title: MLK3 regulates fMLP-stimulated neutrophil motility.
abstract: BACKGROUND
  Mixed lineage kinase 3 (MLK3) is part of the intracellular regulatory system that connects extracellular cytokine or mitogen signals received through G-protein coupled receptors to changes in gene expression. MLK3 activation stimulates motility of epithelial cells and epithelial-derived tumor cells, but its role in mediating the migration of other cell types remains unknown. Since neutrophils play a crucial role in innate immunity and contribute to the pathogenesis of several diseases, we therefore examined whether MLK3 might regulate the motility of mouse neutrophils responding to a chemotactic stimulus, the model bacterial chemoattractant fMLP.
  
  
  METHODS
  The expression of Mlk3 in mouse neutrophils was determined by immunocytochemistry and by RT-PCR. In vitro chemotaxis in a gradient of fMLP, fMLP-stimulated random motility, fMLP-stimulated F-actin formation were measured by direct microscopic observation using neutrophils pre-treated with a novel small molecule inhibitor of MLK3 (URMC099) or neutrophils obtained from Mlk3-/- mice. In vivo effects of MLK3 inhibition were measured by counting the fMLP-induced accumulation of neutrophils in the peritoneum following pre-treatment with URMC099 in wild-type C57Bl/6 or mutant Mlk3-/- mice.
  
  
  RESULTS
  The expression of Mlk3 mRNA and protein was observed in neutrophils purified from wild-type C57Bl/6 mice but not in neutrophils from mutant Mlk3-/- mice. Chemotaxis by wild-type neutrophils induced by a gradient of fMLP was reduced by pre-treatment with URMC099. Neutrophils from C57Bl/6 mice pretreated with URMC099 and neutrophils from Mlk3-/- mice moved far less upon fMLP-stimulation and did not form F-actin as readily as untreated neutrophils from C57Bl/6 controls. In vivo recruitment of neutrophils into the peritoneum by fMLP was significantly reduced in wild-type mice treated with URMC099, as well as in untreated Mlk3-/- mice-thereby confirming the role of MLK3 in neutrophil migration.
  
  
  CONCLUSIONS
  Mlk3 mRNA is expressed in murine neutrophils. Genetic or pharmacologic inhibition of MLK3 blocks fMLP-mediated motility of neutrophils both in vitro and in vivo, suggesting that MLK3 may be a therapeutic target in human diseases characterized by exuberant neutrophil migration.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, 14642 NY, USA. Electronic address: oksana_polesskaya@urmc.rochester.edu.
authors.forename: Oksana
authors.initials: O
authors.lastname: Polesskaya
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, 14642 NY, USA; Carleton College, 1N College Street, Northfield, MN 55057, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Wong
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, 14642 NY, USA.
authors.forename: Luis
authors.initials: L
authors.lastname: Lebron
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, 14642 NY, USA.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Chamberlain
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, 14642 NY, USA; Center for Neural Development and Disease, and Departments of Pediatrics and Neurology, University of Rochester Medical Center, Rochester 14642, NY, USA.
authors.forename: Harris A
authors.initials: HA
authors.lastname: Gelbard
authors.affiliation: Califia Bio Inc., 11575 Sorrento Valley Road, San Diego, CA, USA.
authors.forename: Val
authors.initials: V
authors.lastname: Goodfellow
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, 14642 NY, USA; David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, 14642 NY, USA.
authors.forename: Minsoo
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, 14642 NY, USA; Center for Musculoskeletal Research, and Department of Orthopaedics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 672, Rochester 14642, NY, USA.
authors.forename: John L
authors.initials: JL
authors.lastname: Daiss
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, 14642 NY, USA. Electronic address: stephen_dewhurst@urmc.rochester.edu.
authors.forename: Stephen
authors.initials: S
authors.lastname: Dewhurst
mesh_terms: D000818:Animals; D002464:Cell Migration Inhibition / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002630:Chemotactic Factors / Q000494:pharmacology*; D007154:Immune System Diseases / Q000139:chemically induced* / Q000235:genetics; D007960:Leukocyte Disorders / Q000139:chemically induced* / Q000235:genetics; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D018375:Neutrophil Activation / Q000187:drug effects / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
chemical_list: D002630:Chemotactic Factors; D020930:MAP Kinase Kinase Kinases; D009240:N-Formylmethionine Leucyl-Phenylalanine; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011758:Pyrroles; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C583037:URMC-099
keywords: AD; AKT; APC; CRIB; Chemotaxis; DMSO; EDTA; ERK; EtBr; FITC; G-protein coupled receptor; GAPDH; GPCR; GTP; HBSS; Hank's buffered salt solution; ICAM; JIP; JNK; JNK interacting protein; MKK; MLK3; Mixed lineage kinase 3; Mouse; N-formyl-l-methionyl-l-leucyl-l-phenylalanine; Neutrophil; PBS; PCR; PE; PEG; PI3K; RT-PCR; SH3; Src homology domain 3; TBS; TNF; accumulated distance; allophycocyanin; c-jun N-terminal protein kinase; cdc42/Rac-interactive binding; dimethyl sulfoxide; ethidium bromide; ethylenediaminotetraacetic acid; extracellular signal-related kinase; fMLP; fluorescein isothiocyanate; glyceraldehyde-3-phosphate dehydrogenase; guanosine triphosphate; intercellular adhesion molecule; mitogen-activated protein kinase kinase; mixed lineage kinase 3; phosphate-buffered saline; phosphoinositide-3-kinase; phycoerythrin; polyethyleneglycol; polymerase chain reaction; protein kinase B; reverse transcription-polymerase chain reaction; tris-buffered saline; tumor necrosis factor