Hypoxia reduces the output of matrix metalloproteinase-9 (MMP-9) in monocytes by inhibiting its secretion and elevating membranal association

Abstract

Cellular hypoxia, characterizing tumors, ischemia, and inflammation induce recruitment of monocytes/macrophages, immobilize them at the hypoxic site, and alter their function. To migrate across the extracellular matrix and as part of their inflammatory functions, monocytes and macrophages secrete proteases, including matrix metalloproteinase-9 (MMP-9), whose expression is induced by proinflammatory cytokines [e.g., tumor necrosis factor alpha (TNF-alpha)]. We show that hypoxia (<0.3% O2 for 48 h) reduced the output of TNF-alpha-induced proMMP-9 by threefold (P < 0.01) in the U937 monocytic cell line and in primary human monocytes. TNF-alpha induced MMP-9 transcription by threefold, but no significant difference was observed in MMP-9 mRNA steady-state between normoxia and hypoxia, which inhibited the trafficking of proMMP-9 via secretory vesicles and increased the intracellular accumulation of proMMP-9 in the cells by 47% and 62% compared with normoxia (P < 0.05), as evaluated by zymography of cellular extracts and confocal microscopy, respectively. Secretion of proMMP-9 was reduced by the addition of cytochalazin B or nocodazole, which inhibits the polymerization of actin and tubulin fibers, or by the addition of the Rho kinase inhibitor Y27632, suggesting the involvement of the cytoskeleton and the Rho GTPases in the process of enzyme secretion. Furthermore, attachment of proMMP-9 to the cell membrane increased after hypoxia via its interactions with surface molecules such as CD44. In addition, the reduced migration of monocytes in hypoxia was shown to be mediated, at least partially, by secreted MMP-9. Thus, hypoxia post-translationally reduced the secreted amounts of proMMP-9 by using two mutually nonexclusive mechanisms: mostly, inhibition of cellular trafficking and to a lesser extent, attachment to the membrane.

Expression, pharmacology, and functional role of somatostatin receptor subtypes 1 and 2 in human macrophages

“…Using this method, which enables the detection of the zymogenic and the activated forms of MMP-9, only the latent form of the enzyme (proMMP-9, 92 kDa band) could be observed (Fig. 6), as reported recently in human PBMC-derived monocytes [39]. In particular, LPStreated macrophages exhibited a distinct band corresponding to proMMP-9 which was unaffected by cell treatment with 1 M SRIF-14, CH-275, SMS 201-995, or L-779,976, suggesting that activation of SRIF receptors did not lead to secretion of MMP-9.…”

“…Using published protocols [39], matrix metalloproteinase (MMP)-9 secretion by macrophages cultured for 24 h in six-well plates was measured by SDS-PAGE zymography, which enables the detection of the zymogenic and the activated forms of MMP-9. Briefly, 20 l samples of conditioned supernatants were electrophoresed at 4°C by the Bio-Rad Mini-PROTEAN 3 system on 7.5% polyacrylamide gels containing 10% SDS and gelatine (1 mg/ml) as substrate under nonreducing conditions and without boiling.…”

Classically Activated Macrophages Use Stable Microtubules for Matrix Metalloproteinase-9 (MMP-9) Secretion

“…Blocking of MMP-9 by monoclonal antibodies inhibits the migration of bone marrow stem cells into the circulation (28), and MMP-9 has been shown to assist in monocyte migration (29,30). Additionally, a role for MMP-9 in macrophage migration during the inflammatory response was demonstrated in vivo, where inhibition of MMP-9 activation resulted in the abolished ability of macrophages to migrate to the elicited site of inflammation (23).…”

Kalopanaxsaponin A Inhibits the Invasion of Human Oral Squamous Cell Carcinoma by Reducing Metalloproteinase-9 mRNA Stability and Protein Trafficking

“…37) Exocytic trafficking of secretory vesicles is controlled by small Rab GTPases, which are regulators of membrane traffic and are common to all eukaryotic cells. 38,39) More than 60 human Rab proteins have been identified; this high number of different Rab proteins reflects their importance in the regulation of vesicle trafficking processes, including vesicle formation, motility, tethering and fusion to the acceptor membrane. 40) Defective Rab or associated proteins have been implicated in rare monogenic inherited diseases as well as common multifactorial human conditions.…”