Abstract
BackgroundAn underlying state of inflammation is thought to be an important cause of cardiovascular disease. Among cells involved in the early steps of atherosclerosis, monocyte-derived dendritic cells (Mo-DCs) respond to inflammatory stimuli, including platelet-activating factor (PAF), by the induction of various cytokines, such as interleukin 6 (IL-6). PAF is a potent phospholipid mediator involved in both the onset and progression of atherosclerosis. It mediates its effects by binding to its cognate G-protein coupled receptor, PAFR. Activation of PAFR-induced signaling pathways is tightly coordinated to ensure specific cell responses.ResultsHere, we report that PAF stimulated the phosphatase activity of both the 45 and 48 kDa isoforms of the protein tyrosine phosphatase non-receptor type 2 (PTPN2). However, we found that only the 48 kDa PTPN2 isoform has a role in PAFR-induced signal transduction, leading to activation of the IL-6 promoter. In luciferase reporter assays, expression of the 48 kDa, but not the 45 kDa, PTPN2 isoform increased human IL-6 (hIL-6) promoter activity by 40% after PAF stimulation of HEK-293 cells, stably transfected with PAFR (HEK-PAFR). Our results suggest that the differential localization of the PTPN2 isoforms and the differences in PAF-induced phosphatase activation may contribute to the divergent modulation of PAF-induced IL-6 promoter activation. The involvement of PTPN2 in PAF-induced IL-6 expression was confirmed in immature Mo-DCs (iMo-DCs), using siRNAs targeting the two isoforms of PTPN2, where siRNAs against the 48 kDa PTPN2 significantly inhibited PAF-stimulated IL-6 mRNA expression. Pharmacological inhibition of several signaling pathways suggested a role for PTPN2 in early signaling events. Results obtained by Western blot confirmed that PTPN2 increased the activation of the PI3K/Akt pathway via the modulation of protein kinase D (PKD) activity. WT PKD expression counteracted the effect of PTPN2 on PAF-induced IL-6 promoter transactivation and phosphorylation of Akt. Using siRNAs targeting the individual isoforms of PTPN2, we confirmed that these pathways were also active in iMo-DCs.ConclusionTaken together, our data suggest that PTPN2, in an isoform-specific manner, could be involved in the positive regulation of PI3K/Akt activation, via the modulation of PKD activity, allowing for the maximal induction of PAF-stimulated IL-6 mRNA expression.
Highlights
Chronic inflammation is characterized by the continuous activation of signaling pathways involved in cell survival and promotion of leukocyte recruitment, associated with angiogenesis and reactive-oxygen species (ROS) production, all linked to the progression of atherosclerosis [1, 2]
To investigate regulation of PAFinduced inter‐ leukin 6 (IL-6) expression, we concentrated on protein tyrosine phosphatase non-receptor type 2 (PTPN2), in order to determine whether one, or both, isoforms of PTPN2 were involved in regulation of IL-6 promoter activation induced by platelet-activating factor (PAF)
PAF increased enzymatic activity of both PTPN2 isoforms but only the 48 kDa PTPN2 upregulated PAF-induced IL-6 promoter transactivation, and this increase was mediated by a protein kinase D (PKD)/PI3K/Akt pathway, where PKD could be the primary target of PTPN2
Summary
Chronic inflammation is characterized by the continuous activation of signaling pathways involved in cell survival and promotion of leukocyte recruitment, associated with angiogenesis and reactive-oxygen species (ROS) production, all linked to the progression of atherosclerosis [1, 2]. PAFR is expressed in a wide assortment of cells involved in atherosclerosis, from leucocytes such as neutrophils, macrophages, dendritic cells and monocytes to smooth muscles cells and endothelial cells [6, 7]. Only the 45 kDa isoform sees its localization changed during stimulation: it can shuttle out of the nucleus during cell stress to interact with its cytoplasmic substrates [31, 36] The fact that their localization is tightly regulated, combined with the differences in regulatory mechanisms and substrate specificity, supports the idea that each isoform has a different cellular function, making them an interesting target for study in pathways activated by PAFR. Among cells involved in the early steps of atherosclerosis, monocyte-derived dendritic cells (Mo-DCs) respond to inflammatory stimuli, including platelet-activating factor (PAF), by the induction of various cytokines, such as inter‐ leukin 6 (IL-6). Activation of PAFR-induced signaling pathways is tightly coordinated to ensure specific cell responses
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