Susan R. Rittling

Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size

Stem cells reside in a specialized niche that regulates their abundance and fate. Components of the niche have generally been defined in terms of cells and signaling pathways. We define a role for a matrix glycoprotein, osteopontin (OPN), as a constraining factor on hematopoietic stem cells within the bone marrow microenvironment. Osteoblasts that participate in the niche produce varying amounts of OPN in response to stimulation. Using studies that combine OPN-deficient mice and exogenous OPN, we demonstrate that OPN modifies primitive hematopoietic cell number and function in a stem cell–nonautonomous manner. The OPN-null microenvironment was sufficient to increase the number of stem cells associated with increased stromal Jagged1 and Angiopoietin-1 expression and reduced primitive hematopoietic cell apoptosis. The activation of the stem cell microenvironment with parathyroid hormone induced a superphysiologic increase in stem cells in the absence of OPN. Therefore, OPN is a negative regulatory element of the stem cell niche that limits the size of the stem cell pool and may provide a mechanism for restricting excess stem cell expansion under conditions of niche stimulation.

Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro.

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.

Role of osteopontin in tumour progression

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.

Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice

Rheumatoid arthritis is one of the most critical diseases that impair the quality of life of patients, but its pathogenesis has not yet been fully understood. Osteopontin (OPN) is an extracellular matrix protein containing Arg-Gly-Asp (RGD) sequence, which interacts with αvβ3 integrins, promotes cell attachment, and cell migration and is expressed in both synovial cells and chondrocytes in rheumatoid arthritis; however, its functional relationship to arthritis has not been known. Therefore, we investigated the roles of OPN in the pathogenesis of inflammatory process in a rheumatoid arthritis model induced by a mixture of anti-type II collagen mAbs and lipopolysaccharide (mAbs/LPS). mAbs/LPS injection induced OPN expression in synovia as well as cartilage, and this expression was associated with joint swelling, destruction of the surface structures of the joint based on scanning electron microscopy, and loss of toluidine blue-positive proteoglycan content in the articular cartilage in wild-type mice. In contrast, OPN deficiency prevented the mice from such surface destruction, loss of proteoglycan in the articular joint cartilage, and swelling of the joints even when the mice were subjected to mAbs/LPS injection. Furthermore, mAbs/LPS injection in wild-type mice enhanced the levels of CD31-positive vessels in synovia and terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive chondrocytes in the articular cartilage, whereas such angiogenesis as well as chondrocyte apoptosis was suppressed significantly in OPN-deficient mice. These results indicated that OPN plays a critical role in the destruction of joint cartilage in the rheumatoid arthritis model in mice via promotion of angiogenesis and induction of chondrocyte apoptosis.

Osteopontin function in pathology: lessons from osteopontin-deficient mice.

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.

Unloading Induces Osteoblastic Cell Suppression and Osteoclastic Cell Activation to Lead to Bone Loss via Sympathetic Nervous System

Osteoporosis is one of the major health problems in our modern world. Especially, disuse (unloading) osteoporosis occurs commonly in bedridden patients, a population that is rapidly increasing due to aging-associated diseases. However, the mechanisms underlying such unloading-induced pathological bone loss have not yet been fully understood. Since sympathetic nervous system could control bone mass, we examined whether unloading-induced bone loss is controlled by sympathetic nervous tone. Treatment with β-blocker, propranolol, suppressed the unloading-induced reduction in bone mass. Conversely, β-agonist, isoproterenol, reduced bone mass in loaded mice, and under such conditions, unloading no longer further reduced bone mass. Analyses on the cellular bases indicated that unloading-induced reduction in the levels of osteoblastic cell activities, including mineral apposition rate, mineralizing surface, and bone formation rate, was suppressed by propranolol treatment and that isoproterenol-induced reduction in these levels of bone formation parameters was no longer suppressed by unloading. Unloading-induced reduction in the levels of mineralized nodule formation in bone marrow cell cultures was suppressed by propranolol treatment in vivo. In addition, loss of a half-dosage in the dopamine β-hydroxylase gene suppressed the unloading-induced bone loss and reduction in mineralized nodule formation. Unloading-induced increase in the levels of osteoclastic activities such as osteoclast number and surface as well as urinary deoxypyridinoline was all suppressed by the treatment with propranolol. These observations indicated that sympathetic nervous tone mediates unloading-induced bone loss through suppression of bone formation by osteoblasts and enhancement of resorption by osteoclasts.

Osteopontin Deficiency Attenuates Atherosclerosis in Female Apolipoprotein E-Deficient Mice

Objective—Osteopontin (OPN), a noncollagenous adhesive protein, is implicated in atherosclerosis, in which macrophages within atherosclerotic plaques express OPN. However, it is not known whether the elevated OPN expression is a cause or result of atherosclerosis. Methods and Results—We generated mice that lacked OPN and crossed them with apolipoprotein (apo) E–deficient mice and analyzed these mice with a mixed C57BL/6×129 background after 36 weeks on a normal chow diet. In female mice, OP+/−E−/− and OP−/−E−/− mice had significantly smaller atherosclerotic and inflammatory lesions compared with OP+/+E−/− mice, and that was reflected by smaller area of MOMA-2–positive staining. In male mice, however, there was no significant difference in the atherosclerosis lesion areas among 3 genotypes. In both OP−/−E−/− and OP+/+E−/− mice, typical atherosclerotic lesions were detected, which include necrotic core, foamy cell collections, and cholesterol clefts. However, we found that vascular mineral-deposited areas in 60-week-old male OP−/−E−/− mice were significantly increased compared with those in OP+/+E−/− male mice. Conclusions—These results suggest that OPN plays a promoting effect in atherosclerosis and inhibitory effect in vascular calcification. The suppression of OPN expression in females should be considered a therapeutic possibility in atherosclerosis.

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.

Role of Osteopontin in Cardiac Fibrosis and Remodeling in Angiotensin II-Induced Cardiac Hypertrophy

Osteopontin (OPN) is upregulated in several experimental models of cardiac fibrosis and remodeling. However, its direct effects remain unclear. We examined the hypothesis that OPN is important for the development of cardiac fibrosis and remodeling. Moreover, we examined whether the inhibitory effect of eplerenone (Ep), a novel aldosterone receptor antagonist, was mediated through the inhibition of OPN expression against cardiac fibrosis and remodeling. Wild-type (WT) and OPN-deficient mice were treated with angiotensin II (Ang II) for 4 weeks. WT mice receiving Ang II were divided into 2 groups: a control group and an Ep treatment group. Ang II treatment significantly elevated blood pressure and caused cardiac hypertrophy and fibrosis in WT mice. Ep treatment and OPN deficiency could reduce the Ang II–induced elevation of blood pressure and ameliorate the development of cardiac fibrosis, whereas Ep-only treatment abolished the development of cardiac hypertrophy. Most compelling, the reduction of cardiac fibrosis led to an impairment of cardiac systolic function and subsequent left ventricular dilatation in Ang II–treated OPN-deficient mice. These results suggest that OPN has a pivotal role in the development of Ang II–induced cardiac fibrosis and remodeling. Moreover, the effect of Ep on the prevention of cardiac fibrosis, but not cardiac hypertrophy, might be partially mediated through the inhibition of OPN expression.

Inhibition of Growth and Metastasis of Mouse Mammary Carcinoma by Selective Inhibitor of Transforming Growth Factor-β Type I Receptor Kinase In vivo

Purpose: Transforming growth factor-β (TGF-β) suppresses tumor development by inhibiting cellular proliferation, inducing differentiation and apoptosis, and maintaining genomic integrity. However, once tumor cells escape from the tumor-suppressive effects of TGF-β, they often constitutively overexpress and activate TGF-β, which may promote tumor progression by enhancing invasion, metastasis, and angiogenesis and by suppressing antitumor immunity. The purpose of this study was to test this hypothesis using TGF-β pathway antagonists. Experimental Design: We examined the effects of selective TGF-β type I receptor kinase inhibitors, SD-093 and SD-208, on two murine mammary carcinoma cell lines (R3T and 4T1) in vitro and in vivo. Results: Both agents blocked TGF-β-induced phosphorylation of the receptor-associated Smads, Smad2 and Smad3, in a dose-dependent manner, with IC50 between 20 and 80 nmol/L. TGF-β failed to inhibit growth of these cell lines but stimulated epithelial-to-mesenchymal transdifferentiation, migration, and invasiveness into Matrigel in vitro. These effects were inhibited by SD-093, indicating that these processes are partly driven by TGF-β. Treatment of syngeneic R3T or 4T1 tumor-bearing mice with orally given SD-208 inhibited primary tumor growth as well as the number and size of metastases. In contrast, SD-208 failed to inhibit R3T tumor growth or metastasis in athymic nude mice. Moreover, in vitro anti-4T1 cell cytotoxic T-cell responses of splenocytes from drug-treated animals were enhanced compared with cells from control animals. In addition, SD-208 treatment resulted in a decrease in tumor angiogenesis. Conclusion: TGF-β type I receptor kinase inhibitors hold promise as novel therapeutic agents for metastatic breast cancer.