NFATc3 regulates the transcription of genes involved in T-cell activation and angiogenesis

Abstract

The nuclear factor of activated T cells (NFAT) family of transcription factors plays important roles in many biologic processes, including the development and function of the immune and vascular systems. Cells usually express more than one NFAT member, raising the question of whether NFATs play overlapping roles or if each member has selective functions. Using mRNA knock-down, we show that NFATc3 is specifically required for IL2 and cyclooxygenase-2 (COX2) gene expression in transformed and primary T cells and for T-cell proliferation. We also show that NFATc3 regulates COX2 in endothelial cells, where it is required for COX2, dependent migration and angiogenesis in vivo. These results indicate that individual NFAT members mediate specific functions through the differential regulation of the transcription of target genes. These effects, observed on shortterm suppression by mRNA knock-down, are likely to have been masked by compensatory effects in gene-knockout studies. (Blood. 2011;118(3):795-803) IntroductionThe nuclear factor of activated T cells (NFAT) family of transcription factors consists of 4 members regulated by the Ca 2ϩ -calmodulin-dependent phosphatase calcineurin (CN). NFAT proteins were initially identified in T cells, and their functions in the immune system have been analyzed extensively. 1 In resting T cells, NFATs are present in the cytoplasm in a hyperphosphorylated and inactive form. Increases in the levels of intracellular Ca 2ϩ activate CN, which dephosphorylates NFAT. This promotes the translocation of NFATs to the nucleus, where they generally cooperate with other transcription factors to regulate an array of genes involved in the functions of the immune system, 2-5 including IL2 6 and cyclooxygenase 2 (COX2). 7 Expression of NFATc1, NFATc2, and NFATc3 (but not NFATc4) has been detected in normal and transformed T cells. 8 The co-occurrence of different NFAT members in the same cells raises the question of whether there is a degree of functional redundancy or if the immunoregulatory functions of each member are unique. The NFAT DNA-binding domain is highly conserved among members, 2,3 and the immune phenotypes of mice deficient in a single NFAT member are relatively mild, 1 which supports the existence of functional redundancy in T-cell activation. The use of Nfatc1 knockout (KO) cells in complementation experiments in Rag 2 Ϫ/Ϫ mice indicated that NFATc1 plays a role in the expansion of mature peripheral B and T lymphocytes and in Th2 cytokine production, but is not required for earlier differentiation and activation events. 9,10 Similarly, Nfatc2 Ϫ/Ϫ mice have mild defects in the immune response 11 and impaired Th2 differentiation due to overproduction of IL4. 12,13 Nfatc3-deficient mice show impaired thymic development, characterized by a loss of CD4 ϩ /CD8 ϩ double-positive cells through programmed cell death. 14 More recently, the use of conditional NFATc3 KO mice has revealed a role in both positive selection and double-negative development, but these mice showed no detectable alter...

Plasma Membrane Calcium ATPase Isoform 4 Inhibits Vascular Endothelial Growth Factor–Mediated Angiogenesis Through Interaction With Calcineurin

“…Although our results do not directly demonstrate that reduction of these cellular functions is linked to PMCA4-mediated downregulation of RCAN1.4 and Cox-2, both proteins have been described to promote endothelial cell migration and tube formation, 17,18,31 suggesting that they likely play a significant role in the PMCA4-directed inhibition of angiogenesis. In agreement with this idea, PMCA4-deficient MLECs presented higher expression of Cox-2 ( Figure The results of our gain-or loss-of-PMCA4 function experiments might be particularly relevant in the context of pathophysiological processes associated with changes in the expression of PMCA proteins.…”

“…4 and Cox-2, whose products actively participate in the onset of angiogenesis. [5][6][7][17][18][19] The inhibitory effect produced by PMCA4 on the VEGF-induced activity of the calcineurin/NFAT pathway in endothelial cells prompted us to next examine the role of PMCA4 as a negative regulator of angiogenesis gene expression.…”

C/EBPβ and Nuclear Factor of Activated T Cells Differentially Regulate Adamts-1 Induction by Stimuli Associated with Vascular Remodeling

“…2B), despite efficient blockade of CN signaling by CsA, as confirmed by CsA-mediated inhibition of the genes for cyclooxygenase 2 (Cox-2) and RCAN1.4 ( Fig. 2), two known CN/NFAT targets (19,24). These results suggest that CN signaling is implicated in VEGF-dependent induction of Adamts-1 expression but not in Ang-II-promoted Adamts-1 upregulation.…”

“…Chromatin immunoprecipitation (ChIP) assays were performed essentially as described previously (19). HUVECs (1.5 ϫ 10 7 ) were stimulated with 50 ng/ml VEGF or 10 Ϫ5 M Ang-II for 30 min and then fixed with 1% formaldehyde for 5 min at 4°C.…”

Eukaryotic elongation factor 2 controls TNF-α translation in LPS-induced hepatitis

“…Lentiviruses were produced as described (52). Transient calcium phosphate cotransfection of HEK-293 cells was done with the pGIZP empty vector or pGIZP.shEF2 vector ( Thermo Scientific) together with pΔ8.9 and pVSV-G.…”