Background
Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel which mediates pain and hyperalgesia [
1,
2] and functions as a chemosensor of noxious compounds [
3‐
5]. TRPA1 was first discovered in 1999 [
6] and has since then been found to be widely expressed in afferent sensory neurons, especially in Aδ and C fibers of nociceptors [
7,
8]. In addition to pain, TRPA1 also has a role in mediating neurogenic inflammation [
9,
10]. More recently, TRPA1 has been found to be expressed also in some nonneuronal cells such as keratinocytes [
11] and synoviocytes [
12] but the functional roles of nonneuronal expression remain to be studied.
TRPA1 is activated by numerous exogenous pungent compounds such as allyl isothiocyanate (AITC) from mustard oil [
5], acrolein from exhaust fumes and tobacco smoke [
9], and allicin from garlic [
3]. Interestingly, TRPA1 is also activated and sensitized by agents formed endogenously in inflammatory reactions, such as nitric oxide [
13], hydrogen peroxide [
14] and nitro-oleic acid [
15]. The activation of TRPA1 causes an influx of cation ions, particularly Ca
2+, into the activated cells [
16] and this elevation of intracellular Ca
2+ has been shown to trigger an action potential in neuronal cells [
16,
17]. Interestingly, among the many regulatory effects of the alterations of intracellular Ca
2+ concentration, its increase has also been shown to affect the gene expression of inflammatory mediators [
18‐
20].
Recent evidence suggests TRPA1 to have a role in inflammation through exogenous activation by TRPA1 agonists and also through endogenous mechanisms. TRPA1 has been shown to mediate carrageenan-induced inflammatory edema [
21], tumor necrosis factor (TNF)-triggered hyperalgesia [
22], airway hyperreactivity and inflammation [
23,
24], and to relate to acute gouty arthritis [
25,
26]. Very recently we found that TRPA1 has a role in mediating acute inflammation, cartilage destruction, and joint pain in monosodium iodoacetate (MIA)-induced inflammation and osteoarthritis in the mouse [
27].
Osteoarthritis (OA) is the most common cause of musculoskeletal disability and pain worldwide and its prevalence is constantly increasing as the population ages. OA is a degenerative disease of the joints, which is characterized by inflammation and hypoxia within the joint, leading to cartilage degradation, joint deformity, disability, and pain [
28,
29]. OA-related cartilage degradation is caused by a growing imbalance between the production of catabolic, anabolic, and inflammatory mediators within the joint driven by the increased expression of matrix-degrading metalloproteinases and proinflammatory mediators such as interleukin (IL)-6 and prostaglandin E
2 (PGE
2) [
28].
TRPA1 has not previously been investigated in chondrocytes. However, factors involved in hypoxia and inflammation, such as hydrogen peroxide (H
2O
2), nitric oxide (NO), and IL-6 have been shown to upregulate the expression or activation of TRPA1 in some other cells [
12‐
14]. Furthermore, the activation of TRPA1 has been reported to enhance the production of inflammatory factors [
12,
21,
26,
30]. Since there is a hypoxic and inflammatory state in OA joints [
28,
31], and TRPA1 has been shown to be involved in the mediation of acute inflammation and cartilage degradation in MIA-induced osteoarthritis [
27], we hypothesized that TRPA1 is expressed in the chondrocytes in osteoarthritic joints, where its activation could play a vital part in the inflammation and pathogenesis of OA. In the present study, we tested that hypothesis by measuring the expression and function of TRPA1 in primary human OA chondrocytes.
Discussion
The findings of the present study suggest a hitherto unknown role for TRPA1 in the pathogenesis of OA. We have shown for the first time the expression of the TRPA1 channel in primary human OA chondrocytes and in the human T/C28a2 chondrocyte cell line. We showed the expression of TRPA1 mRNA and protein by qRT-PCR and Western blot, respectively. We were also able to show that the expressed TRPA1 was functional, as evidenced by Ca2+-influx measurements. Further, we found TRPA1 to have a role in mediating the production of OA-related factors MMP-1, MMP-3, MMP-13, IL-6, and PGE2 as evidenced by pharmacological inhibition and genetic depletion of TRPA1.
TRPA1 was first discovered in 1999 in fetal lung fibroblasts [
6]. Since then it has been mainly studied in different afferent sensory neurons such as Aδ and C fibers of nociceptors [
7,
8]. More recently, however, TRPA1 has also been found to be expressed in some nonneuronal cells such as keratinocytes [
11,
37,
38], synoviocytes [
12,
39] and airway epithelial and smooth muscle cells [
30]. It is noteworthy, that not all of these studies have shown functionality of the TRPA1 ion channel and some have only reported the expression of TRPA1 at the mRNA level. In the present study, we have comprehensively shown the expression and activation of TRPA1 in human chondrocytes, to support the criteria set by Fernandes et al. [
40]. We were able to show for the first time the expression of both TRPA1 mRNA and protein and the functionality of the TRPA1 channel in primary human OA chondrocytes and in human T/C28a2 chondrocyte cell line. This finding is particularly interesting as in OA joints there is a hypoxic [
31] and inflammatory [
28,
41] state and related factors, H
2O
2, NO, and IL-6, have previously been shown to upregulate the expression and activation of TRPA1 [
12‐
14]. According to Hatano et al. [
12] the human
TRPA1 promoter has at least six putative nuclear factor kappa B (NF-kB) binding sites and ten core hypoxia response elements (HREs), which are binding sites for hypoxia-inducible factor (HIF) transcription factors. HIFs are known to mediate adaptive responses to hypoxia as well as to be activated by inflammation [
42,
43] and the binding of HIFs to consensus HREs on their target genes regulates gene transcription.
After discovering TRPA1 expression in chondrocytes, we aimed to investigate whether inflammatory factors/mechanisms related to the pathogenesis of OA [
28,
29] regulate expression of TRPA1, which would indicate a role for TRPA1 as a mediator in OA. IL-1β is considered as a major player in OA associated with cartilage destruction. IL-1β is elevated in OA joints and it suppresses type II collagen and aggrecan expression, stimulates the release of MMP-1, MMP-3, and MMP-13, and induces the production of IL-6 and some other cytokines as well as PGE
2 [
28]. In part IL-17 feeds forward these mechanisms as it further induces IL-1β, TNF, and IL-6 production, upregulates NO and MMPs and downregulates proteoglycan levels related to the pathogenesis of OA [
28]. Based on our results, IL-1β and IL-17 both also induce TPRA1 expression and intriguingly, some of the IL-1β-induced inflammatory and catabolic effects are partly mediated by TRPA1. In OA the innate immune system and in particular toll-like receptors (TLRs) activated by cartilage matrix degradation products, also play a significant part in disease progression. Chondrocytes express TLRs, which trigger major inflammatory pathways and are activated by bacterial lipopolysaccharide (LPS) and damage-associated molecular patterns [
29], and also the adipocytokine resistin known to be expressed in OA joints [
44] has been shown to transduce its effects through toll-like receptor 4 [
45]. In the present study, we found that both LPS and resistin increased expression of TRPA1 in human chondrocytes, suggesting a TLR-mediated mechanism to enhance TRPA1 expression in OA cartilage. In support of the present results, Hatano et al. showed that TRPA1 gene expression was enhanced in synoviocytes by inflammatory factors TNF-α and IL-1 [
12], and the present study together with that of Hatano et al. [
12] suggests a previously unrecognized mechanism that links TRPA1 as an inducible factor to joint inflammation.
Activation of TRPA1 results in a substantial influx of Ca
2+ into the stimulated cells [
46]. Here we verified the functionality and activation of the TRPA1 channel in human chondrocytes by measuring Ca
2+ influx using the TRPA1 agonist AITC as well as the TRPA1 antagonist HC-030031. As shown previously, elevated intracellular Ca
2+ concentration may affect the expression of inflammatory genes both in a direct or indirect manner [
20]. In the present study, we found that TRPA1 regulated the production of inflammatory and catabolic factors, namely MMP enzymes, IL-6, and PGE
2 in chondrocytes. IL-1-induced MMP-3, IL-6, and PGE
2 production in the cartilage from TRPA1-deficient mice was less than half of that found in the cartilage from wild-type mice. Accordingly, the selective TRPA1 antagonist HC-030031 reduced IL-1-induced MMP-1, MMP-3, MMP-13, IL-6, and PGE
2 production by 25–45 % in primary human OA chondrocytes. In the latter experiment, the cells were incubated in the presence of IL-1 and HC-030031 for 24 h; therefore the result may be an underestimate of the effect of total inhibition of TRPA1 in OA chondrocytes because HC-030031 is a reversible TRPA1 antagonist with a relatively short half-life [
47]. These findings are supported by previous studies indicating that TRPA1 activation regulates the production of IL-1 in keratinocytes [
38], IL-6 and IL-8 in synoviocytes [
12], and PGE
2 along with leukotriene B
4 in fibroblasts and keratinocytes [
48]. We have recently found that TRPA1 also regulates the expression of cyclooxygenase-2 (COX-2) [
21,
27] and the production of monocyte chemotactic protein-1 (MCP-1), IL-6, IL-1β, myeloperoxidase (MPO), MIP-1α and MIP-2 in inflammatory conditions [
26]. The detailed molecular mechanisms of this regulation remain, however, to be studied.
TRPA1 is shown to be involved in pain, hyperalgesia, and neurogenic inflammation [
10,
16,
49,
50]. In OA-related pain, the role of TRPA1 has been investigated in studies by Moilanen et al. [
27] McGaraughty et al. [
51] and Okun et al. [
52] using the MIA-model of OA. The two first-mentioned studies [
27,
51] concluded TRPA1 to contribute to joint pain in experimental OA. In addition, Moilanen et al. [
27] reported that TRPA1-deficient mice developed less severe cartilage changes following MIA injections. Accordingly, we showed here that TRPA1 is functionally expressed in chondrocytes. We also examined the possible functions of the channel by treating primary chondrocyte cultures with IL-1β and the selective antagonist HC-030031 [
2,
53,
54]. Our results suggest an inflammatory and catabolic role for TRPA1 in human chondrocytes, as we found inhibition of TRPA1 to suppress the production of OA-related factors MMP-1, MMP-3, MMP-13, IL-6, and PGE
2. These results were supported by experiments with cartilage from WT and TRPA1-deficient mice: following stimulation with IL-1β MMP-3, IL-6, and PGE
2 production was lower in the cartilage from TRPA1-deficient mice than from WT animals. These results together suggest that TRPA1-activating factors are present in OA joints, and that TRPA1 mediates, at least partly, OA-related pain, inflammation, and cartilage destruction in neuronal and nonneuronal cells in the joint.
Abbreviations
AITC, allyl isothiocyanate; ANOVA, analysis of variance; COX-2, cyclooxygenase-2; ELISA, enzyme-linked immunosorbent assay; H2O2, hydrogen peroxide; HIF, hypoxia-inducible factor; HRE, hypoxia response element; IL, interleukin; KO, knockout; LPS, lipopolysaccharide; MCP-1, monocyte chemotactic protein-1; MIA, monosodium iodoacetate; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; MPO, myeloperoxidase; NF-kB, nuclear factor-kappa B; NO, nitric oxide; OA, osteoarthritis; PGE2, prostaglandin E2; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean; TLR, toll-like receptor; TNF, tumor necrosis factor; TRPA1, transient receptor potential ankyrin 1; WT, wild-type
Acknowledgements
We wish to thank Ms Salla Hietakangas, Terhi Salonen, and Ella Lehto for excellent technical assistance and Ms Heli Määttä for skillful secretarial help.