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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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Alternative splicing of PMCA in PMCA2- or PMCA3-deficient PC12 cells upon NFAT inhibition.Alternative splicing pattern at sites A and C of mRNA transcripts of PMCAs: Atp21b1 (PMCA1) (A), Atp21b2 (PMCA2) (B), Atp21b3 (PMCA3) (C), Atp21b4 (PMCA4) (D) 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 Gapdh and normalized to control cells, expressed as y = 1, both for non-treated (E) and 11R-VIVIT-treated cells (F). 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.
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pone-0099118-g003: Alternative splicing of PMCA in PMCA2- or PMCA3-deficient PC12 cells upon NFAT inhibition.Alternative splicing pattern at sites A and C of mRNA transcripts of PMCAs: Atp21b1 (PMCA1) (A), Atp21b2 (PMCA2) (B), Atp21b3 (PMCA3) (C), Atp21b4 (PMCA4) (D) 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 Gapdh and normalized to control cells, expressed as y = 1, both for non-treated (E) and 11R-VIVIT-treated cells (F). 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.

Mentions: Diversity of PMCAs is not only due to the fact that these calcium pumps are encoded by four separate genes but mostly due to alternative splicing of mRNA. Thus, following the above findings suggesting increased NFAT activity and contribution to PMCAs expression profiling, a detailed analysis of the alternative splicing pattern of PMCA transcripts was performed. In order to obtain full information on the composition of PMCA splice variants the PCR method with primers flanking the appropriate splicing sites was applied. PMCA splicing variant composition was tested in control cells and in cells with a reduced content of neurospecific isoforms PMCA2 or PMCA3. This study was performed in order to verify whether a reduction in the content of neurospecific PMCAs might be compensated by other PMCA splicing variants. It revealed that downregulation of PMCA2 or PMCA3 did not statistically significantly influence the expression of PMCA1 (Atp2b1), and that the PMCA1x/b variant was abundantly and predominately expressed in PC12 cells (Fig. 3A, left). These outcomes were in accordance with the known data on PC12 cells [62], [63]. The expression of Atp2b1 transcript was in agreement with the protein level of PMCA1, detected with a specific antibody recognizing the PMCA1b form (Fig. S1A). In the case of Atp2b2 expression pattern, the PMCA2x/b variant predominated in all cell lines and its level was elevated in a compensatory mechanism in cells with a reduced amount of PMCA3 (Fig. 3B, left). This finding was verified at a protein level with a specific antibody recognizing the PMCA2b form (Fig. S1A). Regarding the expression pattern of Atp2b3, the most abundant variant was PMCA3x/a and in cells with a reduced amount of PMCA2 a compensatory increase in PMCA3x/a level was observed (Fig. 3C, left). PMCA3x/a transcript expression also correlated with the content of a protein recognized by specific anti-PMCA3a antibody (Fig. S1A). PMCA4x/b expression increased significantly in both PMCA2- and PMCA3-deficient cells (Fig. 3D, left). This was confirmed at a protein level by (Fig. S1A).


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)

Alternative splicing of PMCA in PMCA2- or PMCA3-deficient PC12 cells upon NFAT inhibition.Alternative splicing pattern at sites A and C of mRNA transcripts of PMCAs: Atp21b1 (PMCA1) (A), Atp21b2 (PMCA2) (B), Atp21b3 (PMCA3) (C), Atp21b4 (PMCA4) (D) 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 Gapdh and normalized to control cells, expressed as y = 1, both for non-treated (E) and 11R-VIVIT-treated cells (F). 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.
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Related In: Results  -  Collection

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pone-0099118-g003: Alternative splicing of PMCA in PMCA2- or PMCA3-deficient PC12 cells upon NFAT inhibition.Alternative splicing pattern at sites A and C of mRNA transcripts of PMCAs: Atp21b1 (PMCA1) (A), Atp21b2 (PMCA2) (B), Atp21b3 (PMCA3) (C), Atp21b4 (PMCA4) (D) 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 Gapdh and normalized to control cells, expressed as y = 1, both for non-treated (E) and 11R-VIVIT-treated cells (F). 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.
Mentions: Diversity of PMCAs is not only due to the fact that these calcium pumps are encoded by four separate genes but mostly due to alternative splicing of mRNA. Thus, following the above findings suggesting increased NFAT activity and contribution to PMCAs expression profiling, a detailed analysis of the alternative splicing pattern of PMCA transcripts was performed. In order to obtain full information on the composition of PMCA splice variants the PCR method with primers flanking the appropriate splicing sites was applied. PMCA splicing variant composition was tested in control cells and in cells with a reduced content of neurospecific isoforms PMCA2 or PMCA3. This study was performed in order to verify whether a reduction in the content of neurospecific PMCAs might be compensated by other PMCA splicing variants. It revealed that downregulation of PMCA2 or PMCA3 did not statistically significantly influence the expression of PMCA1 (Atp2b1), and that the PMCA1x/b variant was abundantly and predominately expressed in PC12 cells (Fig. 3A, left). These outcomes were in accordance with the known data on PC12 cells [62], [63]. The expression of Atp2b1 transcript was in agreement with the protein level of PMCA1, detected with a specific antibody recognizing the PMCA1b form (Fig. S1A). In the case of Atp2b2 expression pattern, the PMCA2x/b variant predominated in all cell lines and its level was elevated in a compensatory mechanism in cells with a reduced amount of PMCA3 (Fig. 3B, left). This finding was verified at a protein level with a specific antibody recognizing the PMCA2b form (Fig. S1A). Regarding the expression pattern of Atp2b3, the most abundant variant was PMCA3x/a and in cells with a reduced amount of PMCA2 a compensatory increase in PMCA3x/a level was observed (Fig. 3C, left). PMCA3x/a transcript expression also correlated with the content of a protein recognized by specific anti-PMCA3a antibody (Fig. S1A). PMCA4x/b expression increased significantly in both PMCA2- and PMCA3-deficient cells (Fig. 3D, left). This was confirmed at a protein level by (Fig. S1A).

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