In osteoclasts (OCs) podosomes are organized in a belt, a feature critical for bone resorption. Although microtubules (MTs) promote the formation and stability of the belt, the MT and/or podosome molecules that mediate the interaction of the two systems are not identified. Because the growing "plus" ends of MTs point toward the podosome belt, plus-end tracking proteins (؉TIPs) might regulate podosome patterning. Among the ؉TIPs, EB1 increased as OCs matured and was enriched in the podosome belt, and EB1-positive MTs targeted podosomes. Suppression of MT dynamic instability, displacement of EB1 from MT ends, or EB1 depletion resulted in the loss of the podosome belt. We identified cortactin as an Src-dependent interacting partner of EB1. Cortactin-deficient OCs presented a defective MT targeting to, and patterning of, podosomes and reduced bone resorption. Suppression of MT dynamic instability or EB1 depletion increased cortactin phosphorylation, decreasing its acetylation and affecting its interaction with EB1. Thus, dynamic MTs and podosomes interact to control bone resorption.

Bone remodeling requires osteoclasts to generate and maintain an acidified resorption compartment between the apical membrane and the bone surface to solubilize hydroxyapatite crystals within the bone matrix. This acidification process requires (i) apical proton secretion by a vacuolar H + -ATPase, (ii) actin cytoskeleton reorganization into a podosome belt that forms a gasket to restrict lacunar acid leakage, and (iii) basolateral chloride uptake and bicarbonate extrusion by an anion exchanger to provide Cl − permissive for apical acid secretion while preventing cytoplasmic alkalinization. Here we show that osteoclast-targeted deletion in mice of solute carrier family 4 anion exchanger member 2 (Slc4a2) results in osteopetrosis. We further demonstrate a previously unrecognized consequence of SLC4A2 loss of function in the osteoclast: dysregulation of calpain-dependent podosome disassembly, leading to abnormal actin belt formation, cell spreading, and migration. Rescue of SLC4A2-deficient osteoclasts with functionally defined mutants of SLC4A2 indicates regulation of actin cytoskeletal reorganization by anion-exchange activity and intracellular pH, independent of SLC4A2's long N-terminal cytoplasmic domain. These data suggest that maintenance of intracellular pH in osteoclasts through anion exchange regulates the actin superstructures required for bone resorption.

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Tartrate-resistant acid phosphatase, although encoded by a single gene, exists as two isoforms in human serum, TRAP 5a and 5b, differing in post-translational modifications such as proteolytic processing and kinetic properties including pH optimum and specific activity. The biogenetic relationship between the TRAP isoforms was assessed in a stably transfected breast cancer epithelial MDA-MB-231 cell subline overexpressing 5a- and 5b-like TRAP isoforms intracellularly, with only the monomeric 5a-like isoform being secreted. As judged by immunolocalization and comparative N-glycan profiling by Con A lectin chromatography and glycanase analysis, the majority of the intracellular monomeric TRAP was destined for secretion, while a minor portion provided the putative precursor for the intracellular 5b-like isoform. Brefeldin A blocked secretion of 5a-like TRAP isoform as well as appearance of its putative intracellular precursor, and augmented the intracellular level of proteolytically processed 5b-like isoform, indicating a common early biosynthetic precursor for TRAP isoforms 5a and 5b. The cysteine proteinase inhibitor E64 partially blocked formation of the 5b-like isoform while augmenting the level of its putative monomeric precursor, but did not alter the levels of secreted TRAP or its intracellular precursor, suggesting that distinct precursors for secreted TRAP 5a and intracellular 5b-like isoform are segregated in the ER or Golgi prior to proteolytic processing. In conclusion, these data provide evidence that distinct monomeric TRAP populations are diverted early in the secretory pathway either giving rise to a secreted, monomeric 5a-like TRAP isoform or to an intracellular, proteolytically processed 5b-like TRAP isoform.

Tartrate-resistant acid phosphatase (TRAP) exists in human serum as two major isoforms, monomeric 5a and proteolytically processed enzymatically active 5b. The 5b isoform is secreted by osteoclasts and has recently been advocated as a serum marker for bone metastasis in breast cancer patients. The 5a isoform, on the other hand, is not bone-derived and has been proposed to be a marker of activated macrophages and chronic inflammation. In this study, expression of TRAP protein and enzymatic activity in bone metastases from different primary sites was examined. TRAP activity was high in bone metastases from prostate cancer, intermediate in breast cancer, and low in lung and kidney cancers. The partially purified TRAP from breast cancer bone metastasis samples exhibited the enzymatic characteristics of purple acid phosphatase. Both 5a and 5b isoforms were expressed in bone metastases of different histogenetic origins, i.e. prostate, breast, lung and kidney, and also a novel previously unreported 42 kDa variant of the TRAP 5a isoform was identified in bone metastases. This novel TRAP 5a isoform was absent in human bone, indicating that the 42 kDa variant is specific to metastatic cancer tissue. Immunohistochemistry revealed that metastatic cancer cells were the predominant source of TRAP 5a, whereas tumor-associated macrophages and occasionally multinucleated giant cells in the tumor stroma preferentially expressed the proteolytically processed TRAP 5b variant. Our results indicate the presence of a previously unstudied variant of monomeric TRAP 5a in cancer cells, which may have functional and diagnostic implications. Moreover, the presence of TRAP-positive macrophages in bone metastases could, together with cancer cells and osteoclasts, contribute to the elevated levels of serum TRAP activity observed in patients with bone metastases.

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