Heat shock protein gene expression profile may differentiate between rheumatoid arthritis, osteoarthritis, and healthy controls

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1 354 Scand J Rheumatol 211;4: SRHE Heat shock protein gene expression profile may differentiate between rheumatoid arthritis, osteoarthritis, and healthy controls Sedlackova et al L Sedlackova 1, A Sosna 2, P Vavrincova 3, J Frýdl 4, V Guerriero 5,6, DA Raynes 5, I Hromadnikova 1 1 Department of Molecular Biology and Cell Pathology, 3rd Faculty of Medicine, Charles University, 2 1st Clinic of Orthopaedics, University Hospital Motol, 1st Medical Faculty, Charles University, 3 Outpatient Department of Rheumatology, University Hospital Motol, and 4 Department of Children and Adult Orthopaedics and Traumatology, 2nd Medical Faculty of Charles University and University Hospital Motol, Prague, Czech Republic, and Departments of 5 Animal Sciences, and 6 Molecular and Cellular Biology, University of Arizona, Tucson, AZ, USA Objective: Heat shock proteins (Hsps) have been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this work was to study Hsp mrna and protein levels to determine whether they can be used to differentiate between RA, osteoarthritis (OA), and healthy controls. Methods: Hsp27, Hsp6, Hsp7, Hsp9a, and HspBP1 mrna expression was analysed using real-time quantitative reverse transcription polymerase chain reaction (qrt-pcr) in 24 RA, 11 OA, and 21 healthy controls. Hsp7 and HspBP1 protein levels were measured in serum using an enzyme-linked immunosorbent assay (ELISA). Results: Hsp gene expression profiles differ significantly between inflammatory (RA) and non-inflammatory (OA) joint diseases, showing significantly increased Hsp27 and Hsp9a mrna levels in RA synovial tissues. Up-regulated Hsp6 and Hsp9a together with down-regulated Hsp7 and elevated HspBP1/Hsp7 mrna ratios can be used to differentiate between RA patients and healthy individuals through analysis of peripheral blood samples. Despite increased HspBP1 levels in RA sera, Hsp7 levels and the HspBP1/Hsp7 protein ratio remained identical in the RA patients and healthy individuals, which may contribute to the inhibition of Hsp7 anti-apoptotic activity. Conclusion: Hsp gene expression analysis can be implemented as a new diagnostic approach to facilitate differentiation between RA, OA, and healthy controls. Heat shock proteins (Hsps) have been implicated in the pathogenesis of rheumatoid (RA) and juvenile idiopathic arthritis (JIA) (1). Activated cells of the synovium produce pro-inflammatory and matrix-degrading molecules that maintain the inflammation and lead to the destruction of the joints. Such stress leads to the up-regulation of self-hsp expression in the synovial tissue, which protects synovial cells from apoptotic death (2). Furthermore, in patients with RA or JIA, augmented Hsp expression has been observed in the inflamed synovium and found to be important for proper functioning of Hsp-specific regulatory T cells (3). Our group previously reported higher expression of Hsp7 in synovial cells derived from patients with severe RA and JIA (4). A novel Hsp7 cochaperone known as Hsp7 binding protein 1 (HspBP1) binds to the ATPase domain of Hsp7 and inhibits the ability of Hsp7 to refold denatured proteins. The levels of both Hsp7 and HspBP1 are Lucie Sedlackova, Department of Molecular Biology and Cell Pathology, 3rd Faculty of Medicine, Charles University in Prague, Ruska 87, 1 Prague 1, Czech Republic. lucie.sedlackova@lf3.cuni.cz Accepted 4 January 211 elevated in numerous tumours (5), but there are no available data regarding HspBP1 in autoimmune arthritis. The molar ratio of HspBP1 to Hsp7 in cells may be an important determinant relative to the interaction between these two proteins and of the functioning of the complex. In the current study we examined the gene expression of Hsp27, Hsp6, Hsp7, Hsp9α, and HspBP1 proteins and determined the HspBP1/Hsp7 ratio with the aim of differentitating between RA, osteoarthritis (OA), and healthy controls. Furthermore, we use peripheral blood analysis in an attempt to differentiate between RA patients and healthy individuals on the basis of different Hsp gene expression profiles. In RA patients, we studied also gene expression in autologous skin dermis obtained during the synovectomy. Moreover, we analysed levels of Hsp7 and HspBP1 in sera of RA patients and healthy controls and determined the HspBP1/Hsp7 protein ratio. Materials and methods Patients and controls Ethics committee approval and informed consent were obtained from all participants. The cohort consisted of 211 Taylor & Francis on license from Scandinavian Rheumatology Research Foundation DOI: 1.319/

2 Hsp gene expression may differ in RA and OA rheumatoid factor (RF)-positive polyarthritis patients (23 females, one male, median age 56.5 years) with disease duration ranging from 3 to 32 years (median 17 years). All patients had active disease determined using the 28-joint count Disease Activity Score (DAS28). Patients had been treated, depending on the stage of the disease, with non-steroidal anti-inflammatory drugs, corticosteroids, and/or disease-modifying anti-rheumatic drugs. Synovial tissues were derived from one finger joint, seven metacarpophalangeal joints, one elbow joint, one shoulder joint, six metatarsophalangeal joints, two ankle joints, three knee joints, and three hip joints during synovectomy. Skin samples were taken from the surface of the synovectomy surgical site. Synovial tissues of 11 patients (four males and seven females, median age 73 years) with gonarthrosis (GA) were used as the noninflammatory joint disease control group. Twenty-one healthy controls (18 females, three males, median age 53 years) were included in the study. RNA isolation Total RNA was extracted from all biological samples using the QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany). Analysis of Hsp gene expression All polymerase chain reactions (PCRs) were performed as described previously (6). A comparative threshold cycle (Ct) method was used to interpret the data. Enzyme-linked immunosorbent assay (ELISA) Hsp7 was measured using an Hsp7 High Sensitivity Enzyme Immunometric Assay Kit (Assay Desgins, USA) and an ELISA plate reader (Sunrise, Tecan Trading AG, Switzerland). HspBP1 serum levels were analysed as described previously (7). Results Analysis of Hsp gene expression The levels of Hsp27 and Hsp9a were found to be increased in RA synovial tissue compared to OA patients (p =.5, Figure 1). Concerning the juxtaposition of peripheral blood samples, higher expression of Hsp6 (p <.2) and Hsp9a (p <.1) was found in RA patients when compared with healthy controls (Figure 2). By contrast, Hsp7 showed lower expression in RA patients (p <.1). Of note, all Hsps tested showed higher expression in RA dermis samples compared with autologous synovial tissues (Figure 1). Relative gene expression p =.2 p' = *Hsp p <.1 p' =.23 Hsp6 p <.1 p' =.63 Hsp7 p =.2 p' =.5 Hsp9 p <.1 p' =.34 HspBP1 RA (SC) RA (DF) OA (SC) median p =.5 p' =.89 HspBP1/ Hsp7 Figure 1. Hsp gene expression in synovial tissues derived from RAaffected and OA-affected joints and dermal fibroblasts of RA nonaffected skin dermis. The difference (ΔCt) between the Ct values of Hsp and b-actin was calculated for each sample after real-time RT- PCR analysis. The comparative ΔΔCt calculation involved finding the difference between each sample s ΔCt and the reference s ΔCt. Finally, the ΔΔCt values were transformed to absolute values using the formula 2 ΔΔCt. The ΔCt of a randomly selected healthy control was used as the reference for each comparison calculated as 2 ΔΔCt. The reference control value is then 2 ΔΔCt = 1 and the result of each Hsp system is expressed as a multiple of the reference healthy control. *As the Hsp27 expression results were 1-fold higher than other Hsp expression data, the graph has two y axes with different scales. Hsp gene expression was compared between the patients cohorts using the Mann Whitney U-test. p values represent statistical differences between synovial and skin dermis tisssues in patients with RA. p values compare synovial tissue Hsp gene expression data between patients with RA and OA. The significance level was set at p <.5. SC, synovial cells; DF, dermal fibroblasts. Relative gene expression p =.24 p =.2 p <.1 p <.1 p =.29 p <.1 Hsp27 Hsp6 Hsp7 Hsp9 HspBP1 HspBP1/ Hsp7 RA (PBL) healthy control (PBL) median Figure 2. Hsp gene expression in peripheral blood leucocytes (PBL) of RA patients and healthy controls. Hsp gene expression was compared between RA patients and healthy controls using the Mann Whitney U-test. The significance level was set at p <.5. Evaluation of HspBP1/Hsp7 gene expression ratio No difference in the ratio (p =.89) was identified in synovial tissues between RA and OA, indicating comparable expression of Hsp7 (p =.63) and HspBP1 (p =.34) (Figure 1). Of note, a higher ratio was observed in RA peripheral blood samples compared to healthy controls (p <.1) (Figure 2). However, the ratio was lower

3 356 L Sedlackova et al Serum levels (ng/ml) p =.15 p =.3 p =.16 Hsp7 HspBP1 HspBP1/Hsp7 RA patients Healthy controls median Figure 3. Serum levels of Hsp7 and HspBP1 in RA patients and healthy controls. The levels of inducible Hsp7 and HspBP1 in serum samples of patients with RA and healthy controls were measured using ELISA and compared by the Mann Whitney U-test. The significance level was set at p <.5. Hsp7 and HspBP1 concentrations from serum samples were quantified by interpolating absorbance readings from a standard curve and expressed as nanograms per millilitre. Serum samples of RA patients were diluted 1:4 with the assay buffer. Samples below the detection limit of the standard curve range were reanalysed using a 1:1 dilution. Serum samples of healthy controls were diluted 1:1 with the assay buffer. in dermis than in synovial tissue samples in RA patients (p =.5). Analysis of Hsp7 and HspBP1 serum levels The levels of Hsp7 (p =.15) and the HspBP1/Hsp7 ratio (p =.16) in RA patients and healthy controls remained without any difference. However, the levels of HspBP1 were higher in sera of RA patients (p =.3) (Figure 3). Discussion It has become clear that synovial cells are involved in the pathogenesis of RA. The resistance of synovial cells to stress is associated with the expression of Hsps in synovial tissues, which can protect the cells from apoptotic death (2). Hsps also possess immunoregulatory attributes and are therefore exploited for immunomodulation of various disorders. In experimental models of inflammatory diseases, administration of exogenous Hsp or Hsp-derived peptides was able to suppress disease processes through the induction of T cells that specifically recognized Hsp6 or Hsp7 (3). In recent clinical trials, in patients with diabetes and RA, administration of Hsp-derived peptides has been successful (3). The aim of this study was to expand the previously described Hsp7 expression, in a RA cohort, to gene expression of other Hsps. This is the first study comparing Hsp gene expression profiles between inflammatory and non-inflammatory joint disease that shows increased Hsp27 and Hsp9a mrna levels in RA synovial tissues. These observations suggest that Hsp27 and Hsp9a may be used as new diagnostic markers in cases where a clear diagnosis is uncertain. Overexpressed Hsp27 protects against apoptotic cell death triggered by various stimuli (8). Similarly, we demonstrated that up-regulated Hsp6 and Hsp9a together with down-regulated Hsp7 and a higher HspBP1/Hsp7 ratio can differentiate between RA patients and healthy individuals using solely analysis of peripheral blood samples. Hsps seem to be promising targets for therapeutic intervention in various diseases. Our data suggest the initiation of testing of agents targeting Hsp9 as a potential form of treatment of RA, although inhibition of Hsp9, as a treatment strategy, has already been evaluated primarily in cancer (9). Aberrant cytokine and receptor signalling, angiogenesis, and cellular invasion are common to both cancer and inflammation. Natural inhibitors of Hsp9 block activation of the nuclear factor kappa B (NF-kB) pathway, leading to loss of cytokine production. Our Hsp6 expression data are in concordance with other studies. Hsp6 was found to be expressed in the synovial membrane of RA patients; however, the synovial tissue of normal and OA patients also expressed considerable amounts of Hsp6 (1). Hsp7 inhibits apoptosis, thereby increasing the survival of cells exposed to otherwise lethal stimuli. Another study (11) has demonstrated in vitro protection of chondrocytes from cellular injuries after Hsp7 gene transfer. However, our study showed no difference in Hsp7 gene expression in synovial tissues between RA and OA and therefore Hsp7 protective features could not be exerted. It seems that Hsp expression is up/down-regulated differentially in various types of tissue such as synovia, skin, and peripheral blood leucocytes in RA patients. In RA non-affected dermis samples, taken from the operative site, we observed the highest expression of all Hsp genes tested compared with RA affected synovial tissues. We suggest that this phenomenon is not associated with the disease itself but might represent a physiological response of an external barrier to potentially harmful environmental factors, including the production of protective Hsps that prevent skin cell apoptosis (12). Furthermore, we determined the HspBP1/Hsp7 ratio at the level of gene expression in peripheral blood and at the level of proteins in the sera of RA patients and healthy controls. With regard to proteins in serum samples, the ratio did not reach statistical significance, although increased HspBP1 concentrations were found in RA sera. However, with regard to gene expression, the ratio was higher in RA patients ( Hsp7, HspBP1) compared with controls. Nevertheless, the correlation between mrna and protein abundances in the cell has been reported to be poor (13). The precise mechanism for control of Hsp7 expression has not been completely delineated. It was previously suggested that Hsp7 protein may negatively regulate Hsp7 gene transcription to prevent overproduction of Hsp7 (14).

4 Hsp gene expression may differ in RA and OA 357 Regulation of HspBP1 expression has not yet been described. It is well known that tumour cells with a high HspBP1/ Hsp7 protein ratio are more susceptible to anti-cancer drugs than those with a low ratio. High levels of Hsp7 have been correlated with shorter survival times for patients (15). Unfortunately, despite increased levels of HspBP1 in RA sera, the Hsp7 levels and the HspBP1/ Hsp7 protein ratio remained identical in both RA patients and healthy individuals, which may contribute to the inhibition of Hsp7 anti-apoptotic activity in RA. We are aware that this study involves too small cohort of patients to draw any definitive conclusions. However, the data resulting from this study suggest the potential of Hsps to differentiate between RA/OA patients and healthy controls and these interesting findings should be further confirmed in larger cohorts. Acknowledgements We thank Associate Professor Stanislav Popelka, Dr David Veigl, and Associate Professor Jan Pech, from The First Clinic of Orthopaedics, University Hospital Motol, Prague, for providing tissue samples and their kind collaboration. We would also thank Roman Volchenkov (currently at The Gade Institute, University of Bergen, Norway), who was employed from April 28 to June 29 as an early stage researcher on the TRANSNET project, for his participation in the designing and testing of TaqMan systems for the detection of Hsps. Thanks also to Zuzana Imryskova, who participated in testing of patients with osteoarthritis. This project was supported by TRANSNET (No. MRTN-CT ) and STEMDIAGNOSTICS (No. LSHB-CT ). References 1. Arvonen M, Tikanmaki M, Vahasalo P, Karttunen TJ. Heat shock protein expression is low in intestinal mucosa in juvenile idiopathic arthritis: a defect in immunoregulation. Scand J Rheumatol 21;39: Schett G, Tohidast-Akrad M, Steiner G, Smolen J. The stressed synovium. Arthritis Rheum 21;3: van Eden W, van der Zee R, Prakken Bl. Heat-shock proteins induce T-cell regulation of chronic inflammation. Nat Rev Immunol 25;5: Sedlackova L, Nguyen TTH, Zlacka D, Sosna A, Hromadnikova I. Cell surface and relative mrna expression of heat shock protein 7 in human synovial cells. Autoimmunity 29;42: Raynes DA, Graner MW, Bagatell R, McLellan C, Guerriero V. Increased expression of HspBP1 in tumors. Tumor Biol 23;24: Sedlackova L, Spacek M, Holler E, Imryskova Z, Hromadnikova I. Heat-shock protein expression in leukemia. Tumour Biol 21;32: Raynes DA, Thomson CA, Stroster J, Newton T, Cuneo P, Guerriero V. Human serum contains detectable levels of the Hsp72 cochaperone HspBP1 and antibodies bound to HspBP1. J Immunoassay Immunochem 26;27: Mehlen P, Schulze-Osthoff K, Arrigo AP. Small stress proteins as novel regulators of apoptosis. Heat shock protein 27 blocks Fas/ APO-1- and stautosporine-induced cell death. J Biol Chem 1996;271: Neckers L. Heat shock protein 9: the cancer chaperone. J Biosci 27;32: Sharif M, Worall JG, Singh B, Gupta RS, Lydyard PM, Lambert C, et al. The development of monoclonal antibodies to the human mitochondrial 6-kd heat shock protein, and their use in studying the expression of the protein in rheumatoid arthritis. Arthritis Rheum 1992;35: Grossin L, Cournil-Henrionnet C, Pinzano A, Gaborit N, Dumas D, Etienne S, et al. Gene transfer with HSP 7 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis. FASEB J 26;2: Trautinger F, Trautinger I, Kindas-Mugge I, Metze D, Luger TA. Human keratinocytes in vivo and in vitro constitutively express the 72-kD heat shock protein. J Invest Dermatol 1993; 11: Maier T, Guell M, Serrano L. Correlation of mrna and protein in complex biological samples. FEBS Lett 29;583: Ding XZ, Tsokos GC, Kiang JG. Overexpression of HSP-7 inhibits the phosphorylation of HSF1 by activating protein phosphatase and inhibiting protein kinase C activity. FASEB J 1998;12: Souza AP, Albuquerque C, Torronteguy C, Frasson A, Maito F, Pereira L, et al. HspBP1 levels are elevated in breast tumor tissue and inversely related to tumor aggressiveness. Cell Stress Chaperones 29;14:31 1.

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