Syk Plays a Critical Role in the Expression and Activation of IRAK1 in LPS-Treated Macrophages

Abstract

To address how interleukin-1 receptor-associated kinase 1 (IRAK1) is controlled by other enzymes activated by toll-like receptor (TLR) 4, we investigated the possibility that spleen tyrosine kinase (Syk), a protein tyrosine kinase that is activated at an earlier stage during TLR4 activation, plays a central role in regulating the functional activation of IRAK1. Indeed, we found that overexpression of myeloid differentiation primary response gene 88 (MyD88), an adaptor molecule that drives TLR signaling, induced IRAK1 expression and that piceatannol, a Syk inhibitor, successfully suppressed the MyD88-dependent upregulation of IRAK1 under LPS treatment conditions. Interestingly, in Syk-knockout RAW264.7 cells, IRAK1 activity was almost completely blocked after LPS treatment, while providing a Syk-recovery gene to the knockout cells successfully restored IRAK1 expression. According to our measurements of IRAK1 mRNA levels, the transcriptional upregulation of IRAK1 was induced by LPS treatment between 4 and 60 min, and this can be suppressed in Syk knockout cells, providing an effect similar that that seen under piceatannol treatment. The overexpression of Syk reverses this effect and leads to a significantly higher IRAK1 mRNA level. Collectively, our results strongly suggest that Syk plays a critical role in regulating both the activity and transcriptional level of IRAK1.

Figure 1

Piceatannol impacts IRAK1 activation during LPS treatment in RAW264.7 cells. (a, b, and c) Protein levels of IRAK1, IRAK4, MyD88, and β-actin as determined by lysate immunoblotting prepared from LPS- (1 μg/ml) treated RAW264.7 cells or MyD88-overexpressed HEK293 cells. (d and e) HEK293 cells cotransfected with NF-κB-Luc (1 μg/ml) and β-gal (as a transfection control) plasmid constructs were treated with piceatannol (40 μM) in the presence or absence of an adaptor molecule (MyD88) or IRAK1 for 12 h. Luciferase activity was determined via luminometry. All data are expressed as the mean ± SD of three independent experimental replicates. ^∗∗p < 0.01 compared to the control group.

Figure 2

Syk impacts the regulation of IRAK1 activation in LPS-treated RAW264.7 cells. (a and c) Total or phospho-protein levels of IRAK1, Syk, Myc, TAK1, and β-actin as determined by lysate immunoblotting prepared from LPS- (1 μg/ml) treated RAW264.7-WT, RAW264.7-Syk^−/− cells, or RAW264.7-Syk^−/− cells transfected for 24 h with Myc-tagged Syk (5 × 10⁶ cells/ml). (b) Syk mRNA levels from RAW264.7-WT or RAW264.7-Syk^−/− cells treated with LPS (1 μg/ml) as determined using real-time PCR. All data are expressed as the mean ± SD of three independent replicate experiments.

Figure 3

Syk impacts the transcriptional regulation of IRAK1 in LPS-treated RAW264.7 cells. (a and b) IRAK1 mRNA levels from RAW264.7-WT or RAW264.7-Syk^−/− cells with or without LPS (1 μg/ml) treatment, as determined by real-time PCR. (c) IRAK1 mRNA levels from HEK293 cells transfected with Syk, as determined by real-time PCR. All data are expressed as the mean ± SD of three independent replicate experiments. ^∗p < 0.05 and ^∗∗p < 0.01 compared to the control group (LPS alone) or normal group.

Figure 4

Schematic pathway for Syk regulation of IRAK1 expression and activity.