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Osteoclast differentiation and function are highly dependent on the assembly and turnover of actin filaments, but little is known about the roles of actin binding proteins in these processes. Adseverin (Ads), a member of the gelsolin superfamily of actin capping and severing proteins, regulates actin filament turnover and can regulate the turnover of cortical actin filaments of chromaffin cells during exocytosis. Using a conditional Ads knockout mouse model, we confirmed our previous finding in cultured cells that Ads plays a role in osteoclastogenesis (OCG) and actin cytoskeletal organization in osteoclasts. Here we show that Ads is required for osteoclast formation and that when alveolar bone resorption is experimentally induced in mice, genetic deletion of Ads prevents osteoclast-mediated bone loss. Further, when Ads-null osteoclasts are cultured, they exhibit defective OCG, disorganized podosome-based actin filament superstructures, and decreased bone resorption. Reintroduction of Ads into Ads-null osteoclast precursor cells restored these osteoclast defects. Collectively, these data demonstrate a unique and osteoclast-specific role for Ads in OCG and osteoclast function. [ABSTRACT FROM AUTHOR]

In mechanically loaded tissues such as weight-bearing joints, myocardium, and periodontal ligament, pathophysiological forces can disrupt cell-matrix contacts, which can induce cell death, leading to tissue and organ dysfunction. Protection against force-induced cell death may be mediated by filamin A (FLNa), an actin-binding protein that regulates β1 integrin-mediated cell adhesion. We examined the affect of filamin expression on collagen distribution and cell death in the periodontal ligament, a force-loaded tissue. Conditional deletion of FLNa in fibroblasts was associated with 2-fold increase of acellular areas in periodontal ligament and 7-fold higher proportions of apoptotic cells. In cultured fibroblasts with FLNa knockdown, we examined the affect of supraphysiological forces (1 pN/µm² cell area; applied through the ²1 integrin) on recruitment of talin and vinculin to focal adhesions and on apoptosis. Compared with the wild type, FLNa-knockdown cells exhibited 3-fold increases in floating cells after overnight force application and a 2-fold increase in cell detachment. Force induced time-dependent reductions (P<0.05) in the numbers of activated ²1 integrin-, talin-, and vinculin-stained adhesions in FLNa-knockdown compared with those in wild-type cells. We conclude that FLNa protects against apoptosis in force-loaded cells, and this protection is mediated by enhanced formation an maturation of matrix adhesions. [ABSTRACT FROM AUTHOR]

IL-Iβ is a prominent proinflammatory cytokine that mediates degradation of extracellular matrix proteins through increased expression of matrix metalloproteinases, which involves a signaling pathway in adherent cells that is restricted by focal adhesions. Currently, the mechanism by which IL-lβ affects cell adhesion to matrix proteins is not defined, and it is not known whether degraded matrix proteins affect IL-1β signaling. We examined adhesion-related IL-1β signaling in fibroblasts attaching to native or MMP3-degraded fibronectin. IL-1β increased cell attachment, resistance to shear force and the numbers of focal adhesions containing activated β1 integrins. IL-1β-enhanced attachment required FAK, kindlins 1/2, and talin. MMP3-degraded fibronectin-inhibited IL-1β-enhanced cell adhesion and promoted spontaneous ERK activation that was independent of IL-Iβ treatment. We conclude that IL-1β enhances the adhesion of anchorage-dependent cells to MMP3-degraded fibronectin, which, in turn, is associated with deregulated cellular responses to IL-1β. These data point to a novel role of IL-Iβ as a proadhesive signaling molecule in inflammation that employs kindlins and talin to regulate adhesion. [ABSTRACT FROM AUTHOR]

The role of adhesion-associated actin-binding proteins in cell migration is not well defined. In mouse fibroblasts we screened for focal adhesion-associated proteins that were isolated with collagen-coated beads and detected by tandem mass spectrometry. We identified flightless I (FliI) as an actin-binding protein in focal adhesion fractions, which was verified by immuno-blotting. By confocal microscopy most FliI was distributed throughout the cytosol and in focal adhesions. By sedimentation assays and in vitro binding assays, we found that FliI associates with actin filaments and actin monomers. Assays using purified proteins showed that FliI inhibits actin polymerization and caps but does not sever actin filaments. Cells with FliI knockdown or cells overexpress-ing FliI migrated more or less rapidly, respectively, than wild-type controls. Compared with controls, cells with Fill knockdown were less adherent than wild-type cells, exhibited reduced numbers of focal adhesions containing activated pi integrins and vinculin, and exhibited increased incorporation of actin monomers into nascent filaments at focal adhesions. These data indicate that FliI regulates cell migration through its localization to focal adhesions and its ability to cap actin filaments, which collectively affect focal adhesion maturation. [ABSTRACT FROM AUTHOR]

In connective tissue cells, IL-1-induced ERK activation leading to matrix metalloproteinase (MMP)-3 expression is dependent on cooperative interactions between focal adhesions and the endoplasmic reticulum (ER). As Ras can be activated on the ER, we investigated the role of Ras in IL-1 signaling and focal adhesion formation. We found that constitutively active H-Ras, K-Ras or N-Ras enhanced focal adhesion maturation and β1-integrin activation. IL-1 promoted the accumulation of Ras isoforms in ER and focal adhesion fractions, as shown in cells cotransfected with GFP-tagged Ras isoforms and YFP-ER protein and by analysis of subcellular fractions enriched for ER or focal adhesion proteins. Dominant-negative H-Ras or K-Ras reduced accumulation of H-Ras and K-Ras in focal adhesions induced by IL-1 and also blocked ERK activation and focal adhesion maturation. Ras-GRF was enriched constitutively in focal adhesion fractions and was required for Ras recruitment to focal adhesions. We conclude that Ras activation and IL-1 signaling are interactive processes that regulate the maturation of focal adhesions, which, in turn, is required for ERK activation. [ABSTRACT FROM AUTHOR]

Presents a study which examined the role of mitochondrial function in the regulation of interleukin (IL)-1-induced ERK activation in human fibroblasts. Analysis of IL-1 signaling; Information on ERK activation; Mechanism of mitochondrial function.

The cellular mechanisms that modulate IL-1 signaling and lead to production of inflammatory mediators are not defined. The aim of this study was to determine the role of cell adhesion in regulating specific steps of Ca&sup2+; handling in the IL-1 signals that lead to ERK activation in human fibroblasts. [ABSTRACT FROM AUTHOR]