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Flagellin and lipopolysaccharide stimulate the MEK-ERK signaling pathway in chicken heterophils through differential activation of the small GTPases, Ras and Rap1.
Mol Immunol. 2007 Mar; 44(7):1729-36.MI

Abstract

The TLR agonists, flagellin (FLG) and lipopolysaccharide (LPS) stimulate functional activation and cytokine gene expression via the extracellular signal regulated kinase 1/2 (ERK1/2) MAP kinase cascade. However, the upstream mechanisms of these signaling events remain unknown. In mammals, the small GTP-binding protein Ras mediates ERK1/2 activation through activation of downstream effectors Raf-1-MEK1/2-ERK1/2 in response to a variety of stimuli. It is not clear whether this classic Ras cascade plays a role in TLR signaling in avian cells. In the present study, we investigated the role of Ras in FLG- and LPS-mediated signaling in ERK activation in chicken heterophils. Treatment of heterophils with LPS caused a rapid (within 5min) activation of Ras-GTP. The role of Ras activation in LPS-induced stimulation of ERK1/2 was corroborated when the specific Ras inhibitor, FTI-277, inhibited ERK1/2 activation. The classic Ras-mediated pathway of ERK1/2 activation by LPS was confirmed when the specific Raf-1 inhibitor, GW 5074, and the MEK1/2 inhibitor, U0126, both reduced ERK activation by 51-60%. Of more interest was that treatment of the heterophils with FLG did not activate Ras-GTP. Likewise, neither FTI-277 nor GW 5074 had any effect on FLG-mediated activation of ERK1/2. Another small GTPase, Rap1, has been shown to play a role in mammalian neutrophil function. Using a Rap1-GTP pull-down assay, we found that FLG stimulation, but not LPS, of avian heterophils induced a rapid and transient Rap1 activation. Rap1 has been shown to activate the ERK1/2 via a different Raf family member B-Raf whose downstream effector is MEK1/2. We show here that FLG stimulation of heterophils induces the phosphorylation of Rap1. The FLG induction of the Rap1-->B-Raf-->MEK1/2-->ERK1/2 cascade was confirmed by the reduction of ERK1/2 activation by the specific Rap1 inhibitor (GGTI-298) and U0126. The results demonstrate that for the first time that the small GTPase Ras family is involved in TLR signaling of avian heterophils with the TLR agonists LPS (Ras) and FLG (Rap1) inducing differential signaling cascades to activate the downstream ERK MAP kinase.

Authors+Show Affiliations

Laboratory of Avian Innate Immunity, USDA-ARS, Southern Plains Agricultural Research Center, 2881 F&B Road, College Station, TX 77845, USA. kogut@ffsru.tamu.eduNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

17045653

Citation

Kogut, M H., et al. "Flagellin and Lipopolysaccharide Stimulate the MEK-ERK Signaling Pathway in Chicken Heterophils Through Differential Activation of the Small GTPases, Ras and Rap1." Molecular Immunology, vol. 44, no. 7, 2007, pp. 1729-36.
Kogut MH, Genovese KJ, He H. Flagellin and lipopolysaccharide stimulate the MEK-ERK signaling pathway in chicken heterophils through differential activation of the small GTPases, Ras and Rap1. Mol Immunol. 2007;44(7):1729-36.
Kogut, M. H., Genovese, K. J., & He, H. (2007). Flagellin and lipopolysaccharide stimulate the MEK-ERK signaling pathway in chicken heterophils through differential activation of the small GTPases, Ras and Rap1. Molecular Immunology, 44(7), 1729-36.
Kogut MH, Genovese KJ, He H. Flagellin and Lipopolysaccharide Stimulate the MEK-ERK Signaling Pathway in Chicken Heterophils Through Differential Activation of the Small GTPases, Ras and Rap1. Mol Immunol. 2007;44(7):1729-36. PubMed PMID: 17045653.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Flagellin and lipopolysaccharide stimulate the MEK-ERK signaling pathway in chicken heterophils through differential activation of the small GTPases, Ras and Rap1. AU - Kogut,M H, AU - Genovese,K J, AU - He,H, Y1 - 2006/10/12/ PY - 2006/06/15/received PY - 2006/07/21/revised PY - 2006/07/24/accepted PY - 2006/10/19/pubmed PY - 2007/3/14/medline PY - 2006/10/19/entrez SP - 1729 EP - 36 JF - Molecular immunology JO - Mol Immunol VL - 44 IS - 7 N2 - The TLR agonists, flagellin (FLG) and lipopolysaccharide (LPS) stimulate functional activation and cytokine gene expression via the extracellular signal regulated kinase 1/2 (ERK1/2) MAP kinase cascade. However, the upstream mechanisms of these signaling events remain unknown. In mammals, the small GTP-binding protein Ras mediates ERK1/2 activation through activation of downstream effectors Raf-1-MEK1/2-ERK1/2 in response to a variety of stimuli. It is not clear whether this classic Ras cascade plays a role in TLR signaling in avian cells. In the present study, we investigated the role of Ras in FLG- and LPS-mediated signaling in ERK activation in chicken heterophils. Treatment of heterophils with LPS caused a rapid (within 5min) activation of Ras-GTP. The role of Ras activation in LPS-induced stimulation of ERK1/2 was corroborated when the specific Ras inhibitor, FTI-277, inhibited ERK1/2 activation. The classic Ras-mediated pathway of ERK1/2 activation by LPS was confirmed when the specific Raf-1 inhibitor, GW 5074, and the MEK1/2 inhibitor, U0126, both reduced ERK activation by 51-60%. Of more interest was that treatment of the heterophils with FLG did not activate Ras-GTP. Likewise, neither FTI-277 nor GW 5074 had any effect on FLG-mediated activation of ERK1/2. Another small GTPase, Rap1, has been shown to play a role in mammalian neutrophil function. Using a Rap1-GTP pull-down assay, we found that FLG stimulation, but not LPS, of avian heterophils induced a rapid and transient Rap1 activation. Rap1 has been shown to activate the ERK1/2 via a different Raf family member B-Raf whose downstream effector is MEK1/2. We show here that FLG stimulation of heterophils induces the phosphorylation of Rap1. The FLG induction of the Rap1-->B-Raf-->MEK1/2-->ERK1/2 cascade was confirmed by the reduction of ERK1/2 activation by the specific Rap1 inhibitor (GGTI-298) and U0126. The results demonstrate that for the first time that the small GTPase Ras family is involved in TLR signaling of avian heterophils with the TLR agonists LPS (Ras) and FLG (Rap1) inducing differential signaling cascades to activate the downstream ERK MAP kinase. SN - 0161-5890 UR - https://www.unboundmedicine.com/medline/citation/17045653/Flagellin_and_lipopolysaccharide_stimulate_the_MEK_ERK_signaling_pathway_in_chicken_heterophils_through_differential_activation_of_the_small_GTPases_Ras_and_Rap1_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0161-5890(06)00549-9 DB - PRIME DP - Unbound Medicine ER -