Reponen P, Sahlberg C, Munaut C, Thesleff I, Tryggvason K
Biocenter, University of Oulu, Finland.
J Cell Biol. 1994 Mar;124(6):1091-1102. doi: 10.1083/jcb.124.6.1091.
cDNA clones for murine 92 kD type IV collagenase (gelatinase B) were generated for the determination of its primary structure and for analysis of temporal and spatial expression in vivo. The mouse enzyme has 72% sequence identity with the human counterpart, the major difference being the presence of a 16-residue segment absent from the human enzyme. In situ hybridization analyses of embryonic and postnatal mouse tissues revealed intense signals in cells of the osteoclast cell lineage. Clear expression above background was not observed in macrophages, polymorphonuclear leukocytes, monocytes, or epithelial cells which have been shown to express the gene in vitro in cell cultures. Expression of the gene was first observed at early stage of cartilage and tooth development at E13, where signals were seen transiently in surrounding mesenchymal cells. At later developmental stages and postnatally strong expression was seen in large cells at the surface of bones. These cells were presumably osteoclasts as their location correlated with that of TRAP positive cells. Signals above background were not observed in a number of other tissues studied. The results represent the first demonstration of a highly osteoclast specific extracellular proteinase. The results suggest that during normal development of embryonic organs the 92-kD type IV collagenase does not have a major role in basement membrane degradation, but is rather mainly used for the turnover of bone matrix, possibly as a gelatinase required for the removal of denatured collagen fragments (gelatin) generated by interstitial collagenase.
为了确定小鼠92 kD IV型胶原酶（明胶酶B）的一级结构并分析其在体内的时空表达，构建了其cDNA克隆。该小鼠酶与人类对应酶具有72%的序列同一性，主要差异在于人类酶中不存在的一个16个残基的片段。对胚胎期和出生后小鼠组织的原位杂交分析显示，破骨细胞谱系的细胞中有强烈信号。在巨噬细胞、多形核白细胞、单核细胞或上皮细胞中未观察到明显高于背景的表达，这些细胞在体外细胞培养中已被证明可表达该基因。该基因的表达在胚胎第13天软骨和牙齿发育的早期首次被观察到，在周围间充质细胞中可见短暂信号。在发育后期和出生后，在骨表面的大细胞中可见强表达。这些细胞可能是破骨细胞，因为它们的位置与抗酒石酸酸性磷酸酶（TRAP）阳性细胞的位置相关。在研究的许多其他组织中未观察到高于背景的信号。这些结果首次证明了一种高度破骨细胞特异性的细胞外蛋白酶。结果表明，在胚胎器官的正常发育过程中，92-kD IV型胶原酶在基底膜降解中不起主要作用，而是主要用于骨基质的周转，可能作为一种明胶酶，用于去除间质胶原酶产生的变性胶原片段（明胶）。
