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1.
Eur J Cell Biol ; 92(4-5): 139-49, 2013.
Article in English | MEDLINE | ID: mdl-23598086

ABSTRACT

Podosomes are adhesion structures characteristic of the myeloid cell lineage, encompassing osteoclasts, dendritic cells and macrophages. Podosomes are actin-based structures that are dynamic and capable of self-organization. In particular in the osteoclast, podosomes densely pack into a thick ring called the sealing zone. This adhesion structure is typical of osteoclasts and necessary for the resorption of the bone matrix. We thought to explore in more details the role of podosomes during osteoclast differentiation and migration. To this end, we made from soft to stiff substrates that had not been functionalized with extracellular matrix proteins. Such substrates did not support podosome formation in osteoclasts. With such devices, we could show that integrin activation was sufficient to drive podosome assembly, in a substrate stiffness independent fashion. We additionally report here that osteoclast differentiation is a podosome-independent process. Finally, we show that osteoclasts devoid of podosomes can migrate efficiently. Our study further illustrates the great capacity of myeloid cells to adapt to the different environments they encounter during their life cycle.


Subject(s)
Cell Differentiation , Cell Movement , Cell-Matrix Junctions/physiology , Osteoclasts/physiology , Acrylic Resins/metabolism , Actins/metabolism , Agar/metabolism , Animals , Cell Adhesion , Cells, Cultured , Culture Media , Dimethylpolysiloxanes/metabolism , Extracellular Matrix/metabolism , Integrins/metabolism , Mice , Mice, Inbred C57BL , Vinculin/metabolism , src-Family Kinases/metabolism
2.
PLoS One ; 7(9): e45909, 2012.
Article in English | MEDLINE | ID: mdl-23049890

ABSTRACT

Podosomes are dynamic actin-based structures found constitutively in cells of monocytic origin such as macrophages, dendritic cells and osteoclasts. They have been involved in osteoclast cell adhesion, motility and matrix degradation, and all these functions rely on the ability of podosomes to form supra-molecular structures called podosome belts or sealing zones on mineralized substrates. Podosomes contain two distinct domains, an actin-rich core enriched in actin polymerization regulators, surrounded by a ring of signaling and plaque molecules. The organization of podosome arrays into belts is linked to actin dynamics. Cofilin is an actin-severing protein that is known to regulate cytoskeleton architecture and cell migration. Cofilin is present in lamellipodia and invadopodia where it regulates actin polymerization. In this report, we show that cofilin is a novel component of the podosome belt, the mature osteoclast adhesion structure. Time-course analysis demonstrated that cofilin is activated during primary osteoclast differentiation, at the time of podosome belt assembly. Immunofluorescence studies reveal a localization of active cofilin in the podosome core structure, whereas phosphorylated, inactive cofilin is concentrated in the podosome cloud. Pharmacological studies unraveled the role of a specific cofilin phosphatase to achieve cofilin activation during osteoclast differentiation. We ruled out the implication of PP1/PP2A and PTEN in this process, and rather provided evidence for the involvement of SSH1. In summary, our data involve cofilin as a regulator of podosome organization that is activated during osteoclast differentiation by a RANKL-mediated signaling pathway targeting the SSH1 phosphatase.


Subject(s)
Actins/chemistry , Bone Marrow Cells/cytology , Cofilin 1/metabolism , Osteoclasts/metabolism , Animals , Antibodies, Monoclonal/chemistry , Cell Differentiation , Fluorescent Antibody Technique, Indirect/methods , Macrophages/cytology , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence/methods , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , RANK Ligand/metabolism , Retroviridae/metabolism
3.
Mol Biosyst ; 6(4): 648-61, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20237642

ABSTRACT

Movement of individual cells and of cellular cohorts, chains or sheets requires physical forces that are established through interactions of cells with their environment. In vivo, migration occurs extensively during embryonic development and in adults during wound healing and tumorigenesis. In order to identify the molecular events involved in cell movement, in vitro systems have been developed. These have contributed to the definition of a number of molecular pathways put into play in the course of migratory behaviours, such as mesenchymal and amoeboid movement. More recently, our knowledge of migratory modes has been enriched by analyses of cells exploring and moving through three-dimensional (3D) matrices. While the cells' morphologies differ in 2D and 3D environments, the basic mechanisms that put a cellular body into motion are remarkably similar. Thus, in both 2D and 3D, the polarity of the migrating cell is initially defined by a specific subcellular localization of signalling molecules and components of molecular machines required for motion. While the polarization can be initiated either in response to extracellular signalling or be a chance occurrence, it is reinforced and sustained by positive feedback loops of signalling molecules. Second, adhesion to a substratum is necessary to generate forces that will propel the cell engaged in either mesenchymal or ameboid migration. For collective cell movement, intercellular coordination constitutes an additional requirement: a cell cohort remains stationary if individual cells pull in opposite directions. Finally, the availability of space to move into is a general requirement to set cells into motion. Lack of free space is probably the main obstacle for migration of most healthy cells in an adult multicellular organism. Thus, the requirements for cell movement are both intrinsic to the cell, involving coordinated signalling and interactions with molecular machines, and extrinsic, imposed by the physicochemical nature of the environment. In particular, the geometry and stiffness of the support act on a range of signalling pathways that induce specific cell migratory responses. These issues are discussed in the present review in the context of published work and our own data on collective migration of hepatocyte cohorts.


Subject(s)
Cell Movement/physiology , Animals , Cell Adhesion/physiology , Cell Polarity/physiology , Hepatocytes/physiology , Humans , Imaging, Three-Dimensional , In Vitro Techniques , Mechanotransduction, Cellular , Models, Biological , Signal Transduction , Systems Biology
4.
Int J Biochem Cell Biol ; 41(6): 1391-401, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19135548

ABSTRACT

An excess of osteoclastic bone resorption relative to osteoblastic bone formation results in progressive bone loss, characteristic of osteoporosis. Understanding the mechanisms of osteoclast differentiation is essential to develop novel therapeutic approaches to prevent and treat osteoporosis. We showed previously that Wrch1/RhoU is the only RhoGTPase whose expression is induced by RANKL during osteoclastogenesis. It associates with podosomes and the suppression of Wrch1 in osteoclast precursors leads to defective multinucleated cell formation. Here we further explore the functions of this RhoGTPase in osteoclasts, using RAW264.7 cells and bone marrow macrophages as osteoclast precursors. Suppression of Wrch1 did not prevent induction of classical osteoclastic markers such as NFATc1, Src, TRAP (Tartrate-Resistant Acid Phosphatase) or cathepsin K. ATP6v0d2 and DC-STAMP, which are essential for fusion, were also expressed normally. Similar to the effect of RANKL, we observed that Wrch1 expression increased osteoclast precursor aggregation and reduced their adhesion onto vitronectin but not onto fibronectin. We further found that Wrch1 could bind integrin beta3 cytoplasmic domain and interfered with adhesion-induced Pyk2 and paxillin phosphorylation. Wrch1 also acted as an inhibitor of M-CSF-induced prefusion osteoclast migration. In mature osteoclasts, high Wrch1 activity inhibited podosome belt formation. Nevertheless, it had no effect on mineralized matrix resorption. Our observations suggest that during osteoclastogenesis, Wrch1 potentially acts through the modulation of alphav beta3 signaling to regulate osteoclast precursor adhesion and migration and allow fusion. As an essential actor of osteoclast differentiation, the atypical RhoGTPase Wrch1/RhoU could be an interesting target for the development of novel antiresorptive drugs.


Subject(s)
Osteoclasts/cytology , Osteoclasts/metabolism , rho GTP-Binding Proteins/metabolism , Animals , Blotting, Western , Cell Adhesion/physiology , Cell Line , Cell Movement/physiology , Gene Expression Profiling/methods , Humans , Mice , Mice, Inbred C57BL , Phosphorylation , RANK Ligand/metabolism , Vitronectin/metabolism
5.
Eur J Cell Biol ; 87(8-9): 469-77, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18436334

ABSTRACT

Cells from the myeloid lineage, namely macrophages, dendritic cells and osteoclasts, develop podosomes instead of stress fibers and focal adhesions to adhere and migrate. Podosomes share many components with focal adhesions but differ in their molecular organization, with a dense core of polymerized actin surrounded by scaffolding proteins, kinases and integrins. Podosomes are found either isolated both in macrophages and dendritic cells or arranged into superstructures in osteoclasts. When osteoclasts resorb bone, they form an F-actin rich sealing zone, which is a dense array of connected podosomes that firmly anchors osteoclasts to bone. It delineates a compartment in which protons and proteases are secreted to dissolve and degrade the mineralized matrix. Since Rho GTPases have been shown to control F-actin stress fibers and focal adhesions in mesenchymal cells, the question of whether they could also control podosome formation and arrangement in cells from the myeloid lineage, and particularly in osteoclasts, rapidly emerged. This article considers recent advances made in our understanding of podosome arrangements in osteoclasts and how Rho GTPases may control it.


Subject(s)
Osteoclasts/metabolism , Osteoclasts/ultrastructure , rho GTP-Binding Proteins/metabolism , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/ultrastructure , Animals , Cell Adhesion , Humans , Models, Biological , Signal Transduction , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism
6.
Mol Biol Cell ; 19(2): 633-45, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18045996

ABSTRACT

In Rous sarcoma virus (RSV)-transformed baby hamster kidney (BHK) cells, invadopodia can self-organize into rings and belts, similarly to podosome distribution during osteoclast differentiation. The composition of individual invadopodia is spatiotemporally regulated and depends on invadopodia localization along the ring section: the actin core assembly precedes the recruitment of surrounding integrins and integrin-linked proteins, whereas the loss of the actin core was a prerequisite to invadopodia disassembly. We have shown that invadopodia ring expansion is controlled by paxillin phosphorylations on tyrosine 31 and 118, which allows invadopodia disassembly. In BHK-RSV cells, ectopic expression of the paxillin mutant Y31F-Y118F induces a delay in invadopodia disassembly and impairs their self-organization. A similar mechanism is unraveled in osteoclasts by using paxillin knockdown. Lack of paxillin phosphorylation, calpain or extracellular signal-regulated kinase inhibition, resulted in similar phenotype, suggesting that these proteins belong to the same regulatory pathways. Indeed, we have shown that paxillin phosphorylation promotes Erk activation that in turn activates calpain. Finally, we observed that invadopodia/podosomes ring expansion is required for efficient extracellular matrix degradation both in BHK-RSV cells and primary osteoclasts, and for transmigration through a cell monolayer.


Subject(s)
Paxillin/metabolism , Pseudopodia/metabolism , Animals , Calpain/antagonists & inhibitors , Cell Communication/drug effects , Cell Movement/drug effects , Cell Transformation, Viral/drug effects , Cricetinae , Enzyme Activation/drug effects , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , HeLa Cells , Humans , Mice , Mutant Proteins/metabolism , Phosphoproteins/metabolism , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Protein Kinase Inhibitors/pharmacology , Pseudopodia/drug effects , Pseudopodia/enzymology , Rous sarcoma virus/metabolism , Vanadates/pharmacology
7.
J Cell Sci ; 119(Pt 1): 31-46, 2006 Jan 01.
Article in English | MEDLINE | ID: mdl-16339173

ABSTRACT

Human intestinal cell differentiation is mediated by signaling pathways that remain largely undefined. We and others have shown that cell migration and differentiation along the crypt-villus axis is associated with temporal and spatial modulations of the repertoire, as well as with the function of integrins and E-cadherins and their substrates. Cross-talk between integrin and cadherin signaling was previously described and seems to coordinate this differentiation process. Here, we report that engagement of alpha6 and, to a lesser extent, alpha3 integrin subunits after HT-29 cell adhesion on laminin 5 increases the expression of E-cadherin, which then organizes into nascent adherens junctions. We further identify that phosphoinositide 3-kinase (PI 3-kinase) activation plays a key role in this cross-talk. Indeed, integrin-dependent adhesion on laminin 5 stimulates PI 3-kinase activity. Immunofluorescence and immunoprecipitation experiments revealed that activated PI 3-kinase is recruited at cell-cell contacts. Using LY294002, an inhibitor of PI 3-kinase activity, we found that this activation is essential for E-cadherin connection with the cytoskeleton and for biogenesis of adherens junctions. Finally, we demonstrated that PI 3-kinase could signal through Rac1b activation to control adherens junction assembly. Our results provide a mechanistic insight into integrin-cadherin cross-talk and identify a novel role for PI 3-kinase in the establishment of adherens junctions.


Subject(s)
Adherens Junctions/metabolism , Cell Adhesion Molecules/metabolism , Integrin alpha3/metabolism , Integrin alpha6/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/physiology , rac1 GTP-Binding Protein/metabolism , Cadherins/metabolism , Cell Adhesion/physiology , Cell Shape , Chromones/metabolism , Cytoskeleton/metabolism , Enzyme Activation , Enzyme Inhibitors/metabolism , HT29 Cells , Humans , Morpholines/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Protein Subunits/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Kalinin
8.
J Cell Biochem ; 95(5): 1069-80, 2005 Aug 01.
Article in English | MEDLINE | ID: mdl-15962288

ABSTRACT

The actin cytoskeleton is recognized as an important component of both adhesion- and growth factor-dependent signaling, but its role in oncogene-dependent signaling has received much less attention. In this study, we investigated the role played by the acto-myosin cytoskeleton and its main regulators, i.e., myosin light chain kinase and Rho kinase, in oncogenic Ki-Ras-induced signaling. We found that activation of the ERK cascade by Ras is dependent on acto-myosin contractility, under the regulation of myosin light chain kinase but not Rho kinase. Inhibition of myosin II or myosin light chain kinase caused a complete loss of ERK phosphorylation in a time- and dose-dependent manner, but proved dispensable for activation of the PI3K pathway. We also provide evidence that the target of myosin light chain kinase lays at the level of Raf activation. Since myosin light chain kinase is a target of ERK, these results suggest a previously uncharacterized signaling pathway involving Ras-mediated alterations of the actin cytoskeleton, which might play a critical role in ERK activation by the Ras oncogene and contribute to aberrant signaling and enhanced cell motility. In addition, restoration of stress fibers following ectopic expression of tropomyosin 2 resulted in reduced levels of ERK phosphorylation. Finally, these studies suggest that myosin light chain kinase but not Rho kinase plays an essential role in the generation of ERK signaling in transformed cells and indicate distinct cellular roles for Rho-kinase and myosin light chain kinase-dependent functions involving the regulation of acto-myosin contractility.


Subject(s)
Actomyosin/metabolism , Genes, ras/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Muscle Contraction , Myosin-Light-Chain Kinase/metabolism , Animals , Cell Adhesion , Cell Movement , Enzyme Activation , Fluorescent Antibody Technique , Immunoblotting , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Phosphorylation , Proto-Oncogene Proteins c-raf/metabolism , Rats , Retroviridae , Signal Transduction , rho GTP-Binding Proteins/metabolism
9.
Int J Cancer ; 110(3): 368-73, 2004 Jun 20.
Article in English | MEDLINE | ID: mdl-15095301

ABSTRACT

Previous studies of transformed rodent fibroblasts have suggested that specific isoforms of the actin-binding protein tropomyosin (TM) could function as suppressors of transformation, but an analysis of TM expression in patient tumor tissue is limited. The purpose of our study was to characterize expression of the different TM isoforms in human transitional cell carcinoma of the urinary bladder by immunohistochemistry and Western blot analysis. We found that TM1 and TM2 protein levels were markedly reduced and showed >60% reduction in 61% and 55% of tumor samples, respectively. TM5, which was expressed at very low levels in normal bladder mucosa, exhibited aberrant expression in 91% of tumor specimens. The Western blot findings were confirmed by immunohistochemical analysis in a number of tumors. We then investigated the mechanism underlying TM expression deregulation, in the T24 human bladder cancer cell line. We showed that levels of TM1, TM2 and TM3 are reduced in T24 cells, but significantly upregulated by inhibition of the mitogen-activated protein kinase-signaling pathway. In addition, inhibition of this pathway was accompanied by restoration of stress fibers. Overall, changes in TM expression levels seem to be an early event during bladder carcinogenesis. We conclude that alterations in TM isoform expression may provide further insight into malignant transformation in transitional cell carcinomas of the bladder and may be a useful target for early detection strategies.


Subject(s)
Carcinoma, Transitional Cell/metabolism , Tropomyosin/biosynthesis , Tropomyosin/chemistry , Urinary Bladder Neoplasms/metabolism , Blotting, Western , Cell Line, Tumor , Down-Regulation , Enzyme Inhibitors/pharmacology , Humans , Immunohistochemistry , MAP Kinase Signaling System , Mucous Membrane/metabolism , Protein Isoforms , Signal Transduction , Time Factors , Tropomodulin , Up-Regulation , Urinary Bladder/metabolism
10.
J Biol Chem ; 277(30): 26927-33, 2002 Jul 26.
Article in English | MEDLINE | ID: mdl-12011049

ABSTRACT

Cellular transformation by v-Src is believed to be caused by aberrant activation of signaling pathways that are normally regulated by cellular Src. Using normal rat kidney cells expressing a temperature-sensitive mutant of v-Src, we examined the role of the Raf/MEK/ERK, phosphatidylinositol 3-kinase/Akt, and Rho pathways in morphological transformation and cytoskeletal changes induced by v-Src. Activation of v-Src elicited a loss of actin stress fibers and focal contacts. A decrease in the phosphorylation level of cofilin was detected upon v-Src activation, which is indicative of attenuated Rho function. Inhibition of MEK using U0126 prevented v-Src-induced disruption of the cytoskeleton as well as dephosphorylation of cofilin, whereas treatment with a phosphatidylinositol 3-kinase inhibitor had no protective effect. In normal rat kidney cells stably transformed by v-Src, we found that the chronic activation of MEK induces down-regulation of ROCK expression, thereby uncoupling Rho from stress fiber formation. Taken together, these results establish MEK as an effector of v-Src-induced cytoskeleton disruption, participating in v-Src-induced antagonism of the cellular function of Rho.


Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Oncogene Protein pp60(v-src)/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , rho GTP-Binding Proteins/metabolism , Animals , Blotting, Western , Butadienes/pharmacology , Cells, Cultured , Enzyme Inhibitors/pharmacology , Guanosine Triphosphate/metabolism , Intracellular Signaling Peptides and Proteins , Kidney/cytology , Lim Kinases , Microscopy, Fluorescence , Models, Biological , Mutation , Nitriles/pharmacology , Phenotype , Phosphorylation , Rats , Signal Transduction , Temperature , Time Factors , Transfection , rho-Associated Kinases
11.
Mol Biol Cell ; 13(1): 336-47, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11809843

ABSTRACT

Transformation by oncogenic Ras profoundly alters actin cytoskeleton organization. We investigated Ras-dependent signaling pathways involved in cytoskeleton disruption by transfecting normal rat kidney (NRK) cells with different Ras mutants. RasV12S35, a mutant known to activate specifically the Raf/MAPK pathway, led to stress fiber and focal contact disruption, whereas the adherens junctions remained intact. Next, we found that pharmacological inhibition of MEK was sufficient to restore the cytoskeletal defects of ras-transformed NRK cells, including assembly of stress fibers and focal contacts, but it did not induce reorganization of the cell-cell junctions. Investigating the mechanism underlying this phenotypic reversion, we found that the sustained MAPK signaling resulting from Ras-transformation down-regulated the expression of ROCKI and Rho-kinase, two-Rho effectors required for stress fiber formation, at the post-transcriptional level. On MEK inhibition, ROCKI/Rho-kinase expression and cofilin phosphorylation were increased, demonstrating that the Rho-kinase/LIM-kinase/cofilin pathway was functionally restored. Finally, using dominant negative or constitutively active mutants, we demonstrated that expression of ROCKI/Rho-kinase was both necessary and sufficient to promote cytoskeleton reorganization in NRK/ras cells. These findings further establish the Ras/MAPK pathway as the critical pathway involved in cytoskeleton disruption during Ras-transformation, and they suggest a new mechanism, involving alteration in ROCKI/Rho-kinase expression, by which oncogenic Ras can specifically target the actin-based cytoskeleton and achieve morphological transformation of the cells.


Subject(s)
Actin Cytoskeleton/ultrastructure , Down-Regulation/physiology , Fibroblasts/enzymology , Mitogen-Activated Protein Kinase Kinases/metabolism , Oncogene Protein p21(ras)/genetics , Protein Serine-Threonine Kinases/metabolism , Actin Cytoskeleton/drug effects , Amides/pharmacology , Animals , Butadienes/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Line, Transformed , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Fibroblasts/ultrastructure , Flavonoids/pharmacology , Fluorescent Antibody Technique , Gene Expression Regulation, Enzymologic , Intracellular Signaling Peptides and Proteins , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Morpholines/pharmacology , Nitriles/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Pyridines/pharmacology , Rats , Transcription, Genetic , Transfection , rho-Associated Kinases
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