IntroductionIn response to antigen stimulation, cells of the immune system initiate the expression of activation-induced genes through the coordinate action of transcription factors at gene regulatory elements. The nuclear factor of activated T cells (NFAT), originally identified as a nuclear complex binding to the antigen-response element of the IL-2 gene, 1 has been implicated in the regulation of various inducible genes, particularly those encoding cytokines and cell surface receptors. 2 Sequence inspection, in vitro binding assays, and overexpression studies have identified potential binding sites for NFAT in the promoters and enhancers of numerous genes, including those encoding interleukin-2 (IL-2), IL-3, IL-4, IL-5, tumor necrosis factor ␣ (TNF-␣), granulocyte macrophagecolony-stimulating factor (GM-CSF), CD40L, FasL, CD25, b-type natriuretic peptide, and the adipocyte-specific protein aP2. [2][3][4][5] In general, NFAT is thought to act as a positive regulator of gene expression. For instance, IL-2 production is profoundly compromised in patients unable to generate functional NFAT complexes, 6 and peptides based on the calcineurin-docking site of NFAT proteins, which specifically inhibit the activation of NFAT, impair the production of IL-2 and other cytokines in vitro. 7 To date, 4 calcium-regulated members of the NFAT family (NFAT1-4; also known as NFATc1-4 and hereafter abbreviated NFAT), with distinct but overlapping tissue distributions, have been identified. [8][9][10][11][12][13][14][15] A fifth protein, NFAT5/TonEBP, 16,17 is regulated by osmotic shock. NFAT3 is expressed predominantly outside the immune system, whereas NFAT1 (NFATp, NFATc2), NFAT2 (NFATc, NFATc1), and NFAT4 (NFATx, NFATc3) can be found in immune cells including T cells, B cells, natural killer (NK) cells, macrophages, and mast cells. 2,10,[18][19][20][21] Although NFAT proteins are expressed in multiple cell types, their regulation has been extensively studied in T and B cells, where they are present as phosphoproteins in the cytoplasm of resting cells. After cell stimulation, 4 consecutive steps lead to the activation of NFAT: dephosphorylation by the calcium-dependent phosphatase calcineurin, a process inhibited by the immunosuppressive drugs cyclosporin A and FK506 [22][23][24][25][26] ; translocation into the nucleus 18,23,27 ; binding to specific DNA elements in the regulatory regions of target genes, which occurs in physical or functional cooperation with other transcription factors including AP-1 (Fos/Jun), c-Maf (Maf), and GATA-family proteins 2,4,22,[28][29][30] ; and interaction with known or putative coactivator proteins such as p300, CBP, and NIP-45. [31][32][33] After antigen stimulation, CD4 ϩ T cells differentiate into at least 2 types of effector cells that differ as to the pattern of cytokines they produce on restimulation. [34][35][36] Th1 cells are defined by the production of interferon-␥ (IFN-␥) and mediate predominantly cellular immune responses, whereas the signature cytokines of Th2 cells-IL-4, IL-5, and ...

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.

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