Downregulation of heat shock protein 70 protects rheumatoid arthritis fibroblast-like synoviocytes from nitric oxide-induced apoptosis

Introduction Heat shock protein 70 (Hsp70) is a well-known anti-apoptotic protein that blocks multiple steps in the stress-induced apoptotic pathway. Enhanced Hsp70 expression has previously been demonstrated in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). The authors investigated the role of Hsp70 in the survival of RA FLSs in a sodium nitroprusside (SNP)-treated environment. Methods Targeted knock-down of Hsp70 was performed by RNA interference in RA FLSs at passage 3-7. After SNP treatment, the morphological features of apoptosis were observed by phase-contrast microscopy. Cell survival was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and by flow cytometric analysis after propidium iodide (PI) staining. Bcl-2 expression and signaling pathways (Akt, extracellular signal-regulated kinase, p38, c-Jun N-terminal kinase) were examined with or without Hsp70 downregulation. Results Hsp70 downregulation in RA FLSs, induced by small interfering RNA (siRNA), was confirmed by reverse transcriptase-polymerase chain reaction and Western blotting. When treated with SNP, Hsp70 downregulated cells showed markedly less cell blebbing, cytoplasmic condensation, and nuclear shrinkage than non-downregulated control cells. Furthermore, Hsp70 downregulated cells were found to survive better than control cells in MTT assays (mean of absorbance ratio, 4.39 in target cells versus 1.00 in control siRNA-treated cells versus 1.09 in lipofectamine-treated cells, P = 0.001) and according to PI staining results (mean M1 ratio, 0.21 in target cells versus 1.00 in control siRNA-treated cells versus 1.03 in lipofectamine-treated cells, P = 0.001). Bcl-2 expression and Akt phosphorylation were higher in Hsp70 downregulated RA FLSs than in control cells. When cells were treated with LY294002, a potent phosphoinositide 3-kinase inhibitor, Akt phosphorylation and Bcl-2 levels were reduced and Hsp70 downregulation no longer had a cytoprotective effect. Conclusions Knock-down of Hsp70 protects RA FLSs from nitric oxide-induced apoptosis by activating the Akt signaling pathway. These results suggest that Hsp70 has a pro-apoptotic role in RA FLSs.

Heat shock protein 70 (Hsp70) is a molecular chaperone that is rapidly induced by physical and chemical stresses. The antiapoptotic function of Hsp70 depends on its ability to interact with protein substrates that are not always associated with the chaperoning activity. The mechanisms by which Hsp70 exerts its anti-apoptotic function encompass the inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway, caspase activation, mitochondrial cytochrome c release, and apoptosome formation [9]. Although the anti-apoptotic role of Hsp70 has been demonstrated in a number of studies in various cell types and under different conditions, several other studies have shown that the overexpression of Hsp70 promotes cell death, which suggests that Hsp70 has dual functionality depending on cell and stimulus type [10][11][12]. It has been reported that the expression of Hsp70 is higher in both tissue and cultured RA FLSs than in the FLSs of osteoarthritis and that inflammatory cytokines, such as TNF-α and IL-1β, that exist abundantly in RA joint fluid further increase Hsp70 expression in cultured RA FLSs [13]. However, no study has investigated the actual role of Hsp70 in the survival of RA FLSs. In this study, we investigate the effect of Hsp70 downregulation on RA FLS apoptosis induced by sodium nitroprusside (SNP), a nitric oxide (NO) donor.

Materials and methods
Primary culture of fibroblast-like synoviocyte derived from rheumatoid arthritis patient synovium Synovium was obtained from the affected joints of five patients undergoing joint replacement surgery (Table 1). Individual age data were removed to protect the identities of patients. All patients met the 1987 American Rheumatism Association criteria for RA [14]. The isolation and culture of FLSs were per-formed as previously described [5]. Briefly, minced synovium was incubated with 1 mg/mL collagenase type VIII (Sigma-Aldrich, St. Louis, MO, USA) in serum-free RPMI 1640 medium (Gibco, now part of Invitrogen Corporation, Carlsbad, CA, USA) for 1 hour at 37°C, filtered, extensively washed, and cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Corporation) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Corporation). Cells were allowed to adhere overnight, and adherent FLSs were grown in DMEM containing 10% FBS. FLSs at passages 3 to 7 were used for the experiments. This study was approved by the Institutional Review Board of Seoul National University Hospital (#H-0603-134-170), and informed consent was obtained from participants.

Cell treatments
Cells were incubated with TNF-α (R&D Systems, Minneapolis, MN, USA) at 10 ng/mL for 8 hours where indicated. To induce apoptosis, cells were treated with 1 mM SNP (Sigma-Aldrich) for 24 hours in a light-shielded state. LY294002 (Cell Signaling Technology, Inc., Danvers, MA, USA) at 2 to 10 μM was added to culture media to block phosphoinositide 3-kinase (PI3K) activity.

Transfection using small interfering RNA
FLSs were seeded at a density of 1.0 to approximately1.5 × 10 6 /mL and incubated overnight in antibiotics-free DMEM supplemented with 10% FBS. The following day, cells were washed with phosphate-buffered-saline, and then OPTI-MEM reduced serum medium (Invitrogen Corporation) was added to cells. Hsp70-specific small interfering RNA (siRNA) (forward 5'-CGA-CGG-AGA-CAA-GCC-CAA-GTT-3' and reverse 5'-CUU-GGG-CUU-GUC-UCC-GUC-GTT-3'; Invitrogen Corporation) [15] and a nucleic acid transferring agent, lipofectamine 2000 (Invitrogen Corporation), were incubated together in OPTI-MEM reduced serum medium for 15 minutes at room temperature to form an siRNA-lipofectamine complex. The siRNA-lipofectamine complex-containing medium was added to cells to a final siRNA concentration of 25 nM. Six hours later, the complex-containing medium was exchanged with antibiotics-free DMEM supplemented with 10% FBS. The second round of transfection was performed 48 hours after the first transfection, using the same method. Cells transfected twice with 25 nM control siRNA of medium GC content (Invitrogen Corporation) and cells transfected with lipofectamine only were used as controls.

Figure 1
Expression and specific knock-down of heat shock protein 70 (Hsp70) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) Expression and specific knock-down of heat shock protein 70 (Hsp70) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). Hsp70 was expressed in cultured RA FLSs as shown by reverse transcriptase-polymerase chain reaction (a) and Western blotting (b). Hsp70 levels were increased by tumor necrosis factor-alpha (TNF-α) treatment. Gene knock-down was specific for Hsp70; constitutional Hsp70 (Hsc70) levels were unaffected (c). The experiments were performed in RA FLSs from five patients and these are representative figures. GAPDH, glyceraldehyde-3phosphate dehydrogenase; siRNA, small interfering RNA.

Statistical analysis
Continuous values were presented as mean ± standard deviation. Analysis of variance (ANOVA) was used to compare mean values of more than two groups. Two-tailed P values of less than 0.05 were considered significant. All statistical cal-culations were performed using SPSS version 12 (SPSS Inc., Chicago, IL, USA).

Expression of Hsp70 in cultured rheumatoid arthritis fibroblast-like synoviocytes with or without tumor necrosis factor-alpha
Hsp70 expressions in cultured RA FLSs were confirmed by reverse transcriptase-PCR and Western blotting (Figure 1a, b). They were increased after 8 hours of treatment with 10 ng/ mL TNF-α.

Specific knock-down of Hsp70 expression in rheumatoid arthritis fibroblast-like synoviocyte
Targeted knock-down of Hsp70 by specific siRNA was observed in RA FLSs (Figure 1c). Because knock-down was incomplete after a single transfection, a second round of trans- fection was performed 48 hours after the first transfection. Almost complete knock-down of Hsp70 expression was then achieved and sustained for up to 72 hours. The expression of Hsc70 was unaffected (observed at 48 hours after the second transfection) by Hsp70-specific siRNA despite its 86% amino acid homology with Hsp70 (Figure 1c).

Effect of Hsp70 knock-down on nitric oxide-induced apoptosis in rheumatoid arthritis fibroblast-like synoviocyte
After treatment with SNP (1 mM) for 24 hours, cell blebbing and cytoplasmic condensation were markedly lower in Hsp70 downregulated RA FLSs than in control RA FLSs (Figure 2a). MTT assays showed that the cell viability of Hsp70 downregulated FLSs was significantly higher than that of control FLSs (mean of relative absorbance of 4.39 ± 1.82 in target cells versus 1.00 ± 0.00 in control siRNA-treated cells versus 1.09 ± 0.30 in lipofectamine-treated cells, P = 0.001) (Figure 2b). PI analysis also revealed significantly lower apoptotic fractions in Hsp70 downregulated FLSs than in control FLSs (mean of relative M 1 of 0.21 ± 0.16 in target cells versus 1.00 ± 0.00 in control siRNA-treated cells versus 1.03 ± 0.36 in lipofectamine-treated cells, P = 0.001) (Figure 2c).

Intracellular Bcl-2 levels and signaling pathways
Because Bcl-2 is known to block NO-induced apoptosis [16,17], we investigated whether Hsp70 downregulation causes any change in cellular Bcl-2 level. Bcl-2 expression was higher in Hsp70 downregulated cells than in controls with or without SNP treatment (Figure 3a). We then explored the signaling pathways involved in the cytoprotective effect of Hsp70 downregulation. Western blotting revealed that Akt phosphorylation was higher in Hsp70 downregulated RA FLS than in controls. However, the ERK, p38, and JNK pathways were unaffected by Hsp70 downregulation (Figure 3b).
Effect of Akt phosphorylation on the survival of rheumatoid arthritis fibroblast-like synoviocyte LY294002 (10 μM) decreased Akt phosphorylation to the control level, and this inhibition of Akt phosphorylation was LY294002 dose-dependent, which indicated that Akt is phosphorylated via PI3K (Figure 4a). In addition, Bcl-2 levels were found to be closely correlated with phospho-Akt levels ( Figure  4a). Furthermore, Hsp70 downregulated cells did not exhibit resistance to SNP treatment when Akt signaling was inhibited, and this loss of Hsp70 downregulation-induced cytoprotection was dose-dependent of LY294002 (Figure 4b).

Discussion
This study shows that Hsp70 is expressed in RA FLSs and that knock-down of Hsp70 protects RA FLSs from NOinduced apoptosis via the Akt signaling pathway. This result is consistent with that of Schett and colleagues [13], who found that RA FLSs express Hsp70 and that this expression is increased by inflammatory cytokines, such as TNF-α. On the other hand, Schick and colleagues [18] found that Hsc70, and not Hsp70, was overexpressed in RA synovial tissues and cultured FLSs. They suspected that anti-Hsp70 antibody in the study of Schett and colleagues might have cross-reacted with Hsc70. However, the specificity of the anti-Hsp70 antibody used in the present study has been characterized previously [19]. Furthermore, our observation that anti-Hsp70 antibody demonstrated Hsp70 downregulation by siRNA, whereas anti-Hsc70 antibody showed consistent Hsc70 expression, further supports specific antibody reactivity with Hsp70 in the present study.
Unexpectedly, we observed that Hsp70 downregulation protected RA FLSs from NO-induced apoptosis. This finding suggests that Hsp70 may be a pro-apoptotic protein in RA FLSs.
Our results indicate that hsp70 interferes with Akt phosphorylation by binding an upstream protein of the Akt signaling pathway in RA FLSs. However, in vivo, abundant intracellular anti-apoptotic apparatuses generated by external survival/ growth signals appear to overcome the negative effect of Hsp70 on Akt phosphorylation and on cell survival.
The anti-apoptotic effect mediated by Akt signaling pathway activation after Hsp70 knock-down in RA FLSs is consistent with the findings of the previous report, namely, that Akt phosphorylation is an important mechanism to protect RA FLSs from NO-induced apoptosis [20]. Akt phosphorylation has been shown to regulate Hsp70 expression [21,22]. However, to the best of our knowledge, this is the first report to suggest that Hsp70 is involved in the regulation of the Akt signaling pathway. However, the direct molecular mechanism by which Hsp70 knock-down promotes Akt phosphorylation remains to be determined.
Recently, anti-cytokine therapies for RA have entered the spotlight [23][24][25]. However, response to anti-cytokine agents is often only partial and some patients are refractory to these agents. Therefore, treatments with different mechanisms need to be combined. Although blocking anti-apoptotic proteins is a candidate strategy, the results of the present study do not support the notion that the downregulation of Hsp70 promotes the regression of hyperplastic synovium. Alternatively, the in vivo effect of Hsp70 knock-down might differ from that observed in vitro. Recently, the biological role of extracellular Effect of Akt phosphorylation on rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) survival Effect of Akt phosphorylation on rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) survival. LY294002, a potent PI3K (phosphoinositide 3kinase) inhibitor, decreased Akt phosphorylation in a dose-dependent manner, and Bcl-2 levels were found to be closely correlated with phospho-Akt levels (a). When cells were treated with LY294002, the cytoprotective effect of heat shock protein 70 (Hsp70) knock-down disappeared (b). The experiments were performed using three RA FLS samples (a, b) in triplicate (b). The representative figures are shown. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA. hsp70, particularly in terms of immune response, including the cross-presentation of antigenic peptide and maturation of antigen-presenting cells (APCs), was substantively updated [26][27][28]. Because the synovial fluid of patients with RA contains high levels of soluble Hsp70, which interacts with APCs via a plethora of surface receptors [29], the effect of Hsp70 knockdown is likely to be complex in vivo.

Conclusions
In summary, this study shows that Hsp70 is expressed in RA FLSs and that knock-down of Hsp70 protects RA FLSs from NO-induced apoptosis by activating the Akt signaling pathway. These results suggest that Hsp70 has a pro-apoptotic role in RA FLS.