Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by beta3 integrin

The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.     

Integrin and cytoskeleton behaviour in human neuroblastoma cells during hyperthermia-related apoptosis

Hyperthermia induces several cellular responses leading to morphological changes, cell detachment and death. Loss of integrins from the cell surface after acute heat-treatment may block several physiological signalling pathways, but whether the assembly network between integrin and cytoskeletal actin is perturbed during hyperthermic treatment is unknown. In this study we tested this hypothesis by evaluating cell morphology, protein cytoskeletal profile and integrin CD11a content in both adherent and floating SK-N-MC human neuroblastoma cells. Morphological and cytometric analyses confirmed that hyperthermia is an effective apoptotic trigger, revealing the typical chromatin margination, cell shape changes and 7-AAD incorporation. After hyperthermia, cytoskeletal proteins showed an increase of high-molecular-weight aggregates and a significant decrease of both actin and CD11a content with respect to control cells. The integrin CD11a and membrane-bound actin alterations found in detached floating neuroblastoma cells recovered after heat-shock may cause the cytoskeletal abnormalities related to the observed surface cell rounding/blebbing and anoikis, early events of hyperthermia-induced programmed cell death.     

The role of cytoskeleton in glucose regulation

Cytoskeleton plays an important role in glucose regulation, mainly in the following three aspects. First, cytoskeleton regulates insulin secretion by guiding intracellular transport of insulin-containing vesicles and regulating release of insulin. Second, cytoskeleton is involved in insulin action by regulating distribution of insulin receptor substrate, GLUT4 translocation, and internalization of insulin receptor. In addition, cytoskeleton directs the intracellular distribution of glucose metabolism related enzymes including glycogen synthase and many glycolysis enzymes. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 592–597.     

The Spectrin cytoskeleton regulates the Hippo signalling pathway

The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta‐heavy dimers) produces tissue overgrowth and mis‐regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM‐domain protein Expanded (Ex). Apical beta‐heavy Spectrin binds to Ex and co‐localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors.     

血管扩张刺激磷蛋白在细胞骨架调节中的作用

细胞骨架动力学的调节在细胞粘附、细胞变形、细胞移动等生理过程中是必需的。血管扩张刺激磷蛋白（vasodilator-stimulated phosphoprotein,VASP）是一种肌动蛋白结合蛋白。该蛋白包含以下结构域：EVH1（Ena／VASP homolog1）区、EVH2（Ena／VASP homolog2）区及PRR（proline—rich regions）区。近年来,研究发现VASP在与细胞骨架调节有关的各种细胞行为中起着重要作用,如神经细胞轴索的延伸、T细胞的移动、成纤维细胞的迁移等。VASP的磷酸化受PKG（cGMP-dependent protein kinase）和PKA（cAMP—dependent protein kinase）的调控。在粘附斑的形成与脱落过程中,该磷酸化起着一个“开关”的作用。本文将就近20年来VASP的研究成果,特别是近年来的进展情况做一综述。     

Baicalin positively regulates osteoclast function by activating MAPK/Mitf signalling

Activation of osteoblasts in bone formation and osteoclasts in bone resorption is important during the bone fracture healing process. There has been a long interest in identifying and developing a natural therapy for bone fracture healing. In this study, we investigated the regulation of osteoclast differentiation by baicalin, which is a natural molecule extracted from Eucommiaulmoides (small tree native to China). It was determined that baicalin enhanced osteoclast maturation and bone resorption activity in a dose‐dependent manner. Moreover, this involves the activation of MAPK, increased Mitf nuclear translocation and up‐regulation of downstream osteoclast‐related target genes expression. The baicalin‐induced effect on osteoclast differentiation can be mimicked by specific inhibitors of p‐ERK (U0126) and the Mitf‐specific siRNA, respectively. Protein–ligand docking prediction identified that baicalin might bind to RANK, which is the upstream receptor of p‐ERK/Mitf signalling in osteoclasts. This indicated that RANK might be the binding target of baicalin. In sum, our findings revealed baicalin increased osteoclast maturation and function via p‐ERK/Mitf signalling. In addition, the results suggest that baicalin can potentially be used as a natural product for the treatment of bone fracture.     

The coupling of vinculin to the cytoskeleton is not essential for mechano-chemical signaling in F9 cells

It is well established that mechanical forces can regulate cell growth and guide tissue remodeling, yet little is known about how mechanical signals act at the cell surface membrane to produce biochemical changes in the cell. To explore this question, I used a mouse embryonic F9 vinculin-deficient cell line (gamma229), which, unlike wild-type cells, shows no fibronectin-dependent cell spreading. The wild-type cell line exhibited a twofold increase in area over four hours. I observed (i) an earlier rise in intracellular free calcium from approximately 0.2 to approximately 3 microm in wild-type compared with gamma229 cells, thus similar calcium levels after 4 h; (ii) an initial higher ratio of p-MAP/MAP-Kinase for gamma229, but similar FA-Kinase activation; and (iii) a marginal change in intracellular pH [pH](i) in both F9 cell lines. When I applied controlled local stresses directly to integrin receptors using RGD-coated magnetic beads, they displaced to a lesser extent in wild-type than in gamma229 cells. Both F9 cell lines showed a small stress-dependent rise in [Ca2+]i levels and similar PKA-c activity. In summary, the mechanical linkage of integrin-vinculin-cytoskeleton seemed not to be essential for chemical signal transduction.     

Polyphosphoinositides-dependent regulation of the osteoclast actin cytoskeleton and bone resorption

Background  

Gelsolin, an actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament. Previously, we have reported that osteopontin (OPN) binding to integrin α_vβ₃ increased the levels of gelsolin-associated polyphosphoinositides, podosome assembly/disassembly, and actin filament formation. The present study was undertaken to identify the possible role of polyphosphoinositides and phosphoinositides binding domains (PBDs) of gelsolin in the osteoclast cytoskeletal structural organization and osteoclast function.     

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

Mutual regulation between Hippo signaling and actin cytoskeleton

Hippo signaling plays a crucial role in growth control and tumor suppression by regulating cell proliferation, apoptosis, and differentiation. How Hippo signaling is regulated has been under extensive investigation. Over the past three years, an increasing amount of data have supported a model of actin cytoskeleton blocking Hippo signaling activity to allow nuclear accumulation of a downstream effector, Yki/Yap/Taz. On the other hand, Hippo signaling negatively regulates actin cytoskeleton organization. This review provides insight on the mutual regulatory mechanisms between Hippo signaling and actin cytoskeleton for a tight control of cell behaviors during animal development, and points out outstanding questions for further investigations.     

Cell cycle regulation of the microtubular cytoskeleton

The microtubular element of the plant cytoskeleton undergoes dramatic architectural changes in the course of the cell cycle, specifically at the entry into and exit from mitosis. These changes underlie the acquisition of specialized properties and functions involved, for example, in the equal segregation of chromosomes and the correct positioning and formation of the new cell wall. Here we review some of the molecular mechanisms by which the dynamics and the organization of microtubules are regulated and suggest how these mechanisms may be under the control of cell cycle events.     

Antiresorptive agents and osteoclast apoptosis

Antiresorptive agents have proven to be effective therapies for the treatment of bone diseases associated with excessive osteoclast activity. Decreased osteoclast formation, inhibition of osteoclast actions, and reduced osteoclast survival represent mechanisms by which antiresorptive agents could act. The goals of this article are to present the evidence that antiresorptive agents can decrease osteoclast survival through apoptosis, to review the mechanisms by which they are thought to activate the apoptotic process, and to consider whether the actions on apoptosis fully account for the antiresorptive effects. As background, the apoptotic process will be briefly summarized together with the evidence that factors that promote osteoclast survival affect steps in the process. Following this, therapeutic agents that are both antiresorptive and can stimulate osteoclast apoptosis will be discussed. Other bone therapeutic agents that are either antiresorptive or apoptotic, but not both, will be described. Finally, newer antiresorptive compounds that elicit apoptosis and could represent potential therapeutic agents will be noted.     

Fibronectin inhibits osteoclastogenesis while enhancing osteoclast activity via nitric oxide and interleukin‐1β‐mediated signaling pathways

Osteoclasts are bone‐resorbing cells formed by fusion of mononuclear precursors. The matrix proteins, fibronectin (FN), vitronectin (VN), and osteopontin (OPN) are implicated in joint destruction and interact with osteoclasts mainly through integrins. To assess the effects of these matrix proteins on osteoclast formation and activity, we used RAW 264.7 (RAW) cells and mouse splenocytes differentiated into osteoclasts on tissue culture polystyrene (TCP) or osteologic? slides pre‐coated with 0.01–20 µg/ml FN, VN, and OPN. At 96 h, osteoclast number and multinucleation were decreased on VN and FN compared to OPN and TCP in both RAW and splenocytes cell cultures. When early differentiation was assessed, VN but not FN decreased cytoplasmic tartrate‐resistant acid phosphatase activity and pre‐osteoclast number at 48 h. OPN had the opposite effect to FN on osteoclast formation. When RAW cells were differentiated on OPN and treated by FN and OPN, osteoclast number only in the FN treated group was 40–60% lower than the control, while the total number of nuclei was unchanged, suggesting that FN delays osteoclast fusion. In contrast to its inhibitory effect on osteoclastogenesis, FN increased resorption by increasing both osteoclast activity and the percentage of resorbing osteoclasts. This was accompanied by an increase in nitric oxide (NO) levels and interleukin‐1β (IL‐1β). IL‐1β production was inhibited using the NO‐synthase inhibitor only on FN indicating a FN‐specific cross‐talk between NO and IL‐1β signaling pathways. We conclude that FN upregulates osteoclast activity despite inhibiting osteoclast formation and that these effects involve NO and IL‐1β signaling. J. Cell. Biochem. 111: 1020–1034, 2010. © 2010 Wiley‐Liss, Inc.     

WIP: a multifunctional protein involved in actin cytoskeleton regulation

Knowledge of the dynamics of actin-based structures is a major key to understanding how cells move and respond to their environment. The ability to reorganize actin filaments in a spatial and temporal manner to integrate extracellular signals is at the core of cell adhesion and cell migration. Several proteins have been described as regulators of actin polymerization: this review will focus on the role of WASP-interacting protein (WIP), an actin-binding protein that participates in actin polymerization regulation and signal transduction. WIP is widely expressed and interacts with Wiskott-Aldrich syndrome protein (WASP) (a hematopoietic-specific protein) and its more widely expressed homologue neural WASP (N-WASP), to regulate WASP/N-WASP function in Arp2/3-mediated actin polymerization. WIP also interacts with profilin, globular and filamentous actin (G- and F-actin, respectively) and stabilizes actin filaments. In vivo WIP participates in filopodia and lamellipodia formation, in T and B lymphocyte activation, in mast cell degranulation and signaling through the Fcepsilon receptor (FcepsilonR), in microbial motility and in Syk protein stability.     

Cancer as a disease of epithelial-mesenchymal interactions and extracellular matrix regulation

Carcinogenesis - the process of cancer formation - is commonly discussed in terms of genetic alterations that lead to deregulation of cell growth. Recently, there has been a resurgence of interest in epigenetic factors and, in particular, the role of the stromal microenvironment and angiogenesis in tumor formation. In this article, cancer is presented as a disease of the developmental processes that govern how cells organize into tissues and tissues into organs. This histogenetic perspective raises the possibility that epithelial-mesenchymal interactions and the extracellular matrix (basement membrane) that is deposited through these interactions may actively contribute to the carcinogenic process. Experimental work is reviewed that confirms that extracellular matrix plays a key role in normal histodifferentiation during both epitheliogenesis and angiogenesis, and that epigenetic deregulation of cell-matrix interactions may actively promote tumor initiation and progression. The contributions of integrins, cytoskeleton, tensegrity and local variations in extracellular matrix mechanics to these processes are discussed, as are the implications of this work for future studies on cancer formation.     

P2 receptors in macrophage fusion and osteoclast formation

Cells of the mononuclear phagocyte lineage fuse to form multinucleated giant cells and osteoclasts. Several lines of evidence suggest that P2 receptors, in particular P2X₇, are involved in this process, although P2X₇ is not absolutely required for fusion because P2X₇-null mice form multinucleated osteoclasts. Extracellular ATP may be an important regulator of macrophage fusion.