Control of ALOX5 expression in monocytic cells using a synthetic riboswitch

Abstract

The human 5-lipoxygenase (5-LOX), which is encoded by the arachidonate 5-lipoxygenase (ALOX5) gene, has its canonical function in leukotriene (LT) biosynthesis, which controls inflammatory and allergic responses. Besides oxylipin formation from polyunsaturated fatty acids, 5-LOX has several noncanonical functions. It acts as transcriptional regulator in the nucleus but also interacts with Dicer and modulates microRNA expression and processing.

In this study, we employed a tetracycline riboswitch-controlled cassette-exon system to conditionally control ALOX5 expression in the monocytic leukemic cell line MonoMac6. Synthetic riboswitches are gaining increasing interest as a means of controlling transgene expression, with applications in functional genomics and potential therapeutic strategies. We designed an artificial ALOX5 gene that contains two cassette exons with premature termination codons (PTCs), thus only being expressed when both synthetic exons are skipped. The switchable ALOX5 gene was transduced into MonoMac6 5-LOX knock-out (KO) cells, thereby enabling the tetracycline-dependent re-expression of 5-LOX proteins. The newly established cell line was characterized in terms of tetracycline dose dependency and switching kinetics. Induction of ALOX5 exerted the non-canonical 5-LOX effects on prostaglandin-endoperoxide synthase 2 (PTGS2) and L-kynureninase (KYNU) gene expression. This allowed us to demonstrate the outstanding advantages of a riboswitch-controlled system in terms of time dependency and gene function. The novel MonoMac6 cell line now provides a perfect tool for further research into the non-canonical functions of 5-LOX.