High Osteopontin Research Articles

Streptozotocin (STZ) mediates acute upregulation of serum and pancreatic osteopontin (OPN): a novel islet-protective effect of OPN through inhibition of STZ-induced nitric oxide production

Osteopontin (OPN) is a secreted acidic phosphoprotein that binds to a cell-surface integrin-binding motif and is involved in many inflammatory and immune-modulating disorders. There is compelling evidence that soluble OPN can in a variety of situations help cells survive an otherwise lethal insult. In this study we show that OPN is localized in the rat pancreatic islets and ducts. Staining of pancreatic serial sections with islet hormone antibodies showed that all islet cells express OPN. Rats treated with a single dose of streptozotocin (STZ; 50 mg/kg) showed acute upregulation of serum OPN levels and pancreatic OPN mRNA and protein. Serum OPN dropped by the end of day 7 but was still higher than prediabetic levels. Pancreatic mRNA and protein showed a similar pattern. Twenty-four hours after STZ injection, the intensified OPN expression was localized towards the periphery of the islets and surrounded the remaining insulin-positive cells. To explore the significance of OPN acute upregulation, freshly isolated islets were pretreated with OPN (0.15-15 nM) before addition of STZ. OPN significantly reduced the STZ-induced NO levels in the islets through an Arg-Gly-Asp (RGD)-dependent reduction of inducible NO synthase (iNOS) mRNA levels. Addition of OPN to freshly isolated mildly diabetic islets (blood glucose <300 mg/dl) significantly improved their glucose-stimulated insulin secretion and reduced their NO levels. Next we investigated the regulation of OPN in beta-cells. When STZ (5 mM) was added to the beta-cell line RINm5F it significantly increased OPN mRNA levels within 6 h. To distinguish between the effect of STZ and high glucose on OPN transcription, RINm5F cells were transfected with luciferase-labeled rat OPN promoter and treated with STZ (0.05-5 mM) or with glucose (5-25 mM). STZ induced upregulation of OPN promoter activity within 3 h, while high glucose induced upregulation of OPN promoter activity after 48 h. Our data introduce OPN as a novel islet protein that is differentially regulated by STZ and glucose in the islets. OPN initial upregulation after diabetes induction was probably due to STZ-induced toxicity, while maintenance of the high OPN levels might be due to hyperglycemia. The acute induction of OPN after STZ-induced diabetes might represent an endogenous mechanism to protect the islets against STZ-induced cytotoxicity, partly via an RGD-dependent NO regulatory mechanism.

Osteopontin Expression and Prognostic Significance in Non–Small Cell Lung Cancer

The survival rate of non-small cell lung cancer patients is very low, and knowledge of predictors of outcome is inadequate. To improve the curability of lung cancer, we need to identify new specific molecules involved in tumorigenesis and progression. The purpose of this study was to better define the role of osteopontin in non-small cell lung cancer biology by determining its prognostic significance. Osteopontin expression was evaluated by immunohistochemistry, as percentage of neoplastic cells with cytoplasmic immunoreactivity, in a wide series of patients with stage I-IIIA non-small cell lung cancer (207 cases). The median value of this series (20% of positive cells) was used as the cutoff value to distinguish tumors with low (<20%) from tumors with high (> or =20%) osteopontin expression. Taking the series of patients as a whole (207 cases), osteopontin expression was associated with neither overall survival (P = 0.14) nor disease-free survival (P = 0.074). However, among patients with at least 6 years of follow-up (163 cases), 6-year overall survival and disease-free survival were significantly reduced if osteopontin expression was high (P = 0.0085 for overall survival, P = 0.0023 for disease-free survival). Moreover, a statistically significant correlation between high levels of osteopontin and shorter overall survival (P = 0.034) and disease-free survival (P = 0.011) in patients with stage I tumors (136 cases) was shown. Our results support the hypothesis of an association between high osteopontin expression and poor survival of patients with stage I non-small cell lung cancer, suggesting that osteopontin could be a candidate target for cancer therapy.

Prognostic significance of osteopontin expression in early-stage non-small-cell lung cancer

Osteopontin (OPN) is a multifunctional protein, which has recently been shown to be linked to tumorigenesis, progression and metastasis in different malignancies. Since non-small-cell lung cancer (NSCLC)'s prognosis remains bad, with few predictors of outcome, the purpose of this study was to evaluate if OPN might be involved in NSCLC's biology and therefore represent a prognostic marker and a target for new therapeutic trials. Immunohistochemistry was used to detect OPN expression, evaluated as percentage of neoplastic cells with cytoplasmic immunoreactivity, in a wide cohort of patients with stage I NSCLC (136 cases). The median value of this series (20% of positive cells) was used as the cutoff value to distinguish tumours with low (<20%) from tumours with high (⩾20%) OPN expression. A statistically significant correlation between high levels of OPN and shorter overall (P=0.034) and disease-free (P=0.011) survival in our patients was shown. Our results support the hypothesis that high OPN expression is a significantly unfavourable prognostic factor for the survival of patients with stage I NSCLC. This conclusion has notable importance in terms of the biological characterization of early-stage tumours and therapeutic opportunities.

Clinical Significance of Osteopontin in Esophageal Squamous Cell Carcinoma: Comparison with Common Tumor Markers

Objective: Osteopontin (OPN) is a secreted integrin-binding glycophosphoprotein that may have a role in head and neck squamous cell carcinoma (SCC). To evaluate the clinical significance of OPN in esophageal squamous cell carcinoma (ESCC), we compared plasma OPN levels with those of common tumor markers. Methods: Preoperative plasma OPN levels were measured by enzyme immunoassay in 103 ESCC patients. Serum SCC antigen, Cyfra 21-1, and carcinoembryonic antigen (CEA) levels were also measured routinely at admission by radioimmunoassay. Results: Plasma OPN levels ranged from 82.8 to 1,980 ng/ml. High OPN level was associated with lymph node metastasis (p = 0.05), but not with tumor histology or depth of invasion. The overall survival of the patients with high OPN levels was worse than that of those with low OPN levels (p = 0.02). SCC antigen and Cyfra 21-1 levels were associated with the depth of tumor invasion, the tumor diameter, lymph node metastasis, and the overall survival, but CEA was not associated with these clinicopathological factors. Combined evaluation of OPN plus Cyfra 21-1 or OPN plus SCC antigen was useful as an independent prognostic indicator. Conclusion: Measurement of the plasma OPN level, as well as serum SCC antigen and Cyfra 21-1, may help to predict the progression of ESCC.

Elevation of plasma osteopontin level in patients with undifferentiated nasopharyngeal carcinoma

Aims We evaluated the clinicopathologic relevance of plasma osteopontin (OPN) level in nasopharyngeal carcinoma patients. Methods Seventy-two plasma samples were collected from patients with undifferentiated nasopharyngeal carcinoma (NPC) before radiotherapy. Plasma OPN level was determined by quantitative sandwich enzyme immunoassay. The plasma OPN level was evaluated for its clinicopathologic relevance. Results The mean plasma OPN level was significantly higher in NPC patients than in normal controls (184.66 vs 75.89 ng/ml, p<0.001). In addition, high OPN level was found in the patients with advanced cancer and was correlated with neck node metastasis ( p<0.05). Conclusions Our findings indicated a potential role of OPN in the pathogenesis and nodal metastasis of undifferentiated NPC.

Osteopontin expression in ductal adenocarcinomas and undifferentiated carcinomas of the pancreas

Osteopontin (OPN) is a non-collagenous extracellular matrix protein with pleiotropic functions, including mediation of cell adhesion and migration. Recently, high OPN serum levels were found in pancreatic ductal adenocarcinomas (PDACs) in which OPN mRNA was identified in macrophages. We investigated OPN expression at the protein level in 15 PDACs and 10 undifferentiated pancreatic carcinomas (UCs), 4 of them with osteoclast-like giant cells (OCGCs), to find out whether the degree of OPN expression is related to tumor infiltration by macrophages and the adhesive capacity of tumor cells. With regard to its potential adhesive function, we compared OPN expression in PDACs and UCs with that of E-cadherin and beta-catenin, two well-known adhesive molecules. OPN positivity was observed in two-thirds of PDACs (10/15) and in 7 UCs (7/10), including all 4 UCs with OCGCs. Apart from tumor cells, OPN was expressed in macrophages and OCGCs. When we assessed the relationship between the number of OPN-positive macrophages and tumor cells, we did not find any statistically significant correlation. There was also no correlation, either positive or negative, between OPN expression and E-cadherin and beta-catenin expression. The results demonstrated that, in PDACs and UCs, OPN is expressed in both tumor-associated macrophages and tumor cells. The biological significance of this dual expression pattern is not yet known. It is, however, unlikely that OPN has an adhesive function, since its expression pattern differs distinctly from that of E-cadherin or beta-catenin.

Osteopontin but not osteonectin messenger RNA expression is a prognostic marker in curatively resected non-small cell lung cancer.

The purpose of this study was to better define the role of osteopontin (OPN) and osteonectin [also known as secreted protein acidic and rich in cysteine (SPARC)] in lung tumorigenesis by comparing the expressions of these genes in lung tumor tissue and matched normal tissue and by determining the prognostic significance of the gene expressions. Quantitative real-time reverse transcription-PCR was used to analyze OPN and SPARC mRNA expression in normal lung tissue and matching tumor samples from 82 patients with non-small cell lung cancer. Gene expression data for each patient were matched to survival data. The overall median mRNA expression level of OPN was about 20-fold higher in tumor tissues than in matching normal lung tissues (P < 0.001), whereas SPARC gene expression was not significantly different in both tissue types. Forty of 82 patients had high (>or=4.1) intratumoral OPN expression, and 15 of 82 patients had high (>or15.5) SPARC expression. High OPN expression in the tumor tissue was associated with inferior survival (P = 0.014), whereas high SPARC expression showed a trend toward longer survival (P = 0.095). The impact of high OPN and low SPARC expression on patient survival was additive (P = 0.001). The large increase in OPN expression in tumors compared with normal tissue and its association with survival suggest a role for OPN in lung tumorigenesis.

Osteopontin is up-regulated in human decidual stromal cells

Objective To analyze expression and regulation of osteopontin in human stromal endometrial and decidual cells. Design Study by immunohistochemistry, RNase protection assay, and in vitro study. Setting Academic research unit. Patient(s) Twenty-five fertile women. Intervention(s) Expression of osteopontin mRNA and protein was analyzed by RNase protection assay (RPA) and immunohistochemistry in secretory endometrium and decidua. Regulation of osteopontin expression in endometrial epithelial cell and stromal cells was analyzed by incubation with 17β-estrogen, progesterone, IL-1β, IL-6, and LIF and by incubation with cell culture supernatants of decidual natural killer cells. Furthermore, osteopontin expression was studied in stromal cells, decidualized in vitro. Main outcome measure(s) mRNA and protein expression and regulation of endometrial stromal osteopontin. Result(s) Osteopontin mRNA and protein are expressed at increasing concentrations in human secretory endometrium and in decidua. High concentrations in total decidua were due to high osteopontin concentrations in decidualized stromal cells. Osteopontin mRNA and protein expression was not regulated by incubation of endometrial epithelial and stromal cells with steroids, cytokines, and natural killer cell supernatant for 6 and 24 hours. In contrast, decidualization of stromal cells for 7, 14, and 21 days resulted in significant up-regulation of osteopontin mRNA and protein. Conclusion(s) Osteopontin is expressed at high concentrations in secretory endometrium in epithelial cells and in decidua in stromal cells. Decidual stromal expression of osteopontin is up-regulated by decidualization.

Correlation of Osteopontin Protein Expression and Pathological Stage across a Wide Variety of Tumor Histologies

Osteopontin (OPN) is an integrin-binding protein overexpressed in various experimental models of malignancy and appears to be involved in tumorigenesis and metastasis. Although various studies have assessed OPN protein levels in several tumor types, a broad survey of OPN expression in human neoplasia under the same experimental conditions has not been carried out. We used immunohistochemistry to detect OPN in a selection of 350 human tumors and 113 normal tissues, from a variety of body sites, using stage-oriented human cancer tissue arrays. Tumors included malignancies from breast (26), ovary (22), endometrium (14), esophagus (10), stomach (11), pancreas (16), bile duct (1), liver (9), colon (20), kidney (53), bladder (33), prostate (28), head and neck (60), salivary glands (14), lung (17), skin (6), and brain (10). High cytoplasmic OPN staining was observed in 100% of gastric carcinomas, 85% of colorectal carcinomas, 82% of transitional cell carcinomas of the renal pelvis, 81% of pancreatic carcinomas, 72% of renal cell carcinomas, 71% of lung and endometrial carcinomas, 70% of esophageal carcinomas, 58% of squamous cell carcinomas of the head and neck, and 59% of ovarian carcinomas. Although OPN expression was identified in a good number of bladder, prostate, and brain tumors, the majority of 6 skin cancers, 11 of 14 salivary gland cancers, 2 thyroid carcinomas, and 23 of 26 breast cancers revealed low OPN positivity or were negative. When considering all sites, OPN expression significantly correlated with tumor stage (Spearman's correlation coefficient, P = 0.0002). OPN score and stage were also significantly correlated for specific cancer sites including bladder (P = 0.01), colon (P = 0.004), kidney (P = 0.0001), larynx (P = 0.035), mouth (P = 0.046), and salivary gland (P = 0.011). This study reports the broad distribution of OPN in human tumors from different body sites, suggesting involvement of this protein in tumor formation. The strong correlation between pathological stage and OPN across multiple tumor types suggests a role for OPN in tumor progression.

Differential expression of osteopontin and bone sialoprotein in bone metastasis of breast and prostate carcinoma.

Breast and prostate cancer often metastasise to the skeleton. Interestingly, the histopathological characteristics of the bone lesions that arise from these two cancer types differ. Breast tumours give rise to metastases in the skeleton with a mixed lytic/sclerotic pattern, whereas a predominantly sclerotic pattern is seen in metastases from prostate tumours. Osteopontin (OPN) and bone sialoprotein (BSP) are bone matrix proteins that have been implicated in the selective affinity of cancer cells for bone. In the present study, 21 patient cases with skeletal metastasis and their respective primary tumours (12 with breast cancer, 9 with prostate cancer) were investigated by immunohistochemistry in order to assess the level of OPN and BSP. Moderate to strong OPN expression was found in 42% of all breast tumours and in 56% of all prostate tumours. Significantly more breast cancer bone metastases exhibited high OPN expression, 83%, as compared with prostate tumour bone metastases, 11% (P = 0.0019). In contrast, moderate to strong BSP expression was found in 33% of breast tumours and in 89% of prostate tumours. In the bone lesions, only 33% of breast tumour metastases showed moderate/strong BSP expression compared to 100% of prostate tumour metastases (P = 0.0046). This divergent pattern of OPN/BSP expression could be an important determinant for the different characteristics of these two types of bone metastasis, i.e., lytic vs. sclerotic, consistent with the proposed role of OPN in differentiation and activation of osteoclasts and of BSP as a stimulator of bone mineralisation.

Differences in osteopontin up-regulation between proximal and distal tubules after renal ischemia/reperfusion

Osteopontin (OPN) is a highly acidic phosphoprotein containing an arginine-glycine-aspartic acid (RGD) cell adhesion motif. High OPN expression has been found in tissues with high cell turnover, and OPN up-regulation has been demonstrated in several models of renal injury, suggesting a possible role in tissue remodeling and repair. However, its exact function in the kidney remains unknown. In this study, the possible contribution of OPN to regeneration and repair in the kidney was explored by studying the time course and subcellular localization of OPN up-regulation after renal ischemia/reperfusion injury in different nephron segments and by investigating its relationship with tubular morphology. Rats that underwent 60 minutes of left renal ischemia and a right nephrectomy sacrificed at 10 different time points (from 1 hr to 10 days after reperfusion) were compared with uninephrectomized rats at each time point. In renal tissue sections immunostained for OPN, proximal (PTs) and distal tubules (DTs) in both the renal cortex and outer stripe of the outer medulla (OSOM) were scored for the degree of OPN expression and tubular morphology. Kidneys of uninephrectomized rats showed no injury, and the localization and intensity of their OPN expression remained unaltered compared with normal rats. After ischemia/reperfusion, morphological damage was most severe in PTs of the OSOM, but all examined nephron segments showed a significant increase in OPN expression. The time course of OPN up-regulation was different in PTs and DTs. DTs in both cortex and OSOM rapidly increased their OPN expression, with a maximum at 24 hours after reperfusion followed by a slow decrease. In contrast, PTs showed a delayed increase in OPN staining, with a maximum after five to seven days, higher in the OSOM than in the cortex. In OSOM PTs, OPN expression was predominantly associated with morphological regeneration, whereas DTs showed a substantial OPN up-regulation without major morphological damage. PTs and DTs displayed a different subcellular OPN staining pattern: OPN staining in DTs was located to the apical side of the cell; PTs, however, presented a vesicular, perinuclear staining pattern. Our study found a different pattern of OPN up-regulation after renal ischemia/reperfusion in PTs versus DTs, both with regard to time course and subcellular localization. DTs show an early and persistent increase in OPN staining in the absence of major morphological injury, whereas OPN staining in PTs is delayed and is mostly associated with morphological regeneration. PTs show a vesicular, perinuclear OPN staining pattern, whereas DTs show OPN staining at the apical cell side.

Osteopontin expression in angiotensin II-induced tubulointerstitial nephritis

Osteopontin is an arginine-glycine-aspartate (RGD) containing secreted phosphoprotein recently shown to stimulate a local macrophage influx when injected subcutaneously in mice. We examined the effect of angiotensin II infusion on renal injury and osteopontin expression in the rat kidney by in situ hybridization and immunohistochemistry. Preceding pathologic changes in tubular and interstitial cells, a dramatic increase in renal osteopontin protein and mRNA levels was observed primarily in epithelial cells of the distal tubules, collecting ducts and Bowman's capsule. Although both cortex and medulla showed increased osteopontin levels, the effect was most pronounced in the renal cortex which normally showed very little constitutive osteopontin expression. Interestingly, regions of the kidney expressing high osteopontin levels correlated with sites of monocyte/macrophage accumulation. These observations, coupled with recent findings that osteopontin may be a pro-inflammatory protein, suggests that osteopontin over-expression may facilitate monocyte/macrophage accumulation at the sites of renal tubulointerstitial injury.

Induction of Rapid Osteoblast Differentiation in Rat Bone Marrow Stromal Cell Cultures by Dexamethasone and BMP-2

Adult vertebrates require a continuous supply of osteoblasts for both bone remodeling and regeneration during fracture repair. This implies the existence of a reservoir of cells in the body capable of osteogenesis. One source of these osteoprogenitors is the stem cells within the fibroblastic component of bone marrow stroma. Mature osteoblasts are characterized by high alkaline phosphatase and osteopontin levels, combined with expression of the bone-specific matrix proteins osteocalcin and bone sialoprotein and the capacity for matrix mineralization. We have used these markers to define the conditions permitting rapid osteoblast differentiation from cultured bone marrow stromal cells. Osteoblastic differentiation was induced by continuous culture with 10- 5 M dexamethasone (dex) which stimulated alkaline phosphatase (AP) activity and mRNA levels as well us osteopontin, bone sialoprotein, and osteocalcin mRNA by Day 8 of culture; coaddition of 10 -5 M 1,25-dihydroxyvitamin D 3 (vitamin D) with dex was essential for high osteocalcin mRNA expression. Recombinant bone morphogenetic protein-2 (BMP-2) exerted similar effects to dex and acted in synergy with dex to yield greatly elevated AP activity as well as increased levels of osteoblastic mRNAs. Using in situ hybridization to detect the presence of mRNAs in individual cells, it was shown that appearance of osteopontin mRNA preceded AP mRNA, and was expressed in dex-treated cell colonies as early as Day 4. Quantitation of cell surface AP protein by flow cytometry indicated that culture with dex or BMP-2 produced a mixed population of cells with low AP (dim cells) and cells with high AP levels, while the combination of dex + BMP-2 yielded very few dim cells and a population of cells containing higher AP levels than with either inducer alone. When the dim population from dex-treated cells was sorted and recultured with inducers, these cultures developed high AP levels and were able to deposit a mineralized matrix. Thus, treatment of marrow stromal cells with inducer results in a population of mature osteoblasts as well as a population of undifferentiated cells which retains the capacity for osteoblastic differentiation with further exposure to inducers. These data demonstrate that stem cells within the stromal compartment of bone marrow are capable of rapidly acquiring osteoblast features and suggest a potential role for glucocorticoids in combination with BMP-2 and vitamin D in stages of osteogenic development.

Osteopontin overexpression is associated with arterial smooth muscle cell proliferation in vitro.

To isolate the genes involved in the cell cycle G1 phase progression of arterial smooth muscle cells (SMCs), a cDNA clone (M11) was previously selected by differential hybridization screening of a mid-G1 serum-stimulated SMC cDNA library. The delay of induction after mitogenic stimulation, time of expression, and need for new protein synthesis for full expression made it possible to classify this gene in the "delayed early" gene group. Determination of the partial M11 cDNA sequence showed full homology with the osteopontin gene (secreted phosphoprotein 1, 2ar), an Arg-Gly-Asp-containing extracellular matrix protein. Osteopontin mRNA was also detected in the aorta at levels as high as in the kidney but lower than in bone, two tissues in which it has been previously detected. In vitro analysis of osteopontin expression in serum-stimulated quiescent SMCs and asynchronously cycling SMCs demonstrated that osteopontin overexpression was associated with SMC proliferation. In view of our results, the high osteopontin expression observed by others in the injured carotid artery could be explained by the involvement of SMCs in the proliferative process. Taken together, these results suggest that osteopontin may play an important role in pathological processes that are associated with arterial SMC proliferation, such as atherosclerosis or restenosis.