Microtubule dynamic instability controls podosome patterning in osteoclasts through EB1, cortactin, and Src.

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.

SLC4A2-mediated Cl-/HCO3- exchange activity is essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts.

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.

Differential expression of tartrate-resistant acid phosphatase isoforms 5a and 5b by tumor and stromal cells in human metastatic bone disease.

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.

Long bone osteoclasts display an augmented osteoclast phenotype compared to calvarial osteoclasts.

Osteoclasts are multinucleated cells specialized in degrading bone and characterized by high expression of the enzymes tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CtsK). Recent studies show that osteoclasts exhibit phenotypic differences depending on their anatomical site of action. Using immunohistochemistry, RT-qPCR, FPLC chromatography and immunoblotting, we compared TRAP expression in calvaria and long bone. TRAP protein and enzyme activity levels were higher in long bones compared to calvaria. In addition, proteolytic processing of TRAP was more extensive in long bones than calvaria which correlated with higher cysteine proteinase activity and protein expression of CtsK. These two types of bones also exhibited a differential expression of monomeric TRAP and CtsK isoforms. Analysis of CtsK(-/-) mice revealed that CtsK is involved in proteolytic processing of TRAP in calvaria. Moreover, long bone osteoclasts exhibited higher expression of not only TRAP and CtsK but also of the membrane markers CD68 and CD163. The results suggest that long bone osteoclasts display an augmented osteoclastic phenotype with stronger expression of both membranous and secreted osteoclast proteins.

Biogenesis of tartrate-resistant acid phosphatase isoforms 5a and 5b in stably transfected MDA-MB-231 breast cancer epithelial cells.

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.

Proteolytic processing and polarized secretion of tartrate-resistant acid phosphatase is altered in a subpopulation of metaphyseal osteoclasts in cathepsin K-deficient mice.

Tartrate-resistant acid phosphatase (TRAP) is an enzyme highly expressed in osteoclasts and thought to participate in osteoclast-mediated bone turnover. Cathepsin K (Ctsk) is the major collagenolytic cysteine proteinase expressed in osteoclasts and has recently been shown to be able to proteolytically process and activate TRAP in vitro. In this study, 4-week-old Ctsk(-/-) mice were analysed for TRAP expression at the mRNA, protein and enzyme activity levels to delineate a role of cathepsin K in TRAP processing in osteoclasts in vivo. The absence of cathepsin K in osteoclasts was associated with increased expression of TRAP mRNA, monomeric TRAP protein and total TRAP activity. Proteolytic processing of TRAP was not abolished but prematurely arrested at an intermediate stage without changing enzyme activity, a finding confirmed with RANKL-differentiated osteoclast-like cell line RAW264.7 treated with the cysteine proteinase inhibitor E-64. Thus, the increase in total TRAP activity was mainly due to increased cellular content of monomeric TRAP. The increase in monomeric TRAP expression was more pronounced in osteoclasts of the distal compared to the proximal part of the metaphyseal trabecular bone, suggesting a site-dependent role for cathepsin K in TRAP processing. Moreover, intracellular localization of monomeric TRAP was altered in distal metaphyseal osteoclasts from Ctsk(-/-) mice. Additionally, TRAP was secreted into the ruffled border as the processed form in osteoclasts of Ctsk(-/-) mice, unlike in osteoclasts from wild-type mice which secreted TRAP to the resorption lacuna as the monomeric form. The results demonstrate that cathepsin K is not only involved in proteolytic processing but also affects the intracellular trafficking of TRAP, particularly in osteoclasts of the distal metaphysis. However, contribution by other yet unidentified protease(s) to TRAP processing must also be invoked since proteolytic cleavage of TRAP is not abolished in Ctsk(-/-) mice. Importantly, this study highlights functional differences between bone-resorbing clasts within the trabecular metaphyseal bone, suggesting potentially important differences in the regulation of differentiation and activation depending on the precise anatomical localization of the clast population.