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The Ca2+-permeable cation channel transient receptor potential vanilloid 4 (TRPV4) was first cloned in 2000 (Strotmann et al., 2000) and belongs to the TRP superfamily of channels. TRPV4 expression shows a broad tissue distribution, with expression identified in epithelia of the skin, lung and kidney, in vascular endothelium and smooth muscle and in the brain by a combination of immunohistochemistry, in situ hybridization and functional studies (Strotmann et al., 2000; Jia et al., 2004; Cohen, 2005; Watanabe et al., 2008).
Its physiological roles are equally diverse, with suggested roles in osmoregulation (Liedtke and Friedman, 2003), stretch sensation in the bladder (Gevaert et al., 2007), flow sensation in kidney tubules (Wu et al., 2007) and osteoclast maturation (Masuyama et al., 2008), amongst others. Expression and activation of TRPV4 in the peripheral nervous system is implicated in mechanical nociception, particularly mechanical hypersensitivity following inflammation or neuropathy. It is less clear whether TRPV4 contributes to basal mechanosensation. Sensory deficits in response to noxious pressure (Suzuki et al., 2003) and bladder stretch (Gevaert et al., 2007) have been described in TRPV4 knockout mice. However, other studies have failed to identify basal differences in the response to mechanical stimuli between wild-type and knockout animals (e.g. Grant et al., 2007; Alessandri-Haber et al., 2008).
In common with other TRP channels, TRPV4 is activated by a wide range of physical and chemical stimuli. It was originally identified as a calcium channel responding to extracellular hypo-osmolarity and consequent cell swelling (Liedtke et al., 2000). Application of positive or negative pressure to membranes of TRPV4-expressing HEK293 cells in cell-attached patch clamp experiments did not alter channel activity, which suggested that TRPV4 did not directly respond to swelling-induced membrane stretch (Strotmann et al., 2000). An indirect mechanism of activation was proposed by Watanabe et al. (2003), who suggested swelling activates phospholipase A2-mediated release of arachidonic acid, which is then metabolized to form a TRPV4 agonist 5′,6′-epoxyeicosatrienoic acid (5′,6′-EET). However, the results of two recent studies suggest that direct mechanical gating of TRPV4 by membrane stretch (Loukin et al., 2010) or by force applied to the extracellular matrix (Matthews et al., 2010) can activate the channel. The first synthetic TRPV4 agonist to be identified was 4α-phorbol 12,13-didecanoate (4αPDD), a non-PKC activating phorbol ester with an EC50 around 200 nM at heterologously expressed human and murine TRPV4 (Watanabe et al., 2002a). Direct interaction of 4αPDD with a ligand-binding pocket formed by transmembrane regions 3 and 4 opens the channel (Vriens et al., 2007). The most potent small molecule agonist at TRPV4 identified to date is GSK1016790A, with a structure distinct from that of the phorbol esters. It has an EC50 around 18 nM at heterologously expressed murine TRPV4 (Thorneloe et al., 2008; Willette et al., 2008).
We recently identified TRPV4 expression in rat dorsal root ganglia neurons using RT-PCR and immunohistochemistry. In addition, we demonstrated that TRPV4 was sensitized by protease activated receptor 2 (PAR2) activation in cultured cells, and PAR2 activation in vivo produced a TRPV4-dependent mechanical hypersensitivity in mice (Grant et al., 2007). The increase in intracellular calcium in response to hypotonic challenge was diminished in neurons from TRPV4 knockout mice and was not sensitized by inflammatory ‘soup’, in contrast to the response in wild-type neurons (Alessandri-Haber et al., 2006). Inhibition of tetrodotoxin-resistant sodium currents by hypotonic challenge was reduced in trigeminal ganglia sensory neurons from TRPV4 knockout mice (Chen et al., 2009). Small diameter osmosensitive hepatic neurons were absent in TRPV4 knockout mice. Neurons from these mice only showed a delayed, irreversible increase in intracellular calcium in response to 4αPDD, whereas some wild-type neurons exhibited a rapid, reversible response. The authors interpreted the former response as a non-selective, toxic effect (Lechner et al., 2011). The number of neurons that showed an increase in intracellular calcium after 4αPDD treatment and the size of the increase were diminished in DRG neurons innervating the colon after administration of siRNA directed against TRPV4 (Cenac et al., 2008).
Various laboratories have also carried out studies linking TRPV4 activation to the mechanical hyperalgesia observed in chemotherapeutic and diabetic neuropathies (Alessandri-Haber et al., 2008), colonic inflammation (Sipe et al., 2008), chronic constriction injury of sensory nerves (Zhang et al., 2008) and pancreatitis (Ceppa et al., 2010). Altered nerve growth factor (NGF) signalling is implicated as a causal mechanism of the mechanical hyperalgesia in many neuropathic and inflammatory pain models, via modulation of ion channel expression and activity in sensory neurons (Pezet and McMahon, 2006; Watson et al., 2008). NGF increases the membrane presentation and activity of TRPV1 via phosphorylation at Y200, part of a phosphorylation sequence that is conserved in TRPV4 (Zhang et al., 2005). Thus, it is possible that NGF treatment will similarly enhance TRPV4 activity.
To determine whether sensitization of TRPV4 in DRG neurons is a general mechanism underlying mechanical hyperalgesia, we investigated whether NGF sensitization of mechanosensation is dependent on TRPV4 and studied the TRPV4 agonist-induced changes in [Ca2+]i in neurons from wild-type and TRPV4 knockout mice. We hypothesized that TRPV4 knockout mice would show reduced mechanical hyperalgesia after NGF treatment, and neurons from wild-type animals would show increased responses to TRPV4 activators in the presence of NGF, whilst TRPV4 knockout neurons would not respond to these stimuli.
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These studies suggest that TRPV4 protein is expressed within dorsal root ganglia, and that deletion of the TRPV4 gene can affect mechanosensation in a whole animal. However, our experiments investigating TRPV4 activity in cultured DRG and TG neurons did not identify any differences in the responses of neurons collected from wild-type and TRPV4 knockout mice to various TRPV4 activators. Additionally, these findings do not support previous studies that claim that 4α-PDD is a selective activator of TRPV4.
Our Western blots, carried out with two different TRPV4 antibodies, confirm the presence of TRPV4 protein in whole DRG lysates. A doublet band, with molecular weights of approximately 98 and 104 kDa was observed. It is likely that the doublet is due to glycosylation of the mature TRPV4 protein, as demonstrated by Xu et al. (2006). It was absent in lysates from TRPV4 –/– mice, confirming its identity as TRPV4. However, it is noticeable that both antibodies also recognized bands at lower molecular weights in both wild-type and knockout lysates, suggesting that the antibodies are not truly selective for TRPV4. This has implications for using these antibodies in immunohistochemical study of its localization, as a positive reaction on tissue sections may occur in the absence of TRPV4.
TRPV4 wild-type and knockout mice showed no differences in their baseline sensitivity to radiant heat, and both showed a significant sensitization to a heat stimulus after treatment with NGF. TRPV4 does not play a major role in inflammatory thermal hyperalgesia (Huang et al., 2011), so this result was as expected. NGF promotes increased expression and sensitization of thermosensitive TRPV1 (Ji et al., 2002; Zhang et al., 2005), which probably underlies the observed hyperalgesia. Baseline responses to punctate mechanical stimulation with von Frey hairs and to application of pressure to the paw didn't vary between genotypes. This is in keeping with the majority of previous studies that found no basal difference in somatic sensation in TRPV4 –/– mice (e.g. Grant et al., 2007; Alessandri-Haber et al., 2008). Interestingly, both wild-type and knockout mice showed increased sensitivity to punctate stimuli after NGF, whereas only wild-type mice showed an increase in sensitivity to noxious pressure. This suggests that von Frey hairs and noxious pressure activate different patterns of neuronal and non-neuronal mechanosensory cells in the paw, with TRPV4 activation only required during the detection of pressure. The reason why the sensitization of responses to von Frey hairs by NGF is unaffected by TRPV4 deletion, in contrast to previous studies in chronic pain models where von Frey sensitivity is reduced (e.g. Alessandri-Haber et al., 2008), is unclear. Broadly, TRPV4 activation during mechanosensation seems to be more relevant to pathological conditions of hyperalgesia, such as during inflammation, rather than normal physiological sensation.
The ability to sense and respond to alterations in extracellular osmolarity is a fundamental homeostatic process, so redundancy in these pathways is unsurprising. Hypotonic challenge only produces an increase in [Ca2+]i in HEK cells (Xu et al., 2003; Grant et al., 2007) and CHO cells (Liedtke et al., 2000) after heterologous TRPV4 expression, confirming that decreased extracellular osmolarity can indeed activate the channel. In this study, our finding that hypotonic solutions increase [Ca2+]i as effectively in neurons from TRPV4 knockout mice as in wild-type neurons confirms that mechanisms other than TRPV4 activation can detect extracellular hypotonicity. A recent study by Lechner et al. (2011) identified a subpopulation of osmosensory thoracic DRG neurons in wild-type mice that were absent in TRPV4 –/– animals, suggesting that they require TRPV4 for their osmosensory properties. The vast majority of DRG neurons innervate somatic rather than visceral targets, so these specialized neurons may have also been present in our cultures, but not apparent amongst the far greater number of neurons that can apparently respond to extracellular hypo-osmolarity in the absence of TRPV4. When we focused solely on thoracic neurons, significantly more wild-type (17 of 261) than knockout (1 of 252) neurons responded to the TRPV4 agonist GSK1016790A, which may represent some of these rare TRPV4 expressing neurons. Although some trigeminal ganglia neurons are suggested to respond to hypotonicity via TRPV4 activation (Chen et al., 2009), we were unable to find any difference in the response to GSK1016790A or 4α-PDD in cultured TG neurons to support a functional expression of TRPV4 in these neurons.
The activation of TRPV4 by 4α-PDD was first described by Watanabe et al. (2002a). Exposure to 4α-PDD only produced a significant increase in [Ca2+]i in HEK cells and astrocytoma 1321N1 cells after transfection with TRPV4. We, and others, have also demonstrated a gain in responsiveness to 4α-PDD after transfecting TRPV4 into HEK cells that supports its identification as a TRPV4 agonist (Xu et al., 2003; Grant et al., 2007). However, in this study, we have shown that the increase in [Ca2+]i in cultured murine sensory neurons occurs independently of TRPV4, so it does not seem appropriate to describe 4α-PDD as a selective agonist. Of the many previous studies using 4α-PDD, only a few have confirmed the selectivity of its actions in cells or animals with deletion of TRPV4. Deletion of TRPV4 abolished the increase in cytoplasmic Ca2+ following application of 4α-PDD to murine cochlear hair cells (Shen et al., 2006), urothelial cells (Gevaert et al., 2007), ciliated pulmonary epithelial cells (Lorenzo et al., 2008), macrophages (Hamanaka et al., 2010), chondrocytes (Clark et al., 2010) and oesophageal keratinocytes (Mihara et al., 2011), all of which are non-neuronal. Thus 4α-PDD may selectively activate TRPV4 in certain cell types, but does not do so in DRG or TG neurons. In whole animal studies, the sensitization of viscera motor responses to colorectal distension by 4α-PDD (Cenac et al., 2008) and induction of paw oedema following intraplantar 4α-PDD injection (Vergnolle et al., 2010) were both abolished in TRPV4 knockout mice, and this was taken as evidence for a direct effect on sensory neuron TRPV4. However, an equally valid interpretation of these data is that the 4α-PDD is activating a non-neuronal cell to initiate the biological response.
To our knowledge, only one previous study has studied responses to 4α-PDD in sensory neurons from TRPV4 +/+ and –/– mice. Lechner et al. (2011) identified two distinct responses to 10 μM 4α-PDD: a reversible increase in [Ca2+]i and an irreversible increase, which they suggest is a non-specific toxic effect. The reversible increase was only observed in approximately 5% of TRPV4 +/+ neurons and was never seen in –/– neurons. In contrast to this, we found that approximately 30% (44% of the 70% of neurons that responded) of both +/+ and –/– neurons showed transient increases in [Ca2+]i to 10 μM 4α-PDD. When we focused on thoracic DRG neurons, we identified the transient increase in 34.7% of responding wild-type and 32.1% of responding knockout neurons. Lechner and colleagues may have failed to identify TRPV4 knockout neurons that transiently respond to 4α-PDD because of the relatively small number of neurons considered in their study. Overall, our data suggest that although 4α-PDD is a TRPV4 agonist, it is not selective for TRPV4 on sensory neurons.
Another interesting observation is that not only are responses to hypotonic challenge and 4α-PDD present in neurons from TRPV4 –/– mice, but the proportion of cells that respond and mean size of the response are unchanged. This was surprising, as previous studies have proposed the existence of functional TRPV4 in sensory neurons (e.g. Chen et al., 2009; Lechner et al., 2011). Additionally, the Western blots we performed on whole DRG lysates suggested expression of TRPV4 protein, although we were unable to confirm protein expression in individual cultured neurons by immunochemical staining. It would be expected that, even if hypotonic challenge and 4α-PDD do not selectively activate the channel, TRPV4 would contribute at least partially to the observed increase in [Ca2+]i. If this were the case, then a decrease in the number of responding neurons and/or size of their response would be predicted. To investigate this further, we tested the effects of the recently described synthetic TRPV4 agonist GSK1016790A (Thorneloe et al., 2008), on cultured neurons from wild-type mice. At concentrations that were maximally effective at stimulating an increase in [Ca2+]i in a keratinocyte cell line, no changes in [Ca2+]i greater than those in vehicle-treated neurons were observed. However, when we focused solely on neurons from thoracic DRG, a small group of responding neurons that were not present in knockout cells was observed. This suggests that functional TRPV4 is only present in a restricted subset of our cultured neurons, suggesting that the lack of immunostaining may represent a genuine absence of the protein.
One possible explanation for the TRPV4 expression in whole DRG lysates is that it is actually expression in the vasculature of the DRG or in other non-neuronal cells. TRPV4 expression and function has regularly been demonstrated in murine vascular endothelial cells (e.g. Watanabe et al., 2002b; Hartmannsgruber et al., 2007), so the entirety of the protein seen by Western blotting could be of vascular origin. Alternatively, neuronal TRPV4 expression could be lost under culture conditions. The cells were used for Ca2+ imaging and immunostaining within 24 h of collection to minimize this possibility, well within the culture time used in previous studies (e.g. Lechner et al., 2011). It has been suggested that exposure to NGF can increase cell surface presentation and activity of TRPV1, though phosphorylation of a tyrosine residue conserved in TRPV4 (Zhang et al., 2005). To determine whether TRPV4 required NGF for full functional expression, we supplemented our culture medium with 100 ng·mL−1 NGF. However, this had no significant effect on the responses to either hypotonic challenge or 4α-PDD, further supporting the conclusion that the cultured DRG neurons do not express functional TRPV4. Activating mutations in human TRPV4 have been linked to skeletal dysplasias such as brachyolmia (Rock et al., 2008) and spondylometaphyseal dysplasia (Krakow et al., 2009), and to motor neuropathies such as Charcot-Marie tooth disease type 2C (Landoure et al., 2010). These diseases are not associated with sensory abnormalities, suggesting that human sensory nerves also have little, if any, expression of TRPV4.
The mechanisms by which hypotonic solutions and 4αPDD can induce an elevation in neuronal [Ca2+]i independently of TRPV4 are not clear. The TRPV4 agonist 5′,6-EET has recently been shown to modulate neuronal activity through activation of TRPA1, rather than TRPV4 (Sisignano et al., 2012). However, we found that neurons lacking TRPA1 or TRPV1 still responded to both hypotonic buffer and 4αPDD, suggesting that these channels are not responsible. Neuronal expression of 5-HT3 receptors (Linz and Veelken, 2002), TRPC1 (Staaf et al., 2009) and TRPC5 (Gomis et al., 2008) have all previously been suggested to provide sensitivity to hypo-osmotic challenge. However, identification of the precise mechanisms responsible for hypotonic and 4αPDD-induced neuronal activation is beyond the scope of this study.
In conclusion, our data support the hypothesis that TRPV4 is involved in noxious mechanosensation, but this is not necessarily due to an activation of TRPV4 protein on sensory neurons. Indeed, we have failed to find any evidence for functional TRPV4 expression in the vast majority of DRG and TG neurons in culture. If this is also the case in vivo, the effects of TRPV4 agonists such as 4αPDD on nociceptive behaviours should be interpreted based on a non-neuronal site of TRPV4 activity, such as in the Merkel cells (Boulais et al., 2009) or keratinocytes of the skin (Chung et al., 2003), or as an action on an alternative target.