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NFAT1 and NFAT3 cooperate with HDAC4 during regulation of alternative splicing of PMCA isoforms in PC12 cells.

Kosiorek M, Podszywalow-Bartnicka P, Zylinska L, Pikula S - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland; Department of Neurodegenerative Disorders, Laboratory of Neurogenetics, Mossakowski Medical Research Centre PAS, Warsaw, Poland.

ABSTRACT

Background: 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 (Atp2b1, Atp2b2, Atp2b3, Atp2b4), whose transcript products undergo alternative splicing giving almost 30 variants.

Results: 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.

Conclusions: We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.

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Related in: MedlinePlus

NFAT activation in PC12 cells with reduce PMCAs content.PC12 cells were transfected with plasmids encoding firefly luciferase under NFAT-dependent promoter (pGL3-NFAT-luc) and reference plasmids with Renilla 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 Renilla 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 (A). 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). Representative immunoblots of nuclear content of dephosphorylated NFAT1 (C) and NFAT3 (D) are demonstrated. Symbols correspond to: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3).
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pone-0099118-g001: NFAT activation in PC12 cells with reduce PMCAs content.PC12 cells were transfected with plasmids encoding firefly luciferase under NFAT-dependent promoter (pGL3-NFAT-luc) and reference plasmids with Renilla 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 Renilla 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 (A). 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). Representative immunoblots of nuclear content of dephosphorylated NFAT1 (C) and NFAT3 (D) are demonstrated. Symbols correspond to: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3).

Mentions: Luciferase reporter assays performed using constructs containing NFAT-dependent promoter revealed that NFAT transcriptional activity was significantly higher in the cells with a reduced content of PMCA2 or PMCA3. Moreover, this increase in NFAT activation was comparable to the cells overexpressing NFAT (transfected with constructs pGL3-NFAT-luc-+/+NFAT) (Fig. 1A). In addition, a statistically significant increase in the protein content of dephosphorylated NFAT1 and NFAT3 in the nuclei has been detected in PMCA2- and PMCA3-deficient cells, especially under resting conditions. This was demonstrated by densitometrical measurements of the immunoblots of NFAT1 and NFAT3, standardized to the content of nuclear poly (ADP-ribose) polymerase (PARP) and normalized to control cells (y = 1) (Fig. 1B). Increased level of dephosphorylated NFAT1 and NFAT3 in the nuclei in PMCA-deficient cells under resting conditions and in all cell types upon plasma membrane depolarization was shown by representative immunoblots (Fig. 1C and Fig. 1D). These findings concerning NFAT activity reinforce the hypothesis on the existence of a NFAT-PMCA regulatory loop.


NFAT1 and NFAT3 cooperate with HDAC4 during regulation of alternative splicing of PMCA isoforms in PC12 cells.

Kosiorek M, Podszywalow-Bartnicka P, Zylinska L, Pikula S - PLoS ONE (2014)

NFAT activation in PC12 cells with reduce PMCAs content.PC12 cells were transfected with plasmids encoding firefly luciferase under NFAT-dependent promoter (pGL3-NFAT-luc) and reference plasmids with Renilla 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 Renilla 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 (A). 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). Representative immunoblots of nuclear content of dephosphorylated NFAT1 (C) and NFAT3 (D) are demonstrated. Symbols correspond to: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3).
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pone-0099118-g001: NFAT activation in PC12 cells with reduce PMCAs content.PC12 cells were transfected with plasmids encoding firefly luciferase under NFAT-dependent promoter (pGL3-NFAT-luc) and reference plasmids with Renilla 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 Renilla 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 (A). 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). Representative immunoblots of nuclear content of dephosphorylated NFAT1 (C) and NFAT3 (D) are demonstrated. Symbols correspond to: control cells (C), PMCA2-deficient cells (_2), PMCA3-deficient cells (_3).
Mentions: Luciferase reporter assays performed using constructs containing NFAT-dependent promoter revealed that NFAT transcriptional activity was significantly higher in the cells with a reduced content of PMCA2 or PMCA3. Moreover, this increase in NFAT activation was comparable to the cells overexpressing NFAT (transfected with constructs pGL3-NFAT-luc-+/+NFAT) (Fig. 1A). In addition, a statistically significant increase in the protein content of dephosphorylated NFAT1 and NFAT3 in the nuclei has been detected in PMCA2- and PMCA3-deficient cells, especially under resting conditions. This was demonstrated by densitometrical measurements of the immunoblots of NFAT1 and NFAT3, standardized to the content of nuclear poly (ADP-ribose) polymerase (PARP) and normalized to control cells (y = 1) (Fig. 1B). Increased level of dephosphorylated NFAT1 and NFAT3 in the nuclei in PMCA-deficient cells under resting conditions and in all cell types upon plasma membrane depolarization was shown by representative immunoblots (Fig. 1C and Fig. 1D). These findings concerning NFAT activity reinforce the hypothesis on the existence of a NFAT-PMCA regulatory loop.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland; Department of Neurodegenerative Disorders, Laboratory of Neurogenetics, Mossakowski Medical Research Centre PAS, Warsaw, Poland.

ABSTRACT

Background: 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 (Atp2b1, Atp2b2, Atp2b3, Atp2b4), whose transcript products undergo alternative splicing giving almost 30 variants.

Results: 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.

Conclusions: We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.

Show MeSH
Related in: MedlinePlus