Alternative splicing of PMCA in PMCA2- or PMCA3-deficient PC12 cells upon NFAT inhibition.

<p>Alternative splicing pattern at sites A and C of mRNA transcripts of PMCAs: <i>Atp21b1</i> (PMCA1) (<b>A</b>), <i>Atp21b2</i> (PMCA2) (<b>B</b>), <i>Atp21b3</i> (PMCA3) (<b>C</b>), <i>Atp21b4</i> (PMCA4) (<b>D</b>) was determined by RT-PCR in non-treated and 1 µM 11R-VIVIT-treated PC12 cells. RT-PCR product bands were quantified densitometrically, standardized to <i>Gapdh</i> and normalized to control cells, expressed as y = 1, both for non-treated (<b>E</b>) and 11R-VIVIT-treated cells (<b>F</b>). Student’s t-test was used for comparison of control cells with PMCA2- or PMCA3-reduced cells (n = 3). Bars represent mean values ± SEM. *P≤0.05. Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3). Black arrows indicate the PCR product bands for PMCA2 site A and PMCA3 site C. White asterisks on the images of gels indicate the PCR product bands generated by alternative splicing that underwent a significant change upon NFAT inhibition with 11R-VIVIT.</p

NFAT activation in PC12 cells with reduce PMCAs content.

<p>PC12 cells were transfected with plasmids encoding firefly luciferase under NFAT-dependent promoter (pGL3-NFAT-luc) and reference plasmids with <i>Renilla</i> luciferase (pRL-SV40). Negative controls were wild type PC12 cells transfected with promoterless pGL3-luc plasmids and positive controls were wild type PC12 transfected with plasmids overexpressing NFAT together with the pGL3-NFAT-luc (pGL3-NFAT-luc-NFAT+/+). NFAT activity was determined with a luciferase reporter dual assay (Thermo Scientific Pierce) and showed as the ratio of the luminescence signals derived from Firefly and <i>Renilla</i> luciferases. Bars represent mean values ± SEM. Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3) and pGL3-NFAT-luc+/+NFAT – wild type cells overexpressing NFAT. Student’s t-test was used for comparison of control cells with PMCA2- or PMCA3-deficient cells. *P≤0.05, n = 5 (<b>A</b>). Nuclear content of dephosphorylated NFAT1 and NFAT3 was analyzed by immunoblotting. Protein bands were quantified densitometrically, standardized to PARP (nuclear marker) and normalized to control cells, expressed as y = 1. Bars represent mean values ± SEM. Student’s t-test was used for comparison of control cells with PMCA2- or PMCA3-deficient cells. *P≤0.05, n = 6 (<b>B</b>). Representative immunoblots of nuclear content of dephosphorylated NFAT1 (<b>C</b>) and NFAT3 (<b>D</b>) are demonstrated. Symbols correspond to: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3).</p

HDAC4-NFAT1/NFAT3 complex contribution to regulation of PMCA alternative splicing in PC12 cells.

<p>HDAC<b>/</b>NFAT involvement in regulation of alternative splicing of PMCA transcripts was analyzed by qPCR-chromatin immunoprecipitation. The analysis was performed for the splicing at site A and at site for four PMCA isoforms and the PCR products were migrated in 1% agarose gels (<b>A</b>). The qPCR data, were expressed as fold of change (2−ΔΔC) calculated from the difference: ΔCT of output (immunoprecipitated DNA with HDAC<b>/</b>NFATs) – ΔCT of input (total DNA) and statistics were calculated according to nonparametric paired Wilcoxon signed rank test at 95% confidence, where PMCA2-deficient cells (_2) or PMCA3-deficient cells (_3) were compared to control cells assigned to y = 1 value (<b>B</b>). Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3), n = 3.</p

NFAT1 and NFAT3 Cooperate with HDAC4 during Regulation of Alternative Splicing of PMCA Isoforms in PC12 Cells

<div><p>Background</p><p>The bulk of human genes undergo alternative splicing (AS) upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca²⁺-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca²⁺-ATPases (PMCAs), abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells). PMCAs are encoded by four genes (<i>Atp2b1</i>, <i>Atp2b2</i>, <i>Atp2b3, Atp2b4),</i> whose transcript products undergo alternative splicing giving almost 30 variants.</p><p>Results</p><p>In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT) and histone deacetylases (HDACs). Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation.</p><p>Conclusions</p><p>We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.</p></div

Primers for PMCA isoforms expression analysis.

<p>Primers for PMCA isoforms expression analysis.</p

Primers for PMCA alternative splicing analysis.

<p>Primers for PMCA alternative splicing analysis.</p

PMCA isoforms expression pattern versus transcriptional control by NFAT in PC12 cells.

<p>Expression of genes encoding PMCA isoforms: <i>Atp21b1</i> (PMCA1), <i>Atp21b2</i> (PMCA2), <i>Atp21b3</i> (PMCA3), <i>Atp21b4a</i> (PMCA4a), <i>Atp21b4b</i> (PMCA4b) was determined by qPCR for PC12 cells non-treated (<b>A</b>), and treated with NFAT inhibitor (1 µM 11R-VIVIT) (<b>B</b>). Bars represent mean values ± SEM. Wilcoxon test was used for ΔC_t from qPCR data (n = 3) for comparison between control cells (standardized to y = 0) and PMCA2- or PMCA3-deficient cells non-treated with 11R-VIVIT (n = 3). Two-way ANOVA test was used for comparison between non-treated and 11R-VIVIT treated cells. *P≤0.05, **P≤0.01.</p

The interaction of NFAT1 and NFAT3 with HDAC4 isoform in PMCA2- or PMCA3-deficient PC12 cells.

<p>RIPA-total cellular extracts were subjected to immunoblotting to verify HDAC4 protein content and served as inputs of immunoprecipitation (<b>A</b>). The cellular extracts (inputs) were incubated with protein A/G agarose beads and with anti-NFAT1 antibody (<b>B</b>) or with anti-NFAT3 antibody (<b>C</b>) and the obtained immunoprecipitates were subjected to immunoblotting for HDAC4. All immunoblots and immunoprecipitates were measured densitometrically and expressed as % of control cells (<b>D</b>). Student’s t-test was used for comparison of control cells with PMCA2- or PMCA3- deficient cells. *P≤0.05, n = 3. Symbols: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3).</p

Growth of osteosarcoma Saos-2 and 143B cells treated with ascorbic acid and β-glycerophosphate.

<p>(A) Analysis of cell proliferation rate by flow cytometry. Cells were stained with Alexa Fluor 670. Representative plots of fluorescence measurements at the time points after the 1^st and 2^nd division of Saos-2 or 2^nd and 4^th division of 143B cell line. Histograms: dashed black line - time 0, continuous gray line - control, continuous black line - AA/B-GP treatment. (B) Determination of cell cycle progression in osteosarcoma cells. Representative plots showing analysis at the time points after the 1^st and 2^nd division of Saos-2 or 2^nd and 4^th division of 143B cell line. Percentages of cells in the G0/G1, S, and G2–M phases are presented for each experimental group. Bars represent: open-G0/G1 phases, filled -S phase, gray – G2/M phase. Error bars indicate means ± SEM; n = 3, *p≤0.05 by Student’s t-test.</p

Migration of osteosarcoma Saos-2 and 143B cells in the presence of ascorbic acid and β-glycerophosphate.

<p>Uniform scratches were created in confluent cell cultures, which were treated with AA/B-GP over a period of 7 days. The upper panels show representative images of wound closure in control osteosarcoma cells and cells stimulated with AA/B-GP, 12 h after scratching. Wound borders are marked with dashed lines. The lower panels represent the quantification of wound closure in % 4 h, 8 h and 12 h after injury (time 0). Time lapse imaging of wound closure was captured using a Leica AF7000 microscope Live Imaging System at 10× objective. Bars represent: open – 4 h after injury; dotted – 8 h after injury; grey stripped – 12 h after injury. The mean value for three individual experiments ± SEM is shown. Error bars indicate means ± SEM; n = 6, *p≤0.05 by Student’s t-test.</p