Increased expression of calcineurin in human colorectal adenocarcinomas

Colorectal cancer (CRC) is the third most common cause of cancer death in the Western world. Calcineurin (CaN), a Ca2+/calmodulin (CaM)-dependent protein phosphatase, is important for Ca2+-mediated signal transduction. The main objective of this study is to examine the potential role of Ca2+/CaM-dependent protein phosphatase in both normal and in invasive tumor components of human samples. In this study, we carried out 45 cases of CaN activity, 13 cases of CaN protein expression by Western blot analysis, and 6 cases for immunohistochemical analysis in both normal and invasive tumor components of human samples. Immunohistochemical analysis revealed that strong cytoplasmic staining of varying intensity was observed in colon tumors of all patients compared to normal mucosa. In addition, Western blot analysis revealed a prominent overexpressed immunoreactive band with an apparent molecular mass of 60 kDa catalytic alpha subunit (CaN A) as well as CaN Aalpha and beta in colon tumor samples. Elevated CaN protein expression appears to be a possible link between Ca2+ signaling and oncogenic processes.     

In vitro Proteolytic Degradation of Bovine Brain Calcineurin by m-Calpain

A major cause of neuronal dysfunction is due to altered Ca2+ regulation. An increase in Ca2+ influx can activate Ca2+-dependent enzymes including calpains, causing the proteolysis of its specific substrates. In the present study, calcineurin (CaN) was found to be proteolysed by a Ca2+-dependent cysteine protease, m-calpain. In the presence of Ca2+, the 60 kDa subunit (CaN A) was degraded to a 46 kDa immunoreactive fragment, whereas in the presence of Ca2+ /calmodulin (CaM) immunoreactive fragments of 48 and 54 kDa were observed. The beta-subunit (CaN B) was not proteolysed in either condition. The proteolysis of CaN A increased its phosphatase activity and rendered it totally CaM-independent after 10 min of proteolysis. The molecular weight of the proteolytic fragments suggested that the m-calpain cleaved CaN A in the CaN B binding domain. A CaM-overlay experiment revealed that the CaM-binding site was present only in the 54 kDa fragment produced by CaN A proteolysis in the presence of Ca2+ /CaM. Thus, the increase in CaN A phosphatase activity observed in many neuronal disorders, may be due to the action of calpain.     

Activation of calcineurin expression in ischemia-reperfused rat heart and in human ischemic myocardium

Calcineurin (CaN) has been reported as a critical mediator for cardiac hypertrophy and cardiac myocyte apoptosis. In the present study, we investigated the activity and expression of CaN and the effect of calpain in rat heart after ischemia and reperfusion. Rat ischemic heart showed significant increase in CaN activity. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine CaN indicated a prominent immunoreactive band of 60 kDa (CaN A). In ischemic-reperfused hearts, the expression of CaN A was significantly low and immunoreactivity was observed in proteolytic bands of 46 kDa. This may be due to the proteolytic degradation of CaN A in ischemic tissues by m-calpain. We also noticed in vitro proteolysis of bovine cardiac CaN A by m-calpain. Immunohistochemical studies showed strong staining of immunoreactivity in rat hearts that had gone under 30 min ischemia followed by 30 min reperfusion similar to that found in human ischemic heart. Ischemia is associated with multiple alterations in the extracellular and intracellular signaling of cardiomyocytes and may act as an inducer of apoptosis. The increase in CaN activity and strong immunostaining observed in ischemic/perfused rat heart may be due to the calpain-mediated proteolysis of this phosphatase.     

Generation of constitutively active calcineurin by calpain contributes to delayed neuronal death following mouse brain ischemia

Calpain, a Ca(2+)-dependent cysteine protease, in vitro converts calcineurin (CaN) to constitutively active forms of 45 kDa and 48 kDa by cleaving the autoinhibitory domain of the 60 kDa subunit. In a mouse middle cerebral artery occlusion (MCAO) model, calpain converted the CaN A subunit to the constitutively active form with 48 kDa in vivo. We also confirmed increased Ca(2+)/CaM-independent CaN activity in brain extracts. The generation of constitutively active and Ca(2+)/CaM-independent activity of CaN peaked 2 h after reperfusion in brain extracts. Increased constitutively active CaN activity was associated with dephosphorylation of dopamine-regulated phosphoprotein-32 in the brain. Generation of constitutively active CaN was accompanied by translocation of nuclear factor of activated T-cells (NFAT) into nuclei of hippocampal CA1 pyramidal neurons. In addition, a novel calmodulin antagonist, DY-9760e, blocked the generation of constitutively active CaN by calpain, thereby inhibiting NFAT nuclear translocation. Together with previous studies indicating that NFAT plays a critical role in apoptosis, we propose that calpain-induced CaN activation in part mediates delayed neuronal death in brain ischemia.     

A CaMKII/calcineurin switch controls the direction of Ca(2+)-dependent growth cone guidance

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. Here, we report that calcium-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) phosphatase 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.     

Molecular cloning and biochemical characterization of bovine retina calcineurin

Calcineurin (CaN) is a member of ser/thr protein phosphatase family. Earlier, we have reported that CaN is present in all eye tissues, although the activity and protein expression varied (Seitz et al., Invest Opthalmol Vis Sci, 43:15–21, 2002). We have isolated a full-length cDNA encoding bovine retina CaN. The CaN A subunit consists of 511 amino acid residues. A 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni²⁺ was found to be the strongest stimulator, while Zn²⁺ was found to inhibit CaN phosphatase activity. Mn²⁺ was a relatively less effective stimulator compared to Ni²⁺. Fe²⁺ was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe²⁺ slightly inhibited CaN activity from bovine brain. The residues at 97–201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407–456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known autoinhibitory domain at 457–480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics.     

Decreased Expression of Calmodulin Kinase II and Calcineurin Messenger RNAs in the Mouse Hippocampus After Kainic Acid-Induced Seizures

Abstract: The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN) are especially abundant in the mammalian CNS, where they have been implicated repeatedly in different neuronal functions. CaMKII is a holoenzyme that is likely to be constituted of both homomultimers and heteromultimers, CaMKIIα and CaMKIIβ being the most abundant subunits in the brain. CaN is a heterodimer constituted of a catalytic subunit (CaN A) and a regulatory subunit (CaN B), and CaN Aα is the predominant form in the brain. We studied the expression of CaMKIIα, CaMKIIβ, and CaN Aα subunit messenger RNAs in the mouse hippocampus at different times after the administration of a convulsant dose of kainic acid. CaMKIIα and CaN A immunohistochemistry was also performed. We observed a transient decrease in the three messenger RNAs in the kainic acid-treated mice, peaking at 5 or 24 h of treatment. The effect had disappeared completely 8 days after treatment. No significant alterations in CaMKII or CaN immunolabelling were observed in the hippocampus of kainic acid-treated mice. The observed modifications could be due to the neuronal hyperexcitability induced by kainic acid rather than neuronal degeneration, because no areas of neuronal loss were detected. Our results suggest that the expression of CaMKII and CaN mRNAs is down-regulated in neuronal cells in response to the hyperexcitability induced by kainic acid. The transient nature of the effect and the apparent absence of significant modifications in the amount of their corresponding proteins may be related to the absence of neuronal damage.     

Na(+)/H(+) exchanger 1 directly binds to calcineurin A and activates downstream NFAT signaling, leading to cardiomyocyte hypertrophy

The calcineurin A (CaNA) subunit was identified as a novel binding partner of plasma membrane Na(+)/H(+) exchanger 1 (NHE1). CaN is a Ca(2+)-dependent phosphatase involved in many cellular functions, including cardiac hypertrophy. Direct binding of CaN to the (715)PVITID(720) sequence of NHE1, which resembles the consensus CaN-binding motif (PXIXIT), was observed. Overexpression of NHE1 promoted serum-induced CaN/nuclear factor of activated T cells (NFAT) signaling in fibroblasts, as indicated by enhancement of NFAT promoter activity and nuclear translocation, which was attenuated by NHE1 inhibitor. In neonatal rat cardiomyocytes, NHE1 stimulated hypertrophic gene expression and the NFAT pathway, which were inhibited by a CaN inhibitor, FK506. Importantly, CaN activity was strongly enhanced with increasing pH, so NHE1 may promote CaN/NFAT signaling via increased intracellular pH. Indeed, Na(+)/H(+) exchange activity was required for NHE1-dependent NFAT signaling. Moreover, interaction of CaN with NHE1 and clustering of NHE1 to lipid rafts were also required for this response. Based on these results, we propose that NHE1 activity may generate a localized membrane microdomain with higher pH, thereby sensitizing CaN to activation and promoting NFAT signaling. In cardiomyocytes, such signaling can be a pathway of NHE1-dependent hypertrophy.     

Overactivation of calcineurin induced by amyloid-beta and prion proteins

Amyloid-beta protein (A beta) and the scrapie isoform of prion protein (PrPSs) have a central role in the pathogenesis of Alzheimer's disease (AD) and prion-related encephalopathies (PRE), respectively. In both disorders, the deposition of these misfolded proteins is accompanied by apoptotic neuronal loss. However, the pathogenesis and molecular basis of A beta- and PrPSc-neurotoxic effects are not completely understood. The Ca2+/calmodulin-dependent phosphatase calcineurin (CaN), through the dephosphorylation of the proapoptotic protein BAD, may be the link between Ca2+homeostasis deregulation and apoptotic neuronal death. In this study we used primary cultures of rat brain cortical neurons in order to investigate whether A beta and PrP affect CaN activity. We observed that synthetic peptides of A beta (A beta 25-35 and A beta 1-40) and PrP (PrP106-126) increased CaN activity, but did not affect the levels of this protein phosphatase. Moreover, we found that these peptides reduced the levels of BAD phosphorylated at serine residue 112, and this effect was prevented by the CaN inhibitor FK506. Since dephosphorylated BAD translocates to mitochondria, where it triggers cytochrome c release, we determined the levels of BAD in mitochondrial and cytosolic fractions. The data obtained showed that A beta- and PrP-treated neurons had higher levels of BAD in mitochondria than control neurons. This increase in mitochondrial BAD levels was matched by a decrease in cytochrome c. FK506 prevented the alterations of mitochondrial BAD and cytochrome c levels induced by A beta and PrP peptides. Taken together the data suggest that A beta and PrP increased CaN activity, inducing BAD dephosphorylation and translocation to mitochondria and, subsequently, cytochrome c release that may trigger an apoptotic cascade. Therefore, therapeutic strategies targeting CaN might be valuable for these neurodegenerative disorders.     

Role of calcineurin in neurodegeneration produced by misfolded proteins and endoplasmic reticulum stress

A hallmark event in neurodegenerative diseases is the accumulation of misfolded aggregated proteins in the brain leading to neuronal dysfunction and disease. Compelling evidence suggests that misfolded proteins damage cells by inducing endoplasmic reticulum (ER) stress and alterations in calcium homeostasis. Changes in cytoplasmic calcium concentration lead to unbalances on several signaling pathways. Recent data suggest that calcium-mediated hyperactivation of calcineurin (CaN), a key phosphatase in the brain, triggers synaptic dysfunction and neuronal death, the two central events responsible for brain degeneration in neurodegenerative diseases. Therefore, blocking CaN hyper-activation might be a promising therapeutic strategy to prevent brain damage in neurodegenerative diseases.     

突触中磷蛋白的生后发育变化

为了研究在突触功能中起重要作用的磷蛋白状况,利用高分辩率的放射自显影、梯度电泳和双向电泳,以及抗ＣａＮ多克隆抗体封闭ＣａＮ磷酸酶活力等技术,并运用计算机图象处理系统,对大鼠大脑皮层突触体中磷蛋白生后发育变化进行定量分析．结果表明,大鼠出生后（ＰＮＤ）３ｄ、７ｄ、２１ｄ、和成年磷蛋白表达有很大不同,在出生后早期对应突触主要形成时期,磷蛋白呈高表达;从ＰＮＤ２１ｄ开始至成年,底物蛋白磷酸化状态逐渐降低,同时研究了突触主要形成时期有显著变化的钙调神经磷酸酶,它的内源底物及其在其生后发育所发生的变化．     

Calcium sensing receptor regulates cardiomyocyte function through nuclear calcium

Nuclear Ca2+ plays a pivotal role in the regulation of gene expression. IP3 (inositol-1,4,5-trisphosphate) is an important regulator of nuclear Ca2+. We hypothesized that the CaR (calcium sensing receptor) stimulates nuclear Ca2+ release through IICR (IP3-induced calcium release) from perinuclear stores. Spontaneous Ca2+ oscillations and the spark frequency of nuclear Ca2+ were measured simultaneously in NRVMs (neonatal rat ventricular myocytes) using confocal imaging. CaR-induced nuclear Ca2+ release through IICR was abolished by inhibition of CaR and IP3Rs (IP3 receptors). However, no effect on the inhibition of RyRs (ryanodine receptors) was detected. The results suggest that CaR specifically modulates nuclear Ca2+ signalling through the IP3R pathway. Interestingly, nuclear Ca2+ was released from perinuclear stores by CaR activator-induced cardiomyocyte hypertrophy through the Ca2+-dependent phosphatase CaN (calcineurin)/NFAT (nuclear factor of activated T-cells) pathway. We have also demonstrated that the activation of the CaR increased the NRVM protein content, enlarged cell size and stimulated CaN expression and NFAT nuclear translocation in NRVMs. Thus, CaR enhances the nuclear Ca2+ transient in NRVMs by increasing fractional Ca2+ release from perinuclear stores, which is involved in cardiac hypertrophy through the CaN/NFAT pathway.     

癫痫大鼠与正常大鼠脑中钙调神经磷酸酶及其底物的研究

报道了听源性癫痫大鼠发作后其脑内钙调神经磷酸酶（Ｃａｌｃｉｎｅｕｒｉｎ,ＣａＮ）及其底物蛋白磷酸化水平的改变。以ＰＮＰＰ为底物测ＣａＮ的活力,用间接ＥＬＩＳＡ测ＣａＮ的含量,ＳＤＳ－ＰＡＧＥ和２－Ｄ－ＰＡＧＥ并放射自显影的方法研究脑内蛋白质磷酸化水平,发现与正常大鼠相比,听源性癫痫大鼠发作后,脑内ＣａＮ的含量并没有改变,但比活力下降,其底物的磷酸化状态也有改变,其中一个３０ｋＤ蛋白磷酸化程度明显降低。实验结果提示,大鼠听源性癫痫与ＣａＮ及其调控的底物有相关性。     

Inhibition of excitatory neuronal cell death by cell-permeable calcineurin autoinhibitory peptide

In glutamate-mediated excitatory neuronal cell death, immunosuppressants (FK506, Cys-A) are powerful agents that protect neurons from apoptosis. Immunosuppressants inhibit two types of enzyme, calcium/calmodulin-dependent protein phosphatase (calcineurin: CaN), and peptidyl-prolyl cis-trans-isomerase (PPIase) activity such as the FKBP family. In this study, we used a protein transduction approach to determine the functional role of CaN and to produce a potential therapeutic agent for glutamate-mediated neuronal cell death. We created a novel cell-permeable CaN autoinhibitory peptide using the 11 arginine protein transduction domain. This peptide was highly efficient at transducing into primary culture neurons, potently inhibited CaN phosphatase activities, and inhibited glutamate-mediated neuronal cell death. These results showed that CaN plays an important role in excitatory neuronal cell death and cell-permeable CaN autoinhibitory peptide could be a new drug to protect neurons from excitatory neuronal death.     

肿瘤坏死因子-α诱导心肌肥大中CaN信号通路的作用

目的:研究钙调神经磷酸酶(CaN)信号通路在肿瘤坏死因子-α(TNF-α)诱导心肌细胞肥大中的作用。方法:Lowry法测心肌细胞蛋白含量;计算机图象分析系统测心肌细胞体积;[3H]-亮氨酸掺入法测心肌细胞蛋白合成;Till阳离子测定系统观察胞内[Ca2+]i瞬变;Western blot法测定CaN的表达。结果:①CaN特异性抑制剂CsA(0.2μmol/L)明显抑制TNF-α(100μg/L)诱导的心肌细胞蛋白含量、蛋白合成和细胞体积增大,但对正常心肌细胞生长无影响。②CaN特异性抑制剂CsA(0.2μmol/L)明显降低TNF-α诱导的心肌细胞内钙离子浓度([Ca2+]i)瞬变幅度增高。③TNF-α明显增强心肌细胞内CaN的表达。结论:TNF-α可能通过引起心肌细胞[Ca2+]i升高,促进CaN表达诱导心肌细胞肥大。     

Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells

Neuronal cell death as a result of apoptosis is associated with cerebrovascular stroke and various neurodegenerative disorders. Pharmacological agents that maintain normal intracellular Ca2+ levels and inhibit cellular oxidative stress may be effective in blocking abnormal neuronal apoptosis. In this study, a spontaneous (also referred to as age-induced) model of apoptosis consisting of rat cerebellar granule cells was used to evaluate the antiapoptotic activities of voltage-sensitive Ca2+ channel blockers and various antioxidants. The results of these experiments demonstrated that the charged, dihydropyridine Ca2+ channel blocker amlodipine had very potent neuroprotective activity in this system, compared with antioxidants and neutral Ca2+ channel blockers (nifedipine and nimodipine). Within its effective pharmacological range (10-100 nM), amlodipine attenuated intracellular neuronal Ca2+ increases elicited by KCl depolarization but did not affect Ca2+ changes triggered by N-methyl-D-aspartate receptor activation. Amlodipine also inhibited free radical-induced damage to lipid constituents of the membrane in a dose-dependent manner, independent of Ca2+ channel modulation. In parallel experiments, spontaneous neuronal apoptosis was inhibited in dose- and time-dependent manners by antioxidants (U-78439G, alpha-tocopherol, and melatonin), nitric oxide synthase inhibitors (N-nitro-L-arginine and N-nitro-D-arginine), and a nitric oxide chelator (hemoglobin) in the micromolar range. These results suggest that spontaneous neuronal apoptosis is associated with excessive Ca2+ influx, leading to further intracellular Ca2+ increases and the generation of reactive oxygen species. Agents such as amlodipine that block voltage-sensitive Ca2+ channels and inhibit cellular oxidative stress may be effective in the treatment of cerebrovascular stroke and neurodegenerative diseases associated with excessive apoptosis.     

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide. Evidence for a dithiol-disulfide equilibrium and implications for redox regulation.

Calcineurin (CaN) is a Ca2+-and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe-Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3-8 microM and the inactivation was reversed by 2, 3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+-Zn2+ dinuclear centre. Upon H2O2-mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.     

Blocking gastrin and CCK-B autocrine loop affects cell proliferation and apoptosis in vitro

Since its initial discovery as Ca²⁺/calmodulin (CaM)-dependent serine/threonine protein phosphatase, calcineurin (CaN) has been extensively studied in many mammalian tissues. CaN has been shown to be involved in various biological and Ca²⁺-dependent signal transduction pathways. Over the last decade, our laboratory has been interested and has carried out numerous experiments on this specific protein phosphatase. While, a lot of research has been performed studying CaN’s involvement in ischemia, the immune system, and various mammalian tissues, not much is known about the potential role of CaN in various eye diseases. This review focuses on the studies that have been carried out in our laboratory on CaN, and specifically CaN’s involvement in the eye. We demonstrated that CaN is localized in various eye tissues (cornea, iris, ciliary body, vitreous body, retina, choroid, sclera, and optic nerve) and that both its protein expression and activity were observed in high amounts in the retina, optic nerve and cornea. Recently, we have cloned and characterized the CaN A and B subunits in the bovine retina. These initial findings suggest that CaN may play a potential role in visual transduction and various ocular diseases, including cancer.