The endothelial transcription factor ETS (Erythroblast‐transformation specific)‐related gene (ERG) regulates canonical TGFβ‐SMAD signaling, by repressing SMAD3 activity. SMAD proteins function as ...important mediators of intracellular signal transduction of TGF‐β. Following their phosphorylation by TGF‐β receptor‐I, Receptor‐regulated SMADs (including SMAD2 and SMAD3 proteins) form a heteromeric complex with co‐SMAD and then translocate into the nucleus where they bind and regulate the expression of multiple target genes. Recent studies from our lab have demonstrated that ERG is a critical regulator of permeability in human lung microvascular endothelial cells (HLMECs) where ERG gene activation provided protection to the barrier. We have hypothesized that the protective effects of ERG on barrier integrity may be mediated via inhibiting TGF‐β/SMAD3 signaling pathway. To study the role of SMAD3 in microvascular endothelial barrier functions and permeability, HLMECs were grown on Transwell inserts and other cell culture platforms. Monolayer permeability was evaluated based on FITC‐albumin extravasation and measurement of its fluorescence intensity. TGF‐β (1‐50 ng/ml) induced a dose‐dependent increase in monolayer hyperpermeability. TGF‐β‐induced hyperpermeability was attenuated by a SMAD3 pharmacological inhibitor (SIS3) or CRISPR/cas9‐mediated knockdown of SMAD3. Immunoblot analysis demonstrated highly efficient SMAD3 knockdown by SMAD3 CRISPR/Cas9. TGF‐β treatment resulted in significant increase in matrixmetalloproteinase‐9 (MMP‐9) activity and the effect was decreased by SMAD3 inhibition. MMP‐9 is known to proteolytically breakdown the adherens junction proteins in microvascular endothelial cells, resulting in hyperpermeability. These results demonstrate that SMAD3 is a mediator of TGF‐β signaling‐induced loss of endothelial barrier integrity and permeability in microvascular endothelial cells. It also suggests that one of the downstream effects of TGFβ‐SMAD signaling is the activation of MMP‐9 which mediates loss of adherens junctional integrity.
ERG (ETS-related gene) is a member of the ETS (Erythroblast-transformation specific) family of transcription factors abundantly present in vascular endothelial cells. Recent studies demonstrate that ...ERG has important roles in blood vessel stability and angiogenesis. However, it is unclear how ERG is potentially involved in microvascular barrier functions and permeability. A wide variety of diseases and clinical conditions including trauma-hemorrhagic shock and burn injury are associated with microvascular dysfunctions, which causes excessive microvascular permeability, tissue edema and eventually, multiple organ dysfunction and death. The main purpose of this study was to determine the specific role of ERG in regulating microvascular permeability in human lung microvascular endothelial cells (HLMEC) and to evaluate if exogenous ERG will protect the barrier. The HLMECs were grown on Transwell inserts as monolayers and were transfected with ERG CRISPR/cas9 knockdown plasmid, ERG CRISPR activation plasmid, recombinant ERG protein or their respective controls. Recombinant vascular endothelial growth factor (VEGF) was used as an inducer of permeability for evaluating the effect of ERG activation on permeability. Changes in barrier integrity and permeability were studied using monolayer permeability assay and immunofluorescence of adherens junction proteins (VE-cadherin and β-catenin) respectively. CRISPR/cas9-based ERG knockdown as well as VEGF treatment induced monolayer hyperpermeability, VE-cadherin, and β-catenin junctional relocation and cytoskeletal F-actin stress fiber formation. CRISPR based ERG activation and recombinant ERG transfection attenuated VEGF-induced monolayer hyperpermeability. ERG activation preserved the adherens junctions and cytoskeleton. These results demonstrate that ERG is a potent regulator of barrier integrity and permeability in human lung microvascular endothelial cells and endogenously or exogenously enhancing ERG provides protection against barrier dysfunction and hyperpermeability.
Class II invariant chain peptide (CLIP) expression has been demonstrated to play a pivotal role in the regulation of B cell function after nonspecific polyclonal expansion. Several studies have shown ...vitamin D3 helps regulate the immune response. We hypothesized that activated vitamin D3 suppresses CLIP expression on activated B-cells after nonspecific activation or priming of C57BL/6 mice with CpG. This study showed activated vitamin D3 actively reduced CLIP expression and decreased the number of CLIP+ B-lymphocytes in a dose and formulation dependent fashion. Flow cytometry was used to analyze changes in mean fluorescent intensity (MFI) based on changes in concentration of CLIP on activated B-lymphocytes after treatment with the various formulations of vitamin D3. The human formulation of activated vitamin D (calcitriol) had the most dramatic reduction in CLIP density at an MFI of 257.3 baseline of 701.1 ( P value = 0.01). Cholecalciferol and alfacalcidiol had no significant reduction in MFI at 667.7 and 743.0, respectively. Calcitriol seemed to best reduce CLIP overexpression in this ex vivo model. Bioactive vitamin D3 may be an effective compliment to other B cell suppression therapeutics to augment downregulation of nonspecific inflammation associated with many autoimmune disorders. Further study is necessary to confirm these findings.
CREB binding protein (CBP) is a transcriptional cofactor with intrinsic histone acetyl transferase activity (HAT). We have observed that CBP interacts with BRCA2 and mediates post-translational ...glycosylation of BRCA2. The binding of CBP to the amino-terminal region of BRCA2 is necessary for the glycosylation at residue 272 of BRCA2. Digestion with peptide N-glycosidase F indicates that the glycosylation of BRCA2 is N-linked. It is possible that this novel CBP-mediated post-translational N-glycosylation activity alters the conformation of CBP-interacting proteins, leading to regulation of gene expression, cell growth and differentiation.
RISAT-1 is the first indigenous active Radar Imaging Satellite launched by Polar Satellite Launch Vehicle (PSLV) in April 2012 from Sriharikota. It carries C-band Synthetic Aperture Radar (SAR) ...payload which can operate at various values of resolution and swath for various applications. RISAT-1 is the heaviest and high-power satellite with many new technologies to support the SAR payload and the associated elements. RISAT-1 operates at polar sun-synchronous orbit of 536 km altitude with the inclination of 97.554° and it is designed for 5 years lifetime. Performance of the spacecraft system and the SAR payload is satisfactory. This article outlines the architecture, design and on-orbit performance of RISAT-1.
Triple negative breast cancer (TNBC) is a heterogeneous disease and has a higher rate of recurrence and distant metastasis. African-American (AA) women have a higher frequency of BRCA1 mutations and ...TNBC compared to other populations. Basal-like tumors have a higher rate of brain, lung and distant nodal metastasis more than other TNBC subtypes, contributing to higher mortality rate. Our previous work suggested Ubc9, a SUMO E2-conjugating enzyme to induce proliferation and migration of BRCA1-incompetent TNBC cells and TNBC cell lines established from the pleural effusion metastasis of a woman with TNBC. To understand the downstream signaling axis involved in distant metastasis we have used clinically relevant BRCA1 mutant and lung metastatic TNBC cell lines and our results show deregulated expression of caveolin-1, VEGF and SIRT1 in these cells compared to normal mammary epithelial cells by immunofluorescence analysis. We observed SIRT1 to be induced by wild type BRCA1a and BRCA1a I26A mutant unlike the disease associated Ubc9 binding mutants in TNBC cells. Knock down of Ubc9 induced SIRT1 expression in TNBC and ER-α expression in breast cancer cells. This is the first report demonstrating a role for Ubc9 in repressing both SIRT1 and ER-α expression in BRCA1 associated TNBC cells. It also suggests that the BARD-dependent E3 Ubiquitin ligase and HR (homologous recombination) activity of BRCA1 may not be required for inducing SIRT1 expression. Our results suggest for the first time that in BRCA1 mutant TNBC Ubc9-mediated induction of VEGF, inhibition of caveolin-1, SIRT1 and ER-α expression as a novel molecular mechanism underlying TNBC EMT (epithelial mesenchymal transition) leading to lung metastasis with pleural effusion. Drugs that target Ubc9 to both induce SIRT1 and ER-α or using SIRT1 agonists in combination with chemotherapy can be used as a promising targeted therapeutic approach for treating basal-like metastatic BRCA1-linked TNBC thus reducing the mortality in patients with TNBC.
The incidence of anal carcinoma has increased over the last few decades especially in African Americans (AA) despite the use of highly active anti-retroviral therapy (HAART). Here, we retrospectively ...review oncologic outcomes of AA patients with anal squamous cell carcinoma (SCC) with and without HIV to further examine the cause of this trend.
All adult AA patients diagnosed with anal SCC from 2000 to 2007 who met inclusion were examined. All patients were staged according to the American Joint Committee on Carcinoma (AJCC) sixth edition staging classification. Patients were divided into two cohorts: HIV (-) and HIV (+). Demographics, comorbidities, and oncologic outcomes were analyzed.
Twenty-two AA patients with anal SCC were analyzed. Fifteen (68.%) were HIV (+) and seven (32%) were negative. Seventy-four percent of HIV (+) patients were on HAART therapy at the time of diagnosis. The HIV (+) cohort was significantly younger, mostly male, and had more comorbidities compared to the negative cohort. There was no difference in tumor, nodal or metastasis (TNM) stage for both cohorts. HIV (+) patients were more likely to receive non-operative therapy. The 5-year survival rate for HIV negative and positive patients was 57% and 58%, respectively. AJCC stage was the only factor predictive of survival after performing Cox hazard proportional regression analysis, HR: 1.96 (95% CI, 0.987 to 3.881).
In the HAART era, HIV (+) AA patients are at high risk of developing anal SCC. However, the prognosis of HIV (+) AA with anal SSC is similar to that of their HIV (-) counterparts. Carcinoma stage is the only factor predictive of survival.
Two ets family members, namely erg and Fli-1 are fused with two EWS family members namely EWS and TLS/FUS as a result of chromosome translocation in human solid tumors and leukemias. EWS-erg and ...EWS-Fli-1, which are involved in greater than 95% of Ewing family of tumors, were shown to function as transcriptional activators. TLS/FUS-erg, which is involved in human myeloid leukemias also functions as a transcriptional activator. Expression of these fusion proteins (EWS-erg and EWS-Fli-1) are shown to be essential for maintaining the oncogenic and tumorigenic properties of tumor cells. Cancer is thought to be caused not only by uncontrolled cell proliferation but also by deregulation of programmed cell death. Therefore, we have studied the role of normal (Fli-1 and erg) and aberrant fusion proteins (EWS-erg, EWS-Fli-1 and TLS/FUS-erg) in apoptosis. We have found that expression of normal (Fli-1 and erg) and aberrant fusion proteins inhibit the apoptosis of NIH3T3 cells induced by either serum deprivation or by treatment with calcium ionophore. We have also observed similar suppression of apoptosis in Ewing's sarcoma cells expressing EWS-Fli-1 and EWS-erg proteins suggesting that these fusion proteins may be responsible for the decreased ability of these tumor cells to undergo apoptosis. Inhibition of the expression of these aberrant fusion proteins by antisense RNA technique resulted in increased susceptibility to apoptosis leading to the death of tumor cells. Therefore, our results suggest that one can use therapeutic agents which can down regulate the expression of fusion proteins in combination with chemotherapeutic agents as an effective treatment for these human solid tumors and leukemias.
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