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

Abstract

BackgroundThe 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.ResultsIn 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.ConclusionsWe suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.

HDAC2 promotes the EMT of colorectal cancer cells and via the modular scaffold function of ENSG00000274093.1

“…And HDAC2/HDAC1 complex plays a bridge role in the binding process of EZH2 and Snail 17 . Moreover, small molecules could play a significant role in the process of the function of HDACs 18‐20 . Therefore, we evaluated whether RNA was needful for HDAC2/HDAC1 and EZH2 binding.…”

“…17 Moreover, small molecules could play a significant role in the process of the function of HDACs. [18][19][20] Therefore, we evaluated whether RNA was needful for HDAC2/HDAC1 and EZH2 binding. Co-Immunoprecipitation experiments showed that the binding of HDAC2/ HDAC1 and EZH2 decreased after RNase treatment in HCT116 cells ( Figure 3A).…”

Differential expression of PMCA2 mRNA isoforms in a cohort of Spanish patients with breast tumor types

“…The detection of high levels of PMCA2 mRNA is consistent with the data of previous studies demonstrating that PMCA2 overexpression results in the reduced transcription activity of nuclear factor of activated T cells (NFAT) in breast cancer ( 2 , 43 ). Furthermore, previous studies conducted in PC12 cells indicated that NFAT inhibition is involved in the regulation of the PMCA2× splice variant ( 44 ). These results highlighted the complex association that exists between PMCA2 and NFAT signaling in breast cancer.…”

The plethora of PMCA isoforms: Alternative splicing and differential expression

“…With respect to the discussion before concerning PMCA1a expression it is interesting that Zacharias & Strehler not only noticed that this change of the PMCA2 splice variants was Ca 2+ -dependent but also that the phosphorylation of probably a splicing regulating factor must be involved in this process [59] as mentioned before for the regulation of PMCA1a expression. In this context it is interesting that Kosiorek et al recently reported on the regulation of PMCA2 alternative splicing in PC12 cells through the cooperation of NFAT1/3 and HDAC4 [60]. The authors suggested that this influence on the isoform composition may be important for altered dopamine secretion by PC12 cells [60].…”

“…In this context it is interesting that Kosiorek et al recently reported on the regulation of PMCA2 alternative splicing in PC12 cells through the cooperation of NFAT1/3 and HDAC4 [60]. The authors suggested that this influence on the isoform composition may be important for altered dopamine secretion by PC12 cells [60].…”