Localization of CD44 at the Invasive Margin of Glioblastomas by Immunoelectron Microscopy

Glioblastoma multiforme is a highly invasive primary brain tumor, which is known to strongly express the CD44 cell adhesion receptor. A number of experimental studies suggest that the interaction of this receptor with extracellular matrix (ECM) proteins such as hyaluronic acid may in part mediate human glioma cell adhesion and invasion of brain tissue. Although the expression of CD44 and its spliced variants in brain tumors have been extensively studied, there have been no reports localizing its expression to the invasive margin of the tumor. The authors used immunoelectron microscopy to investigate the expression patterns of CD44 in an in vitro organotypic invasion assay. Tumor spheroids initiated from the U373 MG human glioblastoma line were confronted with fetal rat brain aggregates in a spheroid coculture system. The CD44 expression appeared at the interface between glioblastoma tumor spheroids and brain tissue, as well as in the spheroid itself. CD44 immunoreactivity was not detectable in mature 21-day fetal brain aggregates. The findings provide direct evidence that CD44 is expressed at the confrontational invasive border between glioblastomas and brain tissue, further supporting its role in glioma cell-ECM recognition and attachment.     

A soluble Fn14-Fc decoy receptor reduces infarct volume in a murine model of cerebral ischemia

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily. TWEAK acts on responsive cells via binding to a small cell surface receptor named Fn14. Recent studies have demonstrated that TWEAK can stimulate numerous cellular responses including cell proliferation, migration, and proinflammatory molecule production, but the role of this cytokine in cardiovascular disease and stroke has not been established. The present study investigated whether TWEAK or Fn14 expression was regulated in a murine model of cerebral ischemia and whether TWEAK played a role in ischemia-mediated cell death. We found that TWEAK and Fn14 were expressed by primary mouse cerebral cortex-derived astrocytes and neurons cultured in vitro. Also, both the TWEAK and Fn14 proteins were present at elevated levels in the ischemic penumbra region after middle cerebral artery occlusion. Finally, we report that intracerebroventricular injection of a soluble Fn14-Fc decoy receptor immediately after middle cerebral artery occlusion significantly reduced infarct volume and the extent of microglial cell activation and apoptotic cell death in the ischemic penumbra. We conclude that the cytokine TWEAK may play an important role in ischemia-induced brain injury and that inhibition of TWEAK expression or function in the brain may represent a novel neuroprotective strategy to treat ischemic stroke.     

TWEAK反义寡核苷酸对人结肠癌细胞系SW480增殖及侵袭能力的影响

目的观察肿瘤坏死因子样凋亡微弱诱导剂(TWEAK)对人结肠癌细胞系SW480增殖及侵袭能力的影响。方法合成TWEAK反义寡核苷酸(ASODN)、错义寡核苷酸(SCODN)后转染人结肠癌细胞系SW480,将细胞分为空白对照组、脂质体(LF)对照组、ASODN组、SCODN组;采用MTT法检测肿瘤细胞增殖抑制情况,流式细胞术(FCM)检测肿瘤细胞周期分布,ELISA法检测细胞培养液上清中可溶性TWEAK及其受体成纤维细胞生长因子诱导早期反应蛋白14(Fn14)的表达,Western blotting法、免疫荧光细胞化学法(IF)检测细胞中的TWEAK及Fn14蛋白的表达,运用Transwell侵袭实验观测结肠癌细胞侵袭能力的变化。结果空白对照组、LF对照组、SCODN组之间结肠癌细胞的增殖情况无明显差异(P0.05),与对照组相比ASODN组肿瘤细胞增殖受到明显抑制(P0.05),且呈剂量-时间依赖关系;转染TWEAK ASODN后,G2+M期细胞比例明显高于对照组(P0.05),TWEAK及其受体Fn14在结肠癌细胞培养液及细胞内的表达均低于对照组,差异均有统计学意义(P0.05);转染TWEAK ASODN后肿瘤细胞侵袭抑制率为31.39%,明显高于对照组(P0.05);TWEAK及其受体Fn14在结肠癌细胞培养液及细胞内的表达均密切相关(P0.05)。结论 TWEAK ASODN可能通过抑制TWEAK与其受体Fn14结合干扰肿瘤的发生发展,抑制TWEAK表达能抑制人结肠癌细胞系SW480的增殖与侵袭。     

Platelet-derived growth factor receptor-beta is induced during tumor development and upregulated during tumor progression in endothelial cells in human gliomas.

BACKGROUND: Endothelial cells proliferate during brain development, are quiescent in normal adult brain but proliferate again under pathologic conditions such as glioma growth. The vascular phenotype of low grade glioma is comparable to normal brain, however high grade gliomas are focally highly vascularized and there is associated prominent endothelial cell proliferation. The mechanisms of this change in vascular phenotype are unknown but there is evidence that growth factors play an important role in this process as well as in normal angiogenesis and vascular differentiation. EXPERIMENTAL DESIGN: To investigate whether endothelial cells become activated during tumorigenesis and progression of human gliomas by a platelet-derived growth factor (PDGF) dependent pathway, we analyzed platelet-derived growth factor receptor-beta (PDGFR-beta) expression by in situ hybridization and immunocytochemistry in normal human brain, astrocytoma (grade II), anaplastic oligo-astrocytoma (grade III), and glioblastoma multiforme (grade IV). RESULTS: PDGFR-beta mRNA was not detectable in the vessels of normal human brain, but was expressed in the vasculature of low and high grade gliomas, particularly in endothelial cell proliferations in glioblastomas. The expression of the receptor in the tumor microvessels, was confirmed by double immunofluorescence in which the staining appeared to be in the endothelial cells. Primary cultures of endothelial cells derived from glioblastoma multiforme maintained receptor expression for 2 days in vitro, whereas it was not detectable in vitro in endothelial cells derived from normal brain. Tumor cells in all grades of glioma expressed very little PDGFR-beta mRNA in situ. CONCLUSIONS: Our results indicate that the malignant phenotype in human glial tumors is associated with an upregulation of the PDGFR-beta on endothelial cells of vessels which vascularize the tumor. These findings may contribute to our understanding of the mechanisms that regulate vessel growth and differentiation in normal and pathologic states.     

Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 during cardiac remodelling in rats

Aim: Accumulating evidence supports the concept that proinflammatory cytokines play an essential role in the failing heart. We examined the concomitant tumour necrosis factor-like weak inducer of apoptosis (TWEAK)/Fn14 expression in myocytes in vitro as well as in vivo in cardiac remodelling. Methods: We assessed TWEAK and its receptor Fn14 expression in response to angiotensin (Ang) II, myocardial infarction (MI) as well as to local adenovirus-mediated p38 gene transfer in vivo. The effect of various hypertrophic factors and mechanical stretch was studied in neonatal rat ventricular myocyte cell culture. Results: Ang II increased Fn14 levels from 6 h to 2 weeks, the greatest increase in mRNA levels being observed at 6 h (6.3-fold, P < 0.001) and protein levels at 12 h (4.9-fold, P < 0.01). TWEAK mRNA and protein levels remained almost unchanged during Ang II infusion. Likewise, a rapid and sustained elevation of Fn14 mRNA and protein levels in the left ventricle was observed after experimental MI. Moreover, local p38 gene transfer increased Fn14 mRNA and protein but not TWEAK levels. Fn14 immunoreactive cells were mainly proliferating non-myocytes in the inflammation area while TWEAK immunoreactivity localized to cardiomyocytes and endothelial cells of the coronary arteries. Hypertrophic agonists and lipopolysaccharide increased Fn14 but not TWEAK gene expression in neonatal rat myocytes, while mechanical stretch upregulated Fn14 and downregulated TWEAK gene expression. Conclusions: In conclusion, the cardiac TWEAK/Fn14 pathway is modified in response to myocardial injury, inflammation and pressure overload. Furthermore, our findings underscore the importance of Fn14 as a mediator of TWEAK/Fn14 signalling in the heart and a potential target for therapeutic interventions.     

Participation of host cells: resistance or collaboration

Matrix metalloproteinases play an important regulatory role in tissue morphogenesis, cell differentiation and motility, and tumor cell invasiveness. We have recently demonstrated elevated activity of the 92 kDa type IV collagenase (MMP-9) in human glioblastoma and in the present study examine the relative amounts of MMP-9 protein and mRNA in human gliomas and as well as the distribution of MMP-9 in human glioma tumors in vivo. Using an enzyme-linked immunosorbent assay for the quantitative determination of MMP-9 protein, we found that levels were significantly higher in malignant astrocytomas, especially in glioblastoma multiforme, than in normal brain tissues and low-grade gliomas. In addition, the amount of MMP-9 mRNA, as determined by northern blot analysis was higher in anaplastic astrocytomas and glioblastoma multiforme than in normal brain tissue and low-grade gliomas. Immunocytochemical staining for MMP-9 showed strong cytoplasmic immunoreactivity in the tumor cells and the proliferating endothelial cells of glioblastoma multiforme and anaplastic astrocytomas. The staining intensity was lower in low-grade astrocytomas, and was undetectable or very low in normal brain astrocytes. The results indicate that expression of MMP-9 is dramatically upregulated in highly malignant gliomas and correlates with the highly malignant progression of human gliomas in vivo, and support a role for the MMP-9 in facilitating the invasiveness seen in malignant gliomas in vivo.     

TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice

Neuropsychiatric disease is one of the most common manifestations of human systemic lupus erythematosus, but the mechanisms remain poorly understood. In human brain microvascular endothelial cells in vitro, TNF-like weak inducer of apoptosis (TWEAK) decreases tight junction ZO-1 expression and increases the permeability of monolayer cell cultures. Furthermore, knockout (KO) of the TWEAK receptor, Fn14, in the MRL/lpr lupus mouse strain markedly attenuates neuropsychiatric disease, as demonstrated by significant reductions in depressive-like behavior and improved cognitive function. The purpose of the present study was to determine the mechanisms by which TWEAK signaling is instrumental in the pathogenesis of neuropsychiatric lupus (NPSLE). Evaluating brain sections of MRL/lpr Fn14WT and Fn14KO mice, we found that Fn14KO mice displayed significantly decreased cellular infiltrates in the choroid plexus. To evaluate the integrity of the blood brain barrier (BBB) in MRL/lpr mice, Western blot for fibronectin, qPCR for iNOS, and immunohistochemical staining for VCAM-1/ICAM-1 were performed. We found preserved BBB permeability in MRL/lpr Fn14KO mice, attributable to reduced brain expression of VCAM-1/ICAM-1 and iNOS. Additionally, administration of Fc-TWEAK intravenously directly increased the leakage of a tracer (dextran-FITC) into brain tissue. Furthermore, MRL/lpr Fn14KO mice displayed reduced antibody (IgG) and complement (C3, C6, and C4a) deposition in the brain. Finally, we found that MRL/lpr Fn14KO mice manifested reduced neuron degeneration and hippocampal gliosis. Our studies indicate that TWEAK/Fn14 interactions play an important role in the pathogenesis of NPSLE by increasing the accumulation of inflammatory cells in the choroid plexus, disrupting BBB integrity, and increasing neuronal damage, suggesting a novel target for therapy in this disease.     

TWEAK/Fn14 axis: The current paradigm of tissue injury-inducible function in the midst of complexities

TNF-like weak inducer of apoptosis (TWEAK), a TNF family ligand, and its only known signaling receptor, FGF-inducible molecule-14 (Fn14), have emerged as a key molecular pathway regulating tissue responses after acute tissue injury and in contexts of chronic injury and disease, including autoimmunity, chronic inflammation, fibrosis, and malignancy. Usually dormant due to the low level of Fn14 expression in healthy tissues, this axis is specifically activated by the upregulation of Fn14 expression locally within injured tissues, thereby triggering a wide range of activities in tissue parenchymal and stromal cells as well as tissue progenitor cells. Current evidence supports that although transient TWEAK/Fn14 pathway activation may be beneficial for tissue repair after acute injury, excessive or sustained TWEAK/Fn14 activation due to repeated injury or chronic disease mediates significant tissue damage and pathological tissue remodeling. This paradigm for the dichotomous function of the TWEAK/Fn14 pathway is discussed, highlighting emerging findings, complexities, and implications for the treatment of tissue damage-associated pathologies and cancer.     

Tumor necrosis factor-like weak inducer of apoptosis and fibroblast growth factor-inducible 14 mediate cerebral ischemia-induced poly(ADP-ribose) polymerase-1 activation and neuronal death

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Fn14) are expressed in neurons. Here we demonstrate that TWEAK induces a dose-dependent increase in neuronal death and that this effect is independent of tumor necrosis factor alpha (TNF-α) and mediated by nuclear factor-kappa B (NF-κB) pathway activation. Incubation with TWEAK induces apoptotic cell death in wild-type (Wt) but not in Fn14 deficient (Fn14(-/-)) neurons. Intracerebral injection of TWEAK induces accumulation of poly(ADP-ribose) polymers (PAR) in Wt but not in Fn14(-/-) mice. Exposure to oxygen-glucose deprivation (OGD) conditions increases TWEAK and Fn14 mRNA expression in Wt neurons, and decreases cell survival in Wt but not in Fn14(-/-) or TWEAK deficient (TWEAK(-/-)) neurons. Experimental middle cerebral artery occlusion (MCAO) increases the expression of TWEAK and Fn14 mRNA and active caspase-3, and the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) with accumulation of PAR in the ischemic area in Wt but not Fn14(-/-) mice. Together, these results suggest a model where in response to hypoxia/ischemia the interaction between TWEAK and Fn14 in neurons induces PARP-1 activation with accumulation of PAR polymers and cell death via NF-κB pathway activation. This is a novel pathway for hypoxia/ischemia-induced TWEAK-mediated cell death and a potential therapeutic target for ischemic stroke.     

Brain angiogenesis inhibitor 1 is differentially expressed in normal brain and glioblastoma independently of p53 expression

Brain angiogenesis inhibitors (BAI) are putative transmembrane proteins containing an extracellular domain with thrombospondin type-1 repeats which can exhibit anti-angiogenic activity. BAI1 mRNA is expressed mainly in the brain, while BAI2 and BAI3 mRNAs are more widely expressed. We hypothesized that the BAI family might have anti-tumoral properties and studied the expression of BAI1 protein in normal human brain and in glioblastoma multiforme. We generated an anti-BAI1 antibody and showed that BAI1 was widely expressed in normal brain but was absent in 28 glioma cell lines and in the majority of human glioblastoma investigated. BAI1 expression did not correlate with TP53 status and we did not confirm previous findings that p53 regulates BAI1 mRNA expression in glioma cells. The finding that expression of BAI proteins may be lost during tumor formation is of special interest as restoration of their function in tumors may be of therapeutic benefit.     

TWEAK/Fn14 pathway: an immunological switch for shaping tissue responses

Our immune system performs the vital function of recognizing and eliminating invading pathogens and malignancies. There is an increasing appreciation that the immune system also actively mediates tissue responses under both physiological and pathological conditions, significantly impacting the inflammatory, fibrogenic, and regenerative components. Likewise, there is a growing understanding of how epithelial, endothelial, and other non-hematopoietic tissue cell types actively contribute to the interplay that shapes tissue responses. While much of the molecular basis underlying the immune regulation of tissue responses remains to be delineated, the tumor necrosis factor (TNF) superfamily ligand/receptor pair of TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible molecule 14 (Fn14) has now emerged as a key piece of this puzzle. In this review, we first discuss how the usually 'dormant' TWEAK/Fn14 pathway becomes activated specifically in injury and disease contexts. We then summarize how TWEAK-mediated Fn14 signaling triggers a wide range of activities in tissue parenchymal and stromal cells as well as progenitor cells. Finally, we review recent experimental evidence that further supports the functional dichotomy of TWEAK/Fn14 activation in physiological versus pathological tissue responses and its potential therapeutic implications. Whereas transient TWEAK/Fn14 activation promotes productive tissue responses after injury, excessive or persistent TWEAK/Fn14 activation drives pathological tissue responses, leading to progressive damage and degeneration.     

No end in site: TWEAK/Fn14 activation and autoimmunity associated- end-organ pathologies

Growing evidence suggests that the tumor necrosis factor superfamily (TNFSF) member TWEAK and its cognate receptor Fn14 play an important role in both physiological and pathological tissue remodeling. Herein, we review the various lines of experimental evidence that support the involvement of this ligand/receptor pair in triggering a wide range of cellular responses crucial to tissue remodeling, including angiogenic, proliferative, and inflammatory responses, and discuss the molecular mechanisms by which TWEAK/Fn14-induced tissue responses can lead to desired vs. undesired consequences in a context-dependent manner. We explore the key features of TWEAK-induced end-organ pathologies in various autoimmune disorders and the potential therapeutic benefits of TWEAK inhibition therein. We submit the viewpoint that TWEAK/Fn14-mediated pathogenic tissue remodeling represents an important, universal mechanism leading to various end-organ pathologies associated with autoimmune and inflammatory disorders. The highly specific and localized nature of its pathogenic contribution, therefore, makes the TWEAK/Fn14 pathway a unique and promising therapeutic target.     

TWEAK和Fn14在腰椎间盘退变髓核组织中的表达及意义

目的 研究TWEAK和Fn14在腰椎间盘退变髓核组织中的表达及其临床意义.方法 用半定量RT-PCR和免疫组织化学法检测实验组(30例退变腰椎间盘)和对照组(10例创伤腰椎间盘)中TWEAK和Fn14 mRNA及蛋白表达.结果 实验组TWEAK mRNA表达显著高于对照组(0.949±0.093 vs 0.653±0.110,P＜0.01),实验组TWEAK蛋白表达显著高于对照组(0.682±0.126 vs 0.397±0.057,P＜0.01),实验组Fn14 mRNA表达显著高于对照组(0.936±0.125vs 0.632±0.059,P＜0.01),Fn14蛋白表达显著高于对照组(0.540±0.051 vs 0.344±0.072,P＜0.01).结论TWEAK和Fn14的表达增加可能参与腰椎间盘退变的进程.     

TWEAK/Fn14 pathway promotes a T helper 2-type chronic colitis with fibrosis in mice

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a TNF superfamily member, induces damage of the epithelial cells (ECs) and production of inflammatory mediaters through its receptor Fn14 in a model of acute colitis. In our current study of chronic colitis induced by repeated rectal injection of a hapten, we found that inflammation, fibrosis, and T helper 2 (Th2)-type immunity were significantly reduced in Fn14 gene knockout (KO) mice when compared with wild-type (WT) control mice. Expression of thymic stromal lymphopoietin (TSLP) was lower in Fn14 KO colon ECs than in WT ECs. TWEAK potentiates the induction of TSLP by interleukin-13 (IL-13) in colon explants from WT but not in Fn14 KO tissue. TSLP receptor KO mice exhibit milder chronic colitis, similar to that in Fn14 KO mice. TWEAK and IL-13 synergistically promote fibroblast proliferation. Thus we propose an IL-13-TWEAK/Fn14-TSLP axis as a key mechanism underlying chronic colitis with fibrosis.     

Anti-TWEAK monoclonal antibodies reduce immune cell infiltration in the central nervous system and severity of experimental autoimmune encephalomyelitis

TWEAK is a member of the TNF family, constitutively expressed in the central nervous system (CNS), with pro-inflammatory, proliferative or apoptotic effects depending upon cell types. Its receptor, Fn14, is expressed in CNS by endothelial cells, reactive astrocytes and neurons. We showed that TWEAK and Fn14 mRNA expression increased in spinal cord during experimental autoimmune encephalomyelitis (EAE). We investigated the role of TWEAK during EAE using neutralizing anti-TWEAK antibody in myelin oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice. We observed a reduction of disease severity and leukocyte infiltration when mice were treated after the priming phase.     

Molecular mechanism of SSFA2 deletion inhibiting cell proliferation and promoting cell apoptosis in glioma

Gliomas are the most common primary brain malignant tumors in humans. Glioblastoma multiforme(GBM) is the most malignant intracranial tumor with a relatively poor prognosis. There promote us to find effective anti-cancer therapies to reduce cancer mortality. By using bioinformatic analysis, we found SSFA2 as a gene with elevated expression in the glioma tissues. We detected the expression of SSFA2 in glioma tissues and in the glioma cell lines, as well as in normal brain tissues. SSFA2 expression was higher in glioma tissues, especially in glioblastoma multiforme than normal brain tissues. Subsequently, we found that down-regulate SSFA2 in glioma cell lines can regulate the cell cycle to reduce the proliferation ability and induce the early apoptosis rate in shSSFA2 cells relative to control cells. Moreover, we found that down-regulate SSFA2 in glioma cell line U87(shSSFA2-U87) inhibited the growth effectiveness compared to the control cell line U87. These result reveals us that SSFA2 may act as oncogene to promote the progression of glioma. For further research specific mechanisms of SSFA2 in gliomas, we used the gene chip to detect the downstream gene in U87. We found that 30 genes also may be as target gene of SSFA2, and we testify the protein expression by western-blot. The result reveal that IL1A, IL1B and CDK6 as target gene of SSFA2 to regulate the progression of glioma. These finding suggest that SSFA2 could be a new therapeutic target for gliomas.     

Endogenous TWEAK is critical for regulating the function of mouse uterine natural killer cells in an immunological model of pregnancy loss

Uterine natural killer (uNK) cells are the most abundant lymphocyte population in the feto–maternal interface during early gestation, and uNK cells play a significant role in the establishment and maintenance of pregnancy‐related vascularization, as well as in tolerance to the fetus. Tumour necrosis factor‐like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor‐inducible molecule (Fn14), are involved in preventing local cytotoxicity and counterbalancing the cytotoxic function of uNK cells. Here, we studied the regulation of TWEAK/Fn14‐mediated innate immunity in the uterus using a lipopolysaccharide (LPS)‐induced model of abortion in pregnant mice. Specifically, we detected the expression of TWEAK and Fn14 in the uterus and in uNK cells following LPS treatment. Our results revealed that TWEAK and Fn14 are expressed by uNK cells in pregnant mice; in particular, it appears that the cytokine TWEAK is primarily derived from uNK cells. Interestingly, the down‐regulation of TWEAK in uNK cells and the up‐regulation of the Fn14 receptor in the uterus in LPS‐treated mice may contribute to the disruption of decidual homeostasis by altering uNK cell cytotoxicity – ultimately leading to fetal rejection. In conclusion, the present study strongly suggests that the TWEAK–Fn14 axis in uNK cells is involved in maintaining the tolerance necessary for successful pregnancy.