The ability of AIF-1 to activate human vascular smooth muscle cells is lost by mutations in the EF-hand calcium-binding region.
Allograft Inflammatory Factor-1 (AIF-1) is a cytoplasmic calcium-binding protein expressed in vascular smooth muscle cells (VSMC) in response to injury or cytokine stimulation. AIF-1 contains a partially conserved EF-hand calcium-binding domain, and participates in VSMC activation by activation of Rac1 and induction of Granulocyte-Colony Stimulating Factor (G-CSF) expression; however, the mechanism whereby AIF-1 mediates these effects is presently uncharacterized. To determine if calcium binding plays a functional role in AIF-1 activity, a single site-specific mutation was made in the EF-hand calcium-binding domain to abrogate binding of calcium (AIF-1DeltaA), which was confirmed by calcium overlay. Functionally, similar to wild-type AIF-1, AIF-1DeltaA was able to polymerize F-actin in vitro. However, in contrast to wild-type AIF-1, over-expression of AIF-1DeltaA was unable to increase migration or proliferation of primary human VSMC. Further, it was unable to activate Rac1, or induce G-CSF expression to the degree as wild-type AIF-1. Taken together, modification of the wild-type EF-hand domain and native calcium-binding activity results in a loss of AIF-1 function. We conclude that appropriate calcium-binding potential is critical in AIF-1-mediated effects on VSMC pathophysiology, and that AIF-1 activity is mediated by Rac1 activation and G-CSF expression.
Autieri
Michael V
MV
Department of Physiology, Cardiovascular Research Center, Temple University School of Medicine, Room 810, MRB 3420 N. Broad Street, Philadelphia PA 19140, USA. mautieri@temple.edu
Chen
Xing
X
D019943:Amino Acid Substitution; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D004268:DNA-Binding Proteins; D020832:EF Hand Motifs / Q000235:genetics*; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
C112986:AIF1 protein, human; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D008840:Microfilament Proteins; D016179:Granulocyte Colony-Stimulating Factor; D020830:rac1 GTP-Binding Protein; D002118:Calcium