Fractalkine-induced endothelial cell migration requires MAP kinase signaling.
OBJECTIVE
Angiogenesis is a well-established characteristic in the rheumatoid arthritis (RA) synovial pannus. We have previously demonstrated that fractalkine (Fkn/ CX3CL1) expression is significantly increased in the RA joint and that fractalkine induces angiogenesis. In this work we studied mechanisms through which Fkn functions as an angiogenic mediator.


METHODS
Human microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs) were stimulated with Fkn and analyzed by Western blotting or stained with Alexa Fluor 488 phalloidin for F-actin to characterize the time frame of cytoskeletal rearrangement. Fkn-induced HUVEC chemotaxis was performed in the presence and absence of MAP kinase inhibitors.


RESULTS
Phalloidin staining of F-actin revealed significant cytoskeletal rearrangements in HUVECs and HMVECs starting as early as 10 min after Fkn stimulation. Western blotting demonstrated that HUVEC and HMVEC stimulation with Fkn for 1-30 min resulted in phosphorylation of JNK. Fkn also induces significant phosphorylation of Erk 1/2 in HUVECs over a time course ranging from 1 to 15 min. A somewhat similar time course (5-15 min) was detected for Erk 1/2 phosphorylation in HMVECs. Inhibitors of either JNK or Erk 1/2 nearly abolish Fkn-induced HUVEC migration.


CONCLUSIONS
We demonstrate that Fkn induces significant alterations in cytoskeletal structure and specifically activates the MAP kinases, JNK and Erk 1/2, both of which appear necessary for endothelial cell migration. Our results suggest that the endogenous Fkn present in the RA joint may induce angiogenesis through activation of the JNK and Erk 1/2 pathways.
Volin
Michael V
MV
Department of Microbiology and Immunology, Midwestern University, Chicago College of Osteopathic Medicine, Downers Grove, IL 60515, USA.
Huynh
Nha
N
Klosowska
Karolina
K
Reyes
Rosemary D
RD
Woods
James M
JM
D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D054428:Chemokine CX3CL1 / Q000378:metabolism* / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology
D000199:Actins; D054428:Chemokine CX3CL1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4