ABSTRACT
Since its initial identification as one of the genes most highly upregulated upon T-cell activation, osteopontin (or Eta-1, as it was designated then) has been demonstrated to have many roles in the regulation of the immune response on multiple levels. It contributes to the development of immune-mediated and inflammatory diseases, and it regulates the host response to infection. In some cases, the mechanisms of these effects have been elucidated, while other mechanistic functions of the protein remain obscure. The protein itself makes these analyses complex, since it binds to a series of different integrins, and in addition to its classically secreted form, an intracellular form of osteopontin has been identified, which participates in several aspects of immune regulation. In this review, we focus on the role of osteopontin in a series of immune-related diseases, particularly those where significant advances have been made in recent years: multiple sclerosis, rheumatoid arthritis, lupus and related diseases, Sjögren's disease, colitis, and 1 area of inflammatory pathology, alcoholic and nonalcoholic liver diseases. A recurring theme in these diseases is a link between osteopontin and pathogenic T cells, particularly T helper 17 cells, where osteopontin produced by dendritic cells supports IL-17 expression, contributing to pathology. In addition, a role for osteopontin in B-cell differentiation is becoming clear. In general, osteopontin contributes to pathology in these diseases, but there are examples where it has a protective role; deciphering the mechanisms underlying these differences and the specific receptors for osteopontin will be a research challenge for the future. Aside from its newly discovered role in the development of Sjögren's disease, the role of osteopontin in inflammatory conditions in the oral cavity is still poorly understood. Elucidation of this role will be of interest.
Subject(s)
Immune System Diseases/physiopathology , Osteopontin/physiology , Arthritis, Rheumatoid/physiopathology , Humans , Inflammatory Bowel Diseases/physiopathology , Lupus Erythematosus, Systemic/physiopathology , Multiple Sclerosis/physiopathology , Sjogren's Syndrome/physiopathologyABSTRACT
BACKGROUND: The integrin-binding protein osteopontin is strongly associated with tumour development, yet is an abundant dietary component as a constituent of human and bovine milk. Therefore, we tested the effect of orally administered osteopontin (o-OPN) on the development of subcutaneous tumours in mice. METHODS: Bovine milk osteopontin was administered in drinking water to tumour-bearing immune-competent mice. Tumour growth, proliferation, necrosis, apoptosis and blood vessel size and number were measured. Expression of the α9 integrin was determined. RESULTS: o-OPN suppressed tumour growth, increased the extent of necrosis, and induced formation of abnormally large blood vessels. Anti-OPN reactivity detected in the plasma of OPN-null mice fed OPN suggested that tumour-blocking peptides were absorbed during digestion, but the o-OPN effect was likely distinct from that of an RGD peptide. Expression of the α9 integrin was detected on both tumour cells and blood vessels. Potential active peptides from the α9 binding site of OPN were identified by mass spectrometry following in vitro digestion, and injection of these peptides suppressed tumour growth. CONCLUSIONS: These results suggest that peptides derived from o-OPN are absorbed and interfere with tumour growth and normal vessel development. o-OPN-derived peptides that target the α9 integrin are likely involved.
Subject(s)
Blood Vessels/drug effects , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/drug therapy , Osteopontin/administration & dosage , Administration, Oral , Animals , Binding Sites , Blood Vessels/metabolism , Cattle , Cell Growth Processes/drug effects , Female , Integrin alpha Chains/metabolism , Mice , Mice, Inbred C57BL , Neoplasms, Experimental/metabolism , Neovascularization, Pathologic/pathology , Oligopeptides/metabolism , Osteopontin/blood , Peptides/blood , Peptides/metabolism , Protein BindingABSTRACT
OBJECTIVE: Apoptosis of chondrocytes in articular cartilage has been observed in rheumatoid arthritis patients. However, molecules involved in such chondrocyte apoptosis in arthritic joints have not been fully understood. We previously observed that apoptosis of chondrocytes is enhanced in a murine arthritis model induced by injection with anti-type II collagen antibodies and lipopolysaccharide (mAbs/LPS), and osteopontin (OPN) deficiency suppresses chondrocyte apoptosis in this arthritis model in vivo. To understand how OPN deficiency renders resistance against chondrocyte apoptosis, we examined the cellular basis for this protection. DESIGN: Chondrocytes were prepared from wild-type and OPN-deficient mouse ribs, and tumor necrosis factor (TNF)-α-induced cell death was examined based on lactate dehydrogenase (LDH) release assay and TUNEL assay. RESULTS: TNF-α treatment induced LDH release in wild-type chondrocytes, while OPN deficiency suppressed such LDH release in the cultures of these cells. TNF-α-induced increase in the number of TUNEL-positive cells was observed in wild-type chondrocytes, while OPN deficiency in chondrocytes suppressed the TNF-α induction of TUNEL-positive cells. OPN deficiency suppressed TNF-α-induced increase in caspase-3 activity in chondrocytes in culture. Furthermore, OPN overexpression in chondrocytes enhanced TNF-α-induced apoptosis. CONCLUSION: These results indicated that the presence of OPN in chondrocytes is involved in the susceptibility of these cells to TNF-α-induced apoptosis.
ABSTRACT
Since its first identification as a transformation-associated protein, osteopontin (OPN) has been recognised as important in the processes of tumorigenicity and metastasis. Here, we review the evidence that OPN might be considered as a candidate prognostic marker in human cancer. In animal systems, evidence from cell injection experiments and genetically manipulated mice suggest an important but complex role for the protein in tumour progression. Moreover, studies in a variety of human cancers associate high levels of OPN expression in tumours or in blood with more advanced cancers. The mechanism of action of OPN in promoting cancer is still unclear, and we consider aspects of OPN biology that can complicate interpretation of human studies. Nevertheless, growing evidence supports a role for OPN as a potential prognostic factor for various human cancers.
Subject(s)
Biomarkers, Tumor/analysis , Cell Transformation, Neoplastic , Glycoproteins/pharmacology , Neoplasms/pathology , Animals , Disease Models, Animal , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Mice , PrognosisABSTRACT
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.
Subject(s)
Hyaluronan Receptors/metabolism , Osteoclasts/metabolism , Sialoglycoproteins/deficiency , Animals , Antibodies/immunology , Bone and Bones/metabolism , Bone and Bones/pathology , Cell Line , Cell Movement/physiology , Hyaluronan Receptors/immunology , Integrin alphaVbeta3/metabolism , Mice , Osteopontin , Sialoglycoproteins/immunology , rhoA GTP-Binding Protein/metabolismABSTRACT
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 beta1- and beta3-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.
Subject(s)
Cell Fusion , Cell Movement/physiology , Hyaluronan Receptors/metabolism , Osteoclasts/metabolism , Sialoglycoproteins/metabolism , Animals , Fluorescent Antibody Technique , Hyaluronan Receptors/physiology , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Osteoclasts/cytology , Osteopontin , Rats , Rats, Wistar , Sialoglycoproteins/physiologyABSTRACT
Multiple sclerosis is a demyelinating disease, characterized by inflammation in the brain and spinal cord, possibly due to autoimmunity. Large-scale sequencing of cDNA libraries, derived from plaques dissected from brains of patients with multiple sclerosis (MS), indicated an abundance of transcripts for osteopontin (OPN). Microarray analysis of spinal cords from rats paralyzed by experimental autoimmune encephalomyelitis (EAE), a model of MS, also revealed increased OPN transcripts. Osteopontin-deficient mice were resistant to progressive EAE and had frequent remissions, and myelin-reactive T cells in OPN-/- mice produced more interleukin 10 and less interferon-gamma than in OPN+/+ mice. Osteopontin thus appears to regulate T helper cell-1 (TH1)-mediated demyelinating disease, and it may offer a potential target in blocking development of progressive MS.
Subject(s)
Gene Expression Profiling , Multiple Sclerosis/genetics , Multiple Sclerosis/metabolism , Sialoglycoproteins/metabolism , Animals , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Expressed Sequence Tags , Gene Deletion , Gene Library , Humans , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Interferon-gamma/genetics , Interferon-gamma/metabolism , Interleukin-10/genetics , Interleukin-10/metabolism , Lymphocyte Activation , Mice , Mice, Knockout , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Oligonucleotide Array Sequence Analysis , Osteopontin , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sialoglycoproteins/deficiency , Sialoglycoproteins/genetics , Spinal Cord/metabolism , Th1 Cells/immunologyABSTRACT
Osteopontin has been implicated in the metastasis of tumors, and human tumors with high metastatic activity often express osteopontin at high levels. Osteopontin contains an arginine-glycine-aspartate (RGD) motif that is recognized by integrin family members to promote various cell activities including attachment to substrate and it is abundant in bone, to which certain tumors preferentially metastasize. Therefore, we investigated the role of osteopontin in the experimental metastasis of tumor cells using recently established osteopontin-deficient mice. B16 melanoma cells, which produce little osteopontin, were injected into the left ventricle of osteopontin-deficient mice or wild-type mice. Animals were killed 2 weeks after injection. The number of tumors was reduced in the bones of osteopontin-deficient mice compared with the bones in wild-type mice. The number of tumors in the adrenal gland also was reduced. To investigate the osteopontin effect on metastases via a different route, we injected B16 melanoma cells into the femoral vein. Through this route, the number of lung tumors formed was higher than in the intracardiac route and was again less in osteopontin-deficient mice compared with wild-type mice. In conclusion, in an experimental metastasis assay, the number of tumors found in bone (after intracardiac injection) and lung (after left femoral vein injection) was significantly reduced in osteopontin-deficient mice compared with wild-type mice. Tumor numbers in other organs examined were small and not significantly different in the two situations.
Subject(s)
Bone Neoplasms/secondary , Lung Neoplasms/secondary , Melanoma, Experimental/secondary , Sialoglycoproteins/deficiency , Animals , Bone Neoplasms/prevention & control , Cell Adhesion , Female , Femoral Vein , Gene Expression Regulation, Neoplastic , Heart Ventricles , Injections , Injections, Intravenous , Lung Neoplasms/prevention & control , Male , Melanoma, Experimental/prevention & control , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Proteins/physiology , Neoplasm Transplantation , Osteopontin , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Sialoglycoproteins/genetics , Sialoglycoproteins/physiologyABSTRACT
Osteopontin (OPN) is a glycosylated phosphoprotein found in all body fluids and in the proteinaceous matrix of mineralized tissues. It can function both as a cell attachment protein and as a cytokine, delivering signals to cells via a number of receptors including several integrins and CD44. Expression of OPN is enhanced by a variety of toxicants, especially those that activate protein kinase C. In its capacity as a signaling molecule, OPN can modify gene expression and promote the migration of monocytes/macrophages up an OPN gradient. It has both inflammatory and anti-inflammatory actions. Some experiments suggest that it may inhibit apoptosis, possibly contributing to the survival of cells in response to toxicant injury. Elevated OPN expression often correlates with malignancy and has been shown to enhance the tumorigenic and/or metastatic phenotype of the cancer cell. Recent studies have revealed that OPN plays critical roles in bone remodeling and cell-mediated immunity.
Subject(s)
Sialoglycoproteins/physiology , Signal Transduction/physiology , Toxins, Biological/toxicity , Animals , Bone Diseases/physiopathology , Humans , Neoplasms/physiopathology , Osteopontin , Receptors, Cytokine/physiologyABSTRACT
Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.
Subject(s)
Bone Resorption/physiopathology , Carrier Proteins/metabolism , Membrane Glycoproteins/metabolism , Osteoclasts/physiology , Osteogenesis/physiology , Parathyroid Hormone/pharmacology , Sialoglycoproteins/physiology , Animals , Calcium/metabolism , Carrier Proteins/pharmacology , Macrophage Colony-Stimulating Factor/pharmacology , Membrane Glycoproteins/pharmacology , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Organ Culture Techniques , Osteoclasts/cytology , Osteoclasts/drug effects , Osteogenesis/drug effects , Osteopontin , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , Sialoglycoproteins/deficiency , Sialoglycoproteins/geneticsABSTRACT
Osteoclastic bone resorption requires a number of complex steps that are under the control of local regulatory molecules. Osteopontin is expressed in osteoclasts and is also present in bone matrix; however, its biological function has not been fully understood. To elucidate the role of osteopontin in the process of osteoclastic bone resorption, we conducted ectopic bone implantation experiments using wild-type and osteopontin knockout mouse. In the wild-type group, bone discs from calvariae implanted ectopically in muscle were resorbed, and their mass was reduced by 25% within 4 weeks. In contrast, the mass of the bone discs from calvariae of osteopontin knockout mice was reduced by only 5% when implanted in osteopontin knockout mice. Histological analyses indicated that the number of osteoclasts associated with the implanted bones was reduced in the osteopontin knockout mice. As osteopontin deficiency does not suppress osteoclastogenesis per se, we further examined vascularization immunohistologically and found that the number of vessels containing CD31-positive endothelial cells around the bone discs implanted in muscle was reduced in the osteopontin knockout mice. Furthermore, sc implantation assays indicated that the length and branching points of the newly formed vasculatures associated with the bone discs were also reduced in the absence of osteopontin. In this assay, tartrate-resistant acid phosphatase-positive area of the bone discs was also reduced in the osteopontin knockout mice, indicating further the link between the osteopontin-dependent vascularization and osteoclast accumulation. The bone resorption defect could be rescued by topical administration of recombinant osteopontin to the bones implanted in muscle. These observations indicate that osteopontin is required for efficient vascularization by the hemangiogenic endothelial cells and subsequent osteoclastic resorption of bones.
Subject(s)
Bone Resorption/physiopathology , Bone and Bones , Choristoma/physiopathology , Neovascularization, Physiologic/physiology , Osteoclasts/pathology , Sialoglycoproteins/physiology , Animals , Bone Resorption/pathology , Cell Division/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout/genetics , Muscle, Skeletal , Muscular Diseases/physiopathology , Osteoclasts/drug effects , Osteopontin , Sialoglycoproteins/genetics , Sialoglycoproteins/pharmacologyABSTRACT
Reduced mechanical stress to bone in bedridden patients and astronauts leads to bone loss and increase in fracture risk which is one of the major medical and health issues in modern aging society and space medicine. However, no molecule involved in the mechanisms underlying this phenomenon has been identified to date. Osteopontin (OPN) is one of the major noncollagenous proteins in bone matrix, but its function in mediating physical-force effects on bone in vivo has not been known. To investigate the possible requirement for OPN in the transduction of mechanical signaling in bone metabolism in vivo, we examined the effect of unloading on the bones of OPN(-/-) mice using a tail suspension model. In contrast to the tail suspension-induced bone loss in wild-type mice, OPN(-/-) mice did not lose bone. Elevation of urinary deoxypyridinoline levels due to unloading was observed in wild-type but not in OPN(-/-) mice. Analysis of the mechanisms of OPN deficiency-dependent reduction in bone on the cellular basis resulted in two unexpected findings. First, osteoclasts, which were increased by unloading in wild-type mice, were not increased by tail suspension in OPN(-/-) mice. Second, measures of osteoblastic bone formation, which were decreased in wild-type mice by unloading, were not altered in OPN(-/-) mice. These observations indicate that the presence of OPN is a prerequisite for the activation of osteoclastic bone resorption and for the reduction in osteoblastic bone formation in unloaded mice. Thus, OPN is a molecule required for the bone loss induced by mechanical stress that regulates the functions of osteoblasts and osteoclasts.
Subject(s)
Osteoblasts/physiology , Osteoclasts/physiology , Sialoglycoproteins/physiology , Amino Acids/urine , Animals , Bone Resorption/pathology , Female , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteoclasts/cytology , Osteopontin , Sialoglycoproteins/genetics , Sialoglycoproteins/metabolism , Stress, MechanicalABSTRACT
Transgenic mice expressing c-myc and v-Ha-ras specifically in the mammary gland under the control of the mammary specific promoter MMTV develop unifocal mammary tumors with a half time of about 46 days, and these tumors express high levels of osteopontin mRNA and protein. In order to evaluate the requirement for osteopontin expression by these tumors, we have crossed transgenic mice expressing these two oncogenes with mice with a targeted disruption of the osteopontin gene. Littermates expressing both myc and ras, and with either wild-type or disrupted OPN alleles were evaluated for tumor incidence and growth rate. Both of these parameters were found to be unaffected by a lack of osteopontin in the whole animal. Ras and myc expression level, measured at the level of mRNA, was not different in tumors of the two genotypes. Macrophage accumulation, while extremely variable among different tumors, did not correlate with the OPN status of the animals. Expression of the related gene BSP was not detected in any of the tumors, and was similar in bones of wildtype and OPN -/- mice. Similarly, the vitronectin gene was expressed at very low levels in tumors of either genotype. These results indicate that despite its high level of expression, OPN is either not required for mammary primary tumor formation and growth in this system, or can be replaced by molecules other than BSP and vitronectin in mice that totally lack osteopontin.
Subject(s)
Genes, myc/genetics , Genes, ras/genetics , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/physiopathology , Membrane Proteins/genetics , Receptors, Virus/genetics , Sialoglycoproteins/pharmacology , Animals , Cell Transformation, Neoplastic , Female , Gene Expression Regulation, Neoplastic , Mice , Mice, Transgenic , Osteopontin , Sialoglycoproteins/biosynthesis , Sialoglycoproteins/deficiencyABSTRACT
The secreted phosphoprotein osteopontin (OPN) is strongly associated with the process of neoplastic transformation, based both on its pattern of expression in vivo and in vitro and on functional analyses. We have used 3T3 cells derived from wildtype and OPN-deficient mice and transformed by transfection with oncogenic ras to assess the role of OPN in transformation in vitro and in tumorigenesis in vivo. There was no effect of an absence of OPN on the ability of the cells to undergo immortalization or to form morphologically transformed foci following ras transfection. Wildtype and OPN-deficient cell lines were established from such foci, and lines with similar ras mRNA levels selected for further analysis. Ras-transformed cell lines from both wildtype and OPN-deficient mice could form colonies in soft agar indicating that this process can occur in the absence of OPN. However, the ability of the OPN-deficient cell lines to form colonies was reduced as compared to wildtype cell lines. Tumorigenesis in syngeneic and nude mice was assessed for a subset of cell lines that formed colonies efficiently in soft agar. Cell lines unable to make OPN formed tumors in these mice much more slowly than wildtype cells, despite similar growth of the cells on plastic and in soft agar. Taken together, these results indicate that maximal transformation by ras requires OPN expression, and implicate increased OPN expression as an important effector of the transforming activity of the ras oncogene.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Genes, ras/physiology , Sialoglycoproteins/physiology , 3T3 Cells , Animals , Mice , Mice, Inbred C57BL , Mice, Nude , Neoplasm Transplantation , Osteopontin , Phenotype , Tumor Stem Cell AssayABSTRACT
Cell-mediated (type-1) immunity is necessary for immune protection against most intracellular pathogens and, when excessive, can mediate organ-specific autoimmune destruction. Mice deficient in Eta-1 (also called osteopontin) gene expression have severely impaired type-1 immunity to viral infection [herpes simplex virus-type 1 (KOS strain)] and bacterial infection (Listeria monocytogenes) and do not develop sarcoid-type granulomas. Interleukin-12 (IL-12) and interferon-gamma production is diminished, and IL-10 production is increased. A phosphorylation-dependent interaction between the amino-terminal portion of Eta-1 and its integrin receptor stimulated IL-12 expression, whereas a phosphorylation-independent interaction with CD44 inhibited IL-10 expression. These findings identify Eta-1 as a key cytokine that sets the stage for efficient type-1 immune responses through differential regulation of macrophage IL-12 and IL-10 cytokine expression.
Subject(s)
Interleukin-10/biosynthesis , Interleukin-12/biosynthesis , Macrophages/immunology , Sialoglycoproteins/immunology , T-Lymphocytes/immunology , Animals , Granuloma/immunology , Herpes Simplex/immunology , Herpesvirus 1, Human/immunology , Hyaluronan Receptors/metabolism , Hypersensitivity, Delayed , Interferon-gamma/biosynthesis , Keratitis, Herpetic/immunology , Listeriosis/immunology , Lymphocyte Activation , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Nude , Osteopontin , Phosphorylation , Receptors, Vitronectin/metabolism , Sialoglycoproteins/metabolism , Sialoglycoproteins/pharmacology , T-Lymphocytes/metabolismABSTRACT
Osteopontin is one of the major noncollagenous bone matrix proteins produced by osteoblasts and osteoclasts, bone cells that are uniquely responsible for the remodeling of mineralized tissues. Osteoclasts express the alphavbeta3 integrin, which is one of the receptors for osteopontin. Recent knockout studies revealed that noncollagenous bone matrix proteins are functionally important in regulation of bone metabolism. However, the significance of the presence of osteopontin in in vivo has not been known. We report here that osteopontin knockout mice are resistant to ovariectomy-induced bone resorption compared with wild-type mice. Microcomputed tomography analysis indicated about 60% reduction in bone volume by ovariectomy in wild-type mice, whereas the osteopontin-deficient mice exhibited only about 10% reduction in trabecular bone volume after ovariectomy. Reduction in uterine weight was observed similarly in both wild-type and osteopontin-deficient mice, indicating the specificity of the effect of osteopontin deficiency on bone metabolism. We propose that osteopontin is essential for postmenopausal osteoporosis in women. Strategies to counteract osteopontin's action may prove effective in suppressing osteoporosis.
Subject(s)
Bone Resorption/physiopathology , Osteoporosis, Postmenopausal/physiopathology , Ovariectomy , Sialoglycoproteins/deficiency , Sialoglycoproteins/physiology , Animals , Bone Resorption/prevention & control , Disease Models, Animal , Female , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Osteopontin , Phosphoproteins/deficiency , Phosphoproteins/physiology , Sialoglycoproteins/genetics , Tibia/diagnostic imaging , Tibia/pathology , Tibia/physiopathology , Tomography, X-Ray ComputedABSTRACT
BACKGROUND: Mice with a targeted disruption of the osteopontin gene through homologous recombination in embryonic stem cells have recently been generated and shown to be characterized by unaltered fertility and normal embryonic and postnatal development, including renal development, but altered osteoclastogenesis from spleen progenitors. The lack of detectable pathological manifestations in kidneys of mice with the targeted disruption of the osteopontin gene (opn -/-) makes them an excellent model for studies of pathophysiological processes that are thought to be accompanied by changes in renal osteopontin expression. It has previously been suggested that osteopontin may play an important role in the pathophysiology of acute renal failure, thus prompting this study. METHODS: Wild-type and opn -/- mice were subjected to 30 minutes of renal ischemia and were studied 24 hours later. RESULTS: Control opn +/+ mice showed a significant retention of blood urea nitrogen and creatinine, which is indicative of the development of ischemic acute renal dysfunction. This was accompanied by a 2.7-fold increase in the immunodetectable osteopontin compared with sham-operated control. Animals with the disrupted osteopontin gene exhibited ischemia-induced renal dysfunction, which was twice as pronounced as that observed in mice with the intact osteopontin response to stress. In addition, the structural damage to the ischemic kidneys obtained from opn -/- mice was more pronounced than that observed in similarly treated wild-type mice. This was associated with the augmented expression of inducible nitric oxide synthase and the prevalence of nitrotyrosine residues in kidneys from opn -/- mice versus wild-type counterparts. In vitro studies with proximal tubular cells subjected to hypoxia in the presence of OPN, but not OPN with deleted arginine-glycine-aspartic acid (RGD) domain, resulted in cytoprotection. CONCLUSIONS: The comparative analysis of functional and morphological sequelae of acute renal ischemia in opn +/+ and opn -/- mice provides strong evidence of renoprotective action of osteopontin in acute ischemia.
Subject(s)
Adaptation, Physiological/physiology , Ischemia/genetics , Ischemia/physiopathology , Renal Circulation/physiology , Sialoglycoproteins/physiology , Acute Disease , Animals , Cell Survival/physiology , Cells, Cultured , Epithelial Cells/metabolism , Female , Gene Targeting , Hypoxia/metabolism , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout/genetics , Osteopontin , Sialoglycoproteins/genetics , Sialoglycoproteins/metabolismABSTRACT
Osteopontin (OPN) is a soluble secreted phosphoprotein that binds with high affinity to several different integrins. While numerous activities have been ascribed to OPN in vitro, and several in vivo functions have been suggested for the molecule, including much attention focused recently on OPN in different pathologies, the actual role that the protein plays in mammalian physiology remains conjectural. Analysis of recently developed strains of mice lacking OPN because of a targeted disruption of its gene promises to provide important information on this issue. Here, we review the data implicating OPN as functioning in a variety of pathologic conditions as well as the initial results generated with the OPN knockout mice, with particular focus on the question of whether OPN has a protective or a damaging effect in different pathologies. These data will be important to the definition of how OPN contributes to mammalian physiology and pathophysiology.
Subject(s)
Cell Adhesion/physiology , Integrins/physiology , Kidney/physiology , Sialoglycoproteins/physiology , Animals , Humans , Kidney/pathology , Kidney/physiopathology , Kidney Neoplasms/pathology , Kidney Neoplasms/physiopathology , Mammals , Mice , Mice, Knockout , Osteopontin , Phosphoproteins/physiology , Sialoglycoproteins/deficiency , Sialoglycoproteins/geneticsABSTRACT
Several different antibodies to mouse and/or rat osteopontin have been developed, and some antisera raised against human osteopontin have been shown to react with mouse osteopontin. We have taken advantage of the lack of osteopontin protein in mice with a targeted disruption of the osteopontin gene to characterize the reactivity and the specificity of several of these antibodies with mouse osteopontin by Western blotting. Our results demonstrate that, with the exception of the rat monoclonal antibody MPIIIB10, which does not recognize mouse osteopontin on Western blots, all the tested reagents do react with mouse osteopontin, but their sensitivity varies widely, and in some cases there is significant cross-reactivity of the antibodies with other proteins found in mouse tissue extracts.
Subject(s)
Sialoglycoproteins/analysis , Animals , Antibodies, Monoclonal , Blotting, Western/methods , Humans , Mice , Osteopontin , Rats , Sialoglycoproteins/immunologyABSTRACT
We have used homologous recombination in embryonic stem cells to generate mice with a targeted disruption of the osteopontin (Opn, or Spp1, for secreted phosphoprotein 1) gene. Mice homozygous for this disruption fail to express osteopontin (OPN) as assessed at both the mRNA and protein level, although an N-terminal fragment of OPN is detectable at extremely low levels in the bones of -/- animals. The Opn -/- mice are fertile, their litter size is normal, and they develop normally. The bones and teeth of animals not expressing OPN are morphologically normal at the level of light and electron microscopy, and the skeletal structure of young animals is normal as assessed by radiography. Ultrastructurally, proteinaceous structures normally rich in OPN, such as cement lines, persist in the bones of the Opn-/- animals. Osteoclastogenesis was assessed in vitro in cocultures with a feeder layer of calvarial osteoblast cells from wild-type mice. Spleen cells from Opn-/- mice cells formed osteoclasts 3- to 13-fold more frequently than did control Opn+/+ cells, while the extent of osteoclast development from Opn -/- bone marrow cells was about 2- to 4-fold more than from the corresponding wild-type cells. Osteoclast development occurred when Opn-/- spleen cells were differentiated in the presence of Opn-/-osteoblasts, indicating that endogenous OPN is not required for this process. These results suggest that OPN is not essential for normal mouse development and osteogenesis, but can modulate osteoclast differentiation.