Abstract
The enzyme 15-lipoxygenase (15-LO) participates in the dioxygenation of polyenoic fatty acids. This activity leads to the degradation of mitochondrial membranes during reticulocyte differentiation, the production of pro- and anti-inflammatory mediators by a variety of cell types, and the oxidation of lipids in atherosclerotic lesions. The cytokines, IL-4 and IL-13, are reported to induce the expression of 15-LO in human peripheral blood monocytes. In this report we explore the signaling mechanisms involved in the IL-13-mediated induction of 15-LO expression. First we demonstrate that the delayed induction of 15-LO requires continuous stimulation of monocytes for a minimum period of 12 h. We also found that tyrosine kinase inhibitors blocked the induction of 15-LO in a dose-dependent manner. By immunoprecipitation and antiphosphotyrosine blotting experiments, IL-13 was shown to induce tyrosine phosphorylation of Jak2 and Tyk2, but not Jak1 or Jak3, within 5 min of treatment in human monocytes. To investigate whether the early induction of tyrosine phosphorylation of both Jak2 and Tyk2 was ultimately involved in 15-LO expression, we generated antisense oligodeoxyribonucleotides (ODNs) against Tyk2 and Jak2. We employed a cationic lipid-mediated delivery technique to transfect the monocytes and found that both antisense ODNs inhibited expression of their target proteins by 75-85%. The treatments were specific and did not affect the expression of each other. Furthermore, the antisense ODNs to Jak2 and Tyk2 both inhibited the induction of expression of 15-LO in monocytes treated with IL-13. Parallel experiments with sense ODNs to Jak2 and Tyk2 did not affect their protein levels or the induction of 15-LO by IL-13, and down-regulation of Jak1 also did not affect expression of 15-LO. Our results suggest the novel finding that IL-13 can induce tyrosine phosphorylation of both Jak2 and Tyk2 in primary human monocytes. This occurs as an early and essential signal transduction event for the IL-13-mediated induction of 15-LO expression. These data represent the first characterization of upstream kinases involved in the induced expression of 15-LO.
Highlights
Products of 15-LO-mediated oxidation of linoleic acid have been identified in human and rabbit atherosclerotic lesions and likely result from the action of 15-LO on lipoprotein lipids [6, 14]
Jak2 and Tyk2 Are Required for IL-13 Induction of 15-LO Expression—Because we already established that IL-13 activated and induced tyrosine phosphorylation of Jak2 and Tyk2, we investigated the necessity of each of these kinases for the IL-13-induced signaling pathways leading to expression of 15-LO in monocytes
The level of expression of IL-4R was up-regulated in response to IL-4 in T-cells, IL-4 had no such effect on the IL-4R expression in monocytes [24]
Summary
Products of 15-LO-mediated oxidation of linoleic acid have been identified in human and rabbit atherosclerotic lesions and likely result from the action of 15-LO on lipoprotein lipids [6, 14]. Attempts to study the molecular signaling mechanisms using monocytic cell lines have not been successful, as the cell lines failed to show similar induction of 15-LO when stimulated with interleukins [22]. Several reports support the view that interleukins exert their effects through the mediation of different combinations of receptor subunits in different cell lines and activate different combinations of Jaks and signal transducers and activators of transcription (STATs) [23,24,25,26]. IL-13 Signaling Mechanisms and 15-LO Expression higher affinity toward IL-13 (KD ϭ 20 –90 pM) having a molecular mass of 45–50 kDa [29], termed IL-13R␣Ј. Different groups have utilized antisense oligodeoxyribonucleotides (ODNs) to inhibit the endogenous level of expression of different enzymes, including the Jaks and STATs, in various cells and cell lines [41,42,43]. Our attempts to inhibit the activity and expression of classical protein kinase C [44] and cytosolic phospholipase A2 [45] using antisense ODNs met with an even higher level of inhibition (up to 80%) as this approach worked more efficiently in nonproliferating, rapidly pinocytosing monocytes
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