Baoqian Zhu

Osteopontin modulates CD44-dependent chemotaxis of peritoneal macrophages through G-protein-coupled receptors: evidence of a role for an intracellular form of osteopontin.

Expression of osteopontin (OPN) by activated T‐cells and macrophages is required for the development of cell‐mediated inflammatory responses. Acting through integrin αvβ3 and CD44 receptors, OPN can promote chemoattraction and pro‐inflammatory cytokine expression by macrophages. In this study, we have used periotoneal macrophages from OPN−/, CD44−/−, and WT mice to study the relationship between OPN and CD44 in macrophage migration. Using confocal microscopy, we show that OPN co‐distributes with CD44 inside macrophages at cell edges and in cell processes in a mutually dependent manner. The existence of an intracellular form of OPN is supported by pulse‐chase studies in which a thrombin‐sensitive, phosphorylated protein immunoprecipitated with OPN antibodies is retained inside macrophages. In OPN−/− and CD44−/− macrophages, the absence of CD44 and OPN, respectively, is associated with the formation of fewer cell processes, reduced cell fusion required to form functional multinucleated osteoclasts in the presence of CSF‐1 and RANKL, and impaired chemotaxis. Whereas the chemotaxis of CD44−/− cells to various chemoattractants is almost completely abrogated, a differential effect is seen with the OPN−/− cells. Thus, OPN−/− cells migrate normally towards CSF‐1 but not towards fMLP and MCP‐1, which signal through G‐protein coupled receptors (GPCRs). That the GPCR‐mediated migration is dependent upon the level of cell‐surface CD44 is indicated by the reduced cell‐surface expression of CD44 in OPN−/− cells and a comparable impairment in the chemotaxis of CD44+/− cells. Although chemotaxis of OPN−/− cells could be rescued by an OPN substratum, or by addition of high levels of OPN in solution, no response is evident with physiological levels of OPN, indicating a requirement for the CD44‐associated intracellular OPN in CD44 cell‐surface expression. These studies indicate, therefore, that the level of cell surface CD44 is critical for GPCR‐mediated chemotaxis by peritoneal macrophages and suggest that a novel intracellular form of OPN may modulate CD44 activities involved in these processes. J. Cell. Physiol. 198: 155–167, 2004.

Colocalization of intracellular osteopontin with CD44 is associated with migration, cell fusion, and resorption in osteoclasts.

Although osteopontin (OPN) is recognized generally as a secreted protein, an intracellular form of osteopontin (iOPN), associated with the CD44 complex, has been identified in migrating fibroblastic cells. Because both OPN and CD44 are expressed at high levels in osteoclasts, we have used double immunofluorescence analysis and confocal microscopy to determine whether colocalization of these proteins has functional significance in the formation and activity of osteoclasts. Analysis of rat bone marrow‐derived osteoclasts revealed strong surface staining for CD44 and β1‐ and β3‐integrins, whereas little or no staining for OPN or bone sialoprotein (BSP) was observed in nonpermeabilized cells. In permeabilized perfusion osteoclasts and multinucleated osteoclasts, staining for OPN and CD44 was prominent in cell processes, including filopodia and pseudopodia. Confocal microscopy revealed a high degree of colocalization of OPN with CD44 in motile osteoclasts. In cells treated with cycloheximide (CHX), perinuclear staining for OPN and BSP was lost, but iOPN staining was retained within cell processes. In osteoclasts generated from the OPN‐null and CD44‐null mice, cell spreading and protrusion of pseudopodia were reduced and cell fusion was impaired. Moreover, osteoclast motility and resorptive activity were significantly compromised. Although the area resorbed by OPN‐null osteoclasts could be rescued partially by exogenous OPN, the resorption depth was not affected. These studies have identified an intracellular form of OPN, colocalizing with CD44 in cell processes, that appears to function in the formation and activity of osteoclasts.

Novel Functions of the Matricellular Proteins Osteopontin and Osteonectin/SPARC

Osteopontin (OPN) and osteonectin/SPARC (ON/SPARC) are prominent matricellular components of the extracellular matrix of mineralized tissues of bones and teeth in which they can regulate the formation and growth of hydroxyapatite crystals and influence a variety of cell activities. OPN regulates cell responses through several integrin receptors and is also a ligand for the CD44 receptor, through which it acts as a chemoattractant. Although a cell-surface receptor for SPARC has not been identified it can block cell-cell and cell-matrix interactions and inhibit cell migration and chemotaxis. OPN and SPARC also appear to function inside cells. Thus, OPN appears to exist in association with the CD44 receptor inside migratory cells, while intracellular SPARC is associated with axonemal tubulin in ciliated epithelial cells. Analyses of fibroblasts and peritoneal macrophages from OPN-null and CD44-null cells show impaired functionality involving migration and cell fusion required for osteoclast formation, while disruption of SPARC expression leads to developmental defects in Xenopus . To gain further insights into the intracellular functions of OPN and SPARC, we have used the yeast two-hybrid system to identify potential interacting molecules. Using full-length SPARC as bait the carboxy-terminal domain, which contains two EF-hand, high-affinity binding sites, was found to have transcriptional activity, while several novel proteins that interact with the amino-terminal domains of SPARC and full-length OPN have been identified. The identification of OPN and SPARC inside specialized cells introduces a novel concept in cellular regulation by matricellular proteins.

Characterisation of the constitutive over-expression of AJ18 in a novel rat stromal bone marrow cell line (D8-SBMC)

OBJECTIVE The elucidation of the molecular pathways involved in osteoblast proliferation and differentiation has been greatly enhanced by the availability of cell culture model systems. However, many of the current bone cell culture systems suffer from disadvantages such as the inability to generate mineralised bone-like nodules, a transformed genetic background, cell heterogeneity, and a relatively long time frame from cell seeding to mineralisation, often in the order of several weeks. Here we describe the establishment and characterisation of a novel bone cell line named D8-SBMC. As a first demonstration of their potential value, D8-SBMC was utilised to further support a role for AJ18 during osteogenesis. DESIGN D8-SBMC was established from a single cell suspension of the previously characterised long term rat stromal bone marrow cells [Kotev-Emeth S, Pitaru S, Pri-Chen S, Savion N. Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2. Connect Tissue Res 2002;43(4):606-12; Pitaru S, Kotev-Emeth S, Noff D, Kaffuler S, Savion N. Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture. J Bone Miner Res 1993;8(8):919-29]. AJ18 was constitutively and stably over-expressed in D8-SBMC and analysed. RESULTS D8-SBMC possesses the ability to form robust mineralised bone-like nodules within 8 days proceeding cell confluency. Interestingly, a cement line-like matrix is also generated between the culture dish and a basal monolayer of cells. Constitutive and stable over-expression of AJ18 resulted in an increase in cell proliferation and mineralisation. Expression of bone marker genes, such as bone sialoprotein, osteopontin, osteocalcin, collage type 1, and osteonectin, was up-regulated by AJ18 over-expression. CONCLUSION A novel bone cell line, D8-SBMC, was established and characterised. D8-SBMC may be a valuable model system for biomineralisation studies. D8-SBMC was utilised to further understand the role of AJ18 in cell proliferation and differentiation during osteogenesis.