Calcineurin Phosphatase Activity Research Articles

Abstract 16984: Role of Inositol 1,4,5-Triphosphate Receptor 2 in Post-Ischemic Heart Failure

Introduction: The type 2 inositol 1,4,5-triphosphate receptor (IP3R2) is the predominant isoform expressed in ventricular cardiomyocytes. IP3R2-mediated Ca 2+ release has been proposed to participate in atrial arrhythmogenesis. However, its involvement in post-ischemic heart failure (HF) has not been elucidated. Methods: To investigate whether myocardial IP3R2 plays a functional role in HF, we generated myocardial specific IP3R2 knock-out (KO) mice by crossing IP3R2 flox/flox mice (obtained by opportunely placing loxP sequences flanking exon 3 of IP3R2 gene) with α-MHC Cre mice. Results: We confirmed cardiomyocyte-specific IP3R2 deletion in IP3R2 flox/flox /α-MHC Cre mice (hereafter referred to as KO) at mRNA and protein levels. The loss of IP3R2 in the heart does not result in embryonic lethality: KO mice survived to adulthood and we did not detect any morphological or functional defects in baseline conditions. Serial cardiac ultrasound analyses performed following coronary artery ligation revealed a significant amelioration in cardiac function post myocardial infarction (MI) in KO mice compared with flox littermates (p<.05, ANOVA rep. measures, n=5-8/group). No significant differences were detected by radiotelemetry in blood pressure. Hemodynamic studies performed post-MI confirmed the improved cardiac contractility (dP/dt max ,KO:6424±424; flox:5794±410 mmHg/s; p<.05, n=6/group). These data are further supported by increased IP3R2 levels detected in HF patients. A markedly reduced percentage of apoptotic cells after MI was revealed by TUNEL assay in cardiac sections from KO mice compared with flox controls, mirrored by an augmented caspase activity and a decreased calcineurin phosphatase activity (p<.05, n=7/group). Furthermore, we observed decreased interstitial cardiac fibrosis in failing hearts from KO mice compared with their flox littermates (p<.05, n=6/group). No obvious difference was noticed examining ultrastructural mitochondria morphology. Conclusions: Taken together our results indicate that myocardial IP3R2 plays a crucial role in the pathophysiology of HF, participating via a mechanism involving the calcineurin pathway in the regulation of fibrosis and apoptosis during post-ischemic cardiac remodeling.

Prdm16 regulates mitochondrial dynamics and hematopoietic stem cell function

Hematopoiesis is sustained over an entire lifetime through continuous maintenance and self-renewal of hematopoietic stem cells (HSCs). However, a coherent picture of how these processes are achieved by the HSC to regulate homeostatic responses of the hematopoietic system in vivo has not yet emerged. Moreover, the molecular mechanisms underlying the recently identified clonal heterogeneity among HSCs in lineage potential and self-renewal capacity are unknown. The transcription factor Prdm16 is required for HSC maintenance and self-renewal. We show that regulation of mitochondrial dynamics is a target of the Prdm16 transcriptional program. Our data show that Prdm16 maintains mitochondrial hyperfusion in HSC through transcriptional activation of mitofusin 2 (Mfn2). HSCs retrovirally transduced with Mfn2 showed enhanced function in competitive and serial repopulation assays compared to control vector. Furthermore, our studies reveal that Mfn2 and mitochondrial hyperfusion facilitates increased steady-state intracellular [Ca2+] buffering in HSCs, leading to decreased baseline [Cai2+] and consequently, lower calcineurin phosphatase activity thereby inhibiting nuclear translocation of the transcription factor, NFAT. Pharmacological inhibition of NFAT in vitro maintains lymphoid-biased HSCs and induces differentiation of myeloid-biased HSCs while activation of NFAT had the opposite effect. This is the first identification of a molecular mechanism regulating the fate of lineage-biased HSCs. Insights from these findings will broadly impact our understanding of HSC biology and advance the field towards improving targeted therapies to treat diseases of HSC origin.

A calcineurin- and NFAT-dependent pathway is involved in α-synuclein-induced degeneration of midbrain dopaminergic neurons.

Parkinson's disease (PD), the most common degenerative movement disorder, is caused by a preferential loss of midbrain dopaminergic (mDA) neurons. Both α-synuclein (α-syn) missense and multiplication mutations have been linked to PD. However, the underlying intracellular signalling transduction pathways of α-syn-mediated mDA neurodegeneration remain elusive. Here, we show that transgenic expression of PD-related human α-syn A53T missense mutation promoted calcineurin (CN) activity and the subsequent nuclear translocation of nuclear factor of activated T cells (NFATs) in mDA neurons. α-syn enhanced the phosphatase activity of CN in both cell-free assays and cell lines transfected with either human wild-type or A53T α-syn. Furthermore, overexpression of α-syn A53T mutation significantly increased the CN-dependent nuclear import of NFATc3 in the mDA neurons of transgenic mice. More importantly, a pharmacological inhibition of CN by cyclosporine A (CsA) ameliorated the α-syn-induced loss of mDA neurons. These findings demonstrate an active involvement of CN- and NFAT-mediated signalling pathway in α-syn-mediated degeneration of mDA neurons in PD.

Tau phosphorylation and tau mislocalization mediate soluble Aβ oligomer‐induced AMPA glutamate receptor signaling deficits

In our previous studies, phosphorylation-dependent tau mislocalization to dendritic spines resulted in early cognitive and synaptic deficits. It is well known that amyloid beta (Aβ) oligomers cause synaptic dysfunction by inducing calcineurin-dependent AMPA receptor (AMPAR) internalization. However, it is unknown whether Aβ-induced synaptic deficits depend upon tau phosphorylation. It is also unknown whether changes in tau can cause calcineurin-dependent loss of AMPARs in synapses. Here, we show that tau mislocalizes to dendritic spines in cultured hippocampal neurons from APPSwe Alzheimer's disease (AD)-transgenic mice and in cultured rat hippocampal neurons treated with soluble Aβ oligomers. Interestingly, Aβ treatment also impairs synaptic function by decreasing the amplitude of miniature excitatory postsynaptic currents (mEPSCs). The above tau mislocalization and Aβ-induced synaptic impairment are both diminished by the expression of AP tau, indicating that these events require tau phosphorylation. The phosphatase activity of calcineurin is important for AMPAR internalization via dephosphorylation of GluA1 residue S845. The effects of Aβ oligomers on mEPSCs are blocked by the calcineurin inhibitor FK506. Aβ-induced loss of AMPARs is diminished in neurons from knock-in mice expressing S845A mutant GluA1 AMPA glutamate receptor subunits. This finding suggests that changes in phosphorylation state at S845 are involved in this pathogenic cascade. Furthermore, FK506 rescues deficits in surface AMPAR clustering on dendritic spines in neurons cultured from transgenic mice expressing P301L tau proteins. Together, our results support the role of tau and calcineurin as two intermediate signaling molecules between Aβ initiation and eventual synaptic dysfunction early in AD pathogenesis.

Calcineurin signaling regulates neural induction through antagonizing the BMP pathway.

Development of the nervous system begins with neural induction, which is controlled by complex signaling networks functioning in concert with one another. Fine-tuning of the bone morphogenetic protein (BMP) pathway is essential for neural induction in the developing embryo. However, the molecular mechanisms by which cells integrate the signaling pathways that contribute to neural induction have remained unclear. We find that neural induction is dependent on the Ca(2+)-activated phosphatase calcineurin (CaN). Fibroblast growth factor (FGF)-regulated Ca(2+) entry activates CaN, which directly and specifically dephosphorylates BMP-regulated Smad1/5 proteins. Genetic and biochemical analyses revealed that CaN adjusts the strength and transcriptional output of BMP signaling and that a reduction of CaN activity leads to an increase of Smad1/5-regulated transcription. As a result, FGF-activated CaN signaling opposes BMP signaling during gastrulation, thereby promoting neural induction and the development of anterior structures.

Ca2+‐related signaling events influence TLR9‐induced IL‐10 secretion in human B cells

Suppressory B-cell function controls immune responses and is mainly dependent on IL-10 secretion. Pharmacological manipulation of B-cell-specific IL-10 synthesis could, thus, be therapeutically useful in B-cell chronic lymphocytic leukemia, transplantation, autoimmunity and sepsis. TLR are thought to play a protagonistic role in the formation of IL-10-secreting B cells. The aim of the study was to identify the molecular events selectively driving IL-10 production in TLR9-stimulated human B cells. Our data highlight the selectivity of calcineurin inhibitors in blocking TLR9-induced B-cell-derived IL-10 transcription and secretion, while IL-6 transcription and release, B-cell proliferation, and differentiation remain unaffected. Nevertheless, TLR9-induced IL-10 production was found to be independent of calcineurin phosphatase activity and was even negatively regulated by NFAT. In contrast to TLR9-induced IL-6, IL-10 secretion was highly sensitive to targeting of spleen tyrosine kinase (syk) and Bruton's tyrosine kinase. Further analyses demonstrated increased phosphorylation of Ca(2+) /calmodulin kinase II (CaMKII) in TLR9-stimulated B cells and selective reduction of TLR9-induced secretion of IL-10 upon treatment with CaMKII inhibitors, with negligible impact on IL-6 levels. Altogether, our results identify calcineurin antagonists as selective inhibitors of IL-10 transcription and syk/Bruton´s tyrosine kinase-induced Ca(2+) /calmodulin- and CaMKII-dependent signaling as a pathway regulating the release of TLR9-induced B-cell-derived IL-10.

Calcineurin Activity Assay Measurement by Liquid Chromatography–Tandem Mass Spectrometry in the Multiple Reaction Monitoring Mode

Blood concentrations of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus are currently measured to monitor immunosuppression in transplant patients. The measurement of calcineurin (CN) phosphatase activity has been proposed as a complementary pharmacodynamic approach. However, determining CN activity with current methods is not practical. We developed a new method amenable to routine use. Using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS), we quantified CN activity by measuring the dephosphorylation of a synthetic phosphopeptide substrate. A stable isotope analog of the product peptide served as internal standard, and a novel inhibitor cocktail minimized dephosphorylation by other major serine/threonine phosphatases. The assay was used to determine CN activity in peripheral blood mononuclear cells (PBMCs) isolated from 20 CNI-treated kidney transplant patients and 9 healthy volunteers. Linearity was observed from 0.16 to 2.5 μmol/L of product peptide, with accuracy in the 15% tolerance range. Intraassay and interassay recoveries were 100.6 (9.6) and 100 (7.5), respectively. Michaelis-Menten kinetics for purified CN were Km = 10.7 (1.6) μmol/L, Vmax = 2.8 (0.3) μmol/min · mg, and for Jurkat lysate, Km = 182.2 (118.0) μmol/L, Vmax = 0.013 (0.006) μmol/min · mg. PBMC CN activity was successfully measured in a single tube with an inhibitor cocktail. Because LC-MRM-MS is commonly used in routine clinical dosage of drugs, this CN activity assay could be applied, with parallel blood drug concentration monitoring, to a large panel of patients to reevaluate the validity of PBMC CN activity monitoring.

FK506 reduces calpain-regulated calcineurin activity in both the cytoplasm and the nucleus.

Excessive immune responses induced by ischemia-reperfusion injury (IRI) are known to lead to necrotic and apoptotic cell death, and calcineurin plays a major role in this process. Calcineurin dephosphorylates the nuclear factor of activated T-cells (NFAT), permitting its translocation into the nucleus. As a result, calcineurin promotes the release of pro-inflammatory cytokines, such as tumor necrosis factor-α. The overproduction of pro-inflammatory cytokines causes renal cell death. Calcineurin activity is regulated by calpain, a cysteine protease present in the nucleus. Calpain-mediated proteolysis increases the phosphatase activity of calcineurin, resulting in NFAT dephosphorylation. This process has been studied in cardiomyocytes but its role in renal IRI is unknown. Thus, we examined whether calpain regulates calcineurin in renal tubule nuclei. We established an in vivo renal IRI model in mice and identified the protective role of a calcineurin inhibitor, FK506, in this process. Calcineurin is expressed in the nucleus, where it is present in its calpain-cleaved form. FK506 reduced nuclear expression of calcineurin and prevented calcineurin-mediated NFAT activation. Our study shows clearly that FK506 reduces calpain-mediated calcineurin activity. Consequently, calcineurin could not maintain NFAT activation. FK506 reduced renal cell death by suppressing the transcription of pro-inflammatory cytokine genes. This study provides evidence that FK506 protects against inflammation in a renal IRI mouse model. We also provided a mechanism of calcineurin action in the nucleus. Therefore, FK506 could improve renal function by decreasing calcineurin activity in both the cytoplasm and the nucleus of renal tubule cells.

Combination Therapy In Mast Cell Neoplasms: Co-Targeting KIT and NFAT Signaling Pathways

Activating KIT mutations are a hallmark of human systemic mast cell disease and have also been identified in gastrointestinal stromal tumors, AML, melanoma, and seminoma. Traditionally, constitutively active KIT mutations have been targeted by tyrosine kinase inhibitors; however, currently available inhibitors have limited activity against resistant mutations and sustainable responses are often not achieved. Our goal has been to identify a synergistic drug combination that increases the effectiveness of KIT inhibitors. A recent study in chronic myelogenous leukemia reported enhanced anti-proliferative/pro-apoptotic effects when combining a BCR-ABL inhibitor with inhibitors targeting a novel Wnt/Ca++/Calcineurin/NFAT pathway. Based on shared downstream signaling mechanisms between BCR-ABL and KIT kinases we hypothesized that a similar combination would work to target KIT mutant kinases.We found that combining a KIT inhibitor with a calcineurin phosphatase inhibitor led to a synergistic decrease (average combination index (CI) values less than 1.0) in cell viability as well as an increase in apoptosis in KIT-mutant cell lines including six mastocytosis cell lines. For example, we found that 1uM of cyclosporine A (CSA) plus 20nM of dasatinib in the p815 cell line consistently reduced cell viability by 50% compared to dasatinib alone. This decrease in cell viability corresponded to a 40 fold increase in capsase 3/7 activity (Figure 1). Similar results were obtained using the other five mastocytosis cell lines. [Display omitted] The activity of NFAT transcription factors is known to be regulated by the phosphatase activity of calcineurin. To confirm our results were calcineurin- and NFAT-dependent, we repeated the previous combination experiments with the NFAT specific inhibitors, rocaglamide and tributylhexadecylphosphonium bromide (THPB). We observed a synergistic decrease (average CI value 0.81 for rocaglamide and 0.43 for THPB) in cell viability and increase in apoptosis when combining these NFAT-specific and KIT kinase inhibitors, indicating NFAT as an important mediator of the observed synergy. Next we characterized NFAT in KIT mutant mast cells and found constitutive NFAT activation in each cell line, as indicated by nuclear localization and dephosphorylation under unstimulated conditions. NFAT-dependent transcriptional activity, as measured by a luciferase reporter system, decreased following treatment with either calcineurin or NFAT inhibitors. Unexpectedly, NFAT-dependent transcriptional activity also decreased following treatment with a number of KIT inhibitors. However, only calcineurin inhibitors and not KIT inhibitors, completely blocked NFAT activation following calcium influx, suggesting a unique mechanism by which KIT inhibition affects NFAT-dependent transcriptional activity. To determine downstream effectors of the observed synergy, we measured changes in RNA expression following mono- and combination therapy. RNA-Seq revealed synergistic decreases in a number of genes including members of the JAK-STAT and MAPK pathways, which are druggable targets downstream of this transcriptional program. Further validation studies of these targets are currently underway and will be reported later.The constitutive activation of NFAT has not been previously reported in KIT mutant cell lines. Further, our data suggest a point of convergence between KIT and NFAT signaling pathways, possibly through the interaction of NFAT with a DNA binding partner. Importantly, our results indicate that combination therapy using an NFAT inhibitor and a KIT kinase inhibitor results in synergistic killing of KIT mutant mast cells. However, existing calcineurin phosphatase inhibitors such as CSA have known chronic long-term toxicities, including immunosuppression. To identify alternative drug targets we used RNA-Seq and found that simultaneous calcineurin and KIT inhibition modulated members of the JAK-STAT and MAPK pathways. Preliminary data indicates that targeting these pathways in combination with dasatinib achieves the same synergistic effect on KIT mutant cancer cells. We are currently testing the potential of these alternative combination therapies for the treatment of advanced mast cell disease. Disclosures:Heinrich: Onyx: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; MolecularMD: Consultancy, Equity Ownership; AROG: Research Funding.

Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII

The fertilising sperm triggers a transient Ca2+ increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/Ccdc20 inhibitor, links release from metaphase II arrest with the Ca2+ transient and its degradation is triggered by the Ca2+-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca2+ transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca2+-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca2+ increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates.

Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII

The fertilising sperm triggers a transient Ca(2+) increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/C(cdc20) inhibitor, links release from metaphase II arrest with the Ca(2+) transient and its degradation is triggered by the Ca(2+)-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca(2+) transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca(2+)-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca(2+) increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates.

Tropisetron attenuates amyloid‐beta‐induced inflammatory and apoptotic responses in rats

Alzheimer's disease (AD) is a neurodegenerative disorder featured by deposition of beta-amyloid (Aβ) plaques in the hippocampus and associated cortices and progressive cognitive decline. Tropisetron, a selective 5-HT3 receptor antagonist, is conventionally used to counteract chemotherapy-induced emesis. Recent investigations describe antiphlogistic properties for tropisetron. It has been shown that tropisetron protects against rat embolic stroke. We investigated protective properties of tropisetron in a beta-amyloid (Aβ) rat model of AD and possible involvement of 5-HT3 receptors. Aβ (1-42) was injected into the hippocampus of male rats. Animals were treated intracerebroventricularly with tropisetron, mCPBG (selective 5-HT3 receptor agonist) or mCPBG plus tropisetron on days 1, 3, 5 and 7. Seven days following Aβ administration, inflammatory markers (TNF-α, COX-2, iNOS and NF-κB), apoptotic markers (caspase 3 cytochrome c release) and calcineurin phosphatase activity were assessed in hippocampus. Seven days following Aβ inoculation, control animals displayed dramatic increase in TNF-α, COX-2, iNOS, NF-κB, active caspase 3, cytochrome c release and calcineurin phosphatase activity in the hippocampus. Tropisetron significantly diminished the elevated levels of these markers and reversed the cognitive deficit. Interestingly, tropisetron was also found to be a potent inhibitor of calcineurin phosphatase activity. The selective 5-HT3 receptor agonist mCPBG, when co-administered with tropisetron, completely reversed the procognitive and anti-apoptotic properties of tropisetron while it could only partially counteract the anti-inflammatory effects. mCPBG alone significantly aggravated Aβ-induced injury. Our findings indicate that tropisetron protects against Aβ-induced neurotoxicity in vivo through both 5-HT3 receptor-dependent and independent pathways.

Abstract 054: Deficiency Of Nuclear Receptor Interaction Protein Causes Cardiac Hypertrophy

Previously, we demonstrate a novel gene, nuclear receptor interaction protein (NRIP, also named as DCAF6 or IQWD1). We also identify NRIP as a Ca2+- dependent calmodulin binding protein that activates calcineurin phosphatase activity. To investigate insights into in vivo function of NRIP, we generated NRIP-null mice and found that loss of NRIP impairs cardiac function and lead to cardiac hypertrophy progressively. Furthermore, NRIP-/- mice display weaker muscle strength, reduced cardiac function, and cardiac fibrosis compared with WT. To verify the regulation mechanism, we found that α-actinin-2 (ACTN2), which is a biomarker of muscular Z-disc complex, is one of NRIP-interacting proteins from the yeast two-hybrid system. ACTN2 cross-links with actin filament to stabilize sarcomeric structure and muscle contraction, which is an essential constituent of sarcomere. Through the in vitro and in vivo binding assays, we further confirm the interaction and define the interacting domains between NRIP and ACTN2. Plus co-localization of NRIP and ACTN2 is discovered in cardiac tissue by immunofluorescence assays, we firstly define NRIP as a Z-disc protein. Although the Z-disc has been viewed as a passive constituent of the sarcomere traditionally, increasing numbers of mutations in Z-disc proteins leading to disruption and malfunction of the contractile apparatus have been shown to cause cardiomyopathies and/or muscular dystrophies. Hence, we analyzed the sarcomeric structure of NRIP-/- cardiomyocytes and found reduction of I-band width and extension of Z-disc. Besides, we know that NRIP is a Ca2+- dependent calmodulin binding protein. In cardiomyocytes, calmodulin interacts with multiple calcium ion channels or proteins to directly or indirectly regulate the variation of calcium concentration during muscle contraction. Therefore, we isolated and measured the calcium transient of cardiomyocytes. Then, we found that deficiency of NRIP decreases the amplitude of calcium transient. In a conclusion, we speculated that loss of NRIP impairs the structure of sarcomere, the amplitude of calcium transient during muscle contraction and the function of muscle contraction resulting in cardiomyopathy.

Protein kinase CK2-dependent phosphorylation of the human Regulators of Calcineurin reveals a novel mechanism regulating the calcineurin–NFATc signaling pathway

Cyclosporine A and FK506 produce immunosuppression by blocking calcineurin phosphatase activity and consequently activation of cytosolic Nuclear Factor of Activated T-cell (NFATc) transcription factor. Due to the chronic toxicity associated with their administration, the development of more specific immunosuppressants is currently an important unmet medical need. In this context, an immunosuppressant peptide derived from the CIC motif of the human Regulators of Calcineurin (RCAN) proteins has been shown to inhibit NFATc signaling without affecting general phosphatase activity of calcineurin. Here we show that protein kinase CK2 phosphorylates a conserved serine residue within the CIC motif of vertebrate RCANs, which increases its affinity for calcineurin and consequently its inhibition of NFATc-dependent gene expression in activated T-cells. Molecular modeling studies have led us to identify a positively charged interaction site on the surface of calcineurin where the phosphorylated serine residue of the CIC motif would normally locate. Finally, we have also identified RCAN3 as a new phosphoprotein with multiple phosphorylation sites. Therefore, our findings reveal for the first time a novel molecular mechanism underlying the regulation of calcineurin–NFATc signaling by means of phosphorylation of the CIC motif of RCAN proteins. The knowledge of how RCAN proteins modulate the calcineurin–NFATc pathway paves the way for the development of potent novel selective immunosuppressant drugs.

Tacrolimus inhibits NF-κB activation in peripheral human T cells.

The calcineurin inhibitor, tacrolimus (TAC), inhibits the protein phosphatase activity of calcineurin, leading to suppression of the nuclear translocation of NFAT and inhibition of T cell activation. Apart from NFAT also the transcription factor NF-κB plays a key functional role in T cell activation. Therefore, blockade of the NF-κB activation cascade by immunosuppressive drugs prevents immune activation. Here we studied whether TAC blocks NF-κB activation in peripheral human T cells. After anti-CD3/CD28-activation of T cells from healthy volunteers, NF-κB (p65) phosphorylation was measured by flow cytometry in CD3+ T cells, CD4+ helper T cells and CD8+ cytotoxic T cells in the absence and presence of TAC 10 ng/mL, sotrastaurin 500 nM (positive control) and mycophenolic acid 10 µg/mL (negative control; n = 6). NF-κB transcriptional activity was measured by ELISA and intracellular TNFα protein, a downstream target, was measured by flow cytometry to assess the functional consequences of NF-κB blockade. Anti-CD3/28-activation induced NF-κB phosphorylation in CD3+ T cells, CD4+ T cells and CD8+ T cells by 34% (mean), 38% and 30% resp. (p<0.01). Sotrastaurin inhibited NF-κB activation in the respective T cell subsets by 93%, 95% and 86% (p<0.01 vs. no drug), while mycophenolic acid did not affect this activation pathway. Surprisingly, TAC also inhibited NF-κB phosphorylation, by 55% (p<0.01) in CD3+ T cells, by 56% (p<0.01) in CD4+ T cells and by 51% in CD8+ T cells (p<0.01). In addition, TAC suppressed NF-κB DNA binding capacity by 55% (p<0.05) in CD3+ T cells and TNFα protein expression was inhibited in CD3+ T cells, CD4+ T cells and CD8+ T cells by 76%, 71% and 93% resp. (p<0.01 vs. no drug), confirming impaired NF-κB signaling. This study shows the suppressive effect of TAC on NF-κB signaling in peripheral human T cell subsets, measured at three specific positions in the NF-κB activation cascade.

Secreted Trypanosome Cyclophilin Inactivates Lytic Insect Defense Peptides and Induces Parasite Calcineurin Activation and Infectivity

The mechanisms by which Trypanosoma cruzi survives antimicrobial peptides and differentiates during its transit through the gastrointestinal tract of the reduviid vector are unknown. We show that cyclophilin, a peptidyl-prolyl isomerase secreted from T. cruzi epimastigotes, binds to and neutralizes the reduviid antimicrobial peptide trialysin promoting parasite survival. This is dependent on a singular proline residue in trialysin and is inhibited by the cyclophilin inhibitor cyclosporine A. In addition, cyclophilin-trialysin complexes enhance the production of ATP and reductase responses of parasites, which are inhibited by both calcineurin-specific inhibitors cyclosporine A and FK506. Calcineurin phosphatase activity of cyclophilin-trialysin-treated parasites was higher than in controls and was inhibited by preincubation by either inhibitor. Parasites exposed to cyclophilin-trialysin have enhanced binding and invasion of host cells leading to higher infectivity. Leishmanial cyclophilin also mediates trialysin protection and metabolic stimulation by T. cruzi, indicating that extracellular cyclophilin may be critical to adaptation in other insect-borne protozoa. This work demonstrates that cyclophilin serves as molecular sensor leading to the evasion and adaptive metabolic response to insect defense peptides.

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 Ca(2+). Thus, it would be clinically relevant to know the targets of this aberrant Ca(2+) signal. We hypothesized that the Ca(2+)-activated phosphatase calcineurin is such a Ca(2+) 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 Ca(2+). 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 Ca(2+) 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.

AKAP150-Anchored Calcineurin Regulates Synaptic Plasticity by Limiting Synaptic Incorporation of Ca2+-Permeable AMPA Receptors

AMPA receptors (AMPARs) are tetrameric ion channels assembled from GluA1-GluA4 subunits that mediate the majority of fast excitatory synaptic transmission in the brain. In the hippocampus, most synaptic AMPARs are composed of GluA1/2 or GluA2/3 with the GluA2 subunit preventing Ca(2+) influx. However, a small number of Ca(2+)-permeable GluA1 homomeric receptors reside in extrasynaptic locations where they can be rapidly recruited to synapses during synaptic plasticity. Phosphorylation of GluA1 S845 by the cAMP-dependent protein kinase (PKA) primes extrasynaptic receptors for synaptic insertion in response to NMDA receptor Ca(2+) signaling during long-term potentiation (LTP), while phosphatases dephosphorylate S845 and remove synaptic and extrasynaptic GluA1 during long-term depression (LTD). PKA and the Ca(2+)-activated phosphatase calcineurin (CaN) are targeted to GluA1 through binding to A-kinase anchoring protein 150 (AKAP150) in a complex with PSD-95, but we do not understand how the opposing activities of these enzymes are balanced to control plasticity. Here, we generated AKAP150ΔPIX knock-in mice to selectively disrupt CaN anchoring in vivo. We found that AKAP150ΔPIX mice lack LTD but express enhanced LTP at CA1 synapses. Accordingly, basal GluA1 S845 phosphorylation is elevated in AKAP150ΔPIX hippocampus, and LTD-induced dephosphorylation and removal of GluA1, AKAP150, and PSD-95 from synapses are impaired. In addition, basal synaptic activity of GluA2-lacking AMPARs is increased in AKAP150ΔPIX mice and pharmacologic antagonism of these receptors restores normal LTD and inhibits the enhanced LTP. Thus, AKAP150-anchored CaN opposes PKA phosphorylation of GluA1 to restrict synaptic incorporation of Ca(2+)-permeable AMPARs both basally and during LTP and LTD.

The Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Heterodimerizes with and Regulates Calmodulin Function

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca(2+)) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca(2+), DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca(2+). In the absence of Ca(2+), DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca(2+)-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 μM as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional (1)H,(15)N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to (15)N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca(2+)-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca(2+) also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH(2)-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.

Regulation of inflammatory responses and fibroblast‐like synoviocyte apoptosis by calcineurin‐binding protein 1 in mice with collagen‐induced arthritis

Calcineurin-binding protein 1 (CABIN-1) regulates calcineurin phosphatase activity as well as the activation, apoptosis, and inflammatory responses of fibroblast-like synoviocytes (FLS), which actively participate in the chronic inflammatory responses in rheumatoid arthritis (RA). However, the mechanism of action of CABIN-1 in FLS apoptosis is not clear. This study was undertaken to define the regulatory role of CABIN-1 in FLS from mice with collagen-induced arthritis (CIA). Transgenic mice overexpressing human CABIN-1 in joint tissue under the control of a type II collagen promoter were generated. Expression of human CABIN-1 (hCABIN-1) in joints and FLS was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of cytokines, matrix metalloproteinases (MMPs), and apoptosis-related genes in FLS was determined by enzyme-linked immunosorbent assay, gelatin zymography, and RT-PCR, respectively. Joints were stained with hematoxylin and eosin and with tartrate-resistant acid phosphatase for histologic analysis. Human CABIN-1-transgenic mice with CIA had less severe arthritis than wild-type mice with CIA, as assessed according to hind paw thickness and histologic features. The milder arthritis was accompanied by significantly enhanced apoptosis in transgenic mice, evidenced by a significantly greater number of TUNEL-positive cells in synovial tissue. Expression of inflammatory cytokines and MMPs in the transgenic mice with CIA was reduced, and they exhibited decreased Akt activation and increased expression of p53, caspase 3, caspase 9, and Bax. Our findings demonstrate that hCABIN-1 plays a critical role in promoting apoptosis of FLS and in attenuating inflammation and cartilage and bone destruction in RA. These results help elucidate the pathogenic mechanisms of RA and suggest that CABIN-1 is a potential target for treatment of this disease.