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Regulation of adhesion of AML14.3D10 cells by surface clustering of β 2 ‐integrin caused by ERK‐independent activation of cPLA 2
Author(s) -
Myou Shigeharu,
Zhu Xiangdong,
Boetticher Evan,
Qin Yimin,
Myo Saori,
Meliton Angelo,
Lambertino Anissa,
Munoz Nilda M.,
Hamann Kimm J.,
Leff Alan R.
Publication year - 2002
Publication title -
immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.297
H-Index - 133
eISSN - 1365-2567
pISSN - 0019-2805
DOI - 10.1046/j.1365-2567.2002.01486.x
Subject(s) - protein kinase c , mapk/erk pathway , integrin , integrin alpha m , microbiology and biotechnology , cell adhesion , phorbol , chemistry , protein kinase a , kinase , biology , biochemistry , cell
Summary We examined the role of cell surface clustering of β 2 ‐integrin caused by protein kinase C (PKC)‐activated‐cPLA 2 in adhesion of eosinophilic AML14.3D10 (AML) cells. Phorbol 12‐myristate 13‐acetate (PMA) caused time‐ and concentration‐dependent adhesion of AML cells to plated bovine serum albumin (BSA), which was blocked by anti‐CD11b or anti‐CD18 monoclonal antibodies (mAb) directed against β 2 ‐integrin. Inhibition of PKC with Ro‐31‐8220 or rottlerin blocked PMA‐induced cell adhesion in a concentration‐dependent fashion. Inhibition of cytosolic phospholipase A 2 (cPLA 2 ) with trifluoromethyl ketone or methyl arachidonyl fluorophosphonate also blocked PMA‐induced cell adhesion. PMA caused time‐dependent p42/44 mitogen‐activated protein kinase (MAPK) (ERK) phosphorylation in these cells. U0126, a MAPK/extracellular signal‐regulated protein kinase kinase (MEK) inhibitor, at the concentrations that blocked PMA‐induced ERK phosphorylation, had no effect on PMA stimulated AML cell adhesion. Neither p38 MAPK nor c‐Jun N‐terminal kinase (JNK) was phosphorylated by PMA. PMA also caused increased cPLA 2 activity, which was inhibited by Ro‐31‐8220, but not U0126. Confocal immunofluorescence microscopy showed that PMA caused clustering of CD11b on the cell surface, which was blocked by either PKC or cPLA 2 inhibition. PMA stimulation also caused up‐regulation of CD11b on the AML cell surface. However, this up‐regulation was not affected by cPLA 2 ‐ or PKC‐inhibition. Using the mAb, CBRM1/5, we also demonstrated that PMA does not induce the active conformation of CD11b/CD18. Our data indicate that PMA causes AML cell adhesion through β 2 ‐integrin by PKC activation of cPLA 2 . This pathway is independent of MEK/ERK and does not require change of CD11b/CD18 to its active conformation. We find that avidity caused by integrin surface clustering – rather than conformational change or up‐regulation of CD11b/CD18 – causes PMA stimulated adhesion of AML cells.