Ron Zohar

Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration.

Osteopontin (OPN) is a secreted glycoprotein with mineral‐ and cell‐binding properties that can regulate cell activities through integrin receptors. Previously, we identified an intracellular form of osteopontin with a perimembranous distribution in migrating fetal fibroblasts (Zohar et al., J Cell Physiol 170:88–98, 1997). Since OPN and CD44 expression are increased in migrating cells, we analyzed the relationship of these proteins with immunofluorescence and confocal microscopy. A distinct co‐localization of perimembranous OPN and cell‐surface CD44 was observed in fetal fibroblasts, periodontal ligament cells, activated macrophages, and metastatic breast cancer cells. The co‐localization of OPN and CD44 was prominent at the leading edge of migrating fibroblasts, where OPN also co‐localized with the ezrin/radixin/moesin (ERM) protein ezrin, as well as in cell processes and at attachment sites of hyaluronan‐coated beads. The subcortical location of OPN in these cells was verified by cell‐surface biotinylation experiments in which biotinylated CD44 and non‐biotinylated OPN were isolated from complexes formed with hyaluronan‐coated beads and identified with immunoblotting. That perimembranous OPN represents secreted protein internalized by endocytosis or phagocytosis appeared to be unlikely since exogenous OPN that was added to cell cultures could not be detected inside the cells. A physical association with OPN, CD44, and ERM, but not with vinculin or α‐actin, was indicated by immunoadsorption and immunoblotting of cell proteins in complexes extracted from hyaluronan‐coated beads. The functional significance of OPN in this complex was demonstrated using OPN−/− and CD−/− mouse fibroblasts which displayed impaired migration and a reduced attachment to hyaluronan‐coated beads. These studies indicate that OPN exists as an integral component of a hyaluronan‐CD44‐ERM attachment complex that is involved in the migration of embryonic fibroblasts, activated macrophages, and metastatic cells. J. Cell. Physiol. 184:118–130, 2000.

Role of osteopontin in neutrophil function

Osteopontin (OPN) is important for the function of fibroblasts, macrophages and lymphocytes during inflammation and wound healing. In recent studies of experimental colitis we demonstrated exacerbated tissue destruction in OPN‐null mice, associated with reduced tumour necrosis factor‐α expression and increased myeloperoxidase activity. The objective of this investigation therefore was to determine the importance of OPN expression in neutrophil function. Although, in contrast to macrophages, neutrophils expressed low levels of OPN with little or no association with the CD44 receptor, intraperitoneal recruitment of neutrophils in OPN‐null mice was impaired in response to sodium periodate. The importance of exogenous OPN for neutrophil recruitment was demonstrated by a robust increase in peritoneal infiltration of PMNs in response to injections of native or recombinant OPN. In vitro, OPN–/– neutrophils exhibited reduced chemokinesis and chemotaxis towards N‐formyl methionyl leucyl phenylalanine (fMLP), reflecting a reduction in migration speed and polarization. Exogenous OPN, which was chemotactic for the neutrophils, rescued the defects in polarization and migration speed of the OPN–/– neutrophils. In contrast, the defensive and cytocidal activities of OPN–/– neutrophils, measured by assays for phagocytosis, generation of reactive oxygen species, cytokine production and matrix metalloproteinase‐9, were not impaired. These studies demonstrate that, while exogenous OPN may be important for the recruitment and migration of neutrophils, expression of OPN by neutrophils is not required for their destructive capabilities.

SINGLE CELL ANALYSIS OF INTRACELLULAR OSTEOPONTIN IN OSTEOGENIC CULTURES OF FETAL RAT CALVARIAL CELLS

Osteopontin (OPN), a major component of the bone matrix, is expressed at different stages of bone formation. To determine possible relationships between OPN expression and stages of osteogenic cell differentiation, we have performed single cell analyses of intracellular OPN in early (proliferating), subconfluent (differentiating), and mature (mineralizing) cultures of fetal rat calvarial cells (FRCC) using a combination of flow cytometry and confocal microscopy. At each culture stage, a high proportion (60–98%) of cells were immunoreactive for OPN (OPN+ve). Each of these populations also included a small proportion of OPN‐ve cells which were characterized by their small size, low granularity, high proliferative capacity, and enhanced osteogenic potential. The OPN+ve cells displayed two distinct patterns of intracellular immunostaining: a perinuclear distribution typical of secreted proteins and a perimembrane distribution in which patches of OPN were concentrated at the cell surface. Perimembranous staining predominated in migrant cells, which contained greater than tenfold higher levels of OPN than nonmigrant cells as separated in a Boyden chamber. When cell proliferation was high (day 2), most cells were OPN+ve. At all culture stages the intensity of OPN staining was increased as cells progressed through the cell cycle. As cells differentiated and started to form matrix (days 4 and 6), the mean cell expression of OPN was also increased (fourfold), independent of changes in total cell protein. However, despite the association of OPN with osteogenic cells, we were surprised to find that a high proportion (60%) of fetal skin fibroblasts were also immunoreactive for OPN. The expression of OPN by these cell populations was confirmed by RT‐PCR, and a strong correlation was observed between the quantitative flow cytometry data and Western blot analysis of cell extracts in which the high and low phosphorylated isoforms of OPN were observed. These studies, therefore, have identified several phenotypes in FRCC cultures that are based on OPN expression: small OPN‐ve cell populations enriched in osteogenic precursors, differentiating osteogenic cells that synthesize and secrete OPN, and migrating stromal cells characterized by a perimembranous OPN staining pattern. J Cell Physiol 170:88–100, 1997

Osteopontin and Mucosal Protection

Protection of mucosal tissues of the oral cavity, intestines, respiratory tract, and urogenital tract from the constant challenge of pathogens is achieved by the combined barrier function of the lining epithelia and specialized immune cells. Recent studies have indicated that osteopontin (OPN) has a pivotal role in the development of immune responses and in the tissue destruction and the subsequent repair processes associated with inflammatory diseases. While expression of OPN is increased in immune cells—including neutrophils, macrophages, T- and B-lymphocytes—and in epithelial, endothelial, and fibroblastic cells of inflamed tissues, deciphering the specific functions of OPN has been difficult. In part, this is due to the broad range of biological activities of OPN that are mediated by multiple receptors which recognize several signaling motifs whose activities are influenced by post-translational modifications and proteolytic processing of OPN. Understanding the role of OPN in mucosal inflammation is further complicated by its contributions to the barrier function of the lining epithelia and the complexity of the specialized mucosal immune system. In an attempt to provide some insights into the involvement of OPN in mucosal diseases, this review summarizes current knowledge of the biological activities of OPN involved in the development of inflammatory responses and in wound healing, and indicates how these activities may affect the protection of mucosal tissues.

Exacerbated tissue destruction in DSS-induced acute colitis of OPN-null mice is associated with downregulation of TNF-α expression and non-programmed cell death

Osteopontin (OPN), a pro‐inflammatory mediator, is constitutively expressed in normal gut and is upregulated in inflammatory colitis. To determine the significance of OPN in inflammatory bowel disease, we studied the development of acute, experimental colitis induced by dextran sulfate sodium (DSS) in OPN‐null and wild‐type (WT) mice. OPN expression was markedly increased in WT diseased colons, while a higher disease activity index, including spleen enlargement, bowel shortening, and mucosal destruction, was observed in OPN‐null mice. Although peripheral blood neutrophil numbers were lower in DSS‐treated OPN‐null mice, tissue myeloperoxidase levels, reflecting enhanced neutrophil activity, were increased in the diseased colons. In comparison, lymphocyte numbers in peripheral blood were increased earlier than in DSS‐treated WT mice. Despite a significantly greater spleen enlargement, flow cytometric analysis of splenocytes from the DSS‐treated OPN‐null mice revealed lower numbers of differentiated macrophages and (CD4+ and CD8α+) lymphocytes. Whereas pro‐inflammatory cytokines, including G‐CSF, RANTES, MIP1α, and TNF‐α, were increased <10‐fold in DSS‐treated WT splenocytes, expression of these cytokines was dramatically suppressed in the DSS‐treated OPN‐null splenocytes as well as gut tissues. The suppressed TNF‐α response in OPN‐null mice was reflected in a marked increase in non‐apoptotic cell death in diseased colons. Collectively, these studies demonstrate that OPN is required for mucosal protection in acute inflammatory colitis. J. Cell. Physiol. 208: 629–639, 2006.

Osteopontin attenuation of dextran sulfate sodium- induced colitis in mice

Osteopontin (OPN) is a matricellular cytokine present in most tissues and body fluids; it is known to modulate immune responses. In previous studies using the dextran sulfate sodium (DSS) acute colitis model, we found exacerbated tissue destruction and reduced repair in OPN-null (−/−) mice compared with wild-type (WT) controls. As OPN is normally present in milk, we hypothesized that administration of OPN may protect the intestines from the adverse effects of experimental colitis. A volume of 20 or 2 μg/ml bovine milk OPN, dissolved in drinking water, was given to mice 24 h before, and during administration of DSS. Clinical parameters of colitis and neutrophil functions were analyzed as previously reported. Orally administered OPN was absorbed and detected in the colon mucosa by immunohistochemistry. The 20 μg/ml OPN- and DSS-treated WT mice showed 37% less weight loss and reduced colon shortening and spleen enlargements than control mice (P<0.05). OPN administration also reduced the disease activity index, improved red blood cell counts, and reduced gut neutrophil activity compared with the DSS-treated WT mice that were not administered OPN (P<0.05). Immunohistochemical detection of F4/80-labelled cells (macrophages) was also less frequent. The level of transforming growth factor β1 (TGF-β1) was increased and the levels of pro-inflammatory mediators decreased in colon tissue samples of OPN-treated mice analyzed by ELISA. The reversal of experimental colitis parameters by exogenous OPN was not as robust in the OPN−/− mice. Administration of prokaryotic-expressed recombinant OPN and bovine serum albumin were ineffective. This study shows that administration of a physiological concentration of milk OPN in drinking water ameliorates the destructive host response in DSS-induced acute colitis.

Osteopontin mediates Citrobacter rodentium-induced colonic epithelial cell hyperplasia and attaching-effacing lesions

Although osteopontin (OPN) is up-regulated in inflammatory bowel diseases, its role in disease pathogenesis remains controversial. The objective of this study was to determine the role of OPN in host responses to a non-invasive bacterial pathogen, Citrobacter rodentium, which serves as a murine infectious model of colitis. OPN gene knockout and wild-type mice were infected orogastrically with either C. rodentium or Luria-Bertani (LB) broth. Mouse-derived OPN(+/+) and OPN(-/-) fibroblasts were incubated with C. rodentium and attaching-effacing lesions were demonstrated using transmission electron microscopy and immunofluorescence. Colonic expression of OPN was increased by C. rodentium infection of wild-type mice. Furthermore, colonic epithelial cell hyperplasia, the hallmark of C. rodentium infection, was reduced in OPN(-/-) mice, and spleen enlargement by infection was absent in OPN(-/-) mice. Rectal administration of OPN to OPN(-/-) mice restored these effects. There was an 8- to 17-fold reduction in bacterial colonization in OPN(-/-) mice, compared with wild-type mice, which was accompanied by reduced attaching-effacing lesions, both in infected OPN(-/-) mice and OPN(-/-) mouse fibroblasts. Moreover, adhesion pedestals were restored in OPN(-/-) cells complemented with human OPN. Therefore, lack of OPN results in decreased pedestal formation, colonization, and colonic epithelial cell hyperplasia responses to C. rodentium infection, indicating that OPN impacts disease pathogenesis through bacterial attachment and altered host immune responses.