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 alpha-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.     

Rho-dependent Rho kinase activation increases CD44 surface expression and bone resorption in osteoclasts

Osteoclasts from osteopontin-deficient mice exhibit decreased CD44 surface expression [corrected]. Osteopontin (OPN)/alphavbeta3 generated Rho signaling pathway is required for the surface expression of CD44. In this work we show the Rho effector, Rho kinase (ROK-alpha), to be a potent activator of CD44 surface expression. ROK-alpha activation was associated with autophosphorylation, leading to its translocation to the plasma membrane, as well as its association with CD44. ROK-alpha promoted CD44 surface expression through phosphorylation of CD44 and ezrin-radixin-moesin (ERM) proteins and CD44.ERM.actin complex formation. Osteoclasts from OPN-/- mice exhibited an approximately 55-60% decrease in basal level ROK-alpha phosphorylation as compared with wild type osteoclasts. Furthermore, RhoVal-14 transduction was only partially effective in stimulating ROK-alpha/CD44 phosphorylation, as well as CD44 surface expression, in these osteoclasts. Studies on the inhibition of Rho by C3 transferase or ROK-alpha by the specific inhibitor, Y-27632, showed a decrease in the phosphorylation mediated by ROK-alpha and CD44 surface expression. Neutralizing antibodies to alphav, beta3, or CD44 inhibited the migration and bone resorption of wild type osteoclasts. However, only anti-alphav or -beta3 antibodies blocked OPN-induced phosphorylation of ROK-alpha, CD44, and the ERM proteins. Our results strongly suggest a role for ROK-alpha in alphavbeta3-mediated Rho signaling, which is required for the phosphorylation events and CD44 surface expression. The functional deficiencies in the Rho effector(s) because of the lack of OPN were associated with decreased CD44 surface expression and hypomotility in the OPN-/- osteoclasts. Finally, we find that cooperativity exists between alphavbeta3 and CD44 for osteoclast motility and bone resorption.     

Phosphorylated osteopontin promotes migration of human choriocarcinoma cells via a p70 S6 kinase-dependent pathway

This study examined the role of osteopontin (OPN), a phosphorylated secreted glycoprotein, in the promotion of trophoblastic cell migration, an early event in the embryo implantation process. Three human choriocarcinoma cell lines, namely JAR, BeWo, and JEG-3, were treated with variants of OPN differing in the extent of phosphorylation following sequential dephosphorylation with tartrate-resistant acid phosphatase (TRAP), and their migratory response was measured. The highly phosphorylated human milk form of OPN (OPN-1) strongly triggered migration in all three cell lines, whereas the less phosphorylated variants, OPN-2a and OPN-2b, failed to stimulate migration. JAR cell migration in response to OPN-1 was accompanied by a rapid rearrangement of actin filaments to the cellular membrane. Using broad spectrum protein kinase profiling, we identified p70 S6 kinase as a major signal transduction pathway activated by OPN-1 during the migratory response in JAR cells. Activation was blocked completely by rapamycin and LY294002, thus demonstrating that OPN-1-stimulated migration occurs through mTOR and PI3K pathways, respectively. Conversely, PD98059 did not affect the activation of p70 S6 kinase by OPN-1, therefore, this response does not involve the Ras/ MAPK signaling cascade. Together, these data show that the highly phosphorylated human OPN-1 can stimulate trophoblastic cell migration and provides evidence for the involvement of the PI3K/mTOR/p70 S6 kinase pathway in the JAR cells response. Because both OPN and TRAP are expressed in the uterus during early pregnancy, it is conceivable that extracellular phosphatases such as TRAP may modify OPN charge state and thus modulate cell migration.     

Osteopontin is not required for the development of Th1 responses and viral immunity

Osteopontin (OPN) has been defined as a key cytokine promoting the release of IL-12 and hence inducing the development of protective cell-mediated immunity to viruses and intracellular pathogens. To further characterize the role of OPN in antiviral immunity, OPN-deficient (OPN-/-) mice were analyzed after infection with influenza virus and vaccinia virus. Surprisingly, we found that viral clearance, lung inflammation, and recruitment of effector T cells to the lung were unaffected in OPN-/- mice after influenza infection. Furthermore, effector status of T cells was normal as demonstrated by normal IFN-gamma production and CTL lytic activity. Moreover, activation and Th1 differentiation of naive TCR transgenic CD4+ T cells by dendritic cells and cognate Ag was normal in the absence of OPN in vitro. Contrary to a previous report, we found that OPN-/- mice mounted a normal immune response to Listeria monocytogenes. In conclusion, OPN is dispensable for antiviral immune responses against influenza virus and vaccinia virus.     

Impaired anti-tumor cytotoxicity of macrophages from osteopontin-deficient mice

Osteopontin (OPN) expression in tumors is associated with more aggressive tumor growth; however, several studies have suggested that OPN as a host protein can regulate tumor growth as well. OPN is produced by macrophages and T cells, and reportedly modifies macrophage function. Here, we have investigated the effect of OPN on macrophage function, and its role in host defense against tumor growth. OPN deficient (-/-) and wild-type (WT) peritoneal macrophages were assessed for their ability to mediate cytotoxicity of tumor cells. Thioglycollate-elicited peritoneal exudate cells (PEC) were stimulated in vitro with interferon-gamma and lipopolysaccharide. [(3)H]Thymidine-labeled ras-transformed tumor cells were then added and (3)H release and nitrite accumulation were measured. OPN -/- PEC exhibited as much as a 70% reduction in cytotoxicity as compared to WT PEC. Tumor cell OPN status, on the other hand, had little effect on the extent of cytotoxicity. Production of nitrite by the PEC correlated with their capacity to kill tumor cells. L-929 cells, which are relatively resistant to nitric oxide-induced cytotoxicity and sensitive to that effected by TNF-alpha, were killed equally well by wild-type and OPN-deficient PEC, suggesting that the effect of OPN is not mediated through TNF-alpha. No difference was seen in the cytotoxicity of resident macrophages from mice of different genotypes, indicating that the defect in the OPN-deficient macrophages may result from altered differentiation in vivo. In support of this idea, we show that the expression of the macrophage markers F4/80 in peritoneal cells and of Mac-2 in spleen cells is altered in OPN -/- mice as compared to WT. These data support the hypothesis that host-derived osteopontin may inhibit tumor growth and provide a mechanism for this effect.     

AlphaV-integrins mediate the mechanoprotective action of osteopontin in podocytes

Increased mechanical load in podocytes due to glomerular hypertension is one of the important factors leading to podocyte damage and chronic kidney disease. In previous studies, we have shown that mechanical stretch increases osteopontin (OPN) expression in podocytes and that exogenous OPN is mechanoprotective via facilitating cytoskeletal reorganization of podocytes. In the present study, we asked whether the mechanoprotective effect of OPN in podocytes is mediated through specific integrins and whether endogenous OPN of podocytes is required for mechanoprotection. Conditionally immortalized mouse podocytes and primary podocytes (PP) from OPN-/- and OPN+/+ mice were used. Cyclic biaxial mechanical stretch (0.5 Hz, 7% linear strain) was applied for up to 3 days. Stretch-induced cell loss was ～30% higher in OPN-/- PP compared with OPN+/+ PP. Increased cell loss of OPN-/- PP was rescued by OPN coating. Analysis of integrin expression by RT-PCR, application of RGD and SLAYGLR peptides and anti-integrin antibodies, small-interfering RNA knockdown of integrins, and application of kinase inhibitors identified αV-integrins (αVβ1, αVβ3, and αVβ5) to mediate the mechano-protective effect of OPN in podocytes involving focal adhesion kinase, Src, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Our results demonstrate that endogenous OPN of podocytes plays a nonredundant role in podocyte adaptation to mechanical stretch, and that OPN signaling via α(V)-integrins may represent a relevant therapeutical target in podocytes.     

Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.     

IFN-gamma/lipopolysaccharide activation of macrophages is associated with protein kinase C-dependent down-modulation of the colony-stimulating factor-1 receptor.

IFN gamma/LPS treatment increases macrophage tumoricidal and microbicidal activity and inhibits CSF-1-induced macrophage proliferation. The mechanism underlying the latter effect was investigated in the CSF-1-dependent mouse macrophage cell line, BAC-1.2F5. IFN-gamma and LPS together dramatically reduced the total number of CSF-1 receptors (CSF-1R) via selective degradation of the cell surface form. Processing and transport of intracellular CSF-1R to the cell surface were unaffected. IFN-gamma alone had no effect but significantly enhanced LPS-induced CSF-1R down-regulation. The reduction in CSF-1R number was protein kinase C-dependent and involved changes in serine phosphorylation of the receptor at different sites. CSF-1R down-modulation by this mechanism may be important in switching off the energy-consuming processes of CSF-1R-mediated proliferation and chemotaxis in activated macrophages.     

Requirement for PI 3-kinase gamma in macrophage migration to MCP-1 and CSF-1

Phosphoinositide 3-kinases (PI3Ks) are important regulators of cell migration. The PI3K isoform gamma is primarily expressed in haematopoietic cells, and is activated by G protein-coupled receptors (GPCRs). Here, we investigate the contribution of PI3Kgamma to macrophage responses to chemoattractants, using bone marrow-derived macrophages from wild-type and PI3Kgamma-null mice. We observe that early membrane ruffling induced by MCP-1, which activates a GPCR, or by CSF-1, which activates a tyrosine kinase receptor, is unaltered in PI3Kgamma(-/-) mice, although by 30 min MCP-1-induced cell polarization was strongly reduced in PI3Kgamma(-/-) compared to wild-type macrophages. The migration behaviour of the macrophages was analysed by time-lapse microscopy in Dunn chemotaxis chambers. PI3Kgamma(-/-) macrophages showed reduced migration speed and translocation, and no chemotaxis to MCP-1. Interestingly, there was also a reduction in migration efficiency in PI3Kgamma(-/-) macrophages stimulated with CSF-1 although early CSF-1R signalling was normal. These results indicate that the initial actin reorganization induced by either a GPCR or tyrosine kinase receptor agonist is not dependent on PI3Kgamma, whereas PI3Kgamma is needed for optimal migration of macrophages to either agonist.     

Polarization and directed migration of murine neutrophils is dependent on cell surface expression of CD44

The importance of CD44 in murine neutrophil chemotaxis was studied in a Zigmond chamber. WT neutrophils polarized more rapidly and more extensively than CD44-/- neutrophils, which showed slow random migration and reduced activation of RhoA. CD44+/- neutrophils polarized more slowly, formed fewer directionally polarized cells, and migrated more slowly than WT cells. Antibodies to CD44 decreased polarization of WT neutrophils and reduced directed migration but not migration speed, indicating that CD44 mediates chemotactic signaling and migration through different pathways, while a hyaluronate substratum markedly reduced both the speed and directed migration of WT cells. In contrast to macrophages, the level of cell surface CD44 in neutrophils was not affected by osteopontin expression and CD44 did not co-localize with osteopontin. In polarized neutrophils, CD44 was enriched in uropods while cortical actin was predominant at the leading edge. Thus, both polarization and directed migration of neutrophils are dependent on the expression of CD44 and its interaction with hyaluronan, which could modulate neutrophil migration into inflamed tissues.     

CD148/DEP-1 association with areas of cytoskeletal organisation in macrophages

In macrophages, tyrosine phosphorylation regulates many signalling pathways leading to growth, differentiation, activation, phagocytosis and adhesion. Protein tyrosine phosphatases (PTPs) represent a biochemical counterbalance to the activity of protein tyrosine kinases, thus regulating the dynamic phosphorylation state of a cell. CD148 is a receptor PTP that is highly expressed in macrophages and is further regulated by pro-inflammatory stimuli. CD148 is normally localised to the plasma membrane of macrophages. Following stimulation with CSF-1 or LPS, there was a re-distribution and concentration of CD148 in areas of membrane ruffling. Treatment of macrophages with anti-CD148 monoclonal antibody inhibited CSF-1-induced macrophage spreading, cytoskeletal re-arrangements and chemotaxis, without affecting cell survival. There were no detectable effects on the CSF-1 receptor-akt signalling pathway. These results are consistent with the hypothesis that CD148 is a regulator of macrophage activity.     

Characterization of the expression of variant and standard CD44 in prostate cancer cells: Identification of the possible molecular mechanism of CD44/MMP9 complex formation on the cell surface

CD44 is a glycosylated adhesion molecule and osteopontin is one of its ligand. CD44 undergoes alternative splicing to produce variant isoforms. Our recent studies have shown an increase in the surface expression of CD44 isoforms (sCD44 and v4–v10 variant CD44) in prostate cancer cells over‐expressing osteopontin (PC3/OPN). Formation of CD44/MMP9 complex on the cell surface is indispensable for MMP9 activity. In this study, we have characterized the expression of variant CD44 using RT‐PCR, surface labeling with NHS–biotin, and immunoblotting. Expression of variant CD44 encompassing v4–v10 and sCD44 at mRNA and protein levels are of the same levels in PC3 and PC3/OPN cells. However, an increase in the surface expression of v6, v10, and sCD44 in PC3/OPN cells suggest that OPN may be a ligand for these isoforms. We then proceeded to determine the role of sCD44 in MMP9 activation. Based on our previous studies in osteoclasts, we hypothesized that phosphorylation of CD44 has a role on its surface expression and subsequent activation of MMP9. We have prepared TAT‐fused CD44 peptides comprising unphosphorylated and constitutively phosphorylated serine residues at positions Ser323 and Ser325. Transduction of phosphopeptides at Ser323 and Ser323/325 into PC3 cells reduced the surface levels of CD44, MMP9 activity, and cell migration; but had no effect on the membrane localization of MMP9. However, MMP9 knock‐down PC3 cells showed reduced CD44 at cellular and surface levels. Thus we conclude that surface expression of CD44 and activation of MMP9 on the cell surface are interdependent. J. Cell. Biochem. 108: 272–284, 2009. © 2009 Wiley‐Liss, Inc.     

CD44v6 regulates growth of brain tumor stem cells partially through the AKT-mediated pathway

Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC) has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6) in BTSC of a subset of glioblastoma multiforme (GBM). Patients with CD44(high) GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44(high) GBM but not from CD44(low) GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN), increased expression of phosphorylated AKT in CD44(high) GBM, but not in CD44(low) GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKT pathway.     

The role of C1QBP in CSF-1-dependent PKCζ activation and macrophage migration

Macrophages view as double agents in tumor progression. Trafficking of macrophages to the proximity of tumors is mediated by colony-stimulating factor-1 (CSF-1), a growth factor. In this study, we investigated the role of complement1q-binding protein (C1QBP)/ atypical protein kinase C ζ (PKCζ) in CSF-1-induced macrophage migration. Disruption of C1QBP expression impaired chemotaxis and adhesion of macrophage. Phosphorylation of PKCζ is an essential component in macrophage chemotaxis signaling pathway. C1QBP could interact with PKCζ in macrophage. C1QBP knockdown inhibited CSF-1 induced phosphorylation of PKCζ and integrin-β1. However, C1QBP knockdown didn’t affect the phosphorylation of PKCζ induced by MCP-1. Furthermore, CSF-1 from RCC cell condition medium promoted macrophage chemotaxis and adhesion. Taken together, our results demonstrated that C1QBP plays an essential role in CSF-1 induced migration of macrophages.     

Role of the metastasis‐promoting protein osteopontin in the tumour microenvironment

Osteopontin (OPN) is a secreted protein present in bodily fluids and tissues. It is subject to multiple post‐translational modifications, including phosphorylation, glycosylation, proteolytic cleavage and crosslinking by transglutamination. Binding of OPN to integrin and CD44 receptors regulates signalling cascades that affect processes such as adhesion, migration, invasion, chemotaxis and cell survival. A variety of cells and tissues express OPN, including bone, vasculature, kidney, inflammatory cells and numerous secretory epithelia. Normal physiological roles include regulation of immune functions, vascular remodelling, wound repair and developmental processes. OPN also is expressed in many cancers, and elevated levels in patients’ tumour tissue and blood are associated with poor prognosis. Tumour growth is regulated by interactions between tumour cells and their tissue microenvironment. Within a tumour mass, OPN can be expressed by both tumour cells and cellular components of the tumour microenvironment, and both tumour and normal cells may have receptors able to bind to OPN. OPN can also be found as a component of the extracellular matrix. The functional roles of OPN in a tumour are thus complex, with OPN secreted by both tumour cells and cells in the tumour microenvironment, both of which can in turn respond to OPN. Much remains to be learned about the cross‐talk between normal and tumour cells within a tumour, and the role of multiple forms of OPN in these interactions. Understanding OPN‐mediated interactions within a tumour will be important for the development of therapeutic strategies to target OPN.     

Enhanced killing of Candida albicans by murine macrophages treated with macrophage colony-stimulating factor: evidence for augmented expression of mannose receptors

The effect of macrophage colony-stimulating factor (CSF-1) on killing of Candida albicans by murine peritoneal macrophages was determined. The killing capacity of resident peritoneal macrophages was unaffected by CSF-1. However, proteose-peptone-elicited peritoneal exudate macrophages that had been pretreated with CSF-1 (greater than or equal to 1000 U/ml) for 24 or 48 hr exhibited a significantly enhanced capacity to kill C. albicans. CSF-enhanced killing appeared to be independent of endogenously produced interferon-alpha/beta (IFN) in that enhancement by these two agents differed with regard to onset of the effect, target cell responsiveness, and duration of augmented killing. In addition, a highly specific anti-IFN antiserum that totally neutralized IFN augmentation of candidacidal activity had no effect on CSF-induced enhancement. Evidence was obtained indicating that CSF, unlike IFN, augmented mannose-inhibitable binding and ingestion of C. albicans, suggesting that augmented expression of mannose-receptors by CSF-treated macrophages was at least partially responsible for the enhanced killing.     

Macrophage membrane interleukin 1 regulates the expression of acute phase proteins in human hepatoma Hep 3B cells

To assess the potential role of membrane interleukin 1 (mIL-1) in modulating expression of acute phase proteins, we studied the effect of fixed mouse peritoneal macrophages and isolated cell membranes on the synthesis of C3 and albumin in human hepatoma Hep 3B cells. An increase in C3 synthesis and decrease in albumin synthesis were detected after incubation of Hep 3B cells with fixed stimulated macrophages or with membrane preparations. Estimates of hepatocellular C3 and albumin mRNA indicated that the mIL-1 regulation was exerted at a pretranslational level. The changes in C3 and albumin expression induced by mIL-1 were inhibited by addition of anti-interleukin 1 (IL-1) antiserum to the macrophages, supporting the hypothesis that a form of IL-1 is present on the outer cell membrane of macrophages. Moreover, cell-surface iodination of macrophages allowed the detection of a 33-kDa IL-1 molecule, suggesting that an IL-1 species, similar to the intracellular precursor of IL-1 is present at the outer cell surface of macrophages. The expression of mIL-1 was temporally dissociated from IL-1 release, indicating that the cell-surface associated IL-1 activity is not the result of adsorbed soluble IL-1. These studies provide a basis for further investigation of the role of mIL-1 in modulating monocytes and macrophages function via cell-cell interactions.     

Osteopontin is associated with nuclear factor kappaB gene expression during tail-suspension-induced bone loss

Osteoporosis due to unloading-induced bone loss is a critical issue in the modern aging society. Although the mechanisms underlying this phenomenon are largely unknown, osteopontin (OPN) is one of the critical mediators required for unloading-induced bone loss [M. Ishijima, S.R. Rittling, T. Yamashita, K. Tsuji, H. Kurosawa, A. Nifuji, D.T. Denhardt, and M. Noda, Enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of osteopontin, J Exp Med, 193 (2001) 399-404]. To clarify the molecular bases for OPN actions, we carried out microarray analyses on the genes expressed in the femoral bone marrow cells in wild type and OPN-/- mice. The removal of the mechanical load induced bone loss in wild type, but not in OPN-/- mice, as previously reported. Expression analysis of 9586 cDNAs on a microarray system revealed that OPN deficiency blocked tail-suspension-induced expression of ten genes (group A). This observation was confirmed based on semi-quantitative RT-PCR analyses. On the other hand, expression of four genes (group B) was not altered by tail suspension in wild type but was enhanced in OPN-deficient mice. NF-kappaB p105 subunit gene (Nfkb1) was found in group A and Bax in group B. p53 gene expression was upregulated by tail suspension in wild type mice, but it was no longer observed in OPN-/- mice. These data indicate that OPN acts to mediate mechanical stress signaling upstream to the genes encoding apoptosis-related molecules, and its action is associated with alteration of the genes.     

In vivo osteopontin-induced macrophage accumulation is dependent on CD44 expression

In order to assess the role of osteopontin (OPN) in leukocyte accumulation in inflammatory conditions, native OPN and its thrombin cleaved form (OPN + Thr) were studied in vivo using a rodent subcutaneous air pouch model (AP). Both forms of OPN-induced macrophage infiltration into the AP in wild-type mice. In animals lacking CD44, macrophage numbers were significantly reduced within the cavity, but cells still accumulated along the subcutaneous lining. In animals lacking endogenous OPN, no differences were found in exogenous OPN-induced macrophage accumulation, although macrophage exhibited increased α4 integrin expression. These studies reveal that both OPN and OPN + Thr attract macrophages in vivo through CD44.     

Restoration of podosomes and chemotaxis in Wiskott-Aldrich syndrome macrophages following induced expression of WASp

We used a direct-viewing (Dunn) chemotaxis chamber to analyse the chemotactic responses of human normal and Wiskott-Aldrich syndrome (WAS) macrophages to the cytokine colony stimulating factor-1 (CSF-1). In five patients with classic WAS, where specialised adhesion complexes called podosomes are absent, chemotaxis of macrophages was abolished. The deficient chemotactic responses of WAS macrophages following cytokine stimulation could be correlated with abnormalities in cell polarisation and actin organisation. In a series of cell microinjection studies we found that normal chemotactic responses were restored in WASp macrophages transfected with a full-length human WAS construct. Expression of exogenous WAS protein (WASp) in these cells also restored normal polarised cell morphology and the ability to form podosomes.