was stored at -80 °C for determination of protein concentration with the BioRad Protein Assay Kit (Hercules, CA, USA) according to the manufacturer's instructions. The remaining pellets were lysed in 0.5 M HClO 4 (100 µl) for 20 min on ice. After centrifugation (14000g, 2x10 min, 4 °C), SAM was measured in the supernatants by the reversed-phase HPLC method essentially as described previously [START_REF] Wise | Measuring S-adenosylmethionine in whole blood, red blood cells and cultured cells using a fast preparation method and highperformance liquid chromatography[END_REF]. The concentration of SAM was reported as the amount (nmol) of SAM per mg of protein.

A c c e p t e d M a n u s c r i p t 11

13. TPMT activity

TPMT activity was measured by the reversed-phase HPLC method essentially as described previously [START_REF] Milek | Thiopurine Smethyltransferase pharmacogenetics: genotype to phenotype correlation in the Slovenian population[END_REF][START_REF] Keizer-Garritsen | Measurement of thiopurine S-methyltransferase activity in human blood samples based on high-performance liquid chromatography: reference values in erythrocytes from children[END_REF]. To report the TPMT activity as the amount (pmol) of 6-MMP formed per mg of protein per hour, an aliquot of the reaction mixture was used to determine protein concentration (see above).

14. TPMT protein level

TPMT protein level was determined by flow cytometric analysis of immunofluorescence resulting from cells stained for TPMT. Cells (2.0x10 6 ) were washed twice in PBS and fixed in 0.25% ice-cold paraformaldehyde (Sigma) for 1h. The cells were then pelleted and incubated in permeabilization buffer (0.2% Tween in PBS) for 15 min at 37°C. After incubating briefly in 3% BSA (Sigma), monoclonal anti-TPMT antibody (Abnova, Taipei, Taiwan) diluted in PBS (20 µg/ml) was added. Incubation for 1h on ice was followed by washing the cells in permeabilization buffer, and the pellet was again incubated briefly in 3% BSA, followed by the addition of fluorescein isothiocyanate (FITC)-conjugated secondary goat anti-mouse IgG antibody (Molecular Probes, Invitrogen) diluted in PBS (100 µg/ml). After incubation for 30 min on ice, the cells were washed twice, resuspended in PBS and analyzed on a FACSCalibur flow cytometer (BD Biosciences), detecting the FITC fluorescence intensity at 530 nm (FL1).

For background fluorescence gating, cells were stained with the secondary antibody alone.

TPMT protein expression was reported as the percentage of TPMT-stained cells in the range of <5% false positive cells stained with secondary antibody alone. Data analysis was carried out with CellQuest Pro software (BD Biosciences).

TPMT mRNA level

Total RNA was extracted from 4.0x10 6 cells using Trizol Reagent (Invitrogen) according to the manufacturer's instruction. RNA concentration was determined on an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA), and the integrity was checked by electrophoresis on an Agilent Technologies 2100 bioanalyzer. Reverse transcription (RT) and quantitative PCR were carried out as described previously [START_REF] Lindqvist | Real-time RT-PCR methodology for quantification of thiopurine methyltransferase gene expression[END_REF], by using Omniscript RT kit (Qiagen, Germantown, MD, USA), and a TPMT-specific Taqman Gene Expression assay (AssayID: Hs00909011_m1, Applied Biosystems, Foster City, CA, USA), relative to the expression of human cyclophilin A. Data were analyzed according to the modified comparative C t method of Pfaffl [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF].

16. Statistical analysis

Results were expressed as means ± S.D. from three independent experiments. Student's t-test was used for between-group comparisons, and the Spearman rank test (r) was used for correlations between parameters. All statistical analyses were carried out using Microsoft Office Excel 2003 (Microsoft Corporation) and SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA). A 2-sided P value of 0.05 or less was considered statistically significant.

Results

1. SAM reverses the cytostatic and cytotoxic effects of 6-MP on MOLT cells

A c c e p t e d M a n u s c r i p t 13The cytotoxicity induced by 6-MP was reversed by exogenous SAM in a concentration and time dependent manner in MOLT cells. This cell line serves as a model for 6-MP sensitivity [START_REF] Panetta | Mechanistic mathematical modelling of mercaptopurine effects on cell cycle of human acute lymphoblastic leukaemia cells[END_REF] and exhibits constitutive TPMT activity [START_REF] Keizer-Garritsen | Measurement of thiopurine S-methyltransferase activity in human blood samples based on high-performance liquid chromatography: reference values in erythrocytes from children[END_REF]. The analysis of genomic DNA of these cells revealed the absence of the three most prevalent low-activity mutant TPMT alleles (*2, *3A and *3C; data not shown).

Cell viability and proliferation experiments showed that MOLT cells were sensitive to the presence of 6-MP in the range of 1-20 µM. Proliferation was reduced to approx. 40% of the untreated control after 48-hour incubation with 5 µM 6-MP (Fig. 2A,C) (P<0.01). This concentration was used for all further experiments, since its effects were in accord with other published data [START_REF] Panetta | Mechanistic mathematical modelling of mercaptopurine effects on cell cycle of human acute lymphoblastic leukaemia cells[END_REF]. Approx. 80% of metabolic activity remained when cells were co-treated with 5 µM 6-MP and 25 µM SAM (Fig. 2B,C), indicating a reversal of 6-MP induced growth inhibition. Incubation with only 25 µM SAM had no effect on cell proliferation. This concentration was used for all further experiments.

In addition, we assessed the viability of cells to determine whether the cause of SAM-induced restoration of cell proliferation was also the cause of reduction in cytotoxic effects of 6-MP (Fig. 2D). Approx. 25% reduction in viability was observed when cells were treated with 6-MP for 48 h, while co-treatment with 6-MP and SAM resulted in a protective effect of SAM on 6-MP induced cell death; a 6% reduction in viability was observed when compared to the untreated cells (P<0.01). Therefore, the extent of both cytostatic and cytotoxic effects of 6-MP depends on the presence of exogenous SAM.

2. SAM delays and limits the onset of 6-MP induced programmed cell death

SAM displayed protective effects on 6-MP induced cytotoxicity on the molecular and cellular levels, as confirmed by differences in caspase-3 induction, annexin V staining, cell cycle A c c e p t e d M a n u s c r i p t 14 distribution, and DNA fragmentation (Fig. 3). In 6-MP treated cells, caspase-3 activity peaked after 24 h of incubation, while co-treatment with 6-MP and SAM resulted in maximum caspase-3 activity after 48 h (Fig. 3A). Caspase-3 activity in the co-treated sample after 24 h was approx. 60 % of that observed in cells treated with 6-MP (P<0.01).

To investigate the effect of SAM on the progression of cells to cell death, live cells were double-stained with annexin V and propidium iodide (PI). Flow cytometric analysis revealed that 30 % of 6-MP treated cells were apoptotic after 48 h of incubation, consisting of equal percentages of dead (annexin V + /PI + ) and pro-apoptotic (annexin V + /PI -) cells (Fig. 3B). This effect was partially reversed in the case of simultaneous treatment with SAM, resulting in 20 % of the cells being apoptotic (P=0.04).

Cell cycle distribution after 48 h of incubation with 6-MP and/or SAM was determined by flow cytometric analysis of ethanol-fixed cells stained with PI (Fig. 3C). The histogram representing the cell cycle profile of cells treated with 6-MP reflects the features of apoptotic population, characterized by high proportion of cells in subG 0 /G 1 phase (24 %), increased fraction of cells in the S phase (38 %), and a small population in G 2 /M phase cells (8 %), indicating a 6-MP-induced S phase arrest. However, co-treatment of cells with 6-MP and SAM had a less severe impact. The fraction of cells in the S phase was moderately increased compared to untreated cells (30 % vs 21 %; P<0.02), and additional accumulation of cells in G 2 /M phase was observed (22 % vs 14%; P<0.01). These results demonstrate that the presence of SAM enables the progression of cells through the S phase, a process strongly inhibited when cells are incubated with 6-MP alone.

Nuclear staining (Fig. 3D) revealed numerous cells with fragmented nuclei in 6-MP treated cultures, and only partially condensed but not fragmented nuclei in cultures co-treated with 6-

MP and SAM.

A c c e p t e d M a n u s c r i p t 15 These results suggest that SAM has no direct anti-apoptotic effects but rather delays the onset of downstream apoptotic signalling.

The concentration of cytotoxic 6-MP metabolites is lower in the presence of exogenous SAM

Cytosolic TGNs and MeTIMP are considered to be responsible for the cytotoxic effects of 6-MP, while the metabolic conversion to inactive 6-MMP diminishes these effects (Fig. 1) [START_REF] Yamane | Mismatch repair-mediated G2/M arrest by 6thioguanine involves the ATR-Chk1 pathway[END_REF][START_REF] Tay | Inhibition of phosphoribosyl pyrophosphate amidotransferase from Ehrlich ascites-tumor cells by thiopurine nucleotides[END_REF]. The addition of exogenous SAM influences drug metabolism dynamics and lowers the concentrations of TGNs and MeTIMP (Fig. 4).

Cytosolic TGNs and MeTIMP were determined in cell lysates by HPLC after different times of incubation (Fig. 4). The concentration of TGNs was significantly higher (P<0.01) when cells were incubated with 6-MP alone than in the case of co-treatment with 6-MP and SAM after 8 and 24 h (Fig. 4A). After 48 h of incubation, the concentration of TGNs was also higher in cells treated with 6-MP alone than with both 6-MP and SAM, although the p-value was slightly above the significance threshold (239.6±53,3 vs 171.5±38,1 pmol/10 6 viable cells; P=0,07). Similarly, MeTIMP concentrations were also significantly lower (P<0.05) in the cells treated with both 6-MP and SAM than in cells treated with 6-MP alone, at all times studied (Fig. 4B). Therefore, the reversal of cytostatic and cytotoxic effects, as well as the slower onset of apoptosis in the presence of SAM, can be explained by the lower production of both TGNs and MeTIMP. A significant (P<0.01) reduction in ATP concentration in 6-MP treated cells compared to untreated controls was determined after all times studied (Fig. 5A). This effect was partially reversed by the addition of SAM (P<0.05). Our data show that the concentration of MeTIMP was inversely correlated with ATP levels in the lysates of 6-MP, as well as 6-MP and SAM treated cells (6-MP: r=-0.99; P<0.001; 6-MP and SAM: r=-0.89; P=0.02), while no correlation of ATP and TGN concentrations was observed (6-MP: r=-0.49; P=0.11; 6-MP and SAM: r=-0.52; P=0.08). Therefore, intracellular ATP levels are determined by the concentration of the DNPS inhibitor MeTIMP in both 6-MP treated samples, which shows that exogenous SAM acts upon the processes upstream rather than downstream of MeTIMP and ATP production.

4. The concentration of MeTIMP determines the intracellular levels of ATP and SAM

Endogenous SAM has been shown to be depleted in MOLT cells incubated with 6-MP, since its endogenous synthesis from methionine requires ATP, which is depleted as a result of DNPS inhibition [START_REF] Stet | Decrease in S-adenosylmethionine synthesis by 6-mercaptopurine and methylmercaptopurine ribonucleoside in Molt F4 human malignant lymphoblasts[END_REF]. We observed a significant decrease in intracellular SAM levels (P<0.05) when cells were treated with 6-MP, which is in accord with previously published data (Fig. 5A). However, in cells co-treated with 6-MP and SAM for 24 h, no depletion of intracellular SAM levels was observed (P=0.02), indicating that SAM levels remain unchanged as a result of addition of exogenous SAM. After 48 h, endogenous SAM levels were lowered also in untreated cells, when compared to 24-hour incubation, which may be attributed to the lower availability of SAM precursors (e.g. methionine, folic acid) in culture media.

A c c e p t e d M a n u s c r i p t 17

5. SAM maintains the level of TPMT protein and activity

To address the molecular basis of the regulatory effects of SAM on 6-MP metabolism, TPMT enzyme activity, protein and mRNA levels were analysed after incubation with 6-MP and/or SAM (Fig. 6). Incubation of cells with 6-MP leads to a decreased TPMT activity and protein level, an effect which was reversed in the presence of SAM, indicating that SAM maintains TPMT activity levels.

For TPMT activity, the concentration of 6-MMP formed per unit time was determined by HPLC in samples of cell lysates incubated with the substrate 6-MP (2.25 mM; Fig. 6A).

Lower TPMT activity (P<0.001) was observed in 6-MP treated than in untreated cells.

However, when cells were co-treated with 6-MP and SAM the level of TPMT activity was not significantly different from that in control and SAM-treated cells (P>0.05). The concentration of intracellular SAM (Fig. 5B) correlated with TPMT activity (r=0.481; P=0.02), indicating that TPMT may be regulated by SAM through post-translational stabilisation.

Correspondingly, specific effects of 6-MP and/or SAM were also evident on the protein level, as determined by cytoplasmic staining of TPMT followed by flow cytometric analysis (Fig. 6B). A higher percentage of cells was found in the TPMT-positive range in cells co-treated with 6-MP and SAM for 48h, than with 6-MP alone (59.0 vs 51.1 %). The TPMT protein level was thus less reduced in cells co-treated with 6-MP and SAM than in cells treated with 6-MP alone (P<0.01). It should be noted that protein expression was analysed by flow cytometry since all immunoblotting experiments failed to give a satisfactory signal with low background staining, although several anti-TPMT antibodies from various commercial and custom-made sources were tested.

The decreased TPMT protein level was not due to lower TPMT mRNA expression, as determined by quantitative PCR (Fig. 6C). No significant changes in TPMT expression were

Discussion

The antiproliferative effects of cytotoxic metabolites of 6-MP have been extensively studied, both in vitro [START_REF] Stet | Decrease in S-adenosylmethionine synthesis by 6-mercaptopurine and methylmercaptopurine ribonucleoside in Molt F4 human malignant lymphoblasts[END_REF] and in vivo [START_REF] Lennard | Genetic variation in response to 6-mercaptopurine for childhood acute lymphoblastic leukemia[END_REF][START_REF] Hartford | Differential effects of targeted disruption of thiopurine methyltransferase on mercaptopurine and thioguanine pharmacodynamics[END_REF]. However, very few studies have addressed the impact of endogenous metabolites on drug metabolism [START_REF] Stet | Reversal of methylmercaptopurine ribonucleoside cytotoxicity by purine ribonucleosides and adenine[END_REF]. TPMT is a highly polymorphic enzyme, which catalyzes the S-methylation and consequent deactivation of 6-MP, but may also contribute to cytotoxicity through the formation of MeTIMP, a potent DNPS inhibitor [START_REF] Dervieux | Differing contribution of thiopurine methyltransferase to mercaptopurine versus thioguanine effects in human leukaemic cells[END_REF]. In the S-methylation reaction, SAM acts as a co-substrate by providing the methyl group, but also stabilises the protein structure on binding into the active site of TPMT (Fig. 1) [START_REF] Scheuermann | Consequences of binding an Sadenosylmethionine analogue on the structure and dynamics of the thiopurine methyltransferase protein backbone[END_REF].

Since low TPMT activity is strongly correlated with unfavourable side-effects during thiopurine treatment [START_REF] Weinshilboum | Thiopurine pharmacogenetics: clinical and molecular studies of thiopurine methyltransferase[END_REF], understanding of the role of SAM would enable prediction and modulation of 6-MP induced cytotoxic effects. In this study, human ALL cell line MOLT was used which serves as a relevant model to study the cytotoxic mechanisms and metabolism of thiopurines. We herein propose a novel TPMT-mediated mechanism of SAM-specific effects in the context of 6-MP induced cytotoxicity.

The main mechanism of 6-MP induced apoptosis appears to be a consequence of aberrant mismatch repair pathway signalling, as shown in primary CD4 + T lymphocytes, through the activation of the intrinsic mitochondrial pathway [START_REF] Tiede | CD28-dependent Rac-1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes[END_REF]. We delineated the SAM-specific effects on cell proliferation, viability and apoptosis, and found that the presence of exogenous SAM decreases the extent of both cytostatic and cytotoxic effects of 6-MP (Fig. 2), as well as delaying the onset of 6-MP induced apoptotic signalling (Fig. 3). This led us to hypothesize A c c e p t e d M a n u s c r i p t 19 that SAM does not have direct anti-apoptotic effects, but rather reverses the effects of 6-MP by altering the drug metabolism dynamics and, consequently, the amount of cytotoxic metabolites formed. We confirmed this notion by showing that addition of exogenous SAM to culture media significantly lowered the concentration of both cytotoxic TGN and MeTIMP metabolites (Fig. 4). Furthermore, the concentration of ATP was inversely correlated with MeTIMP concentration, indicating that the extent of MeTIMP induced inhibition of DNPS determines the concentrations of intracellular ATP, and consequently SAM (Fig. 5).

Evidently, the addition of exogenous SAM results not only in less extensive DNPS inhibition due to lower MeTIMP production, resulting in less depleted ATP levels, but also limits the production of TGNs. Therefore, we propose that SAM must act as a positive modulator of TPMT activity, which results in a greater amount of 6-MP being converted to 6-MMP, a process that occurs more extensively than the HPRT catalyzed conversion to TIMP (Fig. 1).

TPMT activity therefore determines the availability of TIMP for the multienzyme conversion to TGNs, as well as for the downstream TPMT-mediated MeTIMP synthesis.

The addition of exogenous SAM also reversed the 6-MP-induced decrease in intracellular SAM (Fig. 5B), TPMT activity and protein levels, but had no effect on TPMT mRNA expression (Fig. 6). Moreover, intracellular SAM levels correlated well with TPMT activity.

Although we have not evaluated the effect of exogenous SAM on TPMT mRNA stabilisation or protein synthesis rate, we propose that SAM has a stabilizing effect on TPMT, and maintains its enzyme activity levels. This notion is supported by reports of a positive effect of SAM on in vitro intrinsic stability of the recombinant wild-type protein [START_REF] Tai | Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT*3A, A c c e p t e d M a n u s c r i p t 24 TPMT*2): mechanisms for the genetic polymorphism of TPMT activity[END_REF]. In addition, the binding of a SAM analogue, sinefungin, into the active site of the bacterial orthologue of TPMT facilitates conformational stabilisation of the enzyme, which prevents intracellular degradation [START_REF] Scheuermann | Consequences of binding an Sadenosylmethionine analogue on the structure and dynamics of the thiopurine methyltransferase protein backbone[END_REF]. We thus assume that the mechanism of SAM-induced TPMT activity involves the SAM-dependent post-translational stabilisation of TPMT. As a result of DNPS A c c e p t e d M a n u s c r i p t 20 inhibition by MeTIMP, ATP is depleted, which prevents SAM re-synthesis through the transmethylation pathway. This causes lower TPMT stability and activity and limits the deactivation of 6-MP to 6-MMP, leading to higher TGN production and cytotoxicity.

However, in cells co-treated with 6-MP and SAM, the intracellular level of SAM remains sufficiently high to maintain high TPMT activity and protein level, resulting in rapid 6-MMP production, which in turn causes low availability of TIMP as a substrate for the production of cytotoxic metabolites.

Due to the pleiotropic properties of SAM, the principal methyl donor in processes such as nucleic acid, protein and phospholipid methylation [START_REF] Chiang | Sadenosylmethionine and methylation[END_REF], other mechanisms that may be responsible for the reversal of cytotoxic properties of 6-MP have to be considered. Due to SAM depletion, DNA was shown to be hypomethylated in MOLT cells treated with 6-MP [START_REF] Abreu | Thiopurine induced disturbance of DNA methylation in human malignant cells[END_REF][START_REF] Hogarth | The effect of thiopurine drugs on DNA methylation in relation to TPMT expression[END_REF], which may cause altered expression profiles of genes involved in cell proliferation and/or cell survival signalling. Although exogenous SAM might prevent DNA hypomethylation following treatment with 6-MP, which remains to be determined, this mechanism would only partly account for the reversal of cytotoxic effects in cells treated with both 6-MP and SAM, since it does not explain the significant decrease in the amounts of cytotoxic MeTIMP and TGNs observed in our study. Similarly, SAM-dependent polyamine synthesis most probably remains largely unaltered [START_REF] Schipper | Polyamines and DNA methylation in childhood leukaemia[END_REF], due to the addition of SAM, ensuring normal polyamine homeostasis, and consequently less compromised growth of 6-MP treated cells. However, no evidence exists for the interaction between metabolic pathways of polyamines and thiopurines. Therefore, we may conclude that the decrease in cytotoxic 6-MP metabolites is the consequence of post-translational protein stabilization of TPMT by SAM, resulting in lower availability of TIMP as the substrate for conversion to cytotoxic metabolites. However, the observed reduction in 6-MP toxicity on the cellular level may be the result of additional direct and/or indirect actions of SAM, which remains to be addressed. a well-designed in vivo approach should be carried out to confirm these findings. In patients receiving 6-MP, a decrease in TPMT activity may be expected after 6-MP administration, due to DNPS inhibition and decreased synthesis of the stabilising factor SAM. In patients with wild-type or heterozygous mutant TPMT genotypes, who exhibit high and intermediate TPMT activity, respectively, a decrease in the enzyme activity may result in an overproduction of TGNs, increasing the risk of undesirable toxicity. On the other hand, high levels of endogenous SAM, as well as potential compensatory responses to SAM depletion, may contribute to the detoxification of the drug, and consequently render the wild-type patients to be non-responders, by decreasing the production of cytotoxic TGNs. The availability of folate pools may also significantly influence 6-MP related cytotoxic effects, since the metabolic fluxes of homocysteine remethylation and downstream SAM synthesis are folate dependent [START_REF] Chiang | Folate restriction and methylenetetrahydrofolate reductase 677T polymorphism decreases adoMet synthesis via folate-dependent remethylation in human-transformed lymphoblasts[END_REF]. The mechanism described in the present study could therefore play an important role in patients receiving folates in dietary supplements during thiopurine treatment, modulating the amount of SAM, and consequently TPMT activity. Further in vivo studies of the correlation of TPMT with the activity of enzymes involved in SAM metabolism, (e.g. methionine adenosyltransferase, S-adenosylmethionine decarboxylase, 5,10methylenetetrahydrofolate reductase), could reveal additional factors influencing treatment with 6-MP. Moreover, the presence of activity-modulating genetic polymorphisms in these enzymes may explain the poor TPMT genotype-to-phenotype correlations observed in some individuals [START_REF] Arenas | Genetic variation in the MTHFR gene influences thiopurine methyltransferase activity[END_REF][START_REF] Kuzelicki | Heterozygosity at the TPMT gene locus, augmented by mutated MTHFR gene, predisposes to 6-MP related toxicities in childhood ALL patients[END_REF].

In conclusion, our results provide new insights into the regulation of 6-MP metabolism by the TPMT-modulating factor SAM, and suggest that SAM status is an important parameter of thiopurine therapy-related toxicity.

  , 6-MP, 6-MMP, MMPR, 6-TG, SAM, N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), dithiothreitol (DTT), DMSO, ATP and propidium iodide (PI) were from Sigma Chemical Co. (St. Louis, MO, USA). For cell culture applications, stock solutions of 6-MP (225 mM in DMSO) and SAM (100 mM in PBS) were filtered (0.2 µm) and diluted in A c c e p t e d M a n u s c r i p t 6 cell culture medium to the appropriate concentration, prior to the addition to cells. In the case of vehicle-treated controls, cell culture media containing the same amount of DMSO or PBS were added. Perchloric acid, ethanol, HPLC-grade acetonitrile and methanol were from Sigma.

  positive control of caspase-3 induction. Data were expressed as increase in fluorescence intensity as a function of time (dF/dt).

A c c e p t e d M a n u s c r i p t 10 2. 11 .

 1011 Intracellular ATP levelsATP concentrations were determined by the CellTiter Glo Luminescent Cell Viability Assay™ (Promega) according to the manufacturer's instruction. After incubation with the chemicals, viable cells were plated into 96-well opaque microtiter plates (NalgeNunc, Roskilde, Denmark) (1.0x10 4 cells per well) and incubated with the CellTiter Glo reagent

  A c c e p t e d M a n u s c r i p t 12 2.

A c c e p t e d M a n u s c r i p t 16 To

 16 investigate the extent of MeTIMP induced inhibition of DNPS on re-synthesis of SAM, the intracellular levels of ATP and SAM were analyzed in cells either treated with 6-MP or co-treated with 6-MP and SAM. Higher concentrations of ATP and SAM were detected in cell lysates of co-treated compared to 6-MP treated cells.

A

  c c e p t e d M a n u s c r i p t 18 observed as a result of treatment of cells with 6-MP or with 6-MP and SAM (P>0.05). SAM thus appears to maintain the level of TPMT activity, most probably by post-translational stabilisation of the protein.

A c c e p t e d M a n u s c r i p t 21 Several

 21 implications of the present study regarding thiopurine treatment are evident, although

Fig. 1 -

 1 Figure captions

Fig. 2 -

 2 Fig. 2 -SAM reverses the cytostatic and cytotoxic effects of 6-MP in MOLT cells.

Fig. 3 -

 3 Fig. 3 -SAM delays the onset of 6-MP induced programmed cell death in MOLT cells.

A c c e p t e d M a n u s c r i p t 29 Apoptotic

 29 features of cells were evaluated after incubation with 6-MP (5 µM) and/or SAM (25 µM) for the designated time periods. The asterisks indicate significant differences from the 6-MP treated sample (t test; *P<0.05; **P<0.01). (A) Caspase-3 activity was determined in whole cell lysates after the indicated time periods by recording continuous fluorescence intensity changes (excitation, 405 nm; emission, 535 nm) resulting from the cleaved Ac-DEVD.AFC substrate. (B) Live cells were stained with annexin V-FITC and PI after 48 hours of incubation to determine the fractions of early apoptotic (annexin V + /PI -) and dead cells (annexin V + /PI + ) by flow cytometric analysis. (C) Cell cycle analysis was performed after 48hour incubation in the presence or absence of 6-MP and/or SAM on ethanol-fixed cells stained with propidium iodide. (D) Representative images after 48 h of incubation of live cells stained with the nucleus-specific Hoechst 33342 dye. Evidence of nuclear fragmentation is indicated by red arrows. Magnification, 960x; scale bar, 20 µm.

Fig. 4 -

 4 Fig. 4 -The concentration of toxic 6-MP metabolites is lower in the presence of

  after the digestion of the cell lysate with alkaline phosphatase. The concentration of MeTIMP present in cells co-treated with 6-MP and SAM was significantly lower than that with 6-MP alone at all times studied [8 h: 15.0±0.5 vs 25.4±1.1 (P=0.007); 24 h: 33.5±10.4 vs 88.6±8.1 (P=0.03); 48 h, 51.2±7.2 vs 114±5.7 pmol/10 6 viable cells (P=0.006)].

Fig. 5 - 3 (

 53 Fig. 5 -Exogenous SAM maintains the intracellular levels of ATP and SAM.

Fig. 6 -

 6 Fig. 6 -SAM maintains the level of TPMT protein and activity.

A

  c c e p t e d M a n u s c r i p t 31 with the substrate 6-MP (2.25 mM) and TPMT activity is reported as the amount of 6-MMP formed after the 2-hour incubation. TPMT activity in the cells treated with 6-MP alone was found to be lower than in untreated control [24 h: 238.3±18.1 vs 381.7±35.0 (P<0.001); 48 h: 212.0±31.1 vs 296.1±11.1 pmol/mg/h (P<0.001)], while the activity of cells with both 6-MP and SAM was unchanged [24 h: 365.5±72.9 (p>0.05); 48 h: 297.4±73.6 pmol/mg/h (P>0.05)]. (B) Representative histograms of flow cytometric analysis of TPMT protein levels after 48 hours of incubation (top). TPMT was detected in permeabilized cells by a secondary FITC-conjugated anti-mouse IgG antibody bound to a monoclonal anti-TPMT antibody.TPMT expression was reported as the percentage of TPMT-stained cells (solid line, transparent peak) in the range (M1) of <5% false positive (control) cells stained with secondary antibody alone (dotted line, grey peak). TPMT protein expression is presented graphically as percent of expression in control cells from three independent experiments ± S.D. (bottom). (C) Total RNA was isolated from cells after 24 and 48h of incubation, the mRNA was reverse transcribed and TPMT cDNA quantified by the Taqman gene expression assay. The expression ratio was calculated as the relative amount in the treated to control samples.

Acknowledgements

We would like to thank Igor Locatelli and Iztok Grabnar for technical assistance and Prof.

Roger H. Pain for proof reading the manuscript. This work was supported by the Slovenian Research Agency grant No. J3-7406.


Javiermegías

Isabel Guillén

Victoria Clérigues

Ana I Rojo

Antonio Cuadrado

Miguel Angel Castejón

Francisco Gomar

María José Alcaraz
email: maria.j.alcaraz@uv.es

Prof M J Alcaraz

Heme oxygenase-1 induction modulates microsomal prostaglandin E synthase-1 expression and prostaglandin E 2 production in osteoarthritic chondrocytes

A c c e p t e d M a n u s c r i p t 2 ABSTRACT Pro-inflammatory cytokines such as interleukin-1 (IL-1may participate in the pathogenesis of cartilage damage in osteoarthritis (OA) through the production of catabolic enzymes and inflammatory mediators. Induction of heme oxygenase-1 (HO-1) has previously been shown to exert anti-inflammatory effects in different cel types. We have investigated whether HO-1 induction may modify chondrocyte viability and the production of relevant mediators such as oxidative stress and prostaglandin E 2 (PGE 2 ) elicited by IL-1 in OA chondrocytes. Chondrocytes were isolated from OA cartilage and used in primary culture. Cells were stimulated with IL-1 in the absence or presence of the HO-1 inducer cobalt protoporphyrin IX (CoPP). Gene expression was assessed by quantitative real-time PCR, protein levels by ELISA and Western blot, apoptosis by laser scanning cytometry using annexin V-FITC and TUNEL assays, and oxidative stress by LSC with dihydrorhodamine 123. HO-1 induction by CoPP enhanced chondrocyte viability and aggrecan content while inhibiting apoptosis and oxidative stress generation. PGE 2 is produced in OA chondrocytes stimulated by IL-1 by the coordinated induction of cyclooxygenase-2 and microsomal PGE synthase 1 (mPGES-1). The production of PGE 2 was decreased by HO-1 induction as a result of diminished mPGES-1 protein and mRNA expression. Transfection with HO-1 small interfering RNA counteracted CoPP effects. In addition, the activation of nuclear factor-B and early growth response-1 was significantly reduced by CoPP providing a basis for its anti-inflammatory effects. These results confirm the protective role of HO-1 induction in OA chondrocytes and suggest the potential interest of this strategy in degenerative joint diseases. The chronic production of inflammatory mediators by articular tissues is an important feature of rheumatoid arthritis and osteoarthritis (OA) contributing to the degenerative process. In these conditions, high levels of pro-inflammatory cytokines such as interleukin(IL)-1have been found in synovial fluids and may participate in collagen and proteoglycan degradation [START_REF] Dodge | Immunohistochemical detection and immunochemical analysis of type II collagen degradation in human normal, rheumatoid, and osteoarthritic articular cartilages and in explants of bovine articular cartilage cultured with interleukin 1[END_REF][START_REF] Van Der Kraan | Anabolic and destructive mediators in osteoarthritis[END_REF]. Moreover, chondrocyte activation by proinflammatory cytokines has been implicated in joint destruction through the production of catabolic enzymes and inflammatory mediators such as reactive oxygen species (ROS) and prostaglandin E 2 (PGE 2 ) [START_REF] Attur | Autocrine production of IL-1 beta by human osteoarthritis-affected cartilage and differential regulation of endogenous nitric oxide, IL-6, prostaglandin E2, and IL-8[END_REF][START_REF] Henrotin | The role of reactive oxygen species in homeostasis and degradation of cartilage[END_REF].

ROS can modulate cell signaling and exert damaging effects on cartilage as a consecuence of extracellular matrix degradation or chondrocyte apoptosis [START_REF] Henrotin | The role of reactive oxygen species in homeostasis and degradation of cartilage[END_REF][START_REF] Burkhardt | Oxygen radicals as effectors of cartilage destruction. Direct degradative effect on matrix components and indirect action via activation of latent collagenase from polymorphonuclear leukocytes[END_REF][START_REF] Clancy | Activation of stress-activated protein kinase in osteoarthritic cartilage: evidence for nitric oxide dependence[END_REF][START_REF] Yoon | Sustained production of H(2)O(2) activates pro-matrix metalloproteinase-2 through receptor tyrosine kinases/phosphatidylinositol 3-kinase/NF-kappa B pathway[END_REF].

Furthermore, studies examining the life span of articular chondrocytes have demonstrated that oxidative stress is related to premature senescence and may be responsible for the development of OA [START_REF] Yudoh | Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function[END_REF][START_REF] Dai | Catabolic stress induces features of chondrocyte senescence through overexpression of caveolin 1: possible involvement of caveolin 1-induced downregulation of articular chondrocytes in the pathogenesis of osteoarthritis[END_REF].

The arachidonic acid metabolite PGE 2 , a mediator of inflammation and pain, is produced in OA chondrocytes stimulated by IL-1 by the coordinated induction of cyclooxygenase-2 (COX-2) and microsomal PGE synthase 1 (mPGES-1) [START_REF] Masuko-Hongo | Up-regulation of microsomal prostaglandin E synthase 1 in osteoarthritic human cartilage: critical roles of the ERK-1/2 and p38 signaling pathways[END_REF]. Previous studies have suggested the interest of this last enzyme as a novel target for OA [START_REF] Masuko-Hongo | Up-regulation of microsomal prostaglandin E synthase 1 in osteoarthritic human cartilage: critical roles of the ERK-1/2 and p38 signaling pathways[END_REF].

Although some studies have reported that mice lacking mPGES-1 do not show significant differences with respect to wild type animals in OA induced by surgical procedures [START_REF] Yamakawa | Association of microsomal prostaglandin E synthase 1 deficiency with impaired fracture healing, but not with bone loss or osteoarthritis, in mouse models of skeletal disorders[END_REF], it is interesting to note that mPGES-1 defficiency results in milder arthritis and lower cartilage degradation [START_REF] Trebino | Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase[END_REF].

Heme oxygenase-1 (HO-1) is induced by oxidative stress (reviewed in [START_REF] Alcaraz | Anti-inflammatory actions of the heme oxygenase-1 pathway[END_REF]) and can inhibit apoptosis in several cell types such as T cells [START_REF] Pae | Roles of heme oxygenase-1 in the antiproliferative and antiapoptotic effects of nitric oxide on Jurkat T cells[END_REF][START_REF] Choi | Overexpression of heme oxygenase (HO)-1 renders Jurkat T cells resistant to fas-mediated apoptosis: involvement of iron released by HO-1[END_REF], endothelial cells [START_REF] Brouard | Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis[END_REF] A c c e p t e d M a n u s c r i p t 5 and enterocytes [START_REF] Busserolles | Heme oxygenase-1 inhibits apoptosis in Caco-2 cells via activation of Akt pathway[END_REF]. In human OA chondrocytes, HO-1 induction by low doses of sodium nitroprusside has been shown to protect against apoptosis induced by high doses of this agent [START_REF] Kim | The mechanism of low-concentration sodium nitroprusside-mediated protection of chondrocyte death[END_REF]. We have reported previously that HO-1 expression in OA chondrocytes is down-regulated by pro-inflammatory cytokines such as IL-1, IL-17 y tumor necrosis factor- but up-regulated by the antiinflammatory cytokine IL-10 [START_REF] Fernandez | Expression of heme oxygenase-1 and regulation by cytokines in human osteoarthritic chondrocytes[END_REF].

Recently, we have also shown a protective effect of HO-1 induction on cartilage degradation [START_REF] Guillen | Heme oxygenase-1 regulates catabolic and anabolic processes in osteoarthritic chondrocytes[END_REF]. We hypothesized that HO-1 could control metabolic processes in chondrocytes relevant in the progression of OA. Therefore, we have investigated whether HO-1 induction by cobalt protoporphyrin IX (CoPP) may modify cell viability and the production of oxidative stress and PGE 2 in OA chondrocytes in primary culture, thus avoiding the phenotypic changes associated with cell culture [START_REF] Von Der | Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture[END_REF].

Material and methods

Reagents

IL-1 was from Peprotech EC Ltd. (London, UK). COX-2 and mPGES-1 polyclonal antibodies were purchased from Cayman Chemical (Ann Arbor, MI, USA) and the HO-1 antibody was from Stressgen (Victoria, Canada). The peroxidase-conjugated IgGs were purchased from Dako (Copenhagen, Denmark). [START_REF] Burkhardt | Oxygen radicals as effectors of cartilage destruction. Direct degradative effect on matrix components and indirect action via activation of latent collagenase from polymorphonuclear leukocytes[END_REF][START_REF] Clancy | Activation of stress-activated protein kinase in osteoarthritic cartilage: evidence for nitric oxide dependence[END_REF][START_REF] Yudoh | Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function[END_REF][START_REF] Yamakawa | Association of microsomal prostaglandin E synthase 1 deficiency with impaired fracture healing, but not with bone loss or osteoarthritis, in mouse models of skeletal disorders[END_REF][START_REF] Trebino | Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase[END_REF][START_REF] Pae | Roles of heme oxygenase-1 in the antiproliferative and antiapoptotic effects of nitric oxide on Jurkat T cells[END_REF][START_REF] Choi | Overexpression of heme oxygenase (HO)-1 renders Jurkat T cells resistant to fas-mediated apoptosis: involvement of iron released by HO-1[END_REF](n)-3 H]PGE 2 was from GE Healthcare (Barcelona, Spain). Pre-designed small interfering RNA (siRNA) oligonucleotides and SiPORT™Amine were purchased from Ambion Inc.

Chondrocyte culture

Cartilage specimens were obtained from 18 patients with the diagnosis of advanced OA undergoing total knee joint replacement. Samples were obtained under the Institutional Ethical Commitee approved protocol. Cartilage slices were removed from the femoral condyles and tibial plateaus and cut into small pieces. Chondrocytes were isolated by sequential enzymatic digestion: 1 h with 0.1 mg/ml hyaluronidase (Sigma) followed by 12 h with 2 mg/ml collagenase (type IA) (Sigma) in DMEM/Ham's F-12 (Sigma) containing penicillin (100 U/ml) and streptomycin (100 g/ml) at 37ºC in 5% CO 2 atmosphere. The digested tissue was filtered through a 70 m nylon mesh, washed and centrifuged. Cell viability was greater than 95 % according to the Trypan blue exclusion test. The isolated chondrocytes were seeded at 2.5 x 10 5 cells/well in six-well plates.

Cells were cultured in growth medium: DMEM/Ham's F-12 supplemented with 10 % human serum, penicillin (100 U/ml) and streptomycin (100 g/ml) in a humidified 5 % CO 2 incubator at 37°. Chondrocytes were allowed to grow until nearly confluence and then incubated with CoPP (10 M) or vehicule for 1 h before stimulation with IL-1 (100 U/ml). In experiments using HO-1 siRNA (100 nM, sense 5'-GGCAGAGAAUGCUGAGUUCtt-3', antisense 5'-GAACUCAGCAUUCUCUGCCtg-3'), transfection was performed in SiPORT™Amine following the manufacturer's recommendations, 24 h before other experimental procedures. A nonspecific siRNA (Ambion) was used as negative control. Determination of aggrecan was performed by ELISA using a kit from Biosource Europe S.A. (Nivelles, Belgium) with sensitivity of 0.9 ng/ml. For lentivirus transduction of chondrocytes, OA chondrocytes isolated as indicated above were cultured in alginate [START_REF] Shakibaei | Differentiation of mesenchymal limb bud cells to chondrocytes in alginate beads[END_REF]. The pellet of primary chondrocytes (1.5x10 6 /ml) was resuspended in sterile alginate (2% in 0.15 M NaCl) and slowly added Chondrocytes in alginate beads were cultured for 10 days in growth medium supplemented with 25 g/ml ascorbic acid.

Transduction with lentiviral vectors

Human embryonic kidney (HEK) 293T cells were grown in DMEM/Ham´s F-12 supplemented with 10% human serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) in a humidified 5% CO 2 incubator at 37 ºC. For lentiviral production we used the at -80ºC. The titers of lentiviral stocks were in the range of 3-5x10 5 IU/ml as determined by immunocytochemical analysis of HEK293T-infected cells [START_REF] Rojo | GSK-3beta down-regulates the transcription factor Nrf2 after oxidant damage: relevance to exposure of neuronal cells to oxidative stress[END_REF]. The three-dimension chondrocyte culture in alginate was infected with 500 µl of each lentiviral stock for 24 h in a humidified 5% CO 2 incubator at 37ºC. After infection and culture in growth medium for two days, alginate beads were stimulated with IL-1 (100 U/ml) for 24 h.

Finally, HO-1 and mPGES-1 expression was measured by immunocytochemical analysis. The beads were fixed with 4% formaldehyde in PBS for 10 minutes and incubated with two antibodies at the same time: anti-FLAG monoclonal antibody (Sigma, 1/400) and anti-mPGES-1 polyclonal antibody (Cayman Chemical, 1/50) for 1.5 h, followed by incubation with the secondary antibodies Alexa Fluor 488 goat antimouse IgG (Invitrogen) and R-phycoerythrin goat anti-rabbit IgG (Invitrogen) for 45 minutes at 37º C. Cell nuclei were counterstained with 4',6'-diamidino-2-phenylindole (DAPI) solution (1/1000). Slides were examined under a fluorescence microscope (Eclipse E800, Nikon Instruments Europe, Amstelveen, The Netherlands). Each experiment was performed in triplicate.

Determination of PGE 2

Chondrocytes in primary culture were stimulated with IL-1 (100 U/ml) or IL-1+CoPP (10 M) for 24 h. Supernatants were harvested and frozen at -80ºC until analysis. PGE 2 was measured by radioimmunoassay [START_REF] Moroney | Selectivity of neutrophil 5-lipoxygenase and cyclo-oxygenase inhibition by an antiinflammatory flavonoid glycoside and related aglycone flavonoids[END_REF]. In another set of experiments, chondrocytes in primary culture were stimulated with IL-1 (100 U/ml) for 24 h to induce COX-2 and mPGES-1, and then cells were washed with medium and incubated with arachidonic acid (10 M) and either CoPP or NS-398 (10 M). After 3 h, supernatants were harvested and PGE 2 measured as above.

A c c e p t e d M a n u s c r i p t 9

Western blotting

After 24 h stimulation with IL-1 (100 U/ml) or IL-1+CoPP (10 M), chondrocytes in primary culture were lysed in 100 μl of buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 10 min at 10,000 x g. Formation of intracellular ROS was detected using DHR which is oxidized to fluorescent rhodamine (excitation at 485 nm and emission at 534 nm). Chondrocytes (75,000/well) were incubated with DMEM without phenol red and DHR (5 M) for 15 min at 37ºC. After washing, fresh medium was added and cells were incubated with CoPP for 4 h and then with IL-1 in the presence or absence of CoPP for other 30 min.

Proteins ( 25
Cultures were washed twice with fresh medium and analyzed by LSC.

Real-time PCR

Chondrocytes in primary culture were stimulated with IL-1 (100 U/ml) or IL-1+CoPP (10 M) for 12 h. Total RNA was extracted using the TRIzol reagent (Life Technologies Inc. Barcelona, Spain) according to the manufacturer's instructions.

Reverse transcription was accomplished on 1 g of total RNA using random primers (TaqMan reverse transcription reagents, Applied Biosystems Spain, Madrid). PCR assays were performed in duplicate on an iCycler Real-Time PCR Detection System using SYBR Green PCR Master Mix (Bio-Rad Laboratories, Richmond, CA, USA) as previously described [START_REF] Guillen | Heme oxygenase-1 regulates catabolic and anabolic processes in osteoarthritic chondrocytes[END_REF]. Primer sequences are shown in table 1. Cycle threshold (CT) values for each gene were corrected using the mean CT value for -actin. Relative gene expression was calculated using the ∆C T method and expressed as fold change (2 -∆∆C T)

relative to the expression values in nonstimulated cells.

Activation of transcription factors

Chondrocytes were seeded into 6-well plates and grown to 50-60 % confluence.

Transient transfection was performed overnight with 2 g of the reporter construct The medium was then replaced and after 18 h cells were treated for 24 h with CoPP in the absence or presence of IL-1 (100 U/ml). After lysis and centrifugation, aliquots of supernatants were used to assay firefly and Renilla luciferase activity using the Dual-Luciferase Reporter Assay System kit (Promega Corporation). Luminescence was measured in a Microbeta counter (Wallac, Turku, Finland) and firefly luciferase activity was normalized to Renilla luciferase activity.

Data analysis

Results are presented as mean ± SEM. Statistical analyses were performed using oneway ANOVA followed by Dunnett's t-test for multiple comparisons and unpaired Student's t-test for dual comparisons.

Results

HO-1 induction increases chondrocyte viability

According to previous studies [START_REF] Guillen | Heme oxygenase-1 regulates catabolic and anabolic processes in osteoarthritic chondrocytes[END_REF], we used CoPP (10 M) to induce HO-1 in OA chondrocytes. To determine whether cell viability is modified in our experimental conditions, we performed LSC experiments. We observed a significant reduction in cell viability after IL-1 treatment for 24 h (Fig. 1A), which was counteracted by CoPP.

We determined whether HO-1 induction by CoPP was able to control apoptosis, an important factor in the evolution of OA [START_REF] Heraud | Apoptosis in normal and osteoarthritic human articular cartilage[END_REF]. Fig. 1B shows that IL-1 weakly induced The ability to synthesize extracellular matrix components is a feature of chondrocyte metabolism. Our results indicate that CoPP treatment was also able to counteract the loss of aggrecan induced by IL-1. Treatment with this cytokine for 24 h reduced aggrecan content in chondrocytes from 11618 ±1664 to 7699 ±567 ng proteoglycan/mg protein (n=6, p<0.05). In contrast, chondrocytes treated with CoPP+IL-1 showed proteoglycan levels similar to those of non-stimulated cells (12063 ±522, ng proteoglycan/mg protein, n=6, p<0.05 with respect to IL-1). Thus, our investigations suggest that overexpression of HO-1 confers to OA chondrocytes an increased resistance to the deleterious effects of IL-1.

HO-1 induction inhibits oxidative stress

Since many of the effects of IL-1 on chondrocyte metabolism are mediated by the generation of ROS, we wanted to determine whether the production of oxidative stress could be modified by HO-1 induction. Stimulation of chondrocytes by this cytokine resulted in a significant level of ROS generation (Fig. 2). Consistent with an antioxidative role for HO-1 induction, oxidative stress was significantly decreased in cells treated with CoPP. The effect of HO-1 induction was confirmed in experiments

Effects on PGE 2 , COX-2 and mPGES-1

As a result of COX-2 and mPGES-1 induction, IL-1strongly increased PGE 2 levels after chondrocyte stimulation for 24 h. Production of PGE 2 was significantly reduced in cells treated with CoPP and stimulated with IL-1 (Fig. 3A). To confirm that CoPP effects were due to the induction of HO-1, we used a siRNA specific for human HO-1.

This agent nearly abolished the induction of HO-1 protein by CoPP, whereas a nonspecific siRNA failed to modify HO-1 protein expression (Fig. 3B). Interestingly, the treatment with the first siRNA reverted the inhibitory effects of CoPP on PGE 2 (Fig. 3A). The possible contribution of inhibitory effects on enzymatic activities regulating 2). To confirm that HO-1 overexpression results in reduced mPGES-1 expression, a three-dimension culture of OA chondrocytes was transduced with a lentiviral HO-1 vector (LV-HO-1). Fig. 4 shows that mPGES-1 was strongly induced by IL-1 in cells transduced with the empty vector LV-. In contrast, in cells transduced with LV-HO-1, mPGES-1 was hardly detected after IL-1 stimulation.

Effects on the activation of transcription factors

Because NF-B is a main regulator of pro-inflammatory genes, we analyzed the contribution of this transcription factor to the effects of CoPP. As shown in Fig. 5A, IL-1 activated NF-B in OA chondrocytes. Although CoPP treatment of cells did not modify basal luciferase activity, it significantly decreased the activation of this transcription factor in the presence of IL-1. In contrast, the activation of AP-1, another transcription factor involved in the expression of pro-inflammatory genes [START_REF] Liacini | Inhibition of interleukin-1stimulated MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NF-kappa B) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes[END_REF], was not influenced by CoPP (Fig. 5B). As EGR-1 is a key transcription factor in regulating the inducible expression of mPGES-1 [START_REF] Cheng | Activation of peroxisome proliferator-activated receptor gamma inhibits interleukin-1beta-induced membrane-associated prostaglandin E2 synthase-1 expression in human synovial fibroblasts by interfering with Egr-1[END_REF], we investigated the effects of CoPP treatment on EGR-1 activation. When IL-1 was incubated with chondrocytes, EGR-1 activity was significantly enhanced. It is interesting that the treatment of chondrocytes with CoPP led to a significant reduction of EGR-1-luc promoter activation in cells stimulated with IL-1 as well as in non-stimulated cells (Fig. 5C).

Discussion

Pro-inflammatory cytokines such as IL-1 induce the formation of intracellular oxidants which may function as second messengers leading to augmentation of gene expression of degradative enzymes [START_REF] Lo | Interleukin-1 beta induction of c-fos and collagenase expression in articular chondrocytes: involvement of reactive oxygen species[END_REF]. In addition, ROS have been implicated in cartilage degeneration in OA through the initiation of chondrocyte apoptosis [START_REF] Del | Nitric oxide-mediated chondrocyte cell death requires the generation of additional reactive oxygen species[END_REF], decreased A c c e p t e d M a n u s c r i p t 15 replicative potential, catabolic changes in matrix cartilage, telomere instability and senescence [START_REF] Yudoh | Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function[END_REF]. It is known that HO-1 induction can decrease the levels of the prooxidant heme and increase the generation of antioxidant molecules (biliverdin and bilirubin) and the anti-apoptotic and anti-inflammatory agent carbon monoxide (reviewed in [START_REF] Alcaraz | Anti-inflammatory actions of the heme oxygenase-1 pathway[END_REF]). In the present study we provide evidence of a protective role for HO-1 induction against oxidative stress in human OA chondrocytes. Our results also suggest that inhibition of ROS generation may contribute to the beneficial effects of CoPP on viability, apoptosis and extracellular matrix metabolism in primary OA chondrocytes.

Therefore, the antioxidant properties of HO-1 may be relevant for its chondroprotective effect.

Previous studies have demonstrated an increased expresion of COX-2 and mPGES-1 in OA cartilage [START_REF] Li | Expression and regulation of microsomal prostaglandin E synthase-1 in human osteoarthritic cartilage and chondrocytes[END_REF] and chondrocytes [START_REF] Grimmer | Hypoxia-inducible factor 1alpha is involved in the prostaglandin metabolism of osteoarthritic cartilage through up-regulation of microsomal prostaglandin E synthase 1 in articular chondrocytes[END_REF]. Experimental evidence indicate that IL-1 induces in chondrocytes large elevations of COX-2 and mPGES-1 leading to increased levels of PGE 2 . Besides its pro-inflammatory effects, this mediator can participate in the catabolic-anabolic imbalance in OA by inhibiting tissue inhibitor of metalloproteinases-1 synthesis or enhancing matrix metalloproteinase production [START_REF] Yamada | Effects of indomethacin on the production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 by human articular chondrocytes[END_REF][START_REF] Shapiro | Induction of macrophage metalloproteinases by extracellular matrix[END_REF] Recently, we have reported that HO-1 protects against cartilage degradation through the inhibition of the expression of several matrix metalloproteinases [START_REF] Guillen | Heme oxygenase-1 regulates catabolic and anabolic processes in osteoarthritic chondrocytes[END_REF]. Here, we show for the first time a regulatory role of HO-1 in PGE 2 production due to mPGES-1 modulation, in primary OA chondrocytes stimulated with IL-1, which may participate in the observed protective effects on cartilage.

NF-B and AP-1 are involved in the transcription of a number of inflammatory genes and MMPs [START_REF] Liacini | Inhibition of interleukin-1stimulated MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NF-kappa B) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes[END_REF][START_REF]Advances in NF-kappaB signaling transduction and transcription[END_REF]. Our data suggest that the inhibitory effects of HO-1 induction on the first transcription factor could play a role in the recently reported inhibition of MMP expresion [START_REF] Guillen | Heme oxygenase-1 regulates catabolic and anabolic processes in osteoarthritic chondrocytes[END_REF]. Although the COX-2 promoter contains multiple regulatory sites including those able to interact with both transcription factors, it has been reported that COX-2 induction by IL-1 in human chondrocytes may be dependent on other transcription factors [START_REF] Thomas | Critical role of C/EBPdelta and C/EBPbeta factors in the stimulation of the cyclooxygenase-2 gene transcription by interleukin-1beta in articular chondrocytes[END_REF], which would be consistent with the lack of effect of CoPP on COX-2 expression.

mPGES-1 is induced by pro-inflammatory cytokines in many cell types and has been proposed as a target in inflammatory diseases [START_REF] Stichtenoth | Microsomal prostaglandin E synthase is regulated by proinflammatory cytokines and glucocorticoids in primary rheumatoid synovial cells[END_REF] and OA [START_REF] Masuko-Hongo | Up-regulation of microsomal prostaglandin E synthase 1 in osteoarthritic human cartilage: critical roles of the ERK-1/2 and p38 signaling pathways[END_REF][START_REF] Kojima | Membraneassociated prostaglandin E synthase-1 is upregulated by proinflammatory cytokines in chondrocytes from patients with osteoarthritis[END_REF]. Recent studies have revealed that EGR-1 could be a key regulator of mPGES-1 transcription in response to cytokine stimulation [START_REF] Naraba | Transcriptional Regulation of the Membrane-associated Prostaglandin E2 Synthase Gene. Essential role of transcription factor Egr-1[END_REF]. Therefore, we sought to establish whether HO-1 induction could regulate this transcription factor. Our data have shown that HO-1 induction results in a significant decrease of EGR-1 activation in primary OA chondrocytes stimulated with IL-1, thus providing a mechanism for the inhibition of mPGES-1 expression. Previous studies from our lab have shown the inhibitory effects

A c c e p t e d M a n u s c r i p t 3

 3 Key words: Heme oxygenase-1, Chondrocyte, Osteoarthritis, Interleukin-1, Prostaglandin E 2 , microsomal PGE synthase-1.Running headline: HO-1 in chondrocytesA c c e p t e

(

  Austin, TX, USA). CoPP was purchased from Frontier Scientific Europe Ltd.(Carnforth, UK). Dihydrorhodamine 123 (DHR) was from Molecular Probes®, (Invitrogen S.A., Barcelona, Spain). The rest of reagents were from Sigma Aldrich (St Louis, MO, USA).

  solution containing 100 mM CaCl 2 (alginate-CaCl 2 , 1/2 (v/v)). The alginate beads polymerized in the presence of CaCl 2 after 10 minutes and they were washed three times with 0.15 M NaCl solution and twice with growth medium.

  expression vectors psPAX2, pMD2G and pWXL (Dr T. Didier, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland). Plasmid pWXL (green fluorescence protein (GFP) minus)Flag-hHO-1 was generated in two steps: first, the GFP coding sequence was replaced by an oligonucleotide cloned and subcloned in the same sites of pWXL (GFP)minus). Lentiviral vector stocks were generated in HEK293T cells by calcium phosphate-mediated transient transfection of three plasmids: the transfer vector plasmid (pWXL-Flag-hHO-1), the packaging plasmid psPAX2, and the VSV-G envelope protein-coding plasmid pMD2G. After transfection for 24 and 48 h, the cellular supernatants were removed, A c c e p t e d M a n u s c r i p t 8 centrifuged at 700 g for 10 min at 4ºC, passed through 45 µm pore size filters and kept

  A c c e p t e d M a n u s c r i p t 10 deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method using an assay kit (Roche Applied Science, Barcelona, Spain).

A c c e p t e d M a n u s c r i p t 11 nuclear

 11 factor-B(NF-B)-luc, activator protein(AP-1)-luc or early growth response(EGR)-1-luc (Stratagene, La Jolla, CA, USA) and 1 g of the internal control pRL-TK (Promega Corporation, Madison, WI, USA) by the Magnetofection™ system (OZ Biosciences, Marseille, France) according to the manufacturer's recommendations.

A c c e p t e d M a n u s c r i p t 12 apoptosis

 12 measured by LSC (annexin V-FITC) and CoPP treatment significantly reduced the rate of apoptosis to basal levels. Our results suggest that CoPP effects on apoptosis were specific, as in cells treated with HO-1 siRNA, CoPP failed to modify this process. Additional experiments using the TUNEL method (Fig.1C) confirmed the beneficial effects of CoPP treatment on chondrocyte apoptosis in the presence of IL-1, although the level of apoptosis induced by this cytokine was a bit higher in this assay, with percentages of 11.4±2.3 (B), 6.2±2.2 (CoPP), 21.2±5.3 (IL-1) and 12.0±2.9 (IL-1+CoPP, n=6, p<0.05 with respect to IL-1).

A c c e p t e d M a n u s c r i p t 13 using

 13 HO-1 siRNA as this agent prevented the down-regulation of ROS production by CoPP treatment.

PGE 2 M a n u s c r i p t 14 (

 214 synthesis was tested by using chondrocytes previously treated with IL-1 for 24 h to induce COX-2 and mPGES-1 and then washed and incubated in fresh medium with CoPP and arachidonic acid for 3 h. In this experimental setting, the production of PGE 2 was dramatically increased in cells treated with IL-1 in comparison with nonstimulated cells (119.3 ±21.6 versus 9.2±1.2 ng/mg protein, n=8, p<0.01). As expected, the COX-2 inhibitor NS-398 (10 M) reduced PGE 2 production to basal levels (5.9±1.2, p<0.01) but CoPP (10 M) was devoid of significant effects (113.5±13.7 ng/mg protein). We investigated whether the effects of HO-1 induction on PGE 2 were the consequence of a reduced expression of COX-2 or mPGES-1. As shown in Fig.3B, IL-1 induced the expression of COX-2 and mPGES-1 in OA chondrocytes. CoPP treatment down-regulated mPGES-1 protein whereas HO-1 siRNA tended to revert this effect. In contrast, COX-2 expression was not significantly modified by CoPP. The ability of CoPP to reduce mPGES-1 expression was also observed at the mRNA levelA c c e p t e d Table

  -[37]. In contrast, low concentrations of PGE 2 may downregulate collagenases and collagen 2A1 cleavage [38]. PGE 2 may enhance chondrocyte death [39] and induce apoptosis in bovine articular chondrocytes through the cAMP pathway [40]. In this study, we investigated the effects of HO-1 induction on IL-1-stimulated PGE 2 production in primary OA chondrocytes. We have shown that these effects were not dependent on the inhibition of COX-2/mPGES-1 enzyme activity. Although the reduction in heme availability may result in COX-2 protein possessing a low activity, our recent data [41] suggest that a product of HO-1 activity such as CO may be responsible for the reductions in PGE 2 production after HO-1 induction. Our results A c c e p t e d M a n u s c r i p t 16 have demonstrated the down-regulation of mPGES-1 gene expression by HO-1 that translates into accompanying declines in PGE 2 production. This may result in the control of PGE 2 overproduction during inflammatory responses without inhibition of other metabolites derived from COX-2 activity, with possible protective effects [42].

A c c e p t e d M a n u s c r i p t 17 of

 17 HO-1 induction on extracellular signal related kinase-1/2 phosphorylation[START_REF] Guillen | Heme oxygenase-1 regulates catabolic and anabolic processes in osteoarthritic chondrocytes[END_REF]. This mechanism may also participate in the down-regulation of mPGES-1 by HO-1 induction, as the activity of mitogen activated protein kinases could be critical for the expression of mPGES-1[START_REF] Masuko-Hongo | Up-regulation of microsomal prostaglandin E synthase 1 in osteoarthritic human cartilage: critical roles of the ERK-1/2 and p38 signaling pathways[END_REF].Therefore, HO-1 induction has shown consistent inhibitory effects toward a number of processes involved in the pathology and progression of OA. The present data revealed new facets of HO-1 actions in OA chondrocytes that could be relevant to cell survival in cartilage degeneration. These studies open the potential for therapy for a range of inflammatory and degenerative conditions and provide an opportunity to clarify HO-1 effects in human articular cells.

A c c e p t e d M a n u s c r i p t 25 FIGURE LEGENDS Fig. 1 .Fig. 2 .Fig. 3 .

 25LEGENDS123 FIGURE LEGENDS

A c c e p t e d M a n u s c r i p t 26 Fig. 4 .Fig. 5 .

 2645 Fig. 4. Effect of HO-1 (after LV-HO-1 transduction) on mPGES-1 protein in OA

A c c e p t e d M a n u s c r i p t

  Figure 1C

Figure 5 Page

 5 Figure 5

Figure 4 -

 4 Figure 4-grey

  μg) in cell lysates were separated by 12.5% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 3% bovine serum albumin and incubated with specific antibodies (1:1000) for 2 h at room

temperature. Finally, membranes were incubated with peroxidase-conjugated goat antirabbit IgG and the immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare) using the AutoChemi image analyzer (UVP Inc., Upland, CA,USA).

2.6. Oxidative stress and apoptosis

Oxidative stress and apoptosis rate were assessed by laser scanning cytometry (LSC) analysis. Chondrocytes (75,000/well were spread in 8-well Lab-tek chambers (Nalge Nunc International, Naperville, IL, USA) with DMEM/Ham's F-12, penicillin (100 U/ml), streptomycin (100 g/ml) and 10% human serum in a humidified 5 % CO 2 incubator at 37°C. Confluent cells were treated with IL-1 (100 U/ml) or IL-1+CoPP (10 M) for 24 h. Analysis of viability and apoptosis was made using LSC Bx50

CompuCyte Olympus, (Cambridge, MA, USA) using the Vybrant Apoptosis Assay Kit (Molecular Probes®, Invitrogen S.A.), according to the manufacturer's protocol. In this assay the externalization of phosphatidylserine, a marker of apoptosis, is detected by annexin V-FITC binding. Apoptotic cell death was also determined by the terminal

27 Table 1 .

 271 Sequences of primers used in real-time PCR experiments

	Forward	Reverse
	______________________________________________________________________
	___
	-actin	M a n u s c r i p t
	A c c e p t e d
		28

Table 2 .

 2 Relative gene expression of OA chondrocytes stimulated with IL-1. ±SEM, n=5) in gene expression relative to control 12 h post stimulation. IL-1: 100 U/ml; CoPP: 10 M. * p<0.05, with respect to IL-1.

	Treatment	HO-1	COX-2	mPGES-1
	_____________________________________________________________________
	IL-1	-1.1±1.5	20.9±2.7	21.8±0.6
	IL-1+CoPP	4.9±0.3*	21.7±1.7	15.7±0.7*
	_____________________________________________________________________
	Mean fold change (

Acknowledgements

This work was supported by grant SAF2007-61769 (Plan Nacional I+D+I-FEDER). J.

Megías thanks Spanish Ministerio de Educación y Ciencia, and Victoria Clérigues thanks Generalitat Valenciana, for predoctoral fellowships.