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...

The zebrafish is a powerful experimental model to investigate the genetic and morphologic basis of vertebrate development. Analysis of skeletogenesis in this fish is challenging as a result of the small size of the developing and adult zebrafish. Many of the bones of small fishes such as the zerbafish and medaka are quite thin, precluding many standard assays of bone quality and morphometrics commonly used on bones of larger animals. Microcomputed tomography (microCT) is a common imaging technique used for detailed analysis of the skeleton of the zebrafish and determination of mutant phenotypes. However, the utility of this modality for analysis of the zebrafish skeleton, and the effect of inherent variation among individual zebrafish, including variables such as sex, age and strain, is not well understood. Given the increased use and accessibility of microCT, we set out to define the sensitivity of microCT methods in developing and adult zebrafish. We assessed skeletal shape and density measures in the developing vertebrae and parasphenoid of the skull base. We found most skeletal variables are tightly correlated to standard length, but that at later growth stages (>3 months) there are age dependent effects on some skeletal measures. Further we find modest strain but not sex differences in skeletal measures. These data suggest that the appropriate control for assessing mutant phenotypes should be age and strain matched, ideally a wild-type sibling. By analyzing two mutants exhibiting skeletal dysplasia, we show that microCT imaging can be a sensitive method to quantify distinct skeletal parameters of adults. Finally, as developing zebrafish skeletons remain difficult to resolve by radiographic means, we define a contrast agent specific for bone that enhances resolution at early stages, permitting detailed morphometric analysis of the forming skeleton. This increased capability for detection extends the use of this imaging modality to leverage the zebrafish model to understand the development causes of skeletal dysplasias.

Atherosclerosis is a complex inflammatory disease involving extensive vascular vessel remodelling and migration of vascular cells. As RCAN1 is implicated in cell migration, we investigated its contribution to atherosclerosis. We show RCAN1 induction in atherosclerotic human and mouse tissues. Rcan1 was expressed in lesional macrophages, endothelial cells and vascular smooth muscle cells and was induced by treatment of these cells with oxidized LDLs (oxLDLs). Rcan1 regulates CD36 expression and its genetic inactivation reduced atherosclerosis extension and severity in Apoe−/− mice. This effect was mechanistically linked to diminished oxLDL uptake, resistance to oxLDL-mediated inhibition of macrophage migration and increased lesional IL-10 and mannose receptor expression. Moreover, Apoe−/−Rcan1−/− macrophages expressed higher-than-Apoe−/− levels of anti-inflammatory markers. We previously showed that Rcan1 mediates aneurysm development and that its expression is not required in haematopoietic cells for this process. However, transplantation of Apoe−/−Rcan1−/− bone-marrow (BM) cells into Apoe−/− recipients confers atherosclerosis resistance. Our data define a major role for haematopoietic Rcan1 in atherosclerosis and suggest that therapies aimed at inhibiting RCAN1 expression or function might significantly reduce atherosclerosis burden.

ObjectiveAlthough physical therapy can help preserve mobility in patients with systemic sclerosis (SSc), stretching has not been used systematically as a treatment to prevent or reverse the disease process. We previously showed in rodent models that stretching promotes the resolution of connective tissue inflammation and reduces new collagen formation after injury. Here, we tested the hypothesis that stretching would impact scleroderma development using a mouse sclerodermatous graft-versus-host disease (sclGvHD) model.MethodsThe model consists in the adoptive transfer (allogeneic) of splenocytes from B10.D2 mice (graft) into Rag2−/− BALB/c hosts (sclGvHD), resulting in skin inflammation followed by fibrosis over 4 weeks. SclGvHD mice and controls were randomized to stretching in vivo for 10 min daily versus no stretching.ResultsWeekly ultrasound measurements of skin thickness and subcutaneous tissue mobility in the back (relative tissue displacement during passive trunk motion) successfully captured the different phases of the sclGvHD model. Stretching reduced skin thickness and increased subcutaneous tissue mobility compared to no stretching at week 3. Stretching also reduced the expression of CCL2 and ADAM8 in the skin at week 4, which are two genes known to be upregulated in both murine sclGvHD and the inflammatory subset of human SSc. However, there was no evidence that stretching attenuated inflammation at week 2.ConclusionDaily stretching for 10 min can improve skin thickness and mobility in the absence of any other treatment in the sclGvHD murine model. These pre-clinical results suggest that a systematic investigation of stretching as a therapeutic modality is warranted in patients with SSc.

In the supplementary information, some of the figures and legends were incorrectly matched.

Increase in intracellular calcium ([Ca(2+) ]i ) is a key mediator of astrocyte signaling, important for activation of the calcineurin (CN)/nuclear factor of activated T cells (NFAT) pathway, a central mediator of inflammatory events. We analyzed the expression of matrix metalloproteinase 3 (Mmp3) in response to increases in [Ca(2+) ]i and the role of the CN/NFAT pathway in this regulation. Astrocyte Mmp3 expression was induced by overexpression of a constitutively active form of NFATc3, whereas other MMPs and tissue inhibitor of metalloproteinases (TIMP) were unaffected. Mmp3 mRNA and protein expression was also induced by calcium ionophore (Io) and 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (Bz-ATP) and Mmp3 upregulation was prevented by the CN inhibitor cyclosporin A (CsA). Ca(2+) -dependent astrocyte Mmp3 expression was also inhibited by actinomycin D, and a Mmp3 promoter luciferase reporter was efficiently activated by increased [Ca(2+) ]i , indicating regulation at the transcriptional level. Furthermore, Ca(2+) /CN/NFAT dependent Mmp3 expression was confirmed in pure astrocyte cultures derived from neural stem cells (Ast-NSC), demonstrating that the induced Mmp3 expression occurs in astrocytes, and not microglial cells. In an in vivo stab-wound model of brain injury, MMP3 expression was detected in NFATc3-positive scar-forming astrocytes. Because [Ca(2+) ]i increase is an early event in most brain injuries, these data support an important role for Ca(2+) /CN/NFAT-induced astrocyte MMP3 expression in the early neuroinflammatory response. Understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain.

Calcium/Calcineurin/Nuclear Factor of Activated T cells (Ca/CN/NFAT) signalling pathway is the main calcium (Ca 2+ ) dependent signalling pathway involved in the homeostasis of brain tissue. Here, we study the presence of NFATc members in human glioma by using U251 cells and a collection of primary human glioblastoma (hGB) cell lines. We show that NFATc3 member is the predominant member. Furthermore, by using constitutive active NFATc3 mutant and shRNA lentiviral vectors to achieve specific silencing of this NFATc member, we describe cytokines and molecules regulated by this pathway which are required for the normal biology of cancer cells. Implanting U251 in an orthotopic intracranial assay, we show that specific NFATc3 silencing has a role in tumour growth. In addition NFATc3 knock-down affects both the proliferation and migration capacities of glioma cells in vitro . Our data open the possibility of NFATc3 as a target for the treatment of glioma.

Macrophages contribute to tissue homeostasis and influence inflammatory responses by modulating their phenotype in response to the local environment. Understanding the molecular mechanisms governing this plasticity would open new avenues for the treatment for inflammatory disorders. We show that deletion of calcineurin (CN) or its inhibition with LxVP peptide in macrophages induces an anti-inflammatory population that confers resistance to arthritis and contact hypersensitivity. Transfer of CN-targeted macrophages or direct injection of LxVP-encoding lentivirus has anti-inflammatory effects in these models. Specific CN targeting in macrophages induces p38 MAPK activity by downregulating MKP-1 expression. However, pharmacological CN inhibition with cyclosporin A (CsA) or FK506 did not reproduce these effects and failed to induce p38 activity. The CN-inhibitory peptide VIVIT also failed to reproduce the effects of LxVP. p38 inhibition prevented the anti-inflammatory phenotype of CN-targeted macrophages, and mice with defective p38-activation were resistant to the anti-inflammatory effect of LxVP. Our results identify a key role for CN and p38 in the modulation of macrophage phenotype and suggest an alternative treatment for inflammation based on redirecting macrophages toward an anti-inflammatory status.