Podosomes Research Articles

Osteoclast differentiation of Raw264.7 cells expressing enhanced green fluorescent protein in vitro

To observe the osteoclast differentiation of Raw264.7 strain stably expressing enhanced green fluorescent protein (EGFP). Raw264.7 cells were transfected with EGFP-Lifeact gene via retrovirus. The G3 cell clone was obtained by limited dilution technique which stably expressed EGFP under the fluorescence microscope. The morphology of G3 cells were observed. The effects of transfection on receptor activator of nuclear factor kappaB ligand (RANKL)--induced osteoclastogenesis and bone resorbing function of G3 cells were examined by tartrate resistant acid phosphatase (TRAP) staining, immunoblotting detection of cathepsin K and bone pit resorption assay. The real-time images of podosome dynamics were taken by laser confocal microscopy during osteoclast differentiation of G3 cells. The Raw264.7 cells were successfully transfected with EGFP-Lifeact gene. The G3-EGFP cloned strain which could stably express EGFP even after 20 passages was constructed. There was no significant difference in morphology between G3-EGFP and wild Raw264.7 cells. The fusion rates of the transfection group and of the wild control group were (35±5)% and (39±5)%, respectively, which were not significantly different (P>0.05). The semi-quantitative ratio of cathepsin K/β-actin in the wild control group and in the transfection group was 0.83±0.07 and 1.02±0.08 (P>0.05), respectively. Bone pit results showed that the total area of the bone resorption was respectively 272,252±36,193 and 262,408±23,243 (P>0.05) and the number of the bone pits was respectively 320±51 and 339±55 (P>0.05). The photos of laser confocal microscopy showed the constant cell-cell fusion during osteoclast differentiation of G3-EGFP cells. In addition, the dynamic self-organized podosome initially assembled podosome clusts, then dynamic rings, finally formed the characteristic podosome belt pattern in mature osteoclasts. Enhanced green fluorescent protein high effectively expressed in Raw264.7. Biological character does not change after transfection.

Early differentiating osteoclast interactions with a well suitable bone-like composite

Osteoclasts, as well as preosteoclasts, show different adhesion features in relationship to the substrate on which cells are grown, i.e. the formation of either podosomes belt or sealing zones. Podosomes belt forms on non bone substrates, i.e. when cells interact with glass coverslips or culture plates, whereas sealing zones form when cells grow on bone-like substrates. Podosomes belt corresponds to numerous F-actin columns arranged at the cell periphery, whereas the sealing zone could be defined as a unique large band of actin [1]. In the study of bone resorption mechanisms, the employ of bone slices is not perfectly suitable to investigate actin rearrangement due to cell-extracellular matrix (ECM) interaction, since it doesn’t allow to obtain high quality preparations to be examined both by light and electronmicroscopy (TEM and SEM). In particular, TEM preparation requires demineralization which could influence the chemical properties of either bone slices or bone-like composites. Moreover, the use of bone slices as scaffold, although extensive, doesn’t allow ultrastructural details that are necessary in the study of mineral resorption by monocytes or preosteoclasts [2,3]. The aim of the present study was to set up an experimental model for the study of cell-ECM interaction between either monocytes or early differentiating osteoclasts and a mineralized ECM. RAW 264.7 cells (a monocyte-macrophage cell line that can differentiate in osteoclasts) were cultured on a composite constituted by calcium phosphate and type I collagen to investigate actin polymerization and podosome formation. This bone-like composite doesn’t present the mechanical bone properties, but it is constituted by the main bone components and exhibits the advantage that collagen glues the mineral phase in clusters that can be either added to cell cultures or applied on coverslips, as well as to the culture medium. Light and fluorescence microscopy, as well as TEM and SEM techniques were employed. Results showed that the use of this bone-like composite allowed to obtain useful morphological information about the resorption activity of RAW 264.7 cell line differentiating towards the osteoclastic phenotype.

Physiology and pathophysiology of the RANKL/RANK system

The bone resorption activity of osteoclasts is regulated at many levels, including differentiation of their monocytes precursors, fusion into multi-nucleated cells, migration to the resorption site, polarization of the mature osteoclasts and assembly of a podosome-based sealing zone. Another function of osteoclasts is relative to the integrity of the actin cytoskeleton, depending on the substratum upon which the osteoclasts are spread. There are two different structures of actin known as podosomes and sealing zone, actived in specialized matrix contacts and delimiting the membrane domain, where the ruffled border is formed. When a dual coculture of murine osteoblasts and murine mononuclear monocytes, in absolute absence of exogenous cytokines and other growth factors, was cultured on glass, the basic architecture of podosomes units and ruffled border was maintained regularly (1). We studied the osteoclast morphology and its behaviour in adhesion and in vesicle traffic by combination of light microscopy immunohistochemistry and transmission electron microscopy (TEM) immunolabeling. The adhesion and the fusion of preosteoclasts were observed by scanner electron microscopy (SEM). The osteoclasts produced by our physiological dual co-culture (without interaction of specific cytokines) are functionally and biologically active TRAP + and multinucleated cells. In fact the role of RANK, expressed by osteoblasts, controls the modulation of OPG bioavailability in the extracellular compartment. The fusion of monocytes is influenced by the presence of osteoblasts, that is based on RANK-RANKL interaction and communication between osteoblasts and preosteoclasts mediated by several molecules (2). Osteoclasts and osteoblasts can make direct contact, allowing membrane-bound ligands and receptors to interact and initiate intracellular signalling. RANKL-RANK complexes are likely internalized via rafts and then degraded in lysosomes. A recent study has shown that membrane-bound RANKL complexes to OPG is internalized by endocytosis process. A potential interaction of RANKL with clathrin components prior to the OPG binding, as shown by the kinetic results, OPG is intracellularly degraded after being internalized: our observation of osteoclast membrane at TEM has shown that immunogold labelled RANKL colocalizes with immunoglod labelled clathrin via the clathrin-coated-pit-mediated (3) pathway and both proteins are degraded by lysosome and proteasome pathway. 1) Nicolin V. et al.,. 2006 J.Mol.Histol.37 :171-177 2) Narducci P., et al.,. Acta Histochem.113(2):73-81. 3) Narducci P., et al., 2010, Eur J Histochem 54(1):e6.

Fimbrin in podosomes of monocyte‐derived osteoclasts

Fimbrin in podosomes of monocyte‐derived osteoclasts