Osteopontin Protein Expression Research Articles

Bone morphogenetic protein-9 effectively induces osteogenic differentiation of reversibly immortalized calvarial mesenchymal progenitor cells

Critical-sized craniofacial defect repair represents a significant challenge to reconstructive surgeons. Many strategies have been employed in an effort to achieve both a functionally and cosmetically acceptable outcome. Bone morphogenetic proteins (BMPs) provide a robust osteoinductive cue to stimulate bony growth and remodeling. Previous studies have suggested that the BMP-9 isoform is particularly effective in promoting osteogenic differentiation of mesenchymal progenitor cells. The aim of this study is to characterize the osteogenic capacity of BMP-9 on calvarial mesenchymal progenitor cell differentiation. Reversibly immortalized murine calvarial progenitor cells (iCALs) were infected with adenoviral vectors encoding BMP-9 or GFP and assessed for early and late stages of osteogenic differentiation in vitro and for osteogenic differentiation via in vivo stem cell implantation studies. Significant elevations in alkaline phosphatase (ALP) activity, osteocalcin (OCN) mRNA transcription, osteopontin (OPN) protein expression, and matrix mineralization were detected in BMP-treated cells compared to control. Specifically, ALP activity was elevated on days 3, 7, 9, 11, and 13 post-infection and OCN mRNA expression was elevated on days 8, 10, and 14 in treated cells. Additionally, treatment groups demonstrated increased OPN protein expression on day 10 and matrix mineralization on day 14 post-infection relative to control groups. BMP-9 also facilitated the formation of new bone in vivo as detailed by gross, microcomputed tomography, and histological analyses. Therefore, we concluded that BMP-9 significantly stimulates osteogenic differentiation in iCALs, and should be considered an effective agent for calvarial tissue regeneration.

Investigation of association between osteopontin and the formation of idiopathic calcium oxalate stone

Objective To investigate the role of osteopontin(OPN)in the formation of idiopathic calcium oxalate stone(ICS)by detecting OPN transcription and translation level in the renal tissue and the urine concentration of OPN in ICS patients. Methods From January 2013 to January 2014, 34 cases of no-function kidneys were collected after the nephrectomy as the case group, 38 cases of normal renal tissuesfar away from the tumor as the control group after the radical nephrectomy,and 19 healthy persons as the normal group in our hospital.The expression of OPN mRNA and protein in the tissues of kidney were detected by real-time quantitative polymerase chain reaction(Real-time PCR), Western blotting and immunohistochemistry, respectively.The concentration of OPN in the urine of the groups was measured by enzyme-linked immunosorbent assay. Results The levels of OPN mRNA and protein in the renal tissues of the case group(2. 040±1. 200,0. 776±0. 071)were higher than that of control group(2. 590±0. 910, 0. 449±0. 085, P< 0. 05). The concentration of OPN in the urine of the patients in the case group [(1. 052±0. 229) mg/L] was lower than that of control group[(1. 579±0. 256) mg/L] and normal group[(1. 498±0. 155) mg/L, P<0. 05]. Conclusion The over-expression of OPN in the renal tissue and the low concentration of OPN in the urine may play an important role in the formation of ICS. Key words: Osteopontin; Idiopathic calcium oxalate stone; Stone causes

Organic Selenium Alleviated the Formation of Ethylene Glycol-Induced Calcium Oxalate Renal Calculi by Improving Osteopontin Expression and Antioxidant Capability in Dogs.

Twenty one-year-old local male dogs were randomly assigned into four groups (five dogs per group). The control and the ethylene glycol (EG) groups were fed basal diets without and with EG, and the EG+sodium selenite (EG+SS) and EG+selenium yeast (EG+SY) groups were fed basal diets with EG containing SS and SY, respectively. Blood, urine, and renal samples were taken after 18 weeks of feeding. The results showed that compared with the control group, the serum calcium levels and antioxidase activities significantly decreased in the EG group. Serum creatinine, urea nitrogen, and malondialdehyde (MDA) levels and urine calcium and oxalate levels significantly increased. Calcium oxalate crystal deposition and osteopontin (OPN) messenger RNA and protein expression in the renal tissues significantly increased. These changes above in the EG group were reversed within limits by adding selenium in the diets (both EG+SS and EG+SY groups). Further, compared with the EG+SS group, the EG+SY group showed better effects in decreasing the formation of EG-induced calcium oxalate renal calculi and OPN expression and improving antioxidant capability in dogs. It indicates that organic selenium has the potential value to alleviate the formation of EG-induced calcium oxalate renal calculi.

The role of osteopontin expression in melanoma progression

It was shown that osteopontin (OPN), a glycophosphoprotein, plays divergent roles in cancer progression. In addition to multiple intra- and extracellular functions, it facilitates migration of tumour cells, has crucial role in cell adhesion and is associated with increased metastasis formation. In previous studies, we performed global gene expression profiling on a series of primary melanoma samples and found that OPN was significantly overexpressed in ulcerated melanomas. The major purpose of this study was to define OPN expression in primary melanomas with differing biological behaviours. OPN mRNA expression was analysed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in primary melanoma tissues. Immunohistochemistry was performed using a tissue microarray. Cox regression tests were used for survival analysis. Greater than 50 % of the tissues exhibited high protein expression that was significantly associated with tumour thickness and metastasis. OPN mRNA expression was significantly increased in thicker melanomas and lesions with an ulcerated surface. Increased expression was primarily detected in advanced-stage tumours. A multivariate Cox regression analysis revealed that high OPN expression, tumour thickness and metastasis were significantly associated with reduced relapse-free survival. In summary, high OPN mRNA and protein expression were associated with a less favourable clinical outcome of primary melanoma patients. We determined that OPN is a significant predictive factor for the survival of primary melanoma patients. Based on our and others data, the high expression of OPN may have a crucial stimulatory role in tumour progression and metastasis formation, which, thus, have been proposed as potential targets for cancer diagnosis and therapy.

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

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

Nanoporous surface topography enhances bone cell differentiation on Ti–6Al–7Nb alloy in bone implant applications

V-free Ti alloys are viewed as a promising alternative to biomedical Ti–Al–V alloys in bone implant applications. However, the biological response to these alloys depends heavily on the surface characteristics of the implant as well as on the choice of material. This study treated Ti–6Al–7Nb alloys with electrochemical anodization in an alkaline solution for the creation of a thin nanoporous layer (<100nm in thickness) to improve bone cell differentiation. The resulting hydrophilic surface nanotopography resulted in anodized Ti–6Al–7Nb alloy with pores 10–100nm. Compared to untreated alloys, the proposed surface treatment was shown to enhance the blood responses, alkaline phosphatase activity, mineralization, and expression of Runx2 transcription factor and various osteogenic markers, including the gene and protein expression of osteopontin, osteocalcin, bone sialoprotein, and type I collagen, from human bone marrow mesenchymal stem cells. The increased expression of osteogenic markers is expected to promote in vivo osseointegration on Ti–6Al–7Nb alloy in bone implant applications. This might be the first report on the enhancement of osteogenic gene and protein expressions on Ti–6Al–7Nb alloy through rapid formation of hydrophilic surface nanotopography.

Microvascular density and vascular endothelial growth factor and osteopontin expression during the implantation window in a controlled ovarian hyperstimulation rat model.

Vascular endothelial growth factor (VEGF) and osteopontin (OPN) are suggested to facilitate angiogenesis and vascular remodeling in endometrial receptivity. Determination of the endometrial microvascular density (MVD) is the commonest method used to indirectly assess the levels of vasculogenesis and angiogenesis; however, the associations among VEGF, OPN and MVD remain unclear. Controlled ovarian hyperstimulation (COH) with the gonadotrophin-releasing hormone agonist-long protocol may impair endometrial receptivity, and Traditional Chinese Medicine (TCM) may exert therapeutic effects to relieve this impairment. The aim of the present study was to investigate the effects of COH on implantation biology and pregnancy outcome, and to explore the potential therapeutic role of the TCM Zi Dan Yin (ZDY). Female Sprague Dawley rats were divided into four groups: Control, COH, ZDY and COH + ZDY. On days 3, 4 and 5 of pregnancy (D3, D4 and D5, respectively), endometrial MVD was measured with cluster of differentiation 34 immunohistochemical detection, and VEGF and OPN protein and mRNA expression was detected through western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. On D10, the average number of implantation sites was observed. Subsequent to conceiving and bearing newborn rats, the number of live births from each group was recorded. COH was shown to have adverse effects on implantation and pregnancy outcome. The MVD was found to be significantly increased in the COH group compared with that in the control, ZDY and COH+ZDY groups. The results of the protein and RT-qPCR analysis of VEGF and OPN revealed the same trend. Conversely, ZDY reversed the changes in endometrial MVD, VEGF and OPN, and was indicated to improve uterine receptivity and pregnancy outcome. No significant difference was observed among the control, ZDY and COH + ZDY groups. In conclusion, since the results for MVD and VEGF and OPN expression were consistent, MVD could be used as an alternative approach to identify the period of receptivity in rats.

Roles of osteopontin and matrix metalloproteinase-7 in occurrence, progression, and prognosis of nonsmall cell lung cancer.

Background:This study detected osteopontin (OPN) and matrix metalloproteinase-7 (MMP-7) expressions to explore the roles of OPN and MMP-7 in the occurrence, progression, and prognosis of nonsmall cell lung cancer (NSCLC).Materials and Methods:A retrospective study was conducted on NSCLC tissues (n = 152; case group) and adjacent nonneoplastic lung parenchyma (adjacent to tumor >5 cm; n = 152; control group) collected from 152 NSCLC patients. The protein expressions of OPN and MMP-7 were detected by immunohistochemistry. OPN and MMP-7 messenger RNA (mRNA) expressions were detected by reverse transcription polymerase chain reaction (RT-PCR).Results:The protein and mRNA expressions of OPN and MMP-7 in NSCLC tissues were evidently higher than those in adjacent nonneoplastic lung parenchyma (all P < 0.05). OPN protein and mRNA expression were associated with the degree of differentiation, tumor node metastasis (TNM) staging, and lymph node metastasis in NSCLC (all P < 0.05). MMP-7 protein expression was associated with TNM staging and lymph node metastasis (both P < 0.05) while MMP-7 mRNA expression was associated with the degree of differentiation, TNM staging, and lymph node metastasis (all P < 0.05). A significantly positive relativity was revealed between OPN expression and MMP-7 expression (protein: r = 0.789, P < 0.001; mRNA: r = 0.377, P < 0.001). Lymph node metastasis, TNM staging, OPN, and MMP-7 protein expressions were independent risk factors for the prognosis of NSCLC (all P < 0.05).Conclusion:High MMP-7 and OPN protein expressions are closely related to the occurrence, progression, and prognosis of NSCLC, and can be served as unfavorable prognostic factors for NSCLC.

Segment-specific targeting via RNA interference mediates down-regulation of OPN expression in hepatocellular carcinoma cells.

Osteopontin (OPN) plays an important role in the metastasis and recurrence of tumors after resection of hepatocellular carcinoma (HCC). In this study, the down-regulation effect on OPN expression in HCC cells of RNA interference (RNAi) molecules designed to target different segments of OPN was investigated to identify a more effective site for OPN knockdown. Specific small interfering RNAs (siRNAs A, B, and C) of OPN were synthesized and transfected into an HCC cell line (HEP-G2; representing the OPNi-A, OPNi-B, and OPNi-C groups). Fluorescent quantitative polymerase chain reaction and immunohistochemical methods were used to detect the mRNA and protein expression of OPN before and after RNAi. Results showed that after transfection, the fluorescence intensity of the OPNi-A group was greater than those of the OPNi-B and OPNi-C groups. After 48 h of transfection, the ΔCT values of OPN mRNA expression in the OPNi-A-C groups increased from 8.31 ± 1.58, 8.78 ± 1.49, and 8.25 ± 1.51 to 12.14 ± 1.43, 10.22 ± 1.97, and 10.48 ± 1.88, respectively (P < 0.05), and the OPN protein levels (immunohistochemistry scores) decreased from 6.44 ± 1.67, 5.43 ± 2.05, and 5.45 ± 2.52 to 2.84 ± 1.52, 4.43 ± 1.65, and 3.95 ± 1.43 points, respectively. These results indicated that RNAi based on different segments of the OPN gene had different down-regulatory effects on OPN expression. Synthesis of targeted siRNA aimed at specific OPN segments might have important significance for dealing with the invasiveness and metastasis of HCC cells.

Effects of Polypropylene Carbonate/Poly(D,L-lactic) Acid/Tricalcium Phosphate Elastic Composites on Improving Osteoblast Maturation.

Bone tissue engineering utilizing biomaterials to improve osteoblast growth has provided de novo consideration for therapy of bone diseases. Polypropylene carbonate (PPC) is a polymer with a low glass transition temperature but high elasticity. In this study, we developed a new PPC-derived composite by mixing poly-lactic acid (PLA) and tricalcium phosphate (TCP), called PPC/PLA/TCP elastic (PPTE) scaffolds. We also evaluated the beneficial effects of PPTE composites on osteoblast growth and maturation and the possible mechanisms. Compared to PPC polymers, PPTE composites had similar pore sizes and porosities but possessed better hydrophilic surface structures. Biological evaluations further revealed that PPTE composites attracted adhesion of mouse osteoblasts, and these bone cells extended along the porous scaffolds to produce accurate fibroblast-like morphologies. In parallel, seeding mouse osteoblasts onto PPTE composites time-dependently increased cell growth. Sequentially, PPTE composites augmented DNA replication and cell proliferation. Consequently, PPTE composites significantly improved osteoblast mineralization. As to the mechanism, treatment with PPTE composites induced osteopontin (OPN) mRNA and protein expression and alkaline phosphatase activity. Taken together, this study showed that PPTE composites with porous and hydrophilic surfaces can stimulate osteoblast adhesion, proliferation, and maturation through an OPN-dependent mechanism. Therefore, the de novo PPTE scaffolds may have biomaterial potential for bone regeneration.

Mangiferin attenuates renal fibrosis through down-regulation of osteopontin in diabetic rats.

This study was designed to investigate the effects of mangiferin on renal fibrosis, osteopontin production, and inflammation in the kidney of diabetic rats. Diabetes was induced through the single administration of streptozotocin (55 mg/kg, i.p.). Diabetic rats were treated with mangiferin (15, 30, and 60 mg/kg/day, i.g.) for 9 weeks. The kidney was fixed in 10% formalin for glomerulus fibrosis examination using Masson trichrome staining. Kidney and blood were obtained for assays of the associated biochemical parameters. Chronic mangiferin treatment prevented renal glomerulus fibrosis evidenced by decreases in Mason-stained positive area of glomeruli, protein expression of type IV collagen, and α-smooth muscle actin in the kidney of diabetic rats, in comparison with decreases in mRNA and protein expression of osteopontin as well as protein expression of cyclooxygenase 2 and NF-кB p65 subunit in the renal cortex of diabetic rats. Moreover, mangiferin reduced the levels of interleukin 1β in both the serum and the kidney of diabetic rats. Our findings demonstrate that mangiferin prevents the renal glomerulus fibrosis of diabetic rats, which is realized through the suppression of osteopontin overproduction and inflammation likely via inactivation of NF-кB.

Association between the hypomethylation of osteopontin and integrin β3 promoters and vascular smooth muscle cell phenotype switching in great saphenous varicose veins.

Lower extremity varicose veins are a common condition in vascular surgery and proliferation of vascular smooth muscle cells (VSMCs) in the intima is a significant pathological feature of varicosity. However, the pathogenesis of varicose veins is not fully understood. Osteopontin (OPN) could promote the migration and adhesion of VSMCs through the cell surface receptor integrin β3 and the cooperation of OPN and integrin β3 is involved in many vascular diseases. However, the role of OPN and integrin β3 in varicosity remains unclear. In the current study, we found that the methylation levels in the promoter regions of OPN and integrin β3 genes in the VSMCs of varicose veins are reduced and the protein expression of OPN and integrin β3 are increased, compared with normal veins. Furthermore, it was observed that VSMCs in the neointima of varicose veins were transformed into the synthetic phenotype. Collectively, hypomethylation of the promoter regions for OPN and integrin β3 genes may increase the expression of these genes in varicosity, which is closely related to VSMC phenotype switching. Hypomethylation of the promoter regions for OPN and integrin β3 genes may be a key factor in the pathogenesis of varicosity.

Detection of human papillomavirus and expression of osteopontin in cervical cancer specimens.

To understand human papillomavirus (HPV) and expression of osteopontin (OPN) in cervical diseased tissues, HPV infection was detected in paraffin-embedded specimens of cervical lesions from 90 patients with cervical cancer. Three polymerase chain reaction (PCR) techniques were used to determine the detectable rate of HPV infection. Expression of HPV OPN protein was detected using immunohistochemical methods. When a pairwise comparison was made among the three PCR methods, χ2 analysis indicated P>0.05 for detection through the methods of MY09/11 and GP5+/6+, and P>0.05 through the methods of MY09/11 and Nested-PCR. This indicated that there was no statistically significant difference in the HPV infection detection sensitivity of these methods. However, χ2 comparison of the methods of GP5+/6+ and Nested-PCR indicated P<0.05, which demonstrated that there was a statistically significant difference. The rate of positive HPV DNA measured with Nested-PCR was significantly higher than that measured using the GP5+/6+ PCR method. The HPV OPN protein is expressed in cervical cancer, and the HPV OPN polypeptide antibody has broad spectrum reaction capacity and significant multivalence for HPV infection. Immunohistochemical detection was performed on tissue specimens using the purified rabbit HPV OPN polypeptide antibody. Sixty-one cases exhibited a positive result and 29 a negative result. The total rate of positive detection was 67.78%. HPV OPN may therefore serve as a candidate target for tumor treatment, including targeted therapies and vaccine development.

Nonpulsed sinusoidal electromagnetic fields as a noninvasive strategy in bone repair: the effect on human mesenchymal stem cell osteogenic differentiation.

In vivo control of osteoblast differentiation is an important process needed to maintain the continuous supply of mature osteoblast cells for growth, repair, and remodeling of bones. The regulation of this process has also an important and significant impact on the clinical strategies and future applications of cell therapy. In this article, we studied the effect of nonpulsed sinusoidal electromagnetic field radiation tuned at calcium-ion cyclotron frequency of 50 Hz exposure treatment for bone differentiation of human mesenchymal stem cells (hMSCs) alone or in synergy with dexamethasone, their canonical chemical differentiation agent. Five days of continuous exposure to calcium-ion cyclotron resonance affect hMSC proliferation, morphology, and cytoskeletal actin reorganization. By quantitative real-time polymerase chain reaction, we also observed an increase of osteoblast differentiation marker expression such as Runx2, alkaline phosphatase (ALP), osteocalcin (OC), and osteopontin (OPN) together with the osteoprotegerin mRNA modulation. Moreover, in these cells, the increase of the protein expression of OPN and ALP was also demonstrated. These results demonstrate bone commitment of hMSCs through a noninvasive and biocompatible differentiating physical agent treatment and highlight possible applications in new regenerative medicine protocols.

Osteopontin is expressed in the oviduct and promotes fertilization and preimplantation embryo development of mouse.

Osteopontin (OPN) is a multifunctional phosphoprotein that is detected in various tissues, including male and female reproductive tracts. In this study, we evaluated OPN expression in mouse oviducts during the estrus cycle, and at days 1-5 of pregnancy and pseudopregnancy by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The mice oocytes, sperm and embryos were treated with different concentrations of anti-OPN antibody in vitro to detect the function of OPN in fertilization and preimplantation embryo development. OPN mRNA and protein expression in mouse oviducts were cyclic dependent throughout the estrous cycle, which was highest at estrous and lowest at diestrous. Such a phenomenon was consistent with the change in estrogen level in mice. The expression levels of OPN in mice oviduct of normal pregnancy and pseudopregnancy were significantly different, which indicated that OPN expression in mouse oviducts was depend on estrogen and preimplantation embryo. Furthermore, anti-OPN antibody treatment could reduce the rates of fertilization, cleavage and blastocyst formation in vitro in a dose-dependent way. Overall, our results indicated that the expression of OPN in mouse oviducts during the estrous cycle and early pregnancy is likely regulated by estrogen and the embryo, and OPN may play a vital role in oocyte fertilization and preimplantation embryo development.

Effects of osteopontin on hypercalciuria-induced attachment of calcium oxalate crystals to rat renal epithelial cells

Objective To address the effects of osteopontin (OPN) on hypercalciuria contributing to the kidney stone formation.Methods The normal rat kidney epithelial cell line NRK cells were divided into three groups,which were cultured in the medium containing calcium of 0.1,0.3 and 0.6 g/L,respectively.The cell proliferation,the content of OPN in the cell culture supernatants and the level of OPN gene expression at 3,6 and 24 h were examined.The lentivirus vector targeting OPN was constructed.The NRK cells were infected with the lentivirus and then cultured in the medium containing calcium of 0.6 g/L for 24 h.Eventually,The inhibitory effects of silencing OPN gene on the adhesion of calcium oxalate crystals to NRK cells were investigated.Results High level calcium could significantly suppress the proliferation of NRK cells and stimulate secretion of OPN as compared with the blank control group (P ＜ 0.05).The expression of OPN mRNA and protein was also significantly increased (P ＜ 0.05).Calcium concentrations of the lysate of infected NRK cells in the three groups after calcium chloride stimulation were (19.25 ±3.71),(27.22 ±3.82) and (31.91 ± 1.15) mg/L,respectively.The silencing of OPN gene expression could significantly inhibit the abilities of attachment of calcium oxalate crystals to NRK cells as compared with negative lentivirus vector and blank control group,respectively (P ＜ 0.05).Conclusion High level calcium could induce the adhesion of calcium oxalate crystals to NRK cells probably mediated by OPN. Key words: Hypercalciuria ; Osteopontin; Renal epithelial cell

Nanocomposite bioactive polymeric scaffold promotes adhesion, proliferation and osteogenesis of rat bone marrow stromal cells

Synthetic degradable polymer scaffolds have often shown poor cell affinity and bone bioactivity. Bioactive inorganic nanoadditives with the composition of calcium silicate in amorphous phase (CSN) were incorporated within poly (lactic acid) (PLA) to develop nanocomposite bone scaffolds. Nanocomposite solutions containing PLA with 10% CSN were porous-structured by a salt-impregnation / leaching method. The nanocomposite scaffold showed higher dynamical mechanical modulus values than the pure PLA equivalent, indicating a proper usage for hard tissue scaffolds. Rat bone marrow stromal cells (rBMSCs) were cultured on the scaffolds and the cell adhesion, proliferation and osteogenic differentiation at gene and protein levels were addressed. Cells adhered better on the nanocomposite scaffold with respect to PLA equivalent, and then proliferated more during the culture periods of up to 7 days. The expression of osteogenic genes, including collagen type I, osteopontin, osteocalcin and alkaline phosphatase, were significantly stimulated on the nanocomposite scaffold, as confirmed by a real time polymerase chain reaction. Furthermore, Western blot analysis indicated higher expression of osteopontin protein of cells by the nanocomposite scaffold. Results demonstrated the bioactive nanocomposite scaffolds provided appropriate matrix conditions for rBMSCs to populate on and to undergo osteogenic processes in vitro.

Expression and significance of osteopontin and cluster of differentiation 44v6 in brain metastatic tumors

Objective To detect the expression of osteopontin (OPN) and cluster of differentiation 44v6 (CD44v6) in brain metastatic tumors,and to explore their role in the formation and invasion of metastases.Methods By reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical (SP) techniques,the expression of OPN and CD44v6 mRNA and protein was detected in brain metastatic tumors,primary tumor and normal brain tissue.The correlation of their expression was assayed by using statistical methods.Results The relative expression levels of OPN and CD44v6 mRNA were 0.82 ± 0.05 and 0.77 ± 0.05 respectively in brain metastases,and 0.75 ± 0.07 and 0.69 ± 0.04 in primary tumors,which were obviously higher than those in human normal brain tissue (P ＜ 0.05).The positive expression rate of OPN and CD44v6 protein was 92％ (46/50) and 88％ (44/50) respectively in brain metastases,which was obviously higher than that in human normal brain tissue too (P ＜ 0.05).The expression of OPN mRNA and protein had a positive correlation with that of CD44v6 in metastases (r =0.632,P ＜ 0.01 ; r =0.915,P ＜ 0.05).Conclusion The over-expression of OPN and CD44v6 played an important biological role in the formation of brain metastatic tumors,and they showed a synergistic effect. Key words: Osteopontin; Cluster of differentiation 44v6 ; Brain metastatic tumor

Osteopontin is up-regulated in chronic hepatitis C and is associated with cellular permissiveness for hepatitis C virus replication

OPN (osteopontin)) is a Hh (Hedgehog)-regulated cytokine that is up-regulated during chronic liver injury and directly promotes fibrosis. We have reported that Hh signalling enhances viral permissiveness and replication in HCV (hepatitis C virus)-infected cells. Hence we hypothesized that OPN directly promotes HCV replication, and that targeting OPN could be beneficial in HCV. In the present study, we compared the expression of OPN mRNA and protein in HCV (JFH1)-infected Huh7 and Huh7.5 cells, and evaluated whether modulating OPN levels using exogenous OPN ligands (up-regulate OPN) or OPN-specific RNA-aptamers (neutralize OPN) leads to changes in HCV expression. Sera and livers from patients with chronic HCV were analysed to determine whether OPN levels were associated with disease severity or response to therapy. Compared with Huh7 cells, Huh7.5 cells support higher levels of HCV replication (15-fold) and expressed significantly more OPN mRNA (30-fold) and protein. Treating Huh7 cells with OPN ligands led to a dose-related increase in HCV (15-fold) and OPN (8-fold) mRNA. Conversely, treating Huh7.5 cells with OPN-specific RNA aptamers inhibited HCV RNA and protein by >50% and repressed OPN mRNA to basal levels. Liver OPN expression was significantly higher (3-fold) in patients with advanced fibrosis. Serum OPN positively correlated with fibrosis-stage (P=0.009), but negatively correlated with ETBCR (end-of-treatment biochemical response), ETVR (end-of-treatment virological response), SBCR (sustained biochemical response) and SVR (sustained virological response) (P=0.007). The OPN fibrosis score (serum OPN and presence of fibrosis ≥F2) may be a predictor of SVR. In conclusion, OPN is up-regulated in the liver and serum of patients with chronic hepatitis C, and supports increased viral replication. OPN neutralization may be a novel therapeutic strategy in chronic hepatitis C.

Expression of Osteogenic Molecules in the Caudate Nucleus and Gray Matter and Their Potential Relevance for Basal Ganglia Calcification in Hypoparathyroidism

Basal ganglia calcification (BGC) is an interesting example of ectopic calcification in patients with hypoparathyroidism. Its pathogenesis and reasons for predilection of calcification at basal ganglia are not clear. To assess the expression of osteogenesis-related molecules in the caudate nucleus and surface gray matter (an area spared from calcification) and discuss potential relevance of the results in context of BGC in idiopathic hypoparathyroidism. Caudate nucleus and gray matter were obtained from 14 autopsies performed in accidental deaths. The mRNA expression of bone transcription factors (RUNX2/osterix), bone morphogenetic proteins (BMPs) 2 and 4, osteonectin, osteopontin, osteocalcin, vitamin D receptor, calcium sensing-receptor, Na phosphate transporters (PiTs) 1 and 2, N-methyl-D-aspartate receptor 2B (NMDAR2B), carbonic anhydrase II (CA-II), PTH1 receptor (PTH1R), PTH2R, and PTHrP were assessed by RT-PCR. Western blot, spot densitometry, and immunohistochemistry were performed to assess protein expression of molecules showing differences in mRNA expression between caudate and gray tissues. The mean mRNA expression of PiT1 (11.0 ± 10.39 vs 32.9 ± 20.98, P = .003) and PTH2R (1.6 ± 1.47 vs 13.7 ± 6.11, P = .001) were significantly lower in the caudate nucleus than the gray matter. The expression of osteonectin, osteopontin, and CA-II were significantly higher in the caudate nucleus than the gray matter (P = .01, .001, and .04, respectively). The mRNA expression of other molecules was comparable in the 2 tissues. The protein expression of both CA-II and osteonectin was 24% higher and PiT1 17% lower in caudate than the gray matter. The differences in the PTH2R and osteopontin protein expression were not appreciable. The presence of several osteogenic molecules in caudate nucleus indicates that BGC would probably be the outcome of an active process. The differences in expression of these molecules in caudate over gray matter could favor BGC at this site in the unique biochemical milieu of hypoparathyroid state.