骨桥蛋白表达和翻译后修饰与肿瘤生物学行为的关系

骨桥蛋白(osteopontin,OPN)是一种分泌型磷酸化糖蛋白。研究表明OPN在肿瘤中高表达,并通过与CD44突变体CD44v6及整联蛋白αvβ3作用参与肿瘤的发生发展。近年来发现OPN存在磷酸化、糖基化、硫酸化、唾液酸化等多种翻译后修饰,在肿瘤发生发展过程中,这些修饰状态的改变和生物学意义已成为研究关注的热点之一。     

骨桥蛋白在肿瘤血管形成中的作用

骨桥蛋白(osteopontin OPN)是一种糖基化磷酸蛋白,许多肿瘤都可以分泌和表达.大量研究显示:OPN在恶性肿瘤转移播散过程中发挥重要作用.而新生血管形成是肿瘤转移进展过程中非常重要的步骤.OPN与肿瘤新生血管关系密切,癌组织中OPN的高表达与肿瘤微血管密度相关.OPN与许多血管形成因子相互影响协同促进血管形成.OPN通过与整合素和CD44受体结合的细胞信号通路作用于血管形成的重要参与者内皮细胞,影响其增殖,迁移,粘附,趋化,凋亡等生物学特性,并降解细胞外基质为内皮细胞在局部组织中延伸形成血管提供基础.最近的研究也显示OPN可以在血管干/祖细胞水平调节其增殖功能,从而影响肿瘤血管新生.本文将对OPN分子结构,OPN在肿瘤血管形成中所起的作用及相关机制进行综述.     

骨桥蛋白RNA干扰对人U251胶质瘤细胞在裸鼠体内生长的影响

研究下调骨桥蛋白(osteopontin,OPN)对人U251胶质瘤细胞在裸鼠体内生长的影响并探讨其对胶质瘤生长、侵袭的可能机制.应用RNA干扰技术,将OPN基因的慢病毒干扰载体LV-OPNshRNA感染U251细胞.将对照和试验组U251细胞分别接种裸鼠,建立裸鼠荷瘤模型.3周后测量肿瘤的体积、瘤重并做肿瘤组织病理切片分析;利用RT-PCR和免疫印迹法检测OPN、尿激酶型纤维蛋白酶原激活物(uPA)、基质金属蛋白酶(MMP-2、MMP-9)的mRNA和蛋白表达;免疫组化法检测肿瘤组织微血管密度和血管内皮生长因子（VEGF）表达情况. 经OPN的RNA干扰后,能显著降低肿瘤组织OPN mRNA水平及蛋白表达,有效抑制肿瘤细胞生长及侵袭能力, 肿瘤体积及重量的减小有统计学意义(P<0.05).感染组uPA、MMP-2和MMP-9的mRNA和蛋白表达明显减少, 肿瘤组织的MVD值和VEGF的表达均显著降低.上述结果表明,抑制OPN的表达能明显抑制人U251胶质瘤细胞在裸鼠体内的生长和侵袭,OPN可能通过激活uPA、MMP-2和MMP-9等蛋白酶降解细胞外基质和促进肿瘤血管生成,参与胶质瘤的生长.     

骨桥蛋白通过NF-κB上调钙蛋白酶小亚基1促进肝癌细胞迁移

骨桥蛋白(osteopontin,OPN)参与调控多种信号途径激活转移相关基因,进而促进细胞迁移.钙蛋白酶小亚基1(calpain small subunit1,Capn4)与肿瘤转移密切相关,在许多肿瘤及其转移组织中高表达.为了探讨OPN促进肝癌细胞迁移的分子机制,应用报告基因检测、RT-PCR、免疫印迹及伤口愈合等方法检测了肝癌细胞中OPN对Capn4的调控作用及其对肝癌细胞迁移的影响.结果显示,在HepG2细胞中过表达OPN后,Capn4的启动子转录活性显著增强,同时mRNA及蛋白质表达水平也明显上调.在HepG2细胞中应用siRNA干扰OPN的表达可导致Capn4启动子转录活性受到明显抑制,同时mRNA及蛋白质表达水平也显著下调.应用核转录因子-κB(NF-κB)的抑制剂PDTC可抑制由过表达OPN导致的HepG2细胞中Capn4的上调.伤口愈合实验显示,OPN可以通过上调Capn4促进肝癌细胞迁移.因此,研究发现,OPN通过NF-κB上调Capn4的表达,进而促进肝癌细胞的迁移,这一发现对进一步阐明肝癌细胞迁移的分子机制具有重要意义.     

骨桥蛋白13肽抑制球囊内皮剥脱术后血管狭窄的实验研究

目的：利用含有骨桥蛋白（OPN）多种功能位点的13肽（Gly^158-Lys^170）,从细胞和整体水平观察其对VSMC和单核巨噬细胞黏附、浸润以及内膜增生的影响,并初步探讨其作用机制。方法：用不同浓度OPN13肽（0,100,200,300mg／L）检测其对体外培养的平滑肌细胞（VSMc）与OPN黏附的抑制作用;以不合黏附序列的6肽分子为对照组,用以确定13肽抑制黏附特异性。用球囊内皮剥脱法建立大鼠内膜增生模型。实验动物分为4组：治疗组大鼠自术前1h及术后静脉滴注13肽,连续给药7d;对照组大鼠给予非特异性对照6肽分子;模型组大鼠给予相同剂量的生理盐水;正常对照组大鼠施假手术。并利用免疫组织化学染色和Western印迹分析方法,检测血管壁中OPN、FAK、ILK的表达变化。结果：OPN13肽能特异性的及浓度依赖性的抑制VSMC与OPN的相互作用,血管内膜剥脱后给予13肽治疗组血管壁单核／巨噬细胞浸润减少,OPN及其下游信号分子ILK,FAK表达下调,内膜增生被明显抑制。结论：含有OPN多功能位点的13肽可通过阻断OPN与膜受体的相互作用而抑制血管炎症的进展和内膜增生。     

Dkk-1在肿瘤中的研究进展

DKK-1是一种分泌型糖蛋白,通过结合细胞表面受体LRP5/6、Kremen1/2在Wnt通路中起负调控作用。Dkk-1的表达受p53、MYCN、β-catenin等基因调控。Dkk-1在细胞内的异位表达能抑制多种肿瘤细胞的增殖,但有时能在促凋亡因子存在时诱发凋亡。Dkk-1在一些肿瘤中低表达,而在另一些肿瘤中高表达。Dkk-1在不同肿瘤的发生、发展以及转移这几个阶段中的表达和功能表现出复杂多重的差异。该文就Dkk-1在肿瘤中表达和功能的研究进展作一综述。     

骨桥蛋白在神经退行性疾病中的作用

骨桥蛋白(OPN)是一种分子量约为60 KDa的糖基化磷蛋白,广泛分布于骨、脑、肾、肺以及肝等多种重要的脏器组织中.该蛋白通过与整合素、CD44V等受体结合,参与应激反应,癌症,骨重建,炎性反应以及感染等多种生理病理性进展.由于早期分泌OPN能够诱发细胞的激活,故OPN也被称为ETA-1(早期T淋巴细胞激活因子-1).目前发现,OPN存在两种形式:一种是分泌型骨桥蛋白(sOPN),另一种是胞内型骨桥蛋白(iOPN).在体内,二者通过不同的作用途径参与免疫调节过程.近年来,随着分子生物学的进展以及对神经退行性疾病研究的不断深入,发现OPN在神经退行性疾病中似乎发挥着双刃剑的作用,即在某些特定情况下,它能够激发神经毒性和神经元的死亡;而在其他情况下,它起到的是神经保护性作用.本文就OPN的结构特点、生物学功能以及在神经退行性病变中的作用进行简要归纳.     

骨桥蛋白对细胞生存的多重作用

骨桥蛋白(osteopontin, OPN)作为一种分泌性蛋白质广泛存在于多种组织细胞中,不仅调控肿瘤细胞的转移、侵袭和增殖,也在炎症反应、血管再生等生理过程中发挥作用。OPN通过与受体结合直接或间接地激活细胞内信号途径,介导细胞与细胞、细胞与细胞外基质之间的相互作用,从而参与调控细胞的生存。大量实验证实,OPN能够促进细胞的增殖,抑制细胞凋亡,尤其是对于肿瘤细胞。但在有些细胞中,OPN对细胞命运的影响却恰恰相反。本文综述了OPN在不同条件下对细胞存活、活化和增殖,细胞自噬和细胞凋亡的多重作用及其作用途径,为进一步研究OPN对不同细胞作用的受体及其信号网络机制提供重要理论基础。     

肝癌细胞中骨桥蛋白基因表达调控的分子机制研究

骨桥蛋白(osteopontin,OPN)是人体内一种重要的分泌蛋白,在细胞黏附、迁移、机体免疫等作用中发挥重要的生理功能。近年来的研究发现,OPN在恶性肿瘤组织中参与诱导细胞外基质的降解及重构,对肿瘤细胞的侵袭能力有重要调控作用。此外,OPN在肝癌及其他多种肿瘤类型中表达均显著上调,但调控机制尚未完全阐明。该研究从肝癌细胞株中克隆了OPN基因SPP1的启动子序列p SPP1,尝试鉴定肝癌中SPP1的主要调控机制。截断突变体及点突变体分析发现,完整的区域(–24~–17)是p SPP1转录活性所必需的,外源表达AP-1转录因子的c-jun亚基,能够显著提高这一区域的转录活性,然而,c-fos没有这种作用效果。凝胶迁移实验进一步证明AP-1转录因子与p SPP1的直接相互作用。利用实时定量PCR和Western blot还发现,外源表达c-jun能够显著提高内源性OPN的表达水平。以上实验结果揭示,AP-1转录因子介导的转录调控很可能是肝癌细胞OPN表达调控的关键机制,这一研究发现有望为肝癌诊断及治疗提供新的思路。     

骨桥蛋白与肝脏发育、肝再生及肝脏疾病

骨桥蛋白(osteopontin,OPN)是一种分泌型磷酸化糖蛋白,结构上与多种胞外基质蛋白相似,功能上具有细胞因子的特点,在多种生理、病理过程中发挥重要作用。在肝中,它可能参与肝脏发育和肝再生,并与急性肝炎、脂肪性肝炎、肝纤维化及肝癌等疾病的发生、发展密切相关。本文综述了OPN在肝脏发育、肝再生和肝脏疾病等中的作用研究进展,为开展OPN在促进肝再生及肝脏疾病诊断、治疗方面的应用提供重要理论基础。     

Suppression of host Th1-type granulomatous inflammation by Taenia solium metacestodes is related to down-regulation of osteopontin gene expression

Inflammation and granuloma formation in human neurocysticercosis has been attributed to Th1-type immune responses of the host. In the present murine model, over 94% of Taenia solium metacestodes were viable and elicited no granulomatous inflammation, whereas parasites killed by praziquantel treatment elicited rapid granuloma formation that calcified within 2weeks. Osteopontin (OPN) is a Th1-related cytokine that is up-stream of IL-12 and which may play an essential role in granuloma formation and calcification. OPN mRNA expression was down-regulated in tissues surrounding viable cysticerci, but was up-regulated in inflammatory tissues surrounding degenerating cysticerci. Moreover, co-culture with a viable cysticercus or ES products from these metacestodes led to a decrease in OPN, IFN-gamma and IL-12 expression, whereas co-culture with somatic proteins enhanced OPN expression by leukocytes. Addition of recombinant mouse OPN (rmOPN) counteracted the down-regulation of IL-12 and IFN-gamma mRNA expression, but not OPN mRNA expression, in leukocyte cultures. Furthermore, injection of rmOPN into the tissues surrounding implanted cysticerci enhanced inflammatory responses while a similar injection of an anti-rmOPN antibody reduced inflammation. These findings suggest that the suppression of host Th1-type granulomatous inflammation by ES products from T. solium metacestodes is related to down-regulation of OPN gene expression.     

Purification and characterization of osteopontin from human milk

Osteopontin (OPN) is expressed in many organs and tissues and has different biological properties related to different molecular forms in respect to size and posttranslational modifications. However, a purification procedure for authentic intact OPN as well as fragments of OPN from an accessible biological source is missing. A four-step procedure was used to purify OPN from human milk, based on its crystal growth inhibitory activity, including anion exchange chromatography, the elimination of casein, hydroxyapatite chromatography, and negative affinity chromatography. Purified OPN was further separated into its different molecular forms by means of a two-step procedure, involving size exclusion chromatography and reverse phase chromatography. A rabbit polyclonal antibody was raised to purified intact OPN and high M(r) OPN components; the immunoreactivity of both forms was almost equal when investigated by enzyme immunoassay (EIA). The procedures facilitate the purification of intact OPN and OPN fragments for purposes of standardization, preparation of monospecific antibodies, and functional studies.     

Osteopontin stimulates autophagy via integrin/CD44 and p38 MAPK signaling pathways in vascular smooth muscle cells

Osteopontin (OPN) exerts pro‐inflammatory effect and is associated with the development of abdominal aortic aneurysm (AAA). However, the molecular mechanism underlying this association remains obscure. In the present study, we compared gene expression profiles of AAA tissues using microarray assay, and found that OPN was the highest expressed gene (>125‐fold). Furthermore, the expression of LC3 protein and autophagy‐related genes including Atg4b, Beclin1/Atg6, Bnip3, and Vps34 was markedly upregulated in AAA tissues. To investigate the ability of OPN to stimulate autophagy as a potential mechanism involved in the pathogenesis of this disease, we treated vascular smooth muscle cells (SMCs) with OPN, and found that OPN significantly increased the formation of autophagosomes, expression of autophagy‐related genes and cell death, whereas blocking the signal by anti‐OPN antibody markedly inhibited OPN‐induced autophagy and SMC death. Furthermore, inhibition of integrin/CD44 and p38 MAPK signaling pathways markedly abrogated the biological effects of OPN on SMCs. These data for the first time demonstrate that OPN sitmulates autophagy directly through integrin/CD44 and p38 MAPK‐mediated pathways in SMCs. Thus, inhibition of OPN‐induced autophagy might be a potential therapeutic target in the treatment of AAA disease. J. Cell. Physiol. 227: 127–135, 2012. © 2011 Wiley Periodicals, Inc.     

Osteopontin expression according to molecular profile of invasive breast cancer: a clinicopathological and immunohistochemical study

Osteopontin (OPN) is a secreted, calcium-binding phosphorylated glycoprotein involved in several physiological and pathological events such as angiogenesis, apoptosis, inflammation, wound healing, vascular remodeling, calcification of mineralized tissues, and induction of cell proteases. There is growing interest in the role of OPN in breast cancer. In an attempt to obtain new insight into the pathogenesis of OPN-associated breast carcinomas, an immunohistochemical panel with 17 primary antibodies including cytokeratins and key regulators of the cell cycle was performed in 100 formalin-fixed paraffin-embedded samples of invasive breast carcinomas. OPN was expressed in 65% of tumors and was negatively correlated with estrogen (p=0.0350) and progesterone (p=0.0069) receptors, but not with the other markers and clinicopathological features evaluated including age, menstrual status, pathological grading, tumor size, and metastasis. There was no correlation between OPN expression and carcinomas of the basal-like phenotype (p=0.1615); however, OPN correlated positively with c-erbB-2 status (p=0.0286) and negatively with carcinomas of the luminal subtype (p=0.0353). It is well known that carcinomas overexpressing c-erbB-2 protein have a worse prognosis than luminal tumors. Here, we hypothesize that the differential expression of OPN in the first subtype of carcinomas may contribute to their more aggressive behavior.     

Osteopontin-Enhanced Hepatic Metastasis of Colorectal Cancer Cells

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.     

Tumor-derived osteopontin is soluble, not matrix associated.

The secreted phosphoprotein osteopontin (OPN), when immobilized on a surface, supports cell adhesion, prevents apoptosis of endothelial cells, and is a ligand for the alpha(v)beta(3) integrin, which is important in endothelial cell biology and neovascularization. OPN synthesized by tumor cells stimulates tumor growth, but the mechanism by which the protein acts remains unclear. One possibility, therefore, is that OPN may exert its effects on tumor growth by enhancing angiogenesis. While OPN is found at high levels in bone, where it is a component of the mineralized matrix, we have asked here whether OPN present in tumors is similarly extracellular matrix associated. We have shown that OPN is detectable in tumor extracts and in serum of tumor-bearing mice, and that the protein in tumors and in serum can be synthesized by both tumor and the host cells. Biochemical fractionation of tumor tissue confirmed that there is little if any association of OPN with the insoluble fraction. Immunochemical analysis of murine mammary tumors shows no co-localization of OPN with the extracellular matrix, identified by laminin staining. Ras-transformed cells in culture produce abundant OPN, however, the protein was found to be associated with the cell fraction but not with the matrix fraction. An enzyme-linked immunosorbent assay was used to demonstrate that OPN in conditioned medium from these cells fails to associate with extracellular matrix components, including laminin and fibronectin, in vitro. Recombinant OPN (GST-OPN) when coated onto a plastic surface can support human umbilical vein endothelial cell adhesion, suppressing apoptosis and allowing cell cycle progression, at concentrations from 1 to 50 microg/ml. Soluble GST-OPN in the same concentration range has no effect on HUVECs held in suspension. Thus, we conclude that OPN associated with tumors is primarily soluble, and that soluble OPN can neither support endothelial cell proliferation nor prevent apoptosis of these cells in the absence of adhesion.     

The functional and clinical roles of osteopontin in cancer and metastasis

Osteopontin (OPN) is a secreted and integrin-binding protein that has been implicated in a number of pathologies. In this review we will focus on the functional and clinical roles of OPN in cancer and metastasis, with a particular emphasis on breast cancer. While much evidence has suggested that OPN is associated with cancer, its functional contribution to cancer remains poorly understood. Here we will review evidence for mechanisms by which OPN may act to enhance malignancy, including evidence that signaling pathways directly induced by OPN, as well as interactions with growth factor receptor pathways, can combine to activate expression of genes and functions that contribute to metastasis. OPN has been shown to be over-expressed in a variety of human tumors and is present in elevated levels in the blood of some patients with metastatic cancers. We also will discuss recent clinical evidence that suggests that OPN is not only associated with several tumor types, but that levels of OPN in cancer patients' blood or tumors may provide prognostic information.     

Overexpression of human osteopontin increases cell proliferation and migration in human embryo kidney-293 cells

Malignant tumors are characterized by dysregulated cell growth and the metastasis of secondary tumors. Numerous studies have documented that osteopontin (OPN) plays a key role in regulating tumor progression and metastasis. Here, we show that the overexpression of OPN in human embryo kidney-293 cells significantly increases both the level of cell proliferation, by provoking the G₁/S transition, and the level of cell migration in vitro. These findings suggest that augmented OPN contributes to cell growth and motility. Inhibiting OPN or the pathway it stimulates may therefore represent a novel approach for the treatment of primary tumors and associated metastases.     

Overexpression of osteopontin in hepatocellular carcinoma and its relationships with metastasis,invasion of tumor cells

Osteopontin (OPN) plays an important role in metastasis and relapse of human cancer. However, the whole story of OPN relating to cancer has been far from clear untill now. To investigate the expression of OPN in hepatocellular carcinoma (HCC) and its relationships with recurrence and metastasis of HCC, normal and malignant liver tissues from patients with HCC were analyzed using immunohistochemical staining. OPN expression was inhibited by small interfering RNA (siRNA) in HCC cells lines, and then colony formation and matrigel invasion were examined. The results showed that expression of OPN was associated with metastasis of HCC with a positive rate of OPN in the tissue of HCC (70.00%), which was highly more obvious than those in paracarcinoma tissue and normal liver tissue (P < 0.01). In HCC cell lines, OPN depletion could reduce formed colony and metastasizing numbers in vitro. In conclusion, Expression of OPN in the tissue of HCC is related to metastasis or metastases. Specific siRNA could decrease expressions of OPN at both mRNA and protein levels, and abates the invasiveness of hepatocellular carcinoma cells, suggesting that OPN might be a promising agent for treatment of metastasis and recurrence of HCC.     

Role of the metastasis‐promoting protein osteopontin in the tumour microenvironment

Osteopontin (OPN) is a secreted protein present in bodily fluids and tissues. It is subject to multiple post‐translational modifications, including phosphorylation, glycosylation, proteolytic cleavage and crosslinking by transglutamination. Binding of OPN to integrin and CD44 receptors regulates signalling cascades that affect processes such as adhesion, migration, invasion, chemotaxis and cell survival. A variety of cells and tissues express OPN, including bone, vasculature, kidney, inflammatory cells and numerous secretory epithelia. Normal physiological roles include regulation of immune functions, vascular remodelling, wound repair and developmental processes. OPN also is expressed in many cancers, and elevated levels in patients’ tumour tissue and blood are associated with poor prognosis. Tumour growth is regulated by interactions between tumour cells and their tissue microenvironment. Within a tumour mass, OPN can be expressed by both tumour cells and cellular components of the tumour microenvironment, and both tumour and normal cells may have receptors able to bind to OPN. OPN can also be found as a component of the extracellular matrix. The functional roles of OPN in a tumour are thus complex, with OPN secreted by both tumour cells and cells in the tumour microenvironment, both of which can in turn respond to OPN. Much remains to be learned about the cross‐talk between normal and tumour cells within a tumour, and the role of multiple forms of OPN in these interactions. Understanding OPN‐mediated interactions within a tumour will be important for the development of therapeutic strategies to target OPN.