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    • The presence of different LO splice variants

    2024-01-19

    The presence of different 5-LO splice variants was first demonstrated in human Pracinostat tumour cells and in dimethyl sulfoxide-differentiated HL-60 cells [40] and a correlation between the 5-LO splicing pattern and the malignancy of the brain tumours was found. Later, other splice variants were identified in TGFβ/calcitriol differentiated and undifferentiated MM6 cells containing premature termination codons (PTC) which seem to be involved in the regulation of 5-LO expression by nonsense mediated mRNA decay (NMD) [41]. Several 5-LO isoforms were detected in polymorphonuclear leukocytes (PMNL) and in human myeloid cell lines. One of them, the 5-LO∆13 isoform, where exon 13 is deleted, does not contain a PTC and thus might represent a 5-LO protein isoform. 5-LO∆13 is catalytically inactive and decreased the activity of 5-LO-WT after co-expression in transiently transfected HEK293 cells and it was suggested that it might act as endogenous inhibitor [42]. Recently, two other novel isoforms were identified, i.e. 5-LO∆4 (lacking exon 4) and 5-LOp12 (lacking part of exon 12) which also are no NMD substrates [43,44].
    Material and methods
    Results
    Discussion LTs are lipid mediators that are part of the innate immune system and play a fundamental role in many inflammatory diseases and in several types of cancer. 5-LO catalyzes the first two steps in the generation of LTs derived from AA. Like with the majority of the other human genes, alternatively spliced transcripts of 5-LO were observed in humans. In 1992 5-LO splice variants were reported in human brain tumors but their sequence was not determined at that time [40]. Recently, several 5-LO splice variants were identified in different B cell lines and primary leukocytes [42,43]. Most of them contain a PTC and are thus NMD targets, so that translation of these mRNAs into proteins is unlikely. However, some alternatively spliced 5-LO transcripts do not contain PTCs, and thus, might encode for potential 5-LO protein isoforms [41,42]. The 5-LO∆13 isoform was identified as a stable and catalytically inactive protein in eukaryotic cells [42]. The isoform 5-LO∆4 was found in B cell lines and in primary B and T cells and does not show 5-LO activity either [44]. In this study, we investigated newly identified 5-LO protein isoforms (5-LO∆4 and 5-LOp12) and compared them with 5-LO∆13. We screened different B and T cell lines as well as primary B and T cells and myeloid cells for their expression of 5-LO-WT and 5-LO isoform mRNAs. On mRNA level, the isoforms 5-LO∆13 and 5-LOp12 were present in different B cell lines and primary B and T cells as well as in myeloid cells. In T cell lines, we detected 5-LO-WT and 5-LO∆13 in Jurkat cells whereas MOLT-4 cells contain 5-LO-WT, but no alternatively spliced transcripts were found. Interestingly, in freshly purified T cells, the full length transcript and both alternative isoforms were detected. For many years, T cells were considered as 5-LO negative but recent results support the existence of 5-LO expression in primary T cells [20]. In a previous study, we could show that the mRNA level of 5-LO in freshly isolated T cells rapidly decreases over time, when taken into cell culture [22]. This might be the reason why we were not able to find any isoform transcripts in the T cell line MOLT-4 and only a small amount of 5-LO-WT and 5-LOΔ13 in cultured Jurkat cells. To evaluate a putative involvement of the isoforms in pathophysiological events, we examined primary monocytes of patients with RA or sepsis. The cells express 5-LO-WT, 5-LO∆13 and 5-LOp12 mRNA, whereas we were not able to detect the isoform 5-LO∆4. Expression of 5-LO-WT and especially of the alternative isoforms is strongly upregulated in both patient groups compared to healthy controls. This is in agreement with previous publications which show that 5-LO mRNA and protein expression is increased in inflammatory diseases and different cancer types [55–58]. Interestingly, especially the expression of the isoforms was strongly upregulated in monocytes from patients with RA or sepsis. Particularly, 5-LO∆13 was induced to a 1:1 ratio with 5-LO-WT in samples of sepsis patients. These findings suggest that the alternative isoforms might have a function as regulators of host defence reactions or inflammatory diseases which is independent of 5-LO catalytic activity.