Interleukin (IL)-1 receptor-associated kinase (IRAK) requirement for optimal induction of multiple IL-1 signaling pathways and IL-6 production

Abstract

Interleukin (IL)-1 is a proinflammatory cytokine with pleiotropic effects in inflammation. IL-1 binding to its receptor triggers a cascade of signaling events, including activation of the stress-activated mitogen-activated protein (MAP) kinases, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase, as well as transcription factor nuclear factor kappaB (NF-kappaB). IL-1 signaling results in cellular responses through induction of inflammatory gene products such as IL-6. One of the earliest events in IL-1 signaling is the rapid interaction of IL-1 receptor-associated kinases, IRAK and IRAK-2, with the receptor complex. The relative roles of IRAK and IRAK-2 in IL-1 signaling pathways and subsequent cellular responses have not been previously determined. To evaluate the importance of IRAK in IL-1 signaling, IRAK-deficient mouse fibroblast cells were prepared and studied. Here we report that IL-1-mediated activation of JNK, p38, and NF-kappaB were all reduced in embryonic fibroblasts deficient in IRAK expression. In addition, IL-6 production in response to IL-1 was also dramatically reduced in IRAK-deficient embryonic fibroblasts and in skin fibroblasts prepared from IRAK-deficient mice. Our results demonstrate that IRAK plays an essential proximal role in coordinating multiple IL-1 signaling pathways for optimal induction of cellular responses.

Figure 1

Lack of IRAK expression in IRAK-deficient cells. (A) Map of the mouse IRAK gene and construct. A genomic DNA fragment containing the 5′ portion of the mouse IRAK gene was used as the homologous region for recombination. Exon 5 to exon 7 of the gene was replaced by a neomycin resistant gene cassette (NEO). A herpes simplex–thymidine kinase gene (HSV-TK) was placed 3′ of the construct. Restriction sites are BamHI (B), EcoRI (E) and SpeI (S). (B) Disruption of the single IRAK allele in male embryonic stem cells. Genomic DNAs from embryonic stem cells with the wild-type (+) or disrupted (−) IRAK gene were digested with EcoRI and hybridized to the DNA probe shown in A. The 12-kb DNA band of the wild-type allele and the 2.6-kb band of the disrupted allele detected in hybridization are also shown in A. (C) Absence of IRAK protein in IRAK-deficient cells. Primary embryonic EF cells were prepared as described in the Materials and Methods. Proteins from control (+) and IRAK-deficient (−) EF cells were separated by 10% SDS-PAGE and IRAK was detected by immunoblotting using an IRAK-specific antibody. Equal loading of proteins was demonstrated by subsequent blotting with an antibody to extracellular signal–regulated kinase 2 (ERK-2).

Figure 1

Lack of IRAK expression in IRAK-deficient cells. (A) Map of the mouse IRAK gene and construct. A genomic DNA fragment containing the 5′ portion of the mouse IRAK gene was used as the homologous region for recombination. Exon 5 to exon 7 of the gene was replaced by a neomycin resistant gene cassette (NEO). A herpes simplex–thymidine kinase gene (HSV-TK) was placed 3′ of the construct. Restriction sites are BamHI (B), EcoRI (E) and SpeI (S). (B) Disruption of the single IRAK allele in male embryonic stem cells. Genomic DNAs from embryonic stem cells with the wild-type (+) or disrupted (−) IRAK gene were digested with EcoRI and hybridized to the DNA probe shown in A. The 12-kb DNA band of the wild-type allele and the 2.6-kb band of the disrupted allele detected in hybridization are also shown in A. (C) Absence of IRAK protein in IRAK-deficient cells. Primary embryonic EF cells were prepared as described in the Materials and Methods. Proteins from control (+) and IRAK-deficient (−) EF cells were separated by 10% SDS-PAGE and IRAK was detected by immunoblotting using an IRAK-specific antibody. Equal loading of proteins was demonstrated by subsequent blotting with an antibody to extracellular signal–regulated kinase 2 (ERK-2).

Figure 1

Lack of IRAK expression in IRAK-deficient cells. (A) Map of the mouse IRAK gene and construct. A genomic DNA fragment containing the 5′ portion of the mouse IRAK gene was used as the homologous region for recombination. Exon 5 to exon 7 of the gene was replaced by a neomycin resistant gene cassette (NEO). A herpes simplex–thymidine kinase gene (HSV-TK) was placed 3′ of the construct. Restriction sites are BamHI (B), EcoRI (E) and SpeI (S). (B) Disruption of the single IRAK allele in male embryonic stem cells. Genomic DNAs from embryonic stem cells with the wild-type (+) or disrupted (−) IRAK gene were digested with EcoRI and hybridized to the DNA probe shown in A. The 12-kb DNA band of the wild-type allele and the 2.6-kb band of the disrupted allele detected in hybridization are also shown in A. (C) Absence of IRAK protein in IRAK-deficient cells. Primary embryonic EF cells were prepared as described in the Materials and Methods. Proteins from control (+) and IRAK-deficient (−) EF cells were separated by 10% SDS-PAGE and IRAK was detected by immunoblotting using an IRAK-specific antibody. Equal loading of proteins was demonstrated by subsequent blotting with an antibody to extracellular signal–regulated kinase 2 (ERK-2).

Figure 2

Defective IL-1–induced p38 and JNK activation in IRAK-deficient EF cells. IRAK-deficient (−) or control (+) EF cells were stimulated with IL-1β (0.1–10 ng/ml) or TNF-α (100 ng/ml) for 10 min at 37°C, and immune complex kinase assays for p38 and JNK1 activity were performed. (A) Phosphorylation of MAPKAPK2 by immunoprecipitated p38 and of c-Jun by immunoprecipitated JNK1. Western blotting for p38 and JNK1 was performed as a loading control. (B) Quantitation of p38 and JNK1 activation. p38 and JNK1 kinase assays were quantitated by PhosphorImager (Molecular Dynamics), and normalized to the amount of p38 or JNK1 detected by Western blotting for each sample and to the activity in unstimulated IRAK-deficient EF cells for each assay. The p38 data are representative of three experiments, and the JNK1 data are expressed as an average of two experiments.

Figure 3

Reduced activation of the NF-κB pathway by IL-1 in IRAK-deficient cells. (A) Inefficient IκB degradation in IRAK-deficient EF cells. Control (+) and IRAK-deficient (−) cells were stimulated with IL-1 for 15 min at 37°C. Proteins in cell lysates were separated on SDS-PAGE and IκB was detected with an IκB-α–specific antibody. Equal amounts of sample loading were demonstrated by subsequent blotting of the same filter with an antibody specific to extracellular signal–regulated kinase 2 (ERK-2). The intensity of the protein bands was quantitated by densitometry. The amount of IκB in the IL-1–stimulated cells is expressed as the percentage of that in unstimulated cells. (B) Decreased activation of NF-κB by IL-1 in IRAK-deficient cells. EF cells were stimulated with IL-1 or TNF-α for 30 min at 37°C. Nuclear extracts were prepared and NF-κB DNA-binding activity was determined with the electrophoretic mobility shift assay. The data are representative of two independent experiments with similar results.

Figure 3

Reduced activation of the NF-κB pathway by IL-1 in IRAK-deficient cells. (A) Inefficient IκB degradation in IRAK-deficient EF cells. Control (+) and IRAK-deficient (−) cells were stimulated with IL-1 for 15 min at 37°C. Proteins in cell lysates were separated on SDS-PAGE and IκB was detected with an IκB-α–specific antibody. Equal amounts of sample loading were demonstrated by subsequent blotting of the same filter with an antibody specific to extracellular signal–regulated kinase 2 (ERK-2). The intensity of the protein bands was quantitated by densitometry. The amount of IκB in the IL-1–stimulated cells is expressed as the percentage of that in unstimulated cells. (B) Decreased activation of NF-κB by IL-1 in IRAK-deficient cells. EF cells were stimulated with IL-1 or TNF-α for 30 min at 37°C. Nuclear extracts were prepared and NF-κB DNA-binding activity was determined with the electrophoretic mobility shift assay. The data are representative of two independent experiments with similar results.

Figure 4

Decreased induction of IL-6 by IL-1 in IRAK-deficient cells. (A) Decreased induction of IL-6 mRNA in IRAK-deficient cells. Control (+) and IRAK-deficient (−) EF cells were treated with IL-1 or TNF-α for 3 h at 37°C. IL-6 mRNA expression in different samples was detected by Northern hybridization and quantitated on a PhosphorImager. (B) Decreased induction of secreted IL-6 in IRAK-deficient cells. Control (+) and IRAK-deficient (−) EF (top) and SF (bottom) cells were stimulated with IL-1 for 8 h at 37°C. The amount of secreted IL-6 in culture supernatants was determined by ELISA.

Figure 4

Decreased induction of IL-6 by IL-1 in IRAK-deficient cells. (A) Decreased induction of IL-6 mRNA in IRAK-deficient cells. Control (+) and IRAK-deficient (−) EF cells were treated with IL-1 or TNF-α for 3 h at 37°C. IL-6 mRNA expression in different samples was detected by Northern hybridization and quantitated on a PhosphorImager. (B) Decreased induction of secreted IL-6 in IRAK-deficient cells. Control (+) and IRAK-deficient (−) EF (top) and SF (bottom) cells were stimulated with IL-1 for 8 h at 37°C. The amount of secreted IL-6 in culture supernatants was determined by ELISA.