title: Polarized osteoclasts put marks of tartrate-resistant acid phosphatase on dentin slices--a simple method for identifying polarized osteoclasts.
abstract: Osteoclasts form ruffled borders and sealing zones toward bone surfaces to resorb bone. Sealing zones are defined as ringed structures of F-actin dots (actin rings). Polarized osteoclasts secrete protons to bone surfaces via vacuolar proton ATPase through ruffled borders. Catabolic enzymes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K are also secreted to bone surfaces. Here we show a simple method of identifying functional vestiges of polarized osteoclasts. Osteoclasts obtained from cocultures of mouse osteoblasts and bone marrow cells were cultured for 48 h on dentin slices. Cultures were then fixed and stained for TRAP to identify osteoclasts on the slices. Cells were removed from the slices with cotton swabs, and the slices subjected to TRAP and Mayer's hematoxylin staining. Small TRAP-positive spots (TRAP-marks) were detected in the resorption pits stained with Mayer's hematoxylin. Pitted areas were not always located in the places of osteoclasts, but osteoclasts existed on all TRAP-marks. A time course experiment showed that the number of TRAP-marks was maintained, while the number of resorption pits increased with the culture period. The position of actin rings formed in osteoclasts corresponded to that of TRAP-marks on dentin slices. Immunostaining of dentin slices showed that both cathepsin K and vacuolar proton ATPase were colocalized with the TRAP-marks. Treatment of osteoclast cultures with alendronate, a bisphosphonate, suppressed the formation of TRAP-marks and resorption pits without affecting the cell viability. Calcitonin induced the disappearance of both actin rings and TRAP-marks in osteoclast cultures. These results suggest that TRAP-marks are vestiges of proteins secreted by polarized osteoclasts.
authors: Institute for Oral Science, Matsumoto Dental University, Shiojiri-shi, Nagano, 399-0781, Japan.
authors: Takahiro
authors: T
authors: Nakayama
authors: Toshihide
authors: T
authors: Mizoguchi
authors: Shunsuke
authors: S
authors: Uehara
authors: Teruhito
authors: T
authors: Yamashita
authors: Ichiro
authors: I
authors: Kawahara
authors: Yasuhiro
authors: Y
authors: Kobayashi
authors: Yoshinori
authors: Y
authors: Moriyama
authors: Saburo
authors: S
authors: Kurihara
authors: Noriyuki
authors: N
authors: Sahara
authors: Hidehiro
authors: H
authors: Ozawa
authors: Nobuyuki
authors: N
authors: Udagawa
authors: Naoyuki
authors: N
authors: Takahashi
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D019386:Alendronate / Q000494:pharmacology; D000818:Animals; D001862:Bone Resorption / Q000473:pathology; D002116:Calcitonin / Q000494:pharmacology; D056657:Cathepsin K / Q000378:metabolism; D016764:Cell Polarity / Q000187:drug effects; D003584:Cytological Techniques / Q000379:methods*; D003804:Dentin / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D051379:Mice; D010010:Osteoclasts / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D000071681:Tartrate-Resistant Acid Phosphatase; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
chemical_list: D000199:Actins; D007527:Isoenzymes; D002116:Calcitonin; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D056657:Cathepsin K; D025262:Vacuolar Proton-Translocating ATPases; D019386:Alendronate