Osteopontin Knockout Mice Research Articles

Osteopontin enhances multi-walled carbon nanotube-triggered lung fibrosis by promoting TGF-\u03b21 activation and myofibroblast differentiation

BackgroundCarbon nanotubes (CNTs) have been used in a variety of applications because of their unique properties and functions. However, many CNTs have been shown to induce lung fibrosis in experimental animals with some at a potency greater than that of silica, raising concern over possible toxic effects of CNT exposure in humans. Research into the mechanisms by which CNTs induce pulmonary fibrosis is warranted in order to facilitate the understanding, monitoring, and treatment of CNT-induced lung lesions that might occur in exposed populations. The current study focuses on investigating the role of osteopontin (OPN) in the development of lung fibrosis upon exposure to multi-walled carbon nanotubes (MWCNTs).MethodsC57BL/6J (WT) and Opn knockout (KO) mice were exposed to MWCNTs by pharyngeal aspiration to examine the acute and chronic effects of MWCNT exposure. The role of OPN and its mode of action in lung fibrosis development were analyzed at the cellular and molecular levels in vivo and in vitro.ResultsOPN was highly and persistently induced in both the acute and chronic phases of the response to MWCNT exposure in mouse lungs. Comparison between WT and Opn KO mice revealed that OPN critically regulated MWCNT-induced lung fibrosis as indicated by reduced fibrotic focus formation and myofibroblast accumulation in Opn KO lungs. At the molecular level, OPN promotes the expression and activation of TGF-β1, stimulates the differentiation of myofibroblasts from fibroblasts, and increases the production of fibrous matrix proteins in lungs and cultured lung cells exposed to MWCNTs.ConclusionOPN is highly induced in CNT-exposed lungs and plays critical roles in TGF-β1 signaling activation and myofibroblast differentiation to promote fibrosis development from MWCNT exposure. This study reveals an OPN-dependent mechanism to promote MWCNT-induced lung fibrosis. The findings raise the possibility of using OPN as a biomarker to monitor CNT exposure and as a drug target to halt fibrosis development.

Osteopontin exacerbates Pseudomonas aeruginosa-induced bacteremia in mice

Osteopontin (OPN) is a multifunctional protein involved in various pathophysiological processes. However, the role of OPN in Pseudomonas aeruginosa-related sepsis is not yet clear. Here, we found that OPN expression was elevated in plasma and spleen samples from P. aeruginosa-infected mice. To determine the function of OPN in sepsis, we used wild-type (WT) and OPN-knockout (KO) mice with P. aeruginosa-induced bacteremia. We found that OPN-KO mice exhibited reduced mortality compared with WT mice and that OPN exacerbated spleen bleeding and functional impairment. OPN-KO mice exhibited reduced secretion of pro-inflammatory cytokines, such as interferon-γ, interleukin (IL)-1β, IL-12, and tumor necrosis factor-α, whereas levels of anti-inflammatory cytokine IL-10 and the leukocyte trafficking mediator macrophage inflammatory protein (MIP)-2 were not altered. Additionally, the percentages and absolute numbers of B cells were elevated in the spleens of OPN-KO mice. Thus, OPN promoted sepsis in P. aeruginosa-infected mice and potentially blocked B cell-dependent immunity.

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.

Cyclic Strain and Hypertension Increase Osteopontin Expression in the Aorta.

Hypertension has a direct impact on vascular hypertrophy and is a known risk factor for the development of atherosclerosis. Osteopontin (OPN) has emerged as an important protein mediator of inflammation and remodeling of large arteries. However, its role and mechanism of regulation in the setting of hypertension is still unknown. Our objectives for this study were therefore to investigate the role of OPN in hypertension-induced vascular remodeling and inflammation. OPN Knockout (KO) and wild type (WT) mice were made hypertensive with angiotensin II (Ang II) infusion for seven days. We observed that OPN KO aortas were protected against Ang II-induced medial hypertrophy and inflammation, despite comparable increases in systolic blood pressure (SBP) in both groups. OPN expression was increased in WT aortas from hypertensive mice (induced by either Ang II or norepinephrine). OPN expression was increased in aortic smooth muscle cells (SMCs) subjected to cyclic mechanical strain suggesting that mechanical deformation of the aortic wall is responsible in part for the increased OPN expression induced by hypertension. Finally, we utilized hypertensive transgenic smooth muscle cell-specific catalase overexpressing (TgSMC-Cat) mice to determine the role of H2O2 in mediating hypertension-induced increases in OPN expression. We also found that the hypertension-induced increase in OPN expression was inhibited in transgenic smooth muscle cell-specific catalase overexpressing (TgSMC-Cat) mice, suggesting that H2O2, plays a vital role in mediating the hypertension-induced increase in OPN expression. Taken together, these results define a potentially important role for OPN in the pathophysiology of hypertension.

Extended time-lapse in vivo imaging of tibia bone marrow to visualize dynamic hematopoietic stem cell engraftment.

Homing, engraftment and proliferation of hematopoietic stem/progenitor cell (HSC/HPCs) are crucial steps required for success of a bone marrow transplant. Observation of these critical events is limited by the opaque nature of bone. Here we demonstrate how individual HSCs engraft in long bones by thinning one side of the tibia for direct and unbiased observation. Intravital imaging enabled detailed visualization of single Sca-1+, c-Kit+, Lineage− (SKL) cell migration to bone marrow niches and subsequent proliferation to reconstitute hematopoiesis. This longitudinal study allowed direct observation of dynamic HSC/HPC activities during engraftment in full color for up to six days in live recipients. Individual SKL cells, but not mature or committed progenitor cells, preferentially homed to a limited number of niches near highly vascularized endosteal regions, and clonally expanded. Engraftment of SKL cells in P-selectin and osteopontin knockout mice showed abnormal homing and expansion of SKL cells. CD150+, CD48− SKL populations initially engrafted in the central marrow region, utilizing only a subset of niches occupied by the parent SKL cells. Our study demonstrates that time-lapse imaging of tibia can be a valuable tool to understand the dynamic characteristics of functional HSC and niche components in various mouse models.

Suppression of osteopontin inhibits chemically induced hepatic carcinogenesis by induction of apoptosis in mice.

Previous clinical reports have found elevated osteopontin (OPN) levels in tumor tissues to be indicative of greater malignancy in human hepatocellular carcinoma (HCC). However, the role of OPN on carcinogenesis and its underlying mechanism remain unclear. In the present study, we investigated the oncogenic role of OPN in diethylnitrosamine (DEN)-induced hepatic carcinogenesis in mice. The overall incidence of hepatic tumors at 36 weeks was significantly lower in OPN knockout (KO) mice than in wild-type (WT) mice. Apoptosis was significantly enhanced in OPN KO mice, and was accompanied by the downregulation of epidermal growth factor receptor (EGFR). In the in vitro study, OPN suppression also led to lower mRNA and protein levels of EGFR associated with the downregulation of c-Jun in Hep3B and Huh7 human HCC cells lines, which resulted in increased apoptotic cell death in both cell lines. Moreover, a positive correlation was clearly identified between the expression of OPN and EGFR in human HCC tissues. These data demonstrate that the OPN deficiency reduced the incidence of chemically induced HCC by suppressing EGFR-mediated anti-apoptotic signaling. An important implication of our findings is that OPN positively contributes to hepatic carcinogenesis.

Osteopontin is a key player for local adipose tissue macrophage proliferation in obesity

ObjectiveRecent findings point towards an important role of local macrophage proliferation also in obesity-induced adipose tissue inflammation that underlies insulin resistance and type 2 diabetes. Osteopontin (OPN) is an inflammatory cytokine highly upregulated in adipose tissue (AT) of obese and has repeatedly been shown to be functionally involved in adipose-tissue inflammation and metabolic sequelae. In the present work, we aimed at unveiling both the role of OPN in human monocyte and macrophage proliferation as well as the impact of OPN deficiency on local macrophage proliferation in a mouse model for diet-induced obesity. MethodsThe impact of recombinant OPN on viability, apoptosis, and proliferation was analyzed in human peripheral blood monocytes and derived macrophages. Wild type (WT) and OPN knockout mice (SPP1KO) were compared with respect to in vivo adipose tissue macrophage and in vitro bone marrow-derived macrophage (BMDM) proliferation. ResultsOPN not only enhanced survival and decreased apoptosis of human monocytes but also induced proliferation similar to macrophage colony stimulating factor (M-CSF). Even in fully differentiated monocyte-derived macrophages, OPN induced a proliferative response. Moreover, proliferation of adipose tissue macrophages in obese mice was detectable in WT but virtually absent in SPP1KO. In BMDM, OPN also induced proliferation while OPN as well as M-CSF-induced proliferation was similar in WT and SPP1KO. ConclusionsThese data confirm that monocytes and macrophages not only are responsive to OPN and migrate to sites of inflammation but also they survive and proliferate more in the presence of OPN, a mechanism also strongly confirmed in vivo. Therefore, secreted OPN appears to be an essential player in AT inflammation, not only by driving monocyte chemotaxis and macrophage differentiation but also by facilitating local proliferation of macrophages.

Osteopontin Is Essential for Type I Collagen Secretion in Reparative Dentin

Osteopontin (OPN) is a highly phosphorylated glycoprotein that is a prominent component of the mineralized extracellular matrix of bone. The secretion of OPN by immunocompetent cells plays a role in the differentiation of odontoblast-like cells during pulpal healing following tooth transplantation. This study aimed to clarify the role of OPN during reparative dentinogenesis. A groove-shaped cavity was prepared on the mesial surface of the upper first molars of wild-type (WT) and Opn knockout (KO) mice, and the samples were collected at intervals of 1 to 14 d. The demineralized sections were processed for immunohistochemistry for Ki67, nestin, OPN, dentin sialoprotein (DSP), integrin αvβ3, and type I collagen; in situ hybridization for Opn, col1a1, and dentin sialophosphoprotein (Dspp); and apoptosis assay. For the loss and gain of function experiments, an in vitro culture assay for evaluating dentin-pulp complex regeneration was performed. On day 1 in WT mice, odontoblasts beneath the affected dentin lost nestin immunoreactivity. On day 3, the expression of Opn was recognized at the mesial dental pulp, and OPN was deposited along the predentin-dentin border. Nestin-positive newly differentiated odontoblast-like cells expressed both Dspp and col1a1 and showed positive immunoreactivity for integrin αvβ3, DSP, and type I collagen. Until day 14, reparative dentin formation continued next to the preexisting dentin at the mesial coronal pulp. In contrast, there was no reparative dentin in the Opn KO mice where nestin- and DSP-positive newly differentiated odontoblast-like cells lacked immunoreaction for type I collagen. The in vitro organ culture demonstrated that the administration of recombinant OPN rescued the type I collagen secretion by odontoblast-like cells in the Opn KO mice. The results suggested that the deposition of OPN at the calcification front is essential for the type I collagen secretion by newly differentiated odontoblast-like cells to form reparative dentin during pulpal healing following cavity preparation.

High fat diet increases melanoma cell growth in the bone marrow by inducing osteopontin and interleukin 6.

The impact of metabolic stress induced by obesity on the bone marrow melanoma niche is largely unknown. Here we employed diet induced obese mice model, where mice received high-fat (HFD) or normal diet (ND) for 6 weeks before challenge with B16F10 melanoma cells. Tumor size, bone loss and osteoclasts numbers were assessed histologically in the tibial bones. For defining the molecular pathway, osteopontin knock-out mice, interleukin 6 neutralizing antibody or Janus kinase 2 inhibition were carried out in the same model. Mechanistic studies such as adipocyte-melanoma co-cultures for defining adipocyte induced changes of tumor cell proliferation and expression profiles were also performed. As results, HFD enhanced melanoma burden in bone by increasing tumor area and osteoclast numbers. This process was associated with higher numbers of bone marrow adipocytes expressing IL-6 in direct vicinity to tumor cells. Inhibition of IL-6 or of downstream JAK2 blocked HFD-induced tumor progression. Furthermore, the phenotypic changes of melanoma cells triggered macrophage and osteoclast accumulation accompanied by increased osteopontin expression. Osteopontin triggered osteoclastogenesis and also exerted a positive feedback loop to tumor cells, which was abrogated in its absence. Metabolic stress by HFD promotes melanoma growth in the bone marrow by an increase in bone marrow adipocytes and IL-6-JAK2-osteopontin mediated activation of tumor cells and osteoclast differentiation.

Osteopontin protects against high phosphate-induced nephrocalcinosis and vascular calcification

Pathologic calcification is a significant cause of increased morbidity and mortality in patients with chronic kidney disease. The precise mechanisms of ectopic calcification are not fully elucidated, but it is known to be caused by an imbalance of procalcific and anticalcific factors. In the chronic kidney disease population, an elevated phosphate burden is both highly prevalent and a known risk factor for ectopic calcification. Here we tested whether osteopontin, an inhibitor of calcification, protects against high phosphate load-induced nephrocalcinosis and vascular calcification. Osteopontin knockout mice were placed on a high phosphate diet for 11 weeks. Osteopontin deficiency together with phosphate overload caused uremia, nephrocalcinosis characterized by substantial renal tubular and interstitial calcium deposition, and marked vascular calcification when compared with control mice. Although the osteopontin-deficient mice did not exhibit hypercalcemia or hyperphosphatemia, they did show abnormalities in the mineral metabolism hormone fibroblast growth factor-23. Thus, endogenous osteopontin plays a critical role in the prevention of phosphate-induced nephrocalcinosis and vascular calcification in response to high phosphate load. A better understanding of osteopontin's role in phosphate-induced calcification will hopefully lead to better biomarkers and therapies for this disease, especially in patients with chronic kidney disease and other at-risk populations.

A strategy to quantitate global phosphorylation of bone matrix proteins

Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured “in bulk” are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.

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.

Ablation of osteopontin suppresses N-methyl-N-nitrosourea and Helicobacter pylori-induced gastric cancer development in mice.

Several clinical studies have reported increased expression of osteopontin (OPN) in various types of human cancer, including gastric cancer. However, the precise mechanisms underlying tumor development remain unclear. In the present study, we investigated the pathogenic roles of OPN in Helicobacter pylori-induced gastric cancer development. Wild-type (WT) and OPN knockout (KO) mice were treated with N-methyl-N-nitrosourea (MNU) and infected with H.pylori. Mice were killed 50 weeks after treatment, and stomach tissues were assessed by histopathological examination, immunohistochemistry, quantitative real-time RT-PCR and western blotting. To clarify the carcinogenic effects of OPN, we also conducted an in vitro study using AGS human gastric cancer cell line and THP-1 human monocytic cell line. The overall incidence of gastric tumors was significantly decreased in OPN KO mice compared with WT mice. Apoptotic cell death was significantly enhanced in OPN KO mice and was accompanied by upregulation of signal transducer and activator of transcription 1 (STAT1) and inducible nitric oxide synthase (iNOS). In vitro study, OPN suppression also caused STAT1 upregulation and iNOS overexpression in AGS and THP-1 cells, which resulted in apoptosis of AGS cells. In addition, a negative correlation was clearly identified between expression of OPN and iNOS in human gastric cancer tissues. Our data demonstrate that loss of OPN decreases H.pylori-induced gastric carcinogenesis by suppressing proinflammatory immune response and augmenting STAT1 and iNOS-mediated apoptosis of gastric epithelial cells. An important implication of these findings is that OPN actually contributes to the development of gastric cancer.

Osteopontin depletion decreases inflammation and gastric epithelial proliferation during Helicobacter pylori infection in mice

Osteopontin (OPN) is a multifunctional protein that plays a role in many physiological and pathological processes, including inflammation and tumorigenesis. Here, we investigated the involvement of OPN in Helicobacter pylori (HP)-induced gastritis using OPN knockout (KO) mice and OPN knockdown (KD) cell lines. HP-infected OPN KO mice showed significantly reduced gastritis compared with wild-type (WT) mice with decreased infiltration of macrophages and a reduction in HP-induced upregulation of IL-1β, TNF-α, and IFN-γ. HP-exposed OPN KD gastric cancer cells and macrophage-like cells showed an attenuated induction of these cytokines. We also demonstrated a reduction in the migration of monocytic and macrophage-like cells toward conditioned media harvested from HP-exposed OPN KD gastric cancer cells as well as reduced migration ability of OPN KD cells itself. In addition, HP-infected OPN KO mice showed decreased epithelial cell proliferation compared with HP-infected WT mice, in association with a reduction in MAPK pathway activation. OPN KD gastric cancer cell lines also showed lower proliferative activity and reduced MAPK activation than shRNA control cells after HP co-culture or after IL-1β and TNF-α treatment. Taken together, these results indicate that OPN exerts a considerable influence on HP-induced gastritis by modulating the production of cytokines and contributing to macrophage infiltration. Moreover, OPN-mediated activation of the MAPK pathway in gastric epithelial cells might contribute to epithelial changes following HP infection.

Osteopontin is indispensible for AP1-mediated angiotensin II-related miR-21 transcription during cardiac fibrosis.

AimsOsteopontin (OPN) is a multifunctional cytokine critically involved in cardiac fibrosis. However, the underlying mechanisms are unresolved. Non-coding RNAs are powerful regulators of gene expression and thus might mediate this process.Methods and resultsOPN and miR-21 were significantly increased in cardiac biopsies of patients with myocardial fibrosis. Ang II infusion via osmotic minipumps led to specific miRNA regulations with miR-21 being strongly induced in wild-type (WT) but not OPN knockout (KO) mice. This was associated with enhanced cardiac collagen content, myofibroblast activation, ERK-MAP kinase as well as AKT signalling pathway activation and a reduced expression of Phosphatase and Tensin Homologue (PTEN) as well as SMAD7 in WT but not OPN KO mice. In contrast, cardiotropic AAV9-mediated overexpression of OPN in vivo further enhanced cardiac fibrosis. In vitro, Ang II induced expression of miR-21 in WT cardiac fibroblasts, while miR-21 levels were unchanged in OPN KO fibroblasts. As pri-miR-21 was also increased by Ang II, we studied potential involved upstream regulators; Electrophoretic Mobility Shift and Chromatin Immunoprecipitation analyses confirmed activation of the miR-21 upstream-transcription factor AP-1 by Ang II. Recombinant OPN directly activated miR-21, enhanced fibrosis, and activated the phosphoinositide 3-kinase pathway. Locked nucleic acid-mediated miR-21 silencing ameliorated cardiac fibrosis development in vivo.ConclusionIn cardiac fibrosis related to Ang II, miR-21 is transcriptionally activated and targets PTEN/SMAD7 resulting in increased fibroblast survival. OPN KO animals are protected from miR-21 increase and fibrosis development due to impaired AP-1 activation and fibroblast activation.

Milk osteopontin may promote brain development via up‐regulating osteopontin expression in vivo

Osteopontin (OPN) is a highly acidic phosphorylated glycoprotein that is involved in a wide range of biological processes, including biomineralization, immune modulatory functions, cell survival and proliferation. OPN is abundantly expressed in the brain and has been suggested to play a role in brain development. OPN is present in high concentration in human milk. In mice, OPN is also expressed in the mammary gland and secreted in milk. However, the functions of milk OPN remain unclear. In this study, wild‐type and OPN knock‐out mice were used to investigate the influence of milk OPN on brain development. To examine if dietary OPN can be absorbed and transferred to the brain, day 20 wild‐type mouse pups were fed I‐125 labeled OPN via oral gavage. Brain radioactivity was measured by gamma counter. After 30 min, 1h and 2h of incubation, radioactivity in the brain was detected at a stable level, around 1.7% of total OPN fed. Two groups, a control and an OPN‐deficient group, were then used to investigate the effects of dietary OPN on brain development. Wild‐type mouse pups were either fed by wild‐type dams (control) or by OPN knock‐out dams (OPN‐deficient group). Brain samples were collected every other day from D1 to D20. OPN mRNA expression was similar from D1 to D20 in both groups. Immunoblotting showed that peak OPN expression occurred at D4, D6 and D8 in both groups, but the control group expressed significantly higher concentrations of OPN compared with the OPN‐deficient group, suggesting post‐transcriptional regulation In addition, genes involved in brain development, including glial cell differentiation regulator and proteolipid protein were up‐regulated in the control group. In conclusion, milk OPN may play a role in brain development during early life. Supported by Mead Johnson Nutrition.

Na(+)/H (+) exchanger isoform 1 induced osteopontin expression in cardiomyocytes involves NFAT3/Gata4.

Osteopontin (OPN), a multifunctional glycophosphoprotein, has been reported to contribute to the development and progression of cardiac remodeling and hypertrophy. Cardiac-specific OPN knockout mice were protected against hypertrophy and fibrosis mediated by Ang II. Recently, transgenic mice expressing the active form of the Na(+)/H(+) exchanger isoform 1 (NHE1) developed spontaneous hypertrophy in association with elevated levels of OPN. The mechanism by which active NHE1 induces OPN expression and contributes to the hypertrophic response remains unclear. To validate whether expression of the active form of NHE1 induces OPN, cardiomyocytes were stimulated with Ang II, a known inducer of both OPN and NHE1. Ang II induced hypertrophy and increased OPN protein expression (151.6 ± 28.19 %, P < 0.01) and NHE1 activity in H9c2 cardiomyoblasts. Ang II-induced hypertrophy and OPN protein expression were regressed in the presence of an NHE1 inhibitor, EMD 87580, or a calcineurin inhibitor, FK506. In addition, our results indicated that activation of NHE1-induced NFAT3 translocation into the nucleus and a significant activation of the transcription factor Gata4 (NHE1: 149 ± 28 % of control, P < 0.05). NHE1-induced activation of Gata4 was inhibited by FK506. In summary, our results suggest that activation of NHE1 induces hypertrophy through the activation of NFAT3/Gata4 and OPN expression.

Defective Initiation of Liver Regeneration in Osteopontin-Deficient Mice after Partial Hepatectomy due to Insufficient Activation of IL-6/Stat3 Pathway.

The initial process in liver regeneration after partial hepatectomy involves the recruitment of immune cells and the release of cytokines. Osteopontin (OPN), a pro-inflammatory protein, plays critical roles in immune cell activation and migration. Although OPN has been implicated in the pathogenesis of many liver diseases, the role of OPN in liver regeneration remains obscure. In the present study, we found that serum and hepatic OPN protein levels were significantly elevated in wild-type (WT) mice after partial hepatectomy (PHx) and that bile ductal epithelia were the major cell source of hepatic OPN. Compared to WT mice, OPN knockout (KO) mice exhibited delayed liver regeneration after PHx. This delay in OPN-/- mice was attributed to impaired hepatic infiltration of macrophages and neutrophils, decreased serum and hepatic IL-6 levels, and blunted activation of macrophages after PHx. Furthermore, we demonstrate that the attenuated activation of macrophages is at least partially due to decreased hepatic and portal vein LPS levels in OPN-/- mice. In response to decreased IL-6 levels, the activation of signal transducer and transcription (Stat) 3 was reduced in hepatocytes of OPN-/- mice compared to WT mice after PHx. Consequently, hepatic activation of the downstream direct targets of IL6/Stat3, such as c-fos, c-jun, and c-myc, was also suppressed post-PHx in OPN-/- mice compared to WT mice. Collectively, these results support a unique role for OPN during the priming phase of liver regeneration, in which OPN enhances the recruitment of macrophages and neutrophils, and triggers hepatocyte proliferation through Kupffer cell-derived IL-6 release and the downstream activation of Stat3.

Effect of osteopontin in regulating bone marrow mesenchymal stem cell treatment of skin wounds in diabetic mice.

We aimed to investigate the role of osteopontin in regulating mesenchymal stem cells transplanted to promote wound healing in diabetic mice. The mesenchymal stem cells of osteopontin knock-out (KO) and wild-type (WT) mice were isolated separately for in vitro culture and characterization. A skin wound on the back of mice was established by skin punching. In 27 osteopontin KO male mice, induced diabetes mellitus was via intraperitoneal injection of streptozotocin. 9 normal mice were used as controls. The mice were divided into four groups and injected with Dulbecco's modified Eagle's medium (DMEM) or mesenchymal stem cells via the tail vein: A (diabetic mice injected with DMEM), B (diabetic mice injected with osteopontin KO mesenchymal stem cells), C (diabetic mice injected with WT mesenchymal stem cells), D (normal mice injected with DMEM). The healing times and closure rates of skin wounds were recorded. The microvessel density of healing wounds was measured, and the localized expression of osteopontin was identified by western blotting and immunohistochemistry. The migration of mesenchymal stem cells was observed on normal mice with skin wound injected with mesenchymal stem cells of C57BL6~GFP transgenic mice, which show green fluorescent under UV light. Compared with normal mice, the healing time of wounds in the mice with diabetes and osteopontin KO was significantly prolonged (p < 0.01). After transplanting osteopontin KO mesenchymal stem cells, the healing time was slightly shorter. Meanwhile, the healing time was significantly shorter after transplanted with WT mesenchymal stem cells and more significant neovascularization at healing wounds (p < 0.05). The expression of osteopontin in local healing wounds after transplantation of WT mesenchymal stem cells was demonstrated with western blotting and immunohistochemistry. After 4 days, the green fluoresces were noted on the wounds of mice injected with mesenchymal stem cells of fluorescent mice. Mesenchymal stem cells can migrate to wound sites, and osteopontin plays a regulatory role in mesenchymal stem cells promoting the healing of diabetic skin wounds.

Tumor-α9β1 integrin-mediated signaling induces breast cancer growth and lymphatic metastasis via the recruitment of cancer-associated fibroblasts.

Tumor-derived matricellular proteins such as osteopontin (OPN) and tenascin-C (TN-C) have been implicated in tumor growth and metastasis. However, the molecular basis of how these proteins contribute to tumor progression remains to be elucidated. Importantly, these matricellular proteins are known to interact with α9β1 integrin. Therefore, we hypothesized that tumor-derived α9β1 integrin may contribute to tumor progression. To clarify the roles of α9β1 integrin in tumor growth and lymphatic metastasis, we used an inhibitory anti-human α9β1 integrin antibody (anti-hα9β1 antibody) and a α9β1 integrin-positive human breast cancer cell line, MDA-MB-231 luc-D3H2LN (D3H2LN), in vitro functional assays, and an in vivo orthotopic xenotransplantation model. In this study, we demonstrated that tumor, but not host α9β1 integrin, contributes to tumor growth, lymphatic metastasis, recruitment of cancer-associated fibroblasts (CAFs), and host-derived OPN production. We also found that CAFs contributed to tumor growth, lymphatic metastasis, and host-derived OPN levels. Consistent with those findings, tumor volume was well-correlated with numbers of CAFs and levels of host-derived OPN. Furthermore, it was shown that the inoculation of D3H2LN cells into mammary fat pads with mouse embryonic fibroblasts (MEFs), obtained from wild type, but not OPN knock-out mice, resulted in enhancement of tumor growth, thus indicating that CAF-derived OPN enhanced tumor growth. These results suggested that tumor α9β1-mediated signaling plays a pivotal role in generating unique primary tumor tissue microenvironments, which favor lymphatic metastasis and tumor growth. Tumor α9β1 integrin promotes lymphatic metastasis through enhancing invasion. Tumor α9β1 integrin promotes tumor growth through CAFs. Tumor α9β1 integrin enhances the recruitment of CAFs into the primary tumor. Tumor cells induce the production of OPN by CAFs in the primary tumor. CAF-derived OPN promotes tumor growth.