Heterotrimeric G_i/G_oproteins modulate endothelial TLR signaling independent of the MyD88-dependent pathway

Abstract

The innate immune recognition of bacterial lipopolysaccharide (LPS) is mediated by Toll-like receptor 4 (TLR4) and results in activation of proinflammatory signaling including NF-B and MAPK pathways. Heterotrimeric G proteins have been previously implicated in LPS signaling in macrophages and monocytes. In the present study, we show that pertussis toxin sensitive heterotrimeric G proteins (G␣i/o) are involved in the activation of MAPK and Akt downstream of TLR2, TLR3, and TLR4 in endothelial cells. G␣ i/o are also required for full activation of interferon signaling downstream of TLR3 and TLR4 but are not required for the activation of NF-B. We find that G␣i/o-mediated activation of the MAPK is independent of the canonical MyD88, interleukin-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor 6 signaling cascade in LPS-stimulated cells. Taken together, the data presented here suggest that heterotrimeric G proteins are widely involved in TLR pathways along a signaling cascade that is distinct from MyD88-TRAF6.lipopolysaccharide; innate immunity; toll-like receptors; heterotrimeric G proteins; myeloid differentiation factor THE INNATE IMMUNE RECOGNITION of bacterial and viral products is mediated by a family of transmembrane receptors known as Toll-like receptors (TLRs). Lipopolysaccharide (LPS), a key component of the outer wall of gram-negative bacteria, initiates endothelial activation through a receptor complex consisting of TLR4, CD14, and MD2 (9). Recruitment of the adaptor proteins TIR-containing adaptor molecule (TIRAP) and myeloid differentiation factor (MyD88) initiates a MyD88-dependent pathway that culminates in the activation of NF-B and MAPK. Activation of these downstream targets requires recruitment of interleukin-1 receptor-associated kinase (IRAK4) and IRAK1 through interaction between the death domains of MyD88 and the IRAKs. The autophosphorylation and activation of IRAK1 results in the ability to bind tumor necrosis factor receptor-associated factor 6 (TRAF6) (7), which leads to oligomerization and polyubiquitination of the TRAF6 molecule (11). This facilitates activation of transforming growth factor-␤ (TGF-␤)-activated kinase 1, which leads to activation of the IKK-NF-B pathway and the three MAPK: p38, ERK, and JNK (39). In addition to the MyD88-dependent pathway, LPS stimulation also results in the activation of a MyD88-independent pathway, through recruitment of the adaptor molecules TIR-containing adaptor inducing IFN-␤ (TRIF) and TRIF-related adaptor molecule. This leads to the late-phase activation of NF-B and interferon regulatory factor 3 (IRF3), as well as activation of the MAPKs and phosphatidylinositol 3-kinase (PI3K) (10).The endothelium plays a major role in the pathogenesis of sepsis. Under normal conditions, the endothelium functions to maintain organ homeostasis through vasoregulation, selective vascular permeability, and providing an anticoagulant surface. During bacterial infection, the normal physiological functions of the endothelium are pert...

Gα i1 and Gα i3 regulate macrophage polarization by forming a complex containing CD14 and Gab1

“…ERK1/2 phosphorylation (10), indicating that TLR4 may transactivate a GPCR to promote signaling (25). Moreover, Dauphinee et al demonstrated that Gα i proteins modulate endothelial TLR signaling independent of TRAF6 (26). Thus, we propose a signaling model of Gα i1 and Gα i3 -mediated activation of the Gab1-PI3K-Akt-NF-κB pathway in response to LPS.…”

Toll-Like Receptor 2–Blocking Antibodies Promote Angiogenesis and Induce ERK1/2 and AKT Signaling via CXCR4 in Endothelial Cells

“…31 CXCR4 is a G i -proteincoupled receptor, and heterotrimeric Gi proteins have recently been shown to induce the activation of MAPK and AKT signaling downstream of TLR2 independent of the adaptor protein MyD88. 32 In the present study, binding of anti-TLR2 ABs to TLR2 resulted in the activation of both ERK1/2 and AKT, the canonical signal transduction pathways of CXCR4. 33 This activation of signaling cascades by anti-TLR2 ABs was similar to signaling induced by direct CXCR4 activation by its ligand SDF-1, and immunoprecipitation in fact revealed an association of CXCR4 with TLR2 on binding of anti-TLR2 ABs.…”

Vascular endothelial cell Toll-like receptor pathways in sepsis

“…[228][229][230][231][232][233][234] Treatment with the TLR2 agonist Pam3Cys has been reported to activate p38-MAPK, JNK and ERK5, but not ERK1/2 in HUVECs, and p38-MAPK, JNK and ERK1/2 in HMVECs. 31,235 Treatment with the TLR4 agonist LPS has been reported to activate p38-MAPK and JNK in HUVECs, and p38-MAPK, JNK and ERK1/2 in HMVECs. 214,235,236 Finally, treatment with the TLR9 agonist CpG DNA has been reported to activate p38-MAPK, but not ERK1/2, in mouse lung endothelial cells.…”

“…31,235 Treatment with the TLR4 agonist LPS has been reported to activate p38-MAPK and JNK in HUVECs, and p38-MAPK, JNK and ERK1/2 in HMVECs. 214,235,236 Finally, treatment with the TLR9 agonist CpG DNA has been reported to activate p38-MAPK, but not ERK1/2, in mouse lung endothelial cells. 9 Because of the importance of the conventional MAPKs in endothelial cell signaling, and because there are some apparent differences in the role of the ERKs in endothelial cells and leukocytes, each is reviewed in more detail below.…”