Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-kappaB p65/RelA subunit

Abstract

The work of Reddy et al. (S. A. Reddy, J. A. Huang, and W. S. Liao, J. Biol. Chem. 272:29167-29173, 1997) reveals that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. We have found that IL-1 stimulates interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression but have no effect on the IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, we found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB.

FIG. 1

IL-1 induces the association of IL-1R AcP with p85. (A) HepG2 cells were stimulated with IL-1β (1 ng/ml) as indicated, and cell lysates were prepared. Polyclonal antibodies against either p85 or IL-1R AcP were used to coimmunoprecipitate the associated proteins. After separation by SDS-PAGE, the immunoprecipitates were immunoblotted with either anti-IL-1R AcP or anti-p85. PC, positive control (human endothelial cell extract). (B) Beads carrying GST fusion proteins with full-length and C-terminally deleted forms of the IL-1R AcP cytoplasmic domain (diagrammed) were bound to proteins in extracts of HepG2 cells. Following binding, the beads were washed, and the bound proteins were analyzed by SDS-PAGE, followed by immunoblotting with anti-p85. ACP, AcP. Mutant proteins are designated by a solid triangle followed by the number of amino acids remaining after deletion; e.g., ▴160 resulted from the deletion of 28 amino acids from the full-length protein (188 amino acids).

FIG. 2

PI3K activity is stimulated by IL-1 and inhibited by LY294,002. Where indicated, HepG2 cells were incubated with 20 μM LY294,002 for 30 min prior to stimulation with IL-1β for the indicated time periods. PI3K activity was measured as the phosphorylation of phosphatidylinositol(4,5)P₂ yielding phosphatidylinositol(3,4,5)P₃ [PI(3,4,5)P₃]. −Ab, no anti-PY20.

FIG. 3

NF-κB-dependent gene expression is stimulated by IL-1 and inhibited by LY294,002. Where indicated, HepG2 cells were incubated with 20 μM LY294,002 for 30 min prior to stimulation with IL-1β for 4 h. (A) Cells were lysed and analyzed for the expression of IL-8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs by the Northern procedure. (B) HepG2 cells were transiently transfected with the NF-κB-dependent reporter plasmid pElam[−143]-luc and analyzed 48 h later. Where indicated, the cells were incubated with 20 μM LY294,002 (LY) for 30 min prior to stimulation with IL-1β for 4 h. Luciferase activity was normalized for transfection efficiency. Data are expressed as fold induction, the ratio between the expression level in the experimental and the level in the untreated control (Con) cells.

FIG. 4

LY294,002 inhibits an NF-κB activation pathway distinct from IκBα degradation. Where indicated, HepG2 cells were incubated with 20 μM LY294,002 (LY) for 30 min prior to stimulation with IL-1β for 30 min for EMSA or for the indicated times for IκBα analysis. (A) EMSA for NF-κB. (B) Immunoblot analysis of IκBα.

FIG. 5

IL-1 stimulates the formation of a p65-p50 heterodimer and a second p65/RelA complex in HepG2 cells. Cells were either left untreated or stimulated with IL-1β for 30 min. The cells were lysed, and the supershift analysis was performed with the antibodies indicated. p65/? is either a homodimer of p65/RelA or a heterodimer of p65/RelA with another NF-κB family member.

FIG. 6

LY294,002 inhibits NF-κB phosphorylation. HepG2 cells were preincubated in phosphate-free medium containing [³²P]orthophosphate (100 μCi/ml). Where indicated, the cells were incubated with 20 μM LY294,002 (LY) for 30 min prior to stimulation with IL-1β for 5 min (A and B) or for the indicated times (C). Cell lysis and immunoprecipitation of the phosphorylated NF-κB complex were performed as described in Materials and Methods. (A) Analysis of phosphorylated NF-κB proteins. pIκB, phosphorylated IκB. (B) Western analysis of phosphorylated p65/RelA proteins. IgG, immunoglobulin G antibody heavy chain; NL, no cell lysate. (C) Analysis of phosphorylated phospho-GSK-3α (serine 21-phosphorylated GSK-3α).

FIG. 7

Involvement of the PI3K pathway in regulating p65/RelA transactivation. (Left) HepG2 cells were cotransfected transiently with the NF-κB-dependent reporter plasmid pElam[−143]-luc and either an empty vector or a plasmid expressing the p110 catalytic subunit of PI3K. The cells were harvested 48 h after transfection. Where indicated, the cells were incubated with 20 μM LY294,002 (LY) for 30 min prior to stimulation with IL-1β for 4 h. Luciferase activity was normalized for transfection efficiency. Data are expressed as fold induction, the ratio between the expression level in the experimental cells and the level in vector-transfected untreated control cells (Con). (Right) HepG2 cells were cotransfected transiently with the pGal4-luc reporter plasmid, pGal4-RelA (expressing the p65-Gal4 fusion protein), and either the vector or a plasmid expressing p110; the cells were harvested 48 h after transfection. Where indicated, the cells were incubated with 20 μM LY294,002 for 30 min prior to stimulation with IL-1β for 4 h. Luciferase activity was normalized for transfection efficiency. The data are expressed as fold induction, the ratio between the expression level in the experimental and the level in vector-transfected untreated control cells (Con).

FIG. 8

Constitutively activated p110 and Akt regulate p65/RelA transactivation. HepG2 cells were cotransfected with the pGal4-luc reporter plasmid, pGal4-RelA 508-550, and either a vector or plasmids expressing wild-type (WT) p110, constitutively activated (CA) p110 or Akt, or their kinase-dead (KD) derivatives. The cells were harvested 48 h after transfection. (A) Luciferase activity was normalized for transfection efficiency. Data are expressed as fold induction, the ratio between the expression level in the experimental cells and the level in vector-transfected control cells. (B) Immunoblot analysis of the Gal4-RelA 508-550 fusion protein.

FIG. 9

Dominant negative Akt blocks IL-1 signaling to an NFκB-dependent promoter. HepG2 cells were transiently cotransfected with the NFκB-dependent reporter plasmid pElam[−143]-luc, together with either a vector or a construct expressing a dominant negative (DN) derivative of Akt. The cells were analyzed 48 h later, with a 4-h stimulation with IL-1β where indicated. Luciferase activity was normalized for transfection efficiency. The data are expressed as fold induction, the ratio of the expression level in the experimental cells and the level in the vector-transfected untreated control cells.