System-level investigation into the regulatory mechanism of the calcineurin/NFAT signaling pathway

Calcineurn/nuclear factor of the activated T cell (CaN/NFAT) signaling pathway plays crucial roles in the development of cardiac hypertrophy, Down's syndrome, and autoimmune diseases in response to pathological stimuli. The aim of the present study is to get a system-level understanding on the regulatory mechanism of CaN/NFAT signaling pathway in consideration of the controversial roles of myocyte-enriched calcineurin interacting protein1 (MCIP1) for varying stress stimuli. To this end, we have developed an experimentally validated mathematical model and carried out computer simulations as well as cell-based experiments. Quantitative overexpression and knock-down experiments in C2C12 myoblasts have revealed that MCIP1 functions only as a calcineurin inhibitor. We have also observed a biphasic response of the NFAT activity with increasing stimuli of isoproterenol. Through extensive in silico simulations, we have discovered that the NFAT activity is primarily modulated by ERK5 and MCIP1 under mild isoproterenol stimuli whereas it is mainly modulated by atrogin1 (muscle atrophy F-box protein) under strong isoproterenol stimuli. This study shows that a system-level analysis may help understanding CaN/NFAT signaling-associated disease.     

PICK1 binds to calcineurin B and modulates the NFAT activity in PC12 cells

In the central nervous system, calcineurin has been implicated in a number of Ca²⁺-sensitive pathways, including the regulation of neurotransmitter release and modulation of synaptic plasticity. PDZ domain-containing proteins also play an important role in the targeting and clustering of synaptic proteins. Using a yeast two-hybrid screen, we herein identified the PDZ domain-containing protein PICK1 as a specific interactor of calcineurin B. The interaction of calcineurin B and PICK1 was confirmed by GST pull-down assay in HEK293 cells and immunoprecipitation using rat brain lysate. Calcineurin B contains the consensus C-terminal peptide sequence required for interacting with the PDZ domain. The deletion of this sequence was sufficient to abolish the interaction between calcineurin B and PICK1. In addition, the knockdown of PICK1 by RNA interference inhibited the calcineurin-dependent activation of NFAT in PC12 cells. These results suggest that PICK1 may be a positive regulator of calcineurin in the central nervous system.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca²⁺]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-κB translocation in human hepatic HepG2 cells, ILY did not affect NF-κB localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca²⁺]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.     

Activation of the Ca(2+)/calcineurin/NFAT2 pathway controls smooth muscle cell differentiation

Cellular mechanisms controlling smooth muscle cells (SMCs) phenotypic modulation are largely unknown. Intracellular Ca2+ movements are essential to ensure SMC functions; one of the roles of Ca2+ is to regulate calcineurin, which in turn induces nuclear localization of the nuclear factor of activated T-cell (NFAT). In order to investigate, during phenotypic differentiation of SMCs, the effect of calcineurin inhibition on NFAT2 nuclear translocation, we used a culture model of SMC differentiation in serum-free conditions. We show that the treatment of cultured SMC with the calcineurin inhibitor cyclosporine A induced their dedifferentiation while preventing their differentiation. These findings suggest that nuclear translocation of NFAT2 is dependent of calcineurin activity during the in vitro SMC differentiation kinetic and that the nuclear presence of NFAT2 is critical in the acquisition and maintenance of SMC differentiation.     

Mitotic regulation of CDK4 by the serine/threonine phosphatase, calcineurin

Calcineurin was demonstrated to regulate the phosphorylation of threonine (T)-172 of CDK4. We further investigated how calcineurin can regulate this essential post-translational modification on CDK4. In this study, we demonstrate that calcineurin can associate predominantly with the cytoplasmic form of CDK4 in the absence of cyclin D. The inhibition of calcineurin phosphatase activity resulted in the specific increase of the phosphorylation and activity levels of CDK4 within the mitotic fraction. The association of calcineurin with CDK4 peaked during the mitotic phase of the cell cycle and coincided with reduction of CDK4 phosphorylation. Using structural mutants to CDK4, we localized the interaction site of calcineurin within the amino terminal residues of CDK4 that are important for both cyclin D and p16INK4a binding. Our data suggest that calcineurin may regulate the kinase activity of CDK4 in a cell cycle-dependent manner and may be an important component of the negative regulation of CDK4.     

Nuclear factor of activated T cells (NFAT) proteins repress canonical Wnt signaling via its interaction with Dishevelled (Dvl) protein and participate in regulating neural progenitor cell proliferation and differentiation

The Ca(2+) signaling pathway appears to regulate the processes of the early development through its antagonism of canonical Wnt/β-catenin signaling pathway. However, the underlying mechanism is still poorly understood. Here, we show that nuclear factor of activated T cells (NFAT), a component of Ca(2+) signaling, interacts directly with Dishevelled (Dvl) in a Ca(2+)-dependent manner. A dominant negative form of NFAT rescued the inhibition of the Wnt/β-catenin pathway triggered by the Ca(2+) signal. NFAT functioned downstream of β-catenin without interfering with its stability, but influencing the interaction of β-catenin with Dvl by its competitively binding to Dvl. Furthermore, we demonstrate that NFAT is a regulator in the proliferation and differentiation of neural progenitor cells by modulating canonical Wnt/β-catenin signaling pathway in the neural tube of chick embryo. Our findings suggest that NFAT negatively regulates canonical Wnt/β-catenin signaling by binding to Dvl, thereby participating in vertebrate neurogenesis.     

NFAT and NF-κB dynamically co-regulate TCR and CAR signaling responses in human T cells

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Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart

The arterial pole of the heart is the region where the ventricular myocardium continues as the vascular smooth muscle tunics of the aorta and pulmonary trunk. It has been shown that the arterial pole myocardium derives from the secondary heart field and the smooth muscle tunic of the aorta and pulmonary trunk derives from neural crest. However, this neural crest-derived smooth muscle does not extend to the arterial pole myocardium leaving a region at the base of the aorta and pulmonary trunk that is invested by vascular smooth muscle of unknown origin. Using tissue marking and vascular smooth muscle markers, we show that the secondary heart field, in addition to providing myocardium to the cardiac outflow tract, also generates prospective smooth muscle that forms the proximal walls of the aorta and pulmonary trunk. As a result, there are two seams in the arterial pole: first, the myocardial junction with secondary heart field-derived smooth muscle; second, the secondary heart field-derived smooth muscle with the neural crest-derived smooth muscle. Both of these seams are points where aortic dissection frequently occurs in Marfan's and other syndromes.     

Normal patterning of the coronary capillary plexus is dependent on the correct transmural gradient of FGF expression in the myocardium

The formation of the coronary vessel system is vital for heart development, an essential step of which is the establishment of a capillary plexus that displays a density gradient across the myocardial wall, being higher on the epicardial than the endocardial side. This gradient in capillary plexus formation develops concurrently with transmural gradients of myocardium-derived growth factors, including FGFs. To test the role of the FGF expression gradient in patterning the nascent capillary plexus, an ectopic FGF-over-expressing site was created in the ventricular myocardial wall in the quail embryo via retroviral infection from E2-2.5, thus abolishing the transmural gradient of FGFs. In FGF virus-infected regions of the ventricular myocardium, the capillary density across the transmural axis shifted away from that in control hearts at E7. This FGF-induced change in vessel patterning was more profound at E12, with the middle zone becoming the most vascularized. An up-regulation of FGFR-1 and VEGFR-2 in epicardial and subepicardial cells adjacent to FGF virus-infected myocardium was also detected, indicating a paracrine effect on induction of vascular signaling components in coronary precursors. These results suggest that correct transmural patterning of coronary vessels requires the correct transmural expression of FGF and, therefore, FGF may act as a template for coronary vessel patterning.