Regulation of Fas/Fas Ligand-Mediated Apoptosis in Megakaryocytes by Nuclear Factor of Activated T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4023-4023
Author(s):  
Laleh S. Arabanian ◽  
Satu Kyttälä ◽  
Ivonne Habermann ◽  
Gerhard Ehninger ◽  
Alexander Kiani
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Fas Ligand ◽  
Jurkat Cells ◽  
Biological Role ◽  
Regulatory Function ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
In Cells

Abstract Abstract 4023 Poster Board III-959 Bone marrow megakaryocytes are the precursors of peripheral blood platelets and therefore constitute an integral part of primary haemostasis, thrombosis and wound healing. Platelets have also been implicated in the regulation of inflammation and other immune responses, but the role of megakaryocytes in this context is less clear. The calcineurin-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) is a master regulator of cytokine production in T lymphocytes and therefore central for T cell-dependent immune reactions. We have previously shown that NFAT is strongly expressed in megakaryocytes and is required for the transcription of specific megakaryocytic genes. The biological role of NFAT in megakaryocytes, however, is unknown. Here we show that activation of the calcineurin/NFAT pathway in either primary megakaryocytes or CMK megakaryocytic cells forces the cells to go into apoptosis. Cell death in megakaryocytes is induced by treating the cells with the calcium ionophore ionomycin and at least partially suppressed by either the pan-caspase inhibitor zVAD or the calcineurin inhibitor cyclosporin A (CsA). Ionomycin stimulation of megakaryocytes leads to the expression of Fas Ligand (FasL), a pro-apoptotic member of the tumor necrosis factor superfamily. Expression of FasL was detectable as early as six hours after stimulation on the membrane of ionomycin-treated megakaryocytes, was augmented in cells stably overexpressing NFATc2, and was suppressed in cells either pretreated with CsA or expressing the specific peptide inhibitor of NFAT, VIVIT. To investigate the physiological relevance of FasL expression on megakaryocytes, we performed cocultures of megakaryocytes with Fas-expressing T lymphocytes, in which CMK cells were either left unstimulated or prestimulated with ionomycin and then added to Jurkat cells. The presence of ionomycin-stimulated CMK cells, but not of unstimulated cells or cells stimulated in the presence of CsA, significantly induced apoptosis in Jurkat cells. Overexpression of NFATc2 in CMK cells enhanced their potency to induce apoptosis in Jurkat cells, while cells expressing VIVIT were less effective. Apoptosis induction of Jurkat cells by stimulated CMK cells was partially blocked by the presence of either a neutralizing antibody against FasL or an antagonistic antibody to Fas during the coculture period, indicating involvement of the FasL/Fas apoptosis pathway. These results have several implications. First, they represent the first clear evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, namely the regulation of Fas/FasL-dependent apoptosis. Second, they underline that the biological role of megakaryocytes is not restricted to the production of proteins and other cellular structures for platelet assembly, but that this population of cells fulfills an independent regulatory function in the context of the surrounding tissue. Our results suggest that this regulatory function may include the induction of Fas/FasL-dependent apoptosis in bystander cells under certain conditions, for example the elimination of activated T cells in inflammatory situations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An Essential Role of the Nuclear Factor of Activated T Cells in the Regulation of the Expression of the Cyclooxygenase-2 Gene in Human T Lymphocytes

2000 ◽  
Vol 275 (31) ◽  
pp. 23627-23635 ◽  
Author(s):  
Miguel A. Íñiguez ◽  
Sara Martı́nez-Martı́nez ◽  
Carmen Punzón ◽  
Juan Miguel Redondo ◽  
Manuel Fresno
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Essential Role ◽  
Cyclooxygenase 2 ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Human T Lymphocytes

scholarly journals The Essential Role of MEKK3 Signaling in Angiotensin II-induced Calcineurin/Nuclear Factor of Activated T-cells Activation

2005 ◽  
Vol 280 (44) ◽  
pp. 36737-36746 ◽  
Author(s):  
Shahrzad Abbasi ◽  
Bing Su ◽  
Rodney E. Kellems ◽  
JianHua Yang ◽  
Yang Xia
Keyword(s):  
T Cells ◽  
Angiotensin Ii ◽  
Essential Role ◽  
Nuclear Factor ◽  
Activated T Cells

Reciprocal Regulation of DSCR1 (Down Syndrome Critical Region 1) Expression and NFAT (Nuclear Factor of Activated T Cells) Activation in Megakaryocytes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2204-2204
Author(s):  
Satu Kyttaelae ◽  
Ivonne Habermann ◽  
Martin Bornhaeuser ◽  
Gerhard Ehninger ◽  
Alexander Kiani
Keyword(s):  
T Cells ◽  
Down Syndrome ◽  
Critical Region ◽  
Fas Ligand ◽  
Nuclear Translocation ◽  
Specific Inhibitor ◽  
Chromosome 21 ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Retroviral Transduction

Abstract NFAT (Nuclear Factor of Activated T cells) is a family of calcium-induced, calcineurin-dependent transcription factors, well characterized as central regulators of inducible gene expression in T lymphocytes but now known to function also in several other cell types in various adaptation and differentiation processes. Activation of NFAT by the phosphatase calcineurin is counteracted by several inhibitory kinases and can be completely blocked by the immunosuppressant Cyclosporin A. The Down syndrome critical region 1 (DSCR1; also termed CSP1, MCIP1 or RCAN1) gene belongs to the calcipressin family of endogenous calcineurin inhibitors and is expressed in several isoforms, one of which (isoform C, coded by exons 4–7) has been described to be a transcriptional target for NFAT in striated muscle, endothelial, and neural cells. The DSCR1 gene is located within the Down syndrome critical region of human chromosome 21 and is, together with 200–300 other genes, overexpressed about 1.5-fold in patients with Down syndrome (DS). Previously, dysregulation of NFAT signaling by overexpression of DSCR1 has been implicated in causing various of the pathophysiological features observed in DS patients. Children with DS also suffer from an about 500-fold increased incidence of acute megakaryocytic leukemia; the respective roles of NFAT or DSCR1 in megakaryocytes of either normal individuals or those with DS, however, has not yet been established. Here we show that DSCR1 is upregulated during megakaryocytic differentiation in a lineage-specific manner, and in mature megakaryocytes is further strongly induced by calcineurin stimulation. DSCR1 expression in megakaryocytes is regulated by NFAT, since overexpression of NFATc2 enhances, while overexpression of the specific inhibitor of NFAT activation, VIVIT, suppresses expression of the gene. We further demonstrate that DSCR1 does not only represent an NFAT target in megakaryocytes, but itself acts an inhibitor of NFAT signaling in these cells. Overexpression of DSCR1 in CMK cells as well as in primary megakaryocytes by retroviral transduction profoundly suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFATc2, as well as transactivation of an NFAT-dependent promoter construct. Finally, overexpression of DSCR1 in megakaryocytes markedly downregulated both the constitutive and induced expression of Fas Ligand, a pro-apoptotic gene recently established as a NFAT target in megakaryocytes. Together, these results suggest that DSCR1 acts as an NFAT-induced NFAT inhibitor in megakaryocytes and, when overexpressed, interferes with the expression of NFAT-dependent megakaryocytic genes.

Cytokine Induced Killer Cells Can Kill Target through Antigen/TCR Dependent and Independent Mechanisms: New Clinical Perspectives of Utilisation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3024-3024
Author(s):  
Alice Pievani ◽  
Gianmaria Borleri ◽  
Camilla Belussi ◽  
Alessandro Rambaldi ◽  
Josee Golay ◽  
...  
Keyword(s):  
T Cells ◽  
Target Cell ◽  
Fas Ligand ◽  
Conflicts Of Interest ◽  
Leukaemic Cell ◽  
Similar Data ◽  
Antigen Specificity ◽  
Activated T Cells ◽  
Cik Cells

Abstract Abstract 3024 Poster Board II-1000 Cytokine induced killer (CIK) cells cultures can easily be obtained by stimulating PBMCs with monoclonal antibody anti-CD3 OKT3, IFNgamma and IL2. After 3-4 weeks at least 3 separate populations are present in the culture: CD3+/CD56-/CD8+ precursors (40.5 ± 19.9%), CD3-CD56+ NK cells (2.5 ± 1.5%) and CD3+/CD56+/CD8+ CIK cells (56.9 ± 21%) which show a T EMRA phenotype (Franceschetti et al., Exp Hematol. 37, 616-628, 2009). CIK cultures are currently used in allogeneic or autologous settings as potential anti-neoplastic effectors for adoptive transfer clinical approaches. We have further characterised the mechanism of target cell recognition and role of activating receptors and of lytic mediators in the cytotoxicity of purified CD3+/CD56+/CD8+ CIK cells. We have observed that CIK cells can kill targets through at least two distinct mechanisms: the first TCR-dependent and antigen-specific and the second non TCR-dependent. Indeed, upon TCR/CD3 crosslinking in CIK cells we observe ERK-1/2 phosphorylation, IFNgamma production (mean 32.6% of positive cells by intracellular staining) and TNFalpha production (mean 19.6%). CD3 ligation by OKT3 results in a significant increase over time in the percentage of CIK cells undergoing degranulation evaluated as CD107a positive cells (respectively 15.5 ± 2.2% at 60', 24.4 ± 1% at 120', 32,9 ± 8.7% at 180' and 34.2 ± 11.1% at 240'). CD3 ligation on CIK cells can induce cytotoxicity in a reverse Ab-dependent killing assay. Addition of OKT3 enhances also the cytotoxicity of CIK cells against K562 leukaemic target (from 16 ± 5% to 50 ± 4 %, E:T 10:1; p<0.001). The TCR/CD3 mediated activation is blocked by pre-treatment with cyclosporine A, confirming the role of calcium-regulated phosphatase calcineurin in the CD3-linked degranulation pathway. Interestingly, CIK cells retain functional activity as antigen-specific memory T cells. Indeed in case of CIK cultures obtained from CMV-seropositive donors, CMV-specific CD3+/CD56+ CIK cells can be generated. CMV-specific CIK cells immunopurified by HLA-peptide tetramers are able to specifically recognise and kill autologous but not allogeneic PHA-blasts pulsed with pp65495-503 but not with irrelevant peptide (average lysis 63 ± 8%, E:T 10:1) and to produce IFNgamma following antigenic stimulation (26.4 ± 7%). Similar data are obtained with EBV-specific T cells. Moreover, CIK cells also show non-MHC-restricted cytotoxicity against numerous leukemic target cell lines. The same CMV-specific purified CIK population shows to posses non MHC-restricted cytotoxic properties against several leukemic targets (average lysis 44 ± 8%, E:T 10:1). NKG2D, described as receptor that can trigger TCR-independent cytotoxicity in activated T cells, is expressed on all CIK cells (mean 99%, MFI 251). Although we can show that NKG2D is functional in these cells since cross-linking with anti-NKG2D mAb leads to ERK phophorylation, blocking of this receptor with mAb does not affect the cytolysis of leukaemic targets. NKG2D crosslinking in CIK cells is not sufficient to induce granule esocytosis and INFgamma or TNFalpha production. No correlation is found between expression and surface density of NKG2D ligands on leukaemic cell targets and their susceptibility to CIK-mediated lysis. Unrestricted CIK-mediated cytotoxicity occurs in the absence of measurable degranulation, is not affected by cyclosporin A, but is inhibited by 70% in presence of brefeldin A, a surface upregulation of glycopolypeptide molecules inhibitor, suggesting a role for Fas-ligand family molecules. CIK cells indeed express TNFalpha (mean 16%, MFI 56), FasL (mean 34%, MFI 34) and TRAIL (mean 37%, MFI 49). The role of this death ligands in leukaemic cell killing is currently under investigation. These data clearly show that CD3+/CD56+/CD8+ CIK cells are activated T EMRA cells which have retained their TCR/CD3 complex usage and their antigen specificity but have acquired unrestricted anti-leukaemic activity. The identification of the molecules involved in this killing is still under investigation. These data open up the possibility of multiple clinical use of CIK cultures, including anti-leukaemic and anti-infective usage particularly in immunodeficient patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dynamic equilibrium between calcineurin and kinase activities regulates the phosphorylation state and localization of the nuclear factor of activated T-cells

1997 ◽  
Vol 324 (2) ◽  
pp. 597-603 ◽  
Author(s):  
John E. SCOTT ◽  
Valerie A. RUFF ◽  
Karen L. LEACH
Keyword(s):  
T Cells ◽  
Nuclear Localization ◽  
T Cell Activation ◽  
Cell Activation ◽  
Kinase Inhibitors ◽  
Dynamic Equilibrium ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Phosphorylation State

The nuclear factor of activated T-cells (NFATp) is a phosphorylated transcription factor that resides in the cytoplasm of unactivated T-cells. T-cell activation results in the activation of the phosphatase calcineurin (CaN), which leads to the dephosphorylation and subsequent nuclear localization of NFATp. We have investigated the role of kinases in the phosphorylation state and subcellular localization of NFATp. The phosphorylation state and nuclear/cytoplasmic location of NFATp were determined in unstimulated murine HT-2 cells treated with a panel of kinase inhibitors. Two of the seven kinase inhibitors, staurosporine (St) and bisindolylmaleimide I (BI), resulted in the dephosphorylation and nuclear localization of NFATp. These St-induced effects were inhibited by pretreatment with FK506, indicating that CaN activity was required for the observed effects on NFATp. Treatment of cells with ionomycin resulted in NFATp dephosphorylation and nuclear localization. Removal of ionomycin from the cells resulted in the reappearance of phosphorylated NFATp in the cytosol. St and BI also inhibited the re-accumulation of NFATp in the cytoplasm and its re-phosphorylation after ionomycin removal. The re-accumulation of NFATp in the cytosol after ionomycin withdrawal was shown to be energy- and temperature-dependent. Taken together, these results suggest that in unstimulated cells NFATp is actively maintained in the cytoplasm by kinases acting in opposition to basal CaN activity.

scholarly journals Mice Lacking the Calcineurin Inhibitor Rcan2 Have an Isolated Defect of Osteoblast Function

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3537-3548 ◽  
Author(s):  
J. H. Duncan Bassett ◽  
John G. Logan ◽  
Alan Boyde ◽  
Moira S. Cheung ◽  
Holly Evans ◽  
...  
Keyword(s):  
T Cells ◽  
Bone Development ◽  
Skeletal Development ◽  
Dominant Negative ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Osteoblast Function ◽  
And Function

Calcineurin-nuclear factor of activated T cells signaling controls the differentiation and function of osteoclasts and osteoblasts, and regulator of calcineurin-2 (Rcan2) is a physiological inhibitor of this pathway. Rcan2 expression is regulated by T3, which also has a central role in skeletal development and bone turnover. To investigate the role of Rcan2 in bone development and maintenance, we characterized Rcan2−/− mice and determined its skeletal expression in T3 receptor (TR) knockout and thyroid-manipulated mice. Rcan2−/− mice had normal linear growth but displayed delayed intramembranous ossification, impaired cortical bone formation, and reduced bone mineral accrual during development as well as increased mineralization of adult bone. These abnormalities resulted from an isolated defect in osteoblast function and are similar to skeletal phenotypes of mice lacking the type 2 deiodinase thyroid hormone activating enzyme or with dominant-negative mutations of TRα, the predominant TR isoform in bone. Rcan2 mRNA was expressed in primary osteoclasts and osteoblasts, and its expression in bone was differentially regulated in TRα and TRβ knockout and thyroid-manipulated mice. However, in primary osteoblast cultures, T3 treatment did not affect Rcan2 mRNA expression or nuclear factor of activated T cells c1 expression and phosphorylation. Overall, these studies establish that Rcan2 regulates osteoblast function and its expression in bone is regulated by thyroid status in vivo.

scholarly journals Human T-Cell Lymphotropic Virus Type 1 p12I Expression Increases Cytoplasmic Calcium To Enhance the Activation of Nuclear Factor of Activated T Cells

2002 ◽  
Vol 76 (20) ◽  
pp. 10374-10382 ◽  
Author(s):  
Wei Ding ◽  
Björn Albrecht ◽  
Robert E. Kelley ◽  
Natarajan Muthusamy ◽  
Seung-Jae Kim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Calcium Release ◽  
Nuclear Factor ◽  
Cytoplasmic Calcium ◽  
Activated T Cells ◽  
Human T Cell ◽  
Nfat Activation

ABSTRACT Human T-cell lymphotropic virus type 1 (HTLV-1) establishes persistent infection and is associated with lymphoproliferative or neurodegenerative diseases. As a complex retrovirus, HTLV-1 contains typical structural and enzymatic genes, as well as regulatory and accessory genes encoded in the pX region. The early events necessary for HTLV-1 to establish infection in lymphocytes, its primary target cells, remain unresolved. Recent studies have demonstrated the importance of regulatory and accessory gene products in determining this virus-host interaction. Among these, pX open reading frame I, which encodes two proteins, p12I and p27I, is required for establishing persistent infection in vivo and for infection in quiescent primary lymphocytes. In addition, p12I localizes in the endoplasmic reticulum (ER) and cis-Golgi apparatus and associates with a calcium binding protein, calreticulin. We recently reported that p12I expression induces the calcium-responsive T-cell transcription factor, nuclear factor of activated T cells (NFAT), in the presence of phorbol ester activation. Based on these studies, we hypothesize that p12I may modulate calcium release from the ER. Here, we report that p12I expression increases basal cytoplasmic calcium and concurrently diminishes calcium available for release from the ER stores. Overexpression of calreticulin, a calcium buffer protein, blocked p12I-mediated NFAT activation independently of its ability to bind p12I. Chemical inhibition studies using inhibitors of inositol 1,4,5-triphosphate receptor and calcium release-activated calcium channels suggest that inositol 1,4,5-triphosphate receptor in the ER membrane and calcium release-activated calcium channels in the plasma membrane contribute to p12I-mediated NFAT activation. Collectively, our results are the first to demonstrate the role of p12I in elevating cytoplasmic calcium, an antecedent to T-cell activation, and further support the important role of this accessory protein in the early events of HTLV-1 infection.

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