Bai Shan, Thummel Ryan, Godwin Alan R, Nagase Hideaki, Itoh Yoshifumi, Li Li, Evans Richard, McDermott Jeffrey, Seiki Motoharu, Sarras Michael P
Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Matrix Biol. 2005 Jun;24(4):247-60. doi: 10.1016/j.matbio.2005.03.007.
Matrix metalloproteinases (MMPs) play key roles in the turnover of extracellular matrix (ECM) and, thereby, function as key regulators of cell-ECM interactions during development. In spite of their importance during developmental processes, relatively little has been reported about the role of these metalloproteinases during limb development and regeneration. To approach the problem of cell-ECM interactions during limb (fin) regeneration, we have utilized zebrafish as an experimental model. Based on previous MMP cloning studies from our laboratory, the current study has focused on the expression of membrane-type 1 metalloproteinase (MT1-MMP), gelatinase A (MMP-2) and endogenous tissue inhibitor 2 of metalloproteinases (TIMP-2) during fin regeneration in adult zebrafish. In situ analysis indicated co-expression of zmt1-mmp, zmmp-2, and ztimp-2 mRNA transcripts in regenerating caudal fins. In situ gelatin-zymography confirmed the presence of active metalloproteinases in regenerating fins. zmt1-mmp, zmmp-2, and ztimp-2 mRNA transcripts were expressed in the blastema and basal epithelium during caudal fin regeneration while expression of type IV collagen [zcol-IV(a5)] transcripts (a basal lamina component) was restricted to the basal epithelium. Fin outgrowth was greatly reduced in the presence of GM6001 (an inhibitor of MMP activity) indicating the importance of these enzymes during fin regeneration. Previous studies by Itoh (EMBO, 2001) indicated that expression of a vertebrate MT1-MMP construct containing only the hemopexin-transmembrane-cytoplasmic domains (MT1HPX) resulted in blockage of MT1-MMP homophilic complex formation and subsequent inhibition of pro-MMP-2 activation. Interference with homophilic complex formation was attributed to expression of the hemopexin domain at the cell surface. Building upon these earlier findings, the current study found that ectopic expression of MT1HPX in fin regenerates inhibited the regeneration process and resulted in a reduction in cell proliferation in the blastema. Taken together, these results indicate that MMPs have an important role during fin regeneration in zebrafish.
基质金属蛋白酶（MMPs）在细胞外基质（ECM）的周转中起关键作用，因此在发育过程中作为细胞与ECM相互作用的关键调节因子。尽管它们在发育过程中很重要，但关于这些金属蛋白酶在肢体发育和再生中的作用报道相对较少。为了解决肢体（鳍）再生过程中的细胞与ECM相互作用问题，我们利用斑马鱼作为实验模型。基于我们实验室之前的MMP克隆研究，本研究聚焦于成年斑马鱼鳍再生过程中膜型1金属蛋白酶（MT1-MMP）、明胶酶A（MMP-2）和金属蛋白酶内源性组织抑制剂2（TIMP-2）的表达。原位分析表明，zmt1-mmp、zmmp-2和ztimp-2 mRNA转录本在再生尾鳍中共表达。原位明胶酶谱分析证实再生鳍中存在活性金属蛋白酶。在尾鳍再生过程中，zmt1-mmp、zmmp-2和ztimp-2 mRNA转录本在芽基和基底上皮中表达，而IV型胶原蛋白[zcol-IV(a5)]转录本（基底膜成分）的表达仅限于基底上皮。在GM6001（一种MMP活性抑制剂）存在的情况下，鳍的生长显著减少，表明这些酶在鳍再生过程中的重要性。Itoh（《欧洲分子生物学组织杂志》，2001年）之前的研究表明，仅包含血红素结合蛋白-跨膜-细胞质结构域（MT1HPX）的脊椎动物MT1-MMP构建体的表达导致MT1-MMP同源复合物形成受阻，并随后抑制前MMP-2的激活。对同源复合物形成的干扰归因于血红素结合蛋白结构域在细胞表面的表达。基于这些早期发现，本研究发现MT1HPX在鳍再生组织中的异位表达抑制了再生过程，并导致芽基中细胞增殖减少。综上所述，这些结果表明MMPs在斑马鱼鳍再生过程中起重要作用。
