Angiopoietin-1 Regulates Brain Endothelial Permeability through PTPN-2 Mediated Tyrosine Dephosphorylation of Occludin

<div><p>Objective</p><p>Blood brain barrier (BBB) breakdown and increased endothelial permeability is a hallmark of neuro-vascular inflammation. Angiopoietin-1 (Ang-1), a Tie-2 receptor agonist ligand, is known to modulate barrier function of endothelial cells; however the molecular mechanisms related to Ang-1 mediated repair of Tight Junctions (TJs) in brain endothelium still remain elusive. In this study, we investigated a novel role of non-receptor protein tyrosine phosphatase N-2 (PTPN-2) in Ang-1 mediated stabilization of tight junction proteins.</p><p>Method and Result</p><p>To study the barrier protective mechanism of Ang-1, we challenged human brain microvascular endothelial cells <i>in-vitro</i>, with a potent inflammatory mediator thrombin. By using confocal microscopy and transwell permeability assay, we show that pretreatment of brain endothelial cells with Ang-1 diminish thrombin mediated disruption of TJs and increase in endothelial permeability. We also found that Ang-1 inhibits thrombin induced tyrosine phosphorylation of Occludin and promote Occludin interaction with Zona Occludens-1 (ZO-1) to stabilize TJs. Interestingly, our study revealed that depletion of PTPN-2 by siRNAs abolishes Ang-1 ability to promote tyrosine dephosphorylation of Occludin, resulting Occludin disassociation from ZO-1 and endothelial hyperpermeability.</p><p>Summary</p><p>Collectively, our findings suggest that in brain endothelial cells blocking PTPN-2 mediated tyrosine phosphorylation of Occludin is a novel mechanism to maintain BBB function, and may offer a key therapeutic strategy for neuro-inflammatory disorders associated with BBB disruption.</p></div

PTPN-2 depletion has no effect on Ang-1 mediated Tie-2 and Erk activation, and on expression of TJ proteins.

<p>Scramble and PTPN-2 siRNA transfected HBMECs were activated with 100ng/ml of Ang-1 for indicated times. (<b>A)</b> Immunoprecipitation of Tie-2 protein followed by immunoblotting with anti-PY-20 antibody; blot was reprobed with anti-Tie-2 antibody. (<b>B)</b> Whole cell lysates were immunoblotted with anti-Phospho-p44/42 (Erk1/2) (Thr202/Tyr204) and anti-Erk1/2 antibody. (<b>C)</b> Whole cell lysates were analyzed by Western blotting to study the expression of major TJ proteins with anti-ZO-1, anti-Occludin, anti-Claudin-5 and anti-PTPN-2 antibody. Actin used for loading control.</p

Ang-1 diminishes thrombin mediated tyrosine phosphorylation of Occludin.

<p>(<b>A)</b> Serum starved HBMECs were treated with 100nM thrombin for indicated time periods. Cellular proteins were immunoprecipitated with anti-Occludin antibody followed by immunoblotting using anti-PY-20 antibody. Blot was reprobed with anti-Occludin antibody. (<b>B)</b> ECs were stimulated with thrombin (100nM for 15 min) or pretreated with 100ng/ml Ang-1 for 15 min followed by thrombin challenge (100nM for 15 min). Cell lysates were immunoprecipitated with anti-Occludin antibody and probed with PY-20 antibody to detect tyrosine phosphorylation of Occludin. The same blot was reprobed for total Occludin.</p

Ang-1 inhibits thrombin induced disassociation of Occludin with ZO-1 and its membrane dis-localization.

<p>Serum starved HBMECs were treated with 100nM thrombin alone or after Ang-1 (100ng/ml for 15 min) for indicated times. Cell lysates were immunoprecipitated with <b>(A)</b> anti-ZO-1 antibody and probed with anti-Occludin antibody, <b>(B)</b> anti-Occludin antibody and probed with anti-ZO-1 antibody. <b>(C)</b> After treating HBMECs with thrombin alone or pretreated with Ang-1 (100ng/ml for 15 min) followed by 100nM thrombin challenged for different times, plasma membrane fractions were prepared, separated on SDS gel and probed for Occludin. Na⁺-K⁺ ATPase β3 was used as a membrane marker.</p

Ang-1 prevents disruption of inter-endothelial junctions and endothelial hyper-permeability induced by thrombin.

<p>(<b>A)</b> Serum starved HBMECs were stimulated with 100ng/ml of Ang-1 for different times. Tyrosine phosphorylation level of Tie-2 was determined by immunoprecipitating Tie-2 followed by Western blotting for PY-20 antibody. (<b>B)</b> Transendothelial EBA tracer permeability was determined in serum starved HBMECs unstimulated or stimulated with Ang-1 (100ng/ml for 15 min) or with thrombin (100nM for 120 min) or incubated with Ang-1 followed by thrombin treatment. Bar graphs show the average ±SEM, *P<0.05. (<b>C)</b> HBMECs were treated with 100nM thrombin alone or after Ang-1 (100ng/ml for 15 min) for indicated times. Cells were fixed, permeabilized and stained for Occludin. Immunofluorescent staining and confocal microscopy was performed as described in Methods. Scale bar = 10μM. <b>(D)</b> Quantification of number of inter-endothelial gap (shown as fold change quantified using Occludin staining described in (<b>C</b>), *P<0.05.</p

Deletion of PTPN-2 abolishes barrier protective function of Ang-1.

<p><b>(A)</b> HBMECs were transfected with 50nM of either scramble or PTPN-2 siRNA. After 48 hours of transfection, cell lysate was collected, and immunoblotted with anti-PTPN-2 antibody to see the knockdown efficiency. The same blot was reprobed for Actin. (<b>B)</b> EBA permeability was determined in HBMECs monolayers transfected by 50nM scramble siRNA or PTPN-2 siRNA. Cells were treated with 100nM of thrombin for 120 min alone and after Ang-1 (100ng/ml for 15 min). Bar graphs show the average ±SEM, *P<0.05. (<b>C)</b> Representative confocal images of Occludin staining to show cell-cell contact in confluent HBMECs transfected with either scramble siRNA or PTPN-2 siRNA. ECs were then pretreated with Ang-1 (100ng/ml for 15 min) followed by thrombin treatment. Scale bar = 10μM. (<b>D)</b> Quantification of number of inter-endothelial gaps is shown in fold change. Data are represented as mean ±SEM *P<0.05. (<b>E)</b> HBMECs were transfected with 50nM scramble siRNA or PTPN-2 siRNA for 48 hr. Serum starved ECs monolayer was stimulated by thrombin (100nM for 15 min) alone and after Ang-1 pretreatment (100ng/ml for 15 min). Level of pY-Occludin and total Occludin was determined by immunoprecipitation and Western blot analysis. (<b>F)</b> ECs were transfected with 50nM of scramble or PTPN-2 siRNA for 48 hr followed by exposure to 100nM of thrombin in absence or presence of Ang-1 pretreatment (100ng/ml for 15 min). Immunoprecipitation was used to study Occludin/ZO-1 interaction.</p

Global Reduction of H3K4me3 Improves Chemotherapeutic Efficacy for Pediatric Ependymomas

BACKGROUND: Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy. METHODS: Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells. RESULTS: H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells increased cell response to chemotherapy. CONCLUSIONS: Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors