Everts V, Delaissé J M, Korper W, Beertsen W
Department of Cell Biology and Histology, Amsterdam, The Netherlands.
J Bone Miner Res. 1998 Sep;13(9):1420-30. doi: 10.1359/jbmr.1998.13.9.1420.
Digestion of calvarial bone by osteoclasts depends on the activity of cysteine proteinases and matrix metalloproteinases (MMPs). It is unknown, however, whether these enzymes act simultaneously or in a certain (time) sequence. In the present study, this was investigated by culturing mouse calvarial bone explants for various time intervals in the presence or absence of selective low molecular weight inhibitors of cysteine proteinases (E-64, Z-Phe-Tyr(O-t-Bu)CHN2 or CA074[Me]) and MMPs (CI-1, CT1166, or RP59794). The explants were morphometrically analyzed at the electron microscopic level. All proteinase inhibitors induced large areas of nondigested demineralized bone matrix adjacent to the ruffled border of actively resorbing osteoclasts. The appearance of these areas proved to be time dependent. In the presence of the cysteine proteinase inhibitors, a maximal surface area of demineralized bone was seen between 4 and 8 h of culturing, whereas the metalloproteinase inhibitors had their maximal effect at a later time interval (between 16 and 24 h). Because different inhibitors of each of the two classes of proteolytic enzymes had the same effects, our data strongly suggest that cysteine proteinases attack the bone matrix prior to digestion by MMPs. In line with the view that a sequence may exist were differences in the amount of proteoglycans (shown with the selective dye cuprolinic blue) in the subosteoclastic demineralized areas induced by the inhibitors. In the presence of the cysteine proteinase inhibitor, relatively high levels of cuprolinic blue precipitates were found, whereas this was less following inhibition of metalloproteinases. These data suggested that cysteine proteinases are important for digestion of noncollagenous proteins. We propose the following sequence in the digestion of calvarial bone by osteoclasts: after attachment of the cell to the mineralized surface an area with a low pH is created which results in dissolution of the mineral, then cysteine proteinases, active at such a low pH, digest part of the bone matrix, and finally, when the pH has increased somewhat, MMPs exert their activity.
破骨细胞对颅骨的消化依赖于半胱氨酸蛋白酶和基质金属蛋白酶（MMPs）的活性。然而，尚不清楚这些酶是同时发挥作用还是按一定（时间）顺序起作用。在本研究中，通过在存在或不存在半胱氨酸蛋白酶（E-64、Z-苯丙氨酸-酪氨酸（叔丁基-O）CHN2或CA074[Me]）和MMPs（CI-1、CT1166或RP59794）的选择性低分子量抑制剂的情况下，将小鼠颅骨外植体培养不同时间间隔来对此进行研究。在电子显微镜水平对这些外植体进行形态计量分析。所有蛋白酶抑制剂均在活跃吸收的破骨细胞的皱褶缘附近诱导出大片未消化的脱矿骨基质。这些区域的出现被证明是时间依赖性的。在存在半胱氨酸蛋白酶抑制剂的情况下，培养4至8小时时可见脱矿骨的最大表面积，而金属蛋白酶抑制剂在较晚的时间间隔（16至24小时之间）具有最大作用。由于两类蛋白水解酶各自的不同抑制剂具有相同的作用，我们的数据强烈表明半胱氨酸蛋白酶在MMPs消化之前攻击骨基质。与可能存在顺序的观点一致的是，抑制剂诱导的破骨细胞下脱矿区域中蛋白聚糖（用选择性染料铜叶绿酸蓝显示）的量存在差异。在存在半胱氨酸蛋白酶抑制剂的情况下，发现铜叶绿酸蓝沉淀水平相对较高，而在抑制金属蛋白酶后则较低。这些数据表明半胱氨酸蛋白酶对非胶原蛋白的消化很重要。我们提出破骨细胞对颅骨消化的以下顺序：细胞附着于矿化表面后，形成一个低pH区域，导致矿物质溶解，然后在如此低pH下有活性的半胱氨酸蛋白酶消化部分骨基质，最后，当pH有所升高时，MMPs发挥其活性。
