Interleukin-13-induced MUC5AC expression is regulated by a PI3K–NFAT3 pathway in mouse tracheal epithelial cells

Interleukin-13 (IL-13) plays a critical role in asthma mucus overproduction, while the mechanisms underlying this process are not fully elucidated. Previous studies showed that nuclear factor of activated T cells (NFAT) is involved in the pathogenesis of asthma, but whether it can directly regulate IL-13-induced mucus (particularly MUC5AC) production is still not clear. Here we showed that IL-13 specifically induced NFAT3 activation through promoting its dephosphorylation in air–liquid interface (ALI) cultures of mouse tracheal epithelial cells (mTECs). Furthermore, both Cyclosporin A (CsA, a specific NFAT inhibitor) and LY294002 (a Phosphoinositide 3-kinase (PI3K) inhibitor) significantly blocked IL-13-induced MUC5AC mRNA and protein production through the inhibition of NFAT3 activity. We also confirmed that CsA could not influence the forkhead Box A2 (Foxa2) and mouse calcium dependent chloride channel 3 (mClca3) expression in IL-13-induced MUC5AC production, which both are known to be important in IL-13-stimulated mucus expression. Our study is the first to demonstrate that the PI3K–NFAT3 pathway is positively involved in IL-13-induced mucus production, and provided novel insights into the molecular mechanism of asthma mucus hypersecretion.     

Urocortin 2 induces tyrosine hydroxylase phosphorylation in PC12 cells

Urocotins (Ucns) are newly discovered members of the corticotropin-releasing factor (CRF) neuropeptide family. Ucn 2 is expressed in the adrenal medulla, and its receptor, CRF2 receptor, is also expressed in the adrenal gland. To predict the physiological significance of Ucn 2 expression in the adrenal medulla, we examined the effects of Ucn 2 on catecholamine secretion and intracellular signaling using PC12 cells, a rat pheochromocytoma cell line. PC12 cells were found to express CRF2 receptor, but not CRF1 receptor. Treatment with Ucn 2 increased noradrenaline secretion and induced phosphorylation of PKA and Erk1/2. Tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, was also phosphorylated by Ucn 2. Pretreatment with a PKA inhibitor blocked Ucn 2-induced NA secretion, and Erk1/2 and TH phosphorylation. Pretreatment with a MEK inhibitor did not block Ucn 2-induced noradrenaline secretion or PKA phosphorylation, although TH phosphorylation was blocked. Thus, Ucn 2 induces noradrenaline secretion and TH phosphorylation through the PKA pathway and the PKA-Erk1/2 pathway, respectively. These results suggest Ucn 2 in the adrenal gland may be involved in the regulation of catecholamine release and synthesis.     

Cyclosporin A promotes growth and invasiveness in vitro of human first-trimester trophoblast cells via MAPK3/MAPK1-mediated AP1 and Ca2+/calcineurin/NFAT signaling pathways

Cyclosporin A (CsA) has provided the pharmacologic foundation for organ transplantation as a calcineurin inhibitor blocking T-cell activation. We have demonstrated that CsA promoted trophoblast viability/proliferation and invasion in vitro. In the present study, we further investigated the intracellular signalling pathways involved in enhancing cell viability/proliferation and invasiveness of the human trophoblast induced by CsA. We showed that blocking mitogen-activated protein kinase 3 (MAPK3)/MAPK1 signaling by U0126 attenuated CsA-increased cell viability and invasiveness of trophoblasts. Cyclosprin A inhibited ionomycin-stimulated nuclear factor of activated T-cells (NFAT) transactivation in JAR cells and reversed the ionomycin-inhibited trophoblast invasiveness. However, either activating calcineurin by ionomycin, resulting in NFAT transactivation, or inhibiting NFAT using an NFAT inhibitor had no effect on trophoblast cell viability/proliferation and apoptosis in vitro. Hence, the CsA-induced promotion of trophoblast growth and invasion occurred by overlapping but independent pathways. The MAPK3/MAPK1 pathway was essential for both trophoblast growth and invasion, whereas the Ca(2+)/calcineurin/NFAT pathway was only involved in the CsA-promoted trophoblast invasiveness. Finally, potential cross-talk between MAPK3/MAPK1 and Ca(2+)/calcineurin/NFAT and its relationship to activator protein 1 activation was investigated. Our findings explored possible signal transduction pathways modulated by CsA, which may lead to the expansion of the clinical applications of this drug.     

Two distinct action mechanisms of immunophilin-ligand complexes for the blockade of T-cell activation

The immunosuppressive effects of cyclosporin A (CsA) and FK506 are mediated through binding to immunophilins. Here we show that FK506–FKBP complex suppresses the activation of JNK and p38 pathways at a level upstream of mitogen-activated protein kinase (MAPK) kinase kinase (MAPKK-K) besides the calcineurin–NFAT pathway. A238L, a viral gene product that binds to immunophilin, also blocks activation of both pathways. In contrast, direct inhibitors of calcineurin, Cabin 1 and FR901725, suppress the activation of NFAT but not the JNK or p38 pathway. We further demonstrate that co-expression of a constitutively active NFAT and a constitutively active MEKK1 renders the interleukin-2 promoter in Jurkat T lymphocytes resistant to CsA and FK506, whereas Jurkat cells expressing a constitutively active NFAT alone are still sensitive to CsA or FK506. Therefore, CsA and FK506 exert their immunosuppressive effects through targeting both the calcineurin-dependent NFAT pathway and calcineurin-independent activation pathway for JNK and p38.     

Regulation of tumor necrosis factor cytotoxicity by calcineurin

Cyclosporin (CsA) inhibits mitochondrial death signaling and opposes tumor necrosis factor (TNF)-induced apoptosis in vitro. However, CsA is also a potent inhibitor of calcineurin, a phosphatase that may participate in cell death. Therefore, we tested the hypothesis that calcineurin regulates TNF cytotoxicity in rat hepatoma cells (FTO2B). TNF-treated FTO2B cells appeared apoptotic by DNA fragmentation, nuclear condensation, annexin V binding, and caspase activation. We studied two calcineurin inhibitors, CsA and FK506, and found that each potently inhibited TNF cytotoxicity. Western blot demonstrated calcineurin in FTO2B homogenates. In a model of mitochondrial permeability transition (MPT), we found that CsA prevented MPT and cytochrome c release, while FK506 inhibited neither. In summary, we present evidence that calcineurin participates in an apoptotic death pathway activated by TNF. CsA may oppose programmed cell death by inhibiting calcineurin activity and/or inhibiting mitochondrial signaling.     

Cyclosporin A-induced hair growth in mice is associated with inhibition of calcineurin-dependent activation of NFAT in follicular keratinocytes

One of the most common side effects of treatment with cyclosporin A (CsA) is hypertrichosis. This study shows that calcineurin activity is associated with hair keratinocyte differentiation in vivo, affecting nuclear factor of activated T cells (NFAT1) activity in these cells. Treatment of nude or C57BL/6 depilated normal mice with CsA inhibited the expression of keratinocyte terminal differentiation markers associated with catagen, along with the inhibition of calcineurin and NFAT1 nuclear translocation. This was associated with induction of hair growth in nude mice and retardation of spontaneous catagen induction in depilated normal mice. Furthermore, calcineurin inhibition blocked the expression of p21(waf/cip1) and p27(kip1), which are usually induced with differentiation. This was also associated with an increase in interleukin-1alpha expression (nude mice), a decrease in transforming growth factor-beta (nude and normal mice), and no change in keratinocyte growth factor expression in the skin. Retardation of catagen in CsA-treated mice was accompanied by significant alterations in apoptosis-related gene product expression in hair follicle keratinocytes. The ratio of the anti-apoptotic Bcl-2 to proapoptotic Bax expression increased, and expression of p53 and interleukin-1beta converting enzyme activity decreased. These data provide the first evidence that calcineurin is functionally active in follicular keratinocytes and that inhibition of the calcineurin-NFAT1 pathway in these cells in vivo by CsA enhances hair growth.     

Cyclosporin A inhibits colon cancer cell growth independently of the calcineurin pathway

Chronic inflammation is a risk factor for the development of colon cancer, providing genotoxic insults, growth and pro-angiogenic factors that can promote tumorigenesis and tumor growth. Immunomodulatory agents can interfere with the inflammation that feeds cancer, but their impact on the transformed cell is poorly understood. The calcium/calcineurin signaling pathway, through activation of NFAT, is essential for effective immune responses, and its inhibitors cyclosporin A (CsA) and FK506 are used in the clinics to suppress immunity. Moreover, the kinases GSK3β and mTOR, modulated by PI-3K/Akt, can inhibit NFAT activity, suggesting a cross-talk between the calcium and growth factor signaling pathways. Both NFAT and mTOR activity have been associated with tumorigenesis. We therefore investigated the impact of calcineurin and PI-3K/mTOR inhibition in growth of human colon carcinoma cells. We show that despite the efficient inhibition of NFAT1 activity, FK506 promotes tumor growth, whereas CsA inhibits it due to a delay in cell cycle progression and induction of necroptosis. We found NFκB activation and mTORC1 activity not to be altered by CsA or FK506. Similarly, changes to mitochondrial homeostasis were equivalent upon treatment with these drugs. We further show that, in our model, NFAT1 activation is not modulated by PI3K/mTOR. We conclude that CsA slows cell cycle progression and induces necroptosis of human carcinoma cell lines in a TGFβ-, NFAT-, NFκB- and PI3K/mTOR-independent fashion. Nevertheless, our data suggest that CsA, in addition to its anti-inflammatory capacity, may target transformed colon and esophagus carcinoma cells without affecting non-transformed cells, promoting beneficial tumoristatic effects.     

Cyclosporin A inhibits colon cancer cell growth independently of the calcineurin pathway

Chronic inflammation is a risk factor for the development of colon cancer, providing genotoxic insults, growth and pro-angiogenic factors that can promote tumorigenesis and tumor growth. Immunomodulatory agents can interfere with the inflammation that feeds cancer, but their impact on the transformed cell is poorly understood. The calcium/calcineurin signaling pathway, through activation of NFAT, is essential for effective immune responses, and its inhibitors cyclosporin A (CsA) and FK506 are used in the clinics to suppress immunity. Moreover, the kinases GSK3β and mTOR, modulated by PI-3K/Akt, can inhibit NFAT activity, suggesting a cross-talk between the calcium and growth factor signaling pathways. Both NFAT and mTOR activity have been associated with tumorigenesis. We therefore investigated the impact of calcineurin and PI-3K/mTOR inhibition in growth of human colon carcinoma cells. We show that despite the efficient inhibition of NFAT1 activity, FK506 promotes tumor growth, whereas CsA inhibits it due to a delay in cell cycle progression and induction of necroptosis. We found NFκB activation and mTORC1 activity not to be altered by CsA or FK506. Similarly, changes to mitochondrial homeostasis were equivalent upon treatment with these drugs. We further show that, in our model, NFAT1 activation is not modulated by PI3K/mTOR. We conclude that CsA slows cell cycle progression and induces necroptosis of human carcinoma cell lines in a TGFβ-, NFAT-, NFκB- and PI3K/mTOR-independent fashion. Nevertheless, our data suggest that CsA, in addition to its anti-inflammatory capacity, may target transformed colon and esophagus carcinoma cells without affecting non-transformed cells, promoting beneficial tumoristatic effects.     

Delphinidin Activates NFAT and Induces IL-2 Production Through SOCE in T Cells

Delphinidin is an anthocyanidin that possesses antioxidant and anti-inflammatory effects; however, some reports suggest that delphinidin has pro-inflammatory properties. For this reason, we assessed the effect of delphinidin on cytokine production in T cells. We demonstrated that delphinidin increased the cytosolic-free Ca²⁺ concentration by releasing Ca²⁺ from intracellular stores and increasing Ca²⁺ entry. The putative Ca²⁺ release activated Ca²⁺ (CRAC) channel inhibitors BTP2 and gadolinium reduced the calcium entry stimulated by the anthocyanidin. Delphinidin induced nuclear factor of activated T cells (NFAT) translocation and NFAT-Luc activity in Jurkat cells and was dependent on the CRAC channel and calcineurin pathway. Delphinidin increased the mRNA expression and production of IL-2 in Jurkat cells and was inhibited by BTP2 and cyclosporine A. Using peripheral blood lymphocytes, we demonstrated that delphinidin increased the production of IL-2 and IFN-γ and was inhibited by BTP2. Taken together, our results suggest that delphinidin exerts immunostimulatory effects on T cells by increasing cytokine production through CRAC channel and NFAT activation.