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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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The interaction of NFAT1 and NFAT3 with HDAC4 isoform in PMCA2- or PMCA3-deficient PC12 cells.RIPA-total cellular extracts were subjected to immunoblotting to verify HDAC4 protein content and served as inputs of immunoprecipitation (A). The cellular extracts (inputs) were incubated with protein A/G agarose beads and with anti-NFAT1 antibody (B) or with anti-NFAT3 antibody (C) and the obtained immunoprecipitates were subjected to immunoblotting for HDAC4. All immunoblots and immunoprecipitates were measured densitometrically and expressed as % of control cells (D). 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).
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pone-0099118-g004: The interaction of NFAT1 and NFAT3 with HDAC4 isoform in PMCA2- or PMCA3-deficient PC12 cells.RIPA-total cellular extracts were subjected to immunoblotting to verify HDAC4 protein content and served as inputs of immunoprecipitation (A). The cellular extracts (inputs) were incubated with protein A/G agarose beads and with anti-NFAT1 antibody (B) or with anti-NFAT3 antibody (C) and the obtained immunoprecipitates were subjected to immunoblotting for HDAC4. All immunoblots and immunoprecipitates were measured densitometrically and expressed as % of control cells (D). 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).

Mentions: As suggested above NFAT might work alone or in complexes with other proteins [64]. NFATs were found to cooperate with HDACs, where NFAT1c mediated HDAC-dependent transcriptional repression [57]. Moreover, both NFATs and HDACs were found to be involved in regulation of alternative splicing [41], [42], [49]. To check whether NFAT cooperates with HDACs in PC12 cells with different PMCA status we first analyzed the presence of various HDACs in total cellular lysates obtained from these cells. This analysis revealed that HDAC4 was predominantly expressed in all examined PC12 cell lines (Fig. 4A). Densitometry analysis showed that in the PMCA2- and PMCA3-reduced cell lines the amount of HDAC4 was significantly higher than in control cells (Fig. 4D). We have tested as well HDAC1, HDAC2, HDAC3, HDAC5 and HDAC6 isoforms, however due to weak signal and very low or residual protein level of these isoforms, and thus, due to low importance these data are not shown in this paper. To study the putative interaction between NFAT1 or NFAT3 and HDAC4 the co-immunoprecipitation assays were performed. These experiments suggested that NFAT might interact with the HDAC4 isoform, both in the case of NFAT1 (ubiquitous) (Fig. 4B) and NFAT3 (neurospecific) (Fig. 4C). The content of immuneprecipitates was similar in all cell lines, as verified densitometrically and expressed as percentage of control cells (Fig. 4D).


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)

The interaction of NFAT1 and NFAT3 with HDAC4 isoform in PMCA2- or PMCA3-deficient PC12 cells.RIPA-total cellular extracts were subjected to immunoblotting to verify HDAC4 protein content and served as inputs of immunoprecipitation (A). The cellular extracts (inputs) were incubated with protein A/G agarose beads and with anti-NFAT1 antibody (B) or with anti-NFAT3 antibody (C) and the obtained immunoprecipitates were subjected to immunoblotting for HDAC4. All immunoblots and immunoprecipitates were measured densitometrically and expressed as % of control cells (D). 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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048221&req=5

pone-0099118-g004: The interaction of NFAT1 and NFAT3 with HDAC4 isoform in PMCA2- or PMCA3-deficient PC12 cells.RIPA-total cellular extracts were subjected to immunoblotting to verify HDAC4 protein content and served as inputs of immunoprecipitation (A). The cellular extracts (inputs) were incubated with protein A/G agarose beads and with anti-NFAT1 antibody (B) or with anti-NFAT3 antibody (C) and the obtained immunoprecipitates were subjected to immunoblotting for HDAC4. All immunoblots and immunoprecipitates were measured densitometrically and expressed as % of control cells (D). 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).
Mentions: As suggested above NFAT might work alone or in complexes with other proteins [64]. NFATs were found to cooperate with HDACs, where NFAT1c mediated HDAC-dependent transcriptional repression [57]. Moreover, both NFATs and HDACs were found to be involved in regulation of alternative splicing [41], [42], [49]. To check whether NFAT cooperates with HDACs in PC12 cells with different PMCA status we first analyzed the presence of various HDACs in total cellular lysates obtained from these cells. This analysis revealed that HDAC4 was predominantly expressed in all examined PC12 cell lines (Fig. 4A). Densitometry analysis showed that in the PMCA2- and PMCA3-reduced cell lines the amount of HDAC4 was significantly higher than in control cells (Fig. 4D). We have tested as well HDAC1, HDAC2, HDAC3, HDAC5 and HDAC6 isoforms, however due to weak signal and very low or residual protein level of these isoforms, and thus, due to low importance these data are not shown in this paper. To study the putative interaction between NFAT1 or NFAT3 and HDAC4 the co-immunoprecipitation assays were performed. These experiments suggested that NFAT might interact with the HDAC4 isoform, both in the case of NFAT1 (ubiquitous) (Fig. 4B) and NFAT3 (neurospecific) (Fig. 4C). The content of immuneprecipitates was similar in all cell lines, as verified densitometrically and expressed as percentage of control cells (Fig. 4D).

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