Transient receptor potential protein family C (TRPC) has been proposed as a candidate for channels involved in capacitative Ca2+ entry (CCE) mechanisms, but the modulation of their gene expression remains unexplored. In this study we show that guinea pig gallbladder smooth muscle contains mRNA encoding TRPC1, TRPC2, TRPC3, and TRPC4 proteins whose abundance depends on cytosolic Ca2+ level ([Ca2+]i). Thus lowering the levels of cellular calcium with the chelators EGTA and BAPTA AM results in a downregulation of TRPC1–TRPC4 gene and protein expression. In contrast, activation of Ca2+ influx through L-type Ca2+ channels and Ca2+ release from intracellular stores induced an increase in TRPC1–TRPC4 mRNA and protein abundance. Activation of Ca2+/calmodulin-dependent kinases (CaMK) and phosphorylation of cAMP-response element binding protein accounts for the increase in TRPC mRNA transcription in response to L-type channel-mediated Ca2+ influx . In addition to this mechanism, activation of TRPC gene expression by intracellular Ca2+ release also involves calcineurin pathway. According to the proposed role for these channels, activation of CCE induced an increase in TRPC1 and TRPC3 mRNA abundance, which depends on the integrity of the calcineurin and CaMK pathways. These findings show for the first time an essential autoregulatory role of Ca2+ in Ca2+ homeostasis at the level of TRPC gene and protein expression.
- transient receptor protein family C channels
- cytosolic calcium levels
calcium (Ca2+) plays a signaling role in many important cellular functions, such as fertilization, embryonic pattern formation, differentiation, proliferation, contraction, secretion, and metabolism (5). Although Ca2+ is a ubiquitous second messenger, cells have found ways to endow distinct Ca2+ signals with specific functions. The versatility of the Ca2+-signaling mechanism in terms of speed, amplitude, and spatiotemporal patterning enables elevations of Ca2+ to specifically regulate many processes of cell activity, including gene expression (23, 26, 44). One potential Ca2+-dependent step in the process of new gene expression is mRNA transcription (6, 17), which also has been shown to be differentially regulated by the pattern of Ca2+ elevation (23). Detection of cytosolic Ca2+ level ([Ca2+]i) increases by specific Ca2+ sensors such as calmodulin (9) or members of the recoverin subfamily of Ca2+-binding proteins (7) transduce the Ca2+ signal into changes in the transcription rate of specific genes through three general mechanisms: activation of phosphorylation/dephosphorylation cascades that modify transcription factors, induction of protein-protein interaction between the Ca2+ sensor and transcription factors, or changes in the binding properties of the Ca2+ sensor to specific sites in the DNA (reviewed in Ref. 26).
Among other genes mediating proliferation and cell survival, mRNA transcription of genes encoding ion channels has been shown to be regulated by Ca2+ (1, 13, 30). Experimental manipulations of Ca2+ entry via L-type channels have shown that increased cytosolic Ca2+ enhances L-type Ca2+ currents either upregulating transcriptional expression of these channels (13) or downregulating Kv mRNA expression (1, 30), thereby causing membrane depolarization, which causes further Ca2+ influx through L-type Ca2+ channels. Ca2+ influx through store-operated channels (SOC) is involved in controlling gene expression during the activation of T cells by antigens in a calcineurin/nuclear factor of activated T cells (NFAT)-dependent manner (for review, see Ref. 18). In keeping with this, it has been shown that transcriptional activation may be regulated by store-operated Ca2+ entry in vascular smooth muscle through changes in cAMP-response element binding protein (CREB)-mediated c-fos expression (32).
At present, the best molecular candidates for SOC are the TRP proteins (so called because of their homology with the transient receptor potential protein that underlies phototransduction in Drosophila). The TRP superfamily has been subdivided into multiple subfamilies on the basis of sequence similarity (27). In the case of SOC, much attention has been focused on the canonical TRP (TRPC) subfamily. Despite considerable effort, it is unclear exactly which of the seven TRPC isoforms are the molecular constituents of endogenous SOC. The mechanisms that regulate TRPC gene expression remain one of the important unexplored areas of research in this field. Transcriptional changes of TRPC gene expression in response to organ culture (4, 14) and to injury (4) have been shown recently in arterial smooth muscle, but the signal pathway and mechanisms mediating the plasticity of TRPC gene expression have not been explored yet. In addition, short-term changes in TRPC expression recently were demonstrated in pulmonary arteries. Thus extracellular ATP induces upregulation of TRPC4 expression, which was mediated by phosphorylation of the transcription factor CREB, probably through Ca2+-independent mechanisms (47). The aim of the present work was to explore the hypothesis that [Ca2+]i participates in the regulation of TRPC transcription and protein expression. If correct, such a proposal increases the complexity of Ca2+ signaling as a consequence of this apparent positive feedback: Ca2+-mediated regulation of Ca2+-permeable TRPC channels.
MATERIALS AND METHODS
Gallbladders were isolated from 300- to 450-g male guinea pigs following cervical dislocation and exsanguination and immediately placed in cold Krebs-Henseleit solution (KHS; for composition, see Solutions). All procedures were reviewed and approved by the office of Animal Care Management at the University of Extremadura.
Total RNA was isolated from gallbladder smooth muscle (GBSM) using the TRI reagent. Residual genomic DNA was removed by treatment with DNA-free (Ambion). Total RNA was quantified with RIBO green. First-strand cDNA was synthesized from 1 μg of total RNA using random decamers and MMLV reverse transcriptase (Ambion). The cDNA was amplified by PCR using known rat-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers and the TRPC isoform-specific primers described by Ong et al. (29) in guinea pig airway smooth muscle. Amplification was carried out using Applied Biosystems AmpliTaq Gold DNA polymerase and a Gene-Amp PCR system 2700. cDNA (2 μl) was added to 23 μl of PCR reaction mixture containing 2.5 μl of 10× PCR buffer, 2.5 μl of 25 mM MgCl2, 1 μl of 10 mM dNTPs, 0.3 μM of each primer (except for the degenerate primer MTA, which was used at 1 μM) and 0.125 μl of Taq polymerase (5 U/μl). The PCR conditions were as followed: 95°C for 7 min, 45 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with final extension of 10 min at 72°C. A negative control reaction (no template) to test for general contamination was included. Under these conditions we never observed unspecific products. Reaction products were separated by electrophoresis on 2% agarose gel stained with ethidium bromide. The obtained RT-PCR products were sequenced either directly by using the same oligonucleotides as primers or after being subcloned into a pGEM-T vector (Promega) by using Applied Biosystems sequencing kits.
Real-time quantitative RT-PCR.
Relative abundance of TRPC1–TRPC4 mRNA was assessed using the 5′ fluorogenic nuclease assay (TaqMan) on an ABI Prism7700 sequence detector system (Applied Biosystems). Primers and probes were designed on gallbladder TRPC1–TRPC4 mRNA. The primers pairs were designed to amplify a short amplicon to which a probe, labeled with 6-FAM reporter dye at the 5′-end and a TAMRA quencher dye at the 3′-end, annealed. The primers and probes sequences of TRPCs and Cavia porcellus GAPDH (used as the endogenous standard) are listed in Table 1. The 25-μl reaction mixtures contained 12.5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems), 200 nM of probe, 300 nM of each specific oligonucleotide primer, and 2 μl of the reverse transcriptase product. The reaction conditions were 10 min at 95°C for enzyme activation and 55 cycles of 30 s at 95°C and 30 s at 59°C. Non-template controls and reactions carried out with 2 μl of total RNA previous to the reverse transcriptase addition, were run routinely for each detector to confirm the absence of unspecific fluorescent signal or contaminant genomic DNA in the samples.
Since standard curves made for all primer pairs on TRPC and GAPDH cDNA revealed an efficiency value close to 2 (fold increase in input cDNA required to decrease the cycle number by 1), the relative amount of RNA was calculated by the comparative cycle threshold (Ct) method (25). This involves comparing the Ct values of the treated samples with a nontreated sample. The Ct values of both treated and nontreated samples were normalized to the endogenous housekeeping gene GAPDH. The normalized values were therefore calculated as RT-PCR determinations were done in three independent experiments, with each experiment containing tissue from three to four different animals.
Western blot analysis for TRPC.
Guinea pig GBSM layers were divided into strips and treated under different experimental conditions. In these protocols, the control and time-course treatments were always performed in strips coming from the same gallbladder. The muscle strips were later ground in a liquid nitrogen-cooled mortar and pestle, homogenized in lysis solution (for composition, see Solutions) using a homogenizer (OMNI International), and then sonicated for 5 s. Lysates were centrifuged at 10,000 g for 15 min at 4°C to remove nuclei and unlysed cells. Protein concentration was measured in the supernatant by using the Bradford method. Protein extracts (25–35 μg) were heat-denaturalized at 95°C for 5 min with DTT, electrophoresed on 7.5% polyacrylamide-SDS gels, and then transferred to a nitrocellulose membrane. For control and treated samples, the same amount of protein was always loaded in the polyacrylamide-SDS gels. Membranes were blocked for 1 h at room temperature using 10% bovine serum albumin (BSA) and incubated overnight at 4°C with affinity-purified polyclonal antibodies for TRPC1, TRPC3, and TRPC4 (1:200; Alomone Labs). A mouse anti-α tubulin monoclonal antibody (1:500; Santa Cruz Biotechnology) was used as load control. After washing, the membranes were incubated for 1 h at room temperature with anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (1:10,000; Bio-Rad Laboratories, Hercules, CA) or anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (1:10,000; Amersham Biosciences). The blots were then detected with the Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). The intensity of the bands was quantified using ImageJ software (NIH, Bethesda, MD) and normalized respect to α-tubulin content. The sample protein loads were in the linear range of detection for TRPC and α-tubulin antibodies (15–60 μg of protein, r = 0.972 and 0.979 for TRPC and α-tubulin, respectively, n = 4)
GBSM layers were fixed with 0.1 M PBS containing 2% paraformaldehyde and 0.2% picric acid and permeabilized with PBS, 0.1% Triton X-100, and 4% normal goat serum. The primary antibody rabbit anti-phospho-CREB (Ser133) (1:200; Cell Signaling Technology) was applied overnight at 4°C. The secondary antibody goat FITC-anti-rabbit IgG (1:200; Santa Cruz Biotechnology) was applied for 1 h at room temperature. Nuclei were stained with the fluorescent nucleic acid dye BOBO-3 iodide (1:10,000; Molecular Probes). Immunostaining for TRPC proteins was performed in isolated cells (see Cell isolation) fixed on the slides with 4% formaldehyde in 0.1 M PBS and permeabilized with 0.2% Triton-X-100 in PBS. The primary (rabbit anti-TRPC1, anti-TRPC3, and anti-TRPC4, diluted 1:60 in 2% BSA for TRPC1 and TRPC4 and 1:200 for TRPC3) and secondary (goat FITC-anti-rabbit IgG; 1:200 ) antibodies were applied under the protocol described above. Primary antibodies were either omitted or incubated with their blocking peptides as negative controls. Stained preparations were examined with a laser scanning confocal microscope (Bio-Rad MRC 1024ES; Bio-Rad Lab, Life Sciences Division).
GBSM cells were dissociated enzymatically using a previously described method (31). Briefly, small pieces of the tissue were incubated in enzyme solution (ES; for composition, see Solutions) supplemented with BSA, papain, and dithioerythritol. The tissue was then transferred to fresh ES containing 1 mg/ml BSA, 1 mg/ml collagenase, and 100 μM CaCl2. The single smooth muscle cells were isolated by several passages of the tissue pieces through the tip of a fire-polished glass Pasteur pipette. All experiments involving isolated cells were performed at room temperature (22°C).
Isolated cells were loaded with 4 μM fura 2-AM (Molecular Probes) in Na+-HEPES solution (for composition, see Solutions) and placed in an experimental chamber mounted on the stage of an inverted microscope. Cells were illuminated at 340 and 380 nm with a monochromator, and the emitted fluorescence was selected using a 500-nm band-pass filter. The emitted fluorescence images were captured with a cooled digital charge-coupled device camera (model C-4742-98; Hamamatsu Photonics) and recorded using dedicated software (MetaFluor; Universal Imaging). The ratio of fluorescence at 340 nm to fluorescence at 380 nm (F340/F380) was calculated pixel by pixel and used to indicate the changes in [Ca2+]i. A calibration of the ratio for [Ca2+] was not performed in view of the many uncertainties related to the binding properties of fura-2 with Ca2+ inside of smooth muscle cells.
The KHS contained (in mM) 113 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 11.5 d-glucose. This solution had a final pH of 7.35 after equilibration with 95% O2-5% CO2. The ES used to disperse cells was made up of (in mM) 10 HEPES, 55 NaCl, 5.6 KCl, 80 Na-glutamate, 2 MgCl2, and 10 d-glucose, with pH adjusted to 7.3 with NaOH. The lysis solution contained 50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.5% (wt/vol) NaN3, 1.5% SDS, 1 mM EGTA, 0.4 mM EDTA, 10 mM benzamidine, 25 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The Na+-HEPES solution contained (in mM) 10 HEPES, 140 NaCl, 4.7 KCl, 2 CaCl2, 2 MgCl2, and 10 d-glucose, with pH adjusted to 7.3 with NaOH. The Ca2+-free Na+-HEPES solution was prepared by substituting EGTA (1 mM) for CaCl2. The high-K+ Na+-HEPES was prepared by isosmotic replacement of NaCl with KCl.
Expression of TRPC family members in GBSM.
TRPC1–TRPC7 expression was evaluated using RT-PCR in GBSM layer. No TRPC5–TRPC7 transcripts were detected in GBSM. Figure 1A shows a 2% agarose gel with ethidium bromide staining of the PCR products for TRPC found in GBSM (n = 5 experiments in 3 animals). All the amplified products were of the predicted sizes: TRPC1, 400 bp; TRPC2, 370 bp; TRPC3, 340 bp; and TRPC4, 370 bp. The cDNA products of TRPC1–TRPC4 were sequenced, and the identities of the amplicons were verified by database homology searches. The partial TRPC sequences found in guinea pig GBSM were deposited at GenBank under the following accession numbers: TRPC1, AY572429; TRPC2, AY574383; TRPC3, AY572430; and TRPC4, AY572431 (Table 1).
Western blot studies using TRPC1, TRPC3, and TRPC4 polyclonal antibodies (Fig. 1B) revealed the expression of a protein for TRPC1 with a molecular mass around 120 kDa. The molecular mass of 120 kDa is in the range previously reported for TRPC1 protein with this commercial antibody in rat (2, 21, 24) but not human tissue (11, 46). Differences in the mobility of the band between humans and other animal species may reflect species differences in the expression of TRPC1 such as glycosylation. For TRPC3 and TRPC4, the molecular masses were ∼90–95 kDa. These sizes are in agreement with previous reports regarding TRPC3 and TRPC4 (2, 11, 12, 24). We did not perform Western blotting for TRPC2 because a specific antibody is currently not available. To further investigate the expression of TRPC proteins, we stained isolated GBSM cells. Immunofluorescence experiments using anti-TRPC1, anti-TRPC3, and anti-TRPC4 polyclonal antibodies showed that TRPC1, TRPC3, and TRPC4 proteins are present in GBSM (Fig. 1C), which agrees with our PCR and Western blot data, and are mainly located in plasma membrane.
To determine the relative abundance of the four TRPC transcripts expressed in GBSM, we performed real-time quantitative RT-PCR using RNA from freshly isolated GBSM and specific primers and probes designed on gallbladder TRPC1–TRPC4 mRNA sequences. Steady-state transcripts were determined relative to the endogenous control housekeeping gene GAPDH. TRPC1 and TRPC2 mRNA were the most highly expressed, whereas TRPC4 was expressed at the lowest level. TRPC expression relative to GAPDH (arbitrary units) was 0.145 ± 0.020, 0.191 ± 0.025, 0.076 ± 0.015, and 0.0012 ± 0.00059 for TRPC1, TRPC2, TRPC3, and TRPC4, respectively (n = 7).
Intracellular Ca2+ regulates TRPC gene transcription and protein expression.
To test the hypothesis that TRPC gene expression is regulated by [Ca2+]i levels, we carried out real-time quantitative RT-PCR on total RNA extracted from GBSM pretreated with the Ca2+ chelators EGTA and BAPTA AM. We have previously shown that both approaches are effective in lowering [Ca2+]i (28). Figure 2A shows that exposure of GBSM to Ca2+-free solution containing 1 mM EGTA for 2 h significantly decreased TRPC1, TRPC2, TRPC3, and TRPC4 mRNA abundance relative to control conditions (n = 5, P < 0.05 for TRPC3 and TRPC4, P < 0.001 for TRPC1 and TRPC2). A similar pattern was also observed when GBSM was treated with 10 μM BAPTA AM for 30 min (n = 4, P < 0.05, Fig. 2B). In agreement with the observed reduction in mRNA content, Ca2+ chelator conditions also reduced the amount of TRPC proteins in GBSM cells (Fig. 2, C and D).
These data suggest that cytosolic Ca2+ ions regulate TRPC gene expression in guinea pig GBSM, but at this stage we cannot rule out the possibility that Ca2+ may just be necessary for gene expression without having a regulatory role in this process. To further test these possibilities, we raised intracellular Ca2+ levels by activation of Ca2+ influx through L-type Ca2+ channels by depolarizing smooth muscle with 60 mM KCl. We have previously demonstrated that L-type Ca2+ channels are the only subtype of Ca2+ voltage-sensitive channels activated by 60 mM KCl depolarization, since the specific blocker of L-type Ca2+ channels, nitrendipine, almost abolished the Ca2+ influx in response to KCl and to the selective L-type channel opener BAY K 8644 (16). Under KCl challenge for 10 min, mRNA encoding TRPC1–TRPC4 proteins were substantially enhanced (69.9, 60.9, 90.4, and 92.9% increase for TRPC1, TRPC2, TRPC3, and TRPC4, respectively, n = 4; Fig. 3A). These increases in TRPC mRNA levels were still detected when GBSM was exposed to KCl for 60 min (Fig. 3A). Even higher increases in TRPC1–TRPC3 mRNA accumulation were observed when GBSM was challenged for 10 and 60 min with 10 nM CCK, a hormone that releases Ca2+ from the intracellular Ca2+ stores (28) (Fig. 3B). Associated with these elevations in mRNA, KCl and CCK also induced a significant increase in TRPC expression at the level of protein, as shown in Fig. 3, C and D. These findings suggest that upregulation of gallbladder TRPC channel expression is mediated by increases in [Ca2+]i and support an active role of [Ca2+] in controlling TRPC gene expression. To ascertain whether the accumulation of TRPC mRNA was caused by transcriptional induction, we carried out a control experiment in which the tissue was previously incubated for 30 min with the transcription inhibitor actinomycin D (5 μg/ml) and then challenged with KCl or CCK. The results, shown as insets in Fig. 3, A and B, indicate that no significant changes in the mRNA levels were detected compared with the controls without actinomycin D (n = 4–6, P > 0.05). Similar results were obtained when smooth muscle layers were exposed to BAPTA (data not shown). This suggests that Ca2+ behaves as a transcriptional inducer (either direct or indirect) of the TRPC gene transcription.
To give some insights into the possible mechanisms by which Ca2+ signal could be transduced into changes in gene activity, and because CaMKs have been suggested to be important signaling molecules in Ca2+-induced changes in gene transcription (19), we examined the effects of KN-93, an inhibitor of CaMK activity, on KCl/CCK-induced increases in TRPC1–TRPC4 gene expression. As shown in Fig. 4, A and B, pretreatment of smooth muscle layer with 30 μM KN-93 for 30 min was able to abolish the increases in TRPC1–TRPC4 gene expression in response to both KCl and CCK (n = 5). It should be noted that even a decrease in TRPC1–TRPC4 mRNA abundance relative to control conditions (expressed as the value of 1) was caused by KN-93, suggesting that CaMKs regulate TRPC gene transcription not only in response to [Ca2+]i increases but also at resting [Ca2+]i levels. This was confirmed when TRPC mRNA abundance was measured in control smooth muscle layers pretreated with KN-93 (59, 60, 56, and 61% inhibition for TRPC1, TRPC2, TRPC3, and TRPC4, respectively, n = 6, P < 0.01).
CREB has been identified as an important signal transduction element in CaMK-mediated gene transcription (37). Thus we performed immunofluorescence experiments to detect changes in CREB phosphorylation in response to KCl and CCK. As can be observed in Fig. 4C, nuclear phospho-CREB was undetectable in resting cells and was only evident under both treatments, suggesting that the phospho-CREB may be involved in enhancing TRPC gene expression. This phosphorylation was dependent on CaMK activation, since the pretreatment with the CaMK inhibitor KN-93 abolished and reduced KCl- and CCK-induced CREB phosphorylation, respectively (Fig. 4C). Another important molecule in mediating Ca2+-induced changes in gene transcription is the Ca2+-dependent transcription factor NFAT. Dephosphorylation of NFAT by the phosphatase calcineurin activates translocation of this transcription factor to the nucleus, where it modulates gene transcription (34). We assayed the effects of the calcineurin inhibitor cyclosporine A (1 μM, for 30 min) in TRPC1–TRPC4 expression in GBSM. As shown in Fig. 4, A and B, this inhibitor only reduced CCK-induced enhancement of TRPC expression without having any effect on KCl-induced changes in TRPC gene transcription (n = 4). These results indicate that activation of the calcineurin pathway selectively mediates modulation of TRPC gene expression in response to Ca2+ release from intracellular stores. Although it is firmly established that NFAT is the calcineurin effector for gene transcription, we studied whether KCl and CCK treatments were associated to the translocation of NFAT to the nucleus. However, our immunofluorescence experiments failed in showing any change in the location of NFATc1 and 3, since NFAT was present in the nucleus and cytosol in all the conditions tested, included resting cells. The presence of NFAT in the nucleus without any stimuli could reflect NFAT-mediated gene transcription in basal state or nonspecificity of the antibodies used. When we repeated the immunostaining in smooth muscle exposed to Ca2+-free medium supplemented with EGTA for 30 min, to avoid calcineurin activation and thereby NFAT translocation, no significant changes were observed compared with control, which may indicate nonspecific binding of the primary antibodies in our preparation. In agreement with this hypothesis, Western blot experiments showed that in addition to the expected band at ∼130 kDa, additional, apparently nonspecific, bands at 100, 85, 75, 70, and 60 kDa also were present, indicating lack of specifity of these antibodies.
Capacitative Ca2+ entry modulates expression of TRPC genes.
We have previously demonstrated that GBSM exhibits capacitative Ca2+ entry (CCE) in response to Ca2+ release from internal stores (28). Given that TRPC family proteins have been suggested to be molecular counterparts of plasma membrane proteins activated by depletion of Ca2+ stores (3, 10), we conducted real-time quantitative RT-PCR experiments to test a possible change in GBSM TRPC gene expression as the result of CCE activation. We activated CCE by using a protocol previously validated in this cellular model (28). Before RNA isolation, Ca2+ stores were depleted by application of the sarco(endo)plasmic Ca2+-ATPase (SERCA) pump inhibitor thapsigargin (1 μM) plus 1 μM nitrendipine (to block L-type Ca2+ channels) in a Ca2+-free medium for 30 min, and then external Ca2+ was reintroduced for 10 or 60 min. TRPC1 and TRPC3 mRNA contents were significantly higher in the CCE-activated group than in the control group (P < 0.05 for both). However, no significant changes were observed in TRPC2 and TRPC4 expression (n = 4, Fig. 5A). These changes are due to transcriptional changes in gene expression, since no changes were detected in smooth muscle layers pretreated with actinomycin D (n = 4, Fig. 5A, inset). In addition, the CCE protocol caused an increase in TRPC1 and TRPC4 proteins at 10 min that did not last for 60 min (Fig. 5, B and C).
The CCE-induced increases in TRPC1 and TRPC3 proteins suggest that these proteins may be subunits of SOC, activated when the stores are depleted. To further explore this, we quantified Ca2+ entry in fura-2-loaded cells following a protocol involving depletion of the pools by a 30-min pretreatment with thapsigargin plus nitrendipine, followed by treatment with two pulses of extracellular Ca2+ separated by a 20-min interval. In control conditions, the [Ca2+]i plateaus induced by the Ca2+ pulses, indicative of CCE, were repetitive (0.059 ± 0.006 vs. 0.057 ± 0.004 ΔF340/F380, n = 7, Fig. 5D, top) in keeping with previously described results (28). However, as shown in Fig. 5D, bottom, when we treated cells with TRPC1 antibody (5 μg/ml) during the interval between the two Ca2+ applications, there was a marked attenuation of CCE (0.056 ± 0.005 vs. 0.023 ± 0.004 ΔF340/F380, n = 12, P < 0.001). TRPC1 antibody treatment did not induce any significant change in TRPC mRNA abundance ( 3.6, 3.9, 6.6, and 10.1% increase for TRPC1–TRPC4, n = 4, P > 0.05), indicating that CCE attenuation would be the result of local effects of TRPC1 antibody on TRPC1 membrane proteins. In fact, this antibody was targeted to a TRPC1-specific peptide that was predicted to be extracellular and located between fifth and sixth membrane-spanning domains. Thus, when we performed immunostaining in nonpermeabilized cells and used this antibody in the same conditions as in the Ca2+ imaging experiments (20-min treatment with the primary antibody), the staining was similar to that obtained in permeabilized cells after overnight treatment with the primary antibody (Figs. 5E and 1B). When we repeated both experiments with the antibody raised against an intracellular epitope of TRPC3, we did not see any specific staining in the immunofluorescence experiments (Fig. 5E). However, in 6 of 15 cells we recorded a reduction in CCE (39% inhibition), for which we do not have any explanation yet. TRPC4 antibody did not cause any change in CCE and was unable to stain nonpermeabilized cells (data not shown).
Similar to the results obtained after the smooth muscle was challenged with CCK, CCE-enhanced TRPC1 and TRPC3 expression was sensitive to the treatment with both KN-93 and cyclosporine A, indicating that Ca2+ influx in response to store depletion activates TRPC expression through CaMK- and calcineurin-mediated pathways (Fig. 6A). CCE activation for 10 min also caused phosphorylation of nuclear CREB that was sensitive to KN-93 pretreatment, as shown in Fig. 6B, which suggests that phospho-CREB can be a mediator of the enhanced TRPC expression. Although the inhibition of mRNA transcription by cyclosporine A indicates that translocation of NFAT may also mediate the positive regulation induced by CCE, we could not perform NFAT immunostaining because of the nonspecificity shown by the commercially available antibodies, as reported above.
Identification of TRPC proteins has received increasing attention due to their possible role as molecular counterparts of some plasma membrane Ca2+ channels, including channels activated by depletion of the intracellular Ca2+ stores. In the present study, we have identified the TRPC isoforms present in GBSM cells and their relative abundance. In addition, we sought to explore the regulation of TRPC gene expression, an area of research that remains unknown in this field. Thus we have demonstrated that intracellular Ca2+ levels participate in the modulation of TRPC gene transcription and protein expression. [Ca2+]i resting levels are necessary to maintain TRPC gene expression, and elevation of [Ca2+]i by different maneuvers, including activation of CCE, is associated with an upregulation of TRPC gene expression, which indicates that this ion autoregulates its levels by positive feedback mechanisms, adding an extra level of complexity to Ca2+ signaling.
mRNA encoding for TRPC1, TRPC2, TRPC3, and TRPC4 but not for TRPC5, TRPC6, and TRPC7 proteins is present in GBSM. TRPC2 and TRPC1 were expressed in the greatest abundance relative to GAPDH, whereas TRPC4 was expressed at the lowest level. We also presented evidences for expression of TRPC1, TRPC3, and TRPC4 proteins by Western blot and immunofluorescence experiments, although we cannot ensure that these are the only ones expressed, because specific antibodies for the TRPC2 isoform are not available. There are conflicting reports whether these proteins function as channels gated by G protein-coupled receptors or by depletion of Ca2+ stores. This has been attributed, in part, to the use of different techniques such as Ca2+ imaging or electrophysiology (see Ref. 33). Inhibition of TRPC1 with the use of TRPC-specific tools such as a functional anti-TRPC1 antibody (Refs. 4, 45; present study) or antisense DNA targeted to TRPC1-encoding mRNA (40) is associated with a decrease in Ca2+ reentry in response to store depletion in smooth muscle (Ref. 45; Fig. 5D), inhibition of contractions with a pharmacological profile similar to that of store-operated Ca2+ entry (4), and reduction in the amplitude of the nonselective cationic current evoked by store depletion (40). In addition, overexpression of TRPC1 enhanced pulmonary artery contraction evoked by depletion of the stores (21). Together, these results support a role for TRPC1 in the store-operated Ca2+ entry phenomenon in smooth muscle. There is ample evidence that TRPC1 forms heteromeric complexes with TRPC4 and TRPC5 but also some involving TRPC3 and TRPC2 (see Ref. 3). Our results do not support or rule out an association of the TRPC isoforms present in our preparation to form nonselective cationic SOC but strongly indicate that TRPC1 is a component of the native channels accounting for Ca2+ influx in response to store depletion.
Although the number of studies focused in TRPC proteins and their functions is increasing, reports concerning the mechanisms that regulate TRPC gene expression are still lacking. The findings reported in the present study demonstrate, for the first time, a link between cytosolic Ca2+ and TRPC gene expression at transcriptional and transductional levels. This hypothesis is based on the observation that lowering the level of cellular Ca2+ with the use of Ca2+ chelators such as EGTA and BAPTA AM leads to a decrease in the expression of all TRPC members found in GBSM, whereas elevations in cellular Ca2+ as a result of Ca2+ influx or store depletion leads to an increase in TRPC gene expression.
It is physiologically relevant that Ca2+ homeostasis -mediated regulation results in changes at the TRPC protein levels, since this open the possibility of the presence of a positive feedback mechanism aimed to enhance Ca2+ signal in response to different stimuli. Another possibility could be that this mechanism participated in prolonging the Ca2+ increase beyond the duration of the stimuli or even that exacerbation of this autoregulation could collaborate in Ca2+ overload and cellular apoptosis. However, this last possibility seems uncertain, since the experimental maneuvers used in this study to increase [Ca2+]i (CCK, KCl, and CCE) do not cause any functional damage to the smooth muscle. It is noteworthy that activation of CCE for 60 min did not cause any increase in TRPC protein expression, although 60-min treatment with KCl and CCK enhanced the expression. This finding could indicate that the TRPC turnover is increased when CCE is specifically stimulated, but it also could reflect that exhaustive and prolonged store depletion with thapsigargin impairs protein synthesis in sarcoplasmic reticulum (42). The requirement of Ca2+ for protein expression is a well-known effect. Ca2+ depletion of endoplasmic reticulum reduces protein synthesis through an active, regulated response (reviewed in Ref. 20). The effect of thapsigargin and Ca2+ depletion in our experiments is in keeping with previous reports of protein synthesis impairment (e.g., Ref. 38, 48). Regarding the turnover of proteins in Ca2+ transport, studies in cardiac ischemia-reperfusion have shown that mRNA for ryanodine receptors, SERCA pumps, phospholamban, and calsequestrin exhibit a significant decrease in response to short protocols (even less than 20 min in duration) (41). In addition, mRNA and protein expression of inositol 1,4,5-trisphosphate type II receptors decrease within 2–3 h in response to Ca2+ depletion (22).
Changes in the [Ca2+]i constitute one of the main routes by which information is transferred from extracellular signals received by animal cells to intracellular sites, including the nucleus (5). This idea is the basis of excitation-transcription coupling (ET coupling), a process by which common signaling pathways that regulate excitation-contraction coupling (EC coupling) also translate into transcriptional gene regulatory events (43). ET coupling represents a potential integrative mechanism whereby short-term regulation of Ca2+ signaling and contraction are transduced into long-term regulation of smooth muscle growth, differentiation, and remodeling. Our study provides novel evidence showing that intracellular signals that regulate short-term contraction also can regulate short-term expression of genes involved in EC coupling, which adds more complexity to the Ca2+ signaling pathway in the control of short- and long-term gene expression.
Two general paradigms in ET coupling in smooth muscle are the dependence on subcellular changes in Ca2+ and the activation of specific transcription factors through different intracellular pathways (43). The underlying Ca2+-dependent signaling mechanisms involved in TRPC gene transcription include CaMK and calcineurin pathways, since the inhibitors KN-93 and cyclosporine A caused a dramatic reduction in the upregulated TRPC mRNA. The Ca2+-mediated regulation is specific for the different modes of Ca2+ signaling: Ca2+ influx through L-type Ca2+ channels induces TRPC upregulation by a CaMK-sensitive pathway, whereas intracellular Ca2+ release and store-mediated Ca2+ influx activate TRPC transcription using both CaMK- and calcineurin-dependent pathways. When [Ca2+]i was increased by a standard Ca2+ reentry protocol after store depletion, the TRPC1 and TRPC3 isoforms were upregulated. As demonstrated in the present study, TRPC1 at least seems to be a component of the channels mediating store-operated Ca2+ entry, which shows the presence of a positive feedback mechanism(s) to ensure Ca2+ entry and replenishment of stores.
Among other kinases activated downstream of Ca2+ increases, CaMK have been shown to phosphorylate CREB (8, 37). The phosphorylation of CREB on Ser133 enables CREB to bind the cAMP-response element (CRE) and to modulate transcription of the genes (e.g., bcl-2, c-fos) whose promoters contain a CRE binding sequence. In the present study, we have shown that Ca2+-induced TRPC upregulation was attenuated when CaMK activity was inhibited. In addition, there was an increase in nuclear CREB phosphorylated on Ser133 associated with those treatments that increase cytosolic Ca2+, which depends on CAMK activation. This result indicates that CaMK-mediated CREB phosphorylation could be responsible, at least in part, for the enhanced TRPC expression. In pulmonary artery smooth muscle cells, it was recently shown that CREB mediated TRPC4 upregulation in response to low concentrations of the mitogen ATP. However, in this study, CREB was phosphorylated by PKA/PKG-dependent pathways via a Ca2+-independent mechanism (47).
We have demonstrated that the calcineurin pathway specifically mediates upregulation evoked by Ca2+ release from intracellular stores and Ca2+ influx activated by depletion of these stores, but it is not activated by depolarization-induced Ca2+ influx. In nonexcitable cells, the sustained increase in intracellular Ca2+ required to effectively translocate to the nucleus, via calcineurin, the transcriptional effector NFAT is provided by Ca2+ influx through SOC (36). In ileal and vascular smooth muscle, it has been reported that elevation of [Ca2+]i by membrane depolarization failed to induce NFAT nuclear accumulation (15, 39). In skeletal myocytes, the ryanodine-sensitive Ca2+ pool is insufficient to maintain NFAT translocation, whereas Ca2+ entering through non-voltage-dependent channels such as TRPC3 is required for NFAT-dependent transactivator function. In addition, expression of TRPC3 is increased in response to activated calcineurin and is limiting to NFAT-dependent transactivation, indicating that TRPC3 channels participate in a positive feed back circuit (35). As commented in results, we could not correlate NFAT nuclear translocation with TRPC mRNA upregulation, probably because of nonspecificity of the antibodies used in our tissue.
We do not know whether TRPC-enhanced expression is due to direct effects of the nuclear transcription factors in the TRPC gene promoters or indirectly through changes in intermediate factors as c-fos or c-jun that subsequently upregulate TRPC gene expression. Pulver et al. (32) recently reported that influx of Ca2+, caused by thapsigargin-induced depletion of intracellular stores in vascular smooth muscle, results in transient phosphorylation of CREB, and transcription of c-fos.
In conclusion, we have shown for the first time that cytosolic free Ca2+ controls TRPC expression through activation of CaMK-dependent CREB phosphorylation and calcineurin-dependent pathway. In addition, we have revealed that expression of TRPC proteins involved in CCE operates as a positive feedback mechanism in Ca2+ homeostasis. These findings provide a basis for further studies investigating the peculiar regulation of TRPC gene in mammalian cells.
This work was supported by the Spanish Ministry of Education and Science grants SAF-2001-0295 and BFU 2004-0637. S. Morales was supported by a Ministry of Education Predoctoral Research Grant.
We thank M. P. Delgado and S. Perez for technical assistance.
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