Cytokine stimulated vascular cell adhesion molecule-1 (VCAM-1) ectodomain release is regulated by TIMP-3.
OBJECTIVE
Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface adhesion molecule involved in the recruitment of leukocytes to endothelial cells on arterial walls during the pathogenesis of atherosclerosis. The soluble ectodomain of VCAM-1 (sVCAM-1) is proteolytically released from the cell surface into the circulation, a process which is up-regulated in patients with cardiovascular or inflammatory disease. Here we investigate mechanisms involved in sVCAM-1 generation in response to cytokine stimulation.


METHODS
VCAM-1 ectodomain release into the conditioned media of MCEC-1 murine endothelial cells and cells grown from primary aortic explants from timp3-/- mice and wild-type littermates was measured by sandwich ELISA and Western blot after stimulation with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), or the phorbol ester PMA. Protease expression was inhibited (knocked down) with siRNA and validated using real-time PCR.


RESULTS
Proinflammatory cytokines IL-1beta and TNFalpha up-regulated VCAM-1 ectodomain release from the MCEC-1 cells, and this was dependant on p38 and mitogen-activated protein kinases (MAP kinases) and inhibited by the matrix metalloproteinase (MMP) inhibitor BB94 and tissue inhibitor of metalloproteinase (TIMP)-3, but not TIMP-1 or TIMP-2. Timp-3-/- cells exhibited greater VCAM-1 ectodomain release following cytokine stimulation than TIMP-3-expressing cells. Additionally, cytokine stimulation of MCEC-1 cells was shown to cause down-regulation of TIMP-3 expression. Knockdown of the metalloproteinase ADAM17, but not ADAM10 or ADAM12, gene expression reduced cytokine-stimulated VCAM-1 shedding.


CONCLUSIONS
TIMP-3 regulates the release of sVCAM-1 from cytokine-stimulated endothelial cells, which is mediated by ADAM17.
Singh
Robert J R
RJ
School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK.
Mason
Justin C
JC
Lidington
Elaine A
EA
Edwards
Dylan R
DR
Nuttall
Robert K
RK
Khokha
Rama
R
Knauper
Vera
V
Murphy
Gillian
G
Gavrilovic
Jelena
J
D000818:Animals; D001011:Aorta; D015152:Blotting, Northern; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D016207:Cytokines / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D048049:Extracellular Signal-Regulated MAP Kinases; D006801:Humans; D007375:Interleukin-1; D051379:Mice; D018345:Mice, Knockout; D047493:PPAR alpha; D013268:Stimulation, Chemical; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha; D014471:Umbilical Veins; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases
D017077:Culture Media, Conditioned; D016207:Cytokines; D007375:Interleukin-1; D047493:PPAR alpha; D019717:Tissue Inhibitor of Metalloproteinase-3; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases