C ALCINEURIN IN CARDIAC AND SKELETAL MUSCLE

The critical role of calcineurin and NFAT in cardiac morphogenesis and cardiac hypertrophy has been recently recognized following the development of transgenic mouse models [Molkentin et al., 1998; Ranger et al., 1998]. NFAT2 gene is critical for the development of cardiac valves and septa, and NFAT2-null mice die in utero from heart

defects [De la Pompa et al., 1998; Ranger et al., 1998]. Transgenic mice overexpressing constitutively active calcineurin in the heart develop cardiac hypertrophy [Molkentin et al., 1998] blocked by calcineurin inhibitors. In later studies targeted expression of CaN inhibiting proteins in the heart prevented cardiac hypertrophy induced by adrenergic agonists, pressure overload or calcineurin overexpression [De Windt et al., 2001; Rothermel et al., 2001; Zou et al., 2001a]. In cultured skeletal myocytes, hypertrophic response to IGF-1 also involves calcineurin [Semsarian et al., 1999; Musaro et al., 1999] and appears to be mediated by NFAT3 interacting with a cardiac-specific transcription factor GATA4 [Molkentin et al., 1998], and in skeletal myocytes by NFAT2 and GATA2 [Musaro et al., 1999]. In addition, calcineurin promotes JNK, ERK and PKCα and θ activation during hypertrophic response in myocytes [De Windt et al., 2000a]. The hypertrophic response caused by calcineurin overexpression involves induction of a distinct set of genes [Aronow et al., 2001], including atrial natriuretic factor, myosin light chain or skeletal actin.

Another role of calcineurin in muscle appears to be the control of the muscle phenotype, which could be classified as either fast or slow twitch, according to muscle contractile and metabolic properties. Calcineurin overexpression causes a fast-to-slow fiber transformation [Chin et al., 1998; Naya et al., 2000]. This process involves cooperation of NFAT and MEF2 transcriptional factors [Wu et al., 2000; Wu et al., 2001]. Additionally, the calcineurin-NFAT4 pathway is involved in skeletal muscle differentiation [Delling et al., 2000].

Recent generation of NFAT3/4 double knock-out mice discovered the role of calcineurin-NFAT pathway in blood vessel morphogenesis [Graef et al., 2001]. NFAT3/4-null mice died in utero due to vessel formation and patterning defects, and a similar phenotype was obtained in mice expressing calcineurin B transgene, which prevents phosphatase activation by calcium.

1.2.11. CALCINEURIN IN THE NERVOUS SYSTEM.

Calcineurin is one of the major brain proteins (up to 1% of the total protein) and is involved in such processes as neurotransmitter release, synaptic plasticity and gene transcription. All major brain regions show some calcineurin immunoreactivity, with particular enrichment in hippocampus, striatum, substantia nigra and cerebellum [Morioka et al., 1999a; Price and Mumby, 1999], while it has a restricted expression in the spinal cord or hindbrain. Calcineurin has a predominantly neuronal localization, and is practically absent

from oligodendroglia. It is also not present in hippocampal interneurons [Sik et al., 1998]. At the subcellular level it localizes not only to the cytoplasm, but also is enriched in postsynaptic densities and dendritic spines [Halpain et al., 1998]. Interestingly, its localization in the brain closely resembles that of immunophilin FKBP12, suggesting a physiological role for calcineurin-FKBP interaction [Steiner et al., 1992].

The first identified neuronal substrate of calcineurin was DARPP-32 [Halpain et al., 1990], a protein inhibitor of protein phosphatase-1 (PP1). Calcineurin-mediated dephosphorylation of DARPP-32 blocks its inhibitory function and results in PP1 activation, explaining the antagonistic action of the NMDA subclass of glutamate receptors on dopaminergic phosphorylation cascades. The list of established calcineurin substrates in the brain extends now into dozens [Morioka et al., 1999a], and several physiological processes under calcineurin control have been elucidated.

Much attention attracted the role of calcineurin in regulation of synaptic plasticity, which governs the processes of learning and memory. Synaptic plasticity is described in terms of two processes, the long-term potentiation (LTP), i.e. synaptic enhancement after brief high-frequency (100Hz) electric stimuli, and its reversal induced by low-high-frequency (1Hz) stimuli – long-term depression (LTD). It was hypothesized that discrimination between both processes occurs on the level of CaMK II (calmodulin-dependent protein kinase) and calcineurin, since only the latter has the calcium/calmodulin affinity high enough to respond to low calcium transients during LTD [Lisman, 1994]. Recent studies involving transgenic mice elucidated a role of calcineurin in regulation of synaptic plasticity. Expression of constitutively active calcineurin in mice hippocampi prevents conversion of so called early LTP to late LTP [Winder et al., 1998] in a PP1-dependent manner. In doing so calcineurin antagonizes PKA action. The same transgenic mice had a specific defect of the long-term memory reversed by prolonged training [Mansuy et al., 1998]. In contrast, expression of dominant-negative calcineurin form enhanced LTP, and also improved short- and long-term memory [Malleret et al., 2001]. In the other study disruption of calcineurin activity in adult mouse forebrain resulted in LTD impairment and performance defects in working and episodic-like memory tasks [Zeng et al., 2001]. In addition, mice with calcineurin Aα knock-out display impaired depotentiation (reversal of LTP after low-frequency stimulation) [Zhuo et al., 1999] with no effect either on LTP or LTD.

Calcineurin affects also the function of several neurotransmitter receptors (reviewed in [Jakel, 1997]). It is involved in the desensitization of NMDA receptors [Lieberman and Mody, 1994; Tong et al., 1995] as well as GABAA receptor channels [Stelzer and Shi, 1994].

Open probability of GluR1 and GluR6 is decreased by calcineurin [Banke et al., 2000;

Traynelis and Wahl, 1997]. Apart from plasma membrane channels one important target of calcineurin is the IP3 receptor (IP3R). Calcineurin binds to IP3R in a complex with FKBP12 [Cameron et al., 1995] and modulates calcium efflux from IP3-sensitive stores probably through dephosphorylation of the receptor. Calcineurin controls also expression levels of IP3R [Genazzani et al., 1999].

The involvement of calcineurin in regulation of neuronal gene expression was first revealed by its participation in the reversible phosphorylation of cAMP-response element-binding protein (CREB). Negative control of CREB phosphorylation through calcineurin/PP1 opposes CAMK-action in stimulus-induced transcription in hippocampus and striatum [Bito et al., 1996; Liu and Graybiel, 1996]. NFAT3 transcription factor was also found in neurons and was shown to be involved in IP3R expression in calcineurin-dependent manner [Graef et al., 1999].

Synaptic vesicle endocytosis is controlled by reversible phosphorylation of several synaptic proteins. A coordinated dephosphorylation of a group of phosphoproteins in calcineurin-dependent manner triggers endocytosis (reviewed in [Cousin and Robinson, 2001]). The involvement of calcineurin in dephosphorylation of dynamin, amphiphisin and synaptojanin was indicated by effects of immunosuppressants [Bauerfeind et al., 1997], and dynamin was shown to be a calcineurin substrate in vitro [Liu et al., 1994]. Calcineurin binding to dynamin is necessary for endocytosis [Lai et al., 1999]. A role for calcineurin in exocytosis and neurotransmitter release was also suggested [Hens et al., 1998]. Recently it has been shown that calcineurin specifically dephosphorylates several serine residues on synapsin I, thus inhibiting exocytosis [Jovanovic et al., 2001].

Several other neuronal substrates of calcineurin were identified. Neuronal NO synthase is dephosphorylated and activated by calcineurin [Dawson et al., 1993], and this process might contribute to glutamate neurotoxicity. Among cytoskeletal proteins, MAP2, tau and tubulin are calcineurin substrates [Morioka et al., 1999a]. In Alzheimer's disease dephosphorylation of abnormally phosphorylated tau by calcineurin [Gong et al., 1994] plays probably a protective role and suggests that calcineurin might be one of the target proteins in the pathophysiology of the disease. This is consistent with the fact that calcineurin β mRNA is the most up-regulated mRNA in brains of the Alzheimer's disease patients as revealed by cDNA microarray technique [Hata et al., 2001]. Dephosphorylation of neuromodulin (GAP-43), a protein involved in neurite outgrowth, by calcineurin [Liu and Storm, 1989] increases its affinity for calmodulin.

The involvement of calcineurin in the pathophysiology of brain-related diseases received significant attention of many researchers. Particularly, its role in ischemia-reperfusion is of great interest since immunosuppressants were successfully tested as neuroprotective agents in ischemic models (reviewed in [Morioka et al., 1999a]). Calcineurin activity in hippocampus is transiently decreased after focal ischemia [Morioka et al., 1999b]

with recovery after 12 h, and this change was not reflected on the level of protein expression.

The target of neuroprotective action of FK506 is, however, disputed. Studies with FK506 analogues indicate that the proteins other than calcineurin are probably involved (reviewed in Snyder et al., 1998]).

1.2.12. CALCINEURIN AND APOPTOSIS.

First indication of calcineurin involvement in apoptosis came from studies on T-lymphocytes. The process of negative selection in the thymus is characterized by the TCR-mediated apoptosis of hyperreactive T-cells. Induction of the nuclear receptor Nur77 is an important part of this process [Woronicz et al., 1994]. This induction requires calcineurin and synergistic activation of NFAT and MEF2 transcriptional pathways [Youn et al., 1999; Youn et al., 2000]. Another contribution of calcineurin-NFAT pathway to T-cell apoptosis is the induction of Fas ligand, a molecule critical for apoptotic signaling in many cell types [Latinis et al., 1997].

Calcineurin function in apoptosis has been established in many other cell types, with both pro-apoptotic and pro-survival effects. Expression of constitutively active calcineurin induces apoptosis in epithelial [Shibasaki and McKeon, 1995] as well as neuronal [Asai et al., 1999] cells. The pro-apoptotic signaling by calcineurin may involve dephosphorylation of Bad, a pro-apoptotic member of Bcl-2 family, which heterodimerizes with Bcl-xL in its unphosphorylated state [Wang et al., 1999a]. Another pro-apoptotic member of Bcl family, Bik is induced by the calcineurin-mediated pathway in B-cells [Jiang and Clark, 2001].

Calcineurin also forms a complex with Bcl-2 itself, and Bcl-2 can suppress phosphatase activity of calcineurin [Shibasaki et al., 1997]. In cardiomyocytes, activation of calcineurin was associated with pro-apoptotic effects of isoproterenol [Saito et al., 2000]. However, other studies reported cytoprotective action of calcineurin in cardiomyocytes [De Windt et al., 2000b; Kakita et al., 2001], possibly by inducing Bcl-2 expression. In summary, calcineurin involvement in apoptosis depends on cellular context and coordinated action of other signaling pathways, e.g. as was demonstrated for the p38 pathway [Lotem et al., 1999].

1.2.13. OTHER CELLULAR FUNCTIONS OF CALCINEURIN.

In addition to the signaling pathways discussed above, other important cellular processes appear to involve calcineurin-dependent steps.

Activation of the NFκB transcription factor plays an important role in inflammation and cell survival. NFκB resides in the cytoplasm as a complex with its inhibitory subunit, IκB, and phosphorylation-triggered proteosomic degradation of IκB leads to NFκB nuclear translocation and activation. Calcineurin was shown to promote NFκB activation in lymphocytes [Frantz et al., 1994] and other cell types [Carballo et al., 1999]. Calcineurin appears to act upstream of IKK, the kinases phosphorylating IκB [Trushin et al., 1999], but the exact substrate is unknown. In addition, calcineurin transcriptionally up-regulates NFκB through NFAT-dependent pathway [Rao et al., 1997]. Calcineurin effect on NFκB signaling pathway is cell type-dependent, with inhibition noted for astrocytes [Conboy et al., 1999;

Pons and Torres-Aleman, 2000] or endothelial cells [Holschermann et al., 2001]. In astrocytes, IGF-1-promoted dephosphorylation of IκB is mediated by calcineurin [Pons and Torres-Aleman, 2000], but whether IκB is a direct calcineurin substrate remains to be elucidated.

Calcineurin can modulate the activity of Elk-1 transcription factor by directly dephosphorylating it [Sugimoto et al., 1997; Tian and Karin, 1999] and preventing its activation. Elk-1 as well as many other transcription factors is also regulated by diverse MAP kinases. Three major MAP kinase families are known: ERK, JNK and p38, and they mediate a plethora of cellular responses to extracellular signals. These kinases are themselves regulated by diverse upstream phosphorylation cascades starting at the receptors on the plasma membrane. Calcineurin appears to modulate the signaling through all of the major MAP kinases. Activation of ERK in cardiomyocytes upon β-adrenergic stimulation is mediated by calcineurin [Zou et al., 2001b; De Windt et al., 2000a]. Calcineurin-mediated activation of JNK is also involved in hypertrophic response [De Windt et al., 2000a], and calcineurin might contribute to JNK activation in lymphocytes [Su et al., 1994]. In addition, calcineurin transgenic mice show decreased p38 activity and calcineurin transfection in myocytes also inhibits p38 [Lim et al., 2001].

NO-mediated signal transduction regulates diverse physiological processes from vasorelaxation to synaptic transmission to bacterial killing. As mentioned above calcineurin

process has been described for endothelial NO synthase (eNOS) [Harris et al., 2001].

Bradykinin induces dephosphorylation of the eNOS on Thr497 in calcineurin-dependent manner, and this dephosphorylation was required for full eNOS activation. Additional research is needed to elucidate the physiological situations, where such type of regulation is involved. Another endothelial-related function of calcineurin could be the regulation of endothelial permeability through a PKC-dependent pathway [Lum et al., 2001].

This list of physiological processes and signaling mechanisms involving calcineurin is far from complete. Identification of calcineurin substrates in vivo and intracellular pathways regulating calcineurin-mediated dephosphorylation should give a better understanding of its involvement in the pathology of disease and provide a rationale for design of drugs specifically targeting detrimental processes without compromising beneficial ones (e.g. see [Kiani et al., 2000]).

1.2.14. CALCINEURIN-INTERACTING PROTEINS.

Apart from calcineurin substrates, several proteins directly interact with calcineurin and function either as calcineurin inhibitors or target calcineurin to distinct subcellular structures. In most of the protein partners of calcineurin these functions are combined.

The interaction of calcineurin with immunophilins was discussed in the previous sections. In addition, several other proteins function as both calcineurin inhibitors and calcineurin anchors. Thus, AKAP79 (A-kinase anchoring protein), a protein functioning as a scaffold for localizing several signal transduction components to subsynaptic densities in neurons, binds calcineurin and inhibits its activity [Coghlan et al., 1995]. AKAP79 also localizes PKA and PKC, thus bringing multiprotein signaling complexes in the vicinity of L-type calcium channels and AMPA receptors.

Cain/cabin was identified in two-hybrid screens for calcineurin-interacting proteins in lymphocytes and neurons [Lai et al., 1998; Sun et al., 1998]. Cain/cabin has a calcineurin-interacting motif similar to that of NFAT, and this interaction inhibits calcineurin activity.

Cain/cabin acts also as a repressor of MEF2 transcriptional activity, and this repression is relieved in a calcium-dependent manner [Youn et al., 1999; Youn and Liu, 2000]. Synaptic vesicle endocytosis might also be regulated by Cain/cabin, probably by inhibiting calcineurin [Lai et al., 2000].

CHP (calcineurin-homologous protein) is a protein of 22 kDa with 43% homology to calcineurin B [Lin and Barber, 1996]. CHP inhibits calcineurin activity and subsequent

downstream signaling [Lin et al., 1999]. It also binds the proteins of the Na⁺/H⁺ exchanger (NHE) family and is necessary for their activity [Pang et al., 2001].

A search for muscle-specific calcineurin-interacting proteins discovered two related proteins, MCIP-1 and –2 (myocyte-enriched calcineurin interacting protein) [Rothermel et al., 2000], whose expression is up-regulated during muscle differentiation. MCIP1 is encoded by DSCR1, a gene located in the Down syndrome critical region. MCIP/DSCR proteins physically interact with calcineurin and inhibit its activity [Fuentes et al., 2000; Rothermel et al., 2000], and the binding is at least partly mediated by the PXIXXT motif of MCIP/DSCR.

MCIP/DSCR homologue in yeast, Rcn1p also inhibits calcineurin when overexpressed [Kingsbury and Cunningham, 2000]. Surprisingly, Rcn1p-null mutants also show a defect of calcineurin signaling, indicating that Rcn1p is probably needed to stabilize calcineurin.

Another interesting observation is that DSCR1 is up-regulated in Alzheimer’s disease and its expression is correlated with the development of neurofibrillary tangles in the pathology of this disease [Ermak et al., 2001]. A two-hybrid screen for cardiac calcineurin-interacting proteins revealed also a family of calsarcins, which tether calcineurin to alpha-actinin at the z-line of the sarcomere of cardiac and skeletal muscle cells [Frey et al., 2000].

In yeast, the stability of calcineurin was shown to be modulated by chaperones. Hsc82, a Hsp90 yeast homologue, suppressed calcineurin activity when overexpressed, but the normal expression level of Hsc82 was needed for calcineurin signaling [Imai and Yahara, 2000]. There is evidence that calcineurin activity could also be modulated by mammalian Hsp70 and Hsp90 [Someren et al., 1999].

The elucidation of protein-protein interaction patterns in the context of intracellular signaling is a topic of tremendous research effort. It is logic to expect that more calcineurin-interacting proteins will appear in this search.

1.2.15. CALCINEURIN REGULATION BY REDOX PROCESSES.

First indications of calcineurin susceptibility to oxidation came from the study showing that some sulfhydryl reagents, including N-ethylmaleimide (NEM) and p-hydroxymercuribenzoic acid, inhibited its metal-dependent and –independent activities [King, 1986; Gupta et al., 1990]. Inactivation of calcineurin could be traced to modification of one or two thiols. Another evidence for redox sensitivity of calcineurin provided the fact that calcineurin was one of the six major proteins in neutrophil homogenate binding to immobilized phenylarsine oxide (PAO), a specific reagent for vicinal thiol groups

[Schmachtel, 1996]. Direct involvement of redox processes in the regulation of calcineurin activity was first shown in the pioneering work of Klee and associates [Wang et al., 1996b].

Investigating the process of calcium- and calmodulin-dependent calcineurin inactivation in rat brain homogenates [Stemmer et al., 1995] Wang et al. found that Cu, Zn-SOD protected calcineurin against this inactivation. In addition, yeast lacking Cu, Zn-SOD had the same phenotype as calcineurin-null strains, and calcineurin activity in lysates of SOD1-mutants was about 1/10 of wild-type strains. Subsequent studies of several groups revealed that exogenous H2O2 could inhibit calcineurin activity in neutrophils [Carballo et al., 1999], NK cells [Furuke et al., 1999] and Jurkat T-lymphocytes [Reiter et al., 1999], and one study on cell lysates and purified calcineurin revealed that in general calcineurin activity is increased by antioxidants and decreased by oxidants [Sommer et al., 2000]. Calcineurin was also inhibited after thiol depletion of the culture medium [Furuke et al., 1999]. H2O2 blocked also downstream calcineurin-NFAT or calcineurin-NFκB signaling in these cases.

The discovery of calcineurin sensitivity to superoxide prompted the efforts to discover the situations, where such an inhibition could take place in vivo. One example provided regulation of gene expression in hippocampus, where the increase of CREB protein phosphorylation after prolonged electrical stimulation of neurons could be traced to the inhibition of calcineurin activity reversed by antioxidants [Bito et al., 1996]. Particular interest focused on the regulation of calcineurin activity during FALS, a disease characterized by mutations of Cu, Zn-SOD. Although one study found no effect of certain FALS mutations on calcineurin activity [Lee et al., 1999], others provided a correlation between the severity of the disease caused by particular mutation and the degree of calcineurin inhibition [Ferri et al., 2000; Volkel et al., 2001; Ferri et al., 2001].

Regarding the mechanism of calcineurin sensitivity towards ROS Klee and associates initially proposed that the iron in the binuclear metal center of calcineurin (postulated to have Fe²⁺-Zn²⁺ composition) is the target as evidenced by the protective effect of ascorbate and the reversal of inactivation by a mix of Fe²⁺ , ascorbate and DTT [Wang et al., 1996]. This hypothesis was however contradicted by the studies of Rusnak and colleagues [Rusnak et al., 1999a; Rusnak and Reiter, 2000]. EPR spectroscopy of the purified bovine calcineurin showed the presence of Fe³⁺ in the binuclear center of calcineurin [Yu et al., 1995; Yu et al., 1997]. This calcineurin was active towards pNPP and RII peptide, although the activity towards RII was lower than reported by Wang et al. Moreover, the reduction of Fe³⁺ by dithionite led to the loss of the EPR signal and the simultaneous loss of calcineurin activity towards pNPP [Yu et al., 1997]. Additionally, a mixed valence Fe³⁺-Fe²⁺ binuclear center of

calcineurin was obtained by metal exchange in the same group [Yu et al., 1995; Yu et al., 1997]. This calcineurin was equally sensitive to H2O2 as well as to dithionite [Yu et al., 1997], and it was proposed that this metal center represents the redox-sensitive calcineurin form in vivo, the hypothesis still contested in favor of the Fe²⁺-Zn²⁺ center [Aramburu et al., 2000].

The involvement of calcineurin thiols in redox regulation of calcineurin remained unattended, although some indications, including reversibility of calcineurin inhibition by DTT [Reiter et al., 1999] and inhibition after cellular thiol depletion [Furuke et al., 1999] pointed to a possible role of calcineurin cysteines in regulation of its activity. It should be noted, however, that site-directed mutagenesis of several calcineurin cysteines (Cys197, Cys228, Cys166 and Cys88) produced protein with the activity equal to that of the wild type enzyme [Reiter et al., 1999]. In summary, although it is recognized that redox reactions modulate calcineurin activity, the mechanism of ROS action remains unclear.

AIMS OF THIS STUDY.

The recognition of the crucial role, which redox modulation of enzyme activity plays in cellular signal transduction, led to a fast development of research concentrated on redox modifications of proteins. Calcineurin represents one of the major protein phosphatases, and is critical for regulation of numerous physiological processes. Therefore elucidation of the mechanisms, by which redox processes modulate calcineurin activity, appears to be of importance for our understanding of how cellular phosphorylation/dephosphorylation cascades respond to environmental changes. Previous research identified two possible redox-sensitive targets on calcineurin, protein thiols and its binuclear metal center, but the exact mechanisms of redox modulation of calcineurin activity remained unclear. In addition, the question, which oxidant is the most relevant redox modulator of calcineurin in vivo, is left

The recognition of the crucial role, which redox modulation of enzyme activity plays in cellular signal transduction, led to a fast development of research concentrated on redox modifications of proteins. Calcineurin represents one of the major protein phosphatases, and is critical for regulation of numerous physiological processes. Therefore elucidation of the mechanisms, by which redox processes modulate calcineurin activity, appears to be of importance for our understanding of how cellular phosphorylation/dephosphorylation cascades respond to environmental changes. Previous research identified two possible redox-sensitive targets on calcineurin, protein thiols and its binuclear metal center, but the exact mechanisms of redox modulation of calcineurin activity remained unclear. In addition, the question, which oxidant is the most relevant redox modulator of calcineurin in vivo, is left