The a3 Isoform of the 100-kDa V-ATPase Subunit Is Highly but Differentially Expressed in Large (≥10 Nuclei) and Small (≤5 Nuclei) Osteoclasts

Abstract

Osteoclasts dissolve bone through acidification of an extracellular compartment by means of a multimeric vacuolar type H+-ATPase (V-ATPase). In mammals, there are four isoforms of the 100-kDa V-ATPase "a" subunit. Mutations in the a3 isoform result in deficient bone resorption and osteopetrosis, suggesting that a3 has a unique function in osteoclasts. It is thus surprising that several studies show a basal level of a3 expression in most tissues. To address this issue, we have compared a3 expression in bone with expression in other tissues. RNA blots revealed that the a3 isoform was expressed highest in bone and confirmed its expression (in decreasing order) in liver, kidney, brain, lung, spleen, and muscle. In situ hybridization on bone tissue sections revealed that the a3 isoform was highly expressed in multinucleated osteoclasts but not in mononuclear stromal cells, whereas the a1 isoform was expressed in both cell types at about the same level. We also found that a3 expression was greater in osteoclasts with 10 or more nuclei as compared with osteoclasts with five or fewer nuclei. We hypothesize that these differences in a3 expression may be associated with previously demonstrated differences between large and small osteoclasts with reference to their resorptive activity.

Tropomyosin 4 regulates adhesion structures and resorptive capacity in osteoclasts

“…Loading control antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta actin, both mouse monoclonal antibodies, were purchased from Abcam (Cambridge, MA). An antibody against V-ATPase subunit a3 was generated in this laboratory and was described previously [14]. Antibodies to paxillin and α-actinin were obtained from BD Transduction Laboratories and Sigma, respectively.…”

Osteopetrosis Mutation R444L Causes Endoplasmic Reticulum Retention and Misprocessing of Vacuolar H+-ATPase a3 Subunit

“…Vph1p is localized to the vacuole, whereas Stv1p is found primarily in Golgi (11,12). The mammalian a subunit has four isoforms, a1-4; a1, a2, and a3 are ubiquitously expressed, but to different degrees in different tissues and organelles, whereas a4 expression appears to be specific to plasma membranes of renal intercalated cells (9,(13)(14)(15). Although ubiquitously expressed, a3 appears to be most highly enriched in osteoclasts (9).…”

“…The mammalian a subunit has four isoforms, a1-4; a1, a2, and a3 are ubiquitously expressed, but to different degrees in different tissues and organelles, whereas a4 expression appears to be specific to plasma membranes of renal intercalated cells (9,(13)(14)(15). Although ubiquitously expressed, a3 appears to be most highly enriched in osteoclasts (9). In actively bone-resorbing osteoclasts, V-ATPases containing the a3 subunit isoform are specifically targeted to the osteoclast ruffled border, where they are involved in acidifying the resorption lacuna to demineralize bone (15).…”

Comparison of expression patterns of cathepsin K and MMP-9 in odontoclasts and osteoclasts in physiological root resorption in the rat molar

“…To determine the morphometric characteristics of three kinds of TRAP-positive cells (odontoclasts, osteoclasts, and their precursor cells), we measured the number of cellular nuclei and the TRAP-positive area, which both correlate to their resorption activities (Teti et al, 1991;Lees et al, 2001;Manolson et al, 2003;Trebec et al, 2007;Hu et al, 2008). Briefl y, the serial sections from maxillae were stained with TRAP after deparaffi nization and dehydration.…”

“…Root resorption lacunae are principally formed by odontoclasts, which develop from the mature monocyte/ macrophage lineage and have cytologic features similar to osteoclasts, including multiple nuclei, ruffl ed borders, clear zones, and tartrate-resistant acid phosphate (TRAP) activity (Domon et al, 2006). It has been proposed that osteoclasts undergoing even more differentiation have a greater number of nuclei with a higher bone resorption activity (Teti et al, 1991;Lees et al, 2001;Manolson et al, 2003). Since odontoclasts normally have fewer nuclei and less clear zones compared with osteoclasts (Tanaka et al, 1990;Ne et al, 1999), we hypothesized that odontoclasts possess less cell differentiation in their matrix resorption characteristics than osteoclasts.…”