Osteoclasts use actin-rich attachment structures in place of focal adhesions for adherence to bone and non-bone substrates. On glass, osteoclasts generate podosomes, foot-like processes containing a core of F-actin and regulatory proteins that undergo high turnover. To facilitate bone resorption, osteoclasts generate an actin-rich sealing zone composed of densely packed podosome-like units. Patterning of both podosomes and sealing zones is dependent upon an intact microtubule system. A role for unconventional myosin X (Myo10), which can bind actin, microtubules, and integrins, was examined in osteoclasts. Immunolocalization showed Myo10 to be associated with the outer edges of immature podosome rings and sealing zones, suggesting a possible role in podosome and sealing zone positioning. Further, complexes containing both Myo10 and ␤-tubulin were readily precipitated from osteoclasts lysates. RNAi-mediated suppression of Myo10 led to decreased cell and sealing zone perimeter, along with decreased motility and resorptive capacity. Further, siRNA-treated cells could not properly position podosomes following microtubule disruption. Osteoclasts overexpressing dominant negative Myo10 microtubule binding domains (MyTH4) showed a similar phenotype. Conversely, overexpression of full-length Myo10 led to increased formation of podosome belts along with larger sealing zones and enhanced bone resorptive capacity. These studies suggest that Myo10 plays a role in osteoclast attachment and podosome positioning by direct linkage of actin to the microtubule network.

Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b+F4/80+Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.

The nonmuscle myosin IIA heavy chain (Myh9) is strongly associated with adhesion structures of osteoclasts. In this study, we demonstrate that during osteoclastogenesis, myosin IIA heavy chain levels are temporarily suppressed, an event that stimulates the onset of cell fusion. This suppression is not mediated by changes in mRNA or translational levels but instead is due to a temporary increase in the rate of myosin IIA degradation. Intracellular activity of cathepsin B is significantly enhanced at the onset of osteoclast precursor fusion, and specific inhibition of its activity prevents myosin IIA degradation. Further, treatment of normal cells with cathepsin B inhibitors during the differentiation process reduces cell fusion and bone resorption capacity, whereas overexpression of cathepsin B enhances fusion. Ongoing suppression of the myosin IIA heavy chain via RNA interference results in formation of large osteoclasts with significantly increased numbers of nuclei, whereas overexpression of myosin IIA results in less osteoclast fusion. Increased multinucleation caused by myosin IIA suppression does not require RANKL. Further, knockdown of myosin IIA enhances cell spreading and lessens motility. These data taken together strongly suggest that base-line expression of nonmuscle myosin IIA inhibits osteoclast precursor fusion and that a temporary, cathepsin B-mediated decrease in myosin IIA levels triggers precursor fusion during osteoclastogenesis.The final stages of osteoclastogenesis involve fusion of differentiated precursors from the monocyte/macrophage lineage (1). Although the membrane structural components regulating preosteoclast fusion are not well understood, in recent years a number of candidate cell surface molecules have been implicated, including receptors CD44 (2, 3), CD47 and its ligand macrophage fusion receptor (also known as signal regulatory protein ␣) (4 -6), the purinergic receptor P2X 7 (7), and the disintegrin and metalloproteinase ADAM8 (8). A recently identified receptor, the dendritic cell-specific transmembrane protein, is essential for osteoclast fusion both in vitro and in vivo (9, 10). More recently, the d2 subunit of proton-translocating vacuolar proton-translocating ATPases, a membrane subunit isoform expressed predominantly in osteoclasts, similarly was demonstrated to be required for fusion in vitro and in vivo (11). However, elucidation of the mechanisms by which these molecules may mediate cell fusion has proved to be difficult.The mammalian class II myosin family consists of distinct isoforms expressed in skeletal, smooth, and cardiac muscle, as well as three nonmuscle forms designated IIA, IIB,. Although all class II molecules are composed of two heavy chains, two essential light chains, and two regulatory chains, their unique activities are a function of their particular heavy chain isoforms. Although the nonmuscle heavy chain isoforms share extensive structural homology, they have been shown to demonstrate distinct patterns of expression (15-18), enzyme kinetics and activ...

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Tropomyosins (Tms) are α-helical dimers that bind and stabilize actin microfilaments while regulating their accessibility to other actin-associated proteins. Four genes encode expression of over forty Tms, most of which are expressed in nonmuscle cells. In recent years, it has become clear that individual Tm isoforms may regulate specific actin pools within cells. In this study, we examined how osteoclast function may be regulated by the tropomyosin isoform Tm-4, which we previously showed to be highly localized to podosomes and sealing zones of osteoclasts. RNAi-mediated knockdown of Tm-4, both in RAW264.7-and mouse marrow-derived osteoclasts, resulted in thinning of the actin ring of the sealing zone. Knockdown of Tm-4 also resulted in diminished bone resorptive capacity and altered resorption pit shape. In contrast, osteoclasts overexpressing Tm-4 demonstrated thickened podosomes on glass as well as thickened, aberrant actin structures on bone, and diminished motility and resorptive capacity. These results indicate that Tm-4 plays a role in regulating adhesion structures of osteoclasts, most likely by stabilizing the actin microfilaments present in podosomes and the sealing zone.