Vääräniemi Jukka, Halleen Jussi M, Kaarlonen Katja, Ylipahkala Hannele, Alatalo Sari L, Andersson Göran, Kaija Helena, Vihko Pirkko, Väänänen H Kalervo
Department of Anatomy, Institute of Biomedicine, University of Turku, Turku FIN-20520, Finland.
J Bone Miner Res. 2004 Sep;19(9):1432-40. doi: 10.1359/JBMR.040603. Epub 2004 Jun 14.
In osteoclasts, TRACP co-localized with cathepsin K in transcytotic vesicles and was activated by cathepsin K in vitro, suggesting that TRACP may degrade organic matrix components in transcytotic vesicles in an event regulated by cathepsin K.
TRACP is an enzyme with unknown biological function. In addition to its phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS). Bone-resorbing osteoclasts contain large amounts of TRACP, and transgenic animal models suggest that TRACP has a role in bone resorption. Osteoclasts resorb bone by secreting acid and lysosomal enzymes such as cathepsin K into an extracellular resorption lacuna between the cell membrane and bone surface. Matrix degradation products are then endocytosed, transcytosed, and secreted through a functional secretory domain in the basolateral membrane facing bone marrow.
We have studied intracellular localization of TRACP in osteoclasts with antibodies against various known endosomal and lysosomal proteins using confocal microscopy. We also studied co-localization of TRACP with cathepsin K and endocytosed bone matrix components and the effect of cathepsin K digestion on the ROS generating activity of TRACP in vitro.
Double-staining experiments of TRACP with endosomal and lysosomal markers showed that, although some endosomal staining was detected, TRACP was not present in lysosomes. However, TRACP was present in transcytotic vesicles, where it co-localized with cathepsin K. Cathepsin K digestion of TRACP in vitro increased the phosphatase activity by 5.6-fold and the ROS generating activity by 2.0-fold.
These results suggest that cathepsin K may activate the ROS-generating activity of TRACP in transcytotic vesicles of resorbing osteoclasts, the ROS being targeted to finalize degradation of organic bone matrix components during their transcytosis.
在破骨细胞中，抗酒石酸酸性磷酸酶（TRACP）与组织蛋白酶K在转胞吞小泡中共定位，并且在体外被组织蛋白酶K激活，这表明TRACP可能在由组织蛋白酶K调节的过程中降解转胞吞小泡中的有机基质成分。
TRACP是一种生物学功能未知的酶。除了其磷酸酶活性外，TRACP还能够产生活性氧（ROS）。骨吸收破骨细胞含有大量TRACP，转基因动物模型表明TRACP在骨吸收中起作用。破骨细胞通过将酸和组织蛋白酶K等溶酶体酶分泌到细胞膜和骨表面之间的细胞外吸收腔中来吸收骨。然后，基质降解产物被内吞、转胞吞，并通过面向骨髓的基底外侧膜中的功能性分泌结构域分泌。
我们使用共聚焦显微镜，用针对各种已知内体和溶酶体蛋白的抗体研究了TRACP在破骨细胞中的细胞内定位。我们还研究了TRACP与组织蛋白酶K和内吞骨基质成分的共定位，以及组织蛋白酶K消化对TRACP体外产生活性氧活性的影响。
TRACP与内体和溶酶体标记物的双重染色实验表明，虽然检测到一些内体染色，但TRACP不存在于溶酶体中。然而，TRACP存在于转胞吞小泡中，在那里它与组织蛋白酶K共定位。体外组织蛋白酶K对TRACP的消化使磷酸酶活性增加了5.6倍，产生活性氧的活性增加了2.0倍。
这些结果表明，组织蛋白酶K可能激活吸收性破骨细胞转胞吞小泡中TRACP的产生活性氧活性，活性氧的作用是在有机骨基质成分转胞吞过程中完成其降解。
