PW01-013 – Localization of alternative pyrin isoforms
© Turanli et al; licensee BioMed Central Ltd. 2013
Published: 8 November 2013
The importance of MEFV gene protein, Pyrin/Marenostrin (P/M) in the inflammatory pathway is well established. P/M is expressed in neutrophils, eosinophils, monocytes, dendritic cells and synovial fibroblasts. There are many MEFV transcripts which are generated by alternative splicing events including deleted exons 2,3,4,5,7 and 8 in several combinations or individually. Some of these transcripts (2a, 2d, 8ext, 2d/8ext and 2d/9ext) were shown to be expressed as protein isoforms in leukocytes. A previous study carried out by our group has shown that exon 2 deleted form (d2) in leukocyte samples of FMF patients is expressed more than 400 fold compared to healthy control samples (p=0.026).
Based on the hypothesis that different localizations and functions for full length and MEFV alternatively spliced transcripts, this study aimed to determine the localization differences between full-length P/M and P/M-d2 protein isoforms in neutrophil-like cells in vitro.
Two GFP-tagged plasmids which are are pCMV6-AC-GFP + MEFV-fl (MEFV-fl-GFP) and pCMV6-AC-GFP + MEFV-d2 (MEFV-d2-GFP) were transfected to HL-60 (Human promyelocytic leukemia cells) cell lines and examined via confocal microscopy. Subsequently, six-day incubation with 1.75% DMSO was performed to differentiate HL-60 cells to neutrophil-like cells. These cells were also transfected with same plasmids and proteins were observed through confocal microscopy technique.
Transfection studies showed that MEFV-fl-GFP was cytoplasmic and MEFV-d2-GFP was nuclear in HL-60 cell line. On the other hand, both MEFV-fl-GFP and MEFV-d2-GFP were localized in cytoplasm of neutrophil-like cells.
In previous studies, cellular localization of P/M-fl and P/M-d2 was investigated in several cell lines through using transfection methods or P/M antibody. Transfection studies showed that full-length P/M was generally cytoplasmic and 2Δ isoform was the only isoform which can enter nucleus but may also localize in cytoplasm. However localization studies using P/M antibodies, which cannot currently distinguish between different isoforms, showed that although native P/M consists of predominantly full-length type, protein was also observed in the nucleus of neutrophils. Our localization results of P/M-fl and P/M-d2 in HL-60 cells were compatible with literature, they were observed in the cytoplasm and nucleus, respectively. On the other hand, both P/M-d2 and P/M-fl isoforms were found to be localized only in cytoplasm not in nucleus in the neutrophil-like cells. These findings had led us to suggest that post-transcriptional modifications for P/M-d2 that may occur during cell differentiation or possibly through inflammation such that its natural localization in the nucleus may point to its role in the inflammation maybe like a transcription factor.
Disclosure of interest
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.