Intracellular machinery for matrix degradation in bone-resorbing osteoclasts.
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.


BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Vääräniemi
Jukka
J
Department of Anatomy, Institute of Biomedicine, University of Turku, Turku FIN-20520, Finland. jukka.vaaraniemi@utu.fi
Halleen
Jussi M
JM
Kaarlonen
Katja
K
Ylipahkala
Hannele
H
Alatalo
Sari L
SL
Andersson
Göran
G
Kaija
Helena
H
Vihko
Pirkko
P
Väänänen
H Kalervo
HK
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D001857:Bone Matrix / Q000378:metabolism* / Q000473:pathology; D001862:Bone Resorption / Q000503:physiopathology*; D056657:Cathepsin K; D002403:Cathepsins / Q000378:metabolism; D002417:Cattle; D018929:Cell Culture Techniques; D011992:Endosomes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid; D007527:Isoenzymes / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D010010:Osteoclasts / Q000473:pathology / Q000502:physiology*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase
D015415:Biomarkers; D007527:Isoenzymes; D017382:Reactive Oxygen Species; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D002403:Cathepsins; D056657:Cathepsin K; C534133:Ctsk protein, rat