The osteoclast proton pump differs in its pharmacology and catalytic subunits from other vacuolar H+-ATPases

Abstract

Osteoclasts are multinucleated cells derived from the mononuclear phagocyte system in the hematopoietic bone marrow. Their function is to resorb bone during skeletal growth and remodeling. They perform this function by acidifying an enclosed extracellular space, the bone resorbing compartment. Analysis of proton transport by inside-out vesicles derived from highly purified chicken osteoclast membranes has revealed the presence of a novel type of multisubunit vacuolar-like H(+)-ATPase. Unlike H(+)-ATPases derived from any other cell type or organelle, proton transport and ATPase activity in osteoclast vesicles are sensitive to two classes of inhibitors, namely V-ATPase inhibitors [N-ethyl-maleimide (NEM) and bafilomycin A1] and vanadate (IC50 100 mumol l-1), an inhibitor previously found to affect only P-ATPases. The osteoclast V-ATPase morphologically resembles vacuolar proton pumps and contains several vacuolar-like subunits (115 x 10(3), 39 x 10(3) and 16 x 10(3)M(r)), demonstrated by Western blot analysis. Subunits A and B of the catalytic domain of the enzyme, however, differ from that of other V-ATPases. In osteoclasts, subunit A has an M(r) of 63 x 10(3) instead of 67 x 10(3)-70 x 10(3); in contrast, monocytes, macrophages and kidney microsomes, which contain a vanadate-insensitive H(+)-ATPase, express the classical subunit A (70 x 10(3)M(r)). Moreover, two types of 57 x 10(3)-60 x 10(3)M(r) B subunits are also found: they are differentially recognized by antibodies and one is expressed predominantly in osteoclasts and the other in bone marrow cells and in kidney microsomes. Preliminary cloning data have indicated that the B subunit expressed in osteoclasts may be similar to the brain isoform. The osteoclast proton pump may, therefore, constitute a novel class of V-ATPase, with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme.

Suppression of AMP‐activated protein kinase reverses osteoprotegerin‐induced inhibition of osteoclast differentiation by reducing autophagy

“…52 BAF binds to the Vo subunits of V-ATPase to inhibit bone resorption activity, which results in OC apoptosis and the impairment of OC endocytosis. 53 BAF suppresses the expression of NF-κB and NFAT during RANKL-induced OC differentiation, resulting in smaller and fewer OC nuclei, due to increased p62 expression. 30 In our study, BAF treatment reduced the number of OCs produced by M-CSF and RANKL treatment, and increased CTSK and NFATc1 expression, which is inconsistent with the results reported by Cai et al 54 CTSK is a lysosomal cysteine protease capable of cleaving the helical and telopeptide regions of type I collagen during OC differentiation.…”

“…Bafilomycin, a late‐stage lysosome inhibitor, could prevent lysosome‐autophagosome fusion, causing autophagic flux disruption . BAF binds to the Vo subunits of V‐ATPase to inhibit bone resorption activity, which results in OC apoptosis and the impairment of OC endocytosis . BAF suppresses the expression of NF‐κB and NFAT during RANKL‐induced OC differentiation, resulting in smaller and fewer OC nuclei, due to increased p62 expression .…”

Structural and mutational analyses of decarboxylated osteocalcin provide insight into its adiponectin‐inducing activity

“…We found that a high-quality crystal was obtained at pH 8.5 using the hanging drop method, and we determined its crystal structure at 1. 4 A resolution (Fig. 1, Table 1).…”

“…During bone destruction, an acidic environment is formed in bone lacunae. This is primarily due to the active secretion of protons through the osteoclastic V-ATPase and by the passive flow of chloride ions through the chloride channel ClC-7 in the osteoclast-bone interface (4)(5)(6)(7). The low pH conditions are essential for the dissolution of hydroxyapatite (8)(9)(10).…”