Down-regulation Of Osteopontin Expression Research Articles

Levels of osteopontin in plasma and tissue in relation to metabolic status in patients with lichen planus

Chronic inflammation with lichen planus (LP) has been suggested as a component of the metabolic syndrome (MetS). LP is assumed to be related closely to dyslipidemia. Plasma osteopontin (OPN) has been reported to be a potential clinical marker for the prediction of atherosclerosis. Selenium (Se), an essential trace element, involved in the defense against oxidative stress, has been hypothesized to prevent cardiovascular disease (CVD). Se compounds are effective in the downregulation of OPN expression. Moreover, an inverse relation has been detected between the levels of plasma OPN and plasma Se in patients with psoriasis. To determine the possible role of OPN and Se in the pathogenesis of LP and their relation to the metabolic status in patients. Thirty patients with LP and 30 controls were included. Participants were assessed for the presence of MetS and/or its components. In all participants, plasma and tissue OPN levels were assessed using an enzyme-linked immunosorbant assay, plasma Se levels were assessed by flame atomic absorption spectrophometry, and high-sensitivity C-reactive protein levels were assessed using a solid-phase enzyme-linked immunosorbant assay. Patients with LP showed a significant association with MetS parameters than the controls. Plasma and tissue OPN were significantly higher in LP patients than those in the control group. Plasma Se levels were significantly lower in patients than those in the control group. In patients, plasma OPN levels were associated positively with the presence of diabetes mellitus, dyslipidemia, and MetS. The presence of LP in patients was associated strongly with MetS parameters, most probably because of long-standing inflammation. OPN may be a critical regulator of chronic inflammation in patients with LP and may explain its association with components of the MetS such as diabetes mellitus and dyslipidemia.

Hairy/enhancer of Split Homologue-1 Suppresses Vascular Endothelial Growth Factor-induced Angiogenesis via Downregulation of Osteopontin Expression

Angiogenesis plays a critical role in the progression and vulnerability of atherosclerotic plaques; however, the orchestration of angiogenesis in atherosclerotic plaque formation remains unclear. The results of microarray analysis, real-time PCR and immunohistochemical analyses showed that Hairy/enhancer of split homologue-1 (Hes-1) expression was significantly decreased, while that of osteopontin (OPN) was increased, in atherosclerotic plaques. Meanwhile, immunofluorescence results demonstrated that both Hes-1 and OPN were expressed in endothelial cells (ECs) of neovessels in atherosclerotic plaques. The results of an in vitro study showed that Hes-1 was downregulated, while OPN was upregulated, in a time- and dose-dependent manner in human umbilical vein endothelial cells (HUVECs) by VEGF treatment. In addition, Hes-1 knockdown was found to have transcriptional promotion effect on OPN expression in HUVECs and enhance OPN-induced angiogenesis in response to VEGF. On the contrary, Hes-1 overexpression inhibited OPN expression in HUVECs and reduced angiogenesis in vitro and in vivo. The results of this study suggest that decreased Hes-1 expression in atherosclerotic plaques exaggerate VEGF-induced angiogenesis by upregulating OPN. Therefore, restoring Hes-1 expression and inhibiting OPN expression may be a promising strategy to prevent vulnerable plaque formation in patients with atherosclerosis.

Interleukin-18 deficiency protects against renal interstitial fibrosis in aldosterone/salt-treated mice.

Interleukin (IL)-18 is a member of the IL-1 family of cytokines and was described originally as an interferon γ-inducing factor. Aldosterone plays a central role in the regulation of sodium and potassium homoeostasis by binding to the mineralocorticoid receptor and contributes to kidney and cardiovascular damage. Aldosterone has been reported to induce IL-18, resulting in cardiac fibrosis with induced IL-18-mediated osteopontin (OPN). We therefore hypothesized that aldosterone-induced renal fibrosis via OPN may be mediated by IL-18. To verify this hypothesis, we compared mice deficient in IL-18 and wild-type (WT) mice in a model of aldosterone/salt-induced hypertension. IL-18(-/-) and C57BL/6 WT mice were used for the uninephrectomized aldosterone/salt hypertensive model, whereas NRK-52E cells (rat kidney epithelial cells) were used in an invitro model. In the present invivo study, IL-18 protein expression was localized in medullary tubules in the WT mice, whereas in aldosterone-infused WT mice this expression was up-regulated markedly in the proximal tubules, especially in injured and dilated tubules. This renal damage caused by aldosterone was attenuated significantly by IL-18 knockout with down-regulation of OPN expression. In the present invitro study, aldosterone directly induced IL-18 gene expression in renal tubular epithelial cells in a concentration- and time-dependent manner. These effects were inhibited completely by spironolactone. IL-18 may be a key mediator of aldosterone-induced renal fibrosis by inducing OPN, thereby exacerbating renal interstitial fibrosis. Inhibition of IL-18 may therefore provide a potential target for therapeutic intervention aimed at preventing the progression of renal injury.

Influence of osteopontin silencing on survival and migration of lung cancer cells

Osteopontin (OPN) is a multifunctional protein overexpressed in many cancers and is involved in tumor progression and metastasis. In lung cancer, elevated OPN expression is associated with an unfavorable prognosis. Therefore, inhibition of OPN is an attractive approach for improving survival. We used siRNA to specifically downregulate OPN expression in A549 lung cancer cells. OPN silencing was evaluated with quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for mRNA levels and with Western blotting for protein levels. Effects on cell proliferation were measured by cell counting. The influence on tumor cell migration was detected using a modified Boyden chamber. Changes in cell cycle distribution were assessed by flow cytometry. Using the colony formation assay, we determined changes in radiosensitivity. A specific and effective downregulation of OPN expression was detected in both RNA and protein levels. Cell proliferation and cell migration were significantly reduced by OPN silencing after 24h and the effects were further increased by the addition of irradiation. The cell cycle distribution showed a reduction in S phase and an increase in cells arrested in both G(0)/G(1) and G(2)/M phases. Specific enhancement of radiosensitivity was clearly shown after OPN knockdown. The combination of OPN silencing and irradiation showed a synergistic effect leading to reduced cell survival.

Short (15 Minutes) Bone Morphogenetic Protein-2 Treatment Stimulates Osteogenic Differentiation of Human Adipose Stem Cells Seeded on Calcium Phosphate Scaffolds In Vitro

A one-step concept for bone regeneration has been postulated in which human adipose stem cells (hASCs) are harvested, triggered to differentiate, seeded on carriers, and implanted in the same operative procedure. Toward this goal it was investigated whether short (minutes) incubation with bone morphogenetic protein-2 (BMP-2) suffices to trigger osteogenic differentiation of hASCs seeded on calcium phosphate carriers. hASCs were treated with or without BMP-2 (10 ng/mL) for 15 min, and seeded on β-tricalcium phosphate granules (β-TCP; sized <0.7 mm or >0.7 mm) or biphasic calcium phosphate (BCP; 60%/40% or 20%/80% hydroxyapatite/β-TCP). Attachment was determined after 10-30 min. Proliferation (DNA content) and osteogenic differentiation (alkaline phosphatase activity, gene expression) were analyzed up to 3 weeks of culture. hASC attachment to the different scaffolds was similar, and unaffected by BMP-2. It stimulated gene expression of the osteogenic markers core binding factor alpha 1, collagen-1, osteonectin, and osteocalcin in hASCs seeded on BCP and β-TCP. Downregulation of osteopontin expression by BMP-2 was seen in BCP-seeded cells only. BMP-2 treatment inhibited expression of the adipogenic marker peroxisome proliferator-activated receptor gamma. In conclusion, 15 min BMP-2 preincubation of hASCs seeded on BCP/β-TCP scaffolds had a long-lasting stimulating effect on osteogenic differentiation in vitro. These results strongly support a one-step clinical concept for bone regeneration.

Down-regulation of osteopontin expression by RNA interference affects cell proliferation and chemotherapy sensitivity of breast cancer MDA-MB-231 cells

To determine the effect of the down-regulation of osteopontin (OPN) expression on the sensitivity of breast cancer cells to doxorubicin, a chemotherapeutic drug, OPN shRNA was used to transfect the OPN-positive breast cancer cell line MDA-MB-231. The expression of OPN mRNA and protein was analyzed by RT-PCR and Western blotting, respectively. The cell cycle distribution and apoptosis were analyzed by flow cytometry. Transfected MDA-MB-231 cells were treated with different concentrations of doxorubicin. Growth of MDA-MB-231 cells and IC50 of doxorubicin were determined by MTT assay. Expression of OPN mRNA and OPN protein of cells transfected with OPN shRNA was significantly decreased when compared to the negative controls (P<0.05). The number of MDA-MB-231 cells in the S phase was significantly increased, from 22.77 to 43.67%. Transfection increased apoptosis from 3.61 to 4.91%. Furthermore, the proliferation of MDA-MB-231 cells transfected with OPN shRNA was significantly decreased when compared to the negative control (transfected with empty vector). Sensitivity of MDA-MB-231 cells to doxorubicin was enhanced after treatment with OPN shRNA. The IC50 values of doxorubicin of the OPN-transfected group, the MDA-MB-231 cells and the negative transfected group were 0.13588, 0.83869 and 0.79652 µg/ml, respectively. Down-regulation of OPN significantly inhibits expression of OPN protein in MDA-MB-231 breast cancer cells, reduces cell proliferation and increases their sensitivity to doxorubicin.

Downregulation of osteopontin is associated with fluoxetine amelioration of monocrotaline-induced pulmonary inflammation and vascular remodelling

1. Osteopontin (OPN) has emerged as a key factor in inflammatory activation and cardiovascular remodelling. The aim of the present study was to investigate the involvement of OPN in fluoxetine amelioration of monocrotaline (MCT)-induced pulmonary inflammation and vascular remodelling in rats. 2. Wistar rats were divided into control, MCT and two fluoxetine-treated groups. Pulmonary arterial hypertension (PAH) was induced by a single injection of MCT (60 mg/kg, i.p.). Fluoxetine (2 and 10 mg/kg) was administered via the intragastric route once a day for 21 days. On Day 22, pulmonary haemodynamic measurements were undertaken, followed by ELISA, western blotting and immunohistochemistry. 3. Monocrotaline caused pulmonary inflammation and vascular remodelling and significantly enhanced OPN expression in the plasma, lungs and pulmonary arteries. Fluoxetine decreased pulmonary arterial pressure and ameliorated pulmonary inflammation and pulmonary vascular remodelling. At 10 mg/kg, fluoxetine significantly inhibited MCT-induced increases in the expression of serotonin transporter (SERT) and phosphorylated extracellular signal-regulated kinase 1/2 and downregulated the expression of OPN, macrophage inflammatory protein-1β and matrix metalloproteinase 2/tissue inhibitor of metalloproteinase 2. Although 2 mg/kg fluoxetine tended to ameliorate some MCT-induced changes in the lung, the differences did not always reach statistical significance. Linear regression analysis showed that there was a positive correlation between plasma OPN concentrations and mean pulmonary arterial pressure, as well as percentage medial wall thickness and percentage wall area in the pulmonary artery. 4. In conclusion, the amelioration by fluoxetine of MCT-induced pulmonary inflammation and vascular remodelling is associated with downregulation of OPN expression in rats.

Osteopontin silencing by small interfering RNA induces apoptosis and suppresses invasion in human renal carcinoma Caki-1 cells

Antiapoptosis and invasion are the causes for the failure of conventional radiotherapy and chemotherapy in human renal carcinomas. Osteopontin (OPN), a ligand for vß3 integrin and CD44 receptors, is a phosphorylated glycoprotein with diverse functions including tumorigenesis and tumor cell metastasis. Recently, OPN has been detected in human renal carcinomas and assessed as a potential prognostic marker of renal carcinomas. However, the function and mechanism of OPN in renal carcinomas remain unknown. In this study, we used OPN siRNA to silence the expression of OPN in renal carcinoma Caki-1 cells. Silent effect showed that sequence-specific siRNA targeting OPN suppressed OPN mRNA expression by 81% and OPN protein level by 91% in vitro. The apoptosis ability was significantly increased while the invasion ability was decreased in Caki-1 cells transfected with OPN siRNA. Western blot demonstrated that the effects of OPN silence were significantly accompanied by the activation of mitochondria-related apoptosis pathway involving cytochrome c, Apaf-1, cleaved caspase-3 and Bcl-2/Bax, and the downregulation of invasion-related proteins of MMP-2 and uPA expression. These results suggest that the downregulation of OPN expression can induce apoptosis increase and invasion suppression in renal carcinoma Caki-1 cells through mitochondria-related apoptosis pathway and MMP-2 and uPA-related invasion proteins, respectively.