Submitted to: Biochemical Pharmacology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/27/2007
Publication Date: 4/1/2008
Citation: Zhao, L., Lee, J.Y., Hwang, D.H. The Phosphatidylinositol 3-Kinase/Akt Pathway Negatively Regulates Nod2-Mediated NF-kB Pathway. 2008. Biochemical Pharmacology. 75:1515-1525, 2008. Interpretive Summary: Evolution of host defense systems against microbial pathogens is the key for the survival of all multicellular organisms. Germ line encoded transmemberane Toll-like receptors (TLRs1) play a crucial role in the detection of invading microbes and the induction of immune and inflammatory responses to defend the host [1,2]. In addition to TLRs, cytosolic proteins, the nucleotide-binding oligomerization domain containing protein family (Nods) recognizes intracellular bacterial products and activates proinflammatory signaling pathways [3,4]. Nod1 and Nod2 protein have been shown to be activated by bacterial components through their leucine rich repeats domain at the COOH terminus. While the minimal PGN structure recognized by Nod1 is a dipeptide, y-D-glutamyl-meso-diaminopimelic acid (iE-DAP) derived mostly from Gram-negative bacteria, the minimal PGN that Nod2 recognizes is the muramyldipeptide, MurNAc-L-Ala-D-isoGln (MDP) from both Gram-positive and Gram-negative bacteria . The activation of Nod2 leads to the recruitment of a CARD-domain containing kinase, RICK/RIP2, through a CARD-CARD interaction, which then leads to the interaction with the regulatory subunit of IKK, IKK'/NEMO, linking to the activation of NF-kB and proinflammatory gene expression [5,6]. However, the downstream signaling pathways of Nods are not fully understood. Recent evidence suggests that phosphatidylinositol 3-kinase (PI3K)/Akt pathway modulate Toll like receptor signaling pathways. PI3K is a heterodimeric protein consisting of p85 regulatory subunit and p110 catalytic subunit. Activation of PI3K occurs through phosphorylation of tyrosine residues in the Src homology 2 domain of p85, which allows association of PI3K to the plasma membrane and increases its catalytic activity through allosteric modifications. Activated PI3K catalyzes the phosphorylation of membrane inositol lipids and the accumulation of phosphatidylinositol 3,4,5-trisphosphate and its phospholipid phosphatase product phosphatidylinositol 3,4-bisphosphate in the membrane, which recruits the lipid kinases phosphatidylinositol-dependent kinase1 (PDK1) and Akt/protein kinase B. After the membrane recruitment, Akt is activated by dual phosphorylation of Ser473 and Thr308 by PDK1 and possibly PDK1 related PDK2 . It has been shown that wortmannin, a specific inhibitor of PI3K, enhanced LPS (TLR4 agonist)-induced nitric-oxide synthase in murine peritoneal macrophages . Activation of PI3K/Akt in human monocytes limited LPS induction of TNF' and tissue factor expression to ensure transient expression of those potent inflammatory mediators . Moreover, inhibition of PI3K enhanced TRIF-dependent NF-'B activation and IFN-k synthesis downstream of Toll-like receptor 3 and 4 . PI3K was also found to negatively regulates flagellin-induced proinflammatory inducible NO synthase, IL-6 and IL-8 expression mediated by TLR5 . In contrast to those negative regulations, PI3K has also been reported to play a critical role in activating cytokine expression mediated by TLR2 and TLR9 [12,13]. TLR2 stimulation by Staphylococcus aureus caused the activation of Rac1 and PI3K pathways, which targets nuclear p65 transcriptional activity, independently of I'B' degradation . CpG DNA (TLR9 agonist) was shown to inhibit dendritic cell apoptosis by up-regulating cellular inhibitor of apoptosis proteins through the PI3K pathway to maintain immune response . However, it has not been reported whether PI3K/Akt pathway is involved in Nod2 activation and how PI3K/Akt might regulate Nod2 signaling pathway. Here, we show that Nod2 activation leads to activation of PI3K/Akt pathway and that PI3K/Akt pathway negatively regulates NF-kB activation and IL-8 expression induced by Nod2 activation.
Technical Abstract: Nucleotide-binding oligomerization domain containing proteins (Nods) are intracellular pattern recognition receptors (PRRs) that recognize conserved moieties of bacterial peptidoglycan and activate downstream signaling pathways including NF-kB pathway. Here, we show that Nod2 agonist MDP induces Akt phosphorylation. Pharmacological inhibitors of PI3K (LY294002 and wortmannin) and dominant negative form of p85 (the regulatory subunit of PI3K) or Akt enhance, while constitutive active form of p110 (the catalytic subunit of PI3K) or Akt inhibit, NF-kB activation and the target gene IL-8 induced by MDP. In addition, both LY 294002 and wortmannin enhance phosphorylation of NF-kB p65 on Ser 529 and Ser 536 residues resulting in enhanced p65 transactivation activity. Furthermore, we show that the negative regulation of PI3K/Akt on MDP-induced NF-kB is at least in part mediated through inactivating glycogen synthase kinase (GSK)-3b. Taken together, our results demonstrate that PI3K/Akt pathway is activated by MDP and negatively regulates NF-kB pathway downstream of Nod2 activation, suggesting that PI3K/Akt pathway may involve in the resolution of inflammatory responses induced by Nod2 activation.