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TRPV1 expression and activity during retinoic acid-induced neuronal differentiation
Neurochemistry International 55 (2009) 768–774
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TRPV1 expression and activity during retinoic acid-induced neuronal differentiation Johanna EL Andaloussi-Lilja, Jessica Lundqvist, Anna Forsby * Department of Neurochemistry, Stockholm University, Svante Arrhenius va¨g 21 A, SE-106 91 Stockholm, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 February 2009 Received in revised form 2 July 2009 Accepted 24 July 2009 Available online 3 August 2009
The transient receptor potential vanilloid subtype 1 (TRPV1) is a Ca2+-permeable channel primarily expressed in dorsal root ganglion neurons. Besides its function in thermogenic nociception and neurogenic inflammation, TRPV1 is involved in cell migration, cytoskeleton re-organisation and in neuronal guidance. To explore the TRPV1 level and activity during conditions for neuronal maturation, TRPV1-expressing SHSY5Y neuroblastoma cells were differentiated into a neuronal phenotype using alltrans-retinoic acid (RA). We show that RA highly up-regulated the total and cell surface TRPV1 protein expression but the TRPV1 mRNA level was unaffected. The up-regulated receptors were localised to the cell bodies and the developed neurites. Furthermore, RA increased both the basal intracellular free Ca2+ concentration by 30% as well as the relative capsaicin-induced Ca2+ influx. The results show that TRPV1 protein expression increases during RA-induced differentiation in vitro, which generates an altered intracellular Ca2+ homeostasis. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: TRPV1 Retinoic acid Differentiation Ca2+ In vitro
1. Introduction The temperature sensitive transient receptor potential, vanilloid subtype 1 (TRPV1) is a non-selective cation channel primarily expressed in C- and Ad fibres of dorsal root ganglion (DRG) neurons. This transmembrane receptor is acting as a sensor for heat, acidic pH and chemical stimuli of which the hot principle in chilli pepper, capsaicin, is the classical agonist (Caterina et al., 1997). When activated, the TRPV1 facilitates entry of predominantly Ca2+ ions into the cytoplasm, thereby stimulating the neuron to evoke an alerting signal to the brain of potential tissue damage. Recently, an alternative function of TRPV1 was suggested when it was shown that TRPV1 is localised in neurites and growth cones where it regulates motility in transiently transfected F11 cells and embryonic DRG neurons (Goswami et al., 2007). Growth cone motility and steering of extending neurites are dynamic
Abbreviations: RA, all-trans-retinoic acid; TRPV1, transient receptor potential vanilloid subtype 1; [Ca2+]i, intracellular free Ca2+ concentration; RFU, relative fluorescence unit; PI(3)K, phosphatidylinositol 3-kinase; MAPK, mitogen activated protein kinase; PKC, protein kinase C; IGF-I, insulin like growth factor type-I; DRG, dorsal root ganglion. * Corresponding author. Tel.: +46 8163598; fax: +46 8161371. E-mail address: [email protected] (A. Forsby). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.07.011
processes involving Ca2+ signals via cell surface receptors. The guidance of developing axons requires an active growth cone, and Zheng (2000) showed that a localised Ca2+ signal in the growth cone is sufficient for both attraction and repulsion. Goswami and Hucho (2007) have also shown that recombinant TRPV1 is involved in the formation of filopodia in both neuronal and nonneuronal cells. In addition, TRPV1 plays a role in cytoskeleton reorganisation (Goswami et al., 2006), cell migration (Waning et al., 2007), and in regeneration of injured neurons (Biggs et al., 2007), processes that are strictly regulated by the intracellular free Ca2+ concentration ([Ca2+]i, reviewed by Zheng and Poo (2007)). These findings suggest an important role for TRPV1 during neuronal differentiation. The aim of this study was to investigate how TRPV1 expression and activity were affected during neuronal differentiation. A stably TRPV1-transfected clone of the SHSY5Y human neuroblastoma cell line was used as a model for developing nervous system and differentiation was induced by the hormone and vitamin A derivative all-trans retinoic acid (RA). We show that the total as well as the cell surface TRPV1 protein level was highly upregulated during RA-induced differentiation. Further on, the basal [Ca2+]i and the capsaicin-induced [Ca2+]i were also increased in the RA-differentiated cells compared to control cells. Additionally, the increased expression seemed to be regulated post-transcriptionally since the TRPV1 mRNA level was sustained during RA exposure.
J. EL Andaloussi-Lilja et al. / Neurochemistry International 55 (2009) 768–774 2. Experimental procedures 2.1. Materials RA, capsaicin, Fura-2/AM, bovine insulin, progesterone, sodium selenite and putrescine were obtained from Sigma–Aldrich (St Louis, MO, CA, USA), bovine apotransferrin from MP biomedical (Solon, OH, USA) and inhibitors for phosphatidyl inositol 3-kinase (PI(3)K, LY294002); mitogen activated protein kinase kinase (MAPK(MEK), PD98059); MAPKp38 (p38 MAP Kinase inhibitor) and protein kinase C (PKC, Bisindolylmaleimide I (BIM)) were purchased from Calbiochem (San Diego, CA, USA). All cell culture media and supplements (except for N2 supplements), Trizol1 reagent, reverse transcriptase, agarose and primers, were purchased from Invitrogen, Carlsbad, CA, USA. Plasticware for cell culturing was obtained from Corning Incorporated (Corning, NY, USA). 2.2. Cells and treatments The maintenance of the native SHSY5Y human neuroblastoma cells that do not express TRPV1 and the generation and maintenance of the TRPV1-SHSY5Y cell clone have been described before (Lilja et al., 2007a,b). Briefly, both the native SHSY5Y and TRPV1-SHSY5Y cells were cultured in Earle’s minimum essential medium with Earle’s salts (referred to as MEM) supplemented with 10% fetal calf serum, 1% nonessential amino acids, 2 mM L-glutamine, 100 mg streptomycin/ml, 100 units penicillin/ml, and for the TRPV1-expressing cells 0.4 mg puromycin/ml. The SHSY5Y cells are frequently used as a neuronal cell model due to its sympathetic feature and low resting membrane potential (Biedler et al., 1978; Tosetti et al., 1998). Additionally, the SHSY5Y cells can be further differentiated to a mature neuronal phenotype by culturing the cells in serum-free medium with RA (Nordin-Andersson et al., 2003; Pahlman et al., 1984). For differentiation, the MEM medium was changed to DMEM:F12 (1:1) medium containing 2 mM L-glutamine,100 IU penicillin/ml, 100 mg streptomycin/ml and the N2 supplements insulin [0.9 mM], progesterone [20 nM], selenite [30 nM], transferrin [100 mg/ml], and putrescine [100 mM] (Bottenstein and Sato, 1979), referred to as N2 medium. 1 mM RA was added to the N2 medium (or the regular MEM medium as control) in order to differentiate the cells (Pahlman et al., 1984). Fresh regular MEM medium or N2 medium with or without RA was changed every 3 days. For TRPV1 mRNA (RT-PCR) and protein (western blot) analyses, 0.2–1 106 cells (depending on exposure conditions) were seeded in 60 mm Petri dishes 1 day before treatment. Cellular RNA was isolated 24 h after the start of RA exposure and proteins were isolated after 12–144 h. For the analysis of any involved signalling pathway, inhibitors for PI(3)K (LY294002, 10 mM); MAPK(MEK) (PD98059, 10 mM); MAPKp38 (p38 MAP Kinase inhibitor, 1 mM) or PKC (BIM, 1 mM) were also added to the dishes and the controls were treated with vehicle (0.1% DMSO). For the biotinylation assay used to assess surface allocated proteins, 2 106 cells were seeded in 100 mm cell culture Petri dishes 2 days before treatments to yield 90–95% confluence when collected. The cells were treated with N2 medium with or without RA for 48 h. For the confocal microscopy study 100,000 cells were seeded on glass cover slips in 12-well plates 1 day before the medium was changed to MEM or N2 medium with or without RA. For TRPV1 activity measurements, 25,000 cells/well were seeded in 96-well plates 1 day before the cells were exposed to N2 medium with or without RA for 48 h before Ca2+ measurements. 2.3. Morphological evaluation 0.5 106 cells were plated in 60 mm Petri dishes and the plating medium was changed to N2 medium with RA the day after. Phase contrast microscopy images were taken at 150 magnification using a CCD camera (Olympus DP50, Olympus, Center Valley, PA, USA) on day 1 and 6 after initiation of RA exposure. 2.4. Reverse transcription polymerase chain reaction (RT-PCR) In this recombinant system the regulation of TRPV1 transcription is provided by a strong constitutively active promoter probably not affected by transcription factors and other DNA regulatory proteins. To confirm this anticipation we studied the TRPV1 mRNA expression by the semi-quantitative method reverse transcription polymerase chain reaction (RT-PCR) as described before (Lilja et al., 2007a). The cells were treated with RA for 24 h as described above, and the total RNA was extracted by Trizol1 reagent. 5 mg (determined spectrophotometrically) was used to amplify mRNA using an oligo-dT primer and cDNA was synthesized using reverse transcriptase, all according to the manufacturer’s protocol. A 400 bp product from the TRPV1-specific primers were analysed after 22 PCR cycles on a 1.5% agarose gel. A 600 bp product of b-actin was used as an internal control for equal loading. 2.5. Western blot The western blot procedure has been described before (Lilja et al., 2007b). In short, protein concentrations in the cell lysates were calculated using Bio Rad Dc
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Protein Assay (Bio Rad Laboratories, Inc., Hercules, CA, USA) and protein separation was carried out on a 7% polyacrylamide gel. TRPV1 proteins were blotted with specific primary antibodies (diluted 1:10,000) from rabbit (Alomone laboratories, Jerusalem, Israel) and secondary rabbit antibodies (diluted 1:3000) from donkey conjugated to horse radish peroxidase (Amersham Biosciences, Buckinghamshire, England). As loading control, tubulin expression was analysed with monoclonal primary antibodies towards atubulin (1:20,000, Sigma) from mouse and secondary mouse antibodies (1:3000, Amersham) from sheep conjugated to horse radish peroxidase. The blot density of 100 kDa, corresponding to TRPV1, was balanced to the density of the 50 kDa tubulin blot in the same lane. Densitometric analysis of visual blots was performed using Science Lab99 Image Gauge 3.46 quantification program (Fujifilm Co. Ltd, Tokyo, Japan). 2.6. Ca2+-measurements The TRPV1 activity was measured by monitoring the [Ca2+]i levels using Fura2/AM fluorescence in a FLEX-Station-II (Molecular Devices, Sunnyvale, CA, USA) as described before (Lilja et al., 2007b) after 48 h in N2 medium with or without RA. Shortly, following Fura-2/AM incubation the cells were rinsed in Hepes Krebs Ringer buffer once and the basal [Ca2+]i and capsaicin-induced [Ca2+]i levels were measured. For the capsaicin-induced [Ca2+]i levels the Ca2+ influx was measured for 1 min every other second before, during and after capsaicin addition, and presented as the absolute relative fluorescence units (RFU) ratio of 340/380 nm excitation, 510 nm emission wavelengths. The 340/380 nm ratio values (corresponding to the Ca2+ influx) peaked immediately after capsaicin addition where after a plateau was reached followed by a slow decrease in [Ca2+]i. The mean peak value from 6 wells in a 96-well plate, from three individual experiments, was registered for each capsaicin concentration (0.3, 0.6, 1, 5, 10, and 100 nM). For the basal [Ca2+]i level measurements in the TRPV1-SHSY5Y and native SHSY5Y cells, values represent the mean of three individual experiments, each in 18 replicates. 2.7. Isolation of cell surface proteins To determine if the TRPV1 receptors were up-regulated at the cell surface, a biotinylation assay was performed after 48 h of treatment in N2 medium with or without RA. Cells from four 100 mm dishes per treatment were biotinylated according to manufacturer’s instructions using Pierce’s pinpoint cell surface protein isolation kit (Pierce, Rockford, IL, USA). The isolated plasma membrane protein concentration was determined and the TRPV1 proteins were analysed by western blot as described above. 2.8. Confocal microscopy To visualise the location of TRPV1 in differentiated cells, the control cells (cultured in MEM) and cells cultured in N2 medium with or without RA, were analysed for their TRPV1 expression using confocal microscopy. After 6 days of treatment the cells were fixed using 4% paraformaldehyde in PBS followed by permeabilisation in 100% methanol. Unspecific binding was blocked with 2% BSA in PBS. Cells were hybridised with the primary TRPV1 antibodies used for western blot (diluted 1:10,000 in 2% BSA/PBS) and blotted with secondary rabbit Alexa fluor1 red 568-conjugated antibodies from goat (diluted 1:1000 in 2% BSA/PBS, Molecular probes, Eugene, OR, USA). Confocal images were captured with Zeiss Axiovert 200 microscope (Zeiss, Bernried, Germany) by stepwise sectioning to the middle of the cells using 568 nm laser and PerkinElmer Ultra VIEW ERS rapid confocal imager (PerkinElmer Life Sciences, Boston, MA, USA). 2.9. Statistics To compare means of all groups, data were analysed using one-way ANOVA followed by Tukey’s Multiple Comparison Test or by Student’s t-test (Figs. 2D, 4 and 5), all in GraphPad Prism 4 software.
3. Results 3.1. Morphology of SHSY5Y and TRPV1-SHSY5Y cells differentiated with RA RA (1 mM) induced morphological differentiation in SHSY5Y and TRPV1-SHSY5Y cells as indicated by rounded cell bodies and the development of a neurite network. At day 6 the TRPV1-SHSY5Y cells had formed aggregates of cell bodies with extensions of neurites between them whilst the native SHSY5Y were still in monolayer (Fig. 1).
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Fig. 1. Morphology of SHSY5Y and TRPV1-SHSY5Y cells during RA-induced differentiation. Phase contrast pictures (originally 150 magnification) of native SHSY5Y (A) and (B) and TRPV1-SHSY5Y cells (C) and (D) cultured in N2 medium with RA for 1 day (A) and (C) and 6 days (B) and (D).
3.2. Effect of RA on TRPV1 expression The total TRPV1 protein level in the SHSY5Y cells cultured in N2 medium with RA was significantly elevated already after 24 h but increased with time and reached a 6-fold higher TRPV1 protein content after 6 days as compared to cells cultured in MEM medium (Fig. 2A and B). Culturing the cells in N2 medium without RA for 6 days induced a more than 2-fold increase in the total TRPV1 protein level as well (Fig. 2A and B). However, the effect of N2 medium on the TRPV1 up-regulation was not visible after 12–48 h of exposure when the effect of RA per se was evident. To exclude the possibility that components in the N2 medium induced the increase in TRPV1 expression, the cells were also treated with RA in the complete MEM medium. The effect of RA in MEM medium was the same as the effect of RA in N2 medium at all time points (48 h shown in Fig. 2C and D). Previously, we showed that insulin and IGF-I induced an increment in TRPV1 proteins that could be blocked by PI(3)K, PKC and MAPK(MEK) inhibitors (Lilja et al., 2007a). To study possible signalling pathways responsible for the RA-induced up-regulation of TRPV1 protein expression, specific inhibitors for PI(3)K (LY294002, 10 mM), MAPK(MEK) (PD98059, 10 mM), MAPK p38 (p38 MAP Kinase inhibitor, 1 mM), and PKC (BIM, 1 mM) were used along with RA treatment. The cells were exposed to RA in N2 medium as above for 48 h with or without inhibitors and the TRPV1 protein level was analysed by western blot. None of the inhibitors used could significantly block the RA-induced upregulation of TRPV1 proteins (Fig. 2E). Because the TRPV1 protein expression was highly elevated already after 24 h and the upregulation was further increased during differentiation, the mRNA was analysed at this time point. The TRPV1 mRNA expression was unaffected by 24 h administration of RA (Fig. 2F).
total TRPV1 protein content was highly increased after 48 h of RA exposure, the biotinylation assay was performed at this time point. The TRPV1 plasma membrane level was also significantly upregulated about 6-fold as compared to cells cultured in N2 medium without RA (Fig. 4A and B). 3.4. Effect of RA on TRPV1 activity The activity of the TRPV1 receptors was measured as capsaicininduced Ca2+ influxes using Fura-2/AM. The basal [Ca2+]i in the TRPV1-SHSY5Y cells was significantly increased by about 30% after 48 h culture in N2 medium with RA as compared to cells cultured in N2 medium without RA (Fig. 5A). The basal [Ca2+]i in the native SHSY5Y cells (lacking TRPV1) was not affected by RA treatment. The relative increased [Ca2+]i in TRPV1-SHSY5Y cells cultured in RA-containing N2 medium for 48 h remained after the addition of capsaicin at all concentrations compared to cells cultured in N2 medium without RA (Fig. 5B). The kinetics of the N2 and N2RA curves were similar but displayed a shift on the y-axis, as a result of different start (basal [Ca2+]i) RFU values. Thus, a comparison of the EC50 values from the equations of the two curves to quantify the TRPV1 receptor activity would be misleading. However, a comparison can be made by displaying the capsaicin concentrations acquired to induce a certain RFU ratio (i.e., the same [Ca2+]i) in the N2- and N2RA-treated cells, respectively. The 50% effect in the N2-treated cells measured 2.2 RFU, which was obtained at 0.8 nM (i.e., EC50) and the capsaicin concentration entailed to induce 2.2 RFU in the N2RA-treated cells was estimated to 0.02 nM. Hence, the capsaicin concentration required to give the same [Ca2+]i in both treatments was reduced 40-fold in N2RA-treated cells as compared to N2 treated cells. 4. Discussion
3.3. Effect of RA on TRPV1 receptor localisation The up-regulated TRPV1 proteins in cells differentiated for 6 days with RA were localised in the cell bodies as well as in the neurites as shown by confocal microscopy (Fig. 3). To study the plasma membrane localisation of the up-regulated TRPV1 receptors, the cell surface TRPV1 proteins were isolated by using a biotinylation assay, and analysed by western blot. Since the
The vanilloid receptor TRPV1 was originally described as a nociceptor localised on sensory neurons where it is activated by noxious stimuli such as heat, protons and capsaicin (Caterina et al., 1997). However, TRPV1 has been shown to be involved in cellular processes beside nociception and inflammatory responses. For example, TRPV1 is a major physiological Ca2+ channel required for the migration of hepatocellular carcinoma (HepG2) cells (Waning
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Fig. 2. Total TRPV1 levels in TRPV1-SHSY5Y cells during RA exposure. Cells were grown in MEM or RA-containing MEM (MEMRA), N2 medium (N2) or RA-containing N2 medium (N2RA). The total protein content was analysed after 12 h, 24 h, 48 h and 144 h by western blot using specific antibodies towards TRPV1 and tubulin. (A and C) One representative western blot picture of TRPV1 (100 kDa) and tubulin (50 kDa) blots after RA exposure. (B and D) The bars represent the density of the 100 kDa TRPV1 blot in relation to the density of the 50 kDa tubulin blot in the same lane. (E) TRPV1-SHSY5Y cells were treated as above for 48 h in N2 medium (referred to as control, C) and RAcontaining N2 medium without inhibitors (RA) or with inhibitors for PI3K, 10 mM (+LY), MAPK 10 mM (+PD), PKC 1 mM (+BIM) or p38MAPK 1 mM (+p38). The bars represent the density of the 100 kDa TRPV1 blot in relation to the corresponding 50 kDa tubulin blot. (F) mRNA expression of TRPV1 after 24 h exposure to N2 medium with (N2RA) or without (N2) RA, measured by RT-PCR. Values in B and E (mean S.E.M., n = 3) were analysed by one-way ANOVA, followed by Tukey’s multiple-comparisons test and values in D were analysed by Student’s t-test.***P < 0.001 compared to N2 (compared to MEM in D), &&P < 0.01 compared to MEM.
et al., 2007) and TRPV1 activity induces maturation and migration of dendritic cells in mice (Basu and Srivastava, 2005). Furthermore, Goswami and Hucho (2007) and Goswami et al. (2007) have shown the importance of TRPV1 protein expression for the development of filopodia and regulation of the growth cone in neurons and nonneuronal cells. Importantly, other TRP channels (of the TRPC family) play a crucial role in the guidance of nerve growth cones (Wang and Poo, 2005; Li et al., 2005). Herein, we have studied the effect on TRPV1 expression and activity during RA-induced neuronal differentiation in stably TRPV1-transfected SHSY5Y cells. The TRPV1-SHSY5Y cells reached a high morphological differentiation with an extensive neurite
network between aggregates of cell bodies after 6 days in culture in N2 medium with 1 mM RA. Native SHSY5Y cells develop a network of neurites after 6 days in N2RA medium as well. However, these cells do not form aggregates until after 2–3 weeks of RA (unpublished observation) indicating a difference in the differentiation process. In accordance with the morphological differentiation in the TRPV1-SHSY5Y cells, the level of TRPV1 total protein expression was dramatically increased, and the upregulated TRPV1 proteins were localised in both the cell bodies and the developed neurites. The elevated TRPV1 protein levels were statistically significant already after 24 h of RA treatment and further increased during differentiation to reach a 6-fold increase
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Fig. 3. Localisation of TRPV1 proteins in TRPV1-SHSY5Y cells during RA-induced differentiation. Cells were seeded on cover slips and treated for 6 days in N2 medium with RA (N2RA) or in N2 medium without RA (N2) and compared to cells grown in regular plating medium (MEM). Cells were fixed and permeabilised in paraformaldehyde and methanol, respectively, and stained with specific TRPV1 antibodies and secondary antibodies conjugated to Alexa fluor1 red 568. The TRPV1 expression was visualised by confocal microscopy from a centred focal point viewing both surface and intracellular fluorescence in the cells.
after 6 days in N2 medium with RA as compared to cells cultured in MEM. Isolation of the plasma membrane proteins after 48 h of RA exposure showed that the same TRPV1 up-regulation was evident at the cell surface. Despite the substantial increase in the TRPV1 protein levels after 24 h to 6 days in culture with RA, the mRNA level remained unaffected. This implies that the increased level of receptors is regulated post-transcriptionally. The function of TRPV1 as a regulator of [Ca2+]i homeostasis during cell migration and growth cone formation can explain the presence of the receptor in the brain and in non-neuronal cell types. Differentiation and neurogenesis are strictly dependent on regulation of the [Ca2+]i, and localisation of Ca2+ permeable plasma membrane receptors like TRPV1 in developing neurons may facilitate maturation of the nervous system. We show that after 48 h of exposure to RA, when the total and plasma membrane TRPV1 protein expression had reached a 6-fold increase, the basal
Fig. 4. Cell surface expression of TRPV1 in TRPV1-SHSY5Y cells during RA exposure. Cells were treated with RA-containing N2 medium (N2RA) and N2 medium without RA (N2) for 48 h and plasma membrane proteins were isolated and analysed with western blot using TRPV1-specific antibodies. (A) One representative western blot image showing plasma membrane-bound TRPV1 in the cells. (B) The bars represent the density of the 100 kDa TRPV1 blot in percent of the total density of all 100 kDa TRPV1 blots on the membrane. Values (mean SEM, n = 3) were analysed by Student’s t-test, ***P < 0.001 compared to N2.
[Ca2+]i was significantly elevated by about 30% in the TRPV1SHSY5Y cells but not in the native SHSY5Y cells. The concomitant up-regulation of cell surface TRPV1 and the increased basal [Ca2+]i in the differentiated TRPV1-SHSY5Y cells indicate an association between the observations. The increased [Ca2+]i might be caused by constitutively, partly active TRPV1 receptors on the cell surface. Since TRPV1 is a voltage-gated ion channel (Voets et al., 2004), the altered resting cell membrane potential, which is accompanied with increased neuronal differentiation, and thereby excitability (Toselli et al., 1996), might cause a controlled, basal leakage of Ca2+ ions into the cells. The importance of TRPV1 as a Ca2+ channel could
Fig. 5. Ca2+ levels in SHSY5Y and TRPV1-SHSY5Y cells during RA exposure. The cells were treated with N2 medium with RA (N2RA) or without RA (N2) and the intracellular [Ca2+]i levels were measured by Fura-2/AM fluorescence. The values represent the ratio of 340/380 nm (excitation wavelengths) absolute RFU. (A) The bars show the relative basal [Ca2+]i in TRPV1-SHSY5Y and native SHSY5Y cells after 48 h of incubation in N2 or N2RA medium. (B) The graph shows the effect of capsaicin-induced [Ca2+]i in TRPV1-SHSY5Y cells after 48 h exposure in N2- or N2RA medium. The values in (A) and (B) represent mean SEM, n = 3 and were analysed by Student’s t-test where *P 0.05, **P 0.01, ***P < 0.001.
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be one explanation for the increased expression during neurite extension. The expression of other Ca2+ channels like L- and N-type Ca2+ channels have also been shown to be up-regulated in RAexposed cells (Gao et al., 1998). The nature of TRPV1 rearranging microtubule (Goswami et al., 2006) could also be of importance during differentiation. The re-organisation of cytoskeletal structures is a process demanded during growth cone motility and guidance also supporting the indication that up-regulation of TRPV1 receptors could play a crucial role for neuronal development (Dent et al., 2003; Gordon-Weeks, 2004). In vivo, RA is involved in neuronal patterning, differentiation and axonal outgrowth as well as the maintenance of the differentiated state of the adult neurons (reviewed by Maden, 2007). The RA hormone binds to the nuclear retinoic acid receptor (RAR) that functions as a dimer with retinoic X receptor (RXR) binding to RA responsive elements (RAREs) in the target genes, activating transcription. In SHSY5Y cells, RA treatment has also shown to have more direct effects on signal transduction activating the PI(3)K and rapidly phosphorylating Akt (Lopez-Carballo et al., 2002). There are several proteins (and mRNAs) that are increased after RA exposure where no RAREs have been found in their respective promoters (Lane and Bailey, 2005) but these non-RARE actions are poorly understood. The effects on the recombinant TRPV1 receptors in these cells, regulated by an artificial and strong promoter which is probably not responding to the original transcription factors, suggest post-transcriptional effects. Indeed, nerve growth factor ((NGF) Ji et al., 2002; Puntambekar et al., 2005), insulin and IGF-I (Lilja et al., 2007a) up-regulate TRPV1 protein expression in a post-transcriptional manner. This may be the reason why we observed that cells cultured for 6 days in N2 medium without RA also increased the total TRPV1 protein level since the N2 medium contains a high concentration of insulin (0.9 mM). The significant effect of RA in N2 medium on TRPV1 expression was verified by treating the cells with RA in regular serum-containing MEM medium, which gave the same effect. However, to induce a terminally differentiated neuronal phenotype of SHSY5Y cells it is desirable to use RA in a defined medium in which the level of growth factors is controlled and where serum proteins are not affecting RA concentration. The regulation of TRPV1 expression and translocation to the plasma membrane has previously been shown to be mediated by the phosphorylation of kinases in the pathways of PI(3)K (Bron et al., 2003; Lilja et al., 2007a; Zhang et al., 2005), MAPK (Ji et al., 2002; Puntambekar et al., 2005) and by PKC (Lilja et al., 2007a; Morenilla-Palao et al., 2004). The PI(3)K pathway and the MAPK pathway are the major pathways involved in neuronal proliferation, differentiation and survival. However, none of the kinase inhibitors used herein could block the effect of RA on TRPV1 expression. Yet, the mechanism behind the RA-induced TRPV1 protein increment in these cells remains unclear. Treating cells with RA for 30 min did not induce an acute potentiating effect on the basal [Ca2+]i, nor the TRPV1 activity (not shown), indicating that the elevated basal [Ca2+]i in the differentiated TRPV1-SHSY5Y cells is probably not due to a sustained effect from immediate phosphorylation of the receptor and/or acute translocation of the receptor to the plasma membrane. Rather, the results imply that the increased [Ca2+]i is connected to the long-term increase in TRPV1 protein levels at the cell surface. TRPV1 has been shown to be expressed in the brain in rat (Mezey et al., 2000; Sanchez et al., 2001; Toth et al., 2005), human (Cortright et al., 2001; Cristino et al., 2006; Mezey et al., 2000; Roberts et al., 2004), mouse (Cristino et al., 2006; Roberts et al., 2004) and monkey (Szabo et al., 2002). Furthermore, Jin et al. (2004) suggested that both the cannabinoid and TRPV1 receptors have roles in adult neurogenesis in mice. The functional role of TRPV1 in brain has not yet been resolved, but one suggestion may
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be that TRPV1 plays a part as a Ca2+ regulator during embryonic development, and also in regeneration and neuronal plasticity. Our new findings support these previous reports suggesting that TRPV1 receptors are involved during the development and differentiation of the nervous system. Acknowledgements This project was financially supported by the Swedish Fund for Research without Animal Experiments and the Swedish Animal Welfare Agency. References Basu, S., Srivastava, P., 2005. Immunological role of neuronal receptor vanilloid receptor 1 expressed on dendritic cells. Proc. Natl. Acad. Sci. U.S.A. 102, 5120– 5125. Biedler, J.L., Roffler-Tarlov, S., Schachner, M., Freedman, L.S., 1978. Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones. Cancer Res. 38, 3751–3757. Biggs, J.E., Yates, J.M., Loescher, A.R., Clayton, N.M., Boissonade, F.M., Robinson, P.P., 2007. Changes in vanilloid receptor 1 (TRPV1) expression following lingual nerve injury. Eur. J. 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TRPV1 expression and activity during retinoic acid-induced neuronal differentiation Johanna EL Andaloussi-Lilja, Jessica Lundqvist, Anna Forsby * Department of Neurochemistry, Stockholm University, Svante Arrhenius va¨g 21 A, SE-106 91 Stockholm, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 February 2009 Received in revised form 2 July 2009 Accepted 24 July 2009 Available online 3 August 2009
The transient receptor potential vanilloid subtype 1 (TRPV1) is a Ca2+-permeable channel primarily expressed in dorsal root ganglion neurons. Besides its function in thermogenic nociception and neurogenic inflammation, TRPV1 is involved in cell migration, cytoskeleton re-organisation and in neuronal guidance. To explore the TRPV1 level and activity during conditions for neuronal maturation, TRPV1-expressing SHSY5Y neuroblastoma cells were differentiated into a neuronal phenotype using alltrans-retinoic acid (RA). We show that RA highly up-regulated the total and cell surface TRPV1 protein expression but the TRPV1 mRNA level was unaffected. The up-regulated receptors were localised to the cell bodies and the developed neurites. Furthermore, RA increased both the basal intracellular free Ca2+ concentration by 30% as well as the relative capsaicin-induced Ca2+ influx. The results show that TRPV1 protein expression increases during RA-induced differentiation in vitro, which generates an altered intracellular Ca2+ homeostasis. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: TRPV1 Retinoic acid Differentiation Ca2+ In vitro
1. Introduction The temperature sensitive transient receptor potential, vanilloid subtype 1 (TRPV1) is a non-selective cation channel primarily expressed in C- and Ad fibres of dorsal root ganglion (DRG) neurons. This transmembrane receptor is acting as a sensor for heat, acidic pH and chemical stimuli of which the hot principle in chilli pepper, capsaicin, is the classical agonist (Caterina et al., 1997). When activated, the TRPV1 facilitates entry of predominantly Ca2+ ions into the cytoplasm, thereby stimulating the neuron to evoke an alerting signal to the brain of potential tissue damage. Recently, an alternative function of TRPV1 was suggested when it was shown that TRPV1 is localised in neurites and growth cones where it regulates motility in transiently transfected F11 cells and embryonic DRG neurons (Goswami et al., 2007). Growth cone motility and steering of extending neurites are dynamic
Abbreviations: RA, all-trans-retinoic acid; TRPV1, transient receptor potential vanilloid subtype 1; [Ca2+]i, intracellular free Ca2+ concentration; RFU, relative fluorescence unit; PI(3)K, phosphatidylinositol 3-kinase; MAPK, mitogen activated protein kinase; PKC, protein kinase C; IGF-I, insulin like growth factor type-I; DRG, dorsal root ganglion. * Corresponding author. Tel.: +46 8163598; fax: +46 8161371. E-mail address: [email protected] (A. Forsby). 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.07.011
processes involving Ca2+ signals via cell surface receptors. The guidance of developing axons requires an active growth cone, and Zheng (2000) showed that a localised Ca2+ signal in the growth cone is sufficient for both attraction and repulsion. Goswami and Hucho (2007) have also shown that recombinant TRPV1 is involved in the formation of filopodia in both neuronal and nonneuronal cells. In addition, TRPV1 plays a role in cytoskeleton reorganisation (Goswami et al., 2006), cell migration (Waning et al., 2007), and in regeneration of injured neurons (Biggs et al., 2007), processes that are strictly regulated by the intracellular free Ca2+ concentration ([Ca2+]i, reviewed by Zheng and Poo (2007)). These findings suggest an important role for TRPV1 during neuronal differentiation. The aim of this study was to investigate how TRPV1 expression and activity were affected during neuronal differentiation. A stably TRPV1-transfected clone of the SHSY5Y human neuroblastoma cell line was used as a model for developing nervous system and differentiation was induced by the hormone and vitamin A derivative all-trans retinoic acid (RA). We show that the total as well as the cell surface TRPV1 protein level was highly upregulated during RA-induced differentiation. Further on, the basal [Ca2+]i and the capsaicin-induced [Ca2+]i were also increased in the RA-differentiated cells compared to control cells. Additionally, the increased expression seemed to be regulated post-transcriptionally since the TRPV1 mRNA level was sustained during RA exposure.
J. EL Andaloussi-Lilja et al. / Neurochemistry International 55 (2009) 768–774 2. Experimental procedures 2.1. Materials RA, capsaicin, Fura-2/AM, bovine insulin, progesterone, sodium selenite and putrescine were obtained from Sigma–Aldrich (St Louis, MO, CA, USA), bovine apotransferrin from MP biomedical (Solon, OH, USA) and inhibitors for phosphatidyl inositol 3-kinase (PI(3)K, LY294002); mitogen activated protein kinase kinase (MAPK(MEK), PD98059); MAPKp38 (p38 MAP Kinase inhibitor) and protein kinase C (PKC, Bisindolylmaleimide I (BIM)) were purchased from Calbiochem (San Diego, CA, USA). All cell culture media and supplements (except for N2 supplements), Trizol1 reagent, reverse transcriptase, agarose and primers, were purchased from Invitrogen, Carlsbad, CA, USA. Plasticware for cell culturing was obtained from Corning Incorporated (Corning, NY, USA). 2.2. Cells and treatments The maintenance of the native SHSY5Y human neuroblastoma cells that do not express TRPV1 and the generation and maintenance of the TRPV1-SHSY5Y cell clone have been described before (Lilja et al., 2007a,b). Briefly, both the native SHSY5Y and TRPV1-SHSY5Y cells were cultured in Earle’s minimum essential medium with Earle’s salts (referred to as MEM) supplemented with 10% fetal calf serum, 1% nonessential amino acids, 2 mM L-glutamine, 100 mg streptomycin/ml, 100 units penicillin/ml, and for the TRPV1-expressing cells 0.4 mg puromycin/ml. The SHSY5Y cells are frequently used as a neuronal cell model due to its sympathetic feature and low resting membrane potential (Biedler et al., 1978; Tosetti et al., 1998). Additionally, the SHSY5Y cells can be further differentiated to a mature neuronal phenotype by culturing the cells in serum-free medium with RA (Nordin-Andersson et al., 2003; Pahlman et al., 1984). For differentiation, the MEM medium was changed to DMEM:F12 (1:1) medium containing 2 mM L-glutamine,100 IU penicillin/ml, 100 mg streptomycin/ml and the N2 supplements insulin [0.9 mM], progesterone [20 nM], selenite [30 nM], transferrin [100 mg/ml], and putrescine [100 mM] (Bottenstein and Sato, 1979), referred to as N2 medium. 1 mM RA was added to the N2 medium (or the regular MEM medium as control) in order to differentiate the cells (Pahlman et al., 1984). Fresh regular MEM medium or N2 medium with or without RA was changed every 3 days. For TRPV1 mRNA (RT-PCR) and protein (western blot) analyses, 0.2–1 106 cells (depending on exposure conditions) were seeded in 60 mm Petri dishes 1 day before treatment. Cellular RNA was isolated 24 h after the start of RA exposure and proteins were isolated after 12–144 h. For the analysis of any involved signalling pathway, inhibitors for PI(3)K (LY294002, 10 mM); MAPK(MEK) (PD98059, 10 mM); MAPKp38 (p38 MAP Kinase inhibitor, 1 mM) or PKC (BIM, 1 mM) were also added to the dishes and the controls were treated with vehicle (0.1% DMSO). For the biotinylation assay used to assess surface allocated proteins, 2 106 cells were seeded in 100 mm cell culture Petri dishes 2 days before treatments to yield 90–95% confluence when collected. The cells were treated with N2 medium with or without RA for 48 h. For the confocal microscopy study 100,000 cells were seeded on glass cover slips in 12-well plates 1 day before the medium was changed to MEM or N2 medium with or without RA. For TRPV1 activity measurements, 25,000 cells/well were seeded in 96-well plates 1 day before the cells were exposed to N2 medium with or without RA for 48 h before Ca2+ measurements. 2.3. Morphological evaluation 0.5 106 cells were plated in 60 mm Petri dishes and the plating medium was changed to N2 medium with RA the day after. Phase contrast microscopy images were taken at 150 magnification using a CCD camera (Olympus DP50, Olympus, Center Valley, PA, USA) on day 1 and 6 after initiation of RA exposure. 2.4. Reverse transcription polymerase chain reaction (RT-PCR) In this recombinant system the regulation of TRPV1 transcription is provided by a strong constitutively active promoter probably not affected by transcription factors and other DNA regulatory proteins. To confirm this anticipation we studied the TRPV1 mRNA expression by the semi-quantitative method reverse transcription polymerase chain reaction (RT-PCR) as described before (Lilja et al., 2007a). The cells were treated with RA for 24 h as described above, and the total RNA was extracted by Trizol1 reagent. 5 mg (determined spectrophotometrically) was used to amplify mRNA using an oligo-dT primer and cDNA was synthesized using reverse transcriptase, all according to the manufacturer’s protocol. A 400 bp product from the TRPV1-specific primers were analysed after 22 PCR cycles on a 1.5% agarose gel. A 600 bp product of b-actin was used as an internal control for equal loading. 2.5. Western blot The western blot procedure has been described before (Lilja et al., 2007b). In short, protein concentrations in the cell lysates were calculated using Bio Rad Dc
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Protein Assay (Bio Rad Laboratories, Inc., Hercules, CA, USA) and protein separation was carried out on a 7% polyacrylamide gel. TRPV1 proteins were blotted with specific primary antibodies (diluted 1:10,000) from rabbit (Alomone laboratories, Jerusalem, Israel) and secondary rabbit antibodies (diluted 1:3000) from donkey conjugated to horse radish peroxidase (Amersham Biosciences, Buckinghamshire, England). As loading control, tubulin expression was analysed with monoclonal primary antibodies towards atubulin (1:20,000, Sigma) from mouse and secondary mouse antibodies (1:3000, Amersham) from sheep conjugated to horse radish peroxidase. The blot density of 100 kDa, corresponding to TRPV1, was balanced to the density of the 50 kDa tubulin blot in the same lane. Densitometric analysis of visual blots was performed using Science Lab99 Image Gauge 3.46 quantification program (Fujifilm Co. Ltd, Tokyo, Japan). 2.6. Ca2+-measurements The TRPV1 activity was measured by monitoring the [Ca2+]i levels using Fura2/AM fluorescence in a FLEX-Station-II (Molecular Devices, Sunnyvale, CA, USA) as described before (Lilja et al., 2007b) after 48 h in N2 medium with or without RA. Shortly, following Fura-2/AM incubation the cells were rinsed in Hepes Krebs Ringer buffer once and the basal [Ca2+]i and capsaicin-induced [Ca2+]i levels were measured. For the capsaicin-induced [Ca2+]i levels the Ca2+ influx was measured for 1 min every other second before, during and after capsaicin addition, and presented as the absolute relative fluorescence units (RFU) ratio of 340/380 nm excitation, 510 nm emission wavelengths. The 340/380 nm ratio values (corresponding to the Ca2+ influx) peaked immediately after capsaicin addition where after a plateau was reached followed by a slow decrease in [Ca2+]i. The mean peak value from 6 wells in a 96-well plate, from three individual experiments, was registered for each capsaicin concentration (0.3, 0.6, 1, 5, 10, and 100 nM). For the basal [Ca2+]i level measurements in the TRPV1-SHSY5Y and native SHSY5Y cells, values represent the mean of three individual experiments, each in 18 replicates. 2.7. Isolation of cell surface proteins To determine if the TRPV1 receptors were up-regulated at the cell surface, a biotinylation assay was performed after 48 h of treatment in N2 medium with or without RA. Cells from four 100 mm dishes per treatment were biotinylated according to manufacturer’s instructions using Pierce’s pinpoint cell surface protein isolation kit (Pierce, Rockford, IL, USA). The isolated plasma membrane protein concentration was determined and the TRPV1 proteins were analysed by western blot as described above. 2.8. Confocal microscopy To visualise the location of TRPV1 in differentiated cells, the control cells (cultured in MEM) and cells cultured in N2 medium with or without RA, were analysed for their TRPV1 expression using confocal microscopy. After 6 days of treatment the cells were fixed using 4% paraformaldehyde in PBS followed by permeabilisation in 100% methanol. Unspecific binding was blocked with 2% BSA in PBS. Cells were hybridised with the primary TRPV1 antibodies used for western blot (diluted 1:10,000 in 2% BSA/PBS) and blotted with secondary rabbit Alexa fluor1 red 568-conjugated antibodies from goat (diluted 1:1000 in 2% BSA/PBS, Molecular probes, Eugene, OR, USA). Confocal images were captured with Zeiss Axiovert 200 microscope (Zeiss, Bernried, Germany) by stepwise sectioning to the middle of the cells using 568 nm laser and PerkinElmer Ultra VIEW ERS rapid confocal imager (PerkinElmer Life Sciences, Boston, MA, USA). 2.9. Statistics To compare means of all groups, data were analysed using one-way ANOVA followed by Tukey’s Multiple Comparison Test or by Student’s t-test (Figs. 2D, 4 and 5), all in GraphPad Prism 4 software.
3. Results 3.1. Morphology of SHSY5Y and TRPV1-SHSY5Y cells differentiated with RA RA (1 mM) induced morphological differentiation in SHSY5Y and TRPV1-SHSY5Y cells as indicated by rounded cell bodies and the development of a neurite network. At day 6 the TRPV1-SHSY5Y cells had formed aggregates of cell bodies with extensions of neurites between them whilst the native SHSY5Y were still in monolayer (Fig. 1).
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Fig. 1. Morphology of SHSY5Y and TRPV1-SHSY5Y cells during RA-induced differentiation. Phase contrast pictures (originally 150 magnification) of native SHSY5Y (A) and (B) and TRPV1-SHSY5Y cells (C) and (D) cultured in N2 medium with RA for 1 day (A) and (C) and 6 days (B) and (D).
3.2. Effect of RA on TRPV1 expression The total TRPV1 protein level in the SHSY5Y cells cultured in N2 medium with RA was significantly elevated already after 24 h but increased with time and reached a 6-fold higher TRPV1 protein content after 6 days as compared to cells cultured in MEM medium (Fig. 2A and B). Culturing the cells in N2 medium without RA for 6 days induced a more than 2-fold increase in the total TRPV1 protein level as well (Fig. 2A and B). However, the effect of N2 medium on the TRPV1 up-regulation was not visible after 12–48 h of exposure when the effect of RA per se was evident. To exclude the possibility that components in the N2 medium induced the increase in TRPV1 expression, the cells were also treated with RA in the complete MEM medium. The effect of RA in MEM medium was the same as the effect of RA in N2 medium at all time points (48 h shown in Fig. 2C and D). Previously, we showed that insulin and IGF-I induced an increment in TRPV1 proteins that could be blocked by PI(3)K, PKC and MAPK(MEK) inhibitors (Lilja et al., 2007a). To study possible signalling pathways responsible for the RA-induced up-regulation of TRPV1 protein expression, specific inhibitors for PI(3)K (LY294002, 10 mM), MAPK(MEK) (PD98059, 10 mM), MAPK p38 (p38 MAP Kinase inhibitor, 1 mM), and PKC (BIM, 1 mM) were used along with RA treatment. The cells were exposed to RA in N2 medium as above for 48 h with or without inhibitors and the TRPV1 protein level was analysed by western blot. None of the inhibitors used could significantly block the RA-induced upregulation of TRPV1 proteins (Fig. 2E). Because the TRPV1 protein expression was highly elevated already after 24 h and the upregulation was further increased during differentiation, the mRNA was analysed at this time point. The TRPV1 mRNA expression was unaffected by 24 h administration of RA (Fig. 2F).
total TRPV1 protein content was highly increased after 48 h of RA exposure, the biotinylation assay was performed at this time point. The TRPV1 plasma membrane level was also significantly upregulated about 6-fold as compared to cells cultured in N2 medium without RA (Fig. 4A and B). 3.4. Effect of RA on TRPV1 activity The activity of the TRPV1 receptors was measured as capsaicininduced Ca2+ influxes using Fura-2/AM. The basal [Ca2+]i in the TRPV1-SHSY5Y cells was significantly increased by about 30% after 48 h culture in N2 medium with RA as compared to cells cultured in N2 medium without RA (Fig. 5A). The basal [Ca2+]i in the native SHSY5Y cells (lacking TRPV1) was not affected by RA treatment. The relative increased [Ca2+]i in TRPV1-SHSY5Y cells cultured in RA-containing N2 medium for 48 h remained after the addition of capsaicin at all concentrations compared to cells cultured in N2 medium without RA (Fig. 5B). The kinetics of the N2 and N2RA curves were similar but displayed a shift on the y-axis, as a result of different start (basal [Ca2+]i) RFU values. Thus, a comparison of the EC50 values from the equations of the two curves to quantify the TRPV1 receptor activity would be misleading. However, a comparison can be made by displaying the capsaicin concentrations acquired to induce a certain RFU ratio (i.e., the same [Ca2+]i) in the N2- and N2RA-treated cells, respectively. The 50% effect in the N2-treated cells measured 2.2 RFU, which was obtained at 0.8 nM (i.e., EC50) and the capsaicin concentration entailed to induce 2.2 RFU in the N2RA-treated cells was estimated to 0.02 nM. Hence, the capsaicin concentration required to give the same [Ca2+]i in both treatments was reduced 40-fold in N2RA-treated cells as compared to N2 treated cells. 4. Discussion
3.3. Effect of RA on TRPV1 receptor localisation The up-regulated TRPV1 proteins in cells differentiated for 6 days with RA were localised in the cell bodies as well as in the neurites as shown by confocal microscopy (Fig. 3). To study the plasma membrane localisation of the up-regulated TRPV1 receptors, the cell surface TRPV1 proteins were isolated by using a biotinylation assay, and analysed by western blot. Since the
The vanilloid receptor TRPV1 was originally described as a nociceptor localised on sensory neurons where it is activated by noxious stimuli such as heat, protons and capsaicin (Caterina et al., 1997). However, TRPV1 has been shown to be involved in cellular processes beside nociception and inflammatory responses. For example, TRPV1 is a major physiological Ca2+ channel required for the migration of hepatocellular carcinoma (HepG2) cells (Waning
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Fig. 2. Total TRPV1 levels in TRPV1-SHSY5Y cells during RA exposure. Cells were grown in MEM or RA-containing MEM (MEMRA), N2 medium (N2) or RA-containing N2 medium (N2RA). The total protein content was analysed after 12 h, 24 h, 48 h and 144 h by western blot using specific antibodies towards TRPV1 and tubulin. (A and C) One representative western blot picture of TRPV1 (100 kDa) and tubulin (50 kDa) blots after RA exposure. (B and D) The bars represent the density of the 100 kDa TRPV1 blot in relation to the density of the 50 kDa tubulin blot in the same lane. (E) TRPV1-SHSY5Y cells were treated as above for 48 h in N2 medium (referred to as control, C) and RAcontaining N2 medium without inhibitors (RA) or with inhibitors for PI3K, 10 mM (+LY), MAPK 10 mM (+PD), PKC 1 mM (+BIM) or p38MAPK 1 mM (+p38). The bars represent the density of the 100 kDa TRPV1 blot in relation to the corresponding 50 kDa tubulin blot. (F) mRNA expression of TRPV1 after 24 h exposure to N2 medium with (N2RA) or without (N2) RA, measured by RT-PCR. Values in B and E (mean S.E.M., n = 3) were analysed by one-way ANOVA, followed by Tukey’s multiple-comparisons test and values in D were analysed by Student’s t-test.***P < 0.001 compared to N2 (compared to MEM in D), &&P < 0.01 compared to MEM.
et al., 2007) and TRPV1 activity induces maturation and migration of dendritic cells in mice (Basu and Srivastava, 2005). Furthermore, Goswami and Hucho (2007) and Goswami et al. (2007) have shown the importance of TRPV1 protein expression for the development of filopodia and regulation of the growth cone in neurons and nonneuronal cells. Importantly, other TRP channels (of the TRPC family) play a crucial role in the guidance of nerve growth cones (Wang and Poo, 2005; Li et al., 2005). Herein, we have studied the effect on TRPV1 expression and activity during RA-induced neuronal differentiation in stably TRPV1-transfected SHSY5Y cells. The TRPV1-SHSY5Y cells reached a high morphological differentiation with an extensive neurite
network between aggregates of cell bodies after 6 days in culture in N2 medium with 1 mM RA. Native SHSY5Y cells develop a network of neurites after 6 days in N2RA medium as well. However, these cells do not form aggregates until after 2–3 weeks of RA (unpublished observation) indicating a difference in the differentiation process. In accordance with the morphological differentiation in the TRPV1-SHSY5Y cells, the level of TRPV1 total protein expression was dramatically increased, and the upregulated TRPV1 proteins were localised in both the cell bodies and the developed neurites. The elevated TRPV1 protein levels were statistically significant already after 24 h of RA treatment and further increased during differentiation to reach a 6-fold increase
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Fig. 3. Localisation of TRPV1 proteins in TRPV1-SHSY5Y cells during RA-induced differentiation. Cells were seeded on cover slips and treated for 6 days in N2 medium with RA (N2RA) or in N2 medium without RA (N2) and compared to cells grown in regular plating medium (MEM). Cells were fixed and permeabilised in paraformaldehyde and methanol, respectively, and stained with specific TRPV1 antibodies and secondary antibodies conjugated to Alexa fluor1 red 568. The TRPV1 expression was visualised by confocal microscopy from a centred focal point viewing both surface and intracellular fluorescence in the cells.
after 6 days in N2 medium with RA as compared to cells cultured in MEM. Isolation of the plasma membrane proteins after 48 h of RA exposure showed that the same TRPV1 up-regulation was evident at the cell surface. Despite the substantial increase in the TRPV1 protein levels after 24 h to 6 days in culture with RA, the mRNA level remained unaffected. This implies that the increased level of receptors is regulated post-transcriptionally. The function of TRPV1 as a regulator of [Ca2+]i homeostasis during cell migration and growth cone formation can explain the presence of the receptor in the brain and in non-neuronal cell types. Differentiation and neurogenesis are strictly dependent on regulation of the [Ca2+]i, and localisation of Ca2+ permeable plasma membrane receptors like TRPV1 in developing neurons may facilitate maturation of the nervous system. We show that after 48 h of exposure to RA, when the total and plasma membrane TRPV1 protein expression had reached a 6-fold increase, the basal
Fig. 4. Cell surface expression of TRPV1 in TRPV1-SHSY5Y cells during RA exposure. Cells were treated with RA-containing N2 medium (N2RA) and N2 medium without RA (N2) for 48 h and plasma membrane proteins were isolated and analysed with western blot using TRPV1-specific antibodies. (A) One representative western blot image showing plasma membrane-bound TRPV1 in the cells. (B) The bars represent the density of the 100 kDa TRPV1 blot in percent of the total density of all 100 kDa TRPV1 blots on the membrane. Values (mean SEM, n = 3) were analysed by Student’s t-test, ***P < 0.001 compared to N2.
[Ca2+]i was significantly elevated by about 30% in the TRPV1SHSY5Y cells but not in the native SHSY5Y cells. The concomitant up-regulation of cell surface TRPV1 and the increased basal [Ca2+]i in the differentiated TRPV1-SHSY5Y cells indicate an association between the observations. The increased [Ca2+]i might be caused by constitutively, partly active TRPV1 receptors on the cell surface. Since TRPV1 is a voltage-gated ion channel (Voets et al., 2004), the altered resting cell membrane potential, which is accompanied with increased neuronal differentiation, and thereby excitability (Toselli et al., 1996), might cause a controlled, basal leakage of Ca2+ ions into the cells. The importance of TRPV1 as a Ca2+ channel could
Fig. 5. Ca2+ levels in SHSY5Y and TRPV1-SHSY5Y cells during RA exposure. The cells were treated with N2 medium with RA (N2RA) or without RA (N2) and the intracellular [Ca2+]i levels were measured by Fura-2/AM fluorescence. The values represent the ratio of 340/380 nm (excitation wavelengths) absolute RFU. (A) The bars show the relative basal [Ca2+]i in TRPV1-SHSY5Y and native SHSY5Y cells after 48 h of incubation in N2 or N2RA medium. (B) The graph shows the effect of capsaicin-induced [Ca2+]i in TRPV1-SHSY5Y cells after 48 h exposure in N2- or N2RA medium. The values in (A) and (B) represent mean SEM, n = 3 and were analysed by Student’s t-test where *P 0.05, **P 0.01, ***P < 0.001.
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be one explanation for the increased expression during neurite extension. The expression of other Ca2+ channels like L- and N-type Ca2+ channels have also been shown to be up-regulated in RAexposed cells (Gao et al., 1998). The nature of TRPV1 rearranging microtubule (Goswami et al., 2006) could also be of importance during differentiation. The re-organisation of cytoskeletal structures is a process demanded during growth cone motility and guidance also supporting the indication that up-regulation of TRPV1 receptors could play a crucial role for neuronal development (Dent et al., 2003; Gordon-Weeks, 2004). In vivo, RA is involved in neuronal patterning, differentiation and axonal outgrowth as well as the maintenance of the differentiated state of the adult neurons (reviewed by Maden, 2007). The RA hormone binds to the nuclear retinoic acid receptor (RAR) that functions as a dimer with retinoic X receptor (RXR) binding to RA responsive elements (RAREs) in the target genes, activating transcription. In SHSY5Y cells, RA treatment has also shown to have more direct effects on signal transduction activating the PI(3)K and rapidly phosphorylating Akt (Lopez-Carballo et al., 2002). There are several proteins (and mRNAs) that are increased after RA exposure where no RAREs have been found in their respective promoters (Lane and Bailey, 2005) but these non-RARE actions are poorly understood. The effects on the recombinant TRPV1 receptors in these cells, regulated by an artificial and strong promoter which is probably not responding to the original transcription factors, suggest post-transcriptional effects. Indeed, nerve growth factor ((NGF) Ji et al., 2002; Puntambekar et al., 2005), insulin and IGF-I (Lilja et al., 2007a) up-regulate TRPV1 protein expression in a post-transcriptional manner. This may be the reason why we observed that cells cultured for 6 days in N2 medium without RA also increased the total TRPV1 protein level since the N2 medium contains a high concentration of insulin (0.9 mM). The significant effect of RA in N2 medium on TRPV1 expression was verified by treating the cells with RA in regular serum-containing MEM medium, which gave the same effect. However, to induce a terminally differentiated neuronal phenotype of SHSY5Y cells it is desirable to use RA in a defined medium in which the level of growth factors is controlled and where serum proteins are not affecting RA concentration. The regulation of TRPV1 expression and translocation to the plasma membrane has previously been shown to be mediated by the phosphorylation of kinases in the pathways of PI(3)K (Bron et al., 2003; Lilja et al., 2007a; Zhang et al., 2005), MAPK (Ji et al., 2002; Puntambekar et al., 2005) and by PKC (Lilja et al., 2007a; Morenilla-Palao et al., 2004). The PI(3)K pathway and the MAPK pathway are the major pathways involved in neuronal proliferation, differentiation and survival. However, none of the kinase inhibitors used herein could block the effect of RA on TRPV1 expression. Yet, the mechanism behind the RA-induced TRPV1 protein increment in these cells remains unclear. Treating cells with RA for 30 min did not induce an acute potentiating effect on the basal [Ca2+]i, nor the TRPV1 activity (not shown), indicating that the elevated basal [Ca2+]i in the differentiated TRPV1-SHSY5Y cells is probably not due to a sustained effect from immediate phosphorylation of the receptor and/or acute translocation of the receptor to the plasma membrane. Rather, the results imply that the increased [Ca2+]i is connected to the long-term increase in TRPV1 protein levels at the cell surface. TRPV1 has been shown to be expressed in the brain in rat (Mezey et al., 2000; Sanchez et al., 2001; Toth et al., 2005), human (Cortright et al., 2001; Cristino et al., 2006; Mezey et al., 2000; Roberts et al., 2004), mouse (Cristino et al., 2006; Roberts et al., 2004) and monkey (Szabo et al., 2002). Furthermore, Jin et al. (2004) suggested that both the cannabinoid and TRPV1 receptors have roles in adult neurogenesis in mice. The functional role of TRPV1 in brain has not yet been resolved, but one suggestion may
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