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1.  The expression of osteopontin and its association with Clara cell 10-kDa protein in allergic rhinitis 
SUMMARY
Background
Osteopontin (OPN) is a multifunctional protein which has recently been linked to allergic diseases. Clara cell 10-kDa protein (CC10) is another protein linked to allergy, and has been suggested to have an inhibitory role in inflammatory airway diseases. At this time, it is not known whether OPN is involved in allergic rhinitis (AR) or if there is any association between CC10 and OPN in AR.
Objective
To study the expression of OPN and its potential association with CC10 in AR.
Methods
The expression of CC10 and OPN in nasal mucosa of AR patients was investigated. AR animal models were established by employing wild-type and CC10-knockout mice. In some experiments, human recombinant CC10 protein was given to AR mice during either sensitization or challenge. The phenotypic changes were examined by histology and real-time RT-PCR. The direct effect of CC10 on OPN expression in spleen mononuclear cells and on OPN-induced inflammatory cytokine expression in BEAS-2B cells was measured through in vitro cell culture.
Results
OPN expression was up-regulated, with a concomitant down-regulation of CC10, in AR patients, showing a significant negative correlation between their expression. Compared with control mice sensitized with PBS, OPN expression was significantly increased in AR mice; such increase was more prominent in CC10-knockout mice, compared to wild-type. Administration of CC10 during both sensitization and challenge could markedly ameliorate Th2-skewed inflammation and OPN expression in nasal mucosa. CC10 administration at the sensitization phase could also reduce spleen OPN expression. The in vitro study showed that CC10 directly down-regulated OPN expression in spleen mononuclear cells stimulated with OVA and suppressed OPN-induced expression of Th2 cytokines and proinflammatory cytokines in BEAS-2B cells.
Conclusion
In the context of allergic airway responses, CC10 can inhibit OPN expression and suppress the Th2 promoting function of OPN, resulting in CC10’s inhibitory biological effects.
doi:10.1111/j.1365-2222.2010.03549.x
PMCID: PMC2948078  PMID: 20553297
allergic rhinitis; Clara cell 10-kDa protein; osteopontin; regulation
2.  Effects of Corticosteroids on Osteopontin Expression in a Murine Model of Allergic Asthma 
Background
Osteopontin (OPN) contributes to the development of T helper 1 (Th1)-mediated immunity and Th1-associated diseases. However, the role of OPN in bronchial asthma is unclear. Corticosteroids reduce airway inflammation, as reflected by the low eosinophil and T-cell counts, and the low level of cytokine expression. We investigated OPN production and the inhibitory effects of corticosteroids on OPN production in a murine model of allergic asthma.
Methods
BALB/c mice were sensitized by intraperitoneal injections of ovalbumin (OVA) with alum. Some mice received daily injections of dexamethasone (DEX) or phosphate-buffered saline for 1 week. All OVA-challenged mice were exposed to aerosolized 1% OVA for 30 min an hour after these injections. After the OVA challenge, the mice were killed, and bronchoalveolar lavage (BAL) fluid and lung tissue were examined.
Results
The levels of OPN protein in BAL fluid and OPN mRNA in lung tissue increased after OVA challenge. Most OPN-expressing cells were CD11c+ cells and some were T cells. DEX decreased the levels of OPN protein in the BAL fluid, and those of OPN mRNA and OPN protein in lung tissue.
Conclusions
OPN may play an important role in allergic bronchial asthma. Corticosteroids inhibit OPN production in mice with allergic asthma. The beneficial effect of corticosteroids in bronchial asthma is partly due to their inhibitory effects on OPN production.
doi:10.1159/000210647
PMCID: PMC2844795  PMID: 19494498
Osteopontin; Corticosteroid; Mouse; Dendritic cells; CD11c; Bronchial asthma
3.  Osteopontin Impairs Host Defense during Established Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis) 
Background
Melioidosis, caused by infection with Burkholderia (B.) pseudomallei, is a severe illness that is endemic in Southeast Asia. Osteopontin (OPN) is a phosphorylated glycoprotein that is involved in several immune responses including induction of T-helper 1 cytokines and recruitment of inflammatory cells.
Methodology and Principal Findings
OPN levels were determined in plasma from 33 melioidosis patients and 31 healthy controls, and in wild-type (WT) mice intranasally infected with B. pseudomallei. OPN function was studied in experimental murine melioidosis using WT and OPN knockout (KO) mice. Plasma OPN levels were elevated in patients with severe melioidosis, even more so in patients who went on to die. In patients who recovered plasma OPN concentrations had decreased after treatment. In experimental melioidosis in mice plasma and pulmonary OPN levels were also increased. Whereas WT and OPN KO mice were indistinguishable during the first 24 hours after infection, after 72 hours OPN KO mice demonstrated reduced bacterial numbers in their lungs, diminished pulmonary tissue injury, especially due to less necrosis, and decreased neutrophil infiltration. Moreover, OPN KO mice displayed a delayed mortality as compared to WT mice. OPN deficiency did not influence the induction of proinflammatory cytokines.
Conclusions
These data suggest that sustained production of OPN impairs host defense during established septic melioidosis.
Author Summary
Melioidosis is a severe tropical disease caused by infection with the bacterium Burkholderia (B.) pseudomallei. In northeast Thailand infection with this bacterium is the major cause of community-acquired septicemia with a mortality rate up to 40%. Extending the knowledge on the mechanisms of host defense against B. pseudomallei infection would be helpful to improve treatment of this severe illness. Osteopontin (OPN) is a cytokine that is involved in several immune responses that occur during bacterial infection. In this study, we investigated levels of OPN in patients with melioidosis, and studied the function of OPN during experimental melioidosis in mice. We found that OPN concentrations were elevated in patients with severe melioidosis, and that high OPN concentrations are associated with poor outcome in patients with melioidosis. In experimental melioidosis in mice plasma and lung OPN levels were also increased. Moreover, mice with melioidosis that were deficient for OPN demonstrated reduced bacterial numbers in their lungs, diminished pulmonary tissue injury, and decreased neutrophil infiltration into the lungs during established melioidosis. Moreover, these mice displayed a delayed mortality as compared to control mice. In conclusion, sustained production of OPN impairs host defense during melioidosis.
doi:10.1371/journal.pntd.0000806
PMCID: PMC2930856  PMID: 20824216
4.  Thrombin-activatable carboxypeptidase B-cleavage of osteopontin regulates neutrophil survival and synoviocyte binding in rheumatoid arthritis 
Arthritis and rheumatism  2009;60(10):2902-2912.
Objective
Osteopontin (OPN) is a pro-inflammatory cytokine important in rheumatoid arthritis (RA). OPN can be cleaved by thrombin, leading to OPN-Arg (OPN-R) and exposing the cryptic C-terminal α4β1 and α9β1 integrin-binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also termed thrombin-activatable fibrinolysis inhibitor (TAFI), removes the C-terminal arginine from OPN-R, generating OPN-Leu (OPN-L) and abrogating its enhanced cell binding. We investigated the roles of OPN-R and OPN-L in: (i) synoviocyte adhesion, which contributes to formation of invasive pannus, and (ii) neutrophil survival, which affects inflammatory infiltrates, in RA.
Methods and Results
We developed ELISAs specific for OPN-R and OPN-L, and demonstrate elevations of OPN-R and OPN-L in RA, but not in osteoarthritis or psoriatic arthritis, synovial fluid samples. OPN-R and OPN-L levels correlated with multiple inflammatory cytokines including TNFα and IL-6. Immunohistochemical analyses demonstrated robust expression of OPN-FL, but minimal OPN-R, in RA synovium, suggesting that cleaved OPN is released into the synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an anti-apoptotic effect on neutrophils. OPN-R, but not OPN-L, augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to α4β1.
Conclusion
Thrombin activation of OPN (OPN-R) and its subsequent inactivation by thrombin-activatable CPB (OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA, by regulating neutrophil viability and reducing synoviocyte adhesion.
doi:10.1002/art.24814
PMCID: PMC3757557  PMID: 19790060
osteopontin; thrombin-activatable carboxypeptidase B; thrombin-activatable fibrinolysis inhibitor; rheumatoid arthritis
5.  Syndecan-4 protects against osteopontin-mediated acute hepatic injury by masking functional domains of osteopontin 
Osteopontin (OPN) is a T helper type 1 immunoregulatory cytokine that plays a critical role in various inflammatory disorders. OPN exerts proinflammatory reactions through interaction with integrin receptors. OPN function can be modulated by protease digestion. However, the molecular mechanisms that regulate OPN function in vivo have not been elucidated. There are two putative heparin-binding domains (HBDs) within the OPN molecule, which may bind both heparin and heparin-like glycosaminoglycans such as syndecan. We show that expression of OPN and syndecan-4 is significantly up-regulated after concanavalin-A (ConA) injection. Syndecan-4 binds to one of the HBDs of OPN, which overlaps with the thrombin cleavage site of OPN. When OPN is associated with syndecan-4, syndecan-4 masks both the thrombin cleavage and the integrin binding sites within OPN. Importantly, syndecan-4–deficient (Syn4KO) mice are more susceptible to hepatic injury, and the thrombin-cleaved form of OPN is significantly elevated in Syn4KO mice as compared with wild-type mice after ConA injection. Finally, we demonstrate that administration of purified syndecan-4 protects mice from ConA-induced hepatic injury. Thus, syndecan-4 is a critical intrinsic regulator of inflammatory reactions via its effects on OPN function and is a potential novel therapeutic tool for treating inflammatory diseases.
doi:10.1084/jem.20071324
PMCID: PMC2234375  PMID: 18158320
6.  Chemokine (C-C Motif) Ligand 20, a Potential Biomarker for Graves' Disease, Is Regulated by Osteopontin 
PLoS ONE  2013;8(5):e64277.
Context
Graves’ disease (GD) is a common autoimmune disease involving the thyroid gland. The altered balance of pro- and anti-inflammatory cytokines plays an important role in the pathogenesis of GD. Chemokine (C-C motif) ligand 20 (CCL20) is important for interleukin-17 (IL-17) signal activation and a potent chemoattractant for Th17 cells. Meanwhile, Osteopontin (OPN), a broadly expressed pleiotropic cytokine, has been implicated in GD through inducing Th1-involved response to enhance the production of proinflammatory cytokines and chemokines, but little is known about the role of OPN in regulating CCL20 and IL-17 signaling.
Objective
This study sought to explore the possibility of CCL20 level as a biomarker for GD, as well as investigate the role of OPN in regulating CCL20 production.
Methods
Fifty untreated GD patients, fifteen euthyroid GD patients, twelve TRAb-negative GD patients and thirty-five healthy control donors were recruited. OPN, CCL20 and other clinical GD diagnosis parameters were measured. CD4+T cells were isolated from peripheral blood mononuclear cells (PBMCs) using antibody-coated magnetic beads. Enzyme-linked immune-sorbent assay and quantitative polymerase chain reaction were used to determine CCL20 expression level.
Results
We found that the plasma CCL20 level was enhanced in GD patients and decreased in euthyroid and TRAb-negative GD patients. In addition, CCL20 level correlated with GD clinical diagnostic parameters and plasma OPN level. Moreover, we demonstrated that recombinant OPN and plasma from untreated GD patients increased the expression of CCL20 in CD4+T cells, which could be blocked by OPN antibody. Furthermore, we found that the effect of OPN on CCL20 expression was mediated by β3 integrin receptor, IL-17, NF-κB and MAPK pathways.
Conclusions
These results demonstrated that CCL20 might serve as a biomarker for GD and suggested the possible role of OPN in induction of CCL20 expression.
doi:10.1371/journal.pone.0064277
PMCID: PMC3661485  PMID: 23717583
7.  Neutralization of Osteopontin Inhibits Obesity-Induced Inflammation and Insulin Resistance 
Diabetes  2010;59(4):935-946.
OBJECTIVE
Obesity is associated with a state of chronic low-grade inflammation mediated by immune cells that are primarily located to adipose tissue and liver. The chronic inflammatory response appears to underlie obesity-induced metabolic deterioration including insulin resistance and type 2 diabetes. Osteopontin (OPN) is an inflammatory cytokine, the expression of which is strongly upregulated in adipose tissue and liver upon obesity. Here, we studied OPN effects in obesity-induced inflammation and insulin resistance by targeting OPN action in vivo.
RESEARCH DESIGN AND METHODS
C57BL/6J mice were fed a high-fat diet to induce obesity and were then intravenously treated with an OPN-neutralizing or control antibody. Insulin sensitivity and inflammatory alterations in adipose tissue and liver were assessed.
RESULTS
Interference with OPN action by a neutralizing antibody for 5 days significantly improved insulin sensitivity in diet-induced obese mice. Anti-OPN treatment attenuated liver and adipose tissue macrophage infiltration and inflammatory gene expression by increasing macrophage apoptosis and significantly reducing c-Jun NH2-terminal kinase activation. Moreover, we report OPN as a novel negative regulator for the activation of hepatic signal transducer and activator of transcription 3 (STAT3), which is essential for glucose homeostasis and insulin sensitivity. Consequently, OPN neutralization decreased expression of hepatic gluconeogenic markers, which are targets of STAT3-mediated downregulation.
CONCLUSIONS
These findings demonstrate that antibody-mediated neutralization of OPN action significantly reduces insulin resistance in obesity. OPN neutralization partially decreases obesity-associated inflammation in adipose tissue and liver and reverses signal transduction related to insulin resistance and glucose homeostasis. Hence, targeting OPN could provide a novel approach for the treatment of obesity-related metabolic disorders.
doi:10.2337/db09-0404
PMCID: PMC2844841  PMID: 20107108
8.  Differentiation, Maturation, and Survival of Dendritic Cells by Osteopontin Regulation 
Dendritic cells (DCs) are antigen-presenting cells with the ability to induce primary immune responses necessary in innate immunity and adaptive immunity. Osteopontin (OPN) is a secreted acidic phosphoprotein containing an arginine-glycine-aspartate sequence and has been suggested to play an important role in early cellular immune responses. The interaction between DCs and OPN has not been clarified. We hypothesized that there is an important interaction between DCs and OPN, which is an indispensable extracellular matrix component in early cellular immune responses. Human monocyte-derived DCs synthesized OPN especially during the differentiation from monocytes to immature DCs. By blocking of OPN with anti-OPN antibody, cultured DCs became smaller and expressed lower levels of costimulatory molecules and major histocompatibility complex class II antigens than untreated DCs. Furthermore, DCs treated with anti-OPN antibody easily underwent apoptosis. These results suggest that human DCs can produce OPN and that OPN may play a role in the differentiation, maturation, and survival of DCs by autocrine and/or paracrine pathways.
doi:10.1128/CDLI.12.1.206-212.2005
PMCID: PMC540203  PMID: 15643009
9.  Osteopontin is Induced by Hedgehog Pathway Activation and Promotes Fibrosis Progression in Nonalcoholic Steatohepatitis 
Hepatology (Baltimore, Md.)  2010;53(1):106-115.
Background
Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH-related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding Osteopontin (OPN), a pro-fibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesized that Hh signaling induces OPN to promote liver fibrosis in NASH.
Methods
Hepatic OPN expression and liver fibrosis were analyzed in wild-type (WT) mice, Patched-deficient (Ptc+/−) (overly-active Hh signaling) mice, and OPN-deficient mice before and after feeding methionine-choline deficient (MCD) diets to induce NASH-related fibrosis. Hepatic OPN was also quantified in human NASH and non-diseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSC) were cultured in medium with different OPN activities to determine effects on HSC phenotype.
Results
When fed MCD diets, Ptc+/− mice expressed more OPN and developed worse liver fibrosis (p<0.05) than WT mice, while OPN-deficient mice exhibited reduced fibrosis (p<0.05). In NASH patients, OPN was significantly up-regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSC, SAG (a Hh agonist) upregulated, while Cyclopamine (a Hh-antagonist) repressed, OPN expression (p<0.005). Cholangiocyte-derived OPN and recombinant OPN promoted fibrogenic responses in HSC (p<0.05); neutralizing OPN with RNA-aptamers attenuated this (p<0.05).
Conclusions
OPN is Hh-regulated and directly promotes pro-fibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis-stage Therefore, OPN inhibition may be beneficial in NASH.
doi:10.1002/hep.23998
PMCID: PMC3025083  PMID: 20967826
hedgehog; non-alcoholic fatty liver disease; osteopontin
10.  Human and Experimental Evidence Supporting a Role for Osteopontin in Alcoholic Hepatitis 
Hepatology (Baltimore, Md.)  2013;58(5):1742-1756.
We identified in the transcriptome analysis of patients with alcoholic hepatitis (AH) osteopontin (OPN) as one of the most up-regulated genes. Here, we used a translational approach to investigate its pathogenic role. OPN hepatic gene expression was quantified in patients with AH and other liver diseases. OPN protein expression and processing were assessed by immmunohistochemistry, Western blotting and ELISA. OPN gene polymorphisms were evaluated in patients with alcoholic liver disease. The role of OPN was evaluated in OPN−/− mice with alcohol-induced liver injury. OPN biological actions were studied in human hepatic stellate cells and in precision-cut liver slices. Hepatic expression and serum levels of OPN were markedly increased in AH compared to normal livers and other types of chronic liver diseases and correlated with short-term survival. Serum levels of OPN also correlated with hepatic expression and disease severity. OPN was mainly expressed in areas with inflammation and fibrosis. Two proteases that process OPN (thrombin and MMP-7) and cleaved-OPN were increased in livers with AH. Patients with AH had a tendency of a lower frequency of the CC genotype of the +1239C SNP of the OPN gene compared to patients with alcohol abuse without liver disease. Importantly, OPN−/− mice were protected against alcohol-induced liver injury and showed decreased expression of inflammatory cytokines. Finally, OPN was induced by LPS and stimulated inflammatory actions in hepatic stellate cells.
Conclusion
Human and experimental data suggest a role for OPN in the pathogenesis of AH. Further studies should evaluate OPN as a potential therapeutic target.
doi:10.1002/hep.26521
PMCID: PMC3877722  PMID: 23729174
alcoholic liver disease; translational research; cytokines; liver fibrosis
11.  Osteopontin deficiency protects against obesity-induced hepatic steatosis and attenuates glucose production in mice 
Diabetologia  2011;54(8):2132-2142.
Aims/hypothesis
Obesity is strongly associated with the development of non-alcoholic fatty liver disease (NAFLD). The cytokine osteopontin (OPN) was recently shown to be involved in obesity-induced adipose tissue inflammation and reduced insulin response. Accumulating evidence links OPN to the pathogenesis of NAFLD. Here we aimed to identify the role of OPN in obesity-associated hepatic steatosis and impaired hepatic glucose metabolism.
Methods
Wild-type (WT) and Opn (also known as Spp1) knockout (Opn−/−) mice were fed a high-fat or low-fat diet to study OPN effects in obesity-driven hepatic alterations.
Results
We show that genetic OPN deficiency protected from obesity-induced hepatic steatosis, at least in part, by downregulating hepatic triacylglycerol synthesis. Conversely, absence of OPN promoted fat storage in adipose tissue thereby preventing the obesity-induced shift to ectopic fat accumulation in the liver. Euglycaemic–hyperinsulinaemic clamp studies revealed that insulin resistance and excess hepatic glucose production in obesity were significantly attenuated in Opn−/− mice. OPN deficiency markedly improved hepatic insulin signalling as shown by enhanced insulin receptor substrate-2 phosphorylation and prevented upregulation of the major hepatic transcription factor Forkhead box O1 and its gluconeogenic target genes. In addition, obesity-driven hepatic inflammation and macrophage accumulation was blocked by OPN deficiency.
Conclusions/interpretation
Our data strongly emphasise OPN as mediator of obesity-associated hepatic alterations including steatosis, inflammation, insulin resistance and excess gluconeogenesis. Targeting OPN action could therefore provide a novel therapeutic strategy to prevent obesity-related complications such as NAFLD and type 2 diabetes.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-011-2170-0) contains supplementary material, which is available to authorised users.
doi:10.1007/s00125-011-2170-0
PMCID: PMC3131508  PMID: 21562757
Gluconeogenesis; High-fat diet; Inflammation; Insulin resistance; Non-alcoholic fatty liver disease
12.  Quantitative expression of osteopontin in nasal mucosa of patients with allergic rhinitis: effects of pollen exposure and nasal glucocorticoid treatment 
Background
Osteopontin (OPN) is a multifunctional cytokine that has been primarily investigated in Th1 diseases. Recently, it has also been implicated in Th2-mediated allergic diseases, such as asthma. The expression of OPN in allergic rhinitis (AR) is currently unknown, as is the effect of intranasal glucocorticosteroids (GCs) on that expression.
Methods
Subjects with AR were randomised to receive treatment with fluticasone propionate (FP) (n = 12) or a placebo (n = 16) over the grass pollen season and nasal biopsies were taken prior to, and during the season. OPN expression in the nasal mucosa was examined with immunohistochemistry. Healthy non-AR controls (n = 5) were used as a comparator.
Results
OPN expression was detected in epithelial cells, subepithelial infiltrating/inflammatory cells and cells lining the vessels and glands of all subjects. Comparison of the pre- and peak-pollen season biopsy sections in placebo treated patients revealed no increase in OPN expression during the grass pollen season (5.7% vs 6.4%). Treatment with a local glucocorticosteroid did not alter the expression of OPN during pollen exposure (6.2% vs 6.7%).
Conclusion
OPN has been increasingly associated with the pathogenesis of various Th2-mediated diseases. However, our finding that the OPN expression in the nasal mucosa of AR patients is not significantly affected by allergen exposure and is comparable to that of the healthy controls, suggests that intracellular OPN is not directly involved in the pathogenesis of allergic rhinitis.
doi:10.1186/1710-1492-6-28
PMCID: PMC2988772  PMID: 21044308
13.  Role of Osteopontin in Systemic Lupus Erythematosus 
Systemic lupus erythematosus (SLE) is a multisystemic disease, caused by a variety of factors, which lead to immunological abnormalities. Osteopontin (OPN) is a pleiotropic protein, important in bone remodeling and immune system signaling. OPN, produced by various cells, including immune cells, plays a key role in regulating T-helper 1/T-helper 2 balance, stimulating B lymphocytes to produce antibodies, regulating macrophages, neutrophils and inducing dendritic cells. OPN expression is influenced by genetic polymorphisms of its promoter, hormones and cytokines. Over expression of OPN has been associated with the pathogenesis of immune-mediated diseases. OPN has been implicated in the development of murine model of lupus and in humans with SLE. In this review, I will present current state of research on the role of OPN and OPN gene polymorphisms in pathogenesis and clinical course of SLE. A better understanding of the role of OPN in SLE will contribute to more precise diagnosis and treatment of the disease.
doi:10.1007/s00005-014-0294-x
PMCID: PMC4244532  PMID: 24917428
Osteopontin; Systemic lupus erythematosus; Gene; Polymorphism
14.  Osteopontin promotes fibrosis in dystrophic mouse muscle by modulating immune cell subsets and intramuscular TGF-β 
The Journal of Clinical Investigation  2009;119(6):1583-1594.
Duchenne muscular dystrophy (DMD) is an X-linked, degenerative muscle disease that is exacerbated by secondary inflammation. Here, we characterized the immunological milieu of dystrophic muscle in mdx mice, a model of DMD, to identify potential therapeutic targets. We identified a specific subpopulation of cells expressing the Vβ8.1/8.2 TCR that is predominant among TCR-β+ T cells. These cells expressed high levels of osteopontin (OPN), a cytokine that promotes immune cell migration and survival. Elevated OPN levels correlated with the dystrophic process, since OPN was substantially elevated in the serum of mdx mice and muscle biopsies after disease onset. Muscle biopsies from individuals with DMD also had elevated OPN levels. To test the role of OPN in mdx muscle, mice lacking both OPN and dystrophin were generated and termed double-mutant mice (DMM mice). Reduced infiltration of NKT-like cells and neutrophils was observed in the muscle of DMM mice, supporting an immunomodulatory role for OPN in mdx muscle. Concomitantly, an increase in CD4+ and FoxP3+ Tregs was also observed in DMM muscle, which also showed reduced levels of TGF-β, a known fibrosis mediator. These inflammatory changes correlated with increased strength and reduced diaphragm and cardiac fibrosis. These studies suggest that OPN may be a promising therapeutic target for reducing inflammation and fibrosis in individuals with DMD.
doi:10.1172/JCI37662
PMCID: PMC2689112  PMID: 19451692
15.  OSTEOPONTIN, AN OXIDANT STRESS-SENSITIVE CYTOKINE, UP-REGULATES COLLAGEN-I VIA INTEGRIN αVβ3 ENGAGEMENT AND PI3K-pAkt-NFκB SIGNALING 
Hepatology (Baltimore, Md.)  2012;55(2):594-608.
Background & Aim
A key feature in the pathogenesis of liver fibrosis is fibrillar collagen-I deposition; yet, mediators that could be key therapeutic targets remain elusive. We hypothesized that osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in hepatic stellate cells (HSC), could drive fibrogenesis by modulating the HSC profibrogenic phenotype and collagen-I expression.
Results
rOPN up-regulated collagen-I protein in primary HSC in a TGFβ-independent fashion whereas it down-regulated matrix metalloprotease-13 (MMP13) thus favoring scarring. rOPN activated primary HSC -confirmed by increased α-smooth muscle actin (α-SMA) expression- and enhanced their invasive and wound-healing potential. HSC isolated from wild type (WT) mice were more profibrogenic than those from Opn-/- mice and infection of primary HSC with an Ad-OPN increased collagen-I, indicating correlation between both proteins. The OPN induction of collagen-I occurred via integrin αvβ3 engagement and activation of the PI3K-pAkt-NFκB signaling pathway, while CD44-binding and mTOR-p70S6K were not involved. Neutralization of integrin αvβ3 prevented the OPN-mediated activation of the PI3K-pAkt-NFκB signaling cascade and collagen-I up-regulation. Likewise, inhibition of PI3K and NFκB blocked the OPN-mediated collagen-I increase. HCV-cirrhotic patients showed co-induction of collagen-I and cleaved OPN compared to healthy individuals. Acute and chronic liver injury by carbon tetrachloride (CCl4)-injection or thioacetamide (TAA)-treatment elevated OPN expression. Reactive oxygen species up-regulated OPN in vitro and in vivo and antioxidants prevented this effect. OpnHEP Tg mice developed spontaneous liver fibrosis compared to WT mice. Lastly, chronic CCl4-injection and TAA-treatment caused more liver fibrosis to WT than to Opn-/- mice and the reverse occurred in OpnHEP Tg mice.
Conclusion
OPN emerges as a cytokine within the ECM protein network driving the increase in collagen-I protein contributing to scarring and liver fibrosis.
doi:10.1002/hep.24701
PMCID: PMC3561739  PMID: 21953216
Extracellular matrix; fibrosis; hepatic stellate cells; oxidant stress
16.  Melanopsin as a Sleep Modulator: Circadian Gating of the Direct Effects of Light on Sleep and Altered Sleep Homeostasis in Opn4−/− Mice 
PLoS Biology  2009;7(6):e1000125.
Analyses in mice deficient for the blue-light-sensitive photopigment melanopsin show that direct effects of light on behavior and EEG depend on the time of day. The data further suggest an unexpected role for melanopsin in sleep homeostasis.
Light influences sleep and alertness either indirectly through a well-characterized circadian pathway or directly through yet poorly understood mechanisms. Melanopsin (Opn4) is a retinal photopigment crucial for conveying nonvisual light information to the brain. Through extensive characterization of sleep and the electrocorticogram (ECoG) in melanopsin-deficient (Opn4−/−) mice under various light–dark (LD) schedules, we assessed the role of melanopsin in mediating the effects of light on sleep and ECoG activity. In control mice, a light pulse given during the habitual dark period readily induced sleep, whereas a dark pulse given during the habitual light period induced waking with pronounced theta (7–10 Hz) and gamma (40–70 Hz) activity, the ECoG correlates of alertness. In contrast, light failed to induce sleep in Opn4−/− mice, and the dark-pulse-induced increase in theta and gamma activity was delayed. A 24-h recording under a LD 1-h∶1-h schedule revealed that the failure to respond to light in Opn4−/− mice was restricted to the subjective dark period. Light induced c-Fos immunoreactivity in the suprachiasmatic nuclei (SCN) and in sleep-active ventrolateral preoptic (VLPO) neurons was importantly reduced in Opn4−/− mice, implicating both sleep-regulatory structures in the melanopsin-mediated effects of light. In addition to these acute light effects, Opn4−/− mice slept 1 h less during the 12-h light period of a LD 12∶12 schedule owing to a lengthening of waking bouts. Despite this reduction in sleep time, ECoG delta power, a marker of sleep need, was decreased in Opn4−/− mice for most of the (subjective) dark period. Delta power reached after a 6-h sleep deprivation was similarly reduced in Opn4−/− mice. In mice, melanopsin's contribution to the direct effects of light on sleep is limited to the dark or active period, suggesting that at this circadian phase, melanopsin compensates for circadian variations in the photo sensitivity of other light-encoding pathways such as rod and cones. Our study, furthermore, demonstrates that lack of melanopsin alters sleep homeostasis. These findings call for a reevaluation of the role of light on mammalian physiology and behavior.
Author Summary
Light affects sleep in two ways: indirectly through the phase adjustment of circadian rhythms and directly through nonvisual mechanisms that are independent of the circadian system. The direct effects of light include the promotion of sleep in night-active animals and of alertness in diurnal species. We analyzed sleep and the electroencephalogram (EEG) under various light–dark regimens in mice lacking melanopsin (Opn4−/−), a retinal photopigment crucial for conveying light-level information to the brain, to determine the role of melanopsin, as opposed to rod and cones, in mediating these direct effects of light. We show that melanopsin mediates the direct effects of light during the subjective dark period, whereas rods and cones contribute to these effects in the light period. Our finding that “sleep-active” (i.e., galanin-positive) neurons of the anterior hypothalamus are not activated by light in Opn4−/− mice suggests that these neurons are part of the circuitry whereby light promotes sleep. Also, the alerting effects of transitions into darkness were less pronounced in Opn4−/− mice judged on the reduced increase in EEG theta and gamma activity. Finally, and unexpectedly, the rate at which the need for sleep, quantified as EEG delta power, accumulated during wakefulness was found to be reduced in Opn4−/− mice both during baseline and sleep deprivation conditions, implicating a photopigment in the homeostatic regulation of sleep. We conclude that melanopsin contributes to the direct effects of light and darkness, and in interaction with circadian and homeostatic drive, determines the occurrence and quality of both sleep and waking. If confirmed in humans, our observations will have applications for the clinical use of light as well as for societal lighting conditions.
doi:10.1371/journal.pbio.1000125
PMCID: PMC2688840  PMID: 19513122
17.  Osteopontin deficiency aggravates hepatic injury induced by ischemia–reperfusion in mice 
Cell Death & Disease  2014;5(5):e1208-.
Osteopontin (OPN) is a multifunctional protein involved in hepatic steatosis, inflammation, fibrosis and cancer progression. However, its role in hepatic injury induced by ischemia–reperfusion (I–R) has not yet been investigated. We show here that hepatic warm ischemia for 45 min followed by reperfusion for 4 h induced the upregulation of the hepatic and systemic level of OPN in mice. Plasma aspartate aminotransferase and alanine aminotransferase levels were strongly increased in Opn−/− mice compared with wild-type (Wt) mice after I–R, and histological analysis of the liver revealed a significantly higher incidence of necrosis of hepatocytes. In addition, the expression levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNFα), interleukin 6 (IL6) and interferon-γ were strongly upregulated in Opn−/− mice versus Wt mice after I–R. One explanation for these responses could be the vulnerability of the OPN-deficient hepatocyte. Indeed, the downregulation of OPN in primary and AML12 hepatocytes decreased cell viability in the basal state and sensitized AML12 hepatocytes to cell death induced by oxygen–glucose deprivation and TNFα. Further, the downregulation of OPN in AML12 hepatocytes caused a strong decrease in the expression of anti-apoptotic Bcl2 and in the ATP level. The hepatic expression of Bcl2 also decreased in Opn−/− mice versus Wt mice livers after I–R. Another explanation could be the regulation of the macrophage activity by OPN. In RAW macrophages, the downregulation of OPN enhanced iNOS expression in the basal state and sensitized macrophages to inflammatory signals, as evaluated by the upregulation of iNOS, TNFα and IL6 in response to lipopolysaccharide. In conclusion, OPN partially protects from hepatic injury and inflammation induced in this experimental model of liver I–R. This could be due to its ability to partially prevent death of hepatocytes and to limit the production of toxic iNOS-derived NO by macrophages.
doi:10.1038/cddis.2014.174
PMCID: PMC4047890  PMID: 24810044
osteopontin; ischemia–reperfusion; hepatic injury; hepatocyte; macrophage
18.  Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes 
PLoS ONE  2014;9(1):e87464.
Osteopontin (OPN) is a secreted phosphoprotein which has been linked to tumor progression and metastasis in a variety of cancers including hepatocellular carcinoma (HCC). Previous studies have shown that OPN is upregulated during liver injury and inflammation. However, the role of OPN in hepatitis C virus (HCV)-induced liver disease pathogenesis is not known. In this study, we determined the induction of OPN, and then investigated the effect of secreted forms of OPN in epithelial to mesenchymal transition (EMT), migration and invasion of hepatocytes. We show the induction of OPN mRNA and protein expression by HCV-infection. Our results also demonstrate the processing of precursor OPN (75 kDa) into 55 kDa, 42 kDa and 36 kDa forms of OPN in HCV-infected cells. Furthermore, we show the binding of secreted OPN to integrin αVβ3 and CD44 at the cell surface, leading to the activation of downstream cellular kinases such as focal adhesion kinase (FAK), Src, and Akt. Importantly, our results show the reduced expression of epithelial marker (E-cadherin) and induction of mesenchymal marker (N-cadherin) in HCV-infected cells. We also show the migration and invasion of HCV-infected cells using wound healing assay and matrigel coated Boyden chamber. In addition, we demonstrate the activation of above EMT markers, and the critical players involved in OPN-mediated cell signaling cascade using primary human hepatocytes infected with Japanese fulminant hepatitis (JFH)-1 HCV. Taken together, these studies suggest a potential role of OPN in inducing chronic liver disease and HCC associated with chronic HCV infection.
doi:10.1371/journal.pone.0087464
PMCID: PMC3909125  PMID: 24498111
19.  Osteopontin/Eta-1 upregulated in Crohn’s disease regulates the Th1 immune response 
Gut  2005;54(9):1254-1262.
Background and aims: The pathogenesis of Crohn’s disease (CD), a chronic inflammatory bowel disease characterised by a Th1 immune response, remains unclear. Osteopontin (OPN) is a phosphoprotein known as an adhesive bone matrix protein. Recent studies have shown that OPN plays an important role in lymphocyte migration, granuloma formation, and interleukin 12 (IL-12) production. The present study investigated expression and the pathophysiological role of OPN in CD.
Methods: Plasma OPN concentration was measured by enzyme linked immunosorbent assay. Expression of OPN in human intestinal mucosa was determined using reverse transcription-polymerase chain reaction and western blot, and localisation of OPN was examined by immunohistochemistry. Expression of integrin β3, an OPN receptor, on lamina propria mononuclear cells (LPMC) was assessed by flow cytometry. Functional activation of OPN in LPMC was investigated by measuring the production of cytokines.
Results: Plasma OPN concentration was significantly higher in patients with CD compared with normal controls or patients with ulcerative colitis (UC). OPN was upregulated in intestinal mucosa from UC and CD patients. OPN producing cells were epithelial or IgG producing plasma cells, or partial macrophages. OPN was detected in areas surrounding granuloma from mucosa in CD. Integrin β3 expressing macrophages infiltrated inflamed mucosa in UC and CD; in contrast, there was no expression of integrin β3 on intestinal macrophages in normal mucosa. OPN induced production of IL-12 from LPMC in CD but not in normal controls or UC.
Conclusions: Increased OPN expression facilitates cytokine production and is closely involved in the Th1 immune response associated with CD.
doi:10.1136/gut.2004.048298
PMCID: PMC1774642  PMID: 16099792
osteopontin; Crohn’s disease; ulcerative colitis; Th1 immune response
20.  Role of Osteopontin in Murine Lyme Arthritis and Host Defense against Borrelia burgdorferi 
Infection and Immunity  2002;70(3):1372-1381.
Several genetic loci in the mouse have been identified that regulate the severity of Lyme arthritis. The region of chromosome 5 including the osteopontin (OPN) gene (Opn) has been identified in intercross populations of C3H/HeN × C57BL/6 and C3H/HeJ × BALB/cAnN mice. OPN is of particular interest as it is involved in the maintenance and remodeling of tissue during inflammation, it regulates production of interleukin-10 (IL-10) and IL-12 (cytokines implicated in Lyme arthritis), it is necessary for host control of certain bacterial infections, and mice displaying different severities of Lyme arthritis possess different alleles of the OPN gene. Macrophages and splenocytes from OPN-deficient mice on mixed C57BL/6J-129S or inbred 129S backgrounds were stimulated with the Pam3Cys modified lipoprotein from Borrelia burgdorferi, OspA. OPN was not required for OspA-induced cytokine production; however, macrophages from 129S-Opn−/− mice displayed a reduced level of IL-10 production. OPN was also not required for resistance to severe arthritis, as B. burgdorferi-infected 129S-Opn−/− mice developed mild arthritis, as did their wild-type littermates. Arthritis was more severe in OPN-deficient mice on the mixed C57BL/6J-129S backgrounds than in inbred mice of either strain. This increase was most likely due to a gene(s) closely linked to Opn on chromosome 5 in conjunction with other randomly assorting genes. Deficiency in OPN did not influence the numbers of spirochetes in tissues from B. burgdorferi-infected mice, indicating OPN is not part of the host defense to this pathogen. Interestingly, there was no alteration in the B. burgdorferi-specific antibody isotypes in OPN-deficient mice, indicating that its effect on helper T-cell responses is not relevant to the host response to B. burgdorferi.
doi:10.1128/IAI.70.3.1372-1381.2002
PMCID: PMC127811  PMID: 11854223
21.  Osteopontin enhances HIV replication and is increased in the brain and cerebrospinal fluid of HIV-infected individuals 
Journal of Neurovirology  2011;17(4):382-392.
Despite effective and widely available suppressive anti-HIV therapy, the prevalence of mild neurocognitive dysfunction continues to increase. HIV-associated neurocognitive disorder (HAND) is a multifactorial disease with sustained central nervous system inflammation and immune activation as prominent features. Inflammatory macrophages, HIV-infected and uninfected, play a central role in the development of HIV dementia. There is a critical need to identify biomarkers and to better understand the molecular mechanisms leading to cognitive dysfunction in HAND. In this regard, we identified through a subtractive hybridization strategy osteopontin (OPN, SPP1, gene) an inflammatory marker, as an upregulated gene in HIV-infected primary human monocyte-derived macrophages. Knockdown of OPN in primary macrophages resulted in a threefold decrease in HIV-1 replication. Ectopic expression of OPN in the TZM-bl cell line significantly enhanced HIV infectivity and replication. A significant increase in the degradation of the NF-κB inhibitor, IκBα and an increase in the nuclear-to-cytoplasmic ratio of NF-κB were found in HIV-infected cells expressing OPN compared to controls. Moreover, mutation of the NF-κB binding domain in the HIV-LTR abrogated enhanced promoter activity stimulated by OPN. Interestingly, compared to cerebrospinal fluid from normal and multiple sclerosis controls, OPN levels were significantly higher in HIV-infected individuals both with and without neurocognitive disorder. OPN levels were highest in HIV-infected individuals with moderate to severe cognitive impairment. Moreover, OPN was significantly elevated in brain tissue from HIV-infected individuals with cognitive disorder versus those without impairment. Collectively, these data suggest that OPN stimulates HIV-1 replication and that high levels of OPN are present in the CNS compartment of HIV-infected individuals, reflecting ongoing inflammatory processes at this site despite anti-HIV therapy.
doi:10.1007/s13365-011-0035-4
PMCID: PMC3331788  PMID: 21556958
HIV-associated neurocognitive disorder; CD44; Nef
22.  RAN GTPase and Osteopontin in Pancreatic Cancer 
Introduction
Pancreatic ductal adenocarcinoma (PDA) has the worst prognosis among cancers, mainly due to the high incidence of early metastases. RAN small GTPase (RAN) is a protein that plays physiological roles in the regulation of nuclear transport and microtubule spindle assembly. RAN was recently shown to mediate the invasive functions of the prometastatic protein osteopontin (OPN) in breast cancer cells. We and others have shown previously that high levels of OPN are present in PDA. In this study, we analyzed the expression and correlation of RAN with OPN in human pancreatic lesions, and explored their regulation in PDA cell lines.
Methods
Real time PCR was used to analyze RAN and OPN mRNA levels in PDA, adjacent non-malignant, and benign pancreatic tissues. Expression levels were correlated with survival and different clinicopathological parameters using different statistical methods. Transient transfection studies using OPN and RAN plasmids, and knockdown experiments using siRNA were used to examine their mutual regulation.
Results
OPN and RAN levels highly correlated with each other (p<0.0001). OPN or RAN levels did not correlate with venous lymphatic invasion, diabetes, obesity, T stage, BMI, or survival. However, we found a significant association between RAN levels and perineural invasion (HR=0.79, 95% CI 0.59, 1.07; p=0.0378.). OPN and RAN colocalized in PDA tissues and cell lines. Increasing RAN expression in PDA cells induced OPN transcription and RAN silencing reduced total OPN levels. OPN did not have any significant effect on RAN transcription.
Conclusions
The high levels of RAN in PDA and its correlation with OPN and with perineural invasion suggest that RAN may contribute to PDA metastasis and progression through the induction of OPN. RAN’s role in the regulation of OPN in PDA is unique and could provide potential novel therapeutic strategies to combat PDA aggressiveness.
PMCID: PMC3989933  PMID: 24749004
Pancreatic cancer; RAN; OPN
23.  The Role of Osteopontin (OPN/SPP1) Haplotypes in the Susceptibility to Crohn's Disease 
PLoS ONE  2011;6(12):e29309.
Background
Osteopontin represents a multifunctional molecule playing a pivotal role in chronic inflammatory and autoimmune diseases. Its expression is increased in inflammatory bowel disease (IBD). The aim of our study was to analyze the association of osteopontin (OPN/SPP1) gene variants in a large cohort of IBD patients.
Methodology/Principal Findings
Genomic DNA from 2819 Caucasian individuals (n = 841 patients with Crohn's disease (CD), n = 473 patients with ulcerative colitis (UC), and n = 1505 healthy unrelated controls) was analyzed for nine OPN SNPs (rs2728127, rs2853744, rs11730582, rs11739060, rs28357094, rs4754 = p.Asp80Asp, rs1126616 = p.Ala236Ala, rs1126772 and rs9138). Considering the important role of osteopontin in Th17-mediated diseases, we performed analysis for epistasis with IBD-associated IL23R variants and analyzed serum levels of the Th17 cytokine IL-22. For four OPN SNPs (rs4754, rs1126616, rs1126772 and rs9138), we observed significantly different distributions between male and female CD patients. rs4754 was protective in male CD patients (p = 0.0004, OR = 0.69). None of the other investigated OPN SNPs was associated with CD or UC susceptibility. However, several OPN haplotypes showed significant associations with CD susceptibility. The strongest association was found for a haplotype consisting of the 8 OPN SNPs rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138 (omnibus p-value = 2.07×10−8). Overall, the mean IL-22 secretion in the combined group of OPN minor allele carriers with CD was significantly lower than that of CD patients with OPN wildtype alleles (p = 3.66×10−5). There was evidence for weak epistasis between the OPN SNP rs28357094 with the IL23R SNP rs10489629 (p = 4.18×10−2) and between OPN SNP rs1126616 and IL23R SNP rs2201841 (p = 4.18×10−2) but none of these associations remained significant after Bonferroni correction.
Conclusions/Significance
Our study identified OPN haplotypes as modifiers of CD susceptibility, while the combined effects of certain OPN variants may modulate IL-22 secretion.
doi:10.1371/journal.pone.0029309
PMCID: PMC3248444  PMID: 22242114
24.  Increased osteopontin expression in dendritic cells amplifies IL-17 production by CD4+ T cells in experimental autoimmune encephalomyelitis and in multiple sclerosis 
Osteopontin (Opn) is a broadly expressed pleiotropic cytokine and has been shown to play an important role in various autoimmune diseases including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). It is reported that Opn exacerbates EAE by skewing T cell differentiation towards IFN-γ producing Th1 cells. Opn expression in dendritic cells (DCs) and its role in IL-17 induction from T cells during EAE or MS is unknown. We found that during EAE Opn expression is elevated in DCs both in the periphery and in the central nervous system. There was increased expression of Opn receptor on T cells and Opn induced IL-17 production by CD4+ T cells via the β3 integrin receptor and Opn inhibited IL-10 production via the CD44 receptor. Furthermore, anti-Opn treatment reduced clinical severity of EAE by reducing IL-17 production. Anti-Opn was also effective in reducing clinical severity of EAE when given after the appearance of clinical symptoms. Analogous to EAE, in subjects with MS we found increased expression of Opn in DCs and increased expression of the Opn receptors CD44, β3 and αv on T cells. Furthermore, Opn stimulated CD4+ T cells from MS patients produced significantly higher amounts of IL-17. Our results demonstrate a role for DC produced Opn both in EAE and MS that is linked to the production of IL-17.
PMCID: PMC2653058  PMID: 19017937
Dendritic cells; T cells; EAE/MS and Osteopontin
25.  Osteopontin in Systemic Sclerosis and its Role in Dermal Fibrosis 
Osteopontin (OPN) is a matricellular protein with proinflammatory and profibrotic properties. Previous reports demonstrate a role for OPN in wound healing and pulmonary fibrosis. Herein, we determined if OPN levels are increased in a large cohort of systemic sclerosis (SSc) patients and if OPN contributes dermal fibrosis. Plasma OPN levels were increased in SSc patients, including patients with limited and diffuse disease, compared to healthy controls. Immunohistology demonstrated OPN on fibroblast-like and inflammatory cells in SSc skin and lesional skin from mice in the bleomycin-induced dermal fibrosis model. OPN deficient (OPN−/−) mice developed less dermal fibrosis compared to wild-type mice in the bleomycin-induced dermal fibrosis model. Additional in vivo studies demonstrated that lesional skin from OPN−/− mice had fewer Mac-3+ cells, fewer myofibroblasts, decreased TGF-beta (TGFβ) and genes in the TGFβ pathway and decreased numbers of cells expressing phosphorylated SMAD2 (pSMAD) and ERK. In vitro, OPN−/− dermal fibroblasts had decreased migratory capacity but similar phosphorylation of SMAD2 by TGFβ. Finally, TGFβ production by OPN deficient macrophages was reduced compared to wild type. These data demonstrate an important role for OPN in the development of dermal fibrosis and suggest that OPN may be a novel therapeutic target in SSc.
doi:10.1038/jid.2012.32
PMCID: PMC3365548  PMID: 22402440

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