Myosin light chain kinase mediates eosinophil chemotaxis in a mitogen-activated protein kinase-dependent manner.
BACKGROUND
Eosinophil migration in the tissue is one characteristic feature of allergic diseases. The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Myosin light chain kinase (MLCK) is known to regulate cytoskeletal rearrangement and cell motility by means of phosphorylation of myosin light chain (MLC).


OBJECTIVE
We have previously shown that mitogen-activated protein (MAP) kinases are important for eosinophil migration. In the present study we hypothesized that MLCK is downstream of MAP kinases, thereby linking the MAP kinase pathway to the activation of cytoskeletal components required for eosinophil chemotaxis.


METHODS
Blood eosinophils were purified by using Percoll and anti-CD16 antibody-coated magnetic beads. We investigated the phosphorylation of MLCK and MLC by using the phosphorous 32-orthophosphates-labeled eosinophils. The kinase activity of MLCK was determined by measuring the phosphotransferase activity for the MLCK-specific peptide substrate. The chemotaxis assay was performed in a 48-well Boyden microchamber.


RESULTS
The phosphotransferase activity of MLCK for a substrate peptide was enhanced in eotaxin-stimulated eosinophils. We also found that eotaxin induced phosphorylation of MLCK in vivo in phosphorous 32-orthophosphate-labeled eosinophils. PD98059 (MAP/extracellular signal-regulated kinase inhibitor) or SB202190 (p38 MAP kinase inhibitor) abrogated the eotaxin-induced phosphorylation of MLCK. The phosphorylation of MLC was upregulated by eotaxin. Eosinophil chemotaxis was inhibited by means of pretreatment of the MLCK inhibitor ML-7.


CONCLUSIONS
These results suggest that eotaxin regulates MLCK through both extracellular signal-regulated kinase 1/2 and p38 MAP kinase. MLCK activation is a critical step in the cytoskeletal rearrangements leading to eosinophil migration.
Adachi
Tetsuya
T
Department of Clinical and Laboratory Medicine, Akita University School of Medicine, Akita, Japan.
Stafford
Susan
S
Kayaba
Hiroyuki
H
Chihara
Junichi
J
Alam
Rafeul
R
D054413:Chemokine CCL11; D019742:Chemokines, CC / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D004804:Eosinophils / Q000201:enzymology* / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000097:blood* / Q000502:physiology*; D009219:Myosin-Light-Chain Kinase / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases
D054413:Chemokine CCL11; D019742:Chemokines, CC; D009219:Myosin-Light-Chain Kinase; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases