Nuclear Translocation Of Angiogenin Research Articles

The secretion of the angiogenic and neurotrophic factor angiogenin is COPII and microtubule dependent

The RNaseA superfamily member Angiogenin (ANG) is a secreted protein involved in neovascularization, cell proliferation and stress response. Dysregulation of ANG expression is found in many cancers with poor prognosis and mutations in ANG are associated with neurodegenerative diseases. While the uptake and nuclear translocation of ANG is relatively well characterised, little is known about how it reaches the plasma membrane and its mode of secretion. We generated SH-SY5Y neuroblastoma cell lines constitutively expressing wild type (WT) Hemagglutinin (HA) epitope tagged mouse Ang1 (mAng1), and two amyotrophic lateral sclerosis associated ANG variants (C39W and K40I). Herein, we show that these cell lines secrete mAng1 into the culture media. Using small molecule inhibitors we probed the route taken between the endoplasmic reticulum and trans-Golgi network during secretion and have characterised it as COPII and microtubule dependent. In addition, we show that disruption by the PI3-kinase inhibitor wortmannin of the later stages of transit to the plasma membrane leads to mAng1 trafficking to lysosomal compartments. This suggests an autophagy dependent regulation of secretion.

Pharmacokinetics of neamine in rats and anti-cervical cancer activity in vitro and in vivo.

To study the pharmacokinetics of neamine in rats and to evaluate its anti-cervical cancer activity. The plasma level of neamine was determined by HPLC-ELSD. Pharmacokinetic parameters were calculated using DAS 2.0 software. Tissue microarray analysis was conducted to examine angiogenin (ANG) expression in normal and cancerous cervical tissues and to determine its correlation with clinical and pathologic presentations of cervical cancers. The anti-cervical cancer activity of neamine was assessed both in vitro and in vivo. After intravenous (i.v.) administration of 15, 30, and 60mgkg(-1) neamine, the pharmacokinetic parameters were as follows: AUC(0-t), 9,398.0±653.4, 19,235.2±2,939.0, and 35,437.7±3,772.2mgL(-1)min; C max, 170.8±13.1, 353.3±15.8, and 464.0±33.1mgL(-1); T 1/2, 34.9±4.1, 46.8±5.1, and 58.0±12.5min, respectively. The bioavailability of neamine administered through intramuscular, subcutaneous, intraperitoneal and intragastric route was 14.0±3.0, 8.4±0.6, 6.5±3.3, and 3.1±0.2%, respectively. Up-regulated ANG expression and increased nuclear translocation were observed in cervical cancers as compared to normal cervical tissues. Moreover, upregulation of ANG was positively correlated with primary tumor invasion. Neamine inhibited ANG-induced HUVEC and HeLa cell proliferation as well as nuclear translocation of ANG. Consistently, neamine inhibited both the establishment and progression of xenograft human cervical cancers in athymic mice. The bioavailability of neamine administered through extravascular routes was low. The half-life of neamine through i.v. administration was short. This suggests that a higher dosing frequency in order to maintain a therapeutic effect. Neamine holds potential against cervical cancer. The mechanisms of neamine inhibition are through blocking nuclear translocation of ANG thereby inhibiting both angiogenesis and cancer cell proliferation.

Identification of Estrogen Receptor-Related Receptor Gamma as a Direct Transcriptional Target of Angiogenin

Nuclear translocation of angiogenin (ANG) is essential for the proliferation of its target cells. ANG promotes rRNA synthesis, while whether it regulates mRNA transcription remains unknown. Using the chromatin immunoprecipitation method, we have identified 12 ANG-binding sequences. One of these sequences lies in the estrogen receptor-related receptor gamma (ERRγ) gene which we designated as ANG-Binding Sequence within ERRγ (ABSE). ABSE exhibited ANG-dependent repressor activity in the luciferase reporter system. Down-regulation of ANG increased ERRγ expression, and active gene marker level at the ABSE region. The expression levels of ERRγ targets genes, p21WAF/CIP and p27KIP1, and the occupation of ERRγ on their promoter regions were increased in ANG-deficient cells accordingly. Furthermore, knockdown of ERRγ promoted the proliferation rate in ANG-deficient breast cancer cells. Finally, immunohistochemistry staining showed negative correlation between ANG and ERRγ in breast cancer tissue. Altogether, our study provides evidence that nuclear ANG directly binds to the ABSE of ERRγ gene and inhibits ERRγ transcription to promote breast cancer cell proliferation.

Abstract WMP31: Neamine, A Angiogenin Inhibitor, Induces Neuroprotection After Acute Ischemic Stroke In Type One Diabetic Rats

Introduction: Angiogenin is a member of the ribonuclease superfamily and promotes degradation of basement membrane and extracellular matrix. After stroke in Type one diabetes (T1DM) rats, Angiogenin is significantly increased and the increased Angiogenin is correlated with worse functional outcome. Neamine, an aminoglycoside antibiotic, blocks nuclear translocation of Angiogenin thereby abolishing the biological activity of Angiogenin. In this study, we therefore investigated the effect and underlying protective mechanisms of Neamine treatment of stroke in T1DM. Methods: T1DM was induced in male Wistar rats by streptozotocin (60mg/kg, ip) and T1DM rats were subjected to embolic middle cerebral artery occlusion (MCAo). Neamine (10 mg/kg i.p.) was administered at 2h, 24h and 48h after MCAo. A battery functional outcome tests were performed. Brain blood barrier (BBB) leakage, lesion volume, brain hemorrhage volume, were evaluated and immunostaining, Western blot and ELISA were performed. Results: Neamine treatment of stroke in T1DM rats significantly decreased Evans Blue dye leakage (Neamine: 16.38±2.14 ng/mg vs contro:30.57± 5.88 ng/mg); and lesion volume (23±2% vs 35±3%) as well as improved functional outcome compared to T1DM-control. Neamine also significantly decreased apoptosis (34.2±0.8 vs 42.5±3.6) and cleaved caspase-3 (28.7±3.7 vs 45.2±3.7) in the ischemic brain. Neamine treatment decreased MIP-1, MCP-1 and MMP9 levels in the ischemic brain measured by ELISA array. Using immunostaining, we found that Neamine treatment significantly decreased nuclear Angiogenin (26.7±1.7 vs 57.1±3.6) number, and the positive area of Angeopoietin-2 (7.4±0.3 vs 10.9±0.5), toll-like receptor 4 (TLR4) (5.4±0.9 vs 10.5±1.2), MMP9 (0.29±0.04 vs 0.48±0.09) expression compared to T1DM-MCAo control rats. In vitro data also show that Neamine treatment significantly decreased tPA, Angiogenin, MMP9 and advanced glycation endproducts receptor (RAGE) expression (2-3 fold) in cultured astrocytes and oligodendrocytes. Conclusion: Neamine treatment of stroke is neuroprotective in T1DM rats. Inhibition of neuroinflammatory factor expression and decrease of BBB leakage may contribute to Neamine induced neuroprotection effects after stroke in T1DM.

Abstract 2389: uPA and uPAR siRNA inhibits angiogenesis by blocking nuclear localization of angiogenin and angiopoietin1 signaling in glioblastoma cell lines

Abstract Transcriptional inactivation of uPA and uPAR system in the glioblastoma cell line SNB19 inhibited angiogenesis both in vitro and in vivo. To decipher this mechanism and extend our studies to other glioblastoma cell lines, U87MG and U87 SPARC-overexpressing cell lines were subjected to uPA, uPAR and U2 (bicistronic construct against uPA and uPAR) downregulation by siRNA. In the present study, the bicistronic construct against uPA and uPAR caused significant inhibition of angiogenesis by in vitro angiogenesis assay and in vivo dorsal skin fold chamber models in both cell lines. Increased apoptosis was seen in siRNA downregulated gliomablastoma cells when compared with control cells by FACS analysis. MTT assay revealed significant inhibition of proliferation by U2 siRNA treatment in both cell lines. Transfection with siRNA against uPA and uPAR in co-cultures of glioma cells with endothelial cells showed significant inhibition of angiopoeitin1, IL-6, GRO and GM-CSF while SPARC-overexpressing U87 cells had decreased secretion of angiogenin, GRO, MCP-1, PDGFBB, VEGF and angiopoietin1 by protein array analysis. Bicistronic construct showed decreased levels of angiogenin by ELISA and Western blot in endothelial cells and in cocultures with cancer cells. Transcriptional inactivation of uPA and uPAR showed significant downregulation of angiogenin-induced phosphorylation of ERK, JNK and Akt signaling in endothelial cells. Nuclear translocation of angiogenin in semi-confluent human umbilical vein endothelial cells treated with conditioned medium from siRNA-transfected U87 SPARC-overexpressing cells was inhibited as shown by Western blot and immunofluorescence analyses. Conditioned medium from U87MG and U87 SPARC-overexpressing cells transfected with uPA, uPAR and U2 siRNA plasmids significantly inhibited the phosphorylation of Tie2 receptor in endothelial cells. uPA and uPAR downregulation significantly downregulated MMP-2 levels by gelatin zymography in co-cultures and in single cultures of cancer cells. Matrix metalloproteinase array analysis showed significant inhibition of MMP-3 in co-cultures of U2-transfected SPARC-overexpressing glioma cells. Overall, our results suggest that the simultaneous downregulation of uPA and uPAR inhibits angiogenesis by inhibition of nuclear localization of angiogenin, angiopoeitin 1 signaling and MMP-2 downregulation in endothelial cells. In summary, these results identify novel mechanisms by which the bicistronic construct against uPA and uPAR inhibits angiogenesis, thereby suggesting the potential use of U2 as an anti-angiogenic agent against glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2389.

Angiogenin Is Translocated to the Nucleus of HeLa Cells and Is Involved in Ribosomal RNA Transcription and Cell Proliferation

Angiogenin is an angiogenic protein known to play a role in rRNA transcription in endothelial cells. Nuclear translocation of angiogenin in endothelial cells decreases as cell density increases and ceases when cells are confluent. Here we report that angiogenin is constantly translocated to the nucleus of HeLa cells in a cell density-independent manner. Down-regulation of angiogenin expression by antisense and RNA interference results in a decrease in rRNA transcription, ribosome biogenesis, proliferation, and tumorigenesis both in vitro and in vivo. Exogenous angiogenin rescues the cells from antisense and RNA interference inhibition. The results showed that angiogenin is constitutively translocated into the nucleus of HeLa cells where it stimulates rRNA transcription. Thus, besides its angiogenic activity, angiogenin also plays a role in cancer cell proliferation.

Identification and characterization of an angiogenin-binding DNA sequence that stimulates luciferase reporter gene expression.

Angiogenin undergoes nuclear translocation in endothelial and smooth muscle cells where it accumulates in the nucleolus and binds to DNA. Nuclear translocation of angiogenin is necessary for its biological activity and is mediated by an endocytotic pathway that is independent of the microtubule system and lysosomal processing. Because the nucleolus is a subnuclear organelle containing clusters of transcriptionally active ribosomal RNA genes, we studied the binding of angiogenin to the intergenic spacer of the ribosomal RNA gene where many of the transcription regulatory elements are located. Here we report that angiogenin binds to CT repeats that are abundant in the nontranscribed region of the ribosomal RNA gene. An angiogenin-binding DNA sequence (CTCTCTCTCTCTCTCTCCCTC) has been identified and designated angiogenin-binding element (ABE). ABE binds angiogenin specifically and exhibits angiogenin-dependent promoter activity in a luciferase reporter system. CT repeats, or inverted GA box, which are abundantly distributed in the eukaryotic genome and are often located in the 5'-flanking region, have been implicated in regulating gene expression. We have previously shown that angiogenin stimulates rRNA synthesis. The present results suggest that the nuclear function of angiogenin may not only be related to rRNA production but also play a role in regulating expression of genes containing CT repeats.

Nuclear translocation of angiogenin in proliferating endothelial cells is essential to its angiogenic activity.

The intracellular pathway of human angiogenin in calf pulmonary artery endothelial (CPAE) cells has been studied by immunofluorescence microscopy. Proliferating CPAE cells specifically endocytose native angiogenin and translocate it to the nucleus, where it accumulates in the nucleoli. Nuclear translocation of angiogenin does not occur in nonproliferative, confluent CPAE cells. These cells were previously found to express an angiogenin-binding protein (AngBP) that was identified as smooth muscle alpha-actin. Exogenous actin, an anti-actin antibody, heparin, and heparinase treatment all inhibit the internalization of angiogenin, suggesting the involvement of cell surface AngBP/actin and heparan sulfate proteoglycans in this process. It has been established that two regions of angiogenin are essential for its angiogenic activity, one is its endothelial cell binding site and the other its catalytic site capable of cleaving RNA. CPAE cells do not internalize four enzymatically active angiogenin derivatives whose cell binding site is modified, but they do internalize two enzymatically inactive mutants whose cell binding site is intact. Thus, the putative cell binding site of angiogenin is necessary for both endocytosis and nuclear translocation, but the catalytic site is not. Three other angiogenic molecules are also translocated to the nucleus of growing CPAE cells. Overall, the results suggest that nuclear translocation of angiogenin and other angiogenic molecules is a critical step in the process of angiogenesis.