The interleukin-1 receptor associated kinase 1 contributes to the regulation of NFAT

Abstract

IRAK-1 is a critical modulator regulating innate immunity signaling processes. However, the physiological substrates for IRAK-1 remain poorly defined. In this report, we have demonstrated that IRAK-1 is a kinase responsible for the constitutive phosphorylation and inactivation of the Nuclear Factor of Activated T-cell (NFAT). Expression of IRAK-1 suppressed NFAT reporter activity. Correspondingly, the levels of both nuclear NFATc1 and NFATc4 were constitutively elevated in IRAK-1 -/-cells. Furthermore, the phosphorylation of NFATc4 at the S 168 PS 170 P site was significantly diminished in IRAK-1 -/-cells. Mechanistically, we observed that IRAK-1 interacted with NFATc4 via the C-terminus of IRAK-1 and the N-terminal NHR region of NFATc4. IRAK-1 mutants that ablated either its kinase activity or its interaction with NFATc4 failed to suppress NFAT reporter activity. The expression level of COX-2, which is under the control of NFAT, was elevated in IRAK-1 -/-cells. Functionally, ApoE -/-/IRAK-1 -/-mice were protected from high-fat-diet induced hypertension and atherosclerosis. Taken together, our findings reveal NFAT molecules as novel physiological targets for IRAK-1.

Low-Dose Endotoxin Induces Inflammation by Selectively Removing Nuclear Receptors and Activating CCAAT/Enhancer-Binding Protein δ

“…Abs against lamin B, b-actin, GAPDH, PPARa, RARa, C/EBPd, NF-kB-p65, and IkB kinase ε (IKKε) were purchased from Santa Cruz Biotechnology. The IkBa-specific Ab was obtained from Cell Signaling Technology, the anti-IRAK-1 Ab was obtained from Millipore, and the pFlag-IRAK-1 and various mutant IRAK-1 expression plasmids were generated as we described previously (12). The IKKε-specific inhibitor IX was purchased from Calbiochem.…”

“…Cell culture, transfection, immunoprecipitation, and Western blot analyses HeLa cells were cultured in complete BMDM medium, as previously described (12). Transfections with various IRAK-1 expression plasmids were performed using Lipofectamine reagents (Invitrogen), per the manufacturer's instruction.…”

“…High-fat diet feeding and related animal studies /IRAK-1 2/2 mice were fed with control chow (Harlan Teklad 2018) or a Western diet (Harlan Teklad 94059) for 3 mo, as described previously (12). Plasma samples were harvested via cardiac puncture to measure levels of selected cytokines using the BIO-RAD multiplex system, as described previously.…”

An Innate Immunity Signaling Process Suppresses Macrophage ABCA1 Expression through IRAK-1-Mediated Downregulation of Retinoic Acid Receptor α and NFATc2

“…Furthermore, IRAK-1 is constitutively active in blood mononuclear cells isolated from patients with atherosclerosis (23,29). Our recent study using ApoE Ϫ/Ϫ /IRAK-1 Ϫ/Ϫ mice also demonstrated that IRAK-1 deletion renders protection from high-fat diet-induced atherosclerosis (56). Despite compelling evidence linking IRAK-1 with the pathogenesis of atherosclerosis, the underlying molecular mechanism is not fully defined.…”

“…In contrast, very limited information has been available regarding its function in macrophages, although NFAT is known to be expressed in these cells (41). We have previously reported that IRAK-1 is critically involved in maintaining NFAT in a phosphorylated and inactive state (56). IRAK-1 physically associates with NFAT and contributes to the phosphorylation of NFAT at its Ser-Pro-rich motif (56).…”