Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype. We showed recently that nicotine, a major risk factor in pancreatic ductal adenocarcinoma (PDA), increases OPN expression in PDA cells. An OPN splice variant, OPNc, supports anchorage independence and maybe the most potent OPN isoform to convey metastatic behavior. In this study, we tested the effect of nicotine on OPNc expression, and analyzed the correlation between total OPN/OPNc levels and patients’ smoking history.
Real time PCR and UV-light-illumination of ethidium-bromide staining were used to examine the mRNA expression in tissue and in PDA cells treated with or without nicotine (3-300 nM). OPN and OPNc were localized by immunohisotchemistry, and ELISA was used to analyze OPN serum levels.
Nicotine treatment of PDA cells selectively induced denovo expression of OPNc. OPNc was found in 87% of invasive PDA lesions, of which 73% were smokers. The levels of OPNc correlated well with higher expression levels of total OPN in the tissue and serum from patients with invasive PDA.
Our data suggest that smoking and nicotine may contribute to PDA metastatic potential through promoting OPNc expression. Although the direct role of OPNc in PDA progression is not defined, OPNc may have value as a diagnostic and prognostic marker, especially in invasive PDA.
pancreatic cancer; nicotine; osteopontin
Background/Aims. Pancreatic ductal adenocarcinoma (PDA) has etiological association with chronic inflammation. Elevated circulating levels of inflammatory mediators, such as monocyte chemoattractant protein-1 (MCP-1), are found in obese individuals. We hypothesized that serum MCP-1 levels are elevated in obese PDA patients. Methods. ELISA was used to analyze MCP-1 serum levels in PDA (n = 62) and intraductal papillary mucinous neoplasms (IPMN) (n = 27). Recursive partitioning statistical analysis investigated the relationship between log MCP-1 and clinicopathological parameters. Results. Log MCP-1 values were significantly (P < 0.05) elevated in patients with BMI ≥ 37.5. In patients with BMI < 37.5, average log MCP-1 values were significantly elevated in PDA patients when compared to IPMN patients. Within the IPMN group, higher log MCP-1 levels correlated with increased age. Recursive partitioning analysis of IPMN versus PDA revealed a strategy of predicting characteristics of patients who are more likely to have cancer. This strategy utilizes log MCP-1 as the primary factor and also utilizes smoking status, gender, and age. Conclusion. MCP-1 is a promising biomarker in pancreatic cancer. The potential of using MCP-1 to distinguish PDA from IPMN patients must be studied in larger populations to validate and demonstrate its eventual clinical utility.
Osteopontin (OPN) is a multifunctional phosphoprotein with an important but poorly understood role in non-small cell lung cancer (NSCLC) pathogenesis. We hypothesize that OPN isoforms (OPNa, OPNb, and OPNc) have divergent roles in NSCLC angiogenesis and divergent impact on vascular endothelial growth factor (VEGF) secretion.
We examined mRNA expression using RT-PCR primers for three OPN isoforms in NSCLC and immortalized bronchial epithelial cell lines, and correlated expression with OPN secretion into media detected by ELISA. Angiogenic properties conferred by OPN isoforms were evaluated by transfecting cDNA plasmids specific to each isoform and controls into NSCLC cell lines, H153 and H358 (low endogenous OPN) and A549 and H460 (high endogenous OPN) and analyzing conditioned media on a bovine capillary endothelial (BCE) platform and measuring VEGF levels by ELISA.
OPNa mRNA expression correlated with OPN secretion in cell lines (r=0.912,p=0.0006). OPNa overexpression significantly increased tubule length compared to controls, OPNb had a similar, but less pronounced effect, while OPNc significantly decreased tubule length compared to controls in each cell line. OPNa overexpression was associated with significant increases in VEGF secretion, while OPNb had no effect and OPNc overexpression was associated with significant decreases in VEGF compared to controls in each cell line.
We demonstrated divergent effects of OPN isoforms on NSCLC angiogenesis and VEGF secretion. OPNa overexpression was associated with increased BCE tubule length and VEGF secretion, while OPNc is associated with decreases in both. These findings may lead to therapeutic strategies for selective isoform inhibition in NSCLC.
Osteopontin is a multifunctional phosphoprotein with an important but poorly understood role in non-small cell lung cancer pathogenesis. The role of the three human isoforms has not been previously reported. We demonstrate that individual osteopontin isoforms have divergent effects on non-small cell lung cancer angiogenesis with associated changes in VEGF.
Osteopontin (OPN) is a matricellular protein that binds to a number of cell surface receptors including integrins and CD44. It is expressed in many tissues and secreted into body fluids including blood, milk and urine. OPN plays important physiological roles in bone remodeling, immune response and inflammation. It is also a tumour-associated protein, and elevated OPN levels are associated with tumour formation, progression and metastasis. Research has revealed a promising role for OPN as a cancer biomarker. OPN is subject to alternative splicing, as well as post-translational modifications such as phosphorylation, glycosylation and proteolytic cleavage. Functional differences have been revealed for different isoforms and post-translational modifications. The pattern of isoform expression and post-translational modification is cell-type specific and may influence the potential role of OPN in malignancy and as a cancer biomarker.
Osteopontin; Cancer; Metastasis; Matricellular protein; Post-translational modification; Alternative splicing
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.
Background. Renal calcium oxalate (CaOx) crystal deposition is associated with epithelial injury and movement of inflammatory cells into the interstitium. We have proposed that oxalate (Ox)- and CaOx crystal-induced injury is most likely caused by reactive oxygen species (ROS) produced by activation of membrane nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.
Methods. Present study was undertaken to determine the effect of NADPH oxidase inhibitor apocynin on the expression of kidney injury molecule-1 (KIM-1) and renal CaOx crystal deposition in rats with hyperoxaluria. We also investigated the urinary excretion of KIM-1, osteopontin (OPN) and monocyte chemoattractant protein-1 (MCP-1) and renal expression of OPN and ED-1. Male Sprague–Dawley rats were fed a diet containing 5% hydroxyl-L-proline (HLP) and 4 mmol apocynin to drink for 28 days. Urine was collected on Days 7, 14, 21 and 28. After that, rats were sacrificed and their kidneys processed for various microscopic and molecular investigations.
Results. HLP consumption produced heavy deposits of CaOx crystals. Renal expression of KIM-1 and OPN and urinary excretion of KIM-1, OPN, H2O2 and MCP-1 was significantly increased. ED-1-positive cells migrated into renal interstitium. Apocynin treatment caused significant reduction of crystal deposits, injured and dilated tubules; renal expression of KIM-1, OPN and ED-1 and urinary excretion of KIM-1, OPN, MCP-1 and H2O2. Apocynin had no effect on the urinary excretion of Ox.
Conclusions. This is the first study of urinary excretion and renal expression of KIM-1 in association with renal CaOx crystal deposition, experimental or clinical. The results indicate that NADPH oxidase inhibition leads to reduction in KIM-1 expression and urinary excretion as well as renal CaOx crystal deposition. KIM-1 is an important marker of renal epithelial injury. The results provide further support to our proposal that renal epithelial injury is critical for crystal retention and that injury is in part caused by the production of ROS with the involvement of NADPH oxidase.
apocynin; calcium oxalate; kidney injury molecule-1; kidney stones; NADPH oxidase; oxalate; oxidative stress
Calcification and fibrosis reduce vascular compliance in arteriosclerosis. To better understand the role of osteopontin (OPN), a multifunctional protein upregulated in diabetic arteries, we evaluated contributions of OPN in male LDLR−/− mice fed high fat diets (HFD).
Methods and Results
OPN had no impact on HFD-induced hyperglycemia, dyslipidemia, or body composition. However, OPN−/−;LDLR−/− mice exhibited an altered time-course of aortic calcium accrual -- reduced during initiation but increased with progression -- vs. OPN+/+;LDLR−/− controls. Collagen accumulation, chondroid metaplasia, and mural thickness were increased in aortas of OPN−/−;LDLR−/− mice. Aortic compliance was concomitantly reduced. Vascular re-expression of OPN (SM22-OPN transgene) reduced aortic Col2A1 and medial chondroid metaplasia, but did not impact atherosclerotic calcification, Col1A1 expression, collagen accumulation, or arterial stiffness. Dosing with the pro-inflammatory OPN fragment SVVYGLR upregulated aortic Wnt and osteogenic gene expression, increased aortic β-catenin, and restored early-phase aortic calcification in OPN−/−;LDLR−/− mice.
OPN exerts stage-specific roles in arteriosclerosis in LDLR−/− mice. Actions phenocopied by the OPN metabolite SVVYGLR promote osteogenic calcification processes with disease initiation. OPN limits vascular chondroid metaplasia, endochondral mineralization and collagen accumulation with progression. Complete deficiency yields a net increase in arteriosclerotic disease, reducing aortic compliance and conduit vessel function in LDLR−/− mice.
Arteriosclerosis; Calcification; Chondroid Metaplasia; Diabetes; Osteopontin
Infectious antigens may be triggers for the exacerbation of systemic lupus erythematosus. The underlying mechanism causing acceleration and exacerbation of lupus nephritis (LN) is largely unknown. Bacterial lipopolysaccharide (LPS) is capable of inducing an accelerated model of LN in NZB/W mice, featuring diffuse proliferation of glomerular resident cells. We hypothesized that mesangial cells (MCs) from LN subjects are more responsive to LPS than normal subjects. Cultured primary NZB/W and DBA/W (nonautoimmune disease-prone strain with MHC class II molecules identical to those of NZB/W) MCs were used. Monocyte chemoattractant protein-1 (MCP-1) and osteopontin (OPN) expressions either in the baseline (normal culture) condition or in the presence of LPS were evaluated by real-time PCR, ELISA, or western blot analysis. NF-κB was detected by ELISA, electrophoresis mobility-shift assay, and immunofluorescence. First, either in the baseline condition or in the presence of LPS, NZB/W MCs produced significantly higher levels of MCP-1 and OPN than the DBA/W MC controls. Second, NZB/W MCs expressed significantly higher levels of Toll-like receptor 4, myeloid differentiation factor 88, and NF-κB than the DBA/W MC controls, both receiving exactly the same LPS treatment. In conclusion, NZB/W MCs are significantly more sensitive than their normal control DBA/W MCs in producing both MCP-1 and OPN. With LPS treatment, the significantly elevated levels of both chemokines produced by NZB/W MCs are more likely due to a significantly greater activation of the Toll-like receptor 4-myeloid differentiation factor 88-associated NF-κB pathway. The observed abnormal molecular events provide an intrarenal pathogenic pathway involved in an accelerated type of LN, which is potentially infection triggered.
Experimental animal model studies suggest that calcium oxalate (CaOx) crystal deposition in the kidneys is associated with the development of oxidative stress, epithelial injury and inflammation. There is increased production of inflammatory molecules including osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1) and various subunits of inter-alpha-inhibitor such as bikunin. What does the increased production of such molecules suggest? Is it a cause or consequence of crystal deposition? We hypothesized that over-expression and increased production of MCP-1 is a result of the interaction between renal epithelial cells and CaOx crystals after their deposition in the renal tubules. We induced hyperoxaluria in MCP-1 null as well as wild type mice and examined pathological changes in their kidneys and urine. Both wild type and MCP-1 null male mice became hyperoxaluric and demonstrated CaOx crystalluria. Neither of them developed crystal deposits in their kidneys. Both showed some morphological changes in their renal proximal tubules. Significant pathological changes such as cell death and increased urinary excretion of LDH were not seen. Results suggest that at least in mice (1) Increase in oxalate and decrease in citrate excretion can lead to CaOx crystalluria but not CaOx nephrolithiasis; (2) MCP-1 does not play a role in crystal retention within the kidneys; (3) Expression of OPN and MCP-1 is not increased in the kidneys in the absence of crystal deposition; (4) Crystal deposition is necessary for significant pathological changes and movement of monocytes and macrophages into the interstitium.
Calcium oxalate; Nephrolithiasis; MCP-1; Inflammation
Osteopontin (OPN) is a secreted glycoprotein that mediates cell-matrix interactions and cellular signaling by binding with integrin (primarily αvβ3) and CD44 receptors. OPN regulates cell adhesion, chemotaxis, macrophage-directed IL-10 suppression, stress-dependent angiogenesis, apoptosis prevention, and anchorage-independent growth of tumor cells. However, the molecular mechanisms that define the role of OPN in tumor progression and metastasis are incompletely understood.
In this study, we use a system of 4T1 and 4T07 murine mammary epithelial tumor cell lines that are divergent in both metastatic phenotype and OPN expression. 4T1 expresses OPN and hematogeneously metastasizes, whereas 4T07 does not express OPN and is highly tumorigenic but fails to metastasize.
Our results demonstrate that OPN regulates Stat1 protein degradation through the ubiquitin-proteasome pathway to alter interferon-γ-dependent growth inhibition and p21 expression. We identify Stat-interacting LIM protein as the critical Stat ubiquitin E3 ligase in this setting.
OPN regulates Stat1-dependent functions, such as growth inhibition and p21 expression, in the murine mammary epithelial cells lines 4T1 and 4T07. This relationship between OPN and Stat1 in the context of tumor biology has not been previously examined.
Ubiquitin; SLIM; proteasome; p21; interferon
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.
Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a characteristic pattern of early metastasis, which is driving a search for biomarkers that can be used to detect the cancer at an early stage. Recently, the actin-associated protein palladin was identified as a candidate biomarker when it was shown that palladin is mutated in a rare inherited form of PDA, and overexpressed in many sporadic pancreas tumors and premalignant precursors. In this study, we analyzed the expression of palladin isoforms in murine and human PDA and explored palladin's potential use in diagnosing PDA. We performed immunohistochemistry and immunoblot analyses on patient samples and tumor-derived cells using an isoform-selective monoclonal antibody and a pan-palladin polyclonal antibody. Immunoblot and real-time quantitative reverse transcription-PCR were used to quantify palladin mRNA levels in human samples. We show that there are two major palladin isoforms expressed in pancreas: 65 and 85–90 kDa. The 65 kDa isoform is expressed in both normal and neoplastic ductal epithelial cells. The 85–90 kDa palladin isoform is highly overexpressed in tumor-associated fibroblasts (TAFs) in both primary and metastatic tumors compared to normal pancreas, in samples obtained from either human patients or genetically engineered mice. In tumor-derived cultured cells, expression of palladin isoforms follows cell-type specific patterns, with the 85–90 kDa isoform in TAFs, and the 65 kDa isoform predominating in normal and neoplastic epithelial cells. These results suggest that upregulation of 85–90 kDa palladin isoform may play a role in the establishment of the TAF phenotype, and thus in the formation of a desmoplastic tumor microenvironment. Thus, palladin may have a potential use in the early diagnosis of PDA and may have much broader significance in understanding metastatic behavior.
Matricellular proteins mediate both tissue morphogenesis and tissue homeostasis in important ways because they modulate cell-matrix and cell-cell interactions. In this study, we found that the matricellular protein osteopontin (Opn) is a novel marker of undifferentiated pancreatic precursors and pancreatic ductal tissues in mice. Our analysis also underscored a specific, dynamic profile of Opn expression in embryonic pancreatic tissues that suggests the participation of this protein’s function in processes involving cell migration, cell-cell interactions, or both. Surprisingly, our analysis of Opn-deficient pancreata did not reveal obvious alterations in the morphology or differentiation of these tissues. Therefore, in embryonic pancreatic tissues, it is possible that other proteins act redundantly to Opn or that this protein’s function is dispensable for pancreas development. Finally, the maintenance of Opn expression in pancreatic tissues of adults argues for a possible function of this protein in injury and pathologic responses.
Pancreas; osteopontin; mouse; matricellular proteins; development; pancreatic ducts; pancreatic precursor; embryo
Osteopontin (OPN) is expressed in atherosclerotic lesions, particularly in diabetic patients. To determine the role of OPN in atherogenesis, ApoE–/–OPN+/+, ApoE–/–OPN+/–, and ApoE–/–OPN–/– mice were infused with Ang II, inducing vascular OPN expression and accelerating atherosclerosis. Compared with ApoE–/–OPN+/+ mice, ApoE–/–OPN+/– and ApoE–/–OPN–/– mice developed less Ang II–accelerated atherosclerosis. ApoE–/– mice transplanted with bone marrow derived from ApoE–/–OPN–/– mice had less Ang II–induced atherosclerosis compared with animals receiving ApoE–/–OPN+/+ cells. Aortae from Ang II–infused ApoE–/–OPN–/– mice expressed less CD68, C-C-chemokine receptor 2, and VCAM-1. In response to intraperitoneal thioglycollate, recruitment of leukocytes in OPN–/– mice was impaired, and OPN–/– leukocytes exhibited decreased basal and MCP-1–directed migration. Furthermore, macrophage viability in atherosclerotic lesions from Ang II–infused ApoE–/–OPN–/– mice was decreased. Finally, Ang II–induced abdominal aortic aneurysm formation in ApoE–/–OPN–/– mice was reduced and associated with decreased MMP-2 and MMP-9 activity. These data suggest an important role for leukocyte-derived OPN in mediating Ang II–accelerated atherosclerosis and aneurysm formation.
Osteopontin (OPN) is a secreted protein of the extracellular matrix. It has been used as a marker for tumor aggressiveness and correlated with clinical outcomes in several solid tumors, such as liver, lung, and breast. We determined the OPN expression and its influence on survival in patients with resected pancreatic adenocarcinoma.
Tissue microarrays were constructed from 245 resected pancreatic adenocarcinomas. Immunohistochemical staining for OPN was undertaken and compared to normal pancreas (n = 12). OPN expression was then correlated with patient demographics, tumor size, grade, node, and margin status. Survival curves were created by the Kaplan–Meier method and compared by log rank analysis.
In total, 181 (74 %) of pancreatic adenocarcinoma tissues expressed OPN compared to 7 (58 %) of normal controls (p = 0.004). Expression was observed predominantly in the cytoplasm of the tumor cells. The median and 2 year overall survival was longer when OPN was expressed (17.1 vs. 11.6 months, and 38 vs. 24 %, respectively, p = 0.04). Multivariate analysis showed OPN expression and T stage to be independent predictors of overall survival, while other histopathologic factors such as tumor grade, tumor size, and nodal status were not.
These results suggest that the presence of OPN expression in pancreatic adenocarcinoma may have a protective effect independent of tumor stage. This emphasizes the importance of the interaction between pancreatic cancer cells and their stromal elements.
Osteopontin (OPN) mediates cancer metastatis. Mechanisms regulating OPN expression in human colorectal cancer are unknown. Using SW480 colon adenocarcinoma cells, we hypothesized that transcription determines OPN expression.
SW480 constitutively express OPN. Transient transfection and deletion analysis of human OPN promoter (full-length 2.1 kb)-luciferase constructs identified cis-regulatory regions. Gelshift and chromatin immunoprecipitation (ChIP) assays identified the trans-regulatory nuclear protein. Using in vitro adhesion, migration and invasion studies, siRNA was utilized to determine the functional effect of decreased nuclear protein expression.
A cis-regulatory promoter region, nt-80 to nt-108, upregulated OPN transcription. Gelshift assays demonstrated specific binding of nuclear proteins. Competition with unlabelled mutant oligonucleotides indicated that the region, nt-94 to nt-104 (TGGGCTGGGC), was essential for protein binding in gelshift assays. Confirmatory ChIP assays showed the corresponding nuclear protein to be Sp1. Sp1 expression was ablated with siRNA (si-Sp1) resulting in decreased OPN dependent adhesion, migration and invasion by 50%, 70% and 65%, respectively. Exogenous addition of OPN to si-Sp1 cells restored adhesion, migration and invasion indices.
In SW480 human colon cancer cells, we conclude that Sp1 mediated expression of the tumor metastasis protein, OPN, regulates in vitro functional correlates of tumor metastasis.
Osteopontin (OPN) is a multifunctional protein with an important but poorly understood role in non-small cell lung cancer (NSCLC) pathogenesis. Moreover, the role of the three known mRNA isoforms (OPNa, OPNb, and OPNc) has not been reported. We hypothesize that OPN isoforms play different roles in determining the metastatic potential of NSCLC.
We amplified mRNA for each OPN isoform in NSCLC tumors and matched normal lung. The functional impact of each isoform was evaluated by transfecting cDNA plasmids specific to each isoform into NSCLC cell lines and comparing behavior to empty vector controls in scratch closure, cell proliferation, soft-agar colony formation and Matrigel™ invasion assays. Gene array was used to evaluate differences in down-stream targets and was compared to a panel of markers epithelial-mesenchymal transition (EMT).
OPNa expression was increased in 91% of NSCLC tumors compared to matched lung. OPNa overexpression significantly increased activity in scratch closure, proliferation, soft-agar colony formation and Matrigel™ invasion assays compared to controls in all cell lines. OPNb overexpression produced a less significant modulation of function. OPNc overexpression significantly decreased activity in proliferation, colony formation and invasion assays compared to controls. Expression arrays revealed an increase in EMT with OPNa overexpression, but not OPNc. Differences were validated by quantitative RT-PCR.
Overexpression of the individual OPN isoforms in NSCLC results in divergent functional phenotypes. OPNa produced an aggressive phenotype while OPNc produced a more indolent phenotype. Exon 4 which is transcribed in OPNa but absent in OPNc may be central to this phenomenon and could serve as a target for isoform-specific inhibition of OPN in NSCLC.
osteopontin; non-small cell lung cancer; mRNA isoforms; tumor progression; epithelial–mesenchymal transition
We investigated the role of osteopontin (OPN) in interleukin-12 (IL-12) production from peripheral blood mononuclear cells (PBMCs) stimulated with Penicillium marneffei. Kinetic studies showed that OPN synthesis preceded that of IL-12 at both mRNA and protein levels when PBMCs were cocultured with P. marneffei. Treatment with anti-OPN monoclonal antibodies (MAb) significantly suppressed IL-12 secretion. Furthermore, native OPN induced a profound level of synthesis of IL-12 from noninfected PBMCs. The major cellular source of OPN was monocytes, because depletion of CD14+ cells resulted in the abrogation of such production. We also examined the regulatory role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in OPN secretion from P. marneffei-stimulated PBMCs. Neutralizing anti-GM-CSF MAb significantly reduced OPN secretion, and treatment with this cytokine induced OPN production from both infected and noninfected PBMCs. Finally, antagonists against the mannose receptor but not the β-glucan receptor almost completely abrogated the production of OPN. Our results demonstrated that OPN secreted from monocytes is involved in the production of IL-12 from PBMCs after stimulation with P. marneffei and that OPN production is regulated by GM-CSF. Our results also indicated the possible involvement of the mannose receptor as a signal-transducing receptor for triggering the secretion of OPN by P. marneffei-stimulated PBMCs.
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.
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.
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.
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.
These results demonstrated that CCL20 might serve as a biomarker for GD and suggested the possible role of OPN in induction of CCL20 expression.
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.
Liver metastasis is a major cause of mortality from colorectal cancer (CRC). However, mechanisms underlying this process are largely unknown. Osteopontin (OPN) is a secreted phosphorylated glycoprotein that is involved in tumor migration and metastasis. The role of OPN in cancer is currently unclear. In this study, OPN mRNA was examined in tissues from CRC, adjacent normal mucosa, and liver metastatic lesions using quantitative real-time PCR analysis. The protein expression of OPN and its receptors (integrin αv and CD44 v6) was detected by using an immunohistochemical (IHC) method. The role of OPN in liver metastasis was studied in established colon cancer Colo-205 and SW-480 cell lines transfected with sense- or antisense-OPN eukaryotic expression plasmids by flow cytometry and cell adhesion assay. Florescence redistribution after photobleaching (FRAP) was used to study gap functional intercellular communication (GJIC) among OPN-transfected cells. It was found that OPN was highly expressed in metastatic hepatic lesions from CRC compared to primary CRC tissue and adjacent normal mucosa. The expression of OPN mRNA in tumor tissues was significantly related with the CRC stages. OPN expression was also detected in normal hepatocytes surrounding CRC metastatic lesions. Two known receptors of OPN, integrin αv and CD44v6 proteins, were strongly expressed in hepatocytes from normal liver. CRC cells with forced OPN expression exhibited increased heterotypic adhesion with endothelial cells and weakened intercellular communication. OPN plays a significant role in CRC metastasis to liver through interaction with its receptors in hepatocytes, decreased homotypic adhesion, and enhanced heterotypic adhesion.
Osteopontin (OPN) is a secreted glycoprotein implicated to function in cancer development and metastasis. Although elevated expression of OPN are observed in cancer cells of various types, in some cases, only the cells in the stromal region surrounding the tumor express OPN, suggesting distinct functional roles for this protein derived from host cells and from cancer cells. To provide a model for addressing the functions and mechanisms of host-derived OPN in cancer progression and metastasis, a cutaneous squamous cell carcinoma cell line (ONSC) that lacks the OPN gene, Spp1, was established. This line of cells was derived from a squamous cell carcinoma that developed in a female, OPN-null mouse subjected to two-stage skin carcinogenesis. Morphologically, ONSC cells resemble epithelial cells, and they express the epithelial markers, K1, K14, and p63, as confirmed by immunohistochemical analyses. Genomic analyses indicate the presence of mutated H-Ras and p53 genes. ONSC cells form colonies in soft agar and, subcutaneously injected into athymic nude mice, develop into squamous cell carcinomas that metastasize to the lungs. Lacking OPN expression, these SCC cells provide a model to address the function of host OPN in the context of cancer progression and metastasis.
Osteopontin; cutaneous squamous cell carcinoma; cell line; tumor progression; metastasis; nude mice
The aggressiveness of pancreatic ductal adenocarcinoma (PDA) is characterized by
its high metastatic potential and lack of effective therapies, which is the
result of a lack of understanding of the mechanisms involved in promoting PDA
metastases. We identified Annexin A2 (ANXA2), a member of the Annexin family of
calcium-dependent phospholipid binding proteins, as a new molecule that promotes
PDA invasion and metastases. We found ANXA2 to be a PDA-associated antigen
recognized by post-treatment sera of patients who demonstrated prolonged
survival following treatment with a PDA-specific vaccine. Cell surface ANXA2
increases with PDA development and progression. Knockdown of ANXA2 expression by
RNA interference or blocking with anti-ANXA2 antibodies inhibits in
vitro invasion of PDA cells. In addition, post-vaccination patient
sera inhibits in vitro invasion of PDA cells, suggesting that
therapeutic anti-ANXA2 antibodies are induced by the vaccine. Furthermore,
cell-surface localization of ANXA2 is tyrosine 23 phosphorylation-dependent; and
tyrosine 23 phosphorylation is required for PDA invasion. We demonstrated that
tyrosine 23 phosphorylation resulting in surface expression of ANXA2 is required
for TGFβ-induced, Rho-mediated epithelial-mesenchymal transition (EMT),
linking the cellular function of ANXA2 which was previously shown to be
associated with small GTPase-regulated cytoskeletal rearrangements, to the EMT
process in PDA. Finally, using mouse PDA models, we showed that shRNA knock-down
of ANXA2, a mutation at tyrosine 23, or anti-ANXA2 antibodies,
inhibit PDA metastases and prolong mouse survival. Thus, ANXA2 is part of a
novel molecular pathway underlying PDA metastases and a new target for
development of PDA therapeutics.
Chronic pancreatitis is a well-known risk factor for pancreatic ductal adenocarcinoma (PDA) development in humans, and inflammation promotes PDA initiation and progression in mouse models of the disease. However, the mechanistic link between inflammatory damage and PDA initiation is unclear. Using a Kras-driven mouse model of PDA, we establish that the inflammatory mediator Stat3 is a critical component of spontaneous and pancreatitis-accelerated PDA precursor formation and supports cell proliferation, metaplasia associated inflammation, and MMP7 expression during neoplastic development. Furthermore, we show that Stat3 signaling enforces MMP7 expression in PDA cells and that MMP7 deletion limits tumor size and metastasis in mice. Finally, we demonstrate that serum MMP7 level in human PDA patients correlated with metastatic disease and survival.
Osteopontin (OPN) binds to CD44 and nuclear factor-κB (NFκB) and OPN mediates tumorigenesis, invasion and metastasis, but the interrelationships between OPN, CD44 and NFκB are not fully understood, and especially in gastric carcinogenesis. We examined the expressions of OPN, CD44, and NFκB in untreated gastric adenocarcinomas.
Materials and Methods
The materials from 211 cases of gastric adenocarcinoma were immunostained for OPN, CD44 and NFκB by using a tissue microarray. The OPN mRNA expression was measured in 10 cases by performing real-time RT-PCR.
The expression of OPN, CD44 and NFκB was noted in 61.7%, 11.4% and 26.6% of the adenocarcinoma tissues, respectively. No significant correlation was detected among the expressions of these proteins. The OPN protein expression was negatively correlated with angioinvasion (p<0.05) and patient survival (p<0.05), whereas the CD44 and NFκB protein expressions were not correlated with any of the clinicopathological factors we examined. The depth of invasion, lymph node status and perineural invasions were prognostic factors based on the Cox analysis. The OPN mRNA expression showed no significant difference between the adenocarcinoma and the paired normal mucosa on real-time RT-PCR.
OPN may have a currently undetermined role in gastric carcinogenesis, and CD44 and NFκB may have minor roles in gastric adenocarcinoma.
Stomach; Adenocarcinoma; Osteopontin; CD44; NFκB