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The link between autoimmune diseases and primary immunodeficiency syndromes has been increasingly appreciated. Immunologic evaluation of a young man with autoimmune enterocolopathy and unexplained infections revealed evidence of immunodeficiency, including IgG subclass deficiency, impaired Ag-induced lymphocyte proliferation, reduced cytokine production by CD8(+) T lymphocytes, and decreased numbers of NK cells. Genetic evaluation identified haploinsufficiency of NFAT5, a transcription factor regulating immune cell function and cellular adaptation to hyperosmotic stress, as a possible cause of this syndrome. Inhibition or deletion of NFAT5 in normal human and murine cells recapitulated several of the immune deficits identified in the patient. These results provide evidence of a primary immunodeficiency disorder associated with organ-specific autoimmunity linked to NFAT5 deficiency.

Hypertensive cardiac hypertrophy (HCH) is a common cause of heart failure (HF), a major public health problem worldwide. However, the molecular bases of HCH have not been completely elucidated. Neuron-derived orphan receptor-1 (NOR-1) is a nuclear receptor whose role in cardiac remodelling is poorly understood. The aim of the present study was to generate a transgenic mouse over-expressing NOR-1 in the heart (TgNOR-1) and assess the impact of this gain-of-function on HCH. The CAG promoter-driven transgenesis led to viable animals that over-expressed NOR-1 in the heart, mainly in cardiomyocytes and also in cardiofibroblasts. Cardiomyocytes from TgNOR-1 exhibited an enhanced cell surface area and myosin heavy chain 7 (Myh7)/Myh6 expression ratio, and increased cell shortening elicited by electric field stimulation. TgNOR-1 cardiofibroblasts expressed higher levels of myofibroblast markers than wild-type (WT) cells (a 1 skeletal muscle actin (Acta1), transgelin (Sm22a)) and were more prone to synthesise collagen and migrate. TgNOR-1 mice experienced an age-associated remodelling of the left ventricle (LV). Angiotensin II (AngII) induced the cardiac expression of NOR-1, and NOR-1 transgenesis exacerbated AngII-induced cardiac hypertrophy and fibrosis. This effect was associated with the up-regulation of hypertrophic (brain natriuretic peptide (Bnp), Acta1 and Myh7) and fibrotic markers (collagen type I a 1 chain (Col1a1), Pai-1 and lysyl oxidase-like 2 (Loxl2)). NOR-1 transgenesis up-regulated two key genes involved in cardiac hypertrophy (Myh7, encoding for ß-myosin heavy chain (β-MHC)) and fibrosis (Loxl2, encoding for the extracellular matrix (ECM) modifying enzyme, Loxl2). Interestigly, in transient transfection assays, NOR-1 drove the transcription of Myh7 and Loxl2 promoters. Our findings suggest that NOR-1 is involved in the transcriptional programme leading to HCH. [ABSTRACT FROM AUTHOR]

Neuron-derived orphan receptor-1 (NOR-1) plays a major role in vascular biology by controlling fibroproliferative and inflammatory responses. Because microRNAs (miRNAs) have recently emerged as key players in the regulation of gene expression in the vasculature, here we have investigated the regulation of NOR-1 by miRNAs in endothelial cells. Computational algorithms suggest that NOR-1 could be targeted by members of the miR-17 family. Accordingly, ectopic over-expression of miR-17 or miR-20a in endothelial cells using synthetic premiRNAs attenuated the up-regulation of NOR-1 expression induced by VEGF (as evidenced by real time PCR, Western blot and immunocitochemistry). Conversely, the antagonism of these miRNAs by specific antagomirs prevented the down-regulation of NOR-1 promoted by miR-17 or miR-20a in VEGF-stimulated cells. Disruption of the miRNA-NOR-1 mRNA interaction using a custom designed target protector evidenced the selectivity of these responses. Further, luciferase reporter assays and seed-sequence mutagenesis confirmed that miR-17 and -20a bind to NOR-1 3’-UTR. Finally, miR-17 and -20a ameliorated the up-regulation of VCAM-1 mediated by NOR-1 in VEGF-stimulated cells. Therefore, miR-17 and -20a target NOR-1 thereby regulating NOR-1-dependent gene expression. [ABSTRACT FROM AUTHOR]

Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance. [ABSTRACT FROM AUTHOR]

PGE2 has been implicated in abdominal aortic aneurysm (AAA) associated hypervascularization. PGE2-metabolism involves 15-hydroxyprostaglandin-dehydrogenase (15-PGDH) the expression of which in AAA is unknown. The aim of this study was to examine the expression and cell distribution of 15-PGDH in AAA. Here, we show that 15-PGDH mRNA levels were significantly higher in aorta samples from patients undergoing AAA repair than in those from healthy multiorgan donors. Consequently, the ratio of metabolized PGE2 secreted by aortic samples was significantly higher in AAA. AAA production of total PGE2 and PGE2 metabolites correlated positively with PGI2 production, while the percentage of metabolized PGE2 correlated negatively with the total amount of PGE2 and with PGI2. Transcript levels of 15-PGDH were statistically associated with leukocyte markers but did not correlate with microvascular endothelial cell markers. Immunohistochemistry revealed 15-PGDH in the areas of leukocyte infiltration in AAA samples, mainly associated with CD45-positive cells, but not in normal aorta samples. We provide new data concerning 15-PGDH expression in human AAA, showing that 15-PGDH is upregulated in AAA and mainly expressed in infiltrating leukocytes. Our data suggest that microvasculature was not involved in PGE2 catabolism, reinforcing the potential role of microvasculature derived PGE2 in AAA-associated hypervascularization. [ABSTRACT FROM AUTHOR]

The Rel-like transcription factors nuclear factor kappa B (NF-κB) and the calcineurin-dependent nuclear factor of activated T cells (NFATc) control specific points of thymocyte maturation. Thymocytes also express a distinct member of the Rel family, the calcineurin-independent, osmostress response regulator NFAT5. Here we show that IKKβ regulates the expression of NFAT5 in thymocytes, which in turn contributes to the survival of T-cell receptor αβ thymocytes and the transition from the β-selection checkpoint to the double-positive stage in an osmostress-independent manner. NFAT5-deficient thymocytes had normal expression and proximal signaling of the pre–T-cell receptor but exhibited a partial defect in β-chain allelic exclusion and increased apoptosis. Further analysis showed that NFAT5 regulated the expression of the prosurvival factors A1 and Bcl2 and attenuated the proapoptotic p53/Noxa axis. These findings position NFAT5 as a target of the IKKβ/NF-κB pathway in thymocytes and as a downstream effector of the prosurvival role of the pre–T-cell receptor. [ABSTRACT FROM AUTHOR]

Background: The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5. Methodology/Principal Findings: Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin. Conclusions/Significance: Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress. [ABSTRACT FROM AUTHOR]

Abstract: The MAPKKK8 Cot/tpl-2, identified as an oncogene (Cot-T), participates in the intracellular signaling activated by members of the TLR and TNFα receptor superfamilies. Here we demonstrate that Cot promotes cell migration by regulating different steps involved in this process, such as cell adhesion and metalloproteinase activity. Indeed, Cot also regulates the cytoskeleton and Cot-T overexpression provokes the polarization of microtubules and the loss of stress fibers. Moreover, and in accordance with the increased Rac-GTP levels observed, Cot-T overexpressing cells develop more lamellipodia than control cells. Conversely, depletion of endogenous Cot increases the formation of stress fibers which is correlated with the high levels of Rho-GTP observed in these cells. In addition, the increase in COX2 expression and the activation of Erk1/2 regulated by Cot are essential for the induction of cell migration. Together, these data provide evidence of a new role for both proto-oncogenic and oncogenic Cot. [Copyright &y& Elsevier]

The transcription factor NFAT5/TonEBP, a member of the NFAT/Rel family of transcription factors, has been implicated in diverse cellular responses, including the response to osmotic stress, integrin-dependent cell migration, T cell activation, and the Ras pathway in Drosophila. To clarify the in vivo role of NFAT5, we generated NFAT5-null mice. Homozygous mutants were genetically underrepresented after embryonic day 14.5. Surviving mice manifested a progressive and profound atrophy of the kidney medulla with impaired activation of several osmoprotective genes, including those encoding aldose reductase, Na+/Cl--coupled betaine/γ-aminobutyric acid transporter, and the Na+/myo-inositol cotransporter. The aldose reductase gene is controlled by a tonicity-responsive enhancer, which was refractory to hypertonic stress in fibroblasts lacking NFAT5, establishing this enhancer as a direct transcriptional target of NFAT5. Our findings demonstrate a central role for NFAT5 as a tonicity-responsive transcription factor required for kidney homeostasis and function. [ABSTRACT FROM AUTHOR]

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-kB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-kB recruitment in response to high LPS doses. Using the transposaseaccessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections. [ABSTRACT FROM AUTHOR]

Recent work has highlighted the role of NR4A receptors in atherosclerosis and inflammation. In vascular smooth muscle cell (VSMC) proliferation, however, NOR-1 (neuron-derived orphan receptor-1) exerts antagonistic effects to Nur77 and Nurr1. The aim of this study was to analyse the effect of NOR-1 in VSMC inflammatory response. We assessed the consequence of a gain-of-function of this receptor on the response of VSMC to inflammatory stimuli. In human VSMC, lentiviral over-expression of NOR-1 reduced lipopolysaccharide (LPS)-induced up-regulation of cytokines (IL-1β, IL-6 and IL-8) and chemokines (MCP-1 and CCL20). Similar effects were obtained in cells stimulated with TNFα or oxLDL. Conversely, siRNA-mediated NOR-1 inhibition significantly increased the expression of pro-inflammatory mediators. Interestingly, in the aortas from transgenic mice that over-express human NOR-1 in VSMC (TgNOR-1), the up-regulation of cytokine/chemokine by LPS was lower compared to wild-type littermates. Similar results were obtained in VSMC from transgenic animals. NOR-1 reduced the transcriptional activity of NFκB sensitive promoters (in transient transfections), and the binding of NFκB to its responsive element (in electrophoretic mobility shift assays). Furthermore, NOR-1 prevented the activation of NFκB pathway by decreasing IκBα phosphorylation/degradation and inhibiting the phosphorylation and subsequent translocation of p65 to the nucleus (assessed by Western blot and immunocytochemistry). These effects were associated with an attenuated phosphorylation of ERK1/2, p38 MAPK and Jun N-terminal kinase, pathways involved in the activation of NFκB. In mouse challenged with LPS, the activation of the NFκB signalling was also attenuated in the aorta from TgNOR-1. Our data support a role for NOR-1 as a negative modulator of the acute response elicited by pro-inflammatory stimuli in the vasculature. [ABSTRACT FROM AUTHOR]

Abstract: Stress, be it from environmental factors or intrinsic to the cell as result of growth and metabolism can be harmful to cells. Mammalian cells have developed numerous mechanisms to respond to diverse forms of stress. These mechanisms combine signaling cascades and activation of gene expression programs to orchestrate an adaptive response that will allow the cell to survive and resume its normal functioning. In this review we will focus on the transcription factor NFAT5, a fundamental regulator of the response to osmotic stress in mammalian cells. Identified in 1999, NFAT5 is the latest addition to the Rel family, which comprises the NF-κB and NFATc proteins. Though in some of its structural and functional features NFAT5 is a hybrid between these two major groups of Rel proteins, it has unique characteristics that make it stand on its own as a third type of Rel transcription factor. Since its discovery, NFAT5 has been studied mostly in the context of the hypertonicity stress response. The advent of mouse models deficient in NFAT5 and other recent advances have confirmed a fundamental osmoprotective role for this factor in mammals, but also revealed features that suggest it may have a wider range of functions. [Copyright &y& Elsevier]