Calcineurin mediates pancreatic growth in protease inhibitor-treated mice

CCK acts on pancreatic acinar cells to increase intracellular Ca(2+) leading to secretion of digestive enzymes and, in the long term, pancreatic growth. Calcineurin (CN) is a serine/threonine-specific protein phosphatase activated by Ca(2+) and calmodulin that recently has been shown to participate in the growth regulation of cardiac and skeletal myocytes. We therefore tested the effect of two different CN inhibitors, cyclosporine A (CsA) and FK506, on mouse pancreatic growth induced by oral administration of the synthetic protease inhibitor camostat, a known stimulator of endogenous CCK release. Mice were fed a powdered diet with or without 0.1% camostat. Pancreatic wet weight, protein, and DNA were increased in response to camostat in a time-dependent manner over 10 days in ICR mice but not in CCK-deficient mice. Both CsA (15 mg/kg) and FK506 (3 mg/kg) given twice daily blocked the increase in pancreatic wet weight and protein and DNA content induced by camostat. The increase in plasma CCK induced by camostat was not blocked by CsA or FK506. Camostat feeding also increased the relative amount of CN protein, whereas levels of MAPKs, ERKs, and p38 were not altered. In summary, 1) CCK released by chronic camostat feeding induces pancreatic growth in mice; 2) this growth is blocked by treatment with both CsA and FK506, indicating a role for CN; 3) CCK stimulation also increases CN protein. In conclusion, activation and possibly upregulation of CN may participate in regulation of pancreatic growth by CCK in mice.     

Calcineurin is required for translational control of protein synthesis in rat pancreatic acini

CCK increases the rate of net protein synthesis in rat pancreatic acini by activating initiation and elongation factors required for translation. The immunosuppressant FK506 inhibits the Ca²⁺-calmodulin-dependent phosphatase calcineurin in pancreatic acinar cells and blocks pancreatic growth induced by chronic CCK treatment. To test a requirement for calcineurin in the activation of the translational machinery stimulated by CCK, we evaluated the effects of FK506 on protein synthesis and on regulatory initiation and elongation factors in rat pancreatic acini in vitro. CCK acutely increased protein synthesis in acini from normal rats with a maximum increase at 100 pM CCK to 170 ± 11% of control. The immunosuppressant FK506 dose-dependently inhibited CCK-stimulated protein synthesis over the same concentration range that blocked calcineurin activity, as assessed by dephosphorylation of the calcineurin substrate calcium-regulated heat-stable protein of 24 kDa. Another immunosuppressant, cyclosporin A, inhibited protein synthesis, but its effects appeared more complex. FK506 also inhibited protein synthesis stimulated by bombesin and carbachol. FK506 did not significantly affect the activity of the initiation factor-2B, or the phosphorylation of the initiation factor-2, ribosomal protein protein S6, or the mRNA cap binding protein eukaryotic initiation factor (eIF) 4E. Instead, blockade of calcineurin with FK506 reduced the phosphorylation of the eIF4E binding protein, reduced the formation of the eIF4F complex, and increased the phosphorylation of eukaryotic elongation factor 2. From these results, we conclude that calcineurin activity is required for protein synthesis, and this action may be related to an effect on the formation of the mRNA cap binding complex and the elongation processes. exocrine pancreas; cholecystokinin; translation initiation factors; protein phosphatase 2B; immunosuppressants     

T-Plastin Expression Downstream to the Calcineurin/NFAT Pathway Is Involved in Keratinocyte Migration

Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT) signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and β6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte migration. These results indicate that T-plastin might be considered as a major actor in the mechanisms underlying calcineurin/NFAT-dependent keratinocyte migration and may explain wound-healing defects observed in patients under calcineurin inhibitor long-term treatment.     

Expression of a constitutively active calcineurin encoded by an intron-retaining mRNA in follicular keratinocytes

Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn). The calcineurin (Cn)/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin Aß CnAß-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnAß-FK was weakly sensitive to Ca²⁺ and dephosphorylated NFATc2 under low Ca²⁺ levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle development.     

A reassessment of the inhibitory capacity of human FKBP38 on calcineurin

The microbial peptidomacrolide FK506 affects many eukaryotic developmental and cell signaling programs via calcineurin inhibition. Prior formation of a complex between FK506 and intracellular FK506-binding proteins (FKBPs) is the precondition for the interaction with calcineurin. A puzzling difference has emerged between the mammalian multidomain protein hFKBP38 and other FKBPs. It was shown that hFKBP38 not only binds to calcineurin but also inhibits the protein phosphatase activity of calcineurin on its own [Shirane, M. and Nakayama, K.I. (2003) Nature Cell Biol. 5, 28-37]. Inherent calcineurin inhibition by hFKBP38 would completely eliminate the need for FK506 in controlling many signal transduction pathways. To address this issue, we have characterized the functional and physical interactions between calcineurin and hFKBP38. A recombinant hFKBP38 variant and endogenous hFKBP38 were tested both in vitro and in vivo. The proteins neither directly inhibited calcineurin activity nor affected NFAT reporter gene activity in SH-SY5Y and Jurkat cells. In addition, a direct physical interaction between calcineurin and hFKBP38 was not detected in co-immunoprecipitation experiments. However, hFKBP38 indirectly affected the subcellular distribution of calcineurin by interaction with typical calcineurin ligands, as exemplified by the anti-apoptotic protein Bcl-2. Our data suggest that hFKBP38 cannot substitute for the FKBP/FK506 complex in signaling pathways controlled by the protein phosphatase activity of calcineurin.     

Calcineurin and NFAT4 induce chondrogenesis

Nuclear factor of activated T-cells (NFAT) and calcineurin are essential regulators of immune cell and mesenchymal cell differentiation. Here we show that elevated intracellular calcium induces chondrogenesis through a calcineurin/NFAT signaling axis that activates bone morphogenetic protein (BMP) expression. The calcium ionophore, ionomycin, induced chondrogenesis through activation of calcineurin. The calcineurin substrate, NFAT4, also induced chondrogenesis and chondrocyte gene expression. Significantly, the BMP antagonist, noggin, or dominant negative BMP receptors blocked the effects of elevated intracellular calcium on chondrogenesis. This suggested that calcineurin/NFAT4 activates BMP expression. Consistent with this, BMP2 gene expression was increased by ionomycin and suppressed by the calcineurin inhibitor, cyclosporine A. Furthermore, activated NFAT4 induced BMP2 gene expression. These results have important implications for the effects of NFATs during development and adaptive responses.     

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.     

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.     

Cyclosporine A- and FK506-induced apoptosis in PC12 cells

The purpose of this study was to examine, using glycogen synthase kinase (GSK) inhibitors, whether GSK-3 is involved in cyclosporine A (CsA)- and FK506-induced apoptosis in PC12 cells. CsA and FK506 increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation or fragmentation. Nerve growth factor (NGF) completely blocked cell death. Caspase-3 activation was accompanied by CsA- and FK506-induced cell death and inhibited by NGF. GSK-3 inhibitors such as alsterpaullone and SB216763 prevented CsA- and FK506-induced apoptosis. These results suggest that CsA and FK506 induce caspase-dependent apoptosis and that GSK-3 activation is involved in CsA- and FK506-induced apoptosis in PC12 cells.     

Bile Acids Induce Pancreatic Acinar Cell Injury and Pancreatitis by Activating Calcineurin

Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca²⁺. Thus, it would be clinically relevant to know the targets of this aberrant Ca²⁺ signal. We hypothesized that the Ca²⁺-activated phosphatase calcineurin is such a Ca²⁺ target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca²⁺. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aβ (CnAβ) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca²⁺ generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.