Regulation of fas/fas ligand-mediated apoptosis by nuclear factor of activated T cells in megakaryocytes.

Signal transduction pathways in megakaryocytes, a rare population of bone marrow cells, are poorly understood. We have previously shown that the calcineurin-dependent transcription factor Nuclear Factor of Activated T cells (NFAT) is 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 megakaryocytes forces the cells to go into apoptosis. Calcineurin/NFAT activation in megakaryocytes leads to membrane expression of Fas ligand (FASLG), a pro-apoptotic member of the tumour necrosis factor superfamily. Expression of FASLG was augmented in cells stably overexpressing NFATC2 and suppressed in cells either pretreated with the calcineurin inhibitor ciclosporin A (CsA) or expressing the specific peptide inhibitor of NFAT, VIVIT. In cocultures with Fas-expressing Jurkat T cells, the presence of activated megakaryocytic cells, but not of unstimulated cells or cells stimulated in the presence of CsA, significantly induced apoptosis in Jurkat cells in a Fas/FASLG- and NFAT-dependent manner. These results represent the first evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, and suggest that the biological role of megakaryocytes may include the induction of apoptosis in bystander cells.

Osteomyelosclerosis, anemia and extramedullary hematopoiesis in mice lacking the transcription factor NFATc2.

BACKGROUND: Nuclear factors of activated T cells (NFAT) are transcription factors that are central to cytokine production in activated T cells and regulate the development and differentiation of various tissues. NFATc2 is expressed in hematopoietic stem cells and regulated during myeloid commitment in a lineage-specific manner. The biological role of NFATc2 in hematopoiesis is, however, unclear. DESIGN AND METHODS: In the present study, we analyzed steady-state hematopoiesis in young (<3 months) and old (>12 months) mice lacking NFATc2. Complete blood counts were performed in the peripheral blood, bone marrow and spleen. Using cytological and histological analyses, the blood cell differential was determined. Colony-formation assays were used to determine the differentiation potential of hematopoietic cells. Bone cell cultures were derived from the bone marrow, and bone remodeling markers were determined in the serum. RESULTS: NFATc2(-/-) mice older than 12 months were anemic and thrombocytopenic. The bone marrows of these mice showed a markedly reduced number of hematopoietic cells, of which megakaryocytic and erythroid lineages were most affected. While the number of hematopoietic progenitor cells in NFATc2-deficent bone marrow was reduced, the myeloid differentiation potential of these cells remained intact. Aged NFATc2(-/-) mice showed ossification of their bone marrow space and developed extramedullary hematopoiesis in the spleen. Ex vivo differentiation assays revealed an intrinsic defect of NFATc2-deficient stromal cells, in which NFATc2(-/-) osteoblasts differentiated more efficiently than wild-type cells, whereas osteoclast differentiation was impaired. CONCLUSIONS: Our data suggest that NFATc2 may play a role in the maintenance of steady-state hematopoiesis and bone remodeling in adult organisms.

Regulation of Down Syndrome Critical Region 1 expression by Nuclear Factor of Activated T cells in megakaryocytes.

As precursors of platelets, megakaryocytes must fulfil the complex tasks of protein synthesis and platelet assembly. Megakaryocytic dysfunction can lead to neoplastic disorders, such as acute megakaryoblastic leukaemia, an entity with a 500-fold increased incidence in children with Down syndrome (DS). Down Syndrome Critical Region 1 (DSCR1), a member of the calcipressin family of calcineurin inhibitors, is overexpressed in DS, and destabilization of the calcineurin/Nuclear Factor of Activated T cells (NFAT) pathway by overexpression of DSCR1 has been implicated in some of the pathophysiological features of the disease. The roles of NFAT and DSCR1 in megakaryocyte signalling and gene expression, however, are unknown. In this study, we show that calcineurin and NFAT are components of a calcium-induced signalling cascade in megakaryocytes. NFAT activation in megakaryocytes was induced by fibrillar collagen type I and was completely sensitive to the calcineurin inhibitor cyclosporin A. We established DSCR1 as a calcium-induced NFAT target gene in these cells and show that overexpression of DSCR1 in megakaryocytes strongly inhibits NFAT activation as well as NFAT-dependent expression of the Fas ligand gene (FASLG). These results suggest that DSCR1 acts as an endogenous feedback inhibitor of NFAT signalling in megakaryocytes, and may have implications for megakaryocytic gene expression in DS.

Expression analysis of nuclear factor of activated T cells (NFAT) during myeloid differentiation of CD34+ cells: regulation of Fas ligand gene expression in megakaryocytes.

OBJECTIVE: Nuclear factor of activated T cells (NFAT) transcription factors belong to a family of five proteins that are primarily known for their central role in the regulation of inducible gene expression in activated T cells. Little information exists on the expression or function of NFAT family members in hematopoietic cells, during myeloid differentiation or in myeloid cells. MATERIALS AND METHODS: In the present study, we establish a comprehensive expression profile of all five NFAT family members in human CD34+ hematopoietic progenitor cells and during their ex vivo differentiation into neutrophil, eosinophil, erythroid, and megakaryocytic lineages. Based on the observed expression pattern, the role of NFAT in Fas ligand gene expression in megakaryocytes was investigated. RESULTS: When CD34+ cells are induced to differentiate into neutrophil granulocytes, expression of all NFAT family members is rapidly suppressed. In contrast, regulation of NFAT expression during eosinophil, erythroid, and megakaryocytic differentiation follows a family member- and lineage-specific pattern. Most obviously, transcript and protein levels of NFATc4 are specifically upregulated about 10-fold during megakaryocytic differentiation, while they remain almost undetectable in neutrophil, eosinophil, and erythroid cells. As a first evidence for a functional role for NFAT in this cell type, NFAT was found to be strictly required for both the constitutive and inducible expression of the Fas ligand gene in megakaryocytes. CONCLUSION: The expression pattern of NFAT and its family member- and lineage-specific regulation during myeloid differentiation will prompt further studies on the role of NFAT in myeloid cells, particularly in megakaryocytes.

Expression and regulation of NFAT (nuclear factors of activated T cells) in human CD34+ cells: down-regulation upon myeloid differentiation.

The calcineurin-dependent, cyclosporin A (CsA)-sensitive transcription factor nuclear factor of activated T cells (NFAT) represents a group of proteins, which is well-characterized as a central regulatory element of cytokine expression in activated T cells. In contrast, little is known about the expression or function of NFAT family members in myeloid cells; moreover, it is unclear whether they are expressed by hematopoietic stem/progenitor cells. Here, we show that NFATc2 (NFAT1) is expressed at high levels in CD34+ cells and megakaryocytes but not in cells committed to the neutrophilic, monocytic, or erythroid lineages. Cytokine-induced in vitro differentiation of CD34+ cells into neutrophil granulocytes results in the rapid suppression of NFATc2 RNA and protein. NFATc2 dephosphorylation/rephosphorylation as well as nuclear/cytoplasmic translocation in CD34+ cells follow the same calcineurin-dependent pattern as in T lymphocytes, suggesting that NFATc2 activation in these cells is equally sensitive to inhibition with CsA. Finally, in vitro proliferation, but not differentiation, of CD34+ cells cultured in the presence of fms-like tyrosine kinase 3 ligand (FLT3L), stem cell factor, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-3, and G-CSF is profoundly inhibited by treatment with CsA in a dose-dependent manner. These results suggest a novel and unexpected role for members of the NFAT transcription factor family in the hematopoietic system.

Normal intrinsic Th1/Th2 balance in patients with chronic phase chronic myeloid leukemia not treated with interferon-alpha or imatinib.

BACKGROUND AND OBJECTIVES: CD4+ T helper cells are an integral part of effective immune responses against various malignancies; however in tumor-bearing patients they are frequently functionally unresponsive. T helper cells of patients with chronic myeloid leukemia (CML), analyzed as part of mononuclear cell fractions, show a loss of signaling molecules, a compromised Th1 cytokine production and a shift towards a non-productive Th2 state. The underlying mechanism is unknown and may involve intrinsic T cell defects as well as indirect effects mediated by leukemia or antigen-presenting cells. The purpose of the present study was to analyze the intrinsic cytokine-producing capacity of purified CML T helper cells in the absence of other cell types. DESIGN AND METHODS: Untouched CD4+ T cells with a purity of more than 90% were isolated from 10 patients with Ph+ chronic phase CML on maintenance treatment with hydroxyurea. The cells were isolated by density gradient centrifugation followed by immunomagnetic depletion of leukemia and accessory cells. The ex vivo cytokine-producing capacity of CML T helper cells in response to polyclonal stimulation with anti-CD3 and anti-CD28 was then compared to that of cells purified from matched healthy volunteers. RESULTS: T helper cells purified from CML patients produced comparable amounts of the Th1 cytokines interleukin (IL)-2 and interferon (IFN)-g as cells purified from healthy volunteers. Likewise, no difference between CML and control T helper cells was found with respect to the Th2 cytokines, IL-4 and IL-13, as well as the immunomodulatory cytokine, IL-10. INTERPRETATION AND CONCLUSIONS: In the absence of leukemia and accessory cells, the intrinsic cytokine-producing capacity of CML T helper cells is normal. A Th2 shift was not detected, and the predominant presence of an IL-10-producing, immunosuppressive T helper cell subset could be excluded.