Henriksen Kim, Sørensen Mette G, Nielsen Rasmus H, Gram Jeppe, Schaller Sophie, Dziegiel Morten H, Everts Vincent, Bollerslev Jens, Karsdal Morten A
Pharmos Bioscience, Nordic Bioscience and Center for Clinical and Basic Research A/S, Herlev, Denmark.
J Bone Miner Res. 2006 Jan;21(1):58-66. doi: 10.1359/JBMR.050905. Epub 2005 Sep 6.
Osteoclasts degrade bone matrix by secretion of hydrochloric acid and proteases. We studied the processes involved in the degradation of the organic matrix of bone in detail and found that lysosomal acidification is involved in this process and that MMPs are capable of degrading the organic matrix in the absence of cathepsin K.
Osteoclasts resorb bone by secretion of acid by the vacuolar H+-adenosine triphosphatase (V-ATPase) and the chloride channel ClC-7, followed by degradation of the matrix, mainly collagen type I, by cathepsin K and possibly by matrix metalloproteinases (MMPs). However, the switch from acidification to proteolysis and the exact roles of both the ion transporters and the proteinases still remain to be studied.
We isolated CD14+ monocytes from human peripheral blood from either controls or patients with autosomal dominant osteopetrosis type II (ADOII) caused by defective ClC-7 function and cultured them in the presence of RANKL and macrophage-colony stimulating factor (M-CSF) to generate osteoclasts. We decalcified cortical bovine bone slices and studied the osteoclasts with respect to morphology, markers, and degradation of the decalcified matrix in the presence of various inhibitors of osteoclast acidification and proteolysis, using normal calcified bone as a reference.
We found that ADOII osteoclasts not only have reduced resorption of the calcified matrix, but also 40% reduced degradation of the organic phase of bone. We found that both acidification inhibitors and cathepsin K inhibitors reduced degradation of the organic matrix by 40% in normal osteoclasts, but had no effect in the ADOII osteoclasts. Furthermore, we showed that inhibition of MMPs leads to a 70% reduction in the degradation of the organic bone matrix and that MMPs and cathepsin K have additive effects. Finally, we show that osteoclastic MMPs mediate release of the carboxyterminal telopeptide of type I collagen (ICTP) fragment in the absence of cathepsin K activity, and therefore, to some extent, are able to compensate for the loss of cathepsin K activity.
These data clearly show that osteoclastic acidification of the lysosomes plays a hitherto nonrecognized role in degradation of the organic matrix. Furthermore, these data shed light on the complicated interplay between acidification dependent and independent proteolytic processes, mediated by cathepsin K and the MMPs, respectively.
破骨细胞通过分泌盐酸和蛋白酶来降解骨基质。我们详细研究了骨有机基质降解过程中涉及的机制，发现溶酶体酸化参与了这一过程，并且基质金属蛋白酶（MMPs）在组织蛋白酶K缺失的情况下能够降解有机基质。
破骨细胞通过液泡型H⁺-腺苷三磷酸酶（V-ATPase）和氯离子通道ClC-7分泌酸，随后主要由组织蛋白酶K以及可能由基质金属蛋白酶（MMPs）降解基质，主要是I型胶原蛋白。然而，从酸化到蛋白水解的转换以及离子转运蛋白和蛋白酶的确切作用仍有待研究。
我们从对照组或由ClC-7功能缺陷引起的常染色体显性遗传性II型骨硬化症（ADOII）患者的人外周血中分离出CD14⁺单核细胞，并在存在核因子κB受体活化因子配体（RANKL）和巨噬细胞集落刺激因子（M-CSF）的情况下培养，以生成破骨细胞。我们对皮质牛骨切片进行脱钙处理，并以正常钙化骨为对照，在存在各种破骨细胞酸化和蛋白水解抑制剂的情况下，研究破骨细胞的形态、标志物以及脱钙基质的降解情况。
我们发现ADOII破骨细胞不仅对钙化基质的吸收减少，而且骨有机相的降解减少了40%。我们发现酸化抑制剂和组织蛋白酶K抑制剂在正常破骨细胞中均使有机基质的降解减少40%，但对ADOII破骨细胞没有影响。此外，我们表明抑制MMPs会导致骨有机基质降解减少70%，并且MMPs和组织蛋白酶K具有累加效应。最后，我们表明在没有组织蛋白酶K活性的情况下，破骨细胞MMPs介导I型胶原蛋白（ICTP）羧基末端肽片段的释放，因此在一定程度上能够补偿组织蛋白酶K活性的丧失。
这些数据清楚地表明，破骨细胞溶酶体的酸化在有机基质降解中发挥了迄今未被认识的作用。此外，这些数据揭示了分别由组织蛋白酶K和MMPs介导的酸化依赖性和非依赖性蛋白水解过程之间复杂的相互作用。
