Calcineurin/NFAT Signaling Represses Genes <i>Vamp1</i> and <i>Vamp2</i> via PMCA-Dependent Mechanism during Dopamine Secretion by Pheochromocytoma Cells

<div><p>Background</p><p>Plasma membrane Ca²⁺-ATPases (PMCA) extrude Ca²⁺ ions out of the cell and contribute to generation of calcium oscillations. Calcium signaling is crucial for transcriptional regulation of dopamine secretion by neuroendocrine PC12 cells. Low resting [Ca²⁺]_c in PC12 cells is maintained mainly by two Ca²⁺-ATPases, PMCA2 and PMCA3. Recently, we found that Ca²⁺ dependent phosphatase calcineurin was excessively activated under conditions of experimental downregulation of PMCA2 or PMCA3. Thus, the aim of this study was to explain if, via modulation of the Ca²⁺/calcineurin-dependent nuclear factor of activated T cells (NFAT) pathway, PMCA2 and PMCA3 affect intracellular signaling in pheochromocytoma/neuronal cells/PC12 cells. Secondly, we tested whether this might influence dopamine secretion by PC12 cells.</p><p>Results</p><p>PMCA2- and PMCA3-deficient cells displayed profound decrease in dopamine secretion accompanied by a permanent increase in [Ca²⁺]_c. Reduction in secretion might result from changes in NFAT signaling, following altered PMCA pattern. Consequently, activation of NFAT1 and NFAT3 transcription factors was observed in PMCA2- or PMCA3-deficient cells. Furthermore, chromatin immunoprecipitation assay indicated that NFATs could be involved in repression of <i>Vamp</i> genes encoding vesicle associated membrane proteins (VAMP).</p><p>Conclusions</p><p>PMCA2 and PMCA3 are crucial for dopamine secretion in PC12 cells. Reduction in PMCA2 or PMCA3 led to calcium-dependent activation of calcineurin/NFAT signaling and, in consequence, to repression of the <i>Vamp</i> gene and deterioration of the SNARE complex formation in PC12 cells.</p></div

Additional file 1: of Whole-body clearing, staining and screening of calcium deposits in the mdx mouse model of Duchenne muscular dystrophy

Figure S1. Implementation of apparatus for macroscopic imaging using light-sheet illumination. (a) General view. (b) Side view. (c) Front view. (1) camera, (2) stepper motor, (3) z-axis line module (here light microscope body, Zeiss), (4) base holder, (5) glass container, (6) line lasers, (7) metal block, (8) laser power supply. Figure S2. Perfusion-based CUBIC cleared rat organs stained with propidium iodide. Bright field images of whole rat organs (a) intestine, (b) kidney, (c) heart, (d) cerebellum and (e) spleen. Single squares in all panels - 5 × 5 mm. This figure has been adapted from the original article “Optimized perfusion-based CUBIC protocol for the efficient whole-body clearing and imaging of rat organs” by P. Matryba et al., J Biophotonics 2017, doi./10.1002/jbio.201700248 . Reproduced with permission 4358370973288. Figure S3. Comparison between optical and standard histopathology sectioning of Alizarin red S stained mdx mouse muscles. (a) Representative optical sections acquired during imaging with custom-made LSFM. Control animals present no staining-positive tissue. (b) Representative histopathology 4 μm sections observed with low and (c) high magnification. It has to be noted that histopathology images stay in line with optical sectioning, presenting petite deposits in triceps brachii and similar amount of deposits between quadriceps femoris and spinalis pars lumborum. White scale bar, 1 mm, black scale bar, 200 μm. Figure S4. Quantitative volume analysis of calcified deposits in mdx mouse. (a) Triceps brachii muscles from left and right side of the animals were compared. Differences between left vs. right side presented as % mineralization of muscle volume were not statistically significant (Wilcoxon test (W = 0.0, number of pairs = 3), p > 0.999). (b) Analysis of volume of muscles replaced by calcific deposits. Percent of mineralization in triceps brachii, quadriceps femoris and spinalis pars lumborum muscles was significantly different (Kruskal-Wallis test (K-W statistic = 5.6), p = 0.05, n = 3 per each muscle group). Figure S5. Perfusion-based CUBIC clearing does not alter microstructure of muscle. Perfusion-based CUBIC cleared triceps brachii and quadriceps femoris muscles of mdx and C57BL/10 control animals were subjected to standard hematoxylin and eosin histological staining. Low (10×) and high (20×) magnification representative images present microstructure preservation. It has to be noted that centrally positioned nuclei, a prominent feature of dystrophy, is maintained during mdx clearing. Scale bar, 100 μm. (ZIP 12014 kb

NFAT1 and NFAT3 binding to the promoters of genes encoding proteins involved in dopamine secretion in PMCA2- or PMCA3-deficient PC12 cells.

<p>The binding of NFATs to the promoter region was analyzed by chromatin immunoprecipitation-qPCR as described in methods. The following genes were analyzed: <i>Snap25</i>, <i>Vamp1</i>, <i>Vamp2</i>, <i>Stx1a</i>. Data are shown as the fold change (2^−ΔΔCt ±SEM) of the promoter occupancy by NFAT1 (<b>A</b>) and by NFAT3 (<b>B</b>). Wilcoxon test for ΔC_t from qPCR data was performed for comparison of the fold of change in NFAT promoter occupancy in control cells (C, standardized to y = 1) with PMCA2- (_2) or PMCA3-deficient cells (_3) (n = 4). *P≤0.05, **P≤0.01. Bars: gray – PMCA2-deficient cells, open – PMCA3-deficient.</p

Characterization of PMCA2- or PMCA3-deficient PC12 cells.

<p>Expression of PMCA2 (<i>Atp21b2</i>) and PMCA3 (<i>Atp2b2</i>) in PC12 was determined by RT-PCR (<b>A</b>). The RT-PCR results were validated by qPCR (<b>B</b>). PMCA2 and PMCA3 protein content was analyzed by immunoblotting, standardized to β-actin level (<b>C</b>). The immunoblotted bands of PMCA2 or PMCA3 were quantified densitometrically, standardized to β-actin and normalized to control cells, expressed as y = 1 (<b>D</b>). Bars represent mean values ± SEM. Student's t-test was used for comparison of control cells with PMCA2- or PMCA3-reduced cells (n = 6). Wilcoxon test was used for ΔC_t from qPCR data (n = 3) for comparison of control cells (ΔC_t expressed as y = 0) with PMCA2- or PMCA3-reduced cells (n = 3). *P≤0.05, **P≤0.01. Bars and symbols: filled – control cells (C), gray – PMCA2-deficient cells (_2), open – PMCA3-deficient cells (_3).</p

Effect of NFAT inhibition on dopamine secretion by PMCA2- or PMCA3-deficient PC12 cells.

<p>The amount of secreted dopamine was measured by RP-HPLC in samples obtained after 1, 5, 10, 15, 20, 30 min of stimulation with 59 mM KCl for non-treated cells (n = 6) (<b>A</b>) and for cells treated with NFAT inhibitor, 1 µM 11R-VIVIT (n = 3) (<b>B</b>). The amount of released dopamine at resting state (5 mM KCl) was compared between cells non-treated and treated with 1 µM 11R-VIVIT for 48 h (n>3) (<b>C</b>). Dopamine secreted after 5 min of stimulation was compared between cells non-treated and treated with 1 µM 11R-VIVIT for 48 h (n>3) using two-way ANOVA test (<b>D</b>). Bars represent mean values ± SEM. Student's t-test was used for comparison of control cells with PMCA2- or with PMCA3-reduced cells (A–C). *P≤0.05, **P≤0.01. Bars and symbols: filled – control cells (C), gray – PMCA2-deficient cells (_2), open – PMCA3-deficient cells (_3).</p

Effects of PMCA2 or PMCA3 deficiency in PC12 cells on [Ca²⁺]_c under resting and depolarizing conditions.

<p>Measurements of [Ca²⁺]_c were performed with Fura-2 AM. [Ca²⁺]_c was measured under resting conditions (5 mM KCl) in the presence of sodium ions or not (+Na⁺ or −Na⁺), 10 µM KB-R7943 (NCX inhibitor) or 20 µM nifedipine (VDCC inhibitor) (<b>A</b>). The effect of 10 µM KB-R7943 (<b>B</b>) or 20 µM nifedipine (<b>C</b>) on Ca²⁺]_c under resting and stimulatory conditions. The statistical analysis was performed for n>10 measurements and calcium entry is expressed as Δ of [Ca²⁺]_c values between depolarizing (59 mM KCl) and resting (5 mM KCl) conditions. Bars represent mean values ±SEM. Student's t-test was used for comparison of control cells with PMCA2- or PMCA3-reduced cells. *P≤0.05, **P≤0.01, n>10. Bars and symbols: filled – control cells (C), gray – PMCA2-deficient cells (_2), open – PMCA3-deficient cells (_3).</p

Dopamine distribution in vesicular fractions from PMCA2- or PMCA3-deficient PC12 cells.

<p>Dopamine content was determined by RP-HPLC in fractions obtained after subcellular fractionation from cells in resting conditions (<b>A</b>) or upon stimulation for 10 min with 59 mM KCl (<b>B</b>). Bars represent mean values±SEM. Student's t-test was used for comparison of the intracellular dopamine content in control cells with PMCA2- or PMCA3-reduced cells (n = 3). *P≤0.05, **P≤0.01. Bars and symbols: filled – control cells (C), gray – PMCA2-deficient cells (_2), open – PMCA3-deficient cells (_3).</p

Primers designed for <i>Rattus norvegicus</i> genome using GenScript real-time qPCR design tool.

a<p>primers used for qPCR.</p>b<p>primers used for RT-PCR.</p>c<p>primers used for chromatin immunoprecipitation assay.</p

Primary antibodies raised against <i>Rattus norvergicus</i>.

<p>Primary antibodies raised against <i>Rattus norvergicus</i>.</p

Effects of NFAT inhibition on expression of selected genes encoding proteins involved in secretion of dopamine in PMCA2- or PMCA3-deficient PC12 cells.

<p>RNA was isolated from non-treated cells or cells treated with 1 µM 11R-VIVIT for 48 h. Expression of genes encoding components of SNARE complex was examined by qPCR: <i>Snap25</i> (<b>A</b>), <i>Stx1a</i> (<b>B</b>), <i>Vamp1</i> (<b>C</b>), <i>Vamp2</i> (<b>D</b>). Bars represent mean values of ΔΔC_t±SEM. Wilcoxon test for ΔC_t from qPCR data was used for comparison of control cells (ΔC_t standardized to y = 1) with PMCA2- or PMCA3-deficient cells (n = 3). *P≤0.05, **P≤0.01. Bars: filled – control, gray – PMCA2-deficient, open – PMCA3-deficient.</p