The Architecture of the Adhesive Apparatus of Cultured Osteoclasts: From Podosome Formation to Sealing Zone Assembly

BACKGROUND: Osteoclasts are bone-degrading cells, which play a central role in physiological bone remodeling. Unbalanced osteoclast activity is largely responsible for pathological conditions such as osteoporosis. Osteoclasts develop specialized adhesion structures, the so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade bone, yet its structural organization is poorly understood. PRINCIPAL FINDINGS: Combining high-resolution scanning electron microscopy with fluorescence microscopy performed on the same sample, we mapped the molecular architecture of the osteoclast resorptive apparatus from individual podosomes to the sealing zone, at an unprecedented resolution. Podosomes are composed of an actin-bundle core, flanked by a ring containing adhesion proteins connected to the core via dome-like radial actin fibers. The sealing zone, hallmark of bone-resorbing osteoclasts, consists of a dense array of podosomes communicating through a network of actin filaments, parallel to the substrate and anchored to the adhesive plaque domain via radial actin fibers. SIGNIFICANCE: The sealing zone of osteoclasts cultured on bone is made of structural units clearly related to individual podosomes. It differs from individual or clustered podosomes in the higher density and degree of inter-connectivity of its building blocks, thus forming a unique continuous functional structure connecting the cell to its extracellular milieu. Through this continuous structure, signals reporting on the substrate condition may be transmitted to the whole cell, modulating the cell response under physiological and pathological conditions

Bone Is Not Essential for Osteoclast Activation

Background: The mechanism whereby bone activates resorptive behavior in osteoclasts, the cells that resorb bone, is unknown. It is known that avb3 ligands are important, because blockade of avb3 receptor signaling inhibits bone resorption, but this might be through inhibition of adhesion or migration rather than resorption itself. Nor is it known whether avb3 ligands are sufficient for resorption the consensus is that bone mineral is essential for the recognition of bone as the substrate appropriate for resorption. Methodology/Principal Findings: Vitronectin- but not fibronectin-coated coverslips induced murine osteoclasts to secrete tartrate-resistant acid phosphatase, as they do on bone. Osteoclasts incubated on vitronectin, unlike fibronectin, formed podosome belts on glass coverslips, and these were modulated by resorption-regulating cytokines. Podosome belts formed on vitronectin-coated surfaces whether the substrates were rough or smooth, rigid or flexible. We developed a novel approach whereby the substrate-apposed surface of cells can be visualized in the scanning electron microscope. With this approach, supported by transmission electron microscopy, we found that osteoclasts on vitronectin-coated surfaces show ruffled borders and clear zones characteristic of resorbing osteoclasts. Ruffles were obscured by a film if cells were incubated in the cathepsin inhibitor E64, suggesting that removal of the film represents substrate-degrading behavior. Analogously, osteoclasts formed resorption-like trails on vitronectin-coated substrates. Like bone resorption, these trails were dependent upon resorbogenic cytokines and were inhibited by E64. Bone mineral induced actin rings and surface excavation only if first coated with vitronectin. Fibronectin could not substitute in any of these activities, despite enabling adhesion and cell spreading. Conclusions/Significance: Our results show that ligands avb3 are not only necessary but sufficient for the induction of resorptive behavior in osteoclasts; and suggest that bone is recognized through its affinity for these ligands, rather than by its mechanical or topographical attributes, or through a putative ‘mineral receptor’

Connexin-mimetic peptide Gap 27 decreases osteoclastic activity

BACKGROUND: Bone remodelling is dependent on the balance between bone resorbing osteoclasts and bone forming osteoblasts. We have shown previously that osteoclasts contain gap-junctional protein connexin-43 and that a commonly used gap-junctional inhibitor, heptanol, can inhibit osteoclastic bone resorption. Since heptanol may also have some unspecific effect unrelated to gap-junctional inhibition we wanted to test the importance of gap-junctional communication to osteoclasts using a more specific inhibitor. METHODS: A synthetic connexin-mimetic peptide, Gap 27, was used to evaluate the contribution of gap-junctional communication to osteoclastic bone resorption. We utilised the well-characterised pit-formation assay to study the effects of the specific gap-junctional inhibitor to the survival and activity of osteoclasts. RESULTS: Gap 27 caused a remarked decrease in the number of both TRAP-positive mononuclear and multinucleated rat osteoclasts cultured on bovine bone slices. The decrease in the cell survival seemed to be restricted to TRAP-positive cells, whereas the other cells of the culture model seemed unaffected. The activity of the remaining osteoclasts was found to be diminished by measuring the percentage of osteoclasts with actin rings of all TRAP-positive cells. In addition, the resorbed area in the treated cultures was greatly diminished. CONCLUSIONS: On the basis of these results we conclude that gap-junctional communication is essential for the action of bone resorbing osteoclasts and for proper remodelling for bone

Roles of hyaluronan in bone resorption

BACKGROUND: Hyaluronan, an unsulfated glycosaminoglycan, while being closely linked to osteoclast function several years ago, has received little attention lately. Given recent new knowledge of hyaluronan's possible cell binding abilities, it is important to re-examine the role of this polysaccharide in bone homeostasis. DISCUSSION: Previously published data demonstrating a linkage between induction of hyaluronan synthesis and osteoclast-mediated bone resorption are reviewed. Suggestions are made involving the cell binding ability of hyaluronan and its potential to mediate osteoclast binding to bone surfaces and its potential to serve as a diffusion barrier and participate in the sealing zone required for osteoclast-mediated bone resorption. SUMMARY: This brief article summarizes previous studies linking HA to bone resorption and suggests roles for hyaluronan in the process of bone resorption

Single nucleotide polymorphisms in bone turnover-related genes in Koreans: ethnic differences in linkage disequilibrium and haplotype

<p>Abstract</p> <p>Background</p> <p>Osteoporosis is defined as the loss of bone mineral density that leads to bone fragility with aging. Population-based case-control studies have identified polymorphisms in many candidate genes that have been associated with bone mass maintenance or osteoporotic fracture. To investigate single nucleotide polymorphisms (SNPs) that are associated with osteoporosis, we examined the genetic variation among Koreans by analyzing 81 genes according to their function in bone formation and resorption during bone remodeling.</p> <p>Methods</p> <p>We resequenced all the exons, splice junctions and promoter regions of candidate osteoporosis genes using 24 unrelated Korean individuals. Using the common SNPs from our study and the HapMap database, a statistical analysis of deviation in heterozygosity depicted.</p> <p>Results</p> <p>We identified 942 variants, including 888 SNPs, 43 insertion/deletion polymorphisms, and 11 microsatellite markers. Of the SNPs, 557 (63%) had been previously identified and 331 (37%) were newly discovered in the Korean population. When compared SNPs in the Korean population with those in HapMap database, 1% (or less) of SNPs in the Japanese and Chinese subpopulations and 20% of those in Caucasian and African subpopulations were significantly differentiated from the Hardy-Weinberg expectations. In addition, an analysis of the genetic diversity showed that there were no significant differences among Korean, Han Chinese and Japanese populations, but African and Caucasian populations were significantly differentiated in selected genes. Nevertheless, in the detailed analysis of genetic properties, the LD and Haplotype block patterns among the five sub-populations were substantially different from one another.</p> <p>Conclusion</p> <p>Through the resequencing of 81 osteoporosis candidate genes, 118 unknown SNPs with a minor allele frequency (MAF) > 0.05 were discovered in the Korean population. In addition, using the common SNPs between our study and HapMap, an analysis of genetic diversity and deviation in heterozygosity was performed and the polymorphisms of the above genes among the five populations were substantially differentiated from one another. Further studies of osteoporosis could utilize the polymorphisms identified in our data since they may have important implications for the selection of highly informative SNPs for future association studies.</p