Blockade of focal clustering and active conformation in beta 2-integrin-mediated adhesion of eosinophils to intercellular adhesion molecule-1 caused by transduction of HIV TAT-dominant negative Ras.
We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to beta(2)-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for beta(1)-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for beta(2)-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.
Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, IL 60637, USA.
Shigeharu
S
Myou
Xiangdong
X
Zhu
Evan
E
Boetticher
Saori
S
Myo
Angelo
A
Meliton
Anissa
A
Lambertino
Nilda M
NM
Munoz
Alan R
AR
Leff
D018821:CD18 Antigens / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics; D054413:Chemokine CCL11; D019742:Chemokines, CC / Q000502:physiology; D015536:Down-Regulation / Q000235:genetics; D004804:Eosinophils / Q000378:metabolism* / Q000821:virology; D015696:Gene Products, tat / Q000235:genetics* / Q000494:pharmacology; D011905:Genes, ras / Q000235:genetics / Q000502:physiology*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000737:chemistry / Q000378:metabolism*; D015848:Interleukin-5 / Q000502:physiology; D016177:Macrophage-1 Antigen / Q000096:biosynthesis / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014161:Transduction, Genetic; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
C515456:CCL11 protein, human; D018821:CD18 Antigens; D054413:Chemokine CCL11; D019742:Chemokines, CC; D015696:Gene Products, tat; D015848:Interleukin-5; D016177:Macrophage-1 Antigen; D011993:Recombinant Fusion Proteins; D018799:Intercellular Adhesion Molecule-1; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate