ORIGINAL RESEARCH ARTICLE
Journal of
Cellular Physiology
Functional Role of the KCa3.1 Potassium Channel in Synovial Fibroblasts From Rheumatoid Arthritis Patients KRISTIN FRIEBEL,1 ROLAND SCHÖNHERR,1 RAIMUND W. KINNE,2 1
AND
ELKE KUNISCH2*
Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University of Jena and Jena University Hospital, Jena, Germany
2
Experimental Rheumatology Unit, Department of Orthopedics, Jena University Hospital, Jena, Germany
Rheumatoid arthritis synovial fibroblasts (RA-SFs) show an aggressive phenotype and support joint inflammation and tissue destruction. New druggable targets in RA-SFs would therefore be of high therapeutic interest. The present study shows that the intermediateconductance, calcium-activated potassium channel KCa3.1 (KCNN4) is expressed at the mRNA and protein level in RA-SFs, is functionally active, and has a regulatory impact on cell proliferation and secretion of pro-inflammatory and pro-destructive mediators. Whole-cell patch-clamp recordings identified KCa3.1 as the dominant potassium channel in the physiologically relevant membrane voltage range below 0 mV. Stimulation with transforming growth factor b1 (TGF-b1) significantly increased transcription, translation, and channel function of KCa3.1. Inhibition of KCa3.1 by the selective, pore-blocking inhibitor TRAM-34, (and, in part, by siRNA) significantly reduced cell proliferation, as well as expression and secretion of pro-inflammatory factors (IL-6, IL-8, and MCP1) and the tissue-destructive protease MMP3. These effects were observed in non-stimulated and/or TGF-b1-stimulated RA-SFs. Since small molecule-based interference with KCa3.1 is principally well tolerated in clinical settings, further evaluation of channel blockers in models of rheumatoid arthritis may be a promising approach to identify new pharmacological targets and develop new therapeutic strategies for this debilitating disease. J. Cell. Physiol. 230: 1677–1688, 2015. © 2014 Wiley Periodicals, Inc.
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, which is characterized by pain, swelling, and progressive destruction of multiple joints. Joint destruction in RA is perpetuated by an aggressive, invasive pannus tissue, a vascular and fibrous granulation tissue consisting of macrophages, synovial fibroblasts (SFs), T lymphocytes, and B lymphocytes/plasma cells. Uncontrolled, active RA causes disability, decreases quality of live, and increases comorbidity. The introduction of biologics meant a great improvement in the treatment of RA (Scott, 2011). Although biologics are very effective in the treatment of RA, they carry several disadvantages, e.g., lack of oral availability, the development of unresponsiveness, and high costs. Therefore, many pharmaceutical companies also develop small molecule inhibitors suitable for oral administration. In most cases, these small molecules are directed against the main signaling pathways regulating the transcription of pro-inflammatory genes, e.g., p38, Jak, or Syk (Bonilla-Hernan et al., 2011; Yazici and Regens, 2011). Besides several other molecules (Edmonds et al., 2011; Fleischmann, 2012), potassium channels might be regarded as new therapeutic targets for RA. Recently, Hu et al. showed that blocking of the calcium and voltage-activated Kþ channel KCa1.1 attenuates the pro-inflammatory and invasive behavior of synovial fibroblasts from RA patients (RA-SFs) (Hu et al., 2012). Potassium channels are attractive therapeutic targets, as they can be modulated by a variety of blockers and openers. The localization of channels in the plasma membrane makes them more attractive than intracellular targets, for which multi-drug resistance mechanisms represent a major limitation. The voltage-gated and Ca2þ-activated Kþ channel KCa1.1 belongs to the six-transmembrane-domain superfamily of Kþ channels. Within this group, eight channel-coding genes form the KCa subfamily, but only five members are truly Ca2þactivated channels (Salkoff et al., 2006). KCa1.1 (KCNMA1), © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .
also called “big”-conductance Kþ channel (BK), is activated by membrane depolarization, as well as by direct binding of cytosolic Ca2þ ions. Various potent and selective pharmacologic mediators for this channel have been described, including iberiotoxin (Wei et al., 2005). In contrast, four other members of the subfamily are voltage-independent, but can be activated by intracellular calcium sensed through constitutively bound calmodulin. Three small-conductance calcium-activated Kþ channels (KCa2.1, KCa2.2, KCa2.3; also called KCNN1–3) share sensitivity to the bee toxin apamin, while the
Disclosures: The authors indicate no potential conflict of interest. Contract grant sponsor: German Federal Ministry of Education and Research; Contract grant numbers: 13N9833, 035577D, 0315711A, 01EO1002, 0316205B. Contract grant sponsor: German Research Foundation; Contract grant numbers: KI 439/6–3, KI 439/7–3. Contract grant sponsor: Deutsche Krebshilfe e.V. (German Cancer Aid); Contract grant number: project 109851. *Correspondence to: Elke Kunisch, Experimental Rheumatology Unit, Dept. of Orthopedics, Jena University Hospital, Klosterlausnitzer Str. 81, D-07607 Eisenberg, Germany. E-mail: [email protected] Manuscript Received: 22 December 2013 Manuscript Accepted: 22 December 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 29 December 2014. DOI: 10.1002/jcp.24924
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intermediate-conductance Ca2þ-activated channel KCa3.1 (IKCa1, KCNN4) can be inhibited by clotrimazole and its more specific derivative TRAM-34 (Wulff et al., 2000a). Many years ago, the antifungal agent clotrimazole has been discussed as a potential therapeutic agent in rheumatoid arthritis (Otterness and Niblack, 1976; Wyburn-Mason, 1976) and a later study principally confirmed its beneficial effects in rheumatoid arthritis, such as improvement of grip strength, pain relief, and reduced joint swelling (Wojtulewski et al., 1980). However, the same study also suggested that the gastrointestinal side-effects of clotrimazole represent a contraindication for this therapeutic approach. The present study shows that KCa3.1 is expressed and functional in RA-SFs. Inhibition of KCa3.1 by the selective KCa3.1 inhibitor TRAM-34 or by siRNA reduced the proliferation of non-stimulated and TGF-b1-stimulated RA-SFs. In addition, TRAM-34 (and, in part, siRNA) reduced the production of several pro-inflammatory factors such asIL-6, IL-8, and monocyte chemotactic protein-1 (MCP1) at the mRNA and protein level, indicating that the potassium channel KCa3.1 may be a therapeutic target for RA. Materials and Methods Patients, tissue digestion, and cell culture Synovial tissue was obtained from RA patients fulfilling the ARA criteria (Arnett et al., 1988) during open joint replacement/ arthroscopic synovectomy at the Clinic of Orthopedics, Eisenberg, Germany. The study was approved by the Ethics Committee of the University of Jena, Germany and patient informed consent was obtained. RA synovial samples were digested, subsequently cultured for 7 days, and RA-SFs negatively isolated as previously described (Zimmermann et al., 2001; Hirth et al., 2002). RA-SFs were cultured in the virtual absence of contaminating non-adherent cells and macrophages. Third-passage cells were used for all experiments. Mycoplasma contamination of the cells was excluded by 4’-6-diamidino-2-phenylindole (DAPI) staining. Stimulation of the cells with TGF-b1 (10.0 ng/ml; R&D Systems, Wiesbaden, Germany) was performed in DMEM/10% FCS. To analyze the functional role of the potassium channel KCa3.1, RA-SFs were incubated with TRAM-34 (generous gift from Dr. Heike Wulff, University of California, Davis), in the absence or presence of TGF-b1 for 24 h. To exclude toxicity of the inhibitors, viability of the cells was assessed by fluorescein diacetate/ propidium iodide or ethidium bromide staining. Silencing of KCa3.1 in RA-SFs was performed by the siRNA technique using reverse transfection as previously described (Kunisch et al., 2012). In brief, GeneSolution siRNAi (Qiagen, Hilden, Germany) and LipofectaminTMRNAiMAX (Invitrogen, Darmstadt, Germany) were mixed in Opti-MEM (Invitrogen) and incubated for 20 min at room temperature to allow complex formation. Subsequently, cells previously suspended in DMEM, 10% FCS without antibiotics were added and incubated for 24 h. Thereafter, the cells were either cultivated for further 24 h without stimulation or, alternatively, stimulated with 10 ng/ml TGF-b1 in DMEM, 10% FCS for 24 h. Supernatants of the cells were collected for the analysis of cytokine and protease secretion. Cells were washed with ice-cold PBS and subsequently lysed in buffer for RNA-isolation (Qiagen). Transfection efficiency was analyzed using 10 nM BlockiTTMAlexaFluor1Red Fluorescent Oligo (Invitrogen). Transfection efficiency was greater than 85% (85.4% 2.7 positive cells). Quantitative RT-PCR Total RNA was isolated from RA-SFs and reverse-transcribed as previously described (Pohlers et al., 2007; Pretzel et al., 2009). mRNA expression was analyzed by real-time PCR using a RealPlex1 PCR machine (Eppendorf, Hamburg, Germany) as previously JOURNAL OF CELLULAR PHYSIOLOGY
described. The primer pairs and conditions for IL-6,IL-8, MCP1, MMP-1, and MMP-3 and the house-keeping gene aldolase are listed in Table 1. Primers were purchased from Jena Bioscience GmbH (Jena, Germany). The concentration of IL-6,IL-8, MCP1, MMP-1, and MMP-3 mRNA in each sample was calculated by the RealPlex1 software using an external standard curve. Product specificity of the real-time PCR was confirmed by: (i) melting curve analysis; (ii) agarose gel electrophoresis; and (iii) cycle sequencing of the PCR products. For mRNA quantification of KCa1.1, KCa2.1, KCa2.2, KCa2.3, KCa3.1, B2M and ACTB, QuantiTect1 primer pairs from Qiagen were used in PCR reactions with a Maxima1 SYBR Green/ ROX qPCR Master Mix (Fermentas, St. Leon-Rot, Germany). Efficiency of these primer pairs was tested using standard curves with logarithmic template dilutions. All primers showed close to 100% efficiency in the analyzed range of Ct values (up to 30). Protein detection by immunohistochemistry, ELISA, and Western blot For KCa3.1-immunohistochemistry in RA-SFs, 0.4 105 cells/well (8-chamber slide) were allowed to adhere for 24 h. Thereafter, cells were cultivated for 24 h, followed by fixation with 4% PFA/PBS for 20 min at room temperature. The fixed cells were rinsed with PBS and then incubated for 1 h in blocking buffer (PBS with 5% goat serum and 0.3% Triton-100). The cells were then exposed to the anti-KCa3.1 antibody (H-120, Santa Cruz; 1:100 diluted in PBS, 1% BSA, and 0.3% Triton) overnight in a moist chamber at 4 °C followed by incubation with horseradish-peroxidase-coupled goat anti-rabbit IgG (DAKO, Hamburg, Germany) for 1 h at RT. Cells were washed thoroughly with PBS and stained with DAB. For quantification of KCa3.1 protein, cells were seeded in 96well plates and treated for 24 h with TGF-b1 as described above. KCa3.1 protein was detected as described above with the slight modification of using the soluble HRP-substrate 3,30 ,5,50 Tetramethylbenzidine (TMB) instead of DAB. The absorbance was read in an ELISA Reader (BMG Fluostar). For Western blot detection, cells were lysed in reducing Laemmli buffer, heated to 60 °C for 5 min and subjected to 12% polyacrylamide SDS-PAGE. The proteins were transferred to Immobilon-P1 polyvinylidene difluoride (PVDF) membranes (Merck Millipore) and the membrane was blocked with 5% milk powder in PBS with 0.1% Tween 20. The membranes were then incubated overnight at 4°C with primary antibodies 1:1000 (rabbit anti KCa3.1, H-120, Santa Cruz) or 1:5000 (anti b-actin, mouse monoclonal AC15, Sigma-Aldrich) diluted in PBS-Tween with 1% BSA. For immunodetection, the membranes were incubated for 1 h with horseradish-peroxidase-coupled IgG anti-rabbit and anti-mouse, TABLE 1. Primer sequences and annealing temperatures used in RT-PCR
Gene Aldolase Sense Anti-sense IL-6 Sense Anti-sense IL-8 Sense Anti-sense MCP-1 Sense Anti-sense MMP-1 Sense Anti-sense MMP-3 sense Anti-sense
Annealing temperature (°C)
Melting temperature (°C)
5-tcatcctcttccatgagacactct-3 5-attctgctggcagatactggcataa-3
58
82
5-atgaactccttctccacaagcg-3 5-ctcctttctcagggctgag-3
62
84
5-gccaagagaatatccgaact-3 5-aggcacagtggaacaaggacttgt-3
60
78
5-cagccagatgcaatcaatgcc-3 5-tggaatcctgaacccacttct-3
60
82
5-gacctggaggaaatcttgc-3 5-gttagcttactgtcacacgc-3
58
81
5-gaacaatggacaaaggatacaaca-3 5-aagattgatgctgtttttgaagaa-3
58
81
Primer sequence
FUNCTION OF KCa3.1 IN SYNOVIAL FIBROBLASTS
respectively, and the reaction visualized using an ECL detection kit (GE Healthcare). Electrophysiological recordings Potassium currents were recorded at room temperature in the whole-cell configuration, using an EPC10 patch clamp amplifier (HEKA Elektronik, Germany) controlled by PatchMaster software (HEKA Elektronik). In all experiments, the holding potential was 80 mV. Series-resistance errors were compensated in the range of 70–90%. Patch pipettes were fabricated from borosilicate filament (Kimble Glass, USA) with resistance values in the range of 1.5–3 MV. Off-line analysis of data was performed using Igor Pro (WaveMetrics, USA) and FitMaster software (HEKA Elektronik). The standard bath solution contained 5 mM KCl, 155 mM NaCl, 2 mM CaCl2, and 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES); the pH was adjusted to 7.4 with NaOH. The internal solution contained 130 mM KCl, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPES, and 9.3 mM CaCl2 to yield 1 mM free Ca2þ; the pH was adjusted to 7.4 with KOH. The internal free calcium concentration was calculated in compliance with the WEBMAXC STANDARD software (http://www.stanford.edu/ cpatton/webmaxc/webmaxcS.htm). Currents were elicited by 500 ms voltage ramps, ranging from 100 mV to þ50 mV. In blocking experiments with TRAM-34, iberiotoxin (Alomone Labs) or apamin (Sigma-Aldrich), the same ramp protocol was applied every 5 sec. Mean current densities were determined in the range between 40 mV and 25 mV to analyze KCa2 and KCa3.1 currents and at about þ50 mV to detect primarily KCa1.1 currents. Voltage-gated Kþ currents were elicited by applying 100 ms pulses in 50 mV increments from 100 mV to þ150 mV. Analysis of functional parameters in RA-SFs by ELISA Proliferation was assessed by BrdU incorporation using a commercial cell proliferation ELISA (Roche) as previously described (Kunisch et al., 2007). Human IL-6, IL-8, MCP1, MMP-1, and MMP-3 proteins were measured in diluted cell culture supernatants as previously described (Kunisch et al., 2012). The resulting color was read at 450 nm in microtitre plates (Fluostar Optima). MMP-1 activity in the supernatants of stimulated cells was measured using a commercially available MMP-1 activity assay (Matrix Metalloproteinase Biotrak Activity Assay System, Amersham Biosciences) according to the manufacturer’s instructions. Statistical analysis Data are presented as means standard error of the mean (SEM). The non-parametric Mann–Whitney U-test was applied to analyze differences between controls and individual stimuli (software program SPSS 13.0TM; SPSS Inc., Chicago, IL). Significant differences were accepted for P 0.05. Results Expression of Kþ channels in RA-SFs
Primary cell cultures of RA-SFs were analyzed for the expression of calcium-activated potassium channels (KCa). In non-stimulated RA-SFs, no mRNA was observed for the small conductance channels KCa2.1, KCa2.2, and KCa2.3. However, mRNA expression was detectable for the intermediate conductance channel KCa3.1 and the big conductance channel KCa1.1 (Fig. 1A). The expression levels of the 2 genes did not differ significantly, ranging between 5% and 10% of the mRNA level of b-actin (ACTB). The expression of KCa3.1 was confirmed at the protein level. In comparison to lack of staining with the isotype control, JOURNAL OF CELLULAR PHYSIOLOGY
Fig. 1. Expression of KCa channel mRNAs in RA-SFs. A) mRNA expression of the KCa channels KCa2.1, KCa2.2, KCa2.3, KCa3.1, and KCa1.1 in non-stimulated RA-SFs (n ¼ 4 donors) was normalized to the expression of b-actin (means SEM). B) Protein expression of KCa3.1 in non-stimulated RA-SFs was assessed by immunohistochemistry (n ¼ 4; one representative result is shown). C) Western Blot detection of KCa3.1 protein in transfected HEK293 cells and RA-SFs. Whole-cell extracts of mock-transfected HEK293 cells, KCa3.1-transfected HEK293, and of RA-SFs in primary culture were analyzed for KCa3.1 protein using a polyclonal rabbit serum. Multiple bands in the molecular weight range of 45 kDa and below in RA-SFs and in the positive control likely represent channel protein fractions with different glycosylation states.
KCa3.1 protein was clearly located in the perinuclear cytoplasm of RA-SFs (Fig. 1B). Specificity of the anti-KCa3.1 antiserum was confirmed by Western blot analysis comparing lysates of KCa3.1-transfected HEK293 cells and RA-SFs (Fig. 1C). The signal in RA-SFs matched the molecular weight of the transfected protein in HEK293 cells, excluding cross-reactions with the
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much larger KCa3.1 protein (47 kDa). No Western blot signal was detected in mock-transfected HEK293 cells. Functional Ca2þ-activated Kþ channels in RA-SFs
The presence of mRNA for the 2 KCa channel family members KCa3.1 and KCa1.1 led to detailed analysis of the electrophysiological properties of RA-SFs. Ionic currents elicited by voltage-ramps from 100 to 50 mV in whole-cell patch-clamp experiments with 1 mM Ca2þ in the pipette reflected the known properties of KCa3.1 and KCa1.1 (Fig. 2A–F). In the negative voltage range, current responses were strictly linear, as expected for a voltage-independent channel such as KCa3.1. In contrast, the increase of the currents in the positive voltage range deviated from the linear slope, a characteristic of depolarization-activated channels such as KCa1.1 (Fig. 2A). Both current components were strongly calcium-dependent, i.e., in the absence of Ca2þ in the pipette solution the mean currents were reduced to
Journal of
Cellular Physiology
Functional Role of the KCa3.1 Potassium Channel in Synovial Fibroblasts From Rheumatoid Arthritis Patients KRISTIN FRIEBEL,1 ROLAND SCHÖNHERR,1 RAIMUND W. KINNE,2 1
AND
ELKE KUNISCH2*
Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University of Jena and Jena University Hospital, Jena, Germany
2
Experimental Rheumatology Unit, Department of Orthopedics, Jena University Hospital, Jena, Germany
Rheumatoid arthritis synovial fibroblasts (RA-SFs) show an aggressive phenotype and support joint inflammation and tissue destruction. New druggable targets in RA-SFs would therefore be of high therapeutic interest. The present study shows that the intermediateconductance, calcium-activated potassium channel KCa3.1 (KCNN4) is expressed at the mRNA and protein level in RA-SFs, is functionally active, and has a regulatory impact on cell proliferation and secretion of pro-inflammatory and pro-destructive mediators. Whole-cell patch-clamp recordings identified KCa3.1 as the dominant potassium channel in the physiologically relevant membrane voltage range below 0 mV. Stimulation with transforming growth factor b1 (TGF-b1) significantly increased transcription, translation, and channel function of KCa3.1. Inhibition of KCa3.1 by the selective, pore-blocking inhibitor TRAM-34, (and, in part, by siRNA) significantly reduced cell proliferation, as well as expression and secretion of pro-inflammatory factors (IL-6, IL-8, and MCP1) and the tissue-destructive protease MMP3. These effects were observed in non-stimulated and/or TGF-b1-stimulated RA-SFs. Since small molecule-based interference with KCa3.1 is principally well tolerated in clinical settings, further evaluation of channel blockers in models of rheumatoid arthritis may be a promising approach to identify new pharmacological targets and develop new therapeutic strategies for this debilitating disease. J. Cell. Physiol. 230: 1677–1688, 2015. © 2014 Wiley Periodicals, Inc.
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, which is characterized by pain, swelling, and progressive destruction of multiple joints. Joint destruction in RA is perpetuated by an aggressive, invasive pannus tissue, a vascular and fibrous granulation tissue consisting of macrophages, synovial fibroblasts (SFs), T lymphocytes, and B lymphocytes/plasma cells. Uncontrolled, active RA causes disability, decreases quality of live, and increases comorbidity. The introduction of biologics meant a great improvement in the treatment of RA (Scott, 2011). Although biologics are very effective in the treatment of RA, they carry several disadvantages, e.g., lack of oral availability, the development of unresponsiveness, and high costs. Therefore, many pharmaceutical companies also develop small molecule inhibitors suitable for oral administration. In most cases, these small molecules are directed against the main signaling pathways regulating the transcription of pro-inflammatory genes, e.g., p38, Jak, or Syk (Bonilla-Hernan et al., 2011; Yazici and Regens, 2011). Besides several other molecules (Edmonds et al., 2011; Fleischmann, 2012), potassium channels might be regarded as new therapeutic targets for RA. Recently, Hu et al. showed that blocking of the calcium and voltage-activated Kþ channel KCa1.1 attenuates the pro-inflammatory and invasive behavior of synovial fibroblasts from RA patients (RA-SFs) (Hu et al., 2012). Potassium channels are attractive therapeutic targets, as they can be modulated by a variety of blockers and openers. The localization of channels in the plasma membrane makes them more attractive than intracellular targets, for which multi-drug resistance mechanisms represent a major limitation. The voltage-gated and Ca2þ-activated Kþ channel KCa1.1 belongs to the six-transmembrane-domain superfamily of Kþ channels. Within this group, eight channel-coding genes form the KCa subfamily, but only five members are truly Ca2þactivated channels (Salkoff et al., 2006). KCa1.1 (KCNMA1), © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .
also called “big”-conductance Kþ channel (BK), is activated by membrane depolarization, as well as by direct binding of cytosolic Ca2þ ions. Various potent and selective pharmacologic mediators for this channel have been described, including iberiotoxin (Wei et al., 2005). In contrast, four other members of the subfamily are voltage-independent, but can be activated by intracellular calcium sensed through constitutively bound calmodulin. Three small-conductance calcium-activated Kþ channels (KCa2.1, KCa2.2, KCa2.3; also called KCNN1–3) share sensitivity to the bee toxin apamin, while the
Disclosures: The authors indicate no potential conflict of interest. Contract grant sponsor: German Federal Ministry of Education and Research; Contract grant numbers: 13N9833, 035577D, 0315711A, 01EO1002, 0316205B. Contract grant sponsor: German Research Foundation; Contract grant numbers: KI 439/6–3, KI 439/7–3. Contract grant sponsor: Deutsche Krebshilfe e.V. (German Cancer Aid); Contract grant number: project 109851. *Correspondence to: Elke Kunisch, Experimental Rheumatology Unit, Dept. of Orthopedics, Jena University Hospital, Klosterlausnitzer Str. 81, D-07607 Eisenberg, Germany. E-mail: [email protected] Manuscript Received: 22 December 2013 Manuscript Accepted: 22 December 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 29 December 2014. DOI: 10.1002/jcp.24924
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intermediate-conductance Ca2þ-activated channel KCa3.1 (IKCa1, KCNN4) can be inhibited by clotrimazole and its more specific derivative TRAM-34 (Wulff et al., 2000a). Many years ago, the antifungal agent clotrimazole has been discussed as a potential therapeutic agent in rheumatoid arthritis (Otterness and Niblack, 1976; Wyburn-Mason, 1976) and a later study principally confirmed its beneficial effects in rheumatoid arthritis, such as improvement of grip strength, pain relief, and reduced joint swelling (Wojtulewski et al., 1980). However, the same study also suggested that the gastrointestinal side-effects of clotrimazole represent a contraindication for this therapeutic approach. The present study shows that KCa3.1 is expressed and functional in RA-SFs. Inhibition of KCa3.1 by the selective KCa3.1 inhibitor TRAM-34 or by siRNA reduced the proliferation of non-stimulated and TGF-b1-stimulated RA-SFs. In addition, TRAM-34 (and, in part, siRNA) reduced the production of several pro-inflammatory factors such asIL-6, IL-8, and monocyte chemotactic protein-1 (MCP1) at the mRNA and protein level, indicating that the potassium channel KCa3.1 may be a therapeutic target for RA. Materials and Methods Patients, tissue digestion, and cell culture Synovial tissue was obtained from RA patients fulfilling the ARA criteria (Arnett et al., 1988) during open joint replacement/ arthroscopic synovectomy at the Clinic of Orthopedics, Eisenberg, Germany. The study was approved by the Ethics Committee of the University of Jena, Germany and patient informed consent was obtained. RA synovial samples were digested, subsequently cultured for 7 days, and RA-SFs negatively isolated as previously described (Zimmermann et al., 2001; Hirth et al., 2002). RA-SFs were cultured in the virtual absence of contaminating non-adherent cells and macrophages. Third-passage cells were used for all experiments. Mycoplasma contamination of the cells was excluded by 4’-6-diamidino-2-phenylindole (DAPI) staining. Stimulation of the cells with TGF-b1 (10.0 ng/ml; R&D Systems, Wiesbaden, Germany) was performed in DMEM/10% FCS. To analyze the functional role of the potassium channel KCa3.1, RA-SFs were incubated with TRAM-34 (generous gift from Dr. Heike Wulff, University of California, Davis), in the absence or presence of TGF-b1 for 24 h. To exclude toxicity of the inhibitors, viability of the cells was assessed by fluorescein diacetate/ propidium iodide or ethidium bromide staining. Silencing of KCa3.1 in RA-SFs was performed by the siRNA technique using reverse transfection as previously described (Kunisch et al., 2012). In brief, GeneSolution siRNAi (Qiagen, Hilden, Germany) and LipofectaminTMRNAiMAX (Invitrogen, Darmstadt, Germany) were mixed in Opti-MEM (Invitrogen) and incubated for 20 min at room temperature to allow complex formation. Subsequently, cells previously suspended in DMEM, 10% FCS without antibiotics were added and incubated for 24 h. Thereafter, the cells were either cultivated for further 24 h without stimulation or, alternatively, stimulated with 10 ng/ml TGF-b1 in DMEM, 10% FCS for 24 h. Supernatants of the cells were collected for the analysis of cytokine and protease secretion. Cells were washed with ice-cold PBS and subsequently lysed in buffer for RNA-isolation (Qiagen). Transfection efficiency was analyzed using 10 nM BlockiTTMAlexaFluor1Red Fluorescent Oligo (Invitrogen). Transfection efficiency was greater than 85% (85.4% 2.7 positive cells). Quantitative RT-PCR Total RNA was isolated from RA-SFs and reverse-transcribed as previously described (Pohlers et al., 2007; Pretzel et al., 2009). mRNA expression was analyzed by real-time PCR using a RealPlex1 PCR machine (Eppendorf, Hamburg, Germany) as previously JOURNAL OF CELLULAR PHYSIOLOGY
described. The primer pairs and conditions for IL-6,IL-8, MCP1, MMP-1, and MMP-3 and the house-keeping gene aldolase are listed in Table 1. Primers were purchased from Jena Bioscience GmbH (Jena, Germany). The concentration of IL-6,IL-8, MCP1, MMP-1, and MMP-3 mRNA in each sample was calculated by the RealPlex1 software using an external standard curve. Product specificity of the real-time PCR was confirmed by: (i) melting curve analysis; (ii) agarose gel electrophoresis; and (iii) cycle sequencing of the PCR products. For mRNA quantification of KCa1.1, KCa2.1, KCa2.2, KCa2.3, KCa3.1, B2M and ACTB, QuantiTect1 primer pairs from Qiagen were used in PCR reactions with a Maxima1 SYBR Green/ ROX qPCR Master Mix (Fermentas, St. Leon-Rot, Germany). Efficiency of these primer pairs was tested using standard curves with logarithmic template dilutions. All primers showed close to 100% efficiency in the analyzed range of Ct values (up to 30). Protein detection by immunohistochemistry, ELISA, and Western blot For KCa3.1-immunohistochemistry in RA-SFs, 0.4 105 cells/well (8-chamber slide) were allowed to adhere for 24 h. Thereafter, cells were cultivated for 24 h, followed by fixation with 4% PFA/PBS for 20 min at room temperature. The fixed cells were rinsed with PBS and then incubated for 1 h in blocking buffer (PBS with 5% goat serum and 0.3% Triton-100). The cells were then exposed to the anti-KCa3.1 antibody (H-120, Santa Cruz; 1:100 diluted in PBS, 1% BSA, and 0.3% Triton) overnight in a moist chamber at 4 °C followed by incubation with horseradish-peroxidase-coupled goat anti-rabbit IgG (DAKO, Hamburg, Germany) for 1 h at RT. Cells were washed thoroughly with PBS and stained with DAB. For quantification of KCa3.1 protein, cells were seeded in 96well plates and treated for 24 h with TGF-b1 as described above. KCa3.1 protein was detected as described above with the slight modification of using the soluble HRP-substrate 3,30 ,5,50 Tetramethylbenzidine (TMB) instead of DAB. The absorbance was read in an ELISA Reader (BMG Fluostar). For Western blot detection, cells were lysed in reducing Laemmli buffer, heated to 60 °C for 5 min and subjected to 12% polyacrylamide SDS-PAGE. The proteins were transferred to Immobilon-P1 polyvinylidene difluoride (PVDF) membranes (Merck Millipore) and the membrane was blocked with 5% milk powder in PBS with 0.1% Tween 20. The membranes were then incubated overnight at 4°C with primary antibodies 1:1000 (rabbit anti KCa3.1, H-120, Santa Cruz) or 1:5000 (anti b-actin, mouse monoclonal AC15, Sigma-Aldrich) diluted in PBS-Tween with 1% BSA. For immunodetection, the membranes were incubated for 1 h with horseradish-peroxidase-coupled IgG anti-rabbit and anti-mouse, TABLE 1. Primer sequences and annealing temperatures used in RT-PCR
Gene Aldolase Sense Anti-sense IL-6 Sense Anti-sense IL-8 Sense Anti-sense MCP-1 Sense Anti-sense MMP-1 Sense Anti-sense MMP-3 sense Anti-sense
Annealing temperature (°C)
Melting temperature (°C)
5-tcatcctcttccatgagacactct-3 5-attctgctggcagatactggcataa-3
58
82
5-atgaactccttctccacaagcg-3 5-ctcctttctcagggctgag-3
62
84
5-gccaagagaatatccgaact-3 5-aggcacagtggaacaaggacttgt-3
60
78
5-cagccagatgcaatcaatgcc-3 5-tggaatcctgaacccacttct-3
60
82
5-gacctggaggaaatcttgc-3 5-gttagcttactgtcacacgc-3
58
81
5-gaacaatggacaaaggatacaaca-3 5-aagattgatgctgtttttgaagaa-3
58
81
Primer sequence
FUNCTION OF KCa3.1 IN SYNOVIAL FIBROBLASTS
respectively, and the reaction visualized using an ECL detection kit (GE Healthcare). Electrophysiological recordings Potassium currents were recorded at room temperature in the whole-cell configuration, using an EPC10 patch clamp amplifier (HEKA Elektronik, Germany) controlled by PatchMaster software (HEKA Elektronik). In all experiments, the holding potential was 80 mV. Series-resistance errors were compensated in the range of 70–90%. Patch pipettes were fabricated from borosilicate filament (Kimble Glass, USA) with resistance values in the range of 1.5–3 MV. Off-line analysis of data was performed using Igor Pro (WaveMetrics, USA) and FitMaster software (HEKA Elektronik). The standard bath solution contained 5 mM KCl, 155 mM NaCl, 2 mM CaCl2, and 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES); the pH was adjusted to 7.4 with NaOH. The internal solution contained 130 mM KCl, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPES, and 9.3 mM CaCl2 to yield 1 mM free Ca2þ; the pH was adjusted to 7.4 with KOH. The internal free calcium concentration was calculated in compliance with the WEBMAXC STANDARD software (http://www.stanford.edu/ cpatton/webmaxc/webmaxcS.htm). Currents were elicited by 500 ms voltage ramps, ranging from 100 mV to þ50 mV. In blocking experiments with TRAM-34, iberiotoxin (Alomone Labs) or apamin (Sigma-Aldrich), the same ramp protocol was applied every 5 sec. Mean current densities were determined in the range between 40 mV and 25 mV to analyze KCa2 and KCa3.1 currents and at about þ50 mV to detect primarily KCa1.1 currents. Voltage-gated Kþ currents were elicited by applying 100 ms pulses in 50 mV increments from 100 mV to þ150 mV. Analysis of functional parameters in RA-SFs by ELISA Proliferation was assessed by BrdU incorporation using a commercial cell proliferation ELISA (Roche) as previously described (Kunisch et al., 2007). Human IL-6, IL-8, MCP1, MMP-1, and MMP-3 proteins were measured in diluted cell culture supernatants as previously described (Kunisch et al., 2012). The resulting color was read at 450 nm in microtitre plates (Fluostar Optima). MMP-1 activity in the supernatants of stimulated cells was measured using a commercially available MMP-1 activity assay (Matrix Metalloproteinase Biotrak Activity Assay System, Amersham Biosciences) according to the manufacturer’s instructions. Statistical analysis Data are presented as means standard error of the mean (SEM). The non-parametric Mann–Whitney U-test was applied to analyze differences between controls and individual stimuli (software program SPSS 13.0TM; SPSS Inc., Chicago, IL). Significant differences were accepted for P 0.05. Results Expression of Kþ channels in RA-SFs
Primary cell cultures of RA-SFs were analyzed for the expression of calcium-activated potassium channels (KCa). In non-stimulated RA-SFs, no mRNA was observed for the small conductance channels KCa2.1, KCa2.2, and KCa2.3. However, mRNA expression was detectable for the intermediate conductance channel KCa3.1 and the big conductance channel KCa1.1 (Fig. 1A). The expression levels of the 2 genes did not differ significantly, ranging between 5% and 10% of the mRNA level of b-actin (ACTB). The expression of KCa3.1 was confirmed at the protein level. In comparison to lack of staining with the isotype control, JOURNAL OF CELLULAR PHYSIOLOGY
Fig. 1. Expression of KCa channel mRNAs in RA-SFs. A) mRNA expression of the KCa channels KCa2.1, KCa2.2, KCa2.3, KCa3.1, and KCa1.1 in non-stimulated RA-SFs (n ¼ 4 donors) was normalized to the expression of b-actin (means SEM). B) Protein expression of KCa3.1 in non-stimulated RA-SFs was assessed by immunohistochemistry (n ¼ 4; one representative result is shown). C) Western Blot detection of KCa3.1 protein in transfected HEK293 cells and RA-SFs. Whole-cell extracts of mock-transfected HEK293 cells, KCa3.1-transfected HEK293, and of RA-SFs in primary culture were analyzed for KCa3.1 protein using a polyclonal rabbit serum. Multiple bands in the molecular weight range of 45 kDa and below in RA-SFs and in the positive control likely represent channel protein fractions with different glycosylation states.
KCa3.1 protein was clearly located in the perinuclear cytoplasm of RA-SFs (Fig. 1B). Specificity of the anti-KCa3.1 antiserum was confirmed by Western blot analysis comparing lysates of KCa3.1-transfected HEK293 cells and RA-SFs (Fig. 1C). The signal in RA-SFs matched the molecular weight of the transfected protein in HEK293 cells, excluding cross-reactions with the
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much larger KCa3.1 protein (47 kDa). No Western blot signal was detected in mock-transfected HEK293 cells. Functional Ca2þ-activated Kþ channels in RA-SFs
The presence of mRNA for the 2 KCa channel family members KCa3.1 and KCa1.1 led to detailed analysis of the electrophysiological properties of RA-SFs. Ionic currents elicited by voltage-ramps from 100 to 50 mV in whole-cell patch-clamp experiments with 1 mM Ca2þ in the pipette reflected the known properties of KCa3.1 and KCa1.1 (Fig. 2A–F). In the negative voltage range, current responses were strictly linear, as expected for a voltage-independent channel such as KCa3.1. In contrast, the increase of the currents in the positive voltage range deviated from the linear slope, a characteristic of depolarization-activated channels such as KCa1.1 (Fig. 2A). Both current components were strongly calcium-dependent, i.e., in the absence of Ca2þ in the pipette solution the mean currents were reduced to
