Matrix metalloproteinase (MMP)-9 plays an important role in cardiovascular events. However, the mechanisms underlying in vivo activation of MMP-9 are largely unknown. We investigated the secretion and activation of MMP-9 under a cell-to-cell interaction, and the effects of
hypoxia and
cytokine. Human umbilical vein endothelial cell (HUVEC) and THP-1 (human monocyte cell line) were cultured individually, or cocultured under normoxic and hypoxic conditions. In a coculture of HUVEC and THP-1,
proMMP-9 secretion was increased twofold compared with individual culture of HUVEC and THP-1, whereas MMP-2 secretion was unchanged. The increase in
proMMP-9 secretion was suppressed by antiadhesion molecule
antibodies and
mitogen-activated protein kinase inhibitors,
PD98059 (MAPK/ERK kinase1 inhibitor) and
SP600125 (
Jun N-terminal kinase inhibitor).
ProMMP-9 secretion was increased by
tumor necrosis factor (TNF)-α at 50 ng/ml (P < 0.05) but was not activated under normoxic (20%) conditions.
ProMMP-9 in coculture was activated under hypoxic (<1%) conditions, and was potentiated by TNF-α (both P < 0.05). To further investigate the mechanism of
hypoxia-induced MMP-9 activation,
heat shock protein (Hsp)90, which was suggested to be related to MMP-9 activation, was measured by Western blot analysis. The ratio of Hsp90 to
glyceraldehyde-3-phosphate dehydrogenase was increased in hypoxic (<1%) coculture conditions with TNF-α (P < 0.05). Treatment with
geldanamycin and
17-DMAG (Hsp90 inhibitor) suppressed the active form of MMP-9. Cell-to-cell interaction between endothelial cells and monocytes promotes
proMMP-9 synthesis and secretion.
Hypoxia and
inflammation are suggested to play an important role in activating
proMMP-9, presumably via Hsp90.