Effect Of Annexin A2and Microtubule On TRPV4in DRG Neurons

BackgroundNeuralgia is a syndrome caused by central and periphery nerve systems, which seriously affects the quality of life of patients with lumbar disc protrusion, etc. Dorsal root ganglion (DRG) neurons are primary sensory neurons that convert external painful stimuli from periphery into internal nerve impulses and transmit the impulses to the spinal cord. Many studies have focused on the role of transient receptor potential ion channels (TRPs) and their function in DRG neurons. As one of the members of TRPs, transient receptor potential vanilloid4(TRPV4, also known as OTRPC4, VRL-2, VR-OAC, and TRP12) in DRG neurons plays an important role in this process. Alessandri-Haber et al. found that TRPV4knockdown resulted in abnormal osmotic modulation and reduced nociceptive responses to hypotonic stimuli. In addition, TRPV4plays a crucial role in mechanical hyperalgesia induced by inflammatory mediators. In models of painful peripheral neuropathy induced by vincristine chemotherapy, alcoholism, diabetes and human immunodeficiency virus, mechanical hyperalgesia was strongly reduced by intrathecal injection of TRPV4antisense oligodeoxynucleotides. Thus, understanding the molecular mechanisms of TRPV4action is beneficial to the further comprehension of the mechanisms involved in the development of pain. Nevertheless, the molecular mechanisms are not exactly understood, especially in relation to the mechanisms regulating TRPV4gene expression and/or function in DRG neurons.Studies showed that the function and/or gene expression of TRPV4can be regulated by several auxiliary proteins such as protein kinase C and casein kinase substrate in neurons protein3(PACSIN3), inositol (1,4,5) triphosphate receptor3(IP3R3) and actin. Chronic compression of the DRG (the procedure named CCD) in animals, which mimics clinical disk hernia and spinal canal stenosis in humans, is a typical model of neuropathic pain. Our previous study demonstrated that TRPV4is involved in the CCD-induced mechanical and thermal hyperalgesia. To further explore the mechanism of TRPV4activity, we investigated the differential protein expression following CCD by proteomic analysis. CCD resulted in the changes of15proteins. Annexin A2acts as one of the targets was up-regulated noticeably compared with normal control. The Annexins family, which expresses on the cytosolic face of the cellular membrane, is a group of Ca2+-dependent phospholipid-binding proteins that play a key role in many biological processes, including the regulation of ion channels. Evidence has demonstrated that the functional complex between Annexin A2and transient receptor potential vanilloid5/6(TRPV5/TRPV6) can promote the trafficking of TRPV5and TRPV6to the plasma membrane. Whether Annexin A2interacts with TRPV4channels and modifies channel function and/or expression, however, has not been demonstrated so far.In this study, we have used immunocytochemistry studies and co-immunoprecipitation assays to investigate the interactions between Annexin A2and TRPV4in DRG neurons. The role of Annexin A2in the regulation of TRPV4gene expression and/or function was further verified by RNA interference (RNAi) and by measurement of intracellular free calcium concentrations ([Ca2+]i).ObjectiveTo investigate the role of Annexin A2in the regulation of TRPV4gene expression and/or function in DRG neuron. Methods1. DRG cell cultureFor rat DRGs, the cell culture protocol was used as previously described with three modifications as follows. Briefly, postnatal day1Wistar rats were obtained from Shandong University Lab Animal Center, and the DRGs from L2to L6were dissected from the spinal cord. Cells were dissociated with1mg/ml Collagenase type I and0.25%trypsin for50min at37℃. After24hours in culture, cells were treated for24hours with5mg/ml cytosine arabinoside to prohibit non-neuronal cell growth. DRG neurons were used for experiments after4days of in vitro culture.2. ImmunofluorescenceDRG neurons were fixed with ice-cold acetone for10min and then blocked with2%bovine serum albumin for30min at37℃. Afterwards, cells were incubated with rabbit polyclonal anti-TRPV4(5ug/ml) and goat polyclonal anti-Annexin A2(20ug/ml) overnight at4℃and subsequently with FITC-and TRITC-labeled secondary antibodies (1:500) at room temperature in the dark for1h. Images were taken with a confocal laser scanning microscope.3. Co-immunoprecipitationDRG neurons were lysed in lysis buffer for30min on ice. The lysates were centrifuged at14,000×g for5min at4℃. The supernatant was collected, and its protein content was measured with the BCA Protein Assay Kit. To reduce non-specific binding, the extract was incubated with normal IgG serum and protein A+G agarose beads for1h at4℃and then centrifuged for2min at14,000×g to pellet the beads. Protein sample was incubated with the corresponding antibody at4℃overnight with gentle agitation on a platform shaker, followed by an incubation with30ul protein A+G agarose beads for2h. After five washes with lysis buffer, samples were resuspended in40ul of sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) sample-loading buffer (1×SDS sample buffer, Beyotime). The proteins remaining attached to the beads were released by boiling for10min at95℃. Thereafter, proteins were analyzed by western blotting analysis. As negative controls, the proteins were incubated with either the nonimmune IgG or agarose beads alone.4. siRNA transfectionShort interfering RNA (siRNA) targeting Annexin A2was purchased from Dhamacon. In the siRNA transfection experiments, a non-targeting Accell siRNA pool was used as a negative control, and a GAPDH Accell siRNA pool was used as a positive control. DRG neurons were seeded at a density of1×105cells/well on96-well culture dishes and, after four days of in vitro culture, were transfected with Accell siRNA probes at a final siRNA concentration of100nmol/L in Accell delivery media for72h..5. Quantitative real-time RT-PCRFor extraction of total RNA, DRG neurons were dissolved in Trizol reagent. Reverse transcription and PCR amplification were carried out using the SYBR(?) PrimeScriptTM RT-PCR Kit. The specific PCR primers for Annexin A2and TRPV4were synthesized by Invitrogen and generated products of82and122bp in length, respectively. The nucleic acid melting temperatures were62℃and58℃, respectively. The amplified fragments were separated by5%(w/v) agarose gel electrophoresis, stained with0.3mg/mL ethidium bromide and analyzed using Lightcycler software4.0.6. Western blottingProtein samples from DRG neurons were prepared on ice as for co-immunoprecipitation. Then, the sample of total protein was separated by7.5%SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes. The membranes were incubated in blocking buffer for2h at room temperature. Then, the membranes were incubated with primary antibody at4℃overnight and afterwards with horseradish peroxidase (HRP)-conjugated secondary antibodies for1h. Primary antibodies were a rabbit anti-TRPV4polyclonal antibody (1:250) and a goat anti-Annexin A2polyclonal antibody (1:1,000). Anti-rabbit (1:5,000) and-goat (1:2,500) secondary antibodies were purchased from JingMei Biotech. The protein signal was visualized using enhanced chemiluminescence in accordance with the manufacturer’s instructions. Photographic films were scanned and subsequently quantified using FluorChem9900and AlphaEaseFC software.7. Measurement of [Ca2+]iIntracellular Ca2+fluorescence was determined as previously described. DRG neurons were used for measurement of [Ca2+]i72h after Annexin A2siRNA transfection. To activate TRPV4and cause an increase in free [Ca2+]i,4a-PDD was used. Neurons were loaded with5umol/L fura-3acetoxymethyl ester (Fura-3AM) and0.02%pluronic f-127for45min at37℃in isotonic solution. Subsequently, neurons were perfused with an isotonic solution for10min to permit hydrolysis of fura-3AM before adding4a-PDD. The volume of the confocal dish was200ul, and perfusion was carried out at20-23℃. The dye was excited at488nm to indicate relative changes in [Ca2+]i. The fluorescence was acquired and analyzed with Zen2009imaging system software.Results1. Localization of Annexin A2and TRPV4in DRG neuronsThe merged image between Annexin A2and TRPV4indicated partial co-localization. Annexin A2has been reported to express mainly at the cytoplasmic face of the cell membrane, but it has also been detected in the cytoplasm. Our experimental data showing immunopositive staining for Annexin A2supported that finding. Our finding regarding the distribution of TRPV4in neurons was also similar to another report showing that TRPV4was distributed in plasma membrane and cytoplasm, but a strong immunolocalization of TRPV4was not observed in DRG neuron. According to reports in the literature, TRPV4mRNA expression level is only equivalent to24.8%of transient receptor potential vanilloid (TRPV1) which was highly expressed in DRG. Perhaps because of the lower expression level of TRPV4, a strong signal of TRPV4was unable to be observed. The double staining for Annexin A2and TRPV4indicated partial co-localization between them at the cell membrane and in the cytoplasm. To test non-specific binding of the secondary antibody, control neurons were incubated without the specific primary antibodies, and no significant staining was evident. 2. Co-immunoprecipitationFirst our studies showed co-immunoprecipitate Annexin A2with TRPV4. Cell lysates prepared from DRG neurons were immunoprecipitated with beads alone, anti-TRPV4antibody or the nonimmune rabbit IgG. Then, Annexin A2was identified by western blot analysis with anti-Annexin A2antibody. Annexin A2was not detected in the beads-alone or the nonimmune rabbit IgG control. The anti-Annexin A2antibody detected a signal at approximately39kDa, corresponding to the molecular mass of Annexin A2, in samples immunoprecipitated with the anti-TRPV4antibody. This observation suggested that Annexin A2is able to be immunoprecipitated using the anti-TRPV4antibody. The procedures of co-immunoprecipitation TRPV4with Annexin A2was the same as stated above except the following one. But there was a weak TRPV4signal was detected. To solve this problem, we optimized the experimental conditions such as protein sample, incubation time and antibody concentration, but the results remain the same as above.3. Gene expression changes of Annexin A2and TRPV4Transcriptional levels of Annexin A2and TRPV4were calculated by real time quantitative RT-PCR. Annexin A2mRNA was significantly decreased by92.3%(p<0.05). We examined the effects of GAPDH and non-targeting siRNA to test the efficiency and stability of the siRNA. The non-targeting siRNA did not downregulate the expression of the two genes concerned, Annexin A2and TRPV4, compared with untreated samples Knockdown of Annexin A2decreased the mRNA expression of TRPV4by35.8%(p>0.05) compared with the untreated samples. Protein expression correlated with mRNA levels for both Annexin A2and TRPV4. Annexin A2protein expression was noticeably reduced by53.4%(p<0.05). Treatment with Annexin A2siRNA reduced TRPV4protein expression by11.4%, compared with non-treated neurons. However, this difference remain non-significant (p>0.05). This finding suggested that downregulation of Annexin A2does not significantly affect expression of TRPV4mRNA and protein in DRG neurons.4. Annexin A2siRNA inhibits agonist-induced [Ca2+]i increaseWe compared the response to TRPV4-activating stimuli between interference and non-treated neurons. Stimulation with10umol/L4α-PDD strongly increased [Ca2+]i in untreated neurons. Compared with untreated neurons, the response to4a-PDD was noticeably decreased but still detectable in Annexin A2siRNA-treated neurons (p<0.05). This partial reduction suggested that Annexin A2was involved in the regulation of TRPV4function.ConclusionsOur results provide evidence that Annexin A2is associated with TRPV4proteins and regulates TRPV4-mediated Ca2+influx in DRG neurons. BackgroundNeuropathic pain is one kind of pain caused or irritated by primary damage and malfunction of nervous systems. Radicular Neuralgia is one of the common clinical types of neuralgia, especially for the pain induced by nociceptive stimulus on dorsal root ganglion (DRG). The nociceptive stimulus include, disc herniation, compression by spinal cord tumors, stimulations by inflammatory substances, etc.. Animal model of chronic compression of DRG (CCD) was established by Hu to investigate the algogenic mechanisms of neuropathic pain, which is used to simulate clinical lumbar disc protrusion and spinal canal stenosis. Spontaneous pain, mechanical allodynia, and thermal hyperalgesia were present after compression on ganglion in rats. Several types of ion channels had been considered to involve in transmitting the nociceptive stimulus in CCD neurons, such as voltage-gated Na+and K+hyperpolarization-activated cation current and transient receptor potential (TRP). As one of the TRP families, transient receptor potential vanilloid4(TRPV4) is a Ca2+-permeable polymodal receptor, which can be activated by various stimuli, including osmo-stimuli, mechano-stimuli, moderate heat, endogenous substances, and chemical compounds such as4a-phorbol12,13-didecanoate (4a-PDD). Our previous study showed a high level of mRNA and protein expression of TRPV4in DRG after CCD, which participates in mechanical and thermal hyperalgesia induced by CCD.As the major component of cytoskeleton, microtubules play a role in many biological processes including pain perception. Studies demonstrated that microtubules dynamic can regulate pain sensitivity. Colchicine is a microtubule depolymerizing agent that can reversibly bind both a and (3tubulin monomers to inhibit tubulin polymerization into microtubules. It has been reported that disruption of microtubules by colchicine can attenuate hyperalgesia in rat paws induced by intradermal epinephrine injection. Taxol, which is a stabilizer of microtubules, can bind to microtubules to stabilize microtubules. Studies showed that taxol treatment increased the nociceptive behavioral responses to mechanical stimulation of the hind paw. Goswami and coworkers have recently found an interaction between TRPV4and microtubules. Evidence demonstrated that the C terminus of TRPV4interacts directly with soluble tubulin by co-immunoprecipitation and pull-down assay. Moreover, stabilization of microtubules by taxol in vivo can result in reduction and delay in Ca2+-influx mediated by TRPV4. Ca2+influx via TRPV4is an indicator of TRPV4channel opening. These suggest that microtubules may participate in regulation of TRPV4function. Owing to its broad expression and the diversity of its activating factors, TRPV4might play different roles in diverse tissues. As a mammalian mechano-or thermo-receptor, TRPV4mediates mechanical and thermal hyperalgesia in neuropathic pain models. However, whether microtubules participates in mechanical and thermal hyperalgesia mediated by TRPV4is still unknown.In this study, mechanical nociceptive withdrawal threshold and paw withdrawal latency were measured to evaluate effect of microtubule on mechanical and thermal hyperalgesia mediated by TRPV4in CCD. The effect was further verified by measurement of intracellular free calcium concentrations ([Ca2+]i).ObjectiveTo investigate the role of microtubulin in the regulation of TRPV4function in DRG neuron after CCD.Methods1. Animal and surgical procedureAdult (200-250g) male Wistar rats were obtained from Shandong University Lab Animal Center and were used for these studies. All experiments complied with the national guidelines and were performed in accordance with the Declaration of Helsinki. Attempts were made to minimize the number of animals used. The rats were housed in clear plastic cages with sawdust bedding under controlled conditions (room temperature23±2℃,12/12h light/dark cycle and50-60%relative humidity) and free access to food and water for7days before surgery. An investigator who was blind to animal treatment performed all the behavioural testing.Rats were randomly divided into CCD and sham groups. Each rat in CCD group received a single unilateral CCD lesion directed at the right L4-L5spinal nerve. To execute this, the rats were anesthetized with300mg/kg chloral hydrate and under clean conditions the transverse process and intervertebral foramina of L4and L5were exposed as previously described. A stainless steel U-shaped rod (0.63mm diameter and4mm length) was inserted into each foramen to form a steady compression against DRG. Then the incision was satured in layers and penicillin was injected to prevent infection. The surgical procedure in sham group was the same as the CCD group, except that the U-shaped rod was not inserted into interveterbral foramen.2. Dorsal root ganglion cell culture2.1. Neogenetic rat DRG culturePostnatal day1rats were obtained from Shandong University Lab Animal Center, and the DRGs from L2to L6were dissected from the spinal cord into cold D-Hanks’ balanced salt solution. Cells were dissociated with1mg/ml collagenase type Ⅰ and0.25%trypsin for50min at37℃. Cells were plated on culture dishes coated with0.1mg/ml poly-L-lysine in neurobasal medium supplemented with2%B27supplement,0.5mM L-glutamine,1%(v/v) penicillin/streptomycin, and20ng/ml2.5S nerve growth factor. After24hours in culture, cells were treated for24hours with5mg/ml cytosine arabinoside to prohibit non-neuronal cell growth. Thereafter, the medium was entirely exchanged every two days. Neogenetic DRG neurons were used for experiments after4days of in vitro culture.2.2. Adult rat DRG cultureThe DRGs from L4to L5in CCD rats were dissected from the spinal cord into D-HBSS. Cells were dissociated with2mg/ml collagenase type Ⅰ for1h at37℃and subsequently with0.25%trypsin for30min at37℃. Cells were plated in neurobasal medium supplemented with2%B27supplement,0.5mM L-glutamine,1%(v/v) penicillin/streptomycin. The medium was exchanged for half dose after24h. After that, the medium was entirely exchanged daily. Adult DRG neurons were used for experiments after3days of in vitro culture.3. Behavioral testingTo assess the effect of different drug treatments, plantar surface of hindpaws of rats were tested for mechanical allodynia and heat hyperalgesia. Tests were carried out on the same day and experimental rats were allowed to adapt to experimental environment for15min before testing began. Baseline withdrawal thresholds were recorded before surgical model preparation and drug treatment.3.1. Mechanical hyperalgesiaMechanical nociceptive withdrawal thresholds were evaluated with calibrated von Frey fibers based on that described by Tal and Bennett. Starting with the lowest filament force, von Frey fibers were pressed against the lateral plantar surface of hindpaws in ascending order with sufficient force to cause slight bending and held for5s. A positive response was noted if the paw was immediately withdrawn. The stimulation of the same force was repeated five times at intervals of5s. If there were no less than three withdrawal responses to any of the five applications, the next lower force fibers was repeated. If less than three withdrawl responses, the process was tried with the next higher force fibers.3.2. Thermal hyperalgesiaHeat hyperalgesia was examined by measuring paw withdrawal latency (PWL) as previously described. Rats were first placed in a plastic chamber atop a6mm thick glass floor. Then, the radiant heat source beneath the glass floor was aim at the mid plantar surface of hindpaws. The paw withdrawal latencies per animal were obtained during five trials at5min intervals. Considering the anomalously long withdrawal latency of the first response, we discarded the related data. Thermal withdrawal latencies were defined as the mean of the last four reading.4. Calcium imagingNeurons were loaded with5uM Fura-3AM and0.02%pluronic f-127for30min at37℃in isotonic solution. After loading, neurons were washed twice with HBSS. Subsequently, neurons were perfused with an isotonic solution for10min to permit hydrolysis of fura-3AM before adding4α-PDD. The volume of the confocal dish was200ul, and perfusion was carried out at20-23℃. The dye was excited at488nm to indicate relative changes in [Ca2+]i. The fluorescence was acquired and analyzed with Zen2009imaging system software.For experiments testing the effect of colchicine, neurons were perfused with isotonic solution containing fura-3AM dye and colchicine for30min, and challenged with isotonic solution containing4α-PDD and colchicine for3min.Results1. Effects of colchicine on CCD-induced mechanical hyperalgesia in DRGTo test the role of microtubules in mechanical hyperalgesia induced by CCD, we injected CCD rats with microtubule destabilizer colchicine at dose from50to250ug. Intrathecal administration with colchicine at dose of250ug and125ug produced a significant (P<0.05) dose-dependent reduction of paw mechanical withdrawal threshold to mechanical stimulation when compared with saline group. The intrathecal injection of colchicine significantly inhibited CCD-induced mechanical hyperalgesia from15min to2h with the peak inhibitory effect at30min post-injection.2. Effects of colchicine on CCD-induced thermal hyperalgesia in DRGTo test the role of microtubules in thermal hyperalgesia induced by CCD, we injected CCD rats with microtubule destabilizer colchicine at dose from50to250ug. Intrathecal administration with colchicine at dose of250ug and125ug produced a significant (P<0.05) dose-dependent reduction of paw withdrawal thermal latency to thermal stimulation when compared with saline group. The intrathecal injection of colchicine significantly inhibited CCD-induced thermal hyperalgesia from15min to2h with the peak inhibitory effect at30min post-injection.3. Effects of4α-PDD on the suppressive effects of colchicine on CCD-induced mechanical hyperalgesiaTo investigate the role of microtubules in mechanical hyperalgesia mediated by TRPV4, we injected CCD rats with microtubule destabilizer colchicine prior to injection of4α-PDD.4α-PDD was unable to attenuate the suppressive effects of colchicine (250ug) on CCD-induced mechanical hyperalgesia when compared to colchicine (250ug) alone (P>0.05).4. Effects of4α-PDD on the suppressive effects of colchicine on CCD-induced thermal hyperalgesiaTo investigate the role of microtubules in thermal hyperalgesia mediated by TRPV4, we injected CCD rats with microtubule destabilizer colchicine prior to injection of4α-PDD.4α-PDD was unable to attenuate the suppressive effects of colchicine (250ug) on CCD-induced thermal hyperalgesia when compared to colchicine (250ug) alone (P>0.05).5. Colchicine inhibits4α-PDD-induced [Ca2+]i increase in normal DRG neuronsTo examine the role of microtubules dynamics disruption in TRPV4channel properties in normal DRG neurons, we measured Ca2+influx via TRPV4as an indicator of TRPV4channel opening. It has been proved that microtubule stabilizer taxol in vivo results in reduction in Ca2+influx via through TRPV4. In the current study, we compared the TRPV4-mediated Ca2+influx in response to4α-PDD in the presence and absence of microtubule destabilizer colchicine. DRG neurons were incubated with colchicine at concentration from0.01to0.1ug/ml for30min and subsequently with10uM4α-PDD for3min. We observed that the average Ca2+-influx due to TRPV4activation was strongly reduced (but not abolished) if the cells were treated with0.1ug/ml colchicine (p<0.001). Colchicine by itself had no effect on Ca2+influx mediated by TRPV4(data not shown).6. Colchicine inhibits4α-PDD-induced [Ca2+]i increase in DRG neurons after CCDTo provide more evidence that TRPV4-mediated hyperalgesia could be inhibited by microtubules dynamics disruption, we tested whether colchicine could prevent the potentiation of Ca2+influx through TRPV4in DRG neurons from CCD rats. Exposure of cultured CCD DRG neurons to0.1ug/ml colchicine in the perfusion bath for30min prior to the application of10uM4α-PDD partially prevented the potentiation of Ca2+influx (P<0.01).Conclusions Our results provide evidence that microtubule regulates TRPV4function in CCD DRG neurons.