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Skeletal muscle fiber type specification is changeable during muscle regeneration following cardiotoxin (CTX) injection; however, the mechanism of muscle fiber shift in regenerating muscle fibers remains unclear. Furthermore, it is unclear as to which factors determine skeletal muscle fiber types in regenerating muscle fibers. Previous studies showed that CTX-induced muscle damage resulted in a temporary hypoxic condition, indicating that hypoxia-inducible factor (HIF)-1α may be involved in muscle fiber type transition. Stabilization of HIF-1α has been shown to result in muscle fiber type transition toward slow-twitch phenotype through the calcineurin/nuclear factor activated T cell 1 (NFATc1) signaling pathway. Therefore, the aim of the present study was to determine whether the calcineurin/NFATc1 pathway is a key mediator of skeletal muscle fiber type transition during muscle regeneration. We found that CTX-induced muscle damage resulted in transient ischemia and HIF-1α expression in skeletal muscle. Additionally, it shifted the muscle fiber type proportion toward a slow-twitch phenotype in the soleus muscle (37.5% in the control muscle vs. 61.3% in the damaged muscle; p < 0.01) three weeks after muscle damage. Moreover, the NFATc1 protein levels increased in damaged muscle, and blockage of the calcineurin/NFATc1 signaling pathway by tacrolimus (FK-506) treatment substantially decreased the number of slow-twitch muscle fibers in the soleus muscle. This study demonstrated that CTX-induced muscle injury results in transient ischemia in hind limb muscle and stabilizes HIF-1α. Moreover, muscle damage increased oxidative phenotype muscle fibers through the calcineurin/NFATc1 signaling pathway during muscle regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The epidermal growth factor receptor (EGFR) is highly expressed or abnormally activated in several types of cancers, such as lung and colorectal cancers. Inhibitors that suppress the tyrosine kinase activity of EGFR have been used in the treatment of lung cancer. However, resistance to these inhibitors has become an issue in cancer treatment, and the development of new therapies that inhibit EGFR is desired. We found that calcineurin, a Ca 2+ /calmodulin-activated serine/threonine phosphatase, is a novel regulator of EGFR. Inhibition of calcineurin by FK506 treatment or calcineurin depletion promoted EGFR degradation in cancer cells. In addition, we found that calcineurin dephosphorylates EGFR at serine (S)1046/1047, which in turn stabilizes EGFR. Furthermore, in human colon cancer cells transplanted into mice, the inhibition of calcineurin by FK506 decreased EGFR expression. These results indicate that calcineurin stabilizes EGFR by dephosphorylating S1046/1047 and promotes tumor growth. These findings suggest that calcineurin may be a new therapeutic target for cancers with high EGFR expression or activation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Delivery of cerebroprotective agents using liposomes has been demonstrated to be useful for treating cerebral ischemia/reperfusion (I/R) injury. We previously reported that intravenous administration of liposomes with diameters of 100 nm showed higher accumulation in the I/R region compared with larger liposomes (>200 nm) by passage through the disintegrated blood-brain barrier, suggesting a size-dependence for liposome-mediated drug delivery. Based on these findings, we hypothesized that regulation of liposomal particle size (<100 nm) may enhance the therapeutic efficacy of encapsulated drugs on cerebral I/R injury. Herein, we prepared lipid nanoparticles (LNP) with particle sizes <100 nm by the microfluidics method and compared their therapeutic potential with LNP exhibiting sizes >100 nm in cerebral I/R model rats. Intravenously administered smaller LNP (ca. 60 nm) exhibited wider accumulation and diffusivity in the brain parenchyma of the I/R region compared with larger LNP (>100 nm). Importantly, treatment with LNP encapsulating the cerebroprotective agent FK506 (FK-LNP) with particle sizes <100 nm showed greater cerebroprotective effects than FK-LNP with sizes >100 nm, and also significantly ameliorated brain injury. These results suggest that particle size regulation of LNP to sizes <100 nm can enhance the therapeutic effect of encapsulated drugs for treatment of cerebral I/R injury, and that FK-LNP could be a promising cerebroprotective agent., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tatsuya Fukuta reports financial support was provided by Takahashi Industrial and Economic Research Foundation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca 2+ ) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca 2+ signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter into CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hyperglycemia promotes podocyte apoptosis and plays an important role in the pathogenesis of diabetic nephropathy (DN). Calcium/calcineurin (CaN) signaling is critical for podocyte apoptosis. Therefore, it is essential to elucidate the mechanisms underlying the regulation of CaN signaling. Recent studies reported that histone deacetylase 4 (HDAC4) is involved in podocyte apoptosis in DN. The aim of this study was to determine whether HDAC4 mediates the regulation of CaN and to elucidate the function of HDAC4 in high glucose (HG)-induced podocyte apoptosis. First, we identified the expression of HDAC4 was upregulated in podocytes of patients with DN. In vitro, the results also indicate that the mRNA and protein expression levels of HDAC4 were increased in HG-cultured podocytes. Silencing and overexpression of HDAC4 markedly decreased and increased CaN expression, respectively. Meanwhile, HG-induced podocyte apoptosis was abrogated by HDAC4-knockdown with subsequent decreased Bax expression and increased Bcl-2 expression. In contrast, overexpression of HDAC4 increased podocyte apoptosis and Bax expression, as well as decreased Bcl-2 expression. In addition, podocyte apoptosis induced by HDAC4 overexpression was effectively rescued by FK506, a pharmacological inhibitor of CaN, which was accompanied by decreased Bax and increased Bcl-2 expression. As a novel finding, HG-induced podocyte apoptosis is mediated by the HDAC4/CaN signaling pathway, which presents a promising target for therapeutic intervention in DN., Competing Interests: Declaration of competing interest There is no conflict of interest among all authors. We also confirm that all the listed authors have participated actively in the study and approved the submitted manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The 12-kDa FK506-binding protein (FKBP12) is the target of the commonly used immunosuppressive drug FK506. The FKBP12-FK506 complex binds to calcineurin and inhibits its activity, leading to immunosuppression and preventing organ transplant rejection. Our recent characterization of crystal structures of FKBP12 proteins in pathogenic fungi revealed the involvement of the 80's loop residue (Pro90) in the active site pocket in self-substrate interaction providing novel evidence on FKBP12 dimerization in vivo. The 40's loop residues have also been shown to be involved in reversible dimerization of FKBP12 in the mammalian and yeast systems. To understand how FKBP12 dimerization affects FK506 binding and influences calcineurin function, we generated Aspergillus fumigatus FKBP12 mutations in the 40's and 50's loop (F37 M/L; W60V). Interestingly, the mutants exhibited variable FK506 susceptibility in vivo indicating differing dimer strengths. In comparison to the 80's loop P90G and V91C mutants, the F37 M/L and W60V mutants exhibited greater FK506 resistance, with the F37M mutation showing complete loss in calcineurin binding in vivo. Molecular dynamics and pulling simulations for each dimeric FKBP12 protein revealed a two-fold increase in dimer strength and significantly higher number of contacts for the F37M, F37L, and W60V mutations, further confirming their varying degree of impact on FK506 binding and calcineurin inhibition in vivo., Competing Interests: Declaration of competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Calcineurin Inhibitors (CNIs) are routinely used for immunosuppression following solid organ transplantation. However, the prolonged use of these agents lead to organ fibrosis which limits their efficacy. CNIs induce TGFβ expression, which is reported to augment endothelial-to-mesenchymal transition (EndMT), but their role in this process is not known. In these studies, we find that the CNIs FK506 and cyclosporine (CsA) are potent to increase endothelial cell (EC) proliferation using established in vitro assays (P < 0.05). Furthermore, using phosphokinase arrays, we find that each CNI activates the MAPK and Akt/mTOR signaling pathways, and that pharmacological inhibition of each pathway targets CNI-induced proliferative responses (P < 0.001). EndMT was evaluated by FACS for N-cadherin and CD31 expression and by qPCR for the expression of α-smooth muscle actin, N-cadherin and Snail. We find that CNIs do not directly induce dedifferentiation, while TGFβ and hypoxia induce EndMT in small numbers of EC. In contrast, the treatment of EC with the inflammatory cytokine TNFα was potent to elicit an EndMT response, and its effects were most notably in EC following proliferation/doubling. Taken together, these observations suggest that CNIs elicit proliferative responses, which enhance EndMT in association with local inflammation. The clinical implications of these findings are that anti-proliferative therapeutics have high potential to target the initiation of this EndMT response., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To investigate the effect of tacrolimus (FK506) on myocardial infarction, and to further explore its function mechanism., Material and Methods: C57BL/6J mice were randomly divided into three groups: the sham group, the control group and the FK506 group. Anterior descending branch ligation was conducted in the control and the FK506 groups, while sham operation was conducted in the sham group. Mice in the sham and the control groups were intragastrical administration with saline, while the FK506 group were with FK506. Heart function were detected by echocardiogram at 3rd day or 21st day after MI. Hearts were harvested at 3rd day or 21st day after MI for the detection of apoptosis, autophagy, mTOR and NF-κB pathway., Results: FK506 treatment increased survival rate and cardiac function in mice after MI. It decreased infarction area, inflammation reaction and apoptosis. To further study the mechanism of FK506 protection effect, we discovered it could increase autophagy via inhibit mTOR pathway., Conclusion: FK506 protect heart function after MI as it improved myocardial cells autophagy process via inhibiting mTOR pathway., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tacrolimus (FK506) has been demonstrated to reduce epidural fibrosis. However, the detailed mechanism of action has not been elucidated. Aberrant miR-429 is involved in many diseases. The aim of this study was to describe the exact mechanism of FK506 induced apoptosis in fibroblasts and the prevention of epidural fibrosis. FK506 induced fibroblast apoptosis was evaluated using CCK-8 assays, flow cytometry, and western blotting. The expression of miR-429 in fibroblasts treated with FK506 was determined by RT-qPCR. Additionally, luciferase activity assays were used to determine the target relationship between miR-429 and RhoE. Flow cytometry and western blot analysis were used to determine the effects of FK506 and miR-429 on fibroblast apoptosis. The effects of FK506 and RhoE on fibroblast apoptosis were determined by CCK-8 assay, flow cytometry, and western blotting. We also evaluate the effects of FK506 and miR-429 on epidural fibrosis in rats by using histological analysis and TUNEL-staining. The results revealed FK506 induces fibroblast apoptosis and significantly downregulates miR-429 expression in fibroblasts. Additionally, miR-429 downregulation caused the apoptosis of fibroblasts. The luciferase activity assay confirmed that RhoE is a direct target of miR-429 and RhoE promotes fibroblast apoptosis. The rat model demonstrated miR-429 inhibition promotes fibroblast apoptosis and epidural fibrosis, which is consistent with the results of FK506 treatment. Our study demonstrates that FK506 induces fibroblast apoptosis and reduces epidural fibrosis by regulating miR-429 expression and its target of RhoE., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2(+) gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis with multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Dedifferentiated fat (DFAT) cells, which are isolated from mature adipocytes using the ceiling culture method, exhibit similar characteristics to mesenchymal stem cells, and possess adipogenic, osteogenic, chondrogenic, and myogenic potentials. Bone morphogenetic protein (BMP)-2 and -9, members of the transforming growth factor-β superfamily, exhibit the most potent osteogenic activity of this growth factor family. However, the effects of BMP-2 and BMP-9 on the osteogenic differentiation of DFAT remain unknown. Here, we examined the effects of BMP-2 and BMP-9 on osteoblastic differentiation of rat DFAT (rDFAT) cells in the presence or absence of FK506, an immunosuppressive agent. Co-stimulation with BMP-9 and FK506 induced gene expression of runx2, osterix, and bone sialoprotein, and ALP activity compared with BMP-9 alone, BMP-2 alone and BMP-2+FK506 in rDFAT cells. Furthermore, it caused mineralization of cultures and phosphorylation of smad1/5/8, compared with BMP-9 alone. The ALP activity induced by BMP-9+FK506 was not influenced by addition of noggin, a BMP antagonist. Our data suggest that the combination of BMP-9 and FK506 potently induces osteoblastic differentiation of rDFAT cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Xenopus tadpoles have high regenerative ability of amputated tails except during the 'refractory period', when the ability is transiently lost. We previously demonstrated that distinct immune responses occur in tail stumps between the refractory and pre/post-refractory regeneration periods. Furthermore, treatment with an immunosuppressant, FK506, restores the tail regenerative ability during the refractory period. Based on these findings, we previously proposed that autoreactive immune cells infiltrate the tail stumps to attack blastema cells as 'non-self' during the refractory period, resulting in the impaired regenerative ability. The immune cells that attack the blastema cells, however, remained unclear. Here we screened for genes whose expression in the tail stumps was altered by FK506 treatment during the refractory period and identified a Xenopus homolog of phytanoyl-CoA dioxygenase (PhyH)-like. XPhyH-like expression transiently increased in tail stumps after amputation during the refractory period, and was reduced by FK506 treatment. XPhyH-like expression in the whole tadpole body specifically increased during the refractory period and was enriched in the blood cell fraction. These findings suggest that XPhyH-like is expressed in autoreactive immune cells that are distributed in the whole body during the refractory period and transiently infiltrate the tail stumps to attack the blastema cells as 'non-self'., (Copyright © 2013 Elsevier Inc. All rights reserved.)

ATBF1 is a transcription factor that regulates genes responsible for repairing tissues and the protection of cells from oxidative stress. Therefore reduction of ATBF1 promotes susceptibility to varieties of human diseases including neurodegenerative diseases and malignant tumors. The instability of the protein was found to be an important background of diseases. Because ATBF1 is composed of a large 404-kDa protein, it can be easily targeted by proteinases. The protein instability should be a serious problem for the function in the cells and practically for our biochemical study of ATBF1. We have found that calpain-1 is a protease responsible for the degeneration of ATBF1. We observed distinct difference between embryo and adult brain derived ATBF1 regarding the sensitivity to calpain-1. The comparative study showed that eight phosphorylated serine residues (Ser1600, Ser2634, Ser2795, Ser2804, Ser2900, Ser3431, Ser3613, Ser3697) in embryonic brain, but only one site (Ser2634) in adult brain. As long as these amino acids were phosphorylated, ATBF1 derived from embryonic mouse brain showed resistance to cleavage; however, treatment with calf intestine alkaline phosphatase sensitized ATBF1 to be digested by calpain-1. An inhibitor (FK506) against calcineurin, which is a serine/threonine specific phosphatase enhanced the resistance of ATBF1 against the digestion by calpain-1. Taken together, these results demonstrate that these phosphorylation sites on ATBF1 function as a defensive shield to calpain-1., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcRγ) and DNAX-activating protein 12kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin (β3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that FK506 treatment significantly (p<0.05) reduced the expression of NFATc1, CathK, OSCAR, FcRγ, TREM2 and DAP12 during the terminal stage of osteoclast formation. VIVIT treatment significantly (p<0.05) decreased CathK, OSCAR, FcRγ, and AnnVIII, gene expression. This data suggest FK506 and VIVIT act differently in targeting the calcineurin-NFAT signalling cascade to suppress key mediators of the ITAM pathway during late stage osteoclast differentiation and this is associated with a reduction in both osteoclast differentiation and activity., (Copyright © 2012. Published by Elsevier Inc.)

Background: Recently, increased cardiac norepinephrine levels were observed in patients who were exposed to irregular stimulation during electrophysiological testing. The molecular mechanisms remain unclear. Intrinsic cardiac adrenergic (ICA) cells are present in mammalian hearts and contain catecholamine-synthesizing enzymes sufficient to produce biologically active norepinephrine levels. Thus, we aimed to investigate the expression of catecholamine-synthesizing enzymes by ICA cells exposed to irregular pacing., Methods: Co-cultures of cardiomyocytes and ICA cells were exposed to irregular pacing for 48h (standard deviation (SD)=5%, 25% and 50% of mean cycle length) at a constant rate of 5Hz. The expression of catecholamine-synthesizing enzymes including tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) were analyzed on mRNA and protein levels., Results: First, immunolabeling identified ICA cells presenting TH and DBH staining around the cell nucleus. Irregular pacing with 25% SD at a constant rate of 5Hz significantly increased the expression of TH and DBH enzyme synthesis. Pharmacological approaches have shown that both metoprolol and losartan reversed the irregular pacing induced DBH increase, whereas the expression of TH was only blocked by metoprolol in a significant manner. Blockade of the endothelin-A receptor by BQ123 or the calcineurin-NFAT pathway by cyclosporine-A, 11R-VIVIT or FK506 revealed a potential role of both cascades in irregular pacing induced catecholamine-synthesizing enzyme expression., Conclusions: ICA cells respond to irregular electrical activation with an increase in catecholamine-synthesizing enzymes. Drugs commonly used in clinical routine significantly influence the expression of TH and DBH by ICA cells via different signaling routes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Glioblastoma multiforme (GBM) is the most aggressive of brain tumors and is extremely insensitive to anticancer drugs. Studies have attributed the ABC transporter Mrp1 (ABCC1, multiple-drug resistance protein 1) with conferring chemoresistance in this tumor by extrusion of a wide spectrum of anticancer drugs. Therefore it is crucial to search for and investigate inhibitors of Mrp1 activity in GBM cells, particularly those that could be safe as chemosensitizers to anticancer drugs in clinical studies. We find that in primary cultured or T98G GBM cells exposed to therapeutic plasma concentrations of FK506 (tacrolimus), the expression of Mrp1 was decreased in a dose-dependent manner. The activity of this transporter, measured by CFDA fluorescent substrate extrusion, decreased significantly in primary cultured GBM cells on exposure to FK506 at concentrations of 15 ng/ml. When GBM cells were exposed to anticancer drugs vincristine, etoposide or taxol, cell viability was not affected. However when the anticancer drugs were assayed in combination with FK506, cell viability was significantly decreased by as much as 50% in GBM primary culture. We conclude that FK506 could be a valuable tool for chemosensitization of GBM cells, offering a possible improvement to the current poor therapy available for high-grade human gliomas., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Calcineurin is the only Ca(2+)-dependent serine/threonine-specific protein phosphatase and is considered a potential regulator of many intracellular signaling events. In this study we identified a novel interaction between calcineurin and the 20S proteasome subunit PSMA6 that increased intracellular proteasomal activity. Using RAW 264.7 macrophage cells, we demonstrated that expression of inflammatory factors was induced by calcineurin, and suppressed by the calcineurin inhibitor FK506. We also found that these calcineurin-activated processes result from activation of NF-κB, and that the interaction of calcineurin with PSMA6 stimulates transcription by NF-κB via degradation of IκB by the ubiquitin-proteasome pathway. These findings indicate that calcineurin is required for expression of inflammatory factors, and reveal a novel process of calcineurin-mediated activation of NF-κB., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mechanical stress can induce cardiac hypertrophy through angiotensin II (AngII) type 1 (AT(1)) receptor independently of AngII, however, the intracellular mechanisms remain largely indeterminate. Since calcineurin, a Ca(2+)-dependent phosphatase, plays a critical role in pressure overload-induced cardiac hypertrophy, we therefore, asked whether calcineurin is involved in the AT(1) receptor-mediated but AngII-independent cardiac hypertrophy. Mechanical stretch failed to elicit hypertrophic responses in COS7 cells co-transfected with plasmid of AT(1) receptor and siRNA of calcineurin. Mechanical stresses for 2weeks in vivo and for 24h in vitro significantly induced upregulation of calcineurin expression and hypertrophic responses, such as the increases in cardiomyocytes size and specific gene expressions, in cardiomyocytes of angiotensinogen gene knockout (ATG(-/-)) mice, both of which were significantly suppressed by a specific calcineurin inhibitor FK506, suggesting a critical role of calcineurin in mechanical stress-induced cardiac hypertrophy in the ATG(-/-) mice. Furthermore, an AT(1) receptor blocker Losartan not only attenuated cardiac hypertrophy but also abrogated upregulation of cardiac calcineurin expression induced by mechanical stresses in the AngII-lacking mice, indicating that calcineurin expression is regulated by AT(1) receptor without the involvement of AngII after mechanical stress. These findings collectively suggest that mechanical stress-evoked but AngII-independent activation of AT(1) receptor induces cardiac hypertrophy through calcineurin pathway., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Cellular ATP level in Saccharomyces cerevisiae was measured during culture growth of strain US50-18C overproducing all major PDR pumps and its isogenic mutants variously deleted in these pumps. It was found to be inversely proportional to the intensity of cell metabolism during different growth phases and to the activity of PDR pumps, which are thus among major ATP consumers in the cells. The ATP level was increased when membrane integrity was affected by 0.5% butanol, and further increased by compound 23.1, a semisynthetic phenol lipid derivative that acts as inhibitor of Pdr5p and Snq2p pumps. The magnitude of increase in cell ATP caused by inhibition of Pdr5p pump by compound 23.1 and the Pdr5p pump inhibitor FK506 used for comparison reflects the activity and hence the energy demand of the pump. The rise in cell ATP caused by different PDR pump inhibitors can be thus used as an indicator of pump activity and the potency of the inhibitor., (Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.)

In this review, we propose that TRESK background K(+) channel could serve as a potential therapeutic target for T-cell mediated immune dysfunction. TRESK has many immune function-related properties. TRESK is abundantly expressed in the thymus, the spleen, and human leukemic T-lymphocytes. TRESK is highly activated by Ca(2+), calcineurin, acetylcholine, and histamine which induce hypertrophy, whereas TRESK is inhibited by immunosuppressants, such as cyclosporin A and FK506. Cyclosporine A and FK506 target the binding site of nuclear factor of activated T-cells (NFAT) to inhibit calcineurin. Interestingly, TRESK possesses an NFAT-like docking site that is present at its intracellular loop. Calcineurin has been found to interact with TRESK via specific NFAT-like docking site. When the T-cell is activated, calcineurin can bind to the NFAT-docking site of TRESK. The activation of both TRESK and NFAT via Ca(2+)-calcineurin-NFAT/TRESK pathway could modulate the transcription of new genes in addition to regulating several aspects of T-cell function.

Intracellular calcium ions (Ca(2+)) have an essential role in the regulation of neurite outgrowth, but how outgrowth is controlled remains largely unknown. In this study, we examined how the mechanisms of neurite outgrowth change during development in chick and mouse dorsal root ganglion neurons. 2APB, a potent inhibitor of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R), inhibited neurite outgrowth at early developmental stages, but not at later stages. In contrast, pharmacological inhibition with Ni(2+), Cd(2+), or dantrolene revealed that ryanodine receptor (RyR)-mediated Ca(2+)-induced Ca(2+) release (CICR) was involved in neurite outgrowth at later stage, but not at early stages. The distribution of IP(3)R and RyR in growth cones also changed during development. Furthermore, pharmacological inhibition of the Ca(2+)-calmodulin-dependent phosphatase calcineurin with FK506 reduced neurite outgrowth only at early stages. These data suggest that the calcium signaling that regulates neurite outgrowth may change during development from an IP(3)R-mediated pathway to a RyR-mediated pathway.

Calcineurin is an important regulator of extracellular matrix (ECM) accumulation in the kidney but functions in a cell-specific manner. Previously, we identified a novel role for calcineurin in mesangial cells where calcineurin activity is required for TGFbeta-mediated induction of fibronectin expression. In this study, we examined the role of the calcineurin substrate NFATc in transcriptional regulation of fibronectin. First, inhibition of calcineurin blocks TGFbeta induction of the fibronectin promoter. Moreover, expression of constitutively active calcineurin in mesangial cells is sufficient to increase fibronectin transcription. Next, inhibition of the calcineurin substrate NFATc1 blocked TGFbeta-mediated activation of the fibronectin promoter. Finally, stable expression of a dominant-negative NFATc protein reduced transcriptional activation of the promoter and inhibited TGFbeta-mediated fibronectin expression. In conclusion, TGFbeta activation of calcineurin in mesangial cells results in regulation of ECM accumulation at least in part by direct transcriptional activity of NFATc on the fibronectin promoter.

Immunosuppressants such as cyclosporinA and FK506 (tacrolimus) are widely prescribed to treat numerous disorders and to treat organ transplant recipients. However, cyclosporine A and FK506 are both known to produce hypomagnesaemia. The mechanism of this effect is still unclear. The present study determined the effects of immunosuppressant treatment on the parathyroid hormone (PTH) mediated Mg(2+) uptake and the mitogen-activated protein kinase (MAPK) activation in mouse distal convoluted tubule (MDCT) cells. The intracellular Ca(2+) and Mg(2+) concentrations in a single MDCT cell were measured by using the fluorescentdye Fura-2 AM and Mag-fura-2 AM, respectively. Cyclosporine A and FK506 illicited a transient increase of intracellular Ca(2+) from a basal level of 99 +/- 16 nM to 685 +/- 105 and 608 +/- 96 nM, respectively. In order to determine the Mg(2+) transport, the MDCT cells were Mg(2+)-depleted by culturing them in Mg(2+)-free media for 16 h, and the Mg(2+) uptake was measured by microfluorescence after placing the depleted cells in 1.5mM MgCl(2). The mean rate of Mg(2+) uptake, d([Mg(2+)](i))/dt, was 140 +/- 16 nM/s in the control MDCT cells. PTH increased the Mg(2+) uptake more than 2 times in this cell. Cyclosporine A (10 microM) and FK506 (0.1 microM) did not affect the basal Mg(2+)uptake (140 +/- 16 and 142 +/- 14 nM/s, respectively), but they inhibited the PTH-stimulated Mg(2+) entry, decreasing it from 248+/-12 to 147 +/- 7 and 148 +/- 14 nM/s, respectively. These effects were inhibited by L685818, which is a potent competitive antagonist of FK506. PTH stimulated the extracellular signal-regulated kinase1/2 (ERK1/2) protein synthesis. This PTH-stimulated ERK1/2 activation was inhibited by cyclosporine A and FK506. In the present study, the role of ERK1/2 activation on the PTH-dependent magnesium uptake was examined in MDCT cells, and we showed that immunosuppressants inhibit the hormone-stimulated Mg(2+) uptake into the MDCT cells by inhibiting the MAPK pathway.

Calcineurin (CN) is thought to play an important role in the immune system by regulating cytokine production, for example, interleukin-2 (IL-2) in T-lymphocytes. We have previously shown that physiological concentrations of Zn2+ inhibit CN activity in vitro [K. Takahashi, E. Akaishi, Y. Abe, R. Ishikawa, S. Tanaka, K. Hosaka, Y. Kubohara, Zinc inhibits calcineurin activity in vitro by competing with nickel, Biochem. Biophys. Res. Commun. 307 (2003) 64-68], in spite of the fact that Zn2+ is an essential element of the CN catalytic domain. In this study, in order to assess whether Zn2+ regulates (suppresses) CN activity in vivo and whether Zn2+ can be used as an anti-inflammatory and/or immunosuppressive drug, we examined the effects of Zn2+ on IL-2 production induced by the mitogen, concanavalin A (ConA), in Jurkat T-cells. Zn2+ at 0.2 mM suppressed ConA-induced IL-2 accumulation in the medium of an in vitro culture of Jurkat cells. Zn2+ at 0.03-0.3 mM dose-dependently suppressed ConA-induced IL-2 mRNA expression in Jurkat cells. Zn2+ also suppressed IL-2 mRNA expression induced by phorbol ester (PMA) and ionomycin. Furthermore, Zn2+ and the immunosuppressant FK506 showed an additive inhibitory effect on ConA-induced IL-2 mRNA expression. These results suggest that exogenously added Zn2+ may disturb (increase) the intracellular Zn2+ concentration and inhibit CN activity, thereby suppressing IL-2 production in Jurkat cells. The present study further indicates that Zn2+ may have therapeutic potential in the treatment of T-cell related inflammation and also that Zn2+ may be utilized as a supplemental drug with FK506.

Pdr5p in Saccharomyces cerevisiae is a functional homologue of mammalian P-glycoprotein implicated in multidrug resistance (MDR). In order to obtain useful inhibitors to overcome MDR in clinical tumors, screening of Pdr5p inhibitors has been carried out. We isolated a fungal strain producing Pdr5p inhibitors using our original assay system, and it was classified as Trichoderma sp. P24-3. The purified inhibitor was identified as isonitrile, 3-(3'-isocyano-cyclopent-2'-enylidene)-propionic acid, a compound whose carboxyl residue is essential for the inhibitory activity. A non-toxic concentration of the isonitrile (41.5 microg/ml, 255 microM) inhibited Pdr5p-mediated efflux of cycloheximide or cerulenin in Pdr5p-overexpressing cells. In addition, addition of the isonitrile led to accumulation of rhodamine 6G, a substrate of Pdr5p, in the Pdr5p-overexpressing cells. The inhibitory profiles of the isonitrile against S1360 mutants (S1360A and S1360F) of Pdr5p were different from those of FK506 and enniatin. The isonitrile did not influence PDR5 gene expression and the amount of Pdr5 protein, nor did it inhibit the function of Snq2p, a homologue of Pdr5p. Interestingly, the isonitrile inhibited the function of Cdr1p and Cdr2p, Pdr5p homologues in pathogenic yeast Candida albicans. Thus, it was found that the isonitrile shows a different inhibitory spectrum from that of FK506 and enniatin as a potent inhibitor for Pdr5p, Cdr1p, and Cdr2p.

Using a cell cluster preparation from the stomach smooth muscle tissue of mice, we measured intracellular Ca(2+) oscillations in interstitial cells of Cajal (ICCs) in the presence of nifedipine. Pacemaker [Ca(2+)](i) activity in ICCs was significantly suppressed by caffeine application and restored after washout. Application of either ryanodine or FK-506 terminated the pacemaker [Ca(2+)](i) activity irreversibly. Immunostaining of smooth muscle tissue showed that c-Kit-immunopositive cells (that form network-like structure cells in the myenteric plexus, equivalent to ICCs) clearly express ryanodine receptors (RyR). RT-PCR revealed that ICCs (identified with c-Kit-immunoreactivity) predominantly express type 3 RyR (RyR3). Furthermore, the FK-binding proteins 12 and 12.6, both of which would interact with RyR3, were detected. In conclusion, we provide first evidence for the essential contribution of RyR to generating pacemaker activity in gastric motility. Similar mechanisms might account for spontaneous rhythmicity seen in smooth muscle tissues distributed in the autonomic nervous system.

Receptor activator of nuclear factor kappaB-ligand (RANKL) transduces a differentiation signal appropriate to osteoclasts likely through induction a receptor homotrimer; however, biological importance of RANK-trimerizarion is unknown. To address the signaling mechanism of the RANK receptor, we analyzed the effect of two different types of homodimer inducers RANK-TM-FKBP36v and hEpoR-RANK-TM on osteoclastogenesis. Dimerizing component FKBP36v or extracellular portion of human erythropoietin receptor (hEpoR) was fused to RANK lacking the extracellular domain, and the dimerization of this fusion protein was induced by addition of the chemical inducer of dimerization AP20187 or erythropoietin, respectively. Such treatment resulted in induction of TRAP-activity, a marker of osteoclast in a dose dependent manner, with an efficiency equivalent to that of induction by RANKL. However, dimerized-RANK-induced osteoclasts showed relatively low levels of multinucleation, pit forming activity, and expression of calcitonin receptor and cathepsin K, compared with osteoclasts which were induced in the presence of RANKL. As expression of nuclear factor of activated T cells 1 (NFATc1) was also reduced in dimerized-RANK-induced osteoclasts, RANK oligomerization by RANKL is a critical event to generate fully matured osteoclasts through upregulation of NFATc1.

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.

We previously reported synergistic induction of apoptosis by IFN-gamma plus either cyclosporin A (CsA) or tacrolimus (FK506) in gastric carcinoma cells. In this study, we aimed to elucidate the mechanism for this synergistic induction of apoptosis. IFN-gamma plus CsA synergistically induced caspase-3 mediated apoptosis in gastric carcinoma cells. Although IFN-gamma induced activation of signal transducer and activator of transcription1 (STAT1) and expression of interferon regulatory factor-1 (IRF-1) mRNA, IFN-gamma alone was not able to induce caspase-3 activation and apoptosis. When gastric carcinoma cells were treated with cyclohexamide, a protein synthesis inhibitor, following IFN-gamma pretreatment, caspase-3 was activated, and apoptosis was markedly induced. These findings suggest the existence of IFN-gamma-induced anti-apoptotic pathway and we evaluated the effect of IFN-gamma and CsA on calcium-sensitive nuclear factor-kappa B (NF-kappa B) activation. IFN-gamma increased intracellular calcium ion concentration ([Ca(2+)](i)) consisting of a spike and a sustained phase, and the latter was completely abrogated by CsA. Activation of NF-kappa B occurred in response to IFN-gamma, and which was markedly inhibited by either CsA or FK506. NF-kappa B decoy also enhanced the cytotoxic effect of IFN-gamma. These results suggest that IFN-gamma may simultaneously induce the STAT1-mediated apoptotic pathway and the anti-apoptotic pathway through calcium-activated NF-kappa B and that inhibition of the latter by CsA may result in dominance of the apoptosis-inducing pathway.

Tacrolimus (Tac) is more immunosuppressive drug compared to cyclosporine (CsA). Our previous studies have demonstrated that CsA induces the expression of p21WAF/CIP1 expression. In this study we explored if like CsA, Tac also induces expression of p21WAF/CIP1. We also determined if induction of p21WAF/CIP1 by Tac is dependent on TGF-beta. Using RT-PCR and Western blot analysis, we studied the induction of p21WAF/CIP1 mRNA and protein in human T cells and A-549 cells (human lung adenocarcinoma cells) by Tac. The stimulation of p21WAF/CIP1 promoter activity was studied by luciferase assay using p21WAF/CIP1-luc, chimeric plasmid DNA containing a p21WAF/CIP1 promoter segment and luciferase reporter gene. Using anti-TGF-beta antibody, we studied if induction of p21WAF/CIP1 by tacrolimus is dependent on TGF-beta. The results demonstrate that Tac induced p21WAF/CIP1 mRNA and protein expression as well as stimulated its promoter activity in T cells and A-549 cells. The induction of p21WAF/CIP1 expression by tacrolimus was dependent on TGF-beta since a neutralizing anti-TGF-beta antibody inhibited induction of p21WAF/CIP1in A-549 cells. These data support the hypothesis that cyclin inhibitor p21WAF/CIP1 might represent a unified mediator of the anti-proliferative effects of Tac and other immunosuppressive agents. Strategies involving p21WAF/CIP1 induction should be considered a viable alternative strategy to achieve immunosuppression possibly with reduced toxicity associated with current immunosuppression.

The calcineurin-mediated pathway is involved in skeletal and cardiac hypertrophy and vascular development in vivo, but the relationship between this pathway and the phenotype of smooth muscle cells (SMCs) remains unknown. Using visceral SMCs in culture as a model system of differentiated SMCs, we investigated the role of the calcineurin-mediated pathway in maintaining the differentiated phenotype of SMCs, which depends on the insulin-like growth factor (IGF-I)-triggered activation of the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB(Akt)) pathway. Treatment with calcineurin inhibitors, cyclosporin A or FK506, or the forced expression of the natural calcineurin inhibitor, CAIN, induced SMC dedifferentiation. Notably, suppression of the promoter activities of the SMC molecular markers caldesmon and alpha1 integrin by blocking the PI3-K/PKB(Akt) pathway was rescued by the forced expression of constitutively active calcineurin Aalpha, suggesting that the calcineurin-mediated pathway is critical for maintaining the differentiated phenotype of SMCs and works downstream of the PI3-K/PKB(Akt) pathway.

Selectins are mediating transient contacts of leukocytes with endothelium during inflammatory processes and in the development of the immune system. L-selectin expressed on almost all leukocytes also functions as a signaling receptor. Recently, we have identified different signaling pathways in T lymphocytes by L-selectin. One signaling cascade leads via the tyrosine kinase p56lck to the small G-proteins Ras and Rac and to MAP-kinases. A second independent pathway results in ceramide release. In this study, an L-selectin-induced translocation of the transcription factor NFAT to the nucleus was identified. Using genetically modified JCaM1.6 cells, pharmacological inhibitors, and antisense molecules, it was shown that L-selectin-induced NFAT activation depends on src-tyrosine kinases, calcineurin and small G-proteins. MAP-kinases and actin filaments were identified as Ras effectors involved in NFAT translocation. We conclude that L-selectin cross-linking results in activation of NFAT by different signaling pathways. The activation of NFAT might modulate the immune response of leukocytes interacting with endothelial cells., (©2002 Elsevier Science (USA).)

FK506 (tacrolimus) is known as an inhibitor for calcineurin and is used in numerous research fields. It is not clear whether intravenously injected FK506 inhibits neuronal calcineurin. We measured the calcineurin activities of normal and postischemic rat hippocampi after intravenous injection of FK506 (3 mg/kg). Intravenously injected FK506 had no inhibitory effect on calcineurin activity in the hippocampi of normal and postischemic rats, whereas FK506 inhibited the calcineurin in vitro (purified enzyme, hippocampal homogenate, and hippocampal slice culture homogenate). Thus, it is considered that intravenously injected FK506 does not act on intraneuronal calcineurin and that several effects of FK506 are not due to the inhibition of neuronal calcineurin., (Copyright 2001 Academic Press.)

A cDNA encoding chicken FK506-binding protein 12 (FKBP12) was isolated and sequenced. The predicted amino acid sequence of the chicken protein shows high homology to those of FKBP12 proteins of other species ranging from human to frog. The possible role of FKBP12 in chick embryonic cardiac development was examined. Northern blot analysis revealed that FKBP12 mRNA is distributed widely in chick embryos, being especially abundant in the heart; the amount of FKBP12 mRNA in the embryonic heart decreased with time. Administration of FK506 to chick embryos at 7 to 9 days resulted in marked cardiac enlargement. FK506 also reduced the expression of myosin, induced a more elongated cell morphology, and impaired network formation in cultured chick embryonic cardiomyocytes. These results suggest that FKBP12 is important in the regulation of contractile function and phenotypic expression in chick cardiomyocytes during embryonic development., (Copyright 2001 Academic Press.)

The transcriptional coactivator CREB-binding protein (CBP) is known to play an important role in coupling signal transduction pathways to changes in gene expression. In many cases, this is achieved by the stimulus-specific recruitment of CBP to promoter-bound transcription factors. However, a number of recent studies have suggested that signal transduction pathways can also directly influence CBP-mediated transcriptional activity. Here we show that in Jurkat cells the activity of the CBP C-terminal transactivation domain is strongly upregulated in response to either T cell receptor stimulation or the combination of ionomycin and phorbol ester. We further show that maximal stimulation of CBP-mediated transcription requires the synergistic activation of both the calcineurin and Ras-MAPK signaling pathways. These results indicate that CBP can function as a T cell activation-inducible transcriptional coactivator and is therefore likely to play an important role in T cell activation-induced gene expression., (Copyright 2001 Academic Press.)

Osteoporosis and vasculopathy are common after organ transplantation and have been largely attributed to the use of immunosuppressants. Osteoprotegerin (OPG) is produced by osteoblastic and arterial cells, and inhibits osteoclast functions by neutralizing receptor activator of NF-kappaB ligand (RANKL). Because OPG-deficient mice develop osteoporosis and arterial calcification, we assessed the effects of immunosuppressants on OPG and RANKL expression by human osteoblastic and coronary artery smooth muscle cells (CASMC). Cyclosporine A, rapamycin, and FK-506 decreased OPG mRNA and protein levels in undifferentiated marrow stromal cells (by 63, 44, and 68%, respectively, P < 0.001). All three immunosuppressants increased RANKL mRNA levels in these cells by 60 to 210%. In contrast to these effects on marrow stromal cells, rapamycin, which may be relatively bone-sparing, increased OPG mRNA and protein production (by 120%, P < 0.001) in mature osteoblastic cells. Cyclosporine A also decreased OPG mRNA and protein production (by 52%, P < 0.001) of CASMC. In conclusion, immunosuppressants decrease OPG mRNA and protein production and increase RANKL gene expression by marrow stromal cells, and cyclosporine suppresses OPG production in CASMC. These studies thus provide a potential mechanism for immunosuppressant-induced bone loss, and the propensity of cyclosporine A to cause vascular disease., (Copyright 2001 Academic Press.)

We have identified an 8.4 kDa minor immunophilin from calf thymus and Jurkat T cells as ubiquitin. It binds tacrolimus and sirolimus with K(d)'s of 0.8 and 0.08 nM, respectively. The binding of this protein to cyclosporin A is negligible. Binding of tacrolimus to two commercial sources of ubiquitin, a bovine product, and a recombinant human ubiquitin, was also demonstrated after HPLC purification of the purchased preparations., (Copyright 2000 Academic Press.)

Leukotriene B(4) (LTB(4)), which is an arachidonic acid metabolite produced by the 5-lipoxygenase pathway and a well-characterized chemical mediator of inflammation, has been proposed to be an immune response modulator. Here we showed the constitutive expression of the LTB(4) receptor (LTB(4)R) in resting and activated T cells. We found that the LTB(4)R antagonist inhibited T cell proliferation induced by Con A, immobilized anti-CD3 mAb, or IL-2. This inhibitory effect was abolished by addition of LTB(4)R agonist. The LTB(4)R antagonist inhibited IL-2, IFN-gamma, and IL-4 production by anti-CD3-stimulated T cells and also inhibited IL-12-induced IFN-gamma production. Moreover, the LTB(4)R antagonist exerted an additive inhibitory effect to FK506 on T cell proliferation. These results suggest that LTB(4) is intrinsically involved in T cell activation to upregulate cytokine production and proliferation, and thus the LTB(4)R antagonist might be useful as an immunosuppressive agent., (Copyright 1999 Academic Press.)

The effects of three immunosuppressants (rapamycin, FK506 and cyclosporin A) on the proliferation and differentiation of rat osteoblasts-like osteosarcoma cell line, ROS 17/2.8 (ROS) cells were examined in vitro. All immunosuppressants showed a direct inhibition on the proliferation of ROS cells with different potencies. Growth inhibition by rapamycin was stronger than that by FK506 or cyclosporin A. Rapamycin caused a significant increase in alkaline phosphatase (ALP) activity and in the expression of osteopontin and osteocalcin mRNAs. FK506 caused a moderate increase in ALP activity and a decreased expression of osteopontin mRNA. Cyclosporin A caused a decrease in ALP activity and in the expression of type 1 alpha 1 collagen mRNA. Our study indicates that rapamycin directly acts on ROS cells and induces osteoblastic differentiation, however, the effect of FK506 and cyclosporin A is weak. Rapamycin significantly enhances the differentiation induced by 1,25(OH)2-vitaminD3.

The immunosuppressant FK506 inhibits thymocyte positive selection. Calcineurin, a FK506-sensitive Ca2+/calmodulin-dependent protein phosphatase, is presumed to be involved in this event without direct evidence. We have previously shown that moderate stimulation of CD4(+)CD8(+) thymocytes with a combination of the calcium ionophore ionomycin and phorbol myristate acetate mimics positive selection events including downregulation of CD8 expression. Moderate stimulation of a CD4(+)CD8(+) T cell line with the same combination of drugs also induced specific downregulation of CD8 expression. FK506 inhibited the CD8 downregulation in both cell types. The T cell line was transfected with an expression vector encoding an active form of calcineurin. The transfectans remained CD4(+)CD8(+), but became CD4(+)CD8(-) or CD4(+)CD8(low) upon stimulation with phorbol myristate acetate alone. The extent of the CD8 downregulation was correlated with the expression level of the mutant calcineurin. These results suggest that the calcium signal for the CD8 downregulation is mainly delivered through calcineurin activation., (Copyright 1998 Academic Press.)

Calcineurin is a Ca2+ calmodulin dependent protein phosphatase which has an important role in the control of intracellular Ca2+ signalling. The protein is a heterodimer of one catalytic (CnA) subunit and one regulatory (CnB) subunit. As suggested by the protein sequence and confirmed by the crystallographic structure, the catalytic subunit of calcineurin (CnA) has high homologies with other protein phosphatases. The regulatory subunit (CnB) belongs to the EF-hand Ca2+ binding protein family. Despite its similarity with calmodulin, it has a different tertiary structure. Calcineurin is the target of two important immunosuppressant drugs: cyclosporin A and FK506. Subsequently, a detailed clarification of the role of calcineurin in the cytokine mediated activation of the T-cells has been possible. The understanding of the role of calcineurin in other cells, in particular neurons, is also progressing rapidly.

Activation of the NFkappaB transcription factor in 16HBE human bronchial epithelial cells was compared with activation of NFkappaB in Jurkat T-cells. An NFkappaB-luciferase reporter gene was activated by phorbol myristyl acetate (PMA) in both cell types. Ionomycin added to PMA (P/I) inhibited NFkappaB activation in epithelial cells and enhanced PMA activation in T-cells. Cyclosporin A (CsA) inhibited calcium signaling in both cell types. Nuclear NFkappaB DNA-binding stimulated with PMA was inhibited with ionomycin in epithelial cells and was enhanced with ionomycin in T-cells; CsA reversed both effects of ionomycin. Cytosolic IkappaB-alpha was regulated identically in both cell types. Thus, calcium activated opposing nuclear signaling pathways in epithelial cells and T-cells. Calcium-mediated repression of NFkappaB in epithelial cells was derepressed by CsA, and this establishes a mechanism through which CsA may exert proinflammatory effects in nonlymphoid cells.

The immunosuppressant rapamycin (RAP) potentiated apoptosis of the murine T lymphoblastoid cell line S49 induced by dexamethasone (DEX), while RAP by itself did not induce apoptosis of the cells. FK506, in contrast, had no effect on DEX-induced apoptosis; moreover, an excess of FK506 reversed the potentiation of apoptosis by RAP, indicating that RAP exerts its effects through binding to FKBP. Both RAP and FK506 enhanced the MMTV promoter activity by dexamethasone, suggesting that the potentiation of apoptosis is not likely explained by the selective enhancement of transcriptional activity of the glucocorticoid receptor. Of interest, the basal activity of c-Jun kinase (JNK), whose activation has been recently suggested to be involved in cell survival signals in lymphocytes, was reduced by RAP in S49 cells. The reduction of JNK activity by RAP was reversed by the addition of an excess of FK506. In summary, we demonstrate for the first time that RAP has the ability to inhibit JNK activity in lymphocytes where the drug enhances apoptosis.

The effects of immunosuppressive agents on the potassium current of human peripheral blood lymphocytes have been studied using the whole-cell patch-clamp technique. Cyclosporin A (10 micrograms/ml), rapamycin (10 micrograms/ml) and FK-506 (2.5 micrograms/ml) reduced the peak K+ current by approximately 40, 30 and 40% of the control, respectively, without any change in the reversal potential of the current. The current inhibition was similar at all membrane potentials studied and was accompanied with an increase in the rate of K+ current inactivation. Membrane potential measurements in current-clamp showed a marked depolarization of the membrane (>10 mV) upon the addition of either immunosuppressor to the cells. Our findings revealed that the voltage-dependent potassium current in human peripheral blood lymphocytes is inhibited by Cyclosporin A and other immunosuppressors, resulting in a depolarized membrane potential.

Two Jurkat cell clones have been stably transfected with a reporter vector for the nuclear factor of activated T cells (NFAT). Upon stimulation, they express high levels of secreted heat stable placental alkaline phosphatase. With these clones, we demonstrated that NFAT activation induced by phorbol 12-myristate 13-acetate and ionomycin was inhibited by both cyclosporin A (CsA) (IC50 = 8 nM) and FK506 (IC50 = 160 pM), presumably by inhibition of calcineurin activity. Selective phosphatase inhibitors for protein phosphatase 1 (PP1) and 2A (PP2A) that do not inhibit calcineurin, such as okadaic acid and calyculin A, also inhibited NFAT activation with IC50s of 87 nM and 4 nM, respectively, suggesting that okadaic acid and related inhibitors may block NFAT activation through the inhibition of PP1, instead of PP2A. NFAT activation was also inhibited by agents that increase cAMP concentrations such as dibutyryl cAMP, forskolin and prostaglandin E2. These stable Jurkat cell clones provide a convenient and sensitive tool to study NFAT regulation.

A 12-kDa inositolphosphate-binding protein has been identified as a component of the human erythrocyte membrane. Its robust peptidylprolyl cis-trans isomerase activity that is strongly inhibited by the immunosuppressant drugs FK506 and rapamycin indicates that it is an immunophilin belonging to the FKBP class. The finding that its peptidylprolyl cis-trans isomerase activity is also strongly inhibited by nanomolar concentrations of the second messengers inositol 1,4,5-trisphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) suggests that IP3 and IP4 could be physiological ligands for this membrane-associated immunophilin.

The molecular complex formed by the immunosuppressant FK506 and the immunophilin protein FKBP12 potently inhibits the Ca2+/calmodulin-activated protein phosphatase calcineurin. This mechanism appears to be common to all types of cell, implying that fundamental physiological modes of calcineurin regulation are exploited by immunosuppressants. The present paper describes a novel adenylyl cyclase regulated by calcineurin that contains an FKBP12-like domain and may thus constitute a physiologically relevant calcineurin docking site mimicked by immunosuppressant-immunophilin complexes. The enzyme messenger RNA is particularly enriched in the cerebral cortex, striatum and hippocampus, where it is localized to neuronal perikarya, indicative of an important role in neuronal function.

Adenovirus-mediated gene transfer into skeletal muscles of adult immune competent animals has been limited by the fact that a cellular immune attack of the host against transduced muscle fibers prevented long-term transgene expression. In this study we treated adult dystrophic [mdx] mice with daily subcutaneous injections of the immunosuppressive drug FK506 (tacrolimus) over 10 and 30 days after adenovirus-mediated reporter gene transfer and compared the transduction level to saline-injected controls. After 30 days, transgene expression was no longer demonstrable in the control group, whereas it remained at about 70% of the 10-day transduction value in the FK506 treated group. In addition, we demonstrated a reduction in the number of CD3 and CD8 positive T-lymphocytes in the muscles of the immunosuppressed group compared to controls.

Previous studies have shown that 4-aminopyridine (4AP) induced Ca-influx effects the release of glutamate from nerve terminals (synaptosomes) isolated from rat cerebral cortex. We now show that the Ca-dependent component of this release is potentiated by preincubation of the synaptosomes with the immunosuppressant, FK506, an inhibitor of protein phosphatase-2B (calcineurin). FK506 did not inhibit the Ca-independent release of glutamate from a cytosolic pool. Examination of the effect of FK506 on the influx of Ca elicited by 4AP indicated that inhibition of calcineurin activity resulted in an increase of voltage-dependent Ca-influx. Based on these results, we suggest that protein dephosphorylation effected by calcineurin may suppress voltage-dependent Ca-channel activity and in so doing inhibits evoked glutamate release. Activation of calcineurin produced by initial Ca-entry may represent a negative feedback to limit the activity of Ca-channels coupled to the release of glutamate.

The effect of recombinant FKBP-59/HBI or of its first N-terminal domain FKBP-59/HBI-I on the phosphatase activity of calcineurin (a Ca(+2)-calmodulin dependent phosphatase) was tested in vitro in the presence or absence of the immunosuppressant drug FK506. Contrarily to the inhibition observed with the immunosuppressant complex FKBP-12-FK506, no significant inhibition was observed with FKBP-59/HBI or FKBP-59/HBI-I in the presence of FK506, even though FKBP-59/HBI-1 is nearly 55% homologous to the immunophilin FKBP-12. Inhibition was tested both with native calcineurin (calcineurin A: Mr 58-59 kDa) and with protease activated, calmodulin independent calcineurin (calcineurin A: Mr 45 kDa). There was no competitive effect of FKBP-59 on the inhibitory activity of the FKBP-12-FK506 complex, even when the molar concentration of FKBP-59/HBI was 100 times higher than that of FKBP-12. Clearly, although the first domain of FKBP-59/HBI displays several structural and functional features of FKBP-12, it does not interact with calcineurin.