Abstract 627: Regulation of Nuclear Factor of Activated T Cells by BMP-Binding Endothelial Regulator Signaling

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Pamela P Lockyer ◽  
Hua Mao ◽  
Xi Li ◽  
Xinchun Pi
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Transcriptional Activity ◽  
Ldl Receptor ◽  
Bmp Signaling ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Morphogenetic Protein ◽  
Endothelial Integrity

Dysfunction of the vascular endothelium results in various cardiovascular, circulatory and blood diseases and exemplifies the importance of endothelial integrity. BMP-binding endothelial regulator (BMPER), a well recognized extracellular modulator of Bone morphogenetic protein (BMP) signaling, has been identified as a vital component in the vascular response to stress. Microarray analysis revealed nuclear factor of activated T cells (NFAT) as one of the genes found to be most highly upregulated by BMPER treatment in mouse endothelial cells (MECs), as well as many genes with NFAT consensus binding sites. Therefore we hypothesize that BMPER is an important regulator of NFAT transcriptional activity. Initially we have investigated the effect of BMPER on NFATc1 activation with MECs and human primary endothelial cells. Our data show that the translocation of NFATc1 from the cytoplasm to the nucleus following BMPER treatment, determined by immunofluorescent analysis. By using the nuclear fractionation assays, we observed the similar result that the translocation of NFATc1 to the nucleus of HUVECs took place after 30 minutes of BMPER treatment. Next, we wanted to determine whether the increased NFATc1 protein level in nucleus results in the enhanced transcriptional activity. Indeed, when HUVECs are treated with BMPER and then analyzed with luciferase reporter assay, a 1.5-fold significant increase in NFAT activity over baseline was observed. Our previous data demonstrate that LDL receptor related protein (LRP1) interacts with BMPER and regulates BMPER’s activity through endocytosis in endothelial cells. Interesting, we observe that LRP1 also interacts with NF45, the 45-kDa subunit of NFAT protein. It strongly suggests that BMPER positively regulates NFAT activity through LRP1. This novel signaling pathway indicates that BMPER may acts as a new ligand and exhibits BMP-independent activity in endothelial cells and therefore contribute to the regulation of vascular homeostasis.

scholarly journals Nuclear Factor of Activated T Cells Regulates Neutrophil Recruitment, Systemic Inflammation, and T-Cell Dysfunction in Abdominal Sepsis

2014 ◽  
Vol 82 (8) ◽  
pp. 3275-3288 ◽  
Author(s):  
Su Zhang ◽  
Lingtao Luo ◽  
Yongzhi Wang ◽  
Maria F. Gomez ◽  
Henrik Thorlacius
Keyword(s):  
T Cells ◽  
T Cell ◽  
Systemic Inflammation ◽  
Abdominal Sepsis ◽  
Neutrophil Recruitment ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Cell Dysfunction ◽  
T Cell Dysfunction

ABSTRACTThe signaling mechanisms regulating neutrophil recruitment, systemic inflammation, and T-cell dysfunction in polymicrobial sepsis are not clear. This study explored the potential involvement of the calcium/calcineurin-dependent transcription factor, nuclear factor of activated T cells (NFAT), in abdominal sepsis. Cecal ligation and puncture (CLP) triggered NFAT-dependent transcriptional activity in the lung, spleen, liver, and aorta in NFAT-luciferase reporter mice. Treatment with the NFAT inhibitor A-285222 prior to CLP completely prevented sepsis-induced NFAT activation in all these organs. Inhibition of NFAT activity reduced sepsis-induced formation of CXCL1, CXCL2, and CXCL5 chemokines and edema as well as neutrophil infiltration in the lung. Notably, NFAT inhibition efficiently reduced the CLP-evoked increases in HMBG1, interleukin 6 (IL-6), and CXCL5 levels in plasma. Moreover, administration of A-285222 restored sepsis-induced T-cell dysfunction, as evidenced by markedly decreased apoptosis and restored proliferative capacity of CD4 T cells. Along these lines, treatment with A-285222 restored gamma interferon (IFN-γ) and IL-4 levels in the spleen, which were markedly reduced in septic mice. CLP-induced formation of regulatory T cells (CD4+CD25+Foxp3+) in the spleen was also abolished in A-285222-treated animals. All together, these novel findings suggest that NFAT is a powerful regulator of pathological inflammation and T-cell immune dysfunction in abdominal sepsis. Thus, our data suggest that NFAT signaling might be a useful target to protect against respiratory failure and immunosuppression in patients with sepsis.

scholarly journals Transcriptional Regulation of BK Virus by Nuclear Factor of Activated T Cells

2009 ◽  
Vol 84 (4) ◽  
pp. 1722-1730 ◽  
Author(s):  
Joslynn A. Jordan ◽  
Kate Manley ◽  
Aisling S. Dugan ◽  
Bethany A. O'Hara ◽  
Walter J. Atwood
Keyword(s):  
T Cells ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Adult Population ◽  
Bk Virus ◽  
Human Polyomavirus ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Transplant Patients

ABSTRACT The human polyomavirus BK virus (BKV) is a common virus for which 80 to 90% of the adult population is seropositive. BKV reactivation in immunosuppressed patients or renal transplant patients is the primary cause of polyomavirus-associated nephropathy (PVN). Using the Dunlop strain of BKV, we found that nuclear factor of activated T cells (NFAT) plays an important regulatory role in BKV infection. Luciferase reporter assays and chromatin immunoprecipitation assays demonstrated that NFAT4 bound to the viral promoter and regulated viral transcription and infection. The mutational analysis of the NFAT binding sites demonstrated complex functional interactions between NFAT, c-fos, c-jun, and the p65 subunit of NF-κB that together influence promoter activity and viral growth. These data indicate that NFAT is required for BKV infection and is involved in a complex regulatory network that both positively and negatively influences promoter activity and viral infection.

scholarly journals Brain Endothelial miR-146a Negatively Modulates T-Cell Adhesion through Repressing Multiple Targets to Inhibit NF-κB Activation

2015 ◽  
Vol 35 (3) ◽  
pp. 412-423 ◽  
Author(s):  
Dongsheng Wu ◽  
Camilla Cerutti ◽  
Miguel A Lopez-Ramirez ◽  
Gareth Pryce ◽  
Josh King-Robson ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Nuclear Translocation ◽  
Leukocyte Adhesion ◽  
Cell Types ◽  
Nuclear Factor ◽  
Autoimmune Encephalomyelitis ◽  
Activated T Cells ◽  
Cytokine Activation

Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in leukocyte adhesion to, and subsequent migration across, brain endothelial cells (BECs), which is crucial for the development of neuroinflammatory disorders such as multiple sclerosis (MS). In contrast, microRNA-146a (miR-146a) has emerged as an anti-inflammatory molecule by inhibiting NF-κB activity in various cell types, but its effect in BECs during neuroinflammation remains to be evaluated. Here, we show that miR-146a was upregulated in microvessels of MS-active lesions and the spinal cord of mice with experimental autoimmune encephalomyelitis. In vitro, TNFα and IFNγ treatment of human cerebral microvascular endothelial cells (hCMEC/D3) led to upregulation of miR-146a. Brain endothelial overexpression of miR-146a diminished, whereas knockdown of miR-146a augmented cytokine-stimulated adhesion of T cells to hCMEC/D3 cells, nuclear translocation of NF-κB, and expression of adhesion molecules in hCMEC/D3 cells. Furthermore, brain endothelial miR-146a modulates NF-κB activity upon cytokine activation through targeting two novel signaling transducers, RhoA and nuclear factor of activated T cells 5, as well as molecules previously identified, IL-1 receptor-associated kinase 1, and TNF receptor-associated factor 6. We propose brain endothelial miR-146a as an endogenous NF-κB inhibitor in BECs associated with decreased leukocyte adhesion during neuroinflammation.

Abstract 15833: LRP1 is a Novel Receptor of Bmper and Required for Pathological Angiogenesis

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Xi Li ◽  
Xinchun Pi
Keyword(s):  
Endothelial Cells ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Bmp Signaling ◽  
Functional Roles ◽  
Oxygen Induced Retinopathy ◽  
Morphogenetic Protein ◽  
Density Lipoprotein Receptor

Low density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional member of the LDL receptor family, impacting a variety of biological processes such as lipid metabolism, endocytosis and signal transduction. However, the role of LRP1 in endothelium was almost unknown. Until recently we discovered that LRP1 is a novel regulator of bone morphogenetic protein (Bmp) signaling through its association with the extracellular modulator-Bmper (Bmp-binding endothelial cell precursor-derived regulator) and regulates zebrafish vascular development. Here we studied the functional roles of LRP1 in mammalian system by performing cell culture studies with endothelial cells (ECs) and analyzing angiogenic defects in oxygen-induced retinopathy model with LRP1flox/flox;Tie2-Cre+/- mice (EC-LRP1 KO). In MECs, we observed that the activation of Src, ERK and tyrosine phosphorylation of multiple proteins were induced by Bmper and this activation was LRP1-dependent since LRP1 knockdown inhibited their activation. However, this Bmper-induced activation was not blocked by Bmp4 neutralized antibody, which suggests that LRP1 is required for Bmp4-independent signaling for Bmper. These data also indicate that Bmper and LRP1 is a novel ligand receptor pair. Moreover, we observed that LRP1 protein was induced in response to 1% hypoxia in both mouse ECs (MECs) and human retinal microvascular endothelial cells, suggesting that LRP1 is required for hypoxia induced endothelial function. In hypoxia condition, EC-LRP1 KO mice resulted in accelerated angiogenesis in retinal endothelial cells, similar to the pattern of Bmper deleted mice. Therefore, we identify that the Bmper/LRP1 signaling is a novel signaling pathway in endothelial cells and their activity regulates angiogenic responses during oxygen-induced retinopathy. This study provides mechanistic insights for angiogenesis-related pathophysiologic conditions.

scholarly journals NFAT regulation of cystathionine γ-lyase expression in endothelial cells is impaired in rats exposed to intermittent hypoxia

2017 ◽  
Vol 312 (4) ◽  
pp. H791-H799 ◽  
Author(s):  
Laura V. Gonzalez Bosc ◽  
Jessica M. Osmond ◽  
Wieslawa K. Giermakowska ◽  
Carolyn E. Pace ◽  
Jennifer L. Riggs ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Sleep Apnea ◽  
Mesenteric Artery ◽  
Intermittent Hypoxia ◽  
Mesenteric Arteries ◽  
Male Rats ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Rat Mesenteric Artery

Sleep apnea is a risk factor for cardiovascular disease, and intermittent hypoxia (IH, 20 episodes/h of 5% O2-5% CO2 for 7 h/day) to mimic sleep apnea increases blood pressure and impairs hydrogen sulfide (H2S)-induced vasodilation in rats. The enzyme that produces H2S, cystathionine γ-lyase (CSE), is decreased in rat mesenteric artery endothelial cells (EC) following in vivo IH exposure. In silico analysis identified putative nuclear factor of activated T cell (NFAT) binding sites in the CSE promoter. Therefore, we hypothesized that IH exposure reduces Ca2+ concentration ([Ca2+]) activation of calcineurin/NFAT to lower CSE expression and impair vasodilation. In cultured rat aortic EC, inhibiting calcineurin with cyclosporine A reduced CSE mRNA, CSE protein, and luciferase activity driven by a full-length but not a truncated CSE promoter. In male rats exposed to sham or IH conditions for 2 wk, [Ca2+] in EC in small mesenteric arteries from IH rats was lower than in EC from sham rat arteries (Δfura 2 ratio of fluorescence at 340 to 380 nm from Ca2+ free: IH = 0.05 ± 0.02, sham = 0.17 ± 0.03, P < 0.05), and fewer EC were NFATc3 nuclear positive in IH rat arteries than in sham rat arteries (IH = 13 ± 3, sham = 59 ± 11%, P < 0.05). H2S production was also lower in mesenteric tissue from IH rats vs. sham rats. Endothelium-dependent vasodilation to acetylcholine (ACh) was lower in mesenteric arteries from IH rats than in arteries from sham rats, and inhibiting CSE with β-cyanoalanine diminished ACh-induced vasodilation in arteries from sham but not IH rats but did not affect dilation to the H2S donor NaHS. Thus, IH lowers EC [Ca2+], NFAT activity, CSE expression and activity, and H2S production while inhibiting NFAT activation lowers CSE expression. The observations that IH exposure decreases NFATc3 activation and CSE-dependent vasodilation support a role for NFAT in regulating endothelial H2S production. NEW & NOTEWORTHY This study identifies the calcium-regulated transcription factor nuclear factor of activated T cells as a novel regulator of cystathionine γ-lyase (CSE). This pathway is basally active in mesenteric artery endothelial cells, but, after exposure to intermittent hypoxia to mimic sleep apnea, nuclear factor of activated T cells c3 nuclear translocation and CSE expression are decreased, concomitant with decreased CSE-dependent vasodilation.

scholarly journals Vascular Endothelial Growth Factor Activates Nuclear Factor of Activated T Cells in Human Endothelial Cells: a Role for Tissue Factor Gene Expression

1999 ◽  
Vol 19 (3) ◽  
pp. 2032-2043 ◽  
Author(s):  
Angel Luis Armesilla ◽  
Elisa Lorenzo ◽  
Pablo Gómez del Arco ◽  
Sara Martínez-Martínez ◽  
Arantzazu Alfranca ◽  
...  
Keyword(s):  
Gene Expression ◽  
Vascular Endothelial Growth Factor ◽  
T Cells ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Vascular Endothelial ◽  
Nuclear Factor ◽  
Promoter Activation ◽  
Activated T Cells ◽  
Endothelial Growth Factor

ABSTRACT Vascular endothelial growth factor (VEGF) is a potent angiogenic inducer that stimulates the expression of tissue factor (TF), the major cellular initiator of blood coagulation. Here we show that signaling triggered by VEGF induced DNA-binding and transcriptional activities of nuclear factor of activated T cells (NFAT) and AP-1 in human umbilical vein endothelial cells (HUVECs). VEGF also induced TF mRNA expression and gene promoter activation by a cyclosporin A (CsA)-sensitive mechanism. As in lymphoid cells, NFAT was dephosphorylated and translocated to the nucleus upon activation of HUVECs, and these processes were blocked by CsA. NFAT was involved in the VEGF-mediated TF promoter activation as evidenced by cotransfection experiments with a dominant negative version of NFAT and site-directed mutagenesis of a newly identified NFAT site within the TF promoter that overlaps with a previously identified κB-like site. Strikingly, this site bound exclusively NFAT not only from nuclear extracts of HUVECs activated by VEGF, a stimulus that failed to induce NF-κB-binding activity, but also from extracts of cells activated with phorbol esters and calcium ionophore, a combination of stimuli that triggered the simultaneous activation of NFAT and NF-κB. These results implicate NFAT in the regulation of endothelial genes by physiological means and shed light on the mechanisms that switch on the gene expression program induced by VEGF and those regulating TF gene expression.

scholarly journals Nuclear factor of activated T cells and early growth response-1 cooperate to mediate tissue factor gene induction by vascular endothelial growth factor in endothelial cells

2007 ◽  
Vol 97 (06) ◽  
pp. 979-987 ◽  
Author(s):  
Gernot Schabbauer ◽  
Bernhard Schweighofer ◽  
Diana Mechtcheriakova ◽  
Markus Lucerna ◽  
Bernd Binder ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Growth Response ◽  
Calcineurin Inhibitor ◽  
Early Growth ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Factor Gene ◽  
Early Growth Response

SummaryBased on the finding that tissue factor belongs to a group of genes upregulated in endothelial cells by VEGF, but not by EGF, we investigated signals selectively triggered by VEGF. Whereas the transcription factor early growth response (EGR)-1, which has previously been shown by us to be essentially involved in tissue factor gene regulation, was similarly induced by both factors, one major difference between VEGF and EGF signaling was the activation of the Ca++-mediated calcineurin/nuclear factor of activated T cells (NFAT) pathway by VEGF. Consistent with the importance of this pathway for tissue factor induction, treatment of endothelial cells with the Ca++ chelator BAPTA-AM, as well as the calcineurin inhibitor cyclosporin A, partially inhibited VEGF induced tissue factor upregulation. Furthermore, tissue factor reporter gene assays revealed a synergistic cooperation of NFAT and EGR-1 in the induction of the TF promoter, and a physical interaction between the two factors was indicated by co-immunoprecipitation assays. Another gene upregulated by VEGF predominantly via NFAT, which is not induced by EGF, is the DSCR-1 gene. The calcineurin inhibitor DSCR-1 seems to be induced by VEGF in a negative feed-back loop to limit NFAT activation. When we tested adenoviral overexpression of DSCR-1, VEGF-mediated induction of tissue factor mRNA was reduced, and complete suppression could be achieved by a combination of viruses expressing DSCR-1 and NAB2, a corepressor of EGR-1. These findings support that both, NFAT and EGR-1, are required for tissue factor upregulation in response to VEGF.

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