Expression of transient receptor potential vanilloid 1 (TRPV1) in synovial fibroblasts from patients with osteoarthritis and rheumatoid arthritis

Biochemical and Biophysical Research Communications 359 (2007) 884–888 www.elsevier.com/locate/ybbrc

Expression of transient receptor potential vanilloid 1 (TRPV1) in synovial fibroblasts from patients with osteoarthritis and rheumatoid arthritis Andrea Engler a

a,b,*

, Andre´ Aeschlimann c, Beat R. Simmen d, Beat A. Michel Renate E. Gay a,b, Steffen Gay a,b, Haiko Sprott a,b

a,b

,

Center of Experimental Rheumatology, Department of Rheumatology and Institute of Physical Medicine, University Hospital, Gloriastrasse 25, Zurich, Switzerland b Center for Integrative Human Physiology (CIHP), University of Zurich, Zurich, Switzerland c RehaClinic Zurzach, CH-5330 Zurzach, Switzerland d Schulthess Clinic, CH-8008 Zurich, Switzerland Received 24 May 2007 Available online 4 June 2007

Abstract The transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel, which is mainly expressed by nociceptive neurons in dorsal root and trigeminal ganglia. However, there is increasing evidence that TRPV1 expression is not limited to primary afferent neurons but that the receptor is expressed in various cell types throughout the body. Here, we demonstrate the expression of TRPV1 in synovial fibroblasts (SF) from patients with symptomatic osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, the mRNA expression of TRPV1 was shown in PBMCs from healthy controls and from OA patients. The presence of TRPV1 was confirmed at the protein level. Stimulation of cultured OA- and RA-SF with the TRPV1 agonist capsaicin led to increased expression of IL-6 mRNA as well as of IL-6 protein in the cell culture supernatants. IL-6 protein expression could be antagonized with capsazepine. Thus, we hypothesize that TRPV1 may play a role in non-neuronal mechanisms that might modulate nociception in symptomatic OA and RA patients.  2007 Elsevier Inc. All rights reserved. Keywords: Transient receptor potential vanilloid 1; Vanilloid receptor; TRPV1; Capsaicin; Capsazepine; Synovial fibroblast; Osteoarthritis; Rheumatoid arthritis; Gene expression; Real-time PCR

The transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel that is predominantely expressed by primary sensory afferents and is activated by noxious heat, protons and vanilloids like capsaicin resulting in calcium influx [1,2]. Besides its role in the nervous system there is increasing evidence that TRPV1 is expressed in various cell types throughout the body, including bladder epithelial cells, keratinocytes, dental pulp and * Corresponding author. Present address: ETH Zurich, Psychology and Behavioral Immunobiology, Turnerstrasse 1, 8092 Zurich, Switzerland. Fax: +41 44 632 13 55. E-mail address: [email protected] (A. Engler).

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.05.178

skin fibroblasts, as well as in peripheral blood mononuclear cells (PBMCs) [3–7]. Activation of non-neuronal TRPV1 by the agonist capsaicin results in the release of interleukin (IL-)8, IL-6 and prostaglandin E2 (PGE2) and increases cyclooxygenase-2 (COX-2) expression in keratinocytes, dental pulp fibroblast and bronichal epithelial cells [4,6,8,9]. In turn, inflammatory mediators, like PGE2 or prostaglandin I2 (PGI2), were shown to increase or sensitize TRPV1 activity in both neuronal and non-neuronal cells, i.e., mouse dorsal root ganglion (DRG) neurons and HEK293 cells [10]. The role of TRPV1 in different pain syndromes has been well documented. For example, TRPV1 deficient mice

A. Engler et al. / Biochemical and Biophysical Research Communications 359 (2007) 884–888

show impaired behavioral responses to noxious thermal stimuli and reduced thermal hyperalgesia after tissue injury [11]. Moreover, silencing of TRPV1 by siRNA reduced both cold allodynia in a rat neuropathic pain model and spontaneous visceral pain behaviour in a mouse model of acute visceral pain [12]. The majority of arthritis patients are affected by pain at some point of the disease and arthritis pain has been recently linked to TRPV1 in animal models. It has been demonstrated that the expression of TRPV1 is increased in the rat iodoacetate osteoarthritis model compared to control animals [13] and that TRPV1 knockout mice exhibit reduced pain behaviour in adjuvant-induced arthritis [14]. DRG neurons, when co-cultured with synovial fibroblasts (SF) from chronically inflamed rat antigen-induced arthritis knee joints displayed an up-regulation of TRPV1 expression, which was not seen when co-cultured with normal SF or without co-culture [15]. Due to the increased attention that is drawn to the various functions of non-neuronal TRPV1 we hypothesize that TRPV1 may play a role in non-neuronal mechanisms that might modulate nociception in arthritis. Therefore, we addressed the question whether TRPV1 is expressed in SF and PBMCs from symptomatic OA and RA patients. Materials and methods Tissue samples and cell culture. Synovial tissues were obtained from patients with OA and RA undergoing joint arthroplasty (Department of Orthopedic Surgery, Schulthess Clinic, Zurich, Switzerland) with approval by the local ethics committee. All OA and RA patients fullfilled the criteria of the American College of Rheumatology for the classification of OA and RA, respectively [16,17] and gave written informed consent. The synovial tissues were minced and digested with dispase at 37 C for 60 min. After washing, cells were grown in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (1:1) (DMEM/F-12) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 10 mM Hepes, 50 IU/ml penicillin–streptomycin and 0.5 lg/ml amphotericin B (all Gibco Invitrogen, Basel, Switzerland). Cell cultures were maintained in a humidified incubator (37 C, 5% CO2) and were used between passages 5 and 9 for all experiments described. Isolation of PBMCs. PBMCs from whole blood was isolated by gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Otelfingen, Switzerland). Blood was diluted 1:2 with PBS, layered on top of the corresponding amount of Ficoll-Paque Plus and centrifuged at 450 · g for 30 min at room temperature. After separation the lymphocytes were transferred to a fresh tube, washed three times with PBS and then subjected to RNA isolation. RNA isolation and reverse transcription. Isolation of total RNA was performed with the RNeasy Mini Kit including on-column DNase I treatment (Qiagen, Hombrechtikon, Switzerland) and reverse transcribed with MultiScribe reverse transcriptase and random hexamer primers (Applied Biosystems, Rotkreuz, Switzerland). The resulting cDNA was used as a template for PCR with TRPV1-specific primers. PCR analysis. Qualitative analysis of TRPV1 receptor expression was done by conventional PCR using forward 5 0 -caacaagatcgcacaggaga-3 0 and reverse 5 0 -tccttgccatcaggtgtgta-3 0 primers (Microsynth, Balgach, Switzerland) that yield a 150 bp product from TRPV1 exons 14 and 15 (NM_080704). cDNA generated from normal human cerebellum, spinal cord and skeletal muscle (all Clontech Europe, France) served as positive controls [18,19]. The amplicon was subcloned into pPCR-Script Amp SK(+) Vector (Stratagene Europe, The Netherlands) and the sequence verified.

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Real-time PCR. Quantitative gene expression analysis was performed by real-time PCR using TaqMan Gene Expression Assays for TRPV1 (Hs00218912_m1) and GAPDH control reagents (Applied Biosystems). IL-6 gene expression was analyzed using a SybrGreen real-time PCR assay with GAPDH as endogenous control. Primer sequences are available in the public RTPrimerDB database http://medgen.UGent.be/rtprimerdb/ (IL-6, RTPrimerDB-ID: 3545 and GAPDH RTPrimerDB-ID: 3), [24]. Relative gene expression was calculated using the comparative threshold cycle (DDCt) method [20]. Western blot. Cells were directly lysed in 2· Laemmli buffer, boiled for 5 min and subjected to SDS–PAGE on a 10% gel. The proteins were transferred onto Protran nitrocellulose membrane (Schleicher & Schu¨ll, Dassel, Germany) at 300 mA for 1 h. The membrane was blocked with 5% non-fat dry milk in PBS over night at 4 C and then incubated with polyclonal antibodies against a synthetic N-terminal TRPV1 peptide (Acris Antibodies, Hiddenhausen, Germany) for 2 h at room temperature followed by 1 h incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Europe, Suffolk, England). Visualization was performed with an enhanced chemiluminescence (ECL) system (GE Healthcare, Otelfingen, Switzerland). Capsaicin/capsazepine stimulation of cells. For stimulation experiments the cells were seeded in 12-well plates at 60,000 cells per well. After 24 h the cells were serum starved in DMEM/F-12 supplemented with 0.5% FCS and polymyxin B (5 lg/ml) for another 24 h prior the addition of 100 lM capsaicin. Capsazepine treatement started with the addition of 10 lM capsazepine 30 min prior capsaicin addition. Capsaicin and capsazepine were obtained from Sigma (Buchs, Switzerland) and were both dissolved in 100% ethanol. Control cells were treated with equal amounts of ethanol. Numbers of different OA- and RA-SF used are indicated in the figure legends. Almost all cell lines were used in at least two independent experiments. IL-6 ELISA. IL-6 production in cell culture supernatants from capsaicin/capsazepine stimulated cells was quantified using the BD OptEIA human IL-6 ELISA Set according to the manufacturer’s instructions (BD Biosciences, Allschwil, Switzerland).

Results Constitutive TRPV1 expression in synovial fibroblasts from OA and RA patients Using RT-PCR we amplified a TRPV1-specific PCR product from total RNA from cultured SF from OA and RA patients. The PCR product was cloned into a vector and the sequence verified. In addition, TRPV1-specific amplicons were obtained from PBMCs from healthy control subjects and from OA patients as well as from spinal cord, cerebellum and skeletal muscle cDNA (Fig. 1A). Western blot analysis revealed the presence of TRPV1 protein in human neuronal tissue (positive control) as well as in SF from OA patients (Fig. 1B). TRPV1 protein was also present in SF from RA patients (data not shown). Real-time PCR analysis of TRPV1 expression Quantitative gene expression analysis by real-time PCR revealed similar expression levels of TRPV1 in OA- and RA-SF (n = 2 each) with cycle threshold (Ct) values around 29. In PBMCs TRPV1 expression was approximately three-fold higher than in OA- and RA-SF. No differences were observed in PBMC expression levels between healthy control subjects (n = 11) and OA-patients (n = 27).

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In general, neuronal tissues displayed remarkably higher expression levels, with 15- and 19-fold higher TRPV1 expression in spinal cord and cerebellum (n = 1 each) than in OA-SF, respectively (Fig. 2).

Capsaicin stimulation of synovial fibroblasts It has been demonstrated previously that activation of TRPV1 promotes the production of pro-inflammatory cytokines in various cell types [21–23,6]. Therefore, we stimulated OA- and RA-SF with the TRPV1 agonist capsaicin and analyzed the cell culture supernatants for IL-6 after 24 h. IL-6 levels in the cell culture supernatants of cells treated with capsaicin were increased by 20% in OASF (n = 5) and 30% in RA-SF (n = 7), whereas capsaicin treated cells that were pre-treated with the TRPV1 antagonist capsazepine (n = 4 each for OA-SF and RA-SF) displayed no increase in IL-6 and had similar IL-6 levels as the untreated controls (Fig. 3A and B). Furthermore, IL-6 gene expression in OA- (n = 5) and RA-SF (n = 7) was increased two-fold and five-fold, respectively, after 24 h capsaicin stimulation, but in contrast to the protein levels IL-6 gene expression was not

relative TRPV1 gene expression (fold change)

Fig. 1. Expression of TRPV1 in synovial fibroblasts. (A) Amplification of TRPV1-specific mRNA by conventional PCR. The input cDNA was derived from total RNA from OA-SF (lane 1), RA-SF (lane 2), healthy control PBMCs (lane 3), OA PBMCs (lane 4), spinal cord (lane 5), cerebellum (lane 6) and skeletal muscle (lane 7). PCR products were resolved on a 2.5% agarose gel and stained with ethidium bromide. (B) Detection of TRPV1 protein in synovial fibroblasts. Western blot analysis of TRPV1 protein from human tissue from the occipital cortex (lanes 1 + 2) and different OA-SF cell cultures (lanes 3–5). Whole-tissue or whole-cell lysates were immunoblotted with anti-human TRPV1 polyclonal antibodies.

20 18 16 14 12 10 8 6 4 2 0 OA-SF

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OA PBMCs spinal cord cerebellum

Fig. 2. Quantitative TRPV1 mRNA expression analysis. Quantitative TRPV1 gene expression was analyzed by real-time PCR using GAPDH as endogenous control. TRPV1 gene expression is presented relative to the expression in OA-SF. Fold changes were calculated according to the comparative threshold cycle (DDCt) method. (n = 2 for OA- and RA-SF each, n = 11 for control PBMCs, n = 27 for OA PBMCs and n = 1 for spinal cord and cerebellum each).

attenuated by antagonist treatment (n = 5 for OA-SF and n = 7 for RA-SF) but was rather enhanced in RA-SF (Fig. 4A and B).

A. Engler et al. / Biochemical and Biophysical Research Communications 359 (2007) 884–888

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Fig. 3. Capsaicin-induced IL-6 synthesis in synovial fibroblasts is inhibited by capsazepine. (A) OA-SF and (B) RA-SF were stimulated with 100 lM capsaicin for 24 h and cell culture supernatants were analyzed for IL-6. IL-6 concentrations were increased in capsaicin treated cells (hatched bars, n = 5 for OA, n = 7 for RA) compared to controls (open bars, n = 5 for OA, n = 7 for RA). Addition of capsazepine blocked the capsaicininduced increase in IL-6 (solid bars, n = 4 each for OA and RA).

12 10 8 6 4 2 0 capsaicin/capsazepine stimulation of RA-SF

Discussion

Fig. 4. IL-6 gene expression in synovial fibroblasts is induced by capsaicin. (A) OA-SF and (B) RA-SF were stimulated with 100 lM capsaicin for 24 h and IL-6 gene expression was analyzed. IL-6 gene expression was increased in capsaicin treated cells (hatched bars, n = 5 for OA, n = 7 for RA) compared to controls (open bars, n = 5 for OA, n = 7 for RA). Addition of capsazepine did not block the capsaicin-induced increase in IL-6 gene expression (solid bars, n = 4 each for OA and RA). In contrast, IL-6 gene expression was further increased in capsazepine treated RA-SF.

In the present study we demonstrated the expression of TRPV1 in synovial fibroblasts (SF) from OA and RA patients by RT-PCR and real-time PCR, which is in line with reports of TRPV1 expression in dental pulp or skin fibroblasts [6,5]. In addition, we could confirm the expression of TRPV1 in PBMCs from healthy individuals. TRPV1 expression levels in PBMCs from OA patients were similar to those in healthy volunteers, with only small differences among individuals in both groups. The physiological role of TRPV1 expression in PBMCs has not been elucidated so far, but it has been suggested that TRPV1 may act as an indicator of noxious stimuli in the blood or inflammatory conditions at secondary sites [7]. However, our data indicate that PBMC TRPV1 mRNA expression levels alone are not sufficient for such predictions and a more detailed analysis of TRPV1 mRNA and protein in different subsets of cells might add valuable information. In order to assess the activation of TRPV1 by capsaicin in SF we analyzed cell culture supernatants from capsaicintreated cells for IL-6 protein and we found increased IL-6 levels in both OA- and RA-SF. The specificity of IL-6 production as a result of TRPV1 activation was demonstrated by attenuated IL-6 protein levels in cells that were treated

with the TRPV1 antagonist capsazepine. However, IL-6 mRNA expression was not found to be attenuated upon antagonist treatment. While our IL-6 protein data are in accordance with other studies performed in dental pulp fibroblasts [6] and upper respiratory epithelial cells [9] our IL-6 mRNA data differ. Currently we cannot rule out, that although in our study, after 24 h of capsaicin/ capsazepine treatment, mRNA expression did not correlate with protein expression levels, IL-6 mRNA and protein expression would have correlated at shorter incubation timepoints, like it was shown in upper respiratory epithelial cells, in which the capsaicin-mediated increase in IL-6 mRNA peaked at 4 h and could be significantly inhibited by capsazepine pre-treatment [9]. Therefore, time-course experiments and the use of other TRPV1 antagonist will be necessary to clarify this issue. Interestingly, the involvement of several signalling pathways in TRPV1 activation has been reported. Miyamoto et al. [6] demonstrated capsaicin-induced IL-6 production through the activation of p38 mitogen-activated protein kinase (MAPK) but not Jun Nterminal kinase (JNK) in dental pulp fibroblast cultures. But at the same time they also suggested a role for the

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extracellular signal-related kinase (ERK) in capsaicin-mediated TRPV1 signalling independent of IL-6 production. This is the first report that TRPV1 is present in synovial fibroblasts from symptomatic OA and RA patients and that the receptor can be activated by stimulation with the vanilloid capsaicin. It is very likely that capsaicin stimulation does not only induce IL-6 production, but also results in the release of other signalling molecules that may transfer the information to other non-neuronal or neuronal cells and thereby contribute to a modulation of nociception in the inflamed joint. However, further studies will be necessary to clarify the possible role of TRPV1 in non-neuronal mechanisms that might modulate nociception or otherwise still unknown functions in OA and RA patients.

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