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Comparative Study
. 2008 Jan 30;28(5):1046-57.
doi: 10.1523/JNEUROSCI.4497-07.2008.

Interaction of transient receptor potential vanilloid 4, integrin, and SRC tyrosine kinase in mechanical hyperalgesia

Nicole Alessandri-Haber  1 , Olayinka A Dina, Elizabeth K Joseph, David B Reichling, Jon D Levine
Affiliations
Comparative Study

Interaction of transient receptor potential vanilloid 4, integrin, and SRC tyrosine kinase in mechanical hyperalgesia

Nicole Alessandri-Haber et al. J Neurosci. .

Abstract

Although the transient receptor potential vanilloid 4 (TRPV4) has been implicated in the process of osmomechanical transduction, it appears to make little contribution to the normal somatosensory detection of mechanical stimuli. However, evidence suggests that it may play an important role in mechanical hyperalgesia. In the present study, we examined the common requirement for TRPV4 in mechanical hyperalgesia associated with diverse pain models and investigated whether the very close association observed between TRPV4 and mechanical hyperalgesia, regardless of etiology, reflects a close functional connection of TRPV4 with other molecules implicated in mechanical transduction. In models of painful peripheral neuropathy associated with vincristine chemotherapy, alcoholism, diabetes, and human immunodeficiency virus/acquired immune deficiency syndrome therapy, mechanical hyperalgesia was markedly reduced by spinal intrathecal administration of oligodeoxynucleotides antisense to TRPV4. Similarly, mechanical hyperalgesia induced by paclitaxel, vincristine, or diabetes was strongly reduced in TRPV4 knock-out mice. We also show that alpha2beta1 integrin and Src tyrosine kinase, which have been implicated in mechanical transduction, are important for the development of mechanical hyperalgesia, and that their contribution requires TRPV4. Furthermore, we establish a direct interaction between TRPV4, alpha2 integrin, and the Src tyrosine kinase Lyn in sensory neurons. We suggest that TRPV4 plays a role in mechanotransduction, as a component of a molecular complex that functions only in the setting of inflammation or nerve injury.

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Figures

Figure 1.
Figure 1.
Contribution of TRPV4 to the hypersensitivity for mechanical and hypotonic stimuli in diverse models of painful peripheral neuropathy. A, Rats treated with vincristine, STZ, ddC, or alcohol showed a decrease in their mechanical nociceptive thresholds (gray bars). Spinal intrathecal treatment with TRPV4 antisense (TRPV4 AS, white bars), compared with mismatch oligodeoxynucleotides (TRPV4 MM, black bars), reversed the mechanical hyperalgesia in alcohol-fed rats and reduced it by 53% in vincristine- and 44% in STZ- and ddC-treated rats, respectively. B, Injection of 10 μl of hypotonic solution induced a significantly higher number of flinches in vincristine-, STZ-, ddC-, and alcohol-treated compared with control rats. TRPV4 antisense, compared with mismatch ODN, reduced the increase in the number of flinches induced by hypotonicity by 89% in vincristine-treated (2.2 ± 1.7 for antisense- vs 19.6 ± 2.4 for mismatch-treated rats; n = 6 for each ODN group; p < 0.0001, unpaired Student's t test), 83% in STZ-treated rats (1.8 ± 0.6 for antisense- vs 12.1 ± 1.2 for mismatch-treated rats; n = 6 for each ODN group; p < 0.0001, unpaired Student's t test), 88% in ddC-treated (1.7 ± 0.3 for antisense- vs 17 ± 2 for mismatch-treated; n = 6 for each ODN group; p < 0.0001, unpaired Student's t test), and 52% in alcohol-fed rats (5.6 ± 0.7 for antisense- vs 14.3 ± 1.2 for mismatch-treated; n = 6 for each ODN group; p < 0.0001, unpaired Student's t test). C, There was a significant decrease in the level of TRPV4 protein expression in saphenous nerve from alcoholic rats treated with antisense compared with mismatch ODN-treated rats (38 ± 8%; n = 6 for each ODN group; p < 0.05, unpaired Student's t test). The amount of protein loaded in each lane was normalized by probing the membrane with an anti-GAPDH antibody. *p < 0.05.
Figure 2.
Figure 2.
Contribution of TRPV4 to neuropathic mechanical hyperalgesia. A, Rats were treated with either TRPV4 antisense (AS) or mismatch (MM) ODN for 6 d. Streptozotocin was administered on day 3. Two-way repeated-measures ANOVA showed a significant group by time interaction (F (4,88) = 22.511; p < 0.001) and a significant main effect of group (F (1,22) = 34.107; p < 0.001). Post hoc one-way ANOVAs (group with 2 levels, antisense and mismatch) revealed that paw withdrawal threshold differed significantly between the two groups from day 4 (the first day after receiving STZ) to day 8. B, TRPV4 antisense or mismatch ODN were administered daily for 14 d starting 3 d before the commencement of ethanol diet. Rats were fed the ethanol diet for four days of the week [day 0 (D0)–D4], and they were fed standard lab chow for the remaining three days (D4–D7). A repeated-measures ANOVA with one within-subjects factor (days with 10 levels) and one between-subjects factor (ODN with 2 levels, antisense and mismatch) demonstrated a significant ODN by days interaction (F (9,90) = 18.448; p < 0.001) and a significant main effect of ODN (F (1,10) = 50.744; p < 0.0001). Post hoc one-way between-subjects ANOVAs showed that the ODN groups differed significantly from day 7 (p < 0.001) to day 15 (p = 0.051).
Figure 3.
Figure 3.
Paclitaxel-, STZ-, and vincristine-induced mechanical hyperalgesia is impaired in TRPV4−/− mice. A, The withdrawal response to von Frey hair mechanical stimuli is similar in naive TRPV4+/+ (solid bars; n = 12) and TRPV4−/− mice (clear bars; n = 12). Four days after a single intraperitoneal injection of STZ (75 mg/kg), vincristine (200 μg/kg), or paclitaxel (6 mg/kg), TRPV4−/− mice exhibit only an ∼1.5-fold increase in withdrawal frequency response compared with the approximately threefold increase present in TRPV4+/+ mice (n = 8 for each group of mice). *p < 0.05. B, Time course of mechanical hyperalgesia after a single intraperitoneal injection of paclitaxel, STZ, or vincristine in TRPV4+/+ (filled circles; n = 8) and TRPV4−/− mice (open squares; n = 8), expressed as mean hindpaw withdrawal frequency (%) ± SEM.
Figure 4.
Figure 4.
Hyperalgesia to hypotonic and mechanical stimuli depend on α2 integrin. A, Treatment with α2 antisense (AS) ODN for 3 d reversed the mechanical hyperalgesia in alcohol-fed [n = 12 for antisense- and n = 10 for mismatch (MM)-treated; p < 0.0001, unpaired Student's t test] and paclitaxel-treated rats (n = 8 for antisense- and mismatch-treated rats; p < 0.0001, unpaired Student's t test). The effect of the antisense was reversible; 4 d after the last ODN injection, the mechanical hyperalgesia induced by alcohol or paclitaxel was not significantly different from the pre-ODN baseline. In contrast, α2 integrin antisense ODN did not affect mechanical thresholds in control-fed rats (n = 10 for antisense- and n = 8 for mismatch-treated; p > 0.05, unpaired Student's t test). Inset, There was a 38 ± 6% decrease in protein expression level in saphenous nerve of alcohol-fed rats treated with α2 integrin antisense- compared with mismatch-treated (n = 5 in each ODN group; p = 0.004, unpaired Student's t test). The amount of protein in both lanes was confirmed to be comparable by probing the membrane with an anti-GAPDH antibody. B, Treatment with α2 integrin antisense markedly reduced the number of flinches induced by intradermal injection of hypotonic solution in both alcohol-fed (n = 12 for antisense- and n = 10 for mismatch-treated rats; p < 0.0001, unpaired Student's t test) and paclitaxel-treated rats (n = 8 for each ODN group; p < 0.0001, unpaired Student's t test). In fact, the number of flinches induced by hypotonicity in alcohol-fed or paclitaxel-treated rats after treatment with α2 integrin antisense ODN was not significantly different from the number of flinches induced by hypotonicity in rats fed control diet (n = 8 for paclitaxel rats treated with α2 integrin AS; n = 12 for alcohol rats treated with α2 integrin AS; n = 10 for rats fed with control diet treated with α2 integrin AS; p > 0.05, ANOVA). *p < 0.05.
Figure 5.
Figure 5.
Enhanced response to hypotonic and mechanical stimuli in alcohol-fed rats depends on Src tyrosine kinase. A, Intradermal injection of PP1 (specific inhibitor of Src family tyrosine kinase) 30 min before measurement of nociceptive thresholds reversed the alcohol-induced mechanical hyperalgesia (mean ± SEM, 32.7 ± 1.8%, n = 12 before and 0.4 ± 1.4%, n = 6 after PP1; p < 0.001, unpaired Student's t test). In contrast, the injection of PCT had no effect on mechanical thresholds (n = 12 before and n = 6 after piceatannol; p > 0.05, unpaired Student's t test). There was no significant difference between the two groups of rats 24 h after the injection of the inhibitors (n = 6 after PP1 or piceatannol; p > 0.05, unpaired Student's t test). Of note, intradermal injection of PP1 did not affect mechanical thresholds in rats fed control diet. B, Intradermal injection of PP1 reduced the number of flinches induced by hypotonicity in alcohol-fed rats by 60% (13.2 ± 0.4, n = 12 before and 5 ± 1, n = 6 after PP1; p < 0.0001, unpaired Student's t test), whereas PCT had no effect. Of note, the number of flinches induced by hypotonicity after PP1 in alcohol-fed rats was not significantly different from the number of flinches induced by hypotonic solution in control diet rats. The number of flinches induced by hypotonicity was not significantly different between the rats treated with PP1 or piceatannol 24 h after the injection of the inhibitors (p > 0.05, unpaired Student's t test). C, Mean [Ca2+]i for neurons first challenged with a 30% hypotonic solution (212 mOsm) and then challenged with a 30% hypotonic solution containing either PP1 (10 μm) or piceatannol (30 μm). PP1 decreased the hypotonicity-induced increase in [Ca2+]i by 26% (1.51 ± 0.14 μm before and 1.11 ± 0.04 μm in presence of PP1; n = 14; p = 0.001, paired Student's t test), whereas piceatannol did not have a significant effect (n = 10; p > 0.05, paired Student's t test). All extracellular solutions contained DIDS (100 μm). *p < 0.05.
Figure 6.
Figure 6.
Src tyrosine kinase-induced mechanical hyperalgesia is TRPV4 dependent. Intradermal injection of the specific activator of Src family tyrosine kinase, YEEIP peptide (100 ng), 30 min before measurement of nociceptive thresholds, induced a decrease in mechanical nociceptive thresholds in control rats (gray bar, 34.4 ± 1.2%; n = 10). Treatment with TRPV4 antisense (AS) markedly reduced the YEEIP-induced mechanical hyperalgesia (6.8 ± 4.3% decrease in mechanical nociceptive threshold for TRPV4 antisense- and 36.2 ± 1.3% for TRPV4 mismatch (MM)-treated rats; n = 6 for each group; p < 0.0001 unpaired Student's t test). Four days after ODN injection, the decrease in nociceptive thresholds induced by intradermal injection of YEEIP peptide was not significantly different between rats previously treated with antisense or mismatch (n = 6 for each group; p > 0.05, unpaired Student's t test). *p < 0.05.
Figure 7.
Figure 7.
Direct interaction of TRPV4 with α2 integrin and Src tyrosine kinase. A, Proteins were extracted from DRG neurons isolated from alcohol-fed rats cultured for 2 d. TRPV4 was immunoprecipitated with anti-TRPV4 antibody (1:500) and immunoprecipitates were subjected to SDS gel electrophoresis. The membrane was then probed with anti-α2 integrin antibody (1:200) or with anti-Lyn antibody (1:300). IP, Immunoprecipitation; IB, immunoblot. B, Intradermal injection of a soup of inflammatory mediators in rat hindpaw induces a decrease in mechanical nociceptive thresholds in the rat. Spinal intrathecal injection of α2 integrin antisense (AS) ODN prevented the induced mechanical hyperalgesia (n = 10) and the effect of the antisense was reversible. Similarly, intradermal injection of anti-α2 integrin antibody (Ab; 100 ng/10 μl) before the intradermal injection of the inflammatory soup markedly reduced the mechanical hyperalgesia (n = 4). C, Small-diameter DRG neurons from TRPV4+/+ and TRPV4−/− mice were first challenged with a 30% hypotonic solution containing the inflammatory mediators PGE2 and 5-HT (10 μm each) for 3 min and then challenged with a 30% hypotonic solution containing PGE2, 5-HT, and the anti-α2 integrin antibody (5 μg/ml). The inflammatory mediator-induced increase in [Ca2+]i is reversed in the presence of anti-α2 integrin antibody in TRPV4+/+ mice (3.71 ± 1.03 before anti-α2 integrin antibody and 1.8 ± 0.3 after; n = 19; p < 0.0001, paired Student's t test), whereas it has no effect on [Ca2+]i in TRPV4−/− mice (1.8 ± 0.2 before and 1.7 ± 0.2 after anti-α2 integrin antibody; n = 30; p > 0.05, paired Student's t test). *p < 0.05.
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