anti-OPN Antibody Research Articles

Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model

Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.

Definition of the contribution of an Osteopontin-producing CD11c+ microglial subset to Alzheimer’s disease

Alzheimer's disease (AD) is the most common form of incurable dementia and represents a critical public health issue as the world's population ages. Although microglial dysregulation is a cardinal feature of AD, the extensive heterogeneity of these immunological cells in the brain has impeded our understanding of their contribution to this disease. Here, we identify a pathogenic microglial subset which expresses the CD11c surface marker as the sole producer of Osteopontin (OPN) in the 5XFAD mouse model of AD. OPN production divides Disease-Associated Microglia (DAM) into two functionally distinct subsets, i.e., a protective CD11c+OPN- subset that robustly ingests amyloid β (Aβ) in a noninflammatory fashion and a pathogenic CD11c+OPN+ subset that produces proinflammatory cytokines and fails to ingest significant amounts of Aβ. Genetic ablation of OPN or administration of monoclonal anti-OPN antibody to 5XFAD mice reduces proinflammatory microglia, plaque formation, and numbers of dystrophic neurites and results in improved cognitive function. Analysis of brain tissue from AD patients indicates that levels of OPN-producing CD11c+ microglia correlate strongly with the degree of cognitive deficit and AD neuropathology. These findings define an OPN-dependent pathway to disease driven by a distinct microglial subset, and identify OPN as a novel therapeutic target for potentially effective immunotherapy to treat AD.

Osteopontin-induced vascular hyperpermeability through tight junction disruption in diabetic retina

Diabetic retinopathy is a major cause of blindness in developed countries, and is characterized by deterioration of barrier function causing vascular hyperpermeability and retinal edema. Vascular endothelial growth factor (VEGF) is a major mediator of diabetic macular edema. Although anti-VEGF drugs are the first-line treatment for diabetic macular edema, some cases are refractory to anti-VEGF therapy. Osteopontin (OPN) is a phosphoglycoprotein with diverse functions and expressed in various cells and tissues. Elevated OPN level has been implicated in diabetic retinopathy, but whether OPN is involved in hyperpermeability remains unclear. Using streptozotocin-induced diabetic mice (STZ mice) and human retinal endothelial cells (HRECs), we tested the hypothesis that up-regulated OPN causes tight junction disruption, leading to vascular hyperpermeability. The serum and retinal OPN concentrations were elevated in STZ mice compared to controls. Intravitreal injection of anti-OPN neutralizing antibody (anti-OPN Ab) suppressed vascular hyperpermeability and prevented decreases in claudin-5 and ZO-1 gene expression levels in the retina of STZ mice. Immunohistochemical staining of retinal vessels in STZ mice revealed claudin-5 immunoreactivity with punctate distribution and attenuated ZO-1 immunoreactivity, and these changes were prevented by anti-OPN Ab. Intravitreal injection of anti-OPN Ab did not change VEGF gene expression or protein concentration in retina of STZ mice. In an in vitro study, HRECs were exposed to normal glucose or high glucose with or without OPN for 48 h, and barrier function was evaluated by transendothelial electrical resistance and Evans blue permeation. Barrier function deteriorated under high glucose condition, and was further exacerbated by the addition of OPN. Immunofluorescence localization of claudin-5 and ZO-1 demonstrated punctate appearance with discontinuous junction in HRECs exposed to high glucose and OPN. There were no changes in VEGF and VEGF receptor-2 expression levels in HRECs by exposure to OPN. Our results suggest that OPN induces tight junction disruption and vascular hyperpermeability under diabetic conditions. Targeting OPN may be an effective approach to manage diabetic retinopathy.

Phosphorylated Osteopontin Secreted from Cancer Cells Induces Cancer Cell Motility.

Osteopontin (OPN) plays a pivotal role in cancer cell invasion and metastasis. Although OPN has a large number of phosphorylation sites, the functional significance of OPN phosphorylation in cancer cell motility remains unclear. In this study, we attempted to investigate whether phosphorylated OPN secreted from cancer cells affect cancer cell migration. Quantitative PCR and Western blot analyses revealed that MDA-MB435S, A549, and H460 cells highly expressed OPN, whereas the OPN expression levels in H358, MIAPaca-2, and Panc-1 cells were quite low or were not detected. Compared with the cancer cell lines with a low OPN expression, the high OPN-expressing cancer cell lines displayed a higher cell migration, and the cell migration was suppressed by the anti-OPN antibody. This was confirmed by the OPN overexpression in H358 cancer cells with a low endogenous OPN. Phos-tag ELISA showed that phosphorylated OPN was abundant in the cell culture media of A549 and H460 cells, but not in those of MDA-MB435S cells. Moreover, the A549 and H460 cell culture media, as well as the MDA-MB435S cell culture media with a kinase treatment increased cancer cell motility, both of which were abrogated by phosphatase treatment or anti-OPN antibodies. These results suggest that phosphorylated OPN secreted from cancer cells regulates cancer cell motility.

Neutralizing antibody against osteopontin attenuates non-alcoholic steatohepatitis in mice.

Previously, we reported that an extracellular matrix protein, osteopontin (OPN), is involved in various autoimmune diseases using a neutralizing polyclonal antibody against OPN generated in rabbits. However, the antibody cannot be used for long-term mouse models of chronic inflammatory disease because of the induction of antibodies against anti-OPN rabbit IgG. In this study, we generated a new antibody, anti-mouse OPN mouse IgG (35B6). 35B6 inhibited the cell adhesion of mouse and human OPN to Chinese Hamster Ovary (CHO) cells or CHO cells expressing α4 or α9 integrin. It was reported that OPN is highly expressed and has an important role in a chronic liver disease, non-alcoholic steatohepatitis (NASH). 35B6 injection twice a week for 8weeks attenuated liver inflammation and fibrosis in a NASH mouse model, suggesting 35B6 is beneficial for the treatment of NASH. 35B6 was preferable to the rabbit anti-OPN antibody for investigating the in vivo function of OPN in mouse models of long-term disease.

Osteopontin mediates murine transfusion-related acute lung injury via stimulation of pulmonary neutrophil accumulation

AbstractTransfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and is characterized by the onset of acute respiratory distress within 6 hours upon blood transfusion. Specific therapies are unavailable. Preexisting inflammation is a risk factor for TRALI and neutrophils (polymorphonuclear neutrophils [PMNs]) are considered to be the major pathogenic cells. Osteopontin (OPN) is a multifunctional protein expressed at sites of inflammation and, for example, is involved in pulmonary disorders, can regulate cellular migration, and can function as a PMN chemoattractant. We investigated whether OPN is involved in TRALI induction by promoting PMN recruitment to the lungs. Using a previously established murine TRALI model, we found that in contrast to wild-type (WT) mice, OPN knockout (KO) mice were resistant to antibody-mediated PMN-dependent TRALI induction. Administration of purified OPN to the OPN KO mice, however, restored the TRALI response and pulmonary PMN accumulation. Alternatively, blockade of OPN in WT mice using an anti-OPN antibody prevented the onset of TRALI induction. Using pulmonary immunohistochemistry, OPN could be specifically detected in the lungs of mice that suffered from TRALI. The OPN-mediated TRALI response seemed dependent on macrophages, likely the cellular source of OPN and OPN polymerization, and independent from the OPN receptor CD44, interleukin 6 (IL-6), and other PMN chemoattractants including macrophage inflammatory protein-2 (MIP-2). These data indicate that OPN is critically required for induction of antibody-mediated murine TRALI through localization to the lungs and stimulation of pulmonary PMN recruitment. This suggests that anti-OPN antibody therapy may be a potential therapeutic strategy to explore in TRALI patients.

Osteopontin Promotes Left Ventricular Diastolic Dysfunction Through a Mitochondrial Pathway

BackgroundPatients with chronic kidney disease (CKD) and coincident heart failure with preserved ejection fraction (HFpEF) may constitute a distinct HFpEF phenotype. Osteopontin (OPN) is a biomarker of HFpEF and predictive of disease outcome. We recently reported that OPN blockade reversed hypertension, mitochondrial dysfunction, and kidney failure in Col4a3−/− mice, a model of human Alport syndrome. ObjectivesThe purpose of this study was to identify potential OPN targets in biopsies of HF patients, healthy control subjects, and human induced pluripotent stem cell–derived cardiomyocytes (hiPS-CMs), and to characterize the cardiac phenotype of Col4a3−/− mice, relate this to HFpEF, and investigate possible causative roles for OPN in driving the cardiomyopathy. MethodsOGDHL mRNA and protein were quantified in myocardial samples from patients with HFpEF, heart failure with reduced ejection fraction, and donor control subjects. OGDHL expression was quantified in hiPS-CMs treated with or without anti-OPN antibody. Cardiac parameters were evaluated in Col4a3−/− mice with and without global OPN knockout or AAV9-mediated delivery of 2-oxoglutarate dehydrogenase-like (Ogdhl) to the heart. ResultsOGDHL mRNA and protein displayed abnormal abundances in cardiac biopsies of HFpEF (n = 17) compared with donor control subjects (n = 12; p < 0.01) or heart failure with reduced ejection fraction patients (n = 12; p < 0.05). Blockade of OPN in hiPS-CMs conferred increased OGDHL expression. Col4a3−/− mice demonstrated cardiomyopathy with similarities to HFpEF, including diastolic dysfunction, cardiac hypertrophy and fibrosis, pulmonary edema, and impaired mitochondrial function. The cardiomyopathy was ameliorated by Opn−/− coincident with improved renal function and increased expression of Ogdhl. Heart-specific overexpression of Ogdhl in Col4a3−/− mice also improved cardiac function and cardiomyocyte energy state. ConclusionsCol4a3−/− mice present a model of HFpEF secondary to CKD wherein OPN and OGDHL are intermediates, and possibly therapeutic targets.

Osteopontin Mediates Murine Transfusion-Related Acute Lung Injury through Stimulation of Pulmonary Neutrophil Accumulation

Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress which develops within 6 hours of blood transfusion. It is the leading cause of transfusion-related deaths and the pathogenesis is complex and incompletely understood. In the majority of the cases, anti-leukocyte antibodies present in the transfused blood product, in combination with recipient predisposing risk-factors such as inflammation, are implicated to be responsible for the onset of TRALI. Unfortunately, no therapies are available for TRALI.Osteopontin (OPN) is an extracellular matrix protein with multiple biological functions. OPN is involved in normal physiological processes, such as cell migration and adhesion, but has also been implicated in a wide range of disease states, including cancer, atherosclerosis, glomerulonephritis, and several chronic inflammatory diseases. Interestingly, OPN is upregulated at sites of inflammation and tissue remodeling. As inflammation is an important risk factor for TRALI development, and as neutrophils (PMNs) are known effector cells in the pathogenesis of TRALI which migrate and accumulate in the lungs during TRALI development, we investigated the potential contribution of OPN in the onset of antibody-mediated TRALI.We utilized a previously established murine TRALI model (Kapur et al, Blood 2017, Blood Advances 2018) in which C57BL/6 mice were first primed with a low dose of lipopolysaccharide (LPS) and depleted of their CD4+ T cells in vivo followed by injection of anti-major histocompatibility complex (MHC) class I antibodies (clones 34-1-2s and AF6-88.5.5.3). The TRALI response was analyzed after 90 minutes by analysis of pulmonary edema (lung wet-to-dry weight ratios, W/Ds) and the levels of pulmonary neutrophils. Wildtype (WT) mice suffered from antibody-mediated TRALI compared to untreated naïve mice, as was shown by their significantly increased lung W/Ds (4.72 vs 4.50, respectively, P<0.0001). This also corresponded to significantly increased levels of pulmonary PMNs compared to untreated naïve mice (34% vs 5%, P<0.0001). In contrast, C57BL/6 OPN knock-out mice were resistant to antibody-mediated TRALI induction as they did not display any significant increase in lung W/Ds levels or pulmonary PMNs compared to untreated naïve OPN knock-out mice (lung W/Ds 4.83 vs 4.75, and pulmonary PMNs 18% vs 16%, respectively). Strikingly, administration of purified recombinant OPN during TRALI induction in C57BL/6 OPN knock-out mice, significantly induced a TRALI reaction (lung W/Ds 5.12 vs 4.75 as compared to untreated naïve OPN knock-out mice, P<0.05). Mechanistically, this TRALI inducing effect of OPN administration to OPN knock-out mice was associated with increased levels of pulmonary PMNs (38% vs 16%, as compared to untreated naïve OPN knock-out mice, P<0.0001). In vivo blocking of OPN in WT mice with an anti-OPN antibody demonstrated decreased lung W/Ds as compared to treatment with an isotype antibody (4.48 vs 4.69, respectively, P<0.05). The OPN blocking response during TRALI was associated with a decreased level of pulmonary PMN accumulation as compared to treatment with an isotype antibody (14% vs 34%, respectively, P<0.0001). As the PMN-chemoattractant macrophage inflammatory protein (MIP)-2 has previously been described to be upregulated in murine antibody-mediated TRALI, we investigated if the OPN-associated pulmonary PMN accumulation and TRALI induction could be related to the levels of MIP-2. We found that plasma MIP-2 levels were increased in mice that were infused with anti-MHC class I antibodies as compared to naïve controls, but that addition or blocking of OPN did not affect these MIP-2 levels. This indicates that the OPN-related PMN responses in TRALI are independent of plasma MIP-2 levels.Collectively, these data indicate OPN as a novel pathogenic factor which enhances antibody-mediated murine TRALI through stimulation of PMN migration towards the lungs, independent of MIP-2. This may suggest that blocking OPN (using an anti-OPN antibody) may prevent TRALI by impairing pulmonary PMN accumulation and could be a therapeutic avenue to explore in combatting this serious adverse complication of blood transfusion. DisclosuresNo relevant conflicts of interest to declare.

The potential role of Osteopontin in the maintenance of commensal bacteria homeostasis in the intestine.

Osteopontin (Opn), a multifunctional extracellular matrix protein, is implicated in the pathogenesis of various inflammatory disorders. Under physiologic conditions, its expression is restricted to certain tissues including bone and kidney tubule. However, cellular activation during disease development induces Opn expression in various immune cells. In this study, using Opn-EGFP knock-in (KI) mice we found that CD8α+ T cells in the intestinal tissues, including Peyer’s patch, lamina propria and epithelium, express Opn under steady state conditions. Therefore, we examined the role of Opn-expressing CD8α+ T cells in intestinal homeostasis. Interestingly, Opn knockout (KO) mice had altered fecal microflora concordant with a reduction of TCRγδ+ intraepithelial lymphocytes (IELs). Consistent with this result, both treatment with anti-Opn blocking antibody and deficiency of Opn resulted in decreased survival of TCRγδ+ and TCRαβ+ IELs. This data suggests that a possibility that Opn may function as a survival factor for IELs in the intestinal tissue. Collectively, these data suggest the possibility that Opn might regulate the homeostasis of intestinal microflora through maintenance of TCRγδ+ IELs, possibly by support of IEL survival.

Osteopontin Blockade Attenuates Renal Injury After Ischemia Reperfusion by Inhibiting NK Cell Infiltration.

Renal ischemia-reperfusion (RIR) injury is a common occurrence after major surgery and shock, leading to acute kidney injury (AKI). Osteopontin (OPN) is a secreted glycoprotein that acts as a proinflammatory cytokine and activator of T lymphocytes. We hypothesized that blockade of OPN reduces the severity of inflammation and injury in RIR. Renal ischemia was induced in adult C57BL/6 mice via bilateral clamping of renal pedicles for 35 min, followed by reperfusion for 24 h. Anti-OPN antibody (Ab), nonimmunized isotype immunoglobulin G, or normal saline was injected intravenously at the time of reperfusion. Blood and kidneys were collected for analysis. At 24 h after RIR, OPN mRNA and protein levels were significantly increased in renal tissue compared with sham mice. In serum, elevated levels of blood urea nitrogen and creatinine were reduced in anti-OPN Ab-treated mice compared with vehicle. Anti-OPN Ab-treated mice also had decreased mRNA levels of injury markers neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 compared with the vehicle. The histologic architecture and apoptosis of renal tissue were improved in the anti-OPN Ab-treated mice. In renal tissue, inflammatory cytokines interleukin 6 and tumor necrosis factor-α protein levels were reduced in the Ab-treated mice. Natural killer (NK) cell infiltration was decreased after anti-OPN Ab treatment, as was neutrophil infiltration, shown by reduced chemokine expression and Gr1 renal immunohistochemical staining. These findings demonstrate a beneficial role of OPN blockade in RIR associated with NK cell-mediated downregulation of inflammatory cytokines and chemokines. Administration of anti-OPN Ab may therefore serve as an immunomodulatory adjunct in the treatment of RIR-induced AKI.

Antibody-mediated molecular-targeted therapy for adult T-cell leukemia: recent progress and future challenges in the treatment of cancers

The role of the secreted matricellular molecule osteopontin (OPN) and its receptor integrins in the pathogenesis of adult T-cell leukemia (ATL) and the possible applications of an anti-OPN monoclonal antibody (mAb) for ATL immunotherapy in NOD/Shi- scid , IL-2R g null (NOG) mice were investigated. Subcutaneous inoculation of ATL cell lines into NOG mice led to increased plasma levels of OPN, correlating well with metastasis of the inoculated cells and survival time. This result suggested that the xenograft NOG mouse model could be a useful system for in vivo assessment of the physiological role of OPN in ATL pathogenesis. Intraperitoneal administration of an anti-OPN mAb resulted in the inhibition of tumor growth, tumor invasion, and metastasis. In addition, the mAb treatment led to reduction in the number of fibroblasts expressing fibroblast activation protein. We have shown here a novel mAb-mediated therapeutic strategy targeting the interaction between OPN from stromal cells and integrins on the tumors of ATL patients. In this editorial research highlight, we also comment on the recent progress in the development of mAbs and their advanced counterparts, the antibody-drug conjugate, for the treatment of cancers.

Osteopontin-integrin interaction as a novel molecular target for antibody-mediated immunotherapy in adult T-cell leukemia.

BackgroundAdult T-cell leukemia (ATL) is a CD4+ T-cell neoplasm with a poor prognosis. A previous study has shown that there is a strong correlation between the secreted matricellular protein osteopontin (OPN) level and disease severity in ATL patients. Here, we investigated the role of OPN in ATL pathogenesis and the possible application of anti-OPN monoclonal antibody (mAb) for ATL immunotherapy in NOD/Shi-scid,IL-2Rgnull (NOG) mice.ResultsSubcutaneous inoculation of ATL cell lines into NOG mice increased the plasma level of OPN, which significantly correlated with metastasis of the inoculated cells and survival time. Administration of an SVVYGLR motif-recognizing anti-OPN mAb resulted in inhibition not only of tumor growth but also of tumor invasion and metastasis. The number of fibroblast activating protein-positive fibroblasts was also reduced by this mAb. We then co-inoculated mouse embryonic fibroblasts (MEFs) isolated from wild-type (WT) or OPN knockout mice together with ATL-derived TL-OmI cells into the NOG mice. The mice co-inoculated with WT MEFs displayed a significant decrease in survival relative to those injected with TL-OmI cells alone and the absence of OPN in MEFs markedly improved the survival rate of TL-OmI-inoculated mice. In addition, tumor volume and metastasis were also reduced in the absence of OPN.ConclusionWe showed that the xenograft NOG mice model can be a useful system for assessment of the physiological role of OPN in ATL pathogenesis. Using this xenograft model, we found that fibroblast-derived OPN was involved in tumor growth and metastasis, and that this tumor growth and metastasis was significantly suppressed by administration of the anti-OPN mAbs. Our findings will lead to a novel mAb-mediated immunotherapeutic strategy targeting against the interaction of OPN with integrins on the tumor of ATL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0225-x) contains supplementary material, which is available to authorized users.

Down-regulation of osteopontin mediates a novel mechanism underlying the cytostatic activity of TGF-β.

Loss of a cytostatic response to TGF-β has been implicated in multiple hyper-proliferative disorders, including cancer. Although several key genes involved in the cytostatic activity of TGF-β have in the past been identified, its exact mode of action is yet to be elucidated. A comprehensive understanding of the mechanisms underlying the cytostatic activity of TGF-β may open up new avenues for the development of therapeutic strategies. Quantitative real-time RT-PCR was used to assess osteopontin (OPN) gene expression in human hepatoma-derived Huh-7 and lung adenocarcinoma-derived A549 cells. Reporter assays using an OPN promoter-luciferase construct and its mutated counterparts were performed to assess its transcriptional activity. Binding of Smad4 to the OPN gene promoter was investigated using chromatin immunoprecipitation (CHIP). The putative role of Smad4 in OPN gene expression down-regulation was also assessed using a shRNA-mediated knockdown strategy. The anti-proliferative effect of TGF-β on different cancer-derived cell lines was determined using the cell proliferation reagent WST-1. We found that the OPN expression levels dose-dependently decreased in TGF-β-treated Huh-7 and A549 cells. Our reporter assays indicated that this TGF-β-induced repression occurred at the transcriptional level, and could largely be abrogated by disruption of an element (TIE2) similar to the TGF-β inhibitory element found in other TGF-β-repressed genes. Our CHIP assay revealed that the Smad protein complex specifically binds to the OPN gene promoter, and that the TGF-β-mediated inhibition of OPN was lost upon shRNA-mediated knockdown of Smad4. Moreover, we found that the deregulation of OPN gene expression by TGF-β occurred concomitantly with loss of the TGF-β anti-proliferative response, whereas a neutralizing anti-OPN antibody partially restored this response. Our results indicate that the OPN gene is a direct target of Smad-mediated TGF-β signaling, implying that OPN expression inhibition serves as a novel mechanism underlying the cytostatic activity of TGF-β.

Mechanical stretch enhances the expression and activity of osteopontin and MMP-2 via the Akt1/AP-1 pathways in VSMC

Hemodynamic forces causing mechanical stretch (MS) of vascular smooth muscle cells (VSMC) play an important role in vascular remodeling, but the underlying mechanism involved is not fully understood. Thus, this study investigated whether osteopontin (OPN) expression in VSMC was induced by MS, and identified the intracellular signaling pathways involved in OPN production. The plasma level of OPN and its expression in aortic tissue were increased in various animal models of hypertension including spontaneous hypertensive rats and hypertensive mice induced by angiotensin II or L-NAME. When aortic VSMC was stimulated with MS, OPN production was increased, which was markedly attenuated in VSMC treated with PI3K/Akt inhibitor as well as in Akt1-depleted cells, but not in Akt2-depleted cells, suggesting a pivotal role of Akt1 isoform in OPN expression in VSMC. In the promoter assay, MS increased OPN promoter activity, which was attenuated when the region harboring AP-1 binding sites was mutated. The MS-enhanced promoter activity and OPN expression were also decreased in cells treated with AP-1 siRNA or inhibitor. Moreover, MS-induced MMP-2 production was attenuated in cells treated with OPN siRNA or anti-OPN antibody as well as in OPN-deficient VSMC cultured from aorta of OPN deficient mice. In in vivo experiments, the expressions of OPN and MMP-2 were increased in the aortic tissues from hypertensive mice, but these increases were markedly attenuated in OPN-deficient mice with hypertension. In conclusion, these results suggested that OPN expression in the hypertensive vasculature was increased via signaling pathways that involve Akt1/AP-1, leading to vascular remodeling by increasing the production of MMP-2.

Neutrophils Modulate Osteoblastic Niche for Hematopoietic Progenitors Via Prostaglandin E2 Production Triggered By Sympathetic Nervous System

The mobilization of hematopoietic stem/progenitor cells (HSC/HPCs) from the bone marrow (BM) to peripheral blood by granulocyte colony-stimulating factor (G-CSF) is an essential method in clinic. We have shown that the suppression of osteoblastic niche by β-adrenergic signal is critical for this phenomenon (Cell 2006; Cell Stem Cell 2013). Because G-CSF administration causes fever and back pain and these symptoms are ameliorated by non-steroidal anti-inflammatory drugs, we investigated the role of prostaglandin E2 (PGE2) in the BM microenvironment during G-CSF treatment. First, HPC (CFU-C) mobilization by G-CSF (125μg/kg/dose, every 12 hours, 8 divided doses) was significantly augmented in microsomal PGE syntase-1-deficient (mPGES-1-/-) mice (42% increase, n=16, p<0.05), and strongly inhibited by exogenous administration of PGE2 (6mg/kg/day for 2 weeks) to wild-type (WT) mice (52%, n=8-9 p<0.05). These data suggest that G-CSF induces mPGES-1-mediated PGE2 production, which suppresses the HPC mobilization. In the early phase (at 4 doses) of G-CSF administration, mPGES-1 mRNA in BM cells was upregulated (34% increase, n=5, p<0.05). Furthermore, WT mice reconstituted with mPGES-1-/- BM showed higher HPC mobilization than control mice reconstituted with mPGES-1+/+ BM (2.2-fold increase, n=4, p<0.05), which indicated that blood cells might be responsible for additional PGE2 synthesis. To address this, we examined the PGE2 production by ELISA from various lineage cell lines, such as neutrophil precursor 32D, macrophage RAW264.7, B cell Ba/F3, and T cell EL4. No increase was observed by G-CSF in any cell lines; whereas, isoproterenol induced PGE2 production significantly only in 32D culture supernatant (2.4-fold increase compared to vehicle treatment, n=4, p<0.05) accompanied with drastic increase of mPGES-1 mRNA in the cells and norepinephrine showed a similar effect. Primary neutrophils sorted from the BM also demonstrated prompt PGE2 production by isoproterenol (3-fold increase compared to vehicle treatment, n=4, p<0.05) but not by G-CSF. These data suggest that G-CSF-triggered high sympathetic tone stimulates the BM neutrophils to lead PGE2 production. We next assessed the exact roles of PGE2 in HPC mobilization. The inhibitory effect of PGE2 on HPC mobilization was completely abrogated in PGE2 receptor EP4-deficient (EP4-/-) mice, and the chimeric model generated by the reciprocal BM transplantation revealed that it was EP4 in microenvironment, but not in hematopoietic cells, that was critical for this effect. Since PGE2 did not change the CXCL12 behavior, we speculated that PGE2 increased another anchor in the niche, osteopontin (OPN). Immunofluorescence staining demonstrated upregulation of OPN by PGE2 and/or G-CSF in the endosteum, which was abolished in EP4-/- mice. Indeed, the inhibitory effect of PGE2 on HPC mobilization was canceled partially in OPN-/- mice and almost completely in anti-OPN antibody-treated WT mice. PGE2 also inhibited AMD3100-induced HPC mobilization, and this effect was canceled by anti-OPN antibody, which confirmed that PGE2-mediated niche modulation was independent of CXCL12 axis. To assess the induction of OPN by PGE2 directly, we fractionated non-hematopoietic (CD45-CD31-Ter119-) cells isolated from adult femur into three populations, i.e. Sca-1+ALCAM- immature mesenchymal cells, Sca-1-ALCAM- preosteoblasts that favorably support HPCs, and Sca-1-ALCAM+ mature osteoblasts that are most potent to maintain quiescent HSCs in vitro. PGE2 upregulated the OPN protein 2-fold in Sca-1+ALCAM- immature mesenchymal cells in cultures and more dramatically (6-fold) in Sca-1-ALCAM- preosteoblasts as assessed by flow cytometry. In sharp contrast, no OPN induction was observed in Sca-1-ALCAM+ mature osteoblasts. PGE2 failed to induce OPN in all three fractions from EP4-/- mice. In contrast to HPCs, the mobilization of long-term (6 months) repopulating HSCs was not altered in mPGES-1-/- and in PGE2-treated WT mice. This was consistent with the OPN induction profile in fractionated osteoblasts. These results suggest that PGE2 selectively regulates the osteoblastic niche for hematopoietic progenitors, but not for stem cells, by the induction of OPN via EP4 receptor. Collectively, we propose the inter-communication between the mature hematopoietic cells and the niche for their immature progenitors governed by the sympathetic nervous system. DisclosuresNo relevant conflicts of interest to declare.

Osteopontin is expressed in the oviduct and promotes fertilization and preimplantation embryo development of mouse.

Osteopontin (OPN) is a multifunctional phosphoprotein that is detected in various tissues, including male and female reproductive tracts. In this study, we evaluated OPN expression in mouse oviducts during the estrus cycle, and at days 1-5 of pregnancy and pseudopregnancy by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The mice oocytes, sperm and embryos were treated with different concentrations of anti-OPN antibody in vitro to detect the function of OPN in fertilization and preimplantation embryo development. OPN mRNA and protein expression in mouse oviducts were cyclic dependent throughout the estrous cycle, which was highest at estrous and lowest at diestrous. Such a phenomenon was consistent with the change in estrogen level in mice. The expression levels of OPN in mice oviduct of normal pregnancy and pseudopregnancy were significantly different, which indicated that OPN expression in mouse oviducts was depend on estrogen and preimplantation embryo. Furthermore, anti-OPN antibody treatment could reduce the rates of fertilization, cleavage and blastocyst formation in vitro in a dose-dependent way. Overall, our results indicated that the expression of OPN in mouse oviducts during the estrous cycle and early pregnancy is likely regulated by estrogen and the embryo, and OPN may play a vital role in oocyte fertilization and preimplantation embryo development.

Role of autocrine osteopontin in promoting multiple functions of murine Nf1+/-osteoclast

Objective To detect the osteopontin （OPN） autocrine function of the osteoclasts in neurofibromatosis type 1 heterozygote （Nfl＋/-） and wild type （Nfl＋/＋） mice. Test the osteoclasts function of neurofibromatosis type 1 heterozygote （Nfl＋/-） and wild type （Nfl＋/＋） mice with exogenous neutralizing OPN antibody, analysis the role of autocrine OPN in the hyperfunction of osteoclast in neurofibromatosis type 1. Methods Culture the low density bone marrow cells from Nfl heterozygote （Nfl＋/-） and wild type （Nfl＋/＋） mice （ 4-6 weeks old ） with macrophage colony-stimulating factor （M-CSF） and receptor activator of NF-KB lig-and（RANKL）, Measure the OPN concentration in osteoclast culture superenant with ELISA. Culture the low density bone marrow ceils from Nfl ＋/- and Nfl ＋/＋ mice with or without exogenous neutralizing antibody for OPN. The function of osteoclasts and os-teoclast progenitors in formation, migration, adhesion, and bone absorption were tested. Results A significantly higher concen- tration of OPN was detected in the Nfl ＋/- osteoclast cuhure media as compared to that of wild type. In control, Osteoclast func-tions, including migration, adhesion, and bone resorption of Nfl＋/-were higher than that of wild type. Addition OPN neutralizing antibody to the Nfl＋/- OCL significantly reduced OCL formation. Neutralizing OPN antibody diminished both wild type and Nfl＋ /- OCL adhensiontion, Anti-OPN minimized OCL migration in both wild type and Nfl＋/- OCL cultures as measured by the tran- swell assays. Neutralizing OPN antibody diminished both wild type and Nfl＋/- OCL pit formation,P〉0.05 for comparing Nfl＋/- vs. wild type OCLs with anti-OPN antibody. Conclusion The hyperfunction of osteoclast in Nfl heterozygote is related with au- tocrine osteopontin, inhibition of OPN may be an effective treatment for bone destruction of neurofibromatosis type 1. Key words: Neurofibromatosis 1; Osteopontin; Osteoclasts; Mice

Osteopontin cytoplasmic immunoexpression is a predictor of poor disease-free survival in thyroid cancer

BackgroundOsteopontin (OPN) is expressed in various malignancies and may play an important role in tumorigenesis, tumour invasion, and metastasis in various malignancies including thyroid cancers. The objective of this study was to investigate the relationship between OPN immunoexpression and clinicopathological characteristics in thyroid lesions. Material and methodsParaffin blocks belonging to 160 patients with thyroid neoplasms were retrieved from the archive of the Department of Pathology at King Abdulaziz University, and King Faisal Specialist Hospital, Jeddah, Saudi Arabia. Immunohistochemistry was performed using anti-OPN antibody. Statistical tests were used to determine the relation of OPN immunoexpression to clinicopathological characteristics and survival. ResultsImmunostaining results showed that OPN was localised in the cytoplasm and nucleus with higher cytoplasmic expression. Cytoplasmic and nuclear OPN was higher in thyroid cancer than other lesions. Microcarcinoma variant of papillary thyroid carcinoma showed a lower OPN cytoplasmic level than other variants. OPN cytoplasmic expression was not associated with most of the clinicopathological parameters tests. However, OPN cytoplasmic overexpression was associated poor survival outcome (p<0.001). ConclusionUpregulation of cytoplasmic OPN was associated with poor survival outcome and was an independent predictor of margin involvement and recurrence. Nuclear OPN expression was lower than cytoplasmic and was associated with extrathyroid extension of thyroid cancer. OPN plays a role in thyroid carcinoma and incoming research has to be focused on the mechanistic association with invasion and metastasis.

Osteopontin alters the functional profile of porcine microgliain vitro

OPN (osteopontin) is a secreted glycoprotein predominantly expressed in bone matrix and kidney tissue. More recently, a neuroprotective role has been attributed to this cytokine since it can be up-regulated by microglia in neurodegeneration and inflammation. We demonstrate the expression of OPN within primary cultured microglia. Microglia incubated in vitro with different concentrations (0.1 fM-1 nM) of recombinant OPN showed increased proliferation at 10 fM. Moreover, conditioned medium of LLC-PK1 cells, a pig renal epithelial cell line and a known source of secreted OPN, more than doubled the rate of proliferation of microglia. Addition of an anti-OPN polyclonal antibody completely reversed this effect. Treatment with OPN dose-dependently also inhibited microglial superoxide production. In contrast, phagocytosis of fluorescent-labelled beads was enhanced by OPN. In conclusion, OPN shifts microglia, at least in vitro, to an alternative functional profile more fit to the immune-balanced microenvironment of the CNS (central nervous system).

Osteopontin-dependent regulation of Th1 and Th17 cytokine responses in Trypanosoma cruzi-infected C57BL/6 mice

Osteopontin (OPN) is a multifunctional protein participating in the regulation of different Th cell lineages and critically involved in the initiation of immune responses to diverse pathogens. Our study goal was to verify whether OPN helps modulate the protective Th1 and Th17 cytokine responses in C57BL/6 mice infected with Trypanosoma cruzi, the etiological agent of Chagas disease. Parasite infection induced OPN release from murine macrophages in vitro and acute Chagas mice displayed enhanced serum levels of this cytokine at the peak of parasitemia. Upon administration of a neutralizing anti-OPN antibody, recently infected mice presented lower Th1 and Th17 responses, increased parasitemia and succumbed earlier and at higher rates to infection than non-immune IgG-receiving controls. The anti-OPN therapy also resulted in reduced circulating levels of IL-12 p70, IFN-γ, IL-17A and specific IgG2a antibodies. Furthermore, antibody-mediated blockade of OPN activity abrogated the ex vivo production of IL-12 p70, IFN-γ and IL-17A, while promoting IL-10 secretion, by spleen macrophages and CD4+ T cells from T. cruzi-infected mice. Th1 and Th17 cytokine release induced by OPN preferentially involved the αvβ3 integrin OPN receptor, whereas concomitant down-modulation of IL-10 production would mostly depend on OPN interaction with CD44. Our findings suggest that, in resistant C57BL/6 mice, elicitation of protective Th1 and Th17 cytokine responses to T. cruzi infection is likely to be regulated by endogenous OPN.