Surface distribution of heterogenous clathrin assemblies in resorbing osteoclasts.

Osteoclasts seeded on either glass coverslips or apatite pellets have at least two morphologically distinct substrate adhesion sites: actin-based adhesion structures including podosome belts and sealing zones, and adjacent clathrin sheets. Clathrin-coated structures are exclusively localized at the podosome belts and sealing zone, in both of which the plasma membrane forms a tight attachment to the substrate surface. When cultured on apatite osteoclasts can degrade the apatite leading to the formation of resorption lacunae. The sealing zone divides the ventral membrane into different domains, outside and inside of the sealing zones. The former facing the smooth-surfaced intact apatite contains relatively solitary or networks of larger flat clathrin structures; and the latter, facing the rough-surfaced degraded apatite in the resorption lacunae contain clathrin in various shapes and sizes. Clathrin assemblies on the membrane domain facing not only a resorption lacuna, or trails but also intact apatite indeed were observed to be heterogeneous in size and intensity, suggesting that they appeared to follow variations in the surface topography of the apatite surface. These results provide a detailed insight into the flat clathrin sheets that have been suggested to be the sites of adhesion and mechanosensing in co-operation with podosomes.

A 15-amino acid C-terminal peptide of beta-defensin-3 inhibits bone resorption by inhibiting the osteoclast differentiation and disrupting podosome belt formation.

Human beta-defensin-3 (HBD3), which is secreted from cells in the skin, salivary gland, and bone marrow, exhibits antimicrobial and immunomodulatory activities. Its C-terminal end contains a 15-amino acid polypeptide (HBD3-C15) that is known to effectively elicit antimicrobial activity. Recently, certain antimicrobial peptides are known to inhibit osteoclast differentiation and, thus, we investigated whether HBD3-C15 hinders osteoclast differentiation and bone destruction to assess its potential use as an anti-bone resorption agent. HBD3-C15 inhibited the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation and formation of resorption pits. In addition, HBD3-C15 disrupted the formation of RANKL-induced podosome belt which is a feature typically found in mature osteoclasts with bone-resorbing capacity. HBD3-C15 downregulated cortactin, cofilin, and vinculin, which are involved in the podosome belt formation. Furthermore, bone loss induced by RANKL was significantly reduced in a mouse calvarial implantation model that was treated with HBD3-C15. Similar inhibitory effects were observed on the osteoclast differentiation and podosome belt formation induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide (AaLPS). Concordantly, HBD3-C15 attenuated the resorption in the calvarial bone of AaLPS-implanted mouse. Collectively, these results suggest that HBD3-C15 has an anti-bone resorption effect in developing osteoclasts and that this occurs via its disruption of podosome belt formation. HBD3-C15 could be a potential therapeutic agent for the inhibition of bone destruction. KEY MESSAGES: HBD3-C15 inhibits osteoclast differentiation and bone resorption capacity. HBD3-C15 disrupts the podosome belt formation in osteoclasts. HBD3-C15 alleviates the bone loss by RANKL or A. actinomycetemcomitans LPS in vivo.

Dynamics of the sealing zone in cultured osteoclasts.

Bone resorption by osteoclasts (OCs) depends on the formation and stability of the sealing zone (SZ), a peripheral belt of actin and integrin-based podosomes. Recent studies demonstrated that the SZ is a highly dynamic structure, undergoing cycles of assembly and disassembly. In this study, we explored the mechanisms underlying the regulation of SZ stability and reorganization in OCs cultured on glass slides, and forming an SZ-like podosome belt (SZL). By monitoring this belt in cultured RAW264.7 cells expressing GFP-tagged actin, we show here that SZL stability is usually locally regulated, and its dissociation, occurring mostly in concave segments, is manifested in the loss of both podosome coherence, and actin belt continuity. Double labeling of cells for actin and tubulin indicated that microtubules (MTs) are mostly confined by the inner aspect of the stable SZL-associated actin belt. However, in unstable regions of the SZL, MTs tend to extend radially, across the SZL, toward the cell edge. Disruption of MTs by nocodazole induces SZ disassembly, without affecting individual podosome stability. Inspection of the MT network indicates that it is enriched along stable SZL regions, while bypassing disorganized regions. These results suggest that the SZL is stabilized by MTs flanking its inner aspect, while disruption or misalignment of MTs leads to SZL destabilization. We further demonstrate that the MT-associated protein dynamin2 is involved in the regulation of SZL stability, and dynamin2 knockdown or inactivation cause SZL destabilization.

Tetraspanin 7 regulates sealing zone formation and the bone-resorbing activity of osteoclasts.

Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement of Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules.