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VASCULAR BIOLOGY

Nuclear translocation of calcineurin A but not calcineurin A by platelet-derived growth factor in rat aortic smooth muscle

¹BHF Laboratories, Department of Medicine, University College London, London, United Kingdom; and ²Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada

Submitted 24 March 2005 ; accepted in final form 9 February 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Calcineurin regulates the proliferation of many cell types through activation of the nuclear factor of activated T cells (NFAT). Two main isoforms of the calcineurin catalytic subunit [calcineurin A (CnA) and CnA] have been identified, although their expression and function are largely unknown in smooth muscle. Western blot analysis and confocal imaging were performed in freshly isolated and cultured rat aortic myocytes to identify these CnA isoforms and elucidate the effect of PDGF on their cellular distribution and interaction with NFAT isoforms. CnA and CnA isoforms displayed differential cellular distribution, with CnA being evenly distributed between the nucleus and cytosol and CnA being restricted to the cytosol. In contrast with the rat brain, we found no evidence for particulate/membrane localization of calcineurin. PDGF caused significant nuclear translocation of CnA and induced smooth muscle cell proliferation, with both effects being abrogated by the calcineurin inhibitor cyclosporin A, the novel NFAT inhibitors A-285222 and inhibitor of NFAT-calcineurin association-6, and the adenylyl cyclase activator forskolin. PDGF also caused cyclosporin A-sensitive translocation of NFATc3, with no apparent effect on either CnA or NFATc1 distribution. Moreover, 87% of nuclear CnA was found to colocalize with NFATc3, consistent with the finding that CnA bound more avidly than CnA to a glutathione S-transferase-NFATc3 fusion protein. Based on their differential distribution in aortic muscle, our results suggest that CnA and CnA are likely to have different cellular functions. However, CnA appears to be specifically activated by PDGF, and we postulate that calcineurin-dependent nuclear translocation of NFATc3 is involved in smooth muscle proliferation induced by this mitogen.

nuclear factor of activated T cells c3; confocal imaging; cell proliferation; inhibitor of nuclear factor activated T cells-calcineurin association-6; A-285222

Calcineurin is a heterodimer composed of a 58- to 61-kDa catalytic A subunit and a 19-kDa Ca²⁺-binding regulatory B subunit (25, 30). The calcineurin A (CnA) subunit is encoded by three separate genes giving rise to CnA (PPP3CA), CnA (PPP3CB), and CnA (PPP3CC) isoforms. In addition, these isoforms show alternative splicing that results in at least two splice variants per isoform (30). So far, the - and -subunits have been found to be coexpressed in all tissues examined to date (30, 34), whereas the -subunit is restricted to specialized organs like the testis (30). Despite an 85% homology between CnA and CnA, different substrate affinities and catalytic activities are displayed by these two isoforms (26). Enzyme specificity may also result from the distinct isoform-specific distribution that has been observed both within and between tissues (30, 34) For example, CnA expression is more abundant than CnA in the brain, heart, and kidney, whereas this pattern is reversed in T cells, the thymus, and the spleen (16).

One of the main targets of calcineurin is a family of transcription factors known as the nuclear factor of activated T cells (NFAT), which is composed of four well-characterized members: NFATc1 (NFAT2), NFATc2 (NFAT1), NFATc3 (NFAT4), and NFATc4 (NFAT3) (14). Once activated, calcineurin binds to and dephosphorylates NFAT, causing its translocation from the cytosol to the nucleus (14, 37). The immunosuppressants cyclosporin A (CysA) and FK506 block this translocation step by inhibiting calcineurin activity (14). A number of kinases, including PKA, are known to phosphorylate NFAT and thus oppose the effects of calcineurin (14, 33). In smooth muscle, nuclear accumulation of NFAT is initiated by a variety of G_q/11-coupled vasoconstrictor agonists and mitogens like PDGF-BB (3, 11, 35, 44). Moreover, NFAT translocation is associated with increased transcriptional activity and the growth of vascular smooth muscle and endothelial cells (3, 17, 35, 44). The role of the different NFAT isoforms in mediating these effects is controversial and proposed to involve either NFATc3 (8, 35) or NFATc1 (3, 17, 44). We show here, for the first time, that CnA - and -isoforms have distinct cellular patterns of expression in rat aortic smooth muscle (RASM) and provide evidence that the -isoform is likely to regulate the nuclear import/export of NFATc3 in response to growth-promoting and -inhibitory agents.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
RASM cells (RASMCs) were dissociated enzymatically from the thoracic aorta of male Sprague-Dawley rats (200–250 g) using a modified method (42). For culturing, RASMCs were resuspended in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin (Invitrogen) and grown at 37°C in a humidified atmosphere of 5% CO₂. Only cells between passages 4 and 9 were used. A smooth muscle phenotype was confirmed as previously described (5).

RT-PCR of CnA isoforms. Total RNA from rat thoracic aortae denuded of the endothelium and adventitia was isolated according to the manufacturer's instructions using the Epicentre Master Pure RNA isolation kit (Epicentre Technologies). cDNA was synthesized using 4 µg of the total RNA and oligonucleotide primers as per the manufacturer's instructions. PCRs were performed using the following sets of oligonucleotide pairs designed against brain sequences. For rat CnA (Genbank Accession No. X57115), the forward primers were 5'-ACGGTGGTTTGTCTCCAGAGA-3' (nt 866–886) and 5'-GAACCACCTGCTTATGG-3' (nt 928–944) and the corresponding reverse primers were 5'-GAGTGAAATGTTCCTGAGTCTT-3' (nt 997–1018) and 5'-GTGGCTCCGTCAAATCCATCTT-3' (nt 1412–1433), respectively. For human CnA (Genbank Accession No. M29551), the forward primer was 5'-CGAGGATTCTCTCCACCACAT-3' (nt 1518–1538) and the reverse primer was 5'-TGCGGTGTTCAGAGAATTGA-3' (nt 1627–1646). Primers were also designed for rat CnA (Genbank Accession No. D90036) around known splice sites reported for rat (31) and human CnA (23), with the forward primer being 5'-GCTCATGAAGCTCAAGATGC-3' (nt 904–923) and the reverse primer being 5'-GCACTATGGTTGCCAGTCC-3' (nt 1592–1610). Amplification was performed using 2 µl of the RT reaction product under the following conditions: 94°C for 2 min; 40 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and a final extension for 5 min at 72°C. PCR products were separated by electrophoresis on a 4% agarose gel and detected by the SYBR green fluorescent stain.

Western blot analysis. Thoracic aortae denuded of endothelial and adventitial layers were washed in cold Tris-buffered saline and snap frozen in liquid nitrogen. Two frozen aortas were placed in 400 µl of cold lysis buffer containing 50 mmol/l Tris·HCl (pH 7.5), 2 mmol/l EGTA, 0.5 mmol/l DTT, 1 mmol/l PMSF, and a Complete protease inhibitor pill (Roche Biologicals) and homogenized on ice for 30 min using a glass tissue grinder (Anachem, Bedford, UK). Frozen rat brains were treated similarly. Lysates were then centrifuged at 1,000 g for 15 min at 4°C, and an aliquot of the supernatant was further purified by centrifugation at 100,000 g (1 h, 4°C) to obtain the cytosolic and particulate fractions. Protein concentrations in the various fractions were determined with Bio-Rad Protein Assay Kit 1 (Bio-Rad, Hertfordshire, UK) using bovine -globulin as the standard. Recombinant rat brain CnA and human brain CnA were purified from Sf21 cells using the baculovirus expression system and calmodulin affinity chromatography as previously described (26). Protein samples were separated using 7.5% high-salt SDS-PAGE and then transferred electrophoretically to nitrocellulose membranes (Amersham Biosciences). Blots were washed in PBS and then incubated overnight at 4°C in blocking buffer (Tropix, Applied Biosciences) before being probed at room temperature for 1 h with primary rabbit polyclonal antibodies against CnA (1:1,000 dilution) and CnA (1:500 dilution) isoforms (Upstate Biotechology). After several washes with PBS containing 0.1% Tween 20 (PBST), blots were incubated for 1 h at room temperature with the secondary antibody, goat anti-rabbit alkaline phosphatase conjugate (dilution 1:5,000, Tropix, Applied Biosciences), followed by several washes in PBST. All primary and secondary antibodies were diluted in blocking buffer. After this procedure, blots were processed and developed using the Western-Star Tropix chemiluminescent immunoblot detection system (Applied Biosciences) and hyperfilm ECLplus (Amersham-Pharmacia Biotech). No CnA bands were observed if blots were probed with the secondary antibody alone.

Confocal immunofluorescence. RASMCs were seeded at a density of 0.5 x 10⁴ cells/ml on poly-L-lysine-coated eight-chambered slides (BD Biosciences, Oxford, UK), grown for 24 h in DMEM containing 10% FBS, and growth arrested by a 48-h incubation in DMEM alone or a 72-h incubation in DMEM containing 0.1% FBS. The immunostaining procedure was carried out using a modified method (6). Briefly, freshly dispersed (see above) or cultured RASMCs were washed at room temperature with HBSS. All subsequent steps were carried out at 4°C unless otherwise stated. RASMCs were fixed with 4% formaldehyde in PBS and permeabilized with either 0.2% Triton X-100 in PBS for 20 min or 0.3% Nonidet P-40 and 1% glycerol in PBS for 10 min. Subsequently, cells were incubated with control blocking solution (2% BSA, 5% goat serum, and 0.1% Triton X-100 in PBS) for 1–2 h. Primary and secondary antibodies were prepared in blocking solution at the dilutions indicated. For double staining, the primary antibody was applied for either 1 h when anti-CnA (1:100), goat anti-NFATc1 (K-18, sc-1149, 1:100, Santa Cruz Biotechnology), or goat anti-NFATc3 (A-17, sc-23814, 1:200, Santa Cruz Biotechnology) antibodies were used or overnight for mouse monoclonal anti-NFATc3 (1:50) antibody (F-1, sc-8405, Santa Cruz Biotechnology). Secondary antibodies were always applied for 1 h at room temperature, which was goat anti-rabbit Alexa 488 (1:200, Molecular Probes, Leiden, The Netherlands) for the CnA isoform experiments and mouse anti-goat or goat anti-mouse IgG FITC-conjugated antibodies for NFATc1 and NFATc3 (1:250, Molecular Probes). To identify cellular nuclei, the fluorescent nucleic acid binding dye TO-PRO-3 (1:300, Molecular Probes) was added with the secondary antibody. For triple labeling, RASMCs were first probed overnight with anti-NFATc3 primary antibody followed by an incubation with goat anti-mouse IgG antibodies for NFATc3. RASMCs were then probed for CnA as described above except that the secondary antibody used was Texas red-conjugated goat anti-rabbit IgG antibody (Alexa 594, 1:250, Molecular Probes). After a final step of washing, slides were air dried and mounted with Vectashield (Vector Laboratories). Slides were viewed and analyzed at x60 magnification (water-immersion lens) using a Bio-Rad Radiance 2000 laser-scanning confocal Nikon TE1000 microscope (Hemel Hempstead, UK). Green fluorescence of FITC/Alexa 488 and blue fluorescence of TO-PRO-111 were measured at an excitation/emission wavelengths of 492/520 or 644/657 nm, respectively. No fluorescence was detected in the presence of either primary or secondary antibodies alone, confirming the specificity of immune staining. Images of RASMCs were taken at a focal plane taken from the middle of the cell using a z-stack of 10 images with 0.1 µm spacing. Nuclear localization was quantified using Bio-Rad LaserPix 4.0 software. Computer visualization of cyan was taken to mean positive localization of either CnA or NFAT within the nucleus or negative if a blue signal was observed. The percent nuclear localization refers to the ratio of the integrated cyan signal to the total blue signal in the unmerged image. In colocalization experiments, yellow reflects positive colocalization of both CnA and NFATc3, which was assessed by measuring the colocalization coefficient of the unmerged images of green (FITC/c3) with red (Texa red, CnA/).

NFATc3-CnA binding assays. Human NFATc3 cDNA (Genbank Accession No. BC001050) was mutated using PCR for in-frame ligation following the codon for amino acid 420 with glutathione-S-transferase (GST). The first 420 amino acids of NFATc3 contains the binding regions for CnA (14). A COOH-terminal GST-NFATc3 1–420 fusion protein was generated using the Gateway system (Invitrogen), bacterial expression, and purification with GSH-Sepharose. The NH₂-terminal fragments of CnA (Genbank Accession No. X57115) and CnA (Genbank Accession No. D90036) were generated using PCR for in-frame ligation following the codon for amino acid 350 (CnA350) or amino acid 360 (CnA360) with a hexa-His affinity tag. The NH₂-terminal catalytic domain of CnA has previously been shown to contain the NFAT binding region (9, 28). COOH-terminal His-CnA350 and His-CnA360 fusion proteins were generated using the Gateway system, bacterial expression, and purification with Ni-Sepharose. CnA350 and CnA360 binding to GST-NFATc3 was assayed by incubating 2 µg of each CnA protein with increasing amounts (0.5–12.5 µg) of GST-NFATc3 and 50 µl GSH-Sepharose in a total volume of 100 µl. The reaction [PBS (pH 7.4) and 0.1% Tween 20] was gently mixed at 4°C for 2 h and microfuged for 10 s at 1,000 g, and the supernatant was removed. Beads were washed three times with binding buffer, and, after the final wash, beads were boiled in 50 µl SDS-PAGE sample buffer. The beads were pelleted, and 30 µl of the supernatant were separated by SDS-PAGE (10% gels), followed by Western blotting onto nitrocellulose sheets. Blots were probed with anti-CnA or anti-CnA antibodies (Chemicon) at 1:5,000 dilutions, followed by horseradish peroxidase-conjugated secondary antibody (1:50,000, Chemicon). CnA protein bands were visualized with ECL Advance (Amersham), and images were collected using a charge-coupled device camera-based detection system (Epi Chem II, UVP Laboratory Products). The collected TIFF images were inverted and analyzed using Adobe Photoshop.

Cell proliferation. RASMCs were plated onto six-well plates at a density of 2 x 10⁴ cells/ml and grown in DMEM containing 10% FBS for 24 h. Prior to drug treatment, cells were growth arrested by incubating them in serum-free DMEM for 48 h. Following this, media were replaced with DMEM containing human PDGF-BB alone or in combination with CysA, cypermethrin, forskolin, adenosine cyclic 3',5'-(hydrogenphosphorothioate)triethylammonium (cAMPS-Sp), or the NFAT inhibitors A-285222 and inhibitor of NFAT-calcineurin association (INCA)-6. After 48 h, cells were counted in a blinded fashion using a hemocytometer or an automated cell counter (Sysmex, Kobe, Japan).

Statistical analysis. Values are given as means ± SE, and n indicates the numbers of cells or experiments. Statistical significance was assessed using Student's t-test or one-way ANOVA with Bonferroni post hoc test. P values of <0.05 were considered statistically significant.

Reagents. PDGF-BB, DTT, papain, BSA, and UTP were all obtained from Sigma-Aldrich, and CysA was from BioMol Research Laboratories. Forskolin and INCA-6 were from Merck Biosciences (Nottingham, UK), whereas cypermethrin, (S)-adenosine, and cAMPS-Sp were from Tocris Cookson (Bristol, UK). A-28522 (compound 19 in Ref. 7) was a gift from Abbott Laboratories (Abbott Park, IL). Stock solutions were made up in DMSO (CysA, forskolin, A-28522, and INCA-6), ethanol (cypermethrin), or distilled water. Aliquots were kept at –20°C, and, on the day of the experiment, solutions were diluted to give the desired concentration.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
CnA isoform expression in native RASM. Analysis of total RNA was performed using primers that specifically amplify CnA or CnA isoforms. With primers designed against different conserved regions in the catalytic A subunit, single bands were detected (Fig. 1, A and B) and shown to be CnA (153 and 506 bp) and CnA (129 bp) by sequence analysis of the PCR products (not shown). With the use of primers flanking known splice sites, three distinct bands were detected for CnA (Fig. 1B). The upper two products shown in Fig. 1B are predicted from full-length CnA and a splice variant previously identified with a 30-bp deletion (23, 31). The third product shown in Fig. 1B may correspond to novel splice variants that we have identified with a 95-bp deletion that may or may not contain the 30-bp deletion as well (unpublished observations). To assess protein expression, Western blot analysis was performed in native RASM homogenates, with purified CnA isoforms and rat brain samples used as positive controls (Fig. 1B). Protein samples were probed using Upstate Biotechnology anti-CnA antibodies raised against different COOH-terminal sequences in rat CnA and CnA. With the use of crude and cytosolic protein from the RASM and rat brain, bands of 57–60 kDa were observed that ran at a similar mobility to purified CnA and CnA protein. The density of these bands was much greater in the rat brain, indicating a higher level of CaN expression compared with smooth muscle. Both CnA isoforms were essentially undetectable in the particulate fraction from the RASM, whereas distinct bands were visible in this fraction from the brain. Similar results were observed in at least nine independent experiments. However, a faster migrating band of 53 kDa was detected with the anti-CnA antibody in the RASM but not in the brain (Fig. 1B, bottom). The specificity of the antibodies was confirmed by the lack of cross reactivity when purified CnA was probed with the anti-CnA antibody and CnA was probed with the anti-CnA antibody (Fig. 1B).

View larger version (52K): [in this window] [in a new window]   Fig. 1. RNA and Western blot analysis of calcineurin A (CnA) isoforms. A: RT-PCR using RNA isolated from rat aorta. Amplification of cDNA was performed using specific primers for CnA and CnA designed around conserved regions and known splice sites in CnA. Expected product sizes for CnA and CnA are shown with possible splice variant sizes given in parentheses for CnA (right). No PCR product was obtained if murine Moloney leukemia virus reverse transcriptase was omitted from the reverse transcription step (lane 4 at left). B: protein expression in different cellular fractions of CnA (top) and CnA (bottom) isoforms confirmed by Western blotting of rat brain and aortic homogenates (20 µg protein/lane). Controls are purified CnA proteins (0.125 µg protein/lane except where indicated).

Differential distribution of CnA and CnA in RASM.

View larger version (40K): [in this window] [in a new window]   Fig. 2. Differential cellular distribution of CnA and CnA isoforms in rat aortic smooth muscle (RASM) cells (RASMCs). A and B: immunofluorescence images showing the distribution of CnA and CnA isoforms (green) in freshly dispersed (A) and growth-arrested cultured (B) RASMCs costained with TO-PRO-3 (blue). Scale bar = 10 µm in this and subsequent figures. C: summary of the percentage of each CnA isoform localized within the nucleus of cells. Numbers above each bar refer to the numbers of cells. ***P < 0.001.

View larger version (49K): [in this window] [in a new window]   Fig. 3. PDGF-BB or cyclosporin A (CysA) do not affect localization of CnA. A: RASMCs immunostained with CnA antibody under control conditions (left) or after a 30-min treatment of cells with PDGF-BB (10 ng/ml; right). B: summary of the percentage of nuclear accumulation of CnA in the absence (control) or presence of either PDGF-BB or CysA (30 min). Numbers within each bar refer to the numbers of cells.

View larger version (39K): [in this window] [in a new window]   Fig. 4. PDGF-BB and UTP induce nuclear accumulation of CnA in cultured RASMCs. A: relative nuclear accumulation of CnA fluorescence in RASMCs after 15, 30, and 60 min of treatment with PDGF-BB (10 ng/ml). B: immunofluorescent images of cells stained with CnA under control conditions and after a 30-min incubation with either 10 ng/ml PDGF-BB (left) or 10 µmol/l UTP (right) shown in the absence and presence of either CysA (1–10 µM) or forskolin (FSK; 1 µM). C and D: summarized data showing percentages of nuclear localization following various treatments. Numbers within the bars refer to the numbers of cells. *P < 0.05 or ***P < 0.001.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Nuclear factor of activated T cells (NFAT) inhibitors block PDGF-BB-induced nuclear accumulation of CnA. A: immunofluorescent images of RASMCs stained with CnA in the absence (left) and presence (right) of PDGF-BB with and without either A-285222 (A-285; 10 µM) or inhibitor of NFAT-calcineurin association 6 (INCA-6; 3 µM) as indicated. The NFAT inhibitors were given 30 min prior to treatment with PDGF-BB. C: summarized data showing percentages of nuclear localization following various treatments. Numbers within the bars refer to the numbers of cells. ***P < 0.001 compared with control.

Effect of NFAT inhibitors on nuclear translocation of CnA. We assessed the role of two novel inhibitors of calcineurin-NFAT signaling on PDGF-BB-induced translocation of CnA. The first agent used was A-285222, a bis-trifluoromethylpyrazole belonging to a novel class of immunosuppressive agents that inhibit nuclear import of NFAT and cytokine production in T cells (7, 39). The second NFAT inhibitor used was INCA-6, a VIVIT-like compound that binds to calcineurin with high affinity and specifically disrupts its interaction with NFAT, blocking both dephosphorylation and nuclear export in T cells (29). Both compounds, however, can be distinguished from calcineurin inhibitors like CysA since they do not inhibit phosphatase activity directly or prevent the dephosphorylation of other well-known calcineurin substrates like the RII subunit of PKA (29, 39). In such experiments, we found that A-285222 (10 µM) and INCA-6 (3 µM) had little effect on the nuclear distribution of CnA in growth-arrested cells, although these agents fully inhibited its nuclear accumulation induced by PDGF-BB (Fig. 5).

Cellular translocation of NFATc3 but not NFATc1 with PDGF-BB. Experiments were performed to determine which NFAT isoform might translocate with CnA. We chose to investigate NFATc1 and NFATc3, since CysA-sensitive nuclear accumulation of these isoforms has been reported with receptor activation in smooth muscle (11, 35, 44). In unstimulated cells, cytoplasmic staining of both NFATc1 and NFATc3 was evident, although NFATc1 staining was most intense around the perinuclear region (Fig. 6). Some basal nuclear staining was also observed with NFAT antibodies, although accumulation was significantly higher (P < 0.001) for NFATc3 than for NFATc1 (Fig. 6C). Moreover, nuclear accumulation of NFATc3, but not NFATc1, was substantially increased in the presence of PDGF-BB (Fig. 6B), and this was prevented by prior treatment with 1 µM CysA (Fig. 7A). In experiments where cells were simultaneously stained with NFATc3 and CnA antibodies, colocalization of these proteins was extremely high in the nucleus and immediately outside the nucleus (Fig. 7B), with 86.7 ± 1.6% of nuclear and 45.2 ± 10.0% of cytosolic CnA staining colocalizing with NFATc3 in the presence of PDGF-BB (n = 47 cells). Interestingly, cytosolic colocalization was much higher in control cells (91.4 ± 0.07%, n = 23), suggesting that PDGF-BB also caused translocation of NFATc3 that was independent of CnA movement. Although strong nuclear staining was consistently observed for CnA, overall this was associated with significantly less NFATc3 colocalization (Fig. 7C) and was not altered by PDGF-BB, averaging 27.6 ± 1.1% (n = 27) in control cells versus 27.9 ± 0.9% (n = 18) with PDGF-BB.

View larger version (46K): [in this window] [in a new window]   Fig. 6. NFATc3, but not NFATc1, translocates to the nucleus in the presence of PDGF-BB. A and B: immunofluorescent images of RASMCs stained with NFATc1 (A) or NFATc3 (B) under control conditions and after a 30-min incubation with 10 ng/ml PDGF-BB. C: summarized data showing percentages of nuclear localization. Numbers within the bars refer to the numbers of cells. ***P < 0.001.

View larger version (92K): [in this window] [in a new window]   Fig. 7. Nuclear localization of NFATc3 is dependent on calcineurin. Immunofluorescent images of RASMCs stained with NFATc3 after a 30-min application of 10 ng/ml PDGF-BB are shown. Cells were pretreated for 1 h with 1 µM CysA (A) or were concurrently stained with TO-PRO-3 and either CnA (B) or CnA (C). Yellow staining in the merged images represents nuclear colocalization of NFATc3 with CnA.

Binding of CnA and CnA to a GST-NFATc3 fusion protein.

View larger version (26K): [in this window] [in a new window]   Fig. 8. Unequal binding of CnA and CnA to NFATc3. GSH-Sepharose beads containing glutathione-S-transferase (GST)-NFATc3 1–420 were incubated with CnA350 or CnA360. Bound CnA proteins were detected by 10% SDS-PAGE and immunoblot (IB) analysis with anti-CnA (left) or anti-CnA (right) antibodies. As a negative control, 10 µg GST was added alone with the primary antibodies.

View larger version (9K): [in this window] [in a new window]   Fig. 9. Calcineurin and NFAT inhibitors as well as cAMP-elevating agents oppose the proliferative effects of PDGF-BB. A: growth-arrested RASMCs were stimulated for 48 h with PDGF-BB (10 ng/ml) in the absence [control (Con)] and presence of CysA (1 µM), cypermethrin (Cyper; 100 nM), FSK (1 µM), or cAMPS-Sp (50 µM). B: growth-arrested RASMCs were treated with DMEM containing INCA-6 (3 µM) or A-285222 (10 µM) either alone or in combination with PDGF-BB. Numbers within the bars refer to the numbers of cells. ***P < 0.001 compared with unstimulated cells and #P = 0.001 compared to PDGF alone (by ANOVA with correction for multiple comparisons).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Western blot experiments revealed that both CnA isoforms are expressed mainly in the cytosolic fraction of RASM with little evidence for membrane localization. This contrasts with our results in the brain, where the distribution was equally split between cytosolic and particulate fractions. The latter agrees well with a previous study (19) showing 58% of total CnA and 47% of total CnA protein is associated with the particulate fraction in the rat cerebrum and other studies (30, 34) showing a high localization of calcineurin to the cytoskeleton. Interestingly, Western blots of RASM lysates show two distinct bands that migrate at 60 and 54 kDa but only the higher band in the brain. Most nucleotide sequences of known CnA splice variants predict proteins of 61–57 kDa (25, 30). However, we have identified a number of novel CnA splice variants including one with a 95-bp deletion (928–1022 bp in sequence D90036 [GenBank] ; unpublished observations) that theoretically could account for the 54-kDa immunoreactive band. Likewise, two GenBank sequences predicted by automated computational analysis of annotated genomic sequences (XM-508174 and XM-857434) would also give rise to 54- to 55-kDa proteins. We cannot at this stage rule out the possibility that abnormal migration might result from some unexplained artifact or from posttranslational modification of CnA, although, so far, this has only been reported for the calcineurin B subunit (30). Furthermore, proteolysis is unlikely to explain the existence of the lower band, since the known tryptic digests for the COOH-terminal antibody we used (Upstate Biotechnology) would give rise to bands ranging from 9 to 16 kDa, with the bulk of the catalytic subunit being resistant to proteolysis (18, 25).

In immunohistochemical experiments, we found clear differences in the subcellular distribution of CnA isoforms, where the -subunit was expressed in the cytosol and the nucleus and the -subunit was essentially cytosolic. Early work reported a similar localization of the - and -subunits in the rat brain, with both isoforms being highly enriched in cytoplasmic fractions and only a small amount (<20%) located in the nuclei (19). In subsequent studies (24, 40), distinct subcellular distribution was reported in rat and mouse neuronal and retinal cell types, with the -isoform being predominately nuclear and the -isoform being predominately cytosolic. What gives rise to the distinct isoform-specific subcellular distribution is unknown but may result from binding to specific A-kinase anchoring proteins or direct tethering to subcellular structures (25). In this regard, it is worth noting that the NH₂- and COOH-termini of CnA isoforms share very little homology so that these domains may be involved in the spatial targeting of calcineurin (25, 30).

It is well known that a rise in intracellular Ca²⁺ causes calcineurin to translocate from the cytosol to the nucleus by forming a complex with NFAT (14, 34). Our results suggest that CnA is unlikely to be the major stimulus facilitating NFAT translocation, since this isoform displayed a predominantly nuclear localization in cultured RASMCs and PDGF-BB failed to increase the nuclear accumulation of CnA. In contrast, CnA exhibited essentially no nuclear localization until cells were exposed to PDGF-BB or UTP. CysA abrogated this nuclear accumulation, as did NFAT inhibitors, further confirming the activation of calcineurin. A previous Western blotting study (21) in cultured RASMCs has reported increases in nuclear calcineurin protein with angiotensin II over a similar time frame, although the specific CnA isoform involved was not invesitigated. The observed equipotency of both UTP and PDGF at inducing CnA translocation in RASM is, however, different from those in previous studies. Thus, UTP along with endothelin were the most potent agonists at inducing the nuclear accumulation of NFATc3 in the cerebral artery, whereas PDGF-BB had no effect in this tissue. Such tissue specificity in agonist-mediated NFAT nuclear translocation may relate to either different receptor densities between tissues and/or in cell culture or receptors coupling to different second messenger pathways. Although PDGF and UTP act via receptor tyrosine kinases (2) and purinergic receptors coupled to various phospholipases (15), respectively, both mitogens elevate intracellular Ca²⁺ in the rat aorta (1, 3) and, hence, would be predicted to activate calcineurin.

The identity, distribution, and function of NFAT isoforms in smooth muscle are still controversial. For example, NFATc3 was the dominant isoform found in native smooth muscle and primary cultured RASMCs, with expression presiding predominately in the cytosol (11, 22, 35). In contrast, NFATc4 appears the only isoform significantly expressed in cultured human pulmonary arterial smooth muscle cells (43). However, other studies (3, 44) in cultured RASMCs have detected significant expression of NFATc1 and NFATc2, but little NFATc3, with NFATc1 showing both cytosolic and nuclear accumulation (3, 44). In our study, we found NFATc1 to be predominately cytosolic, whereas NFATc3 displayed a small but significant amount (20%) of nuclear accumulation in growth-arrested cultured RASMCs. Moreover, nuclear accumulation of CnA was 20% higher in cultured compared with freshly isolated cells, also consistent with our finding of some nuclear colocalization of CnA (27%) with NFATc3. The predominant nuclear localization previously reported for NFATc2 and the lack of NFATc4 expression in RASM (3, 44) suggest that NFATc2 may indeed reside in the nucleus with CnA. The functional significance of this remains unclear, but the presence of significant nuclear CnA in freshly isolated RASMCs may advocate their involvement in maintaining a differentiated smooth muscle phenotype. Moreover, nuclear export of CnA was not promoted by CysA, suggesting that cellular targeting of CnA is regulated by different import and export mechanisms to CnA.

The lack of NFATc1 and CnA translocation and the observed strong nuclear colocalization of CnA with NFATc3 following treatment with PDGF-BB suggest that NFATc3 is a critical downstream effector of calcineurin-mediated stimulation in response to vascular mitogens. Interestingly, both NFATc3 and CnA had a similar uneven distribution within the nucleus and were concentrated together in punctate dots (Fig. 6). The significance of this remains unknown, although punctate intranuclear distribution with different NFAT isoforms has been reported (33, 37) and recent data have shown tiny clusters of calcineurin associated with nucleoli of developing rat skeletal muscle cells (38). Furthermore, increases in nuclear NFATc3 levels after 30 min have been described in ileal and portal vein smooth muscle for PDGF-BB (8, 35). Our data do not concur with Western blot data in RASMCs, where the same agonist caused NAFTc1 translocation but had no effect on NFATc3 distribution (44). The reason for this discrepancy remains unclear but could relate to the different time points at which translocation was measured in this study (30 min) compared with 2 h by Yellaturu and colleagues. However, they did report a threefold increase in NFATc3 protein levels following a 2- to 8-h treatment with PDGF-BB, suggesting a role for this isoform in cell proliferation to this mitogen (44). However, our results are reminiscent of previous findings where receptor stimulation appears associated with selective movement of only one NFAT isoform (17, 44). Thus, NFATs appear under the control of different signaling pathways and are probably differently activated due to localized Ca²⁺ signaling and/or spatial location (11, 14). Indeed, specific cellular functions for different NFATs appear an emerging theme for this family of transcription factors (14, 34, 41). One intriguing finding is that there appears to be movement of NFATc3 into the nucleus independent of CnA. Since continued dephosphorylation appears necessary for NFAT to be retained in the nucleus (14), this suggests the involvement of another phosphatase or the existence of a novel splice variant of CnA. Alternatively, CnA ( or ) may promote the movement of NFAT through its indirect activation of protein phosphatase 1 (25). Indeed, phosphatase activity in RASMCs shows a high level of okadaic-sensitive (55%) activity (unpublished observations). Clearly, future experiments will be required to distinguish between these possibilities. However, NFAT can be activated in T cells through a CysA-resistant pathway, although the exact nature of the pathway remains unclear (10).

The structurally unrelated calcineurin inhibitors CysA and cypermethrin both completely inhibited PDGF-induced cell proliferation, as did the NFAT inhibitors A-285222 and INCA-6, suggesting that both calcineurin and NFAT are crucial for the stimulation of growth through this receptor pathway. Likewise, in cultured RASMCs, CysA inhibited NFAT-mediated transcription induced by PDGF (3) as well as thymidine incorporation and cell proliferation (44). Although calcineurin has many cellular substrates that may be involved in mediating the response to growth factors, our data with NFAT inhibitors suggest that calcineurin dephosphorylation of NFAT alone is sufficient to drive the proliferative response to PDGF. This contrasts with data in fibroblasts showing that PDGF-induced growth was blocked by CysA and FK506, but not by cypermethrin, suggesting the involvement of a calcineurin-independent pathway, at least in this cell type (27).

The fact that both CnA and NFATc3 isoforms were responsive to mitogenic signals and heavily colocalized in the nucleus makes it conceivable that they promote cellular growth and proliferation. Moreover, CnA350 showed only weak binding to the COOH-terminal GST-NFATc3 fusion protein, whereas CnA360 appeared to bind strongly. While it is unclear if such preferential binding occurs in vivo, such findings are supported by functional data elsewhere. For example, hearts from CnA^–/– or NFATc3^–/– mice failed to undergo hypertrophy in response to aortic banding or infusion of angiotensin II or isoproterenol (4, 41). Thus, both CnA and NFATc3 work via a common signaling pathway. Moreover, cardiac hypertrophy is associated with increased message and protein levels for CnA, whereas those of CnA remain unaltered (36). Similarly, we found CnA staining to increase and CnA staining to decease in cultured compared with freshly isolated RASMCs. However, our results do not rule out the possibility that CnA is involved in PDGF-mediated effects on vascular growth. Indeed, CnA activity may increase through changes in nuclear Ca²⁺ or this subunit may regulate ion channel function to enhance Ca²⁺ entry (13, 20, 32, 42) and thus promote growth indirectly. Future experiments involving subtype selective inhibition of CnA as well as NFAT isoforms will be required to determine their respective roles.

In our studies, the PKA activators forskolin and cAMPS-Sp also inhibited cell growth and CnA translocation stimulated by PDGF-BB. It is tempting to speculate that the cAMP/PKA pathway opposes cell growth by preventing CnA-induced NFATc3 translocation. NFAT must be dephosphorylated by calcineurin for nuclear translocation to occur. However, the maintenance of NFAT in the nucleus requires continuous calcineurin signaling to counteract nuclear export kinases like PKA, glycogen synthase kinase 3, and JNK, which phosphorylate NFAT at multiple sites and oppose translocation (14). Thus, a reduction in cell growth may be brought about by PKA-induced phosphorylation of NFAT directly (33) or indirectly by inhibition of calcineurin activity through lowering intracellular Ca²⁺ (e.g., increased movement of Ca²⁺ into internal pools) (2), which are both effects leading to retention of NFAT in the cytosol. Such a mechanism may represent a novel pathway whereby cAMP-elevating agents regulate vascular smooth muscle cell proliferation.

In conclusion, we showed that not only do CnA isoforms have differential cellular distribution but that after stimulation of aortic smooth muscle cells with PDGF, CnA heavily colocalized with NFATc3 in the nucleus. Inhibition of calcineurin or NFAT activity prevented cell growth, and, thus, we postulate that CnA, through its interaction with NFATc3, is an important initiator of vascular smooth muscle proliferation, although at this stage we cannot rule out a contribution from CnA.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was funded by Medical Research Council (MRC) UK Grant G117/440 (to L. H. Clapp) and by National Institute of Neurological Disorders and Stroke Grant NS-36318 (to B. A. Perrino). L. H. Clapp was an MRC Senior Fellow in Basic Science.

	ACKNOWLEDGMENTS

 
We thank Dr Kelly (University College London) and Dr. William Hatton (Reno, NV) for the technical assistance in confocal microscopy and Maggie Elorza (Reno, NV) for obtaining and culturing the aortic smooth muscle cells in some experiments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. H. Clapp, BHF Laboratories, Rayne Bldg., Dept. of Medicine, Univ. College London, 5 University St., London WC1E 6JF, UK (e-mail: l.clapp{at}ucl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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