Osteopontin Deficiency Ameliorates Prostatic Fibrosis and Inflammation.

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

OPN Deficiency Increases the Severity of Osteoarthritis Associated with Aberrant Chondrocyte Senescence and Apoptosis and Upregulates the Expression of Osteoarthritis-Associated Genes.

Objectives: A recent work has reported that the elevated osteopontin (OPN) levels in the articular cartilage and synovial fluid are correlated with the progressive osteoarthritis (OA) joint damage, and OPN has a protective effect against OA by suppressing the expressions of OA-associated genes. The present study examined whether the OPN deficiency was susceptible to OA through the regulation of chondrocyte senescence and apoptosis and the expressions of OA-associated genes. Methods: The mRNA levels of COL2A1 and OPN were compared between human OA chondrocytes and normal chondrocytes. The effects of OPN siRNA on the SA-ß-Gal expressions and the percentage of apoptotic chondrocytes were examined by using SA-ß-Gal staining and apoptosis assay, and the effects on the expressions of COL2A1 and OA-associated genes (COL10A1, IL-1ß, TNF-ɑ, MMP-13, and ADAMTS5) were examined by western blot analysis and quantitative real-time RT-PCR. Furthermore, an in vivo OA model was established to examine the effects of OPN siRNA on the senescence and apoptosis of OA chondrocytes and the expressions of OA-associated genes. Results: The mRNA levels of COL2A1 and OPN were decreased in knee OA chondrocytes in comparison with those in normal chondrocytes. The OPN deficiency enhanced the senescence and apoptosis of OA chondrocytes and increased the expressions of COL10A1, IL-1ß, TNF-ɑ, MMP-13, and ADAMTS5 but decreased the expression of COL2A1. Meanwhile, OPN deficiency could result in severe, accelerated OA in vivo, which was also associated with enhanced senescence and apoptosis of chondrocytes and elevated expressions of OA-associated genes. Conclusions: The findings of this study suggest that the OPN deficiency can result in accelerated OA, which is associated with enhanced senescence and apoptosis of OA chondrocytes and the upregulated expressions of OA-associated genes.

Osteopontin accelerates chondrocyte proliferation in osteoarthritis rats through the NF-κb signaling pathway.

OBJECTIVE: To explore the influence of osteopontin (OPN) on the chondrocyte proliferation in osteoarthritis (OA) rats. MATERIALS AND METHODS: A total of 30 Sprague-Dawley rats were divided in the control group (n=10), model group (n=10), and OPN knockdown group (n=10). No treatment was performed in the control group, while OA rats were administrated with control adenovirus in the model group and OPN knockdown adenovirus in the OPN knockdown group. After sampling, the degree of OA was evaluated via hematoxylin-eosin (HE) staining, and the mRNA expression of OPN was detected. Moreover, the expression of the proliferation-associated protein cyclin D1 was detected using immunohistochemistry. The chondrocytes were isolated from the normal rats, cultured, and transfected with OPN overexpression vector or si-OPN. Methyl thiazolyl tetrazolium (MTT) assay was adopted to determine the proliferative capacity of chondrocytes, and Caspase3 activity was measured to evaluate the changes in the apoptotic capacity of chondrocytes. Meanwhile, Western blotting was performed to verify the influences of OPN on the pathways on chondrocyte proliferation. RESULTS: After the OA model was established, the expression level of OPN significantly increased. According to HE staining results, OPN knockdown effectively inhibited the onset of OA. Compared with that in the control group, the expression level of cyclin D1 in the model group was raised. However, upregulated cyclin D1 in OA rats was repressed in OPN knockdown group. OPN overexpression promoted the proliferation of chondrocytes, but suppressed their apoptosis, while OPN knockdown had the opposite effects. Besides, OPN overexpression upregulated nuclear factor-κB (NF-κB), and NF-κB knockdown eliminated the regulatory effects of OPN on proliferation and apoptosis of chondrocytes. CONCLUSIONS: OPN promotes the expression of NF-κB signals to accelerate chondrocyte proliferation, thereby inducing OA in rats.

Osteopontin is An Important Regulative Component of the Fetal Bone Marrow Hematopoietic Stem Cell Niche.

Osteopontin (OPN) is an important component in both bone and blood regulation, functioning as a bridge between the two. Previously, thrombin-cleaved osteopontin (trOPN), the dominant form of OPN in adult bone marrow (BM), was demonstrated to be a critical negative regulator of adult hematopoietic stem cells (HSC) via interactions with α4ß1 and α9ß1 integrins. We now demonstrate OPN is also required for fetal hematopoiesis in maintaining the HSC and progenitor pool in fetal BM. Specifically, we showed that trOPN is highly expressed in fetal BM and its receptors, α4ß1 and α9ß1 integrins, are both highly expressed and endogenously activated on fetal BM HSC and progenitors. Notably, the endogenous activation of integrins expressed by HSC was attributed to high concentrations of three divalent metal cations, Ca2+, Mg2+ and Mn2+, which were highly prevalent in developing fetal BM. In contrast, minimal levels of OPN were detected in fetal liver, and α4ß1 and α9ß1 integrins expressed by fetal liver HSC were not in the activated state, thereby permitting the massive expansion of HSC and progenitors required during early fetal hematopoiesis. Consistent with these results, no differences in the number or composition of hematopoietic cells in the liver of fetal OPN-/- mice were detected, but significant increases in the hematopoietic progenitor pool in fetal BM as well as an increase in the BM HSC pool following birth and into adulthood were observed. Together, the data demonstrates OPN is a necessary negative regulator of fetal and neonatal BM progenitors and HSC, and it exhibits preserved regulatory roles during early development, adulthood and ageing.

Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model.

Chronic liver inflammation (CLI) is a risk factor for development of hepatocellular carcinoma (HCC). Galectin-1 (Gal1) is involved in the regulation of inflammation, angiogenesis, and tumorigenesis, exhibiting multiple anti-inflammatory and protumorigenic activities. We aimed to explore its regulatory role in CLI and HCC progression using an established model of CLI-mediated HCC development, Abcb4 [multidrug-resistance 2 (Mdr2)]-knockout (KO) mice, which express high levels of Gal1 in the liver. We generated double-KO (dKO) Gal1-KO/Mdr2-KO mice on C57BL/6 and FVB/N genetic backgrounds and compared HCC development in the generated strains with their parental Mdr2-KO strains. Loss of Gal1 increased liver injury, inflammation, fibrosis, and ductular reaction in dKO mice of both strains starting from an early age. Aged dKO mutants displayed earlier hepatocarcinogenesis and increased tumor size compared with control Mdr2-KO mice. We found that osteopontin, a well-known modulator of HCC development, and oncogenic proteins Ntrk2 (TrkB) and S100A4 were overexpressed in dKO compared with Mdr2-KO livers. Our results demonstrate that in Mdr2-KO mice, a model of CLI-mediated HCC, Gal1-mediated protection from hepatitis, liver fibrosis, and HCC initiation dominates over its known procarcinogenic activities at later stages of HCC development. These findings suggest that anti-Gal1 treatments may not be applicable at all stages of CLI-mediated HCC.-Potikha, T., Pappo, O., Mizrahi, L., Olam, D., Maller, S. M., Rabinovich, G. A., Galun, E., Goldenberg, D. S. Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model.

Thrombin cleavage of osteopontin controls activation of hepatic stellate cells and is essential for liver fibrogenesis.

Liver biopsy is the current reliable way of evaluating liver fibrosis. However, no specific sera biomarker could be applied in clinical diagnosis. As the pivotal role of osteopontin (OPN) reported in numerous liver diseases, thrombin-cleaved OPN (Thr-OPN) exposes an integrin-binding motif that promoted biological functions. Herein, we investigated the potential of Thr-OPN in liver fibrosis. Using patient samples, mouse models and hepatic stellate cells (HSCs), we analyzed the involvement of Thr-OPN in liver fibrosis. The result showed that, first, Thr-OPN level was significantly higher in patients with liver cirrhosis than that in patients with chronic hepatitis B and healthy controls. Thr-OPN level was positively correlated with liver fibrosis degree in clinical samples. Then in mouse models, it showed a similar correlation between hepatic Thr-OPN levels and liver fibrosis degree. Thr-OPN peptides exacerbated liver fibrosis in OPN-deficient mice, whereas the neutralization of Thr-OPN alleviated liver fibrosis in wild-type mice. Furthermore, when compared with full-length OPN (FL-OPN), Thr-OPN exhibited a greater ability to promote HSC activation, proliferation, and migration via mitogen-activated protein (MAP) kinase and nuclear factor (NF)-κB pathways. In conclusion, Thr-OPN, not FL-OPN, was critically involved in the exacerbation of liver fibrosis by α9 and α4 integrins via MAP kinase and NF-κB signaling pathway, thus representing a novel diagnostic biomarker and treatment target for liver cirrhosis.

Osteopontin isoforms differentially promote arteriogenesis in response to ischemia via macrophage accumulation and survival.

Osteopontin (OPN) is critical for ischemia-induced neovascularization. Unlike rodents, humans express three OPN isoforms (a, b, and c); however, the roles of these isoforms in post-ischemic neovascularization and cell migration remain undefined. Our objective was to determine if OPN isoforms differentially affect post-ischemic neovascularization and to elucidate the mechanisms underlying these differences. To investigate if human OPN isoforms exert divergent effects on post-ischemic neovascularization, we utilized OPN-/- mice and a loss-of-function/gain-of-function approach in vivo and in vitro. In this study OPN-/- mice underwent hindlimb ischemia surgery and 1.5 × 106 lentivirus particles were administered intramuscularly to overexpress OPNa, OPNb, or OPNc. OPNa and OPNc significantly improved limb perfusion 30.4% ± 0.8 and 70.9% ± 6.3, respectively, and this translated to improved functional limb use, as measured by voluntary running wheel utilization. OPNa- and OPNc-treated animals exhibited significant increases in arteriogenesis, defined here as the remodeling of existing arterioles into larger conductance arteries. Macrophages play a prominent role in the arteriogenesis process and OPNa- and OPNc-treated animals showed significant increases in macrophage accumulation in vivo. In vitro, OPN isoforms did not affect macrophage polarization, whereas all three isoforms increased macrophage survival and decreased macrophage apoptosis. However, OPN isoforms exert differential effects on macrophage migration, where OPNa and OPNc significantly increased macrophage migration, with OPNc serving as the most potent isoform. In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage accumulation in vivo and on macrophage migration and survival, but not polarization, in vitro. Altogether, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in patients with obstructive artery diseases.

Deletion of OPN in BSP knockout mice does not correct bone hypomineralization but results in high bone turnover.

The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.

Atorvastatin provides a new lipidome improving early regeneration after partial hepatectomy in osteopontin deficient mice.

Osteopontin (OPN), a multifunctional cytokine that controls liver glycerolipid metabolism, is involved in activation and proliferation of several liver cell types during regeneration, a condition of high metabolic demands. Here we investigated the role of OPN in modulating the liver lipidome during regeneration after partial-hepatectomy (PH) and the impact that atorvastatin treatment has over regeneration in OPN knockout (KO) mice. The results showed that OPN deficiency leads to remodeling of phosphatidylcholine and triacylglycerol (TG) species primarily during the first 24 h after PH, with minimal effects on regeneration. Changes in the quiescent liver lipidome in OPN-KO mice included TG enrichment with linoleic acid and were associated with higher lysosome TG-hydrolase activity that maintained 24 h after PH but increased in WT mice. OPN-KO mice showed increased beta-oxidation 24 h after PH with less body weight loss. In OPN-KO mice, atorvastatin treatment induced changes in the lipidome 24 h after PH and improved liver regeneration while no effect was observed 48 h post-PH. These results suggest that increased dietary-lipid uptake in OPN-KO mice provides the metabolic precursors required for regeneration 24 h and 48 h after PH. However, atorvastatin treatment offers a new metabolic program that improves early regeneration when OPN is deficient.

IL (Interleukin)-10-STAT3-Galectin-3 Axis Is Essential for Osteopontin-Producing Reparative Macrophage Polarization After Myocardial Infarction.

BACKGROUND: Both osteopontin (OPN) and galectin-3 have been implicated in phagocytic clearance of dead cells and reparative fibrosis during wound healing. CD206+ macrophages are involved in tissue repair through phagocytosis and fibrosis after myocardial infarction (MI). However, the relationship among OPN, galectin-3, and macrophage polarization in the context of MI remains unclear. METHODS: The time course of Spp1 (encoding OPN) expression in the heart after MI showed a strong activation of Spp1 on day 3 after MI. To identify where in the body and in which cells the transcriptional activity of Spp1 increased after MI, we analyzed EGFP (enhanced green fluorescent protein)- Spp1 knockin reporter mice on day 3 after MI. RESULTS: The transcriptional activity of Spp1 increased only in CD206+ macrophages in the infarct myocardium, and most of CD206+ macrophages have strong transcriptional activation of Spp1 after MI. The temporal expression pattern of Lgal3 (encoding galectin-3) in cardiac macrophages after MI was similar to that of Spp1, and OPN is almost exclusively produced by galectin-3hiCD206+ macrophages. Although both interleukin (IL)-4 and IL-10 were reported to promote CD206+ macrophage-mediated cardiac repair after MI, IL-10- but not IL-4-stimulated CD11b+Ly6G- cells could differentiate into OPN-producing galectin-3hiCD206+ macrophages and showed enhanced phagocytic ability. Inhibition of STAT3 tyrosine phosphorylation suppressed IL-10-induced expression of intracellular galectin-3 and transcriptional activation of Spp1. Knockdown of galectin-3 suppressed their ability to differentiate into OPN-producing cells, but not STAT3 activation. The tyrosine phosphorylation of STAT3 and the appearance rate of galectin-3hiCD206+ cells on cardiac CD11b+Ly6G- cells in Spp1 knockout mice were the same as those in wild-type mice. Spp1 knockout mice showed vulnerability to developing post-MI left ventricular chamber dilatation and the terminal deoxynucleo-tidyltransferase 2'-Deoxyuridine-5'-triphosphate nick-end labeling (TUNEL)-positive cells in the infarcted myocardium after MI remained higher in number in Spp1 knockout mice than in wild-type mice. CONCLUSIONS: OPN is almost exclusively produced by galectin-3hiCD206+ macrophages, which specifically appear in the infarct myocardium after MI. The IL-10-STAT3-galectin-3 axis is essential for OPN-producing reparative macrophage polarization after myocardial infarction, and these macrophages contribute to tissue repair by promoting fibrosis and clearance of apoptotic cells. These results suggest that galectin-3 may contribute to reparative fibrosis in the infarct myocardium by controlling OPN levels.

Accounting for tumor heterogeneity when using CRISPR-Cas9 for cancer progression and drug sensitivity studies.

Gene editing protocols often require the use of a subcloning step to isolate successfully edited cells, the behavior of which is then compared to the aggregate parental population and/or other non-edited subclones. Here we demonstrate that the inherent functional heterogeneity present in many cell lines can render these populations inappropriate controls, resulting in erroneous interpretations of experimental findings. We describe a novel CRISPR/Cas9 protocol that incorporates a single-cell cloning step prior to gene editing, allowing for the generation of appropriately matched, functionally equivalent control and edited cell lines. As a proof of concept, we generated matched control and osteopontin-knockout Her2+ and Estrogen receptor-negative murine mammary carcinoma cell lines and demonstrated that the osteopontin-knockout cell lines exhibit the expected biological phenotypes, including unaffected primary tumor growth kinetics and reduced metastatic outgrowth in female FVB mice. Using these matched cell lines, we discovered that osteopontin-knockout mammary tumors were more sensitive than control tumors to chemotherapy in vivo. Our results demonstrate that heterogeneity must be considered during experimental design when utilizing gene editing protocols and provide a solution to account for it.

Osteopontin-deficient progenitor cells display enhanced differentiation to adipocytes.

OBJECTIVE: Osteopontin (OPN, Spp1) is a protein upregulated in white adipose tissue (WAT) of obese subjects. Deletion of OPN protects mice from high-fat diet-induced WAT inflammation and insulin resistance. However, the alterations mediated by loss of OPN in WAT before the obesogenic challenge have not yet been investigated. Therefore, we hypothesised that the lack of OPN might enhance the pro-adipogenic micro environment before obesity driven inflammation. METHODS: OPN deficiency was tested in visceral (V) and subcutaneous (SC) WAT from WT and Spp1-/- female mice. Gene expression for hypoxia, inflammation and adipogenesis was checked in WT vs. Spp1-/- mice (n=15). Adipocytes progenitor cells (APC) were isolated by fluorescence cell sorting and role of OPN deficiency in adipogenesis was investigated by cell images and RT-PCR. RESULTS: We show that Spp1-/- maintained normal body and fat-pad weights, although hypoxia and inflammation markers were significantly reduced. In contrast, expression of genes involved in adipogenesis was increased in WAT from Spp1-/- mice. Strikingly, APC from Spp1-/- were diminished but differentiated more efficiently to adipocytes than those from control mice. CONCLUSIONS: APC from SC-WAT of lean OPN-deficient mice display an enhanced capacity for differentiating to adipocytes. These alterations may explain the healthy expansion of WAT in the OPN-deficient model which is associated with reduced inflammation and insulin resistance.

Visceral Adipose Tissue Drives Cardiac Aging Through Modulation of Fibroblast Senescence by Osteopontin Production.

BACKGROUND: Aging induces cardiac structural and functional changes linked to the increased deposition of extracellular matrix proteins, including OPN (osteopontin), conducing to progressive interstitial fibrosis. Although OPN is involved in various pathological conditions, its role in myocardial aging remains unknown. METHODS: OPN deficient mice (OPN-/-) with their wild-type (WT) littermates were evaluated at 2 and 14 months of age in terms of cardiac structure, function, histology and key molecular markers. OPN expression was determined by reverse-transcription polymerase chain reaction, immunoblot and immunofluorescence. Luminex assays were performed to screen plasma samples for various cytokines/adipokines in addition to OPN. Similar explorations were conducted in aged WT mice after surgical removal of visceral adipose tissue (VAT) or treatment with a small-molecule OPN inhibitor agelastatin A. Primary WT fibroblasts were incubated with plasma from aged WT and OPN-/- mice, and evaluated for senescence (senescence-associated ß-galactosidase and p16), as well as fibroblast activation markers (Acta2 and Fn1). RESULTS: Plasma OPN levels increased in WT mice during aging, with VAT showing the strongest OPN induction contrasting with myocardium that did not express OPN. VAT removal in aged WT mice restored cardiac function and decreased myocardial fibrosis in addition to a substantial reduction of circulating OPN and transforming growth factor ß levels. OPN deficiency provided a comparable protection against age-related cardiac fibrosis and dysfunction. Intriguingly, a strong induction of senescence in cardiac fibroblasts was observed in both VAT removal and OPN-/- mice. The addition of plasma from aged OPN-/- mice to cultures of primary cardiac fibroblasts induced senescence and reduced their activation (compared to aged WT plasma). Finally, Agelastatin A treatment of aged WT mice fully reversed age-related myocardial fibrosis and dysfunction. CONCLUSIONS: During aging, VAT represents the main source of OPN and alters heart structure and function via its profibrotic secretome. As a proof-of-concept, interventions targeting OPN, such as VAT removal and OPN deficiency, rescued the heart and induced a selective modulation of fibroblast senescence. Our work uncovers OPN's role in the context of myocardial aging and proposes OPN as a potential new therapeutic target for a healthy cardiac aging.

The Retina of Osteopontin deficient Mice in Aging.

Osteopontin (OPN) is a secreted glycosylated phosphoprotein that influences cell survival, inflammation, migration, and homeostasis after injury. As the role of OPN in the retina remains unclear, this study issue was addressed by aiming to study how the absence of OPN in knock-out mice affects the retina and the influence of age on these effects. The study focused on retinal ganglion cells (RGCs) and glial cells (astrocytes, Müller cells, and resident microglia) in 3- and 20-month-old mice. The number of RGCs in the retina was quantified and the area occupied by astrocytes was measured. In addition, the morphology of Müller cells and microglia was examined in retinal sections. The deficiency in OPN reduces RGC density by 25.09% at 3 months of age and by 60.37% at 20 months of age. The astrocyte area was also reduced by 51.01% in 3-month-old mice and by 57.84% at 20 months of age, although Müller glia and microglia did not seem to be affected by the lack of OPN. This study demonstrates the influence of OPN on astrocytes and RGCs, whereby the absence of OPN in the retina diminishes the area occupied by astrocytes and produces a secondary reduction in the number of RGCs. Accordingly, OPN could be a target to develop therapies to combat neurodegenerative diseases and astrocytes may represent a key mediator of such effects.

Osteocalcin and osteopontin influence bone morphology and mechanical properties.

Osteocalcin (OC) and osteopontin (OPN) are major non-collagenous proteins (NCPs) involved in bone matrix organization and deposition. In spite of this, it is currently unknown whether OC and OPN alter bone morphology and consequently affect bone fracture resistance. The goal of this study is to establish the role of OC and OPN in the determination of cortical bone size, shape, and mechanical properties. Our results show that Oc-/- and Opn-/- mice were no different from each other or wild type (WT) with respect to bone morphology (P > 0.1). Bones from mice lacking both NCPs (Oc-/- Opn-/- ) were shorter, with thicker cortices and larger cortical areas, compared with the WT, Oc-/- , and Opn-/- groups (P < 0.05), suggesting a synergistic role for NCPs in the determination of bone morphology. Maximum bending load was significantly different among the groups (P = 0.024), while tissue mineral density and measures of stiffness and strength were not different (P > 0.1). We conclude that the removal of both OC and OPN from bone matrix induces morphological adaptation at the structural level to maintain bone strength.

Osteopontin activates mesenchymal stem cells to repair skin wound.

Mesenchymal stem cells (MSCs) are promising candidates for skin wound repair due to their capabilities of accumulating at wounds and differentiating into multiple types of skin cells. However, the underlying mechanisms responsible for these processes remain unclear. In this study, we found that osteopontin (OPN) stimulated the migration of MSCs in vitro, and observed the recruitment of endogenous MSCs to a skin wound and their differentiation into keratinocytes and endothelial cells. In OPN knock-out mice, the recruitment of MSCs to the skin wound was significantly inhibited, and wound closure was hampered after an intradermal injection of exogenous MSCs compared to wild-type mice. Consistent with these observations, the expressions of adhesion molecule CD44 and its receptor E-selectin were significantly decreased in the lesions of OPN knock-out mice compared with wild-type mice suggesting that OPN may regulate the migration of MSCs through its interactions with CD44 during skin wound recovery. In summary, our data demonstrated that OPN played a critical role in activating the migration of MSCs to injured sites and their differentiation into specific skin cell types during skin wound healing.

Osteopontin regulates the cross-talk between phosphatidylcholine and cholesterol metabolism in mouse liver.

Osteopontin (OPN) is involved in different liver pathologies in which metabolic dysregulation is a hallmark. Here, we investigated whether OPN could alter liver, and more specifically hepatocyte, lipid metabolism and the mechanism involved. In mice, lack of OPN enhanced cholesterol 7α-hydroxylase (CYP7A1) levels and promoted loss of phosphatidylcholine (PC) content in liver; in vivo treatment with recombinant (r)OPN caused opposite effects. rOPN directly decreased CYP7A1 levels through activation of focal adhesion kinase-AKT signaling in hepatocytes. PC content was also decreased in OPN-deficient (OPN-KO) hepatocytes in which de novo FA and PC synthesis was lower, whereas cholesterol (CHOL) synthesis was higher, than in WT hepatocytes. In vivo inhibition of cholesterogenesis normalized liver PC content in OPN-KO mice, demonstrating that OPN regulates the cross-talk between liver CHOL and PC metabolism. Matched liver and serum samples showed a positive correlation between serum OPN levels and liver PC and CHOL concentration in nonobese patients with nonalcoholic fatty liver. In conclusion, OPN regulates CYP7A1 levels and the metabolic fate of liver acetyl-CoA as a result of CHOL and PC metabolism interplay. The results suggest that CYP7A1 is a main axis and that serum OPN could disrupt liver PC and CHOL metabolism, contributing to nonalcoholic fatty liver disease progression in nonobese patients.

Osteopontin enhances multi-walled carbon nanotube-triggered lung fibrosis by promoting TGF-ß1 activation and myofibroblast differentiation.

BACKGROUND: Carbon 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). METHODS: C57BL/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. RESULTS: OPN 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-ß1, stimulates the differentiation of myofibroblasts from fibroblasts, and increases the production of fibrous matrix proteins in lungs and cultured lung cells exposed to MWCNTs. CONCLUSION: OPN is highly induced in CNT-exposed lungs and plays critical roles in TGF-ß1 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 deficiency protects mice from cholesterol gallstone formation by reducing expression of intestinal NPC1L1.

Homeostasis of cholesterol is regulated by absorption in the intestine and synthesis in the liver. The authors previously demonstrated that OPN (osteopontin) exhibits the ability to alter hepatic cholesterol metabolism, thus affecting cholesterol gallstone formation in mice. The present study investigated the role of OPN in cholesterol gallstone formation, focusing on its effect on intestinal absorption of cholesterol. OPN gene knockout (OPN­/­) mice and wild­type mice were respectively fed with a chow or lithogenic diet (LD) for 8 weeks. Following an 8­week LD period, the incidence of gallstone, bile composition, level of serum and fecal lipids and the expression of intestinal associated genes were analyzed. OPN­/­ mice were protected from gallstone formation induced by 8 weeks' LD­feeding. This protective effect from OPN deficiency was associated with alterations in bile composition, including a reduced concentration of biliary cholesterol. Additionally, plasma cholesterol level was decreased in LD­fed OPN­/­ mice. The alterations primarily resulted from the decreased expression of intestinal Niemann­Pick C1­like (NPC1 L) 1, which is important in the intestinal absorption of cholesterol. The present study demonstrated that OPN deficiency reduced intestinal absorption of cholesterol by suppressing the expression of NPC1L1, thus protecting mice from cholesterol gallstone formation.

Osteopontin Deficiency Suppresses Intestinal Tumor Development in Apc-Deficient Min Mice.

Osteopontin (OPN) is a secreted phosphoglycoprotein, and is a transcriptional target of aberrant Wnt signaling. OPN is upregulated in human colon cancers, and is suggested to enhance cancer progression. In this study, the effect of deficiency of OPN on intestinal tumor development in Apc-deficient Min mice was investigated. At 16 weeks of age, the number of small intestinal polyps in Min/OPN(+/-) and Min/OPN(-/-) mice was lower than that of Min/OPN(+/+) mice. Colorectal tumor incidences and multiplicities in Min/OPN(+/-) and Min/OPN(-/-) mice were significantly lower than those in Min/OPN(+/+) mice, being 48% and 0.6 ± 0.8, 50% and 0.8 ± 0.9 vs. 80% and 1.6 ± 1.7, respectively. OPN expression in colorectal tumors was strongly upregulated in Min/OPN(+/+) compared to adjacent non-tumor parts, but was decreased in Min/OPN(+/-) and not detected in Min/OPN(-/-). Targets of OPN, matrix metalloproteinases (MMPs)-3, -9, and -13 were lowered by OPN deficiency. Macrophage marker F4/80 in colorectal tumors was also lowered by OPN deficiency. MMP-9 expression was observed in tumor cells and tumor-infiltrating neutrophils. These results indicate that induction of OPN by aberrant Wnt signaling could enhance colorectal tumor development in part by upregulation of MMP-3, -9, and -13 and infiltration of macrophage and neutrophils. Suppression of OPN expression could contribute to tumor prevention, but complete deficiency of OPN may cause some adverse effects.