Rahat Michal A, Marom Barak, Bitterman Haim, Weiss-Cerem Lea, Kinarty Amalia, Lahat Nitza
Immunology Research Unit, Carmel Medical Center, 7 Michal St., Haifa, 34362, Israel.
J Leukoc Biol. 2006 Apr;79(4):706-18. doi: 10.1189/jlb.0605302. Epub 2006 Jan 24.
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.
细胞缺氧是肿瘤、缺血和炎症的特征，可诱导单核细胞/巨噬细胞的募集，将它们固定在缺氧部位，并改变其功能。为了穿过细胞外基质并作为其炎症功能的一部分，单核细胞和巨噬细胞会分泌蛋白酶，包括基质金属蛋白酶-9（MMP-9），其表达由促炎细胞因子[如肿瘤坏死因子α（TNF-α）]诱导。我们发现，缺氧（48小时内氧气含量<0.3%）使U937单核细胞系和原代人单核细胞中TNF-α诱导的前MMP-9产量降低了三倍（P<0.01）。TNF-α使MMP-9转录增加了三倍，但在常氧和缺氧条件下，MMP-9 mRNA稳态未观察到显著差异，缺氧抑制了前MMP-9通过分泌囊泡的运输，并使细胞内前MMP-9的积累量比常氧增加了47%和62%（P<0.05），分别通过细胞提取物的酶谱分析和共聚焦显微镜评估。添加抑制肌动蛋白和微管蛋白纤维聚合的细胞松弛素B或诺考达唑，或添加Rho激酶抑制剂Y27632，可减少前MMP-9的分泌，表明细胞骨架和Rho GTP酶参与了酶分泌过程。此外，缺氧后，前MMP-9通过与CD44等表面分子相互作用增加了与细胞膜的附着。此外，缺氧条件下单核细胞迁移减少至少部分是由分泌的MMP-9介导的。因此，缺氧通过两种互不排斥的机制在翻译后减少了前MMP-9的分泌量：主要是抑制细胞运输，其次是附着于细胞膜。
