The PMC1 gene in S. cerevisiae encodes a vacuolar Ca2+ ATPase required for growth in high-Ca2+ conditions. Previous work has shown that Ca2+ tolerance can be restored to pmc1 mutants by inactivation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase sensitive to the immunosuppressive drug FK506. Calcineurin decreases Ca2+ tolerance of pmc1 mutants by inhibiting the function of VCX1, which encodes a vacuolar H+/Ca2+ exchanger related to vertebrate Na+/Ca2+ exchangers. The contribution of VCX1 in Ca2+ tolerance is low in strains with a functional calcineurin and is high in strains which lack calcineurin activity. In contrast, the contribution of PMC1 to Ca2+ tolerance is augmented by calcineurin activation. Consistent with these positive and negative roles of calcineurin, expression of a vcx1::lacZ reporter is slightly diminished and a pmc1::lacZ reporter is induced up to 500-fold by processes dependent on calcineurin, calmodulin, and Ca2+. It is likely that calcineurin inhibits VCX1 function mainly by posttranslational mechanisms. Activities of VCX1 and PMC1 help to control cytosolic free Ca2+ concentrations because their function can decrease pmc1::lacZ induction by calcineurin. Additional studies with reporter genes and mutants indicate that PMR1 and PMR2A, encoding P-type ion pumps required for Mn2+ and Na+ tolerance, may also be induced physiologically in response to high-Mn2+ and -Na+ conditions through calcineurin-dependent mechanisms. In these situations, inhibition of VCX1 function may be important for the production of Ca2+ signals. It is proposed that elevated cytosolic free Ca2+ concentrations, calmodulin, and calcineurin regulate at least four ion transporters in S. cerevisiae in response to several environmental conditions (Cunningham, 1996).
S. cerevisiae mutants that exhibit phenotypes (calcium resistance and vanadate sensitivity) similar to those of calcineurin-deficient mutants were classified into four complementation groups (crv1,2,3 and 4). Crv1 is allelic to cnb1, a mutation in the regulatory subunit of calcineurin. The nucleotide sequences of mutant crv2 are identical to those of BCK1/SLK1/SKC1/SSP31 and the sequences of mutant crv3 match those of MPK1/SLT2; both genes are involved in the MAP kinase cascade. A calcineurin-deletion mutation (delta cnb1), which by itself has no detectable effect on growth and morphology, enhances some phenotypes (slow growth and morphological abnormality) of crv2 and crv3 mutants. The phenotypes of crv2 and crv3 mutants are partially suppressed by Ca2+ or by overproduction of the calcineurin subunits (Cmp2 and Cnb1). Like the calcineurin-deficient mutant, crv2 and crv3 mutants are defective in recovery from alpha-factor-induced growth arrest. The defect in recovery of the delta cnb1 mutant is suppressed by overexpression of MPK1. These results indicate that the calcineurin-mediated and the Mpk1- (Bck1-) mediated signaling pathways act in parallel to regulate functionally redundant cellular events important for growth (Nakamura, 1996).
The catalytic subunit of Ca2+/calmodulin(CaM)-dependent protein phosphatase (calcineurin A, protein phosphatase 2B) was isolated from Dictyostelium discoideum. A complete cDNA of 2146 bp predicts a protein of 623 amino acids with homology to calcineurin A from other organisms and a similar molecular architecture. However, the Dictyostelium protein contains N-terminal and C-terminal extra domains causing a significantly higher molecular mass than found in any of its known counterparts. Recombinant Dictyostelium calcineurin A was purified from Escherichia coli cells and shows similar enzymatic properties as does the enzyme from other sources. A band of approximately 80 kDa migrates on gels and possesses an endogenous CaM-binding activity. Both the mRNA for calcineurin A and the protein are expressed during the growth phase. During early development the abundance of the protein is reduced and then increases to peak after 10 h of starvation, when tight aggregates have formed (Dammann, 1996).
Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase required for Saccharomyces cerevisiae to respond to a variety of environmental stresses. Calcineurin promotes cell survival during stress by dephosphorylating and activating the Zn-finger transcription factor Crz1p/Tcn1p. Using a high-throughput assay, 119 yeast kinases were screened for their ability to phosphorylate Crz1p in vitro; the casein kinase I homolog Hrr25p was identifed. Hrr25p negatively regulates Crz1p activity and nuclear localization in vivo. Hrr25p binds to and phosphorylates Crz1p in vitro and in vivo. Overexpression of Hrr25p decreases Crz1p-dependent transcription and antagonizes its Ca2+-induced nuclear accumulation. In the absence of Hrr25p, activation of Crz1p by Ca2+/calcineurin is potentiated. These findings represent the first identification of a negative regulator for Crz1p, and establish a novel physiological role for Hrr25p in antagonizing calcineurin signaling (Kafadar, 2003).
Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised patients. Its growth is sensitive to the immunosuppressants FK506 and cyclosporin, which inhibit the Ca2+- calmodulin-activated protein phosphatase calcineurin. Calcineurin is required for growth at 37°C and the virulence of C. neoformans. Calcineurin is also required for mating. FK506 blocks mating of C. neoformans via FKBP12-dependent inhibition of calcineurin, and mutants lacking calcineurin are bilaterally sterile. Calcineurin is not essential for the initial fusion event, but is required for hyphal elongation and survival of the heterokaryon produced by cell fusion. It is also required for hyphal elongation in diploid strains and during asexual haploid fruiting of MATalpha cells in response to nitrogen limitation. Because mating and haploid fruiting produce infectious basidiospores, these studies suggest a second link between calcineurin and the virulence of C. neoformans. Calcineurin regulates filamentation and 37°C growth via distinct pathways. Together with studies revealing that calcineurin mediates neurite extension and neutrophil migration in mammals, these findings indicate that calcineurin plays a conserved role in the control of cell morphology (Cruz, 2001).
A conserved family of calcineurin-regulating proteins whose members have been implicated in several disease models such as Down's syndrome, Alzheimer's disease, and cardiac hypertrophy has been identified in several organisms including yeast, mice, and humans. Caenorhabditis elegans rcn-1 belongs to this family of calcineurin regulators, and shows approximately 40% identity with the human homologue DSCR-1. rcn-1 is expressed in hypodermal cells, nerve cords and various neurons, vulva epithelial and muscle cells, marginal cells of the pharynx, and structures of the male tail. rcn-1 expression is upregulated by calcineurin activity. RCN-1 binds to calcineurin A from C.elegans lysate in a calcium-dependent manner, and inhibits bovine calcineurin phosphatase activity dose-dependently. In addition, overexpression of RCN-1 results in calcineurin-deficient phenotypes such as small body size, cuticle defects, fertility defects, slow growth, and serotonin-resistant egg-laying defects. Moreover, phenotypes observed in gain-of-function calcineurin mutant animals were restored to normal by RCN-1 overexpression. These results demonstrate an effective and specific inhibition of calcineurin in vitro as well as in vivo by RCN-1 (Lee, 2003).
Mutations in calcineurin B2 gene cause the collapse of indirect flight muscles during mid stages of pupal development. Examination of cell fate-specific markers indicates that unlike mutations in genes such as vestigial, calcineurin B2 does not cause a shift in cell fate from indirect flight muscle (IFM) to direct flight muscle (DFM). Genetic and molecular analyses indicate a severe reduction of myosin heavy chain gene expression in calcineurin B2 mutants, which accounts at least in part for the muscle collapse. Myofibrils in calcineurin B2 mutants display a variety of phenotypes, ranging from normal to a lack of sarcomeric structure. Calcineurin B2 also plays a role in the transition to an adult-specific isoform of troponin I during the late pupal stages, although the incompleteness of this transition in calcineurin B2 mutants does not contribute to the phenotype of muscle collapse. Together, these findings suggest a molecular basis for the indirect flight muscle hypercontractility phenotype observed in flies mutant for Drosophila calcineurin B2 (Gajewski, 2005).
This report further characterizes the IFM collapse phenotype of the canB2[EP(2)0774] mutation. Studies of mutations in other loci that produce IFM collapse revealed two major causes for the phenotype: change of cell fate in the adepithelial cells of the 3rd Instar lava, or hypercontraction of the IFM muscle fibers. In mutants that cause a change in cell fate, such as vg[null], a change in muscle cell fate can be clearly demonstrated by the loss of IFM-specific markers, and the ectopic expression of DFM-specific markers. No such changes are observed in canB2 mutant IFM. Unlike vg[null] mutants, the 88Factin-GFP reporter is expressed strongly in canB2 mutants, even after collapse of the IFM. A DFM-specific marker, gD1142.1-lacZ, expressed in a subset of DFM, also showed no alteration of expression pattern in canB2 mutants. The expression of these reporters in the expected places indicates proper fate determination for the precursor cells that form the DFM and IFM (Gajewski, 2005).
Disruption of the myofibrillar structure by mutation of the fli I locus, encoding a member of the gelsolin protein family, involved in the capping, severing, and bundling of actin filaments, partially suppresses the IFM collapse phenotype, pointing to hypercontraction rather than a shift in cell fate as a cause. Addition of two doses of the fli I[3] allele to a canB2 mutant background significantly increases the numbers of uncollapsed DLM. However, the suppression is not complete, and this may be due to the relatively mild effect of the fli I[3] allele. Null alleles of fli I cause lethality in the early embryonic stages. The fli I[3] allele is a less severe mutation, caused by a change of a highly conserved glycine to serine. It is possible that even with the disruptions of the sarcomeric structure, fli I[3] does not completely inhibit IFM contraction (Gajewski, 2005).
A reduction of calcineurin function has a profound effect on the expression of the mhc gene in the IFM. One copy of canB2[EP(2)0774] enhances the severity of IFM defects in flies heterozygous for the antimorphic Mhc[5] allele. mhc transcripts are barely detectable in the IFM of canB2 mutant flies, and many of the mutant myofibrils have greatly reduced or completely absent thick filaments. However, there is no interference with the tissue-specific splicing of the five versions of Mhc exon 11. The reduction of mhc expression is not due to a nonspecific reduction in transcription; the levels of GFP transcript from a reporter driven by mhc upstream sequences (mhc-GFP) are also reduced in a mutant background, but expression of actin88F is unaffected. The simplest explanation is that transcription of mhc is greatly reduced in the absence/reduction of calcineurin function, but further studies will be needed to confirm it (Gajewski, 2005).
The function of calcineurin in transcriptional activation is well documented, for example, its role in regulating transcription factors such as NFAT and Mef2. There are multiple Dmef2 binding sites upstream of the mhc gene, as well as a binding site for the zinc-finger transcription factor CF2. Work in other systems has established that calcineurin can activate Mef2 both directly and indirectly; it is likely that this will also hold true for Drosophila. Whether calcineurin can affect CF2 activity is not yet known, but the phosphorylation state of CF2 has been demonstrated to play a role in its regulation via the EGFR pathway in Drosophila ovaries. Phosphorylated CF2 is found predominantly in the cytoplasm of the anterodorsal follicle cells, where it is fated to be degraded. It is speculated that removal of the phosphate group allows entry in the nucleus CF2 is expressed in all three muscle types of the Drosophila embryo (Bagni, 2002), but it is not yet known if this protein is required for IFM development. It will be of interest to investigate whether CF2 is expressed in the developing IFM, and what effects calcineurin function (or lack thereof) would possibly have on its subcellular localization (Gajewski, 2005).
It is also interesting to note that the lack/reduction of calcineurin has a much more drastic effect on mhc transcript levels in the IFM than it does on the various muscle types of the abdomen. The amount of total mhc transcripts are readily detectable in the mutant abdominal musculature, but not in the mutant IFM under the same PCR conditions. The transcript is not completely missing in the mutant IFM; if extra PCR cycles are done, or extra fly equivalents of cDNA are added for the mutants, a mhc band can be amplified. The reason for greater IFM sensitivity to lack of calcineurin function is unknown, and warrants further investigation. It may be that calcineurin is part of a system to promote maximum expression of mhc. The IFM are the largest muscles in the fly, and their tightly packed hexagonal arrangement of thick and thin filaments (unique in the fly musculature) could require increased expression of myosin and other structural proteins. There are numerous examples of mutations in muscle structural protein genes that result in a flightless phenotype, but do not impair the functions of other types of muscles (Gajewski, 2005).
The myofilament structure of the canB2 mutants reflects the reduction in mhc transcripts. While about 20% of the adult mutant IFM tissue examined resembled wild type, the majority of samples exhibited some degree of defect. Some myofibrils had patches of organized filament structure at the periphery, but have no recognizable structures in focus at the center region. This is likely the result of hypercontraction, which can lead to random myofilament orientation. In the most severely affected myofibrils, no organized structures of any sort could be detected. Examination of longitudinal sections confirmed this range of phenotypes. Some samples resembled the wild type sarcomeric pattern. Mildly affected mutant tissue had broken Z-bands, partial or missing M-lines (indicative of reduced or missing thick filaments), and shorter sarcomeres (indicative of hypercontraction). The most severely affected mutant muscles lacked any Z-bands or M-lines. The mutant pupal samples tended to display the most severe myofibrillar phenotypes. It is likely that using adults for examination selects against the most severe phenotypes; the animals examined in the pupal stage are likely to represent those that would not have successfully eclosed, and a small sample of canB2 mutant pupae could easily display a propensity for the strongest defects. The canB2[EP(2)0774] mutation is semi-lethal; life cycle analysis reveals that many of the animals that die do so in the pupal stages. It may be that the most severe canB2 phenotypes render the animals unable to eclose, although the role, if any, of the IFM is this process is yet to be confirmed. It is also possible that the most severe canB2 phenotypes could impair other muscles (Gajewski, 2005).
The effect of the canB2 mutation on Tn I expression represents a possible novel role for calcineurin, that being in different isoform formation. Although no direct role for calcineurin in the control of RNA splicing has yet been demonstrated, it is interesting to note that phosphorylation status of SR proteins plays a role in their localization within the nucleus, and assembly, disassembly, and activity of the spliceosome may by influenced by a cycle of protein phosphorylation. The degree of phosphorylation is believed to effect protein-protein and protein-RNA interactions in the spliceosomal complexes. The splicing of at least one variant exon of the mouse CD44 gene is coupled to signal transduction via the protein kinase C/ras signaling pathway. Therefore, it is possible that calcineurin helps regulate a system responsible for transition in the pupal IFM from the smaller Tn I isoform to the larger version, by control of the phosphorylation states of one or more proteins in the spliceosome complex that are required for inclusion of the third exon (Gajewski, 2005).
It should be noted that the effects of the canB2 mutation on the relative levels of the two Tn I isoforms in the adult IFM are highly variable. In some PCR experiments, the smaller, exon 3 lacking transcript is predominant, but in others, both forms can be clearly seen. However, the results for the wild type adults are consistent: the larger transcript is clearly present, with little or no smaller form detected, in multiple repeats of the experiment. Thus, it must be considered that the differential formation of the Tn I isoforms may not be a direct result of altered calcineurin control of splicing in the IFM, but an indirect consequence of the physiological status of mutant versus normal muscle. That is, the switch to the larger exon 3 containing isoform may normally occur in a wild type genetic background due to some signal (or muscle state) perceived and transmitted within IFM that is of a proper developmental age and competency. In canB2 mutants, an abnormal cellular environment may exist in some or all IFM that prevents the normal sensing of this signal and subsequent isoform switch. Thus, the variability observed in the relative ratio of the two Tn I mRNA forms may simply reflect a nonequivalent status of collapsed muscles as to their competency to sense and execute this developmental molecular switch (Gajewski, 2005).
Taken together, these results have provided mechanistic insights into the cause of IFM collapse in canB2 mutants. Cell fate changes can be ruled out, as can problems with mhc isoform production. In canB2 mutants, the transition to the adult Tn I splice variant is incomplete at best, but this change occurs after the time when the muscles collapse, so an altered stoichiometry of troponin isoforms cannot contribute to this phenotype. Reduction of calcineurin function in the IFM leads to lower levels of mhc transcripts and a variable reduction in the numbers of thick filaments. This reduction in mhc expression is likely a major contributing factor in the collapse of the canB2 mutant IFM. Heterozygotes of Mhc[1], which is a null allele, have reduced numbers of thick filaments and partial hypercontraction of the IFM. However, there is a striking difference in the collapse phenotypes of canB2 and various mhc mutations. In canB2 mutants, without fail, the collapse of the IFMs is directed towards the posterior of the thorax. In a number of different mhc mutant alleles, the IFM can bunch to either. The most severe myofibrillar phenotypes also suggest problems with more than just mhc. The strongest canB2 phenotypes had no Z-bands or any semblance of sarcomeric structure, an effect seen in some mutations that cause defects in the thin filaments. In animals homozygous for the Tn I allele heldup[3] (hdp[3]), which is functionally a null in the IFM, pupal myofibrils showed diffuse Z-bands at 42 h APF, and no sarcomeric structures by 46-48 h APF. Since no Z-bands in the most severely affected canB2 mutant pupae, it is possible that Z-bands could form and break down in a manner similar to hdp[3] mutants. Therefore, it is quite likely that expression and/or processing of other muscle structural proteins are regulated by calcineurin activity, and these warrant future investigation (Gajewski, 2005).
The protein phosphatase activity of calcineurin (CaN) is activated through calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B subunit is required for either binding to the CaN A subunit or for transducing the effects of B subunit Ca2+ binding to the stimulation of the CaN A subunit phosphatase activity. Interaction of CaN B regulatory subunit with the CaN A catalytic subunit requires hydrophobic residues within the CaN A sequence 328-390. Selected hydrophobic residues within the B subunit were mutated to Glu to Gln. CaN B subunit mutants BE-1 (Val115/Leu116 to Glu), BE-2 (Val156/157/168/169 to Glu), and BQ-2 (Val156/157/168/169 to Gln) were expressed and purified. The three mutant B subunits bind 45Ca2+ normally. Mutants BE-2 and BQ-2 interact with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390); they stimulate the phosphatase activity of the CaN A subunit. Mutant BE-1 has a 3-fold reduced affinity for binding CaN A, and this mutant, even at saturating concentrations, gives very little stimulation of CaN A phosphatase activity. It is concluded that residues Val115/Leu116 in the B subunit participate in high-affinity binding to the A subunit and are required for transducing the effects of B subunit Ca2+ binding in stimulation of CaN A phosphatase activity (Watanabe, 1996).
Using a genetic screen in yeast, a new family of proteins conserved in fungi and animals has been identified that inhibits calcineurin function when overexpressed. Overexpression of the yeast protein Rcn1p or the human homologs DSCR1 or ZAKI-4 inhibits two independent functions of calcineurin in yeast -- the activation of the transcription factor Tcn1p and the inhibition of the H+/Ca2+ exchanger Vcx1p. Purified recombinant Rcn1p and DSCR1 binds calcineurin in vitro and inhibits its protein phosphatase activity. Signaling via calmodulin, calcineurin, and Tcn1p induces Rcn1p expression, suggesting that Rcn1p operates as an endogenous feedback inhibitor of calcineurin. Surprisingly, rcn1 null mutants exhibit phenotypes similar to those of Rcn1p-overexpressing cells. This effect may be due to lower expression of calcineurin in rcn1 mutants during signaling conditions. Thus, Rcn1p levels may fine-tune calcineurin signaling in yeast. The structural and functional conservation between Rcn1p and DSCR1 suggests that the mammalian Rcn1p-related proteins, termed calcipressins, will modulate calcineurin signaling in humans and potentially contribute to disorders such as Down Syndrome (Kingsbury, 2000).
Calcineurin plays a pivotal role in the T cell receptor (TCR)-mediated signal transduction pathway and serves as a common target for the immunosuppressants FK506 and cyclosporin A. A novel endogenous calcineurin binding protein named Cabin 1 has been identified that inhibits calcineurin-mediated signal transduction. The interaction between Cabin 1 and calcineurin is dependent on PKC activation. Overexpression of Cabin 1 or its N-terminal truncation mutants inhibits the transcriptional activation of calcineurin-responsive elements in the interleukin-2 promoter and blocks dephosphorylation of NF-AT upon T cell activation. These results suggest a negative regulatory role for Cabin 1 in calcineurin signaling and provide a possible mechanism of feedback inhibition of TCR signaling through cross-talk between protein kinases and calcineurin (Sun, 1998).
Calcipressin 1 is an endogenous inhibitor of calcineurin, which is a serine/threonine phosphatase under the control of Ca(2+) and calmodulin. Calcipressin 1 is encoded by DSCR1, a gene on human chromosome 21 with seven exons, exons 1-4 are alternative first exons (isoforms 1-4). Calcipressin 1 isoform 1 has an N-terminal coding region longer than that previously described, and this generates a new polypeptide of 252 amino acids. This polypeptide is able to interact with calcineurin A and to inhibit NF-AT-mediated transcriptional activation. Endogenous calcipressin 1 exists as a complex together with the calcineurin A and B heterodimer. Calcipressin 1 is a phosphoprotein that increases its capacity to inhibit calcineurin when phosphorylated at the FLISPP motif, and this phosphorylation also controls the half-life of calcipressin 1 by accelerating its degradation. Additionally, further phosphorylation sites have been detected outside the FLISPP motif and these contribute to the complex phosphorylation pattern of calcipressin 1. Taking these results into consideration it is suggested that phosphorylation of calcipressin 1 is involved in the regulation of the phosphatase activity of calcineurin and can therefore act as a modulator of calcineurin-dependent cellular pathways (Genesca, 2003).
Down's syndrome is one of the major causes of mental retardation and congenital heart malformations. Other common clinical features of Down's syndrome include gastrointestinal anomalies, immune system defects and Alzheimer's disease pathological and neurochemical changes. The most likely consequence of the presence of three copies of chromosome 21 is the overexpression of its resident genes, a fact which must underlie the pathogenesis of the abnormalities that occur in Down's syndrome. DSCR1, the product of a chromosome 21 gene highly expressed in brain, heart and skeletal muscle, is overexpressed in the brain of Down's syndrome fetuses, and interacts physically and functionally with calcineurin A, the catalytic subunit of the Ca(2+)/calmodulin-dependent protein phosphatase PP2B. The DSCR1 binding region in calcineurin A is located in the linker region between the calcineurin A catalytic domain and the calcineurin B binding domain, outside of other functional domains previously defined in calcineurin A. DSCR1 belongs to a family of evolutionarily conserved proteins with three members in humans: DSCR1, ZAKI-4 and DSCR1L2. Overexpression of DSCR1 and ZAKI-4 inhibits calcineurin-dependent gene transcription through the inhibition of NF-AT translocation to the nucleus. Together, these results suggest that members of this newly described family of human proteins are endogenous regulators of calcineurin-mediated signaling pathways and as such, they may be involved in many physiological processes (Fuentes, 2000).
Calcineurin phosphatase activity regulates the nuclear localization of the nuclear factor of activated T cells (NFAT) family of transcription factors during immune challenge. Calcineurin inhibitors, such as the cyclosporin A-cyclophilin A and FK506-FKBP12 complexes, regulate this enzymatic activity noncompetitively by binding at a site distinct from the enzyme active site. A family of endogenous protein inhibitors of calcineurin was recently identified and shown to block calcineurin-mediated NFAT nuclear localization and transcriptional activation. One such inhibitor, Down's Syndrome Critical Region 1 (DSCR1), functions in T cell activation, cardiac hypertrophy, and angiogenesis. A small region of DSCR1, the C-terminal 57 residues encoded by exon 7, has been identified is a potent inhibitor of calcineurin activity in vitro and in vivo (Chan, 2005).
Inhibition of the calcineurin-NFAT signalling pathway is one of the main challenges for immunosuppression therapy to avoid the severe side effects of the current anticalcineurinic drugs, cyclosporin A and FK506. The members of the calcipressin family are endogenous inhibitors of calcineurin. Two independent motifs within human calcipressin 1, the ELHA and the PxIxxT motifs, interact with calcineurin in an independent functional manner. The main finding here is that the ELHA-containing calcineurin-inhibitor CALP1 (CIC) motif is the responsible for the in vivo inhibition of calcineurin-mediated NFAT-dependent cytokine gene expression in human T cells. The identification of the CIC motif could be used as a starting point for the development of new immunosuppressive drugs for use in transplantation and autoimmune diseases (Aubereda, 2006).
A new facet of calcium signaling involves the nuclear import of the NF-AT transcription factors from their dormant position in the cytoplasm. The protein phosphatase calcineurin appears to play an essential role in activating NF-AT nuclear import, as the calcineurin inhibitors cyclosporin A and FK506 block dephosphorylation and nuclear import of NF-AT. Calcium signaling induces an association between NF-AT4 and calcineurin; these molecules are transported, as a complex, to the nucleus, where calcineurin continues to dephosphorylate NF-AT4. It is proposed that a nuclear complex of NF-AT4 and calcineurin maintains calcium signaling by counteracting a vigorous nuclear NF-AT kinase (Shibasaki, 1996).
Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. Two new isoforms of the transcription factor NFAT1 are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive agent cyclosporin A (CsA). Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol ester; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response (Luo, 1996).
It is thought that inositol-1,4,5-trisphosphate-Ca2+ signaling has a function in dorsoventral axis formation in Xenopus embryos; however, the immediate target of free Ca2+ is unclear. The secreted Wnt protein family comprises two functional groups, the canonical Wnt and Wnt/Ca2+ pathways. The Wnt/Ca2+ pathway interferes with the canonical Wnt pathway, but the underlying molecular mechanism is poorly understood. The complementary DNA coding for the Xenopus homolog of nuclear factor of activated T cells (XNF-AT) was cloned. A gain-of-function, calcineurin-independent active XNF-AT mutation (CA XNF-AT) inhibits anterior development of the primary axis, as well as Xwnt-8-induced ectopic dorsal axis development in embryos. A loss-of-function, dominant negative XNF-AT mutation (DN XNF-AT) induces ectopic dorsal axis formation and expression of the canonical Wnt signaling target molecules siamois and Xnr3. Xwnt-5A induces translocation of XNF-AT from the cytosol to the nucleus. These data indicate that XNF-AT functions as a downstream target of the Wnt/Ca2+ and Ins(1,4,5)P3-Ca2+ pathways, and has an essential role in mediating ventral signals in the Xenopus embryo through suppression of the canonical Wnt pathway (Saneyoshi, 2002).
Intracellular calcium is one of the important signals that initiates the myogenic program. The calcium-activated phosphatase calcineurin is necessary for the nuclear import of the nuclear factor of activated T cell (NFAT) family members, which interact with zinc finger GATA transcription factors. Whereas GATA-6 plays a role in the maintenance of the differentiated phenotype in vascular smooth muscle cells (VSMCs), it is unknown whether the calcineurin pathway is associated with GATA-6 and plays a role in the differentiation of VSMCs. The smooth muscle-myosin heavy chain (Sm-MHC) gene is a downstream target of GATA-6, and provides a highly specific marker for differentiated VSMCs. Using immunoprecipitation Western blotting, it has been shown that NFATc1 interacts with GATA-6. Consistent with this, NFATc1 further potentiates GATA-6-activated Sm-MHC transcription. Induction of VSMCs to the quiescent phenotype causes nuclear translocation of NFATc1. In differentiated VSMCs, blockage of calcineurin down-regulates the amount of GATA-6-DNA binding as well as the expression of Sm-MHC and its transcriptional activity. These findings demonstrate that the calcineurin pathway is associated with GATA-6 and is required for the maintenance of the differentiated phenotype in VSMCs (Wada, 2002).
The Notch and Calcineurin/NFAT pathways have both been implicated in control of keratinocyte differentiation. Induction of the p21WAF1/Cip1 gene by Notch 1 activation in differentiating keratinocytes is associated with direct targeting of the RBP-Jκ protein to the p21 promoter. Notch 1 activation functions also through a second Calcineurin-dependent mechanism acting on the p21 TATA box-proximal region. Increased Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin (see Drosophila Sarah), an endogenous Calcineurin inhibitor, through a HES-1-dependent mechanism. Besides control of the p21 gene, Calcineurin contributes significantly to the transcriptional response of keratinocytes to Notch 1 activation, both in vitro and in vivo. In fact, deletion of the Calcineurin B1 gene in the skin results in a cyclic alopecia phenotype, associated with altered expression of Notch-responsive genes involved in hair follicle structure and/or adhesion to the surrounding mesenchyme. Thus, an important interconnection exists between Notch 1 and Calcineurin-NFAT pathways in keratinocyte growth/differentiation control (Mammucari, 2005).
Levels of extra- and intra-cellular calcium play a major role in keratinocyte growth/differentiation control, and the calcium/Calmodulin-dependent phosphatase Calcineurin has been implicated in this process. Calcineurin is the only known serine/threonine phosphatase under calcium/calmodulin control. Among the proteins that are dephosphorylated as a consequence of Calcineurin activation are the nuclear factors of activated T cells (NFATs). Increased Calcineurin activity promotes the localization of NFATs to the nucleus, and its effect is counteracted by the phosphorylation of these factors by a number of both constitutive and inducible kinases such as GSK3, CK1, p38, and JNK1. Such a complexity of regulation is reflected by the fact that induction of NFAT-dependent transcription by Calcineurin activation is not immediately associated with increases in intracellular calcium levels, but requires a prolonged stimulus consistent with an oscillatory and accumulative mechanism of NFAT dephosphorylation and nuclear translocation (Mammucari, 2005).
Studies on the biological function of Calcineurin have been greatly facilitated by the use of the inhibitory drugs Cyclosporin A (CsA) and FK506. Several endogenous Calcineurin inhibitors have also been reported. Among these is Calcipressin (CALP1), also known as the DSCR1 gene product, located in the Down Syndrome Critical Region of human chromosome 21 and mouse chromosome 16. This protein binds directly to the CnA subunit and inhibits its activity. Importantly, Calcipressin gene expression is under direct positive control of Calcineurin/NFAT activity, so that this protein is thought to function as a feedback inhibitor of Calcineurin signaling, with an impact on T cell activation as well as the response to different stress stimuli in cardiac hypertrophy (Mammucari, 2005).
The function of Calcineurin has been elucidated in great detail in T cells, but has also been studied in the hematopoietic, neuronal, myogenic, and vascular systems. Calcineurin/NFAT activity has also been directly implicated in keratinocyte growth/differentiation control and, in vivo, in control of the hair cycle. Molecular analysis of the role of this pathway in keratinocytes has focused on control of p21 gene transcription. Induction of p21(WAF1/Cip1) is one of the earliest regulatory events associated with keratinocyte differentiation, contributing to withdrawal from the cell cycle. In mouse primary keratinocytes, p21 expression is induced by increased extracellular calcium, and the responsive region of the p21 promoter maps to a 78 bp GC-rich region close to the TATA box, containing six Sp1/Sp3 binding sites. Calcineurin induces activation of this promoter through the Calcineurin-dependent association of NFAT with the transcription factors Sp1/Sp3 (Mammucari, 2005).
Notch 1 activation induces p21 transcription not only through direct binding of the RBP-Jκ protein to the p21 promoter, but also through the calcium/Calcineurin-responsive TATA box-proximal region. Underlying this effect, induction of Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin, in opposition to positive control of this gene by Calcineurin/NFAT itself. Besides control of p21 expression, Calcineurin signaling plays a significantly broader role in the transcriptional response of keratinocytes to Notch 1 activation. In particular, inducible deletion of the CnB1 gene in the skin causes a cyclic alopecia phenotype that is linked to altered expression of several Notch-responsive genes involved in hair follicle structure and adhesion to the surrounding mesenchyme (Mammucari, 2005).
Chemoattractants stimulate neutrophil migration by activating signaling pathways including repeated transient increases in intracellular free calcium, [Ca2+]i. A motile neutrophil sends out many pseudopods, some of which adhere to the substrate; to continue moving forward, the cell must release these attachments. Adhesion can be actively regulated, and neutrophils in which [Ca2+]i transients are inhibited become stuck on fibronectin or vitronectin, extracellular matrix proteins that neutrophils encounter in vivo. Function-blocking antibodies to beta 3 integrins or the alpha v beta 3 heterodimer restore motility on vitronectin to [Ca2+]i-buffered cells, indicating that an alpha v beta 3-like integrin is responsible for the [Ca2+]i-sensitive adhesion. The density of alpha v beta 3 integrins in the adherent membrane of neutrophils migrating on vitronectin is much higher at the leading edge than at the rear, but [Ca2+]i buffering or inhibition of Ca2+-calmodulin-activated protein phosphatase 2B (calcineurin) leads to accumulation of alpha v beta 3 on the adherent surface at the rear of the cell. The polarized distribution of alpha v beta 3 integrins in migrating neutrophils is maintained by [Ca2+]i-dependent release of adhesion followed by endocytosis of these integrins and recycling to the leading edge (Lawson, 1995).
Rat brain sodium channels are phosphorylated at multiple serine residues by cAMP-dependent protein kinase. Soluble rat brain phosphatases have been identified that dephosphorylate purified sodium channels. Five separable forms of sodium channel phosphatase activity have been observed. Three forms (two, approximately 234 kDa and one, 192 kDa) are identical or related to phosphatase 2A. The two major peaks of phosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) probably represent calcineurin. Each is relatively insensitive to okadaic acid, is active only in the presence of CaCl2 and calmodulin, and contains a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcineurin. Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A, or with cyclosporin A to inhibit calcineurin, increases apparent phosphorylation of sodium channels at cAMP-dependent phosphorylation sites. These results indicate that both phosphatase 2A and calcineurin dephosphorylate sodium channels in rat brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity (Chen, 1995).
The M current regulates neuronal excitability, with its amplitude resulting from high open probability modal M channel behavior. The M current is affected by changes in intracellular calcium levels. It is proposed that internal calcium acts by regulating M channel modal gating. Intracellular application of a preactivated form of the calcium-dependent phosphatase calcineurin (CaN420) inhibits the macroscopic M current, while its application to excised inside-out patches reduces high open probability M channel activity. Addition of ATP reverses the action of CaN420 on excised patches. The change in M channel gating induced by CaN420 is different from the effect of muscarine. A kinetic model supports the proposition that shifts in channel gating induced by calcium-dependent phosphorylation and dephosphorylation control M current amplitude (Marrion, 1996).
Whole-cell recording in the superficial layers of the developing superior colliculus (sSC) reveals a large drop in NMDA receptor (NMDAR) current decay time synchronized across all neurons and occurring consistently between post-natal (P) day 10 and P11. Blocking the Ca2+/calmodulin-dependent phosphatase calcineurin (CaN) in the postsynaptic neuron can abolish this drop. The regulation is induced prematurely by 1-2 hr of electrical stimulation in P10 collicular slices only if CaN and NMDAR currents can be activated in the neuron. These data suggest that a long-lasting, CaN-mediated control of NMDAR kinetics is rapidly initiated by heightened activity of the NMDAR itself and demonstrate a novel developmental and tonic function of CaN that can play an important role in modulating the plasticity of the developing CNS (Shi, 2000).
It is likely that this developmental regulation of the NMDAR serves functions that are qualitatively different from desensitization in more mature brain. For example, the CaN effect on NMDARCs described here is large. During the synaptogenic period from P6-P21, NMDARC decay time decreases by ~18 ms. Close to half of this decrease is caused by the sudden appearance of CaN activity at P11. By the end of the following week, the slower incorporation of the NR2A subunit into sSC synaptic NMDARs could be responsible for the additional shortening of the NMDARC decay time. Rat eyes open on P13-P14, and the sudden increase in visual activity resulting from pattern visual could readily damage collicular neurons if the mechanism of CaN-dependent NMDARC downregulation did not exist. In addition, the rapid downregulation of NMDARC decay time on P11 probably reduces much of the amplification of synaptic function caused by NMDAR activity. This loss of amplification may account for the sustained decreases in spontaneous spiking activity reported in the sSC after P10 by other investigators, and it may also serve to abruptly limit ongoing synapticplasticity in the sSC. In short, this developmental function of CaN appears to be an exceptionally rapid and potent homeostatic mechanism that uses an increase in Ca2+ through the NMDAR channel to tonically decrease the potency of the NMDAR posttranslationally. This could maintain a nontoxic level of intracellular free Ca2+ in the face of a sudden increase in the activity arriving at collicular neurons. It is likely that in the visual system the increase in CaN activity results from the first powerful activation of the central visual pathway by light. It is also possible that similar sudden increases in activity occur in other regions of the nervous system. This activity-dependent, tonic, CaN-mediated control system may be broadly distributed within the vertebrate CNS (Shi, 2000).
A final significant property of the developmental change in CaN activity reported here is that it is not associated with changes in total CaN protein levels. The only event necessary to initiate phosphatase activity may therefore be the activation exerted by the NMDAR itself. Nevertheless, CaN is an elaborately regulated enzyme. Thus, changes in the activity or the synaptic localization of AKAP proteins, FKBP or cyclophorin family members, DARPP-32, CHP, or Cabin 1 may be mediating an additional level of control. Alternatively, or in addition, a prolonged depression of kinase activity could contribute to both the onset and stability of the CaN effect in sSC NMDARCs. The prolonged CaN activation at sSC synapses may also arise from a fundamentally different mechanism, such as protection of the phosphatases Fe-Zn active center from oxidation by superoxide dismutase. Regardless of the precise mechanism through which the prolongation of synaptic CaN activity is exerted, these developmental data suggest an interaction that may be retained in the mature brain. Prolonged activation of synaptic CaN could protect neurons from excitotoxicity in the face of seizure, ischemia, trauma, or disease-induced tonic increases in NMDAR activity. Thus, interventions that amplify or maintain this response may prove useful in the clinical treatment of a variety of neurological dysfunctions (Shi, 2000).
Small conductance Ca2+-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. In addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels (Bildl, 2004).
Slow- and fast-twitch myofibers of adult skeletal muscles express unique sets of muscle-specific genes, and these distinctive programs of gene expression are controlled by variations in motor neuron activity. It is well established that, as a consequence of more frequent neural stimulation, slow fibers maintain higher levels of intracellular free calcium than fast fibers, but the mechanisms by which calcium may function as a messenger linking nerve activity to changes in gene expression in skeletal muscle have been unknown. Here, fiber-type-specific gene expression in skeletal muscles is shown to be controlled by a signaling pathway that involves calcineurin, a cyclosporin-sensitive, calcium-regulated serine/threonine phosphatase. Activation of calcineurin in skeletal myocytes selectively up-regulates slow-fiber-specific gene promoters (Chin, 1998).
The myoglobin (Mb) and troponin I slow (TnIs) genes are expressed selectively in slow, oxidative skeletal muscle fibers, whereas the muscle creatine kinase (MCK) gene is expressed most abundantly in the fast, glycolytic myofiber subtype. To test whether these genes might respond differently to a calcineurin-stimulated signaling pathway, skeletal myogenic cells were transfected with reporter genes linked to well-characterized control regions from these genes, along with an expression vector encoding a constitutively active (calcium-insensitive) form of calcineurin that retains sensitivity to inhibition by cyclosporin A. Transcriptional activity of the slow-fiber-specific myoglobin and TnIs promoters is stimulated in cultured skeletal myotubes (C2C12) by active calcineurin, as measured by expression of luciferasein cotransfection assays. In contrast, activity of the fast-fiber-specific MCK promoter, or of other strong (CMV) or weak (minimal TATA element) promoters, is unaffected by activated calcineurin. The induction of the myoglobin promoter in the presence of the calcineurin expression plasmid is inhibited by cyclosporin A. This result indicates the specificity of the response, since the effect of cyclosporin A is to bind cyclophilin and form a protein complex that binds calcineurin and inhibits its protein phosphatase activity. The same relative potency of calcineurin-dependent transactivation (myoglobin and TnIs is much more potent than MCK, CMV, or TATA promoters) is observed in Sol8 myotubes, a different myogenic cell line. In contrast, forced expression of activated calcineurin had no effect on promoter activity in undifferentiated myoblasts or in 3T3 fibroblasts, demonstrating a requirement for muscle-specific factors in the calcineurin-stimulated pathway for transcriptional control of the myoglobin and TnIs promoters. Inhibition of calcineurin activity by administration of cyclosporin A to intact animals promotes slow-to-fast fiber transformation (Chin, 1998).
Transcriptional activation of slow-fiber-specific transcription appears to be mediated by a combinatorial mechanism involving proteins of the NFAT and MEF2 families. The finding that the myoglobin and TnIs promoters can be transcriptionally regulated by a calcineurin-dependent mechanism suggests the participation of NFAT transcription factors in the signaling cascade. Examination of the complete nucleotide sequences of these functionally defined transcriptional control regions (2.0 and 4.2 kb, respectively) reveals two 8-bp elements within each that match the consensus-binding sequence for NFAT transcription factors. The response to activated calcineurin of the native promoter sequences was compared to that of mutated promoters in which these putative NFAT recognition elements were ablated by site-directed mutagenesis. Disruption of putative NFAT recognition elements within both the myoglobin and TnIs promoters diminishes the response to activated calcineurin, indicating that the transactivation mechanism is likely to involve DNA binding of NFAT proteins. Transduction of the calcineurin-directed signal to the native myoglobin and TnIs promoters exhibits a saturable dose-response relationship with respect to the activated calcineurin expression plasmid; diminished reporter gene activation was evident across the entire dose range examined. Some degree of calcineurin-dependent transactivation persists after ablation of identifiable NFAT binding sites within these transcriptional control regions. Thus, either cryptic binding sites for NFAT proteins that cannot be recognized by inspection of the DNA sequence are present, or calcineurin-dependent signaling to these promoters can be driven without direct DNA binding of NFAT proteins. Nuclear localization of NFAT proteins in skeletal myocytes is under the control of calcineurin. These results identify a molecular mechanism by which different patterns of motor nerve activity promotes selective changes in gene expression to establish the specialized characteristics of slow and fast myofibers (Chin, 1998).
Calcineurin-dependent pathways have been implicated in the hypertrophic response of skeletal muscle to functional overload (OV). Skeletal muscles overexpressing an activated form of calcineurin (CnA*) exhibit a phenotype indistinguishable from wild-type counterparts under normal weightbearing conditions and respond to OV with a similar doubling in cell size and slow fiber number. These adaptations occur despite the fact that CnA* muscles display threefold higher calcineurin activity and enhance dephosphorylation of the calcineurin targets NFATc1, MEF2A, and MEF2D. Moreover, when calcineurin signaling is compromised with cyclosporin A, muscles from OV wild-type mice display a lower molecular weight form of CnA, originally detected in failing hearts, whereas CnA* muscles are spared this manifestation. OV-induced growth and type transformations are prevented in muscle fibers of transgenic mice overexpressing a peptide that inhibits calmodulin from signaling to target enzymes. Taken together, these findings provide evidence that both calcineurin and its activity-linked upstream signaling elements are crucial for muscle adaptations to OV and that, unless significantly compromised, endogenous levels of this enzyme can accommodate large fluctuations in upstream calcium-dependent signaling events (Dunn, 2000).
Regarding the potential identity of contractile activity-dependent signal transduction events, there is mounting evidence that calcineurin must interact with parallel calcium-sensitive signaling pathways in order to fully activate downstream target genes. For instance, calcineurin synergizes with phorbol ester-dependent pathways to stimulate the IL-2 promoter in T lymphocytes and the expression of atrial natriuretic factor in cardiomyocytes. Similarly, calcineurin acts in conjunction with CaM-dependent kinase IV to fully activate the myoglobin promoter in cultured skeletal myocytes and the Nur77 promoter in T lymphocytes. Moreover, retroviral-mediated gene transfer of CnA* induces skeletal myogenesis in vitro only in the presence of extracellular Ca2+. Additionally, there is evidence that MAP kinase pathways are activated in response to increased contractile activity and play a role in regulation of the slow fiber phenotype. In this context, MEF2 is an enticing candidate as an integrator of calcineurin and other activation-linked signal transduction pathways, since this transcription factor is both dephosphorylated by calcineurin and phosphorylated by various CaM kinases, ERK5, p38, and PKC (Dunn, 2000 and references therein).
An alternative possibility is that calcineurin signaling may converge with other activity-linked pathways via the association of GATA with NFAT. Indeed, activation of calcineurin promotes the association of these two transcription factors via the dephosphorylation of NFATc1 and increased expression of GATA-2 under conditions of skeletal myocyte growth. Consistent with findings from hypertrophic myocytes, this protein is upregulated in the plantaris in response to muscle overload, but not lowered by CsA treatment, suggesting that this transcription factor may be important for growth but not necessarily a gene target of calcineurin. The fact that GATA is also known to associate with MEF2, and that fiber hypertrophy is observed only when NFATc1 and MEF2 are dephosphorylated and GATA-2 increases, leads to the idea that NFAT, MEF2, and GATA proteins act in synergy to transactivate target genes that lead to fiber growth in response to OV. Future studies should help identify the particular permutations of these transcription factors involved in the activation of slow fiber-specific genes versus those modulating adult fiber size (Dunn, 2000 and references therein).
Myf5 is a member of the muscle regulatory factor family of transcription factors and plays an important role in the determination, development, and differentiation of skeletal muscle. However, factors that regulate the expression and activity of Myf5 itself are not well understood. Recently, a role for the calcium-dependent phosphatase calcineurin was suggested in three distinct pathways in skeletal muscle: differentiation, hypertrophy, and fiber-type determination. It is proposed that one downstream target of calcineurin and the calcineurin substrate NFAT in skeletal muscle is regulation of Myf5 gene expression. Myotube cultures were used that contain both multinucleated myotubes and quiescent, mononucleated cells termed 'reserve' cells, which share many characteristics with satellite cells. Treatment of such myotube cultures with the calcium ionophore ionomycin results in an approximately 4-fold increase in Myf5 mRNA levels, but similar effects are not observed in proliferating myoblast cultures, indicating that Myf5 is regulated by different pathways in different cell populations. The increase in Myf5 mRNA levels in myotube cultures requires the activity of calcineurin and NFAT, and can be specifically enhanced by overexpressing the NFATc isoform. Immunohistochemical analyses and fractionation of the cell populations were used to demonstrate that the calcium regulated expression of Myf5 occurs in the mononucleated reserve cells. It is concluded that Myf5 gene expression is regulated by a calcineurin- and NFAT-dependent pathway in the reserve cell population of myotube cultures. These results may provide important insights into the molecular mechanisms responsible for satellite cell activation and/or the renewal of the satellite cell pool following activation and proliferation (Friday, 2001).
Multiple intracellular signaling pathways have been shown to regulate the hypertrophic growth of cardiac myocytes including mitogen-activated protein kinase (MAPK) and calcineurin-nuclear factor of activated T-cells. However, it is uncertain if individual regulatory pathways operate in isolation or if interconnectivity between unrelated pathways is required for the orchestration of the entire hypertrophic response. To this end, the interconnectivity between calcineurin-mediated cardiac myocyte hypertrophy and p38 MAPK signaling was investigated in vitro and in vivo. Calcineurin promotes down-regulation of p38 MAPK activity and enhances expression of the dual specificity phosphatase MAPK phosphatase-1 (MKP-1). Transgenic mice expressing activated calcineurin in the heart are characterized by inactivation of p38 and increased MKP-1 expression during early postnatal development, before the onset of cardiac hypertrophy. In vitro, cultured neonatal cardiomyocytes infected with a calcineurin-expressing adenovirus and stimulated with phenylephrine demonstrate reduced p38 phosphorylation and increased MKP-1 protein levels. Activation of endogenous calcineurin with the calcium ionophore decreases p38 phosphorylation and increases MKP-1 protein levels. Inhibition of endogenous calcineurin with cyclosporin A decreases MKP-1 protein levels and increases p38 activation in response to agonist stimulation. To further investigate potential cross-talk between calcineurin and p38 through alteration in MKP-1 expression, the MKP-1 promoter was characterized and determined to be calcineurin-responsive. These data suggest that calcineurin enhances MKP-1 expression in cardiac myocytes; this expression is associated with p38 inactivation (Lim, 2001).
Increases in the expression of endothelin-1 (ET-1) in cardiac myocytes play a critical role in the development of heart failure in vivo. Whereas norepinephrine (NE) is a potent inducer of ET-1 expression in cardiac myocytes, the signaling pathways that link NE to inducible cardiac ET-1 expression are unknown. Adrenergic stimulation results in an increase in intracellular calcium levels, which in turn activates calcineurin. Stimulation with NE markedly increases the expression of the ET-1 gene in primary cardiac myocytes from neonatal rats. This increase is severely attenuated by a beta-adrenergic antagonist, metoprolol, but not by an alpha-adrenergic antagonist, prazosin. Consistent with these data, the beta-adrenergic agonist isoproterenol (ISO) activates the rat ET-1 promoter activity to an extent that is similar to NE. The ISO-stimulated increase in promoter activity is significantly inhibited by a Ca2+-antagonist, nifedipine, and an immunosuppressant, cyclosporin A, which blocks calcineurin. Mutation analysis indicated that the GATA4 binding site is required for ISO-responsive ET-1 transcription. Stimulation with ISO enhances the interaction between NFATc and GATA4 in cardiac myocytes. Consistent with this interaction, overexpression of GATA4 and NFATc synergistically activates the ET-1 promoter. These findings demonstrate that NE-stimulated ET-1 expression in cardiac myocytes is mediated predominantly via a beta-adrenergic pathway, and that calcium-activated calcineurin-GATA4 plays a role in this process (Morimoto, 2001).
Signaling events controlled by calcineurin promote cardiac hypertrophy, but the degree to which such pathways are required to transduce the effects of various hypertrophic stimuli remains uncertain. In particular, the administration of immunosuppressive drugs that inhibit calcineurin has inconsistent effects in blocking cardiac hypertrophy in various animal models. As an alternative approach to inhibiting calcineurin in the hearts of intact animals, transgenic mice were engineered to overexpress a human cDNA encoding the calcineurin-binding protein, myocyte-enriched calcineurin-interacting protein-1 (hMCIP1) under control of the cardiac-specific, alpha-myosin heavy chain promoter (alpha-MHC). In unstressed mice, forced expression of hMCIP1 results in a 5%-10% decline in cardiac mass relative to wild-type littermates, but otherwise produced no apparent structural or functional abnormalities. However, cardiac-specific expression of hMCIP1 inhibits cardiac hypertrophy, reinduction of fetal gene expression, and progression to dilated cardiomyopathy, all of which otherwise results from expression of a constitutively active form of calcineurin. Expression of the hMCIP1 transgene also inhibits hypertrophic responses to beta-adrenergic receptor stimulation or exercise training. These results demonstrate that levels of hMCIP1 producing no apparent deleterious effects in cells of the normal heart are sufficient to inhibit several forms of cardiac hypertrophy, and suggest an important role for calcineurin signaling in diverse forms of cardiac hypertrophy. The future development of measures to increase expression or activity of MCIP proteins selectively within the heart may have clinical value for prevention of heart failure (Rothermel, 2001).
Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin in vivo has not been unambiguously demonstrated. The up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506 (Serrano, 2001).
The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocks the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression are inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation does not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) is not inhibited by in vivo treatment with FK506 and that there is no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. Different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes (Wang, 1995).
Embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) were prepared to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appears to be normal in CNA alpha -/- mice. CNA alpha -/- T cells respond normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts have a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remain sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. Thus CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA (Zhang, 1996).
Vascular development requires an orderly exchange of signals between growing vessels and their supporting tissues, but little is known of the intracellular signaling pathways underlying this communication. Mice with disruptions of both NFATc4 and the related NFATc3 genes die around E11 with generalized defects in vessel assembly as well as excessive and disorganized growth of vessels into the neural tube and somites. Since calcineurin is thought to control nuclear localization of NFATc proteins, a mutation was introduced into the calcineurin B gene that prevents phosphatase activation by Ca2+ signals. These CnB mutant mice exhibit vascular developmental abnormalities similar to the NFATc3/c4 null mice. Calcineurin function is transiently required between E7.5 and E8.5. Hence, early calcineurin/NFAT signaling initiates the later cross-talk between vessels and surrounding tissues that pattern the vasculature (Graef, 2001).
Primary culture of postnatal cerebellar granule cells provides a model system that recapitulates many molecular events of developing granule cells in vivo. Depolarization of cultured granule cells increases intracellular Ca(2+) and activates Ca(2+)/calmodulin-dependent calcineurin (CaN) phosphatase. This Ca(2+) signaling mimics some of the signaling events for proliferation, migration, and differentiation of granule cells in vivo. This study investigated the genome-wide expression profiles of depolarization- and CaN-regulated genes in cultured mouse granule cells and addressed their relevance to gene regulation in developing granule cells in vivo. Granule cells were cultured under a nondepolarization condition (5 mM KCl) and a depolarization condition (25 mM KCl) with and without the CaN inhibitor FK506. Gene expression profiles between depolarization and nondepolarization and between FK506 treatment and untreatment were analyzed by microarray techniques. Both depolarization and FK506 treatment influence expression levels of a large number of genes, most of which are overlapping, however, are conversely regulated by these two treatments. Importantly, many of the FK506-responsive genes are up- or down-regulated in parallel with gene expression in postnatal granule cells in vivo. The FK506-down-regulated genes are highly expressed in proliferating/premigratory granule cells and many of these genes encode cellular components involved in cell proliferation, migration, and differentiation. In contrast, the FK506-up-regulated genes are predominantly expressed in postmigratory granule cells, including many functional molecules implicated in synaptic transmission and modulation. This investigation demonstrates that the CaN signaling plays a pivotal role in development and synaptic organization of granule cells during the postnatal period (Sato, 2005).
Growth cones generate spontaneous transient elevations of intracellular Ca2+ that regulate the rate of neurite outgrowth. These Ca2+ waves inhibit neurite extension via the Ca2+-dependent phosphatase calcineurin (CN) in Xenopus spinal neurons. Pharmacological blockers of CN (cyclosporin A and deltamethrin) and peptide inhibitors of CN [the Xenopus CN (xCN) autoinhibitory domain and African swine fever virus protein A238L] block the Ca2+-dependent reduction of neurite outgrowth in cultured neurons. Time-lapse microscopy of growing neurites demonstrates directly that the reduction in the rate of outgrowth by Ca2+ transients is blocked by cyclosporin A. In contrast, expression of a constitutively active form of xCN in the absence of waves results in shorter neurite lengths similar to those seen in the presence of waves. The developmental expression pattern of xCN transcripts in vivo coincides temporally with axonal pathfinding by spinal neurons, supporting a role of CN in regulating Ca2+-dependent neurite extension in the spinal cord. Ca2+ wave frequency and Ca2+-dependent expression of GABA are not affected by inhibition or activation of CN. However, phosphorylation of the cytoskeletal element GAP-43, which promotes actin polymerization, is reduced by Ca2+ waves and enhanced by suppression of CN activity. CN ultimately acts on the growth cone actin cytoskeleton, because disrupting actin microfilaments with cytochalasin D or stabilizing them with jasplakinolide negates the effects of suppressing or activating CN. Destabilization or stabilization of microtubules with colcemide or taxol results in Ca2+-independent inhibition of neurite outgrowth. The results identify components of the cascade by which Ca2+ waves act to regulate neurite extension (Lautermilch, 2000).
Axon outgrowth is the first step in the formation of neuronal connections, but the pathways that regulate axon extension are still poorly understood. NFAT proteins belong to the Rel/Dorsal family of transcription factors. Mice deficient in calcineurin-NFAT signaling have dramatic defects in axonal outgrowth, yet have little or no defect in neuronal differentiation or survival. In vitro, sensory and commissural neurons lacking calcineurin function or NFATc2, c3, and c4 are unable to respond to neurotrophins or netrin-1 with efficient axonal outgrowth. Neurotrophins and netrins stimulate calcineurin-dependent nuclear localization of NFATc4 and activation of NFAT-mediated gene transcription in cultured primary neurons. These data indicate that the ability of these embryonic axons to respond to growth factors with rapid outgrowth requires activation of calcineurin/NFAT signaling by these factors. The precise parsing of signals for elongation, turning and survival could allow independent control of these processes during development (Graef, 2003).
NFAT transcription complexes are appealing candidates for regulating aspects of neuronal morphogenesis because they integrate extracellular signals. Cell membrane signaling results in the assembly of NFAT transcription complexes in the nucleus and the activation of genes that are dependent on the cell type in which the signal is received. A rise in intracellular Ca2+ activates the serine/threonine phosphatase calcineurin and rapidly dephosphorylates the four cytoplasmic subunits NFATc1-4. Dephosphorylation of serines in the amino-termini of NFATc proteins by calcineurin exposes nuclear localization sequences leading to their rapid nuclear import. NFATc cytoplasmic subunits require other transcription factors for DNA binding, including AP-1, MEF2, GATA4, and additional factors generically referred to as nuclear partners (NFATn). The nuclear components of NFAT transcription complexes are often regulated by the PKC and Ras/MAPK pathways. Hence, the assembly of NFAT transcription complexes requires that Ca2+/calcineurin signaling be coincident with other signals. Nuclear import of NFATc family members is opposed by rapid export induced by rephosphorylation mediated by the sequential actions of PKA and GSK3. The rapid export of NFATc proteins from the nucleus can make NFAT signaling responsive to receptor occupancy and/or Ca2+ channel dynamics (Graef, 2003 and references therein).
Evidence is provided for an unexpected role for calcineurin and NFATc family members in controlling the outgrowth of embryonic axons. The results suggest that calcineurin/NFAT signaling is required specifically for axon outgrowth stimulated by growth factors like neurotrophins and netrins and provides a potential regulatory site for controlling axonal elongation independent of neuronal survival (Graef, 2003).
Axon pathfinding depends on attractive and repulsive turning of growth cones to extracellular cues. Localized cytosolic Ca2+ signals are known to mediate the bidirectional responses, but downstream mechanisms remain elusive. Calcium-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) phosphatase have been shown to provide a switch-like mechanism to control the direction of Ca(2+)-dependent growth cone turning. A relatively large local Ca2+ elevation preferentially activates CaMKII to induce attraction, while a modest local Ca2+ signal predominantly acts through CaN and phosphatase-1 (PP1) to produce repulsion. The resting level of intracellular Ca2+ concentrations also affects CaMKII/CaN operation: a normal baseline allows distinct turning responses to different local Ca2+ signals, while a low baseline favors CaN-PP1 activation for repulsion. Moreover, the cAMP pathway negatively regulates CaN-PP1 signaling to inhibit repulsion. Finally, CaMKII/CaN-PP1 also mediates netrin-1 guidance. Together, these findings establish a complex Ca2+ mechanism that targets the balance of CaMKII/CaN-PP1 activation to control distinct growth cone responses (Wen, 2004).
Axon guidance by a number of guidance molecules has been shown to depend on localized Ca2+ signals in the growth cone. Using direct focal photoactivated release of caged Ca2+ in the growth cone, it has been found that a localized Ca2+ signal in the growth cone is sufficient to induce growth cone attraction as well as repulsion, depending on the resting level of intracellular Ca2+ concentrations ([Ca2+]i) at the growth cone. Similarly, studies on netrin-1 guidance have indicated that different Ca2+ signals might underlie distinct turning responses induced by netrin-1 gradients. These results not only demonstrate a crucial role for Ca2+ signals in growth cone guidance but also indicate that complex Ca2+ mechanisms may operate to control distinct growth cone responses to a wide spectrum of external molecules (Wen, 2004 and references therein).
How different turning responses are generated by distinct Ca2+ signals, however, remains unknown. It is conceivable that different local Ca2+ signals, integrated with the baseline level of [Ca2+]i in the growth cone, activate distinct pathways to mediate attraction and repulsion, respectively. The complexity of Ca2+ signaling in axon guidance is further increased by a series of recent studies demonstrating that Ca2+-dependent growth cone responses can be further modulated by the cAMP pathway: elevation of cAMP leads to switching of repulsion to attraction and vice versa. Does the cAMP pathway act upstream to modify the characteristics of Ca2+ signals (local and/or global) or target the downstream effectors of Ca2+ for the switching? It was recently shown that cAMP/cGMP can affect L-type Ca2+ channels to alter intracellular Ca2+ signals induced by netrin-1, thus placing cAMP/cGMP upstream of Ca2+ in mediating bidirectional turning responses. However, whether cAMP also plays a role downstream of Ca2+ signaling in guidance remains to be evaluated. Most importantly, the question of what downstream targets mediate distinct Ca2+-dependent turning behaviors is still unanswered. Do attraction and repulsion involve the same or separate downstream signaling cascades? In this study, a direct local Ca2+ elevation approach was used to study downstream mechanisms of Ca2+-dependent bidirectional turning responses of nerve growth cones. The use of direct local elevation of intracellular Ca2+ concentrations through photoactivated release of caged Ca2+ bypasses membrane receptor activation and can largely avoid crosstalk among different signaling pathways, thus allowing a focus on intracellular Ca2+ and its downstream events during distinct turning responses. Evidence suggests that Ca2+-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN)-phosphatase-1 (PP1) mediate attraction and repulsion, respectively. Significantly, CaMKII/CaN-PP1 acts as a bimodal switch to control the direction of growth cone turning in response to different Ca2+ signals (local and global) by preferentially activating one component over the other. It is further shown that the cAMP pathway negatively regulates the CaN-PP1 side of the switch to modulate growth cone responses. Finally, evidence is presented that the CaMKII/CaN-PP1 mechanism also mediates netrin-1 guidance. These findings thus provide significant insights toward the downstream mechanisms underlying various turning behaviors induced by complex Ca2+ signals (Wen, 2004).
The threshold for hippocampal-dependent synaptic plasticity and memory storage is thought to be determined by the balance between protein phosphorylation and dephosphorylation mediated by the kinase PKA and the phosphatase calcineurin. To establish whether endogenous calcineurin acts as an inhibitory constraint in this balance, the effect of genetically inhibiting calcineurin on plasticity and memory was examined. Using the doxycycline-dependent rtTA system to express a calcineurin inhibitor reversibly in the mouse brain, it has been found that the transient reduction of calcineurin activity facilitates LTP in vitro and in vivo. This facilitation is PKA dependent and persists over several days in vivo. It is accompanied by enhanced learning and strengthened short- and long-term memory in several hippocampal-dependent spatial and nonspatial tasks. The LTP and memory improvements are reversed fully by suppression of transgene expression. These results demonstrate that endogenous calcineurin constrains LTP and memory (Malleret, 2001).
The main finding of this study is that the regulated inhibition of the phosphatase CN leads to enhanced LTP both in vitro and in vivo, and to improved learning and memory storage. The parallel in time course of the increased persistence of LTP in awake animals and of the memory improvement strongly suggest a correlation between the duration of LTP and memory storage. Improved cognitive performance was observed both on spatial and nonspatial hippocampal-dependent tasks, consistent with the multipurpose role of the hippocampus in human declarative memory. Moreover, with different tasks, different temporal components of memory were improved. Thus, the complex object recognition task, involving brief training sessions, multiple objects, spatial transfer, and object change, elicit a weak form of memory that is strengthened at early and intermediate time points by the CN inhibitor, but does not persist longer in mutants than in controls. By contrast, a more robust form of memory elicited by a more intense training is maintained and persists for over a week longer in mutants expressing the CN inhibitor when compared to controls (Malleret, 2001).
Facilitated learning and memory was also observed in the Morris water maze and was evident not only with traditional measurements of spatial performance, such as escape latency, but also with more specific aspects of performance such as the precision of navigation. These measures suggest that mutant mice expressing the CN inhibitor retain spatial information more efficiently than controls. The persistent memory for the first platform position associated with the efficient learning of a second platform position suggests an overall enhanced capacity for memory storage with the CN inhibitor. Further, the rapid adaptation to spatial changes observed in mutants expressing the CN inhibitor on both the Morris water maze and the object exploration task suggest increased cognitive flexibility, a process that depends on the hippocampus (Malleret, 2001).
One of the molecular mechanisms allowing transmitted signals to persist or decay is thought to be the balance between phosphatase and kinase activity. Much evidence suggests that PKA and CN specifically regulate this balance and thereby serve as a gate for LTP. In the current study, evidence in support of this model is provided by demonstrating that shifting the endogenous balance away from calcineurin activity positively modulates synaptic plasticity in a PKA-dependent manner. Further, the data indicate that altering CN activity transiently in the adult brain is sufficient to positively or negatively control synaptic plasticity and memory storage. The effects observed suggest that CN is essential both for early events of plasticity and memory and for downstream pathways that contribute to persistent changes in plasticity and memory storage (Malleret, 2001).
Mechanistically, early and transient forms of plasticity and memory are known to rely on the covalent modification of pre-existing proteins while long-term forms require activation of transcription factors such as CREB, and protein synthesis. One possible mechanism for the facilitatory effect of the CN inhibitor may be a decrease in the activity of PP1, a protein phosphatase positively regulated by CN through dephosphorylation of inhibitor-1 (I-1). PP1 inhibition has been shown to promote the induction of LTP, whereas increased PP1 activity, produced by genetic suppression of I-1, has been shown to affect certain forms of LTP in some hippocampal regions. Since PP1 is effective in modulating CaMKII, a kinase critical for the transmission of postsynaptic signals required for the induction of LTP, it is possible that increased CaMKII activity mediated by lower PP1 activity facilitates the induction of LTP. Raising the signal for the induction of LTP, through genetic upregulation of NMDA-R function, has been shown to enhance LTP, learning, and memory. The findings suggest that LTP and memory enhancements can be similarly achieved by relieving a constraint downstream of the NMDA-R and that this constraint is exercised by CN (Malleret, 2001).
The effect of the CN inhibitor on long-lasting changes in plasticity and memory may be mediated by modulation of transcriptional control. Thus, the prolonged maintenance of LTP and of memory may arise from augmented CREB transcriptional activity via reduced CREB dephosphorylation by PP1. In this context, it is important to note that unlike the phenotypes observed in Drosophila mutants expressing active CREB, the CN inhibitor does not convert labile memory into long-lasting memory. It is, however, able to strengthen or prolong different phases of memory, suggesting that CN inhibition modulates rather than mediates memory processes (Malleret, 2001).
The PKA and CN pathways may also interact antagonistically at sites other than CREB. For example, CN can inhibit specific isoforms of adenylyl cyclase required for PKA activation. Similarly, PKA and CN can regulate, in opposite ways, phosphorylation sites on key proteins in synaptic transmission, such as the NMDA-R or AMPA receptor. The effect of inhibiting CN may also occur through processes additional to or independent of the cAMP pathway. For instance, CN inhibitor may modulate Ca2+-dependent kinases such as CaMKII or PKC through control of intracellular Ca2+ mobilization by regulation of inositol 1,4,5-triphosphate receptors. Finally, the PKA/CN gate most likely represents only one of several activator/suppressor mechanisms regulating plasticity and memory (Malleret, 2001).
Several genetic approaches have been used to study the molecular mechanisms of hippocampal functions such as memory. Standard genetic techniques, however, have suffered from the limitation that the genetic modification is permanent. Here, the usefulness of the rtTA system for such studies has been demonstrated by showing that inducible and reversible transgene expression allows temporary improvement of complex cognitive functions and of brain plasticity. The ability to achieve such reversible improvements in the adult animal demonstrates that no permanent changes in neuronal circuits are involved and that the effects result specifically from molecular and biochemical changes elicited by a reduction in CN activity. In this context, the rtTA system could be further exploited to assess the timing of the requirement of CN in such processes, for instance in various stages of memory storage such as memory consolidation or retrieval since CN has been suggested to be involved in both processes. Transgene expression was found in several brain structures and, therefore, the contribution of other structures, in addition to the hippocampus, to the enhancement of long-term memory, cannot be excluded. Overall, however, these results may provide a clear target for potential treatment of learning and memory disorders (Malleret, 2001).
The calcium-dependent phosphatase calcineurin and its downstream transcriptional effector nuclear factor of activated T cells (NFAT) are important regulators of inducible gene expression in multiple cell types. In T cells, calcineurin-NFAT signaling represents a critical event for mediating cellular activation and the immune response. The widely used immunosuppressant agents cyclosporin and FK506 are thought to antagonize the immune response by directly inhibiting calcineurin-NFAT signal transduction in lymphocytes. To unequivocally establish the importance of calcineurin signaling as a mediator of the immune response, the gene encoding the predominant calcineurin isoform expressed in lymphocytes, calcineurin Abeta (CnAbeta), was deleted. CnAbeta-/- mice are viable as adults, but display defective T cell development characterized by fewer total CD3 cells and reduced CD4 and CD8 single positive cells. Total peripheral T cell numbers are significantly reduced in CnAbeta-/- mice and are defective in proliferative capacity and IL-2 production in response to PMA/ionomycin and T cell receptor cross-linking. CnAbeta-/- mice also are permissive to allogeneic tumor-cell transplantation in vivo, similar to cyclosporin-treated wild-type mice. A mechanism for the compromised immune response is suggested by the observation that CnAbeta-/- T cells are defective in stimulation-induced NFATc1, NFATc2, and NFATc3 activation. These results establish a critical role for CnAbeta signaling in regulating T cell development and activation in vivo (Bueno, 2002).
Metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) was investigated in hippocampal CA1 pyramidal neurons of 6- to 8-d-old [postnatal days 6-8 (P6-P8)] and 21- to 25-d-old (P21-P25) rats. In P6-P8 rats, induction of LTD depends on the activity of group II mGluRs. In P21-P25 rats, however, this LTD disappears, and instead, NMDA receptor (NMDAR)-dependent LTD appears. A bath containing a specific calcineurin (CaN) inhibitor restores the group II mGluR-dependent LTD in the neurons of the P21-P25 rats. Although postsynaptic injection of CaN inhibitors suppresses NMDAR-dependent LTD, it does not affect induction of group II mGluR-dependent LTD. These results demonstrate that CaN plays different roles in the induction of two forms of LTD: presynaptic CaN inhibits group II mGluR-dependent LTD, whereas postsynaptic CaN facilitates NMDAR-dependent LTD. These findings are the first demonstration in vitro of group II mGluR-dependent LTD that is negatively regulated by CaN via an age-dependent mechanism (Li, 2002).
Animals sense and adapt to variable environments by regulating appropriate sensory signal transduction pathways. Calcineurin plays a key role in regulating the gain of sensory neuron responsiveness across multiple modalities. C. elegans animals bearing a loss-of-function mutation in TAX-6, a calcineurin A subunit, exhibit pleiotropic abnormalities, including many aberrant sensory behaviors. The tax-6 mutant defect in thermosensation is consistent with hyperactivation of the AFD thermosensory neurons. Conversely, constitutive activation of TAX-6 causes a behavioral phenotype consistent with inactivation of AFD neurons. In olfactory neurons, the impaired olfactory response of tax-6 mutants to an AWC-sensed odorant is caused by hyperadaptation, which is suppressible by a mutation causing defective olfactory adaptation. Taken together, these results suggest that stimulus-evoked calcium entry activates calcineurin, which in turn negatively regulates multiple aspects of sensory signaling (Kuhara, 2002).
Two types of cation channels, TAX-4/TAX-2 and OSM-9, are expressed in the AWC olfactory neurons. Genetic analyses suggest that odor sensing activates primary olfactory transduction through the TAX-4/TAX-2 channel, which allows calcium entry to activate AWC neurons, whereas odor-provoked calcium influx through the OSM-9 channel only affects olfactory adaptation. tax-6 animals are hyperadaptable to AWC-sensed isoamyl alcohol. Exposure to isoamyl alcohol for only 10 min is sufficient for tax-6 animals to adapt. This hyperadaptable phenotype and the partially defective olfactory response of tax-6 mutants to isoamyl alcohol are both completely suppressed by an osm-9 mutation. These results suggest that TAX-6 represses OSM-9-dependent olfactory adaptation in AWC. Taken together, two possible models are proposed for the role of TAX-6 in AWC signaling. TAX-6 could be activated by calcium entry through the primary signal transduction channel TAX-4/TAX-2 upon activation of the odorant (IAA) receptor, and the activated TAX-6 could inhibit the adaptation machinery. Alternatively, TAX-6 could be activated by the odorant (IAA)-evoked calcium influx through the OSM-9 channel, and the activated TAX-6 could negatively regulate opening of the OSM-9 channel that is required for isoamyl alcohol adaptation (Kuhara, 2002).
These models on the role of TAX-6 as a negative regulator for OSM-9-dependent olfactory adaptation in AWC might paradoxically imply that TAX-6 could be a positive regulator for TAX-4/TAX-2-dependent primary sensory signaling. If TAX-6 is a direct positive regulator of AWC primary transduction, at least partially defective olfactory responses to isoamyl alcohol could be expected in osm-9 tax-6 double mutants. It was found, however, that osm-9 tax-6 mutants show completely normal olfactory response to isoamyl alcohol. This result argues against a direct positive role of TAX-6 in AWC primary signaling. The results on osm-9 tax-6 mutants are also inconsistent with a direct negative regulatory role of TAX-6 in AWC primary signaling. If that were true, hyperattractive olfactory responses to isoamyl alcohol would be seen in osm-9 tax-6 mutants (Kuhara, 2002).
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