Mastoparan, a G protein agonist peptide, differentially modulates TLR4- and TLR2-mediated signaling in human endothelial cells and murine macrophages.
Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-kappaB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-alpha, or IL-1beta was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.
Lentschat
Arnd
A
Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201, USA.
Karahashi
Hisae
H
Michelsen
Kathrin S
KS
Thomas
Lisa S
LS
Zhang
Wenxuan
W
Vogel
Stefanie N
SN
Arditi
Moshe
M
D000818:Animals; D042783:Endothelial Cells / Q000276:immunology / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000276:immunology*; D019204:GTP-Binding Proteins / Q000819:agonists; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D018950:Lipopolysaccharide Receptors; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D010455:Peptides; D011956:Receptors, Cell Surface / Q000819:agonists / Q000187:drug effects / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled; D015398:Signal Transduction; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014862:Wasp Venoms / Q000494:pharmacology*
D036341:Intercellular Signaling Peptides and Proteins; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D010455:Peptides; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014862:Wasp Venoms; C037500:mastoparan; D019204:GTP-Binding Proteins