Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development

Abstract

NFAT transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. NFAT is rephosphorylated and inactivated through the concerted action of at least 3 different kinases: CK1, GSK-3, and DYRK. The major docking sites for calcineurin and CK1 are strongly conserved throughout vertebrate evolution, and conversion of either the calcineurin docking site to a high-affinity version or the CK1 docking site to a low-affinity version results in generation of hyperactivable NFAT proteins that are still fully responsive to stimulation. In this study, we generated transgenic mice expressing hyperactivable versions of NFAT1 from the ROSA26 locus. We show that hyperactivable NFAT increases the expression of NFAT-dependent cytokines by differentiated T cells as expected, but exerts unexpected signal-dependent effects during T cell differentiation in the thymus, and is progressively deleterious for the development of B cells from hematopoietic stem cells. Moreover, progressively hyperactivable versions of NFAT1 are increasingly deleterious for embryonic development, particularly when normal embryos are also present in utero. Forced expression of hyperactivable NFAT1 in the developing embryo leads to mosaic expression in many tissues, and the hyperactivable proteins are barely tolerated in organs such as brain, and cardiac and skeletal muscle. Our results highlight the need for balanced Ca/NFAT signaling in hematopoietic stem cells and progenitor cells of the developing embryo, and emphasize the evolutionary importance of kinase and phosphatase docking sites in preventing inappropriate activation of NFAT.T he activities of many signaling proteins and transcription factors are tightly regulated by phosphorylation and dephosphorylation. Protein kinases and phosphatases bind to specific docking sites on these intracellular proteins to allow their activation or inactivation at the appropriate location and time. A well-studied example of a transcription factor regulated in this fashion is nuclear factor of activated T cells (NFAT) (1-3). In resting cells, NFAT proteins are highly phosphorylated and reside in the cytoplasm; upon cell activation, they are dephosphorylated by the calcium/calmodulindependent phosphatase calcineurin and translocate to the nucleus. NFAT transcription factors play a key role in orchestrating diverse developmental programs, including those of the immune, central nervous, cardiovascular, and musculoskeletal systems (4-11). NFAT also is implicated in maintaining the quiescent state of stem cells in the skin (12).NFAT activation is initiated by dephosphorylation of the NFAT regulatory domain, a conserved 300-amino acid region located N-terminal to the DNA-binding domain (Fig. 1A) (2, 13). The phosphorylated residues (serines) in this domain are distributed among several classes of conserved serine...

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

“…Of note, NFATc2 is expressed by both hematopoietic stem cells and stromal cells. 15,26,27 and thus both direct and indirect mechanisms have to be considered as causes for the hematopoietic insufficiency in the bone marrow of NFATc2-deficient animals. Our results suggest that NFATc2 is neither directly nor indirectly involved in the myeloid differentiation of hematopoietic progenitor cells, as the capacity for erythroid and granulocytic differentiation of these cells was maintained in the absence of NFATc2, both within the local bone marrow environment and in extramedullary organs.…”

Regulatory Subunit Myristoylation Antagonizes Calcineurin Phosphatase Activation in Yeast

“…Calcineurin-NFAT signaling must be appropriately regulated in vivo. Mice expressing an NFAT allele with increased affinity for calcineurin exhibit developmental abnormalities (8). Alterations in calcineurin activity have been linked to multiple human diseases including cardiac hypertrophy (9,10), cancer (11,12), and neurodegeneration (13).…”

NFAT control of innate immunity

“…Interestingly, NFAT has now also been detected in stromal stem cells as well as in HSCs. 10,103 NFAT regulation of immune homeostasis is mediated by growth factors, such as GM-CSF and IL-2, which are transcribed after NFAT translocation. 25 Homeostasis of innate immune cells is a tightly regulated process that depends on growth factors, including GM-CSF 104 and Flt3-L. 105 Systemic administration of calcineurin/ NFAT inhibitors may therefore profoundly impact innate immune homeostasis in treated patients.…”