Expression of osteopontin (OPN) by activated T-cells and macrophages is required for the development of cell-mediated inflammatory responses. Acting through integrin alpha(v)beta(3) and CD44 receptors, OPN can promote chemoattraction and pro-inflammatory cytokine expression by macrophages. In this study, we have used peritoneal 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.

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 ␤1-and ␤3-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

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induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN Ϫ/Ϫ ) mice were treated in vitro with H2O2 to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN Ϫ/Ϫ cells but was increased to only 20% in WT cells. In contrast, after 1-8 h of treatment with H2O2, the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN Ϫ/Ϫ cells. Electron microscopy of WT cells treated with H 2O2 showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H 2O2-treated OPN Ϫ/Ϫ cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN Ϫ/Ϫ and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN Ϫ/Ϫ cells by ϳ30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PIpermeable OPN Ϫ/Ϫ cells was not altered. Restoration of OPN expression in OPN Ϫ/Ϫ fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H 2O2 treatment. Thus H2O2-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death. necrosis; reperfusion; myocardium; nuclear fragmentation AFTER MYOCARDIAL INFARCTION, ischemic damage to cardiac tissues causes tissue necrosis and an associated inflammatory response that leads to disorganization of myocardial architecture (1, 14). The death of cardiomyocytes is an integral part of pathological tissue destruction after infarction, but the viability of endothelial cells and fibroblasts is protected, thereby enabling the formation of a reparative matrix. Although matrix production by fibroblasts is important for maintaining the integrity of cardiac tissues, the persistence of activated fibroblasts (5, 10, 15) leads to the formation of a stiff, noncompliant scar tissue that compromises cardiac function. Accordingly, present therapeutic approaches are aimed at minimizing fibrosis during reperfusion (22).During the reperfusion stage after myocardial infarction, there is increased production of oxidants and cytokines including IL-10, transforming growth factor-␤1, and osteopontin (OPN) that stimulate fibroblast proliferation and matrix formation. In postischemic hearts, OPN expression is increased (51, 54, 58); this enhanced expression is considered an import...

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