Calcineurin in development and disease

Abstract

Calcineurin (CaN) is a unique calcium (Ca²⁺) and calmodulin (CaM)-dependent serine/threonine phosphatase that becomes activated in the presence of increased intracellular Ca²⁺ level. CaN then functions to dephosphorylate target substrates including various transcription factors, receptors, and channels. Once activated, the CaN signaling pathway participates in the development of multiple organs as well as the onset and progression of various diseases via regulation of different cellular processes. Here, we review current literature regarding the structural and functional properties of CaN, highlighting its crucial role in the development and pathogenesis of immune system disorders, neurodegenerative diseases, kidney disease, cardiomyopathy and cancer.

Introduction

Calcineurin (CaN, alternate name protein phosphatase 2B) is best characterized as a unique Ca²⁺ and calmodulin (CaM)-dependent serine/threonine phosphatase that activates substrates important in various cellular processes ranging from activation of T cells to regulation of cell apoptosis.1, 2, 3 In the canonical CaN pathway, which was first described in immune cells, CaN becomes activated following an increase in calcium (Ca²⁺) concentration. Following activation, CaN dephosphorylates nuclear factor of activated T-cells (NFAT) allowing its nuclear translocation to regulate the expression of interleukin (IL)-2 and IL-4.^3,4 Meanwhile, within the noncanonical CaN pathway, CaN activation leads to the dephosphorylation of other substrates, including dynamin-related protein 1 (Drp1),⁵ Na⁺/H⁺-exchanger 1 (NHE1),⁶ TWIK-related spinal cord K⁺ channel (TRESK),⁷ calcineurin response zinc finger (CRZ1),³ and kinase suppressor of ras 2 (KSR2).⁸ In this manner, CaN regulates the function of various organ systems, including the immune system, heart, kidney, neurons.9, 10, 11, 12 Meanwhile, CaN dysfunction leads to developmental defects, contributing to the pathogenesis of many common disorders. Accordingly, CaN inhibitors (CNIs), such as cyclosporine A (CsA) and tacrolimus (FK506), have been developed to inhibit CaN functions for the treatment of clinical diseases.^4,13,14

In the 40 years since Claude Klee first described the structure and regulation of Ca²⁺-activated CaN, accumulating studies have revealed that CaN plays an essential role in a myriad of physiological and pathological processes. Published reviews on this topic have been presented in Table S1^3,4,13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29^, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44^, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, however, these articles largely focus on the structure and functions of CaN. In particular, CaN alterations are reportedly associated with the development and the onset and progression of immune system disease, neurodegenerative diseases, kidney disease, cardiomyopathy, and cancer. Here, we provide a comprehensive description of the pivotal roles of CaN in development and disease.

Calcineurin: structure and activation insights

CaN is broadly distributed throughout the cytoplasm of lymphocytes, as well as nerve, cardiac muscle, skeletal muscle, lung, spleen, and liver cells. Its structure is highly conserved from yeast to humans with high homology observed at the nucleotide and amino acid levels.⁶⁰ CaN is a heterodimer, comprising a 60 kDa catalytic A subunit (CnA) and a 19 kDa regulatory B subunit (CnB). Further, it contains a catalytic domain, B subunit regulatory domain, calmodulin-binding domain, and an autoinhibitory domain^3,61,62 (Fig. 1A). The latter three regions together constitute the regulatory region of CnA. Constitutive activation of CnA occurs via removal of its last two regions, which occurs independently of Ca²⁺ signaling.⁶³ Meanwhile, CnB, the regulatory subunit, is structurally homologous to CaM, both of which are Ca²⁺ dependent. CnB contains two Ca²⁺-binding lobes connected by a short linker comprising four Ca²⁺-binding EF-hand motifs (EF hands), which participate in the activation of CnA^61,63 (Fig. 1A). Three isoforms exist for CnA: CnAα (PPP3CA), CnAβ (PPP3CB) and CnAγ (PPP3CC), while CnB exists in two forms (CnB1 and CnB2).⁶³ CnAγ expression is restricted to the testis and the brain, while CnB1 is found only in testis.^63,64 All other CaN isoforms are ubiquitously expressed.⁶³ Moreover, CnAα and CnAβ share 81% similarity,⁶⁵ with the primary difference being the unique repeating proline sequence present in the N-terminus of CnAβ, which is highly associated with substrate recognition. Similarly, CnB1 and CnB2 have 83% sequence homology and appear to exhibit redundant functions.⁶¹

Figure 1.

The structure of CaN and mechanism of activation. (A) Structure of calcineurin, with boxes indicating functional domains and lines indicating intervening amino acid sequence. (B) The proposed mechanism of activation of calcineurin.

The native form of CaN is enzymatically inactive with crystal structure analysis revealing blockade of the catalytic center by the autoinhibitory domain. Meanwhile, Ca²⁺ signal and CaM induce the necessary conformational changes to allow for complete activation of CaN. Specifically, internal and external signals trigger Ca²⁺ transport across the cell membrane by Ca²⁺ pumps and subsequent release of Ca²⁺ from endoplasmic reticulum (ER) stores.⁶⁶ The increased cellular cytosolic Ca²⁺ binds CnB, causing dissociation of the calmodulin-binding domain from the B subunit regulatory domain. Thereafter, CaM binds to the calmodulin-binding domain, resulting in displacement of an autoinhibitory domain from the catalytic center, effectively activating the protein³ (Fig. 1B). However, in the absence of CaM, the increased cytosolic Ca²⁺ binds to the four Ca²⁺-binding EF-hand motifs of CaN, leading to a partially-activated state (Fig. 1B). Activation of CaN results in the dephosphorylation of substrates responsible for regulating various cellular processes.

Calcineurin and development

CaN signaling is required for a broad spectrum of developmental processes in a variety of organ systems, including immune, kidney, heart, nervous, and musculoskeletal systems, as well as the differentiation of bone, cartilage, muscle, skin, and fat.^9,10,34,54 Therefore, inhibition of genes associated with CaN pathway contribute to defects or alterations in kidney maturation, cardiomyocyte maturation, heart valve formation, vascular development, skeletal muscle differentiation and T cell development. In fact, a study found that CnAα–deficient mice exhibited significantly impaired development of T helper type 2 cells (Th2).⁶⁷ Meanwhile, another study reported defects in nephrogenic zone (NZ) and superficial glomeruli development, altered cell cycle in the NZ, and impaired kidney function, leading to progressive kidney failure and a shortened lifespan in CnAα knockout mice.^10,68 Further, CnAβ-deficient mice demonstrated a significant reduction in CD3 positive cells, as well as CD4 and CD8 single positive cells.⁹ The proliferative capacity of T cells in CnAβ^−/−mice as well as IL-2 production was reportedly decreased in response to PMA ionomycin stimulation and T cell receptor cross-linking.⁹ Moreover, CnB1 mutant embryos do not develop beyond E9.5 and display defects in angiogenesis and axonal outgrowth, as evidenced by the lack of fusion and remodeling of the vascular plexus into larger vessels.⁶⁹ Nfatc1 knockout-mice exhibited abnormal heart valves resulting in embryonic lethality.⁷⁰ Still further, Nfatc3 and Nfatc4 deficient embryos displayed cardiovascular abnormalities and heart failure at E10.5 with distinct skeletal muscle defects.⁵⁴ Meanwhile, pharmacological inhibition of CaN signaling affects embryogenesis. In mice, treatment with CsA from E7.5 to E8.5 resulted in defective vascular remodeling.⁶⁹ Meanwhile, FK506 treatment contributed to the development of edema, gut coiling disruption, and teratogenesis in the kidney, heart, gut, liver, and somatic tissue during Xenopus development.⁷¹ Cumulatively, these results indicate that CaN is a necessary component for normal organ development. However, the impact of CaN signaling in development is far more complex than what we yet known and requires further exploration to elucidate precisely how CaN-NFAT signaling regulates the development of different organs.

Calcineurin and disease

CaN is a multifunctional protein that participates in nearly all aspects of cellular functioning. Specifically, it is actively involved in various biological processes under both physiological and pathological conditions, including T cell activation, cell apoptosis, cell cycling, cell proliferation, cell migration, cell invasion, stem cell generation, as well as cell transformation and fate. As such, CaN dysfunction has been found to contribute to the pathogenies of many common disorders, as summarized in Figure S1. The sheer number of pathological conditions associated with CaN implies its central role in the regulation of cell physiology. Below, we summarize the main findings reporting a link between alterations in CaN and several diseases.

Immune system disorders

CaN is considered to be a key enzyme in the immune response with its function originally described in T cells. Increasing intracellular Ca²⁺ concentration in T lymphocytes leads to CaM binding with CaN, which then dephosphorylates NFAT1, NFAT2, NFAT3, and NFAT4, leading to their nuclear translocation. Activation of NFAT upregulates cytokines responsible for T cell activation, including IL-2, IL-4, IL-17, interferon-γ (IFNγ), and tumor necrosis factor-α (TNF-α)^3,72 (Fig. 2). Meanwhile, accumulation of activated T cells in host tissues and organs leads to initiation of the inflammatory process, inducing the onset and progression of chronic inflammatory disease and autoimmune diseases.

Figure 2.

The CaN-NFAT pathway in T cells. Intracellular and extracellular signals trigger an initial cytoplasmic Ca²⁺ increase. Elevated cytoplasmic Ca²⁺ activates CaN, which dephosphorylates the NFAT transcription factors, causing NFATs to translocate into the nucleus and initiate gene transcription. CsA and FK506 inhibit the activity of CaN through their interaction with the immunophilins called cyclophilin A and FK-binding protein 12 (FKBP12) respectively.

Autoimmune diseases are characterized by T cell activation and an increase in interleukin turnover, while the Ca²⁺-CaN-NFAT pathway is reportedly dysregulated in autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Indeed expression of CaN has been discovered in monocytes/macrophages and vascular endothelial cells in RA synovia, in which it promotes the expression of IL-2, IL-17, and TNF-α, thereby contributing to the development of chronic inflammation.¹³ Moreover, Ca²⁺ influx and nuclear NFAT levels are abnormally increased in activated T cells of SLE patients.⁷³ This aberrant activation of the Ca²⁺-CaN-NFAT pathway upregulates CD40 ligand expression, which subsequently induces antibody production and dendritic cell (DC) activation in SLE patients.⁷⁴ Meanwhile, inhibition of the CaN-NFAT pathway suppresses production of inflammatory cytokines and co-stimulatory molecules by T cells. Hence, CaN-NFAT signaling is believed to represent an attractive target for therapeutic approaches to control autoimmune responses. CNIs, including CsA and FK506, inhibit CaN activity through their interaction with immunophilins, namely cyclophilin A, and FK-binding protein 12 (FKBP12), respectively⁴ (Fig. 2), and are widely administered for the treatment of autoimmune diseases. In fact, CNIs effectively improve patient's clinical symptoms and elevate survival rates. However, the use of CsA and FK506 is associated with certain adverse effects, including hypertension, nephrotoxicity, neurotoxicity and metabolic disorders.^4,75 It is, therefore, necessary to conduct further research to identify novel CNIs capable of not only inhibiting CaN activity but also doing so without eliciting serious adverse effects.

While CaN has long been considered a unique regulator of T-cell activity,³⁶ it is also expressed in other immune cells, including B cells,^76,77 macrophages,^78,79 and DCs.^80,81 The CaN-NFAT pathway in myeloid cells participates in mounting immune responses to bacteria,82, 83, 84 fungi,⁸⁵ and viruses.⁸⁶ The molecular mechanism underpinning activation of the CaN-NFAT pathway involves ligand binding of multiple different pattern recognition receptors (PRRs), such as TLRs and C-type lectin receptors, which subsequently activate spleen tyrosine kinase (Syk)/phospholipase (PLC). This signaling cascade promotes an increase in the Ca²⁺ concentration in monocytes, macrophages, and DCs, triggering CaN-NFAT-IL2 signaling upon recognition of complex particulate antigens.³⁶ In fact, a recent study showed that CR3 engagement with Mycobacterium leprae (pathogen responsible for leprosy) activates Syk, inducing CaN-dependent nuclear translocation of the transcription factor NFAT, which selectively augmented the production of IL-2, IL-10, and IL-1β.⁸⁴ Meanwhile, the addition of CsA significantly reduced the levels of these cytokines. Furthermore, human DCs treated with CsA exhibited reduced IFN-γ responses to Sendai virus infection.⁸⁷ Similarly, when bone marrow-derived dendritic cells (BMDCs), depleted of CaN were stimulated with Aspergillus fumigatus, they exhibited reduced expression of Ptx3, a molecule of particular importance in antifungal activity.⁸⁸ Furthermore, the Ca²⁺-CaN-NFAT-IL-2 pathway in DCs modulates Th17 cell expansion in response to Aspergillus-germinated particles; whereas conditional knockout of IL-2 in CD11c⁺ cells impairs fungus recognition, represses phagocytosis, and disrupts Th17 responses to live conidia.⁸⁰ These results indicate that CaN inhibition increases the risk of associated with a myriad of infections. In accordance with these experimental results, transplant patients treated with CNIs exhibited an increased risk of bacterial and fungal infections. Therefore, novel specific CaN inhibitors are required for transplant patients capable of reducing the risk of infection.

Kidney disease

CaN is also an important element in the pathogenesis of glomerular hypertrophy, injury, and sclerosis. Specifically, Gooch and colleagues observed an increase in CaN expression in the glomeruli, and activation of CaN in the renal cortex of rats following induction of diabetes by hyperglycemia.⁸⁹ Meanwhile, inhibition of CaN with CsA reduces whole kidney hypertrophy and completely blocks both glomerular hypertrophy and extracellular matrix (ECM) accumulation,⁹⁰ suggesting that CaN mediates the latter symptoms in diabetic nephropathy in vivo. Compelling evidence has also shown that podocyte injury is a common and typical feature of glomerular injury and sclerosis, and is an initiating factor of the pathogenic process. The disruption of podocyte actin fibers, cytoskeletal damage, and podocyte apoptosis contributes to foot process effacement, glomerular filtration barrier damage, and proteinuria, which ultimately leads to kidney dysfunction.⁹¹ Many studies have been performed to investigate how the activation of Ca²⁺/CaN signaling induces podocyte loss and cytoskeletal damage. Their results suggest that angiotensin II (AngII), or angiotensin II type I receptor agonistic autoantibody (AT1-AA), enhance the expression of transient receptor potential channel 6 (TRPC6), which stimulates Ca²⁺/CaN signaling, ultimately leading to actin fiber damage and podocyte injury via several pathways, as shown in Figure 3.^92,93 One mechanism involves the binding of receptor activator of NF-κB (RANK) to TRPC6, which promotes podocyte loss and impairs glomerular function by stimulating CaN activity and increasing nuclear NFAT accumulation.⁹⁴ AngII also downregulates microRNA-30 (miR-30) family members, resulting in CaN activation, and facilitating dephosphorylation and degradation of the actin-binding protein, synaptopodin (SYNPO), which induces cytoskeletal injury and apoptosis in podocytes.^95,96 Moreover, a recent study revealed that miR-30 deficiency leads to CaN-NFAT signaling activation, which in turn activates the urokinase plasminogen activator receptor-integrin β3 (uPAR-ITGB3) pathway, ultimately altering Rac family small GTPase 1 (RAC1) and cell division cycle 42 (CDC42) activity, and inducing podocyte injury⁹¹ (Fig. 3).

Figure 3.

Signal molecules regulating CaN activity to mediate podocyte injury. (1) AT1-AA induces podocyte injury via activation of the TRPC6- Ca²⁺/CaN pathway. (2) Ang II contributes to podocyte injury by increasing TRPC6 expression, which activates CaN/NFAT signaling. (3) Ang II induces Ca²⁺/CaN signaling and podocyte injury by downregulating miR-30 family members. (4) RANK promotes podocyte injury by activating Ca²⁺/CaN/NFAT signaling. (5) MiR-30 family members inhibit uPAR-ITGB3 signaling activation through the CaN/NFAT pathway.

Cardiomyopathy

Cardiac hypertrophy (CH) occurs in a number of disease states in response to increased cardiac workload and can readily progress to ventricular dilatation, contractile dysfunction, and heart failure. As such, many studies have focused on the molecular mechanisms of cardiac myocyte hypertrophy and have found that myocyte hypertrophy is activated by multiple intracellular signaling pathways including Ca²⁺-dependent signalings.⁹⁷ Upon exposure to hypertrophic stimuli, the increase in intracellular Ca²⁺ activates CaN signaling pathways which promotes activation of hypertrophic gene transcription and subsequently induces the onset and development of pathological hypertrophy. As a critical mediator of CH, CaN is involved in the development of cardiac hypertrophy via regulation of downstream targets including NFAT, myocyte enhancer factor2 (MEF2), GATA binding protein4 (GATA4), nuclear factor-κ-gene binding (NF-κB), and Drp1, as well as interaction with other pathways such as protein kinase C (PKC), mitogen-activated protein kinase (MAPK), calmodulin-dependent protein kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3-K), and Wnt pathways.^56,97,98 Moreover, enhanced CaN activity may positively correlate with CH. For instance, a study reported an increase in serum CaN activity in hypertensive and hypertrophic patients.⁹⁹ Moreover, significantly increased concentration of intracellular Ca²⁺ was found to enhance CaN and NFATc4 expression in cardiomyocyte hypertrophy.¹⁰⁰ Additionally, transgenic mice expressing activated forms of CaN, or downstream targets exhibit CH and heart failure. Specifically, constitutive activation of CnA was reported to induce strong CH resulting in heart failure within the first weeks of life. This result is consistent with previous reports showing enhanced CH in response to overexpression of a constitutively active form of NFAT.¹⁰¹ In contrast, genetic or pharmacological inhibition of CaN signaling significantly attenuates the pathogenesis of CH and dysfunction in response to various stresses.¹⁰⁰ Indeed, a recent study demonstrated that specific deletion of CnB1 in cardiomyocyte reduces AngII-induced increases in ventricular mass, cardiomyocyte cross-sectional area, and left ventricular wall thickness, preventing AngII-induced CH.¹² Previous reports also found that constitutive deficiency of the catalytic subunit CnAβ, or NFATc3 or NFATc2 diminish AngII-mediated heart weight gain.¹² Similarly, overexpression of the endogenous CaN inhibitors (regulator of calcineurin 1, carabin) represses cardiac growth and attenuates heart function.¹⁰²

Neurodegenerative disease

Expression of CaN is particularly high in neurons, accounting for 1% of total neural protein, in which it is localized in the cytosol, presynaptic and postsynaptic terminals. Besides, CaN is also expressed in astrocytes and microglia.⁴⁵ It has been reported that CaN plays a critical role in the maintenance and plasticity of spines, as well as the acquisition of learning, memory, and long-term potentiation (LTP), among other functions.¹⁰³ Alterations in Ca²⁺ homeostasis are related to the accumulation of misfolded protein aggregates in different neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), transmissible spongiform encephalopathies (TSEs), and amyotrophic lateral sclerosis (ALS).^44,104 Meanwhile, accumulation of misfolded/unfolded aggregated proteins leads to sustained ER stress, which dysregulates Ca²⁺ homeostasis in various neurodegenerative diseases and results in activation of CaN.¹⁰⁵ Aberrantly activated CaN is increasingly linked to a variety of pathologic features associated with neurodegenerative disorders, including synaptic dysfunction and loss, neuroinflammation, and neuronal cell death.^106,107

The outcomes associated with aberrant Ca²⁺ dynamics particularly impairs neurotransmission in synaptic spines and hyperactivation of CaN. Both the release and uptake of neurotransmitters including synaptobrevin, synapsin, rabphilin2A, synaptotagmin, and dephosphins via exocytosis and endocytosis rely on CaN activity.¹⁰⁸ Hence, CaN is an important regulator of synaptic transmission in both the pre- and post-synaptic compartments. In presynaptic terminals, hyperactivated CaN dephosphorylates synapsin I, a phosphoprotein that tethers neurotransmitter-containing vesicles to the cytoskeleton¹⁰⁹ (Fig. 4A). Once phosphorylated, synapsin I detaches from the vesicles, which become exocytosed from the cell, thereby releasing neurotransmitters into the synapse.¹¹⁰ Meanwhile, in postsynaptic terminals, CaN has been shown to dephosphorylate and thus inactivate, the N-methyl-d-aspartate receptor (NMDA-R), thereby reducing the amount of time that the ion channel remains open.^111,112 CaN has also been reported to enhance or prolong the desensitization period of other ligand-gated channels, including γ-aminobutyric acid type A receptors (GABAARs)¹¹³ and serotonin.¹¹⁴ Besides, it was observed that CnB1-deficiency in mice slows synaptic vesicle secretion in the hippocampal glutamatergic synapses, which may be related to Ca²⁺ entry via neurotransmitter release triggering N-type Ca²⁺ channels enhanced by CaN.¹¹⁵

Figure 4.

Role of activated CaN in synaptic dysfunction and neuronal death. (A) (1) Activated CaN dephosphorylates CREB, inhibiting its translocation to the nucleus and reducing CREB target gene expression required for neuronal growth and synaptic plasticity. (2) Activated CaN dephosphorylates synapsin I, inhibiting neurotransmitter release by abrogating synaptic vesicle transport. (B) CaN dephosphorylates BAD, which dissociates from scaffolding proteins and forms a dimer with Bcl-2 or Bcl-xL, leading to the release of cytochrome c and the apoptotic cascade that results in neuronal cell death.

Changes in Ca²⁺ regulation during neurodegenerative disease also leads to inhibition of LTP, synaptic dysfunction and memory loss through disruption of CaN signaling cascades. Meanwhile, hyperactivated CaN-mediated endocytosis of the a-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid (AMPA) receptors is associated with Aβ oligomer-induced synaptic dysfunction and memory loss related to LTP repression.¹¹⁶ Furthermore, CaN activates protein phosphatases 1 (PP1) which functions to phosphatase AMPA receptors.¹¹⁷ However, activation of CaN inhibits the activity of NMDA receptors and the enhanced AMPA receptor internalization.¹¹⁸ Meanwhile, CaN is also involved in regulating synaptic plasticity by controlling the expression of target genes via cAMP response element-binding (CREB), which is a major target of CaN.¹¹⁹ Under normal conditions, CREB becomes activated by phosphorylation and nuclear translocation. Phosphorylated CREB (pCREB) then modulates the expression of target genes necessary for neuronal growth and survival, including brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (trkB)120, 121, 122 (Fig. 4A). However, hyperactivated CaN induces the dephosphorylation and inactivation of CREB, thereby inhibiting CREB-target gene expression, and causing synaptic dysfunction and memory loss.¹¹⁹ CaN also interacts with and activates PP1, thereby indirectly promoting PP1-dependent CREB dephosphorylation.⁴⁶ Interestingly, a study showed that pCREB levels are significantly reduced in the hippocampi of AD patients,¹²³ while another group reported that hippocampal pCREB immunoreactivity was decreased in the Tg2576 murine model of AD, however, was restored following FK506 treatment.¹²⁴ Taken together, these data suggest that aberrant CaN activity promotes synaptic dysfunction, highlighting the importance of regulating CaN activity at an appropriate level.

Many forms of injury or disease in the central nervous system (CNS) activate astrocytes and microglia. Astrocytes form a critical part of the neurovascular unit, which support and maintain an appropriate neuronal environment. Activated astrocytes secrete numerous pro-inflammatory cytokines and other factors involved in neuroinflammation⁴⁵ and therefore, serve as a hallmark of this process. Meanwhile, CaN plays a critical role in the neuroinflammatory signaling inherent to astrocytes during neural damage and dysfunction.^45,112 Although it is only weakly expressed in the astrocytes of healthy adult neural tissue, CaN is strongly expressed in activated astrocytes during aging, injury, and/or disease.^45,125 For example, Christopher and colleagues observed that intense CaN expression is localized in activated astrocytes surrounding amyloid plaques in an AD murine model.⁴⁵ Moreover, in vitro, amyloid β-protein (Aβ) stimulates CaN activation in primary rat astrocyte cultures.¹²⁶ In addition, CaN expression is upregulated in astrocytes from gerbil hippocampi subjected to bilateral carotid artery occlusion.⁴⁵ Similarly, numerous CN-positive astrocytes are present in human hippocampi during the very early stages of cognitive decline. Once activated in astrocytes, CaN dephosphorylates NFAT, which translocates from the cytoplasm to the nucleus. In the nucleus, NFAT interacts with distinct DNA binding elements to drive the expression of numerous immune/inflammatory factors.^45,127 A study by Hafiz and colleagues shows that nuclear NFAT1 levels in astrocytes increase in patients with mild cognitive impairment and nuclear localization of NFAT3 in astrocytes is apparent in those with AD.¹²⁷ CN also interacts with NFκB, peroxisome proliferator-activated receptor γ (PPARγ), and/or forkhead box O3 (FOXO3) transcription factors to differentially affect neuroinflammation in astrocytes.⁴⁵ These results suggest that CaN signaling in astrocytes is involved in the neuroinflammatory processes that lead to injury, disease, and aging.

Over-activated CaN has also been implicated in reversible neuronal apoptosis. Hyperactivated CaN dephosphorylates Bcl2-associated death protein (BAD), which is normally bound to the 14-3-3 protein in the cytosol and which is phosphorylated on certain serine residues.¹²⁸ Following dephosphorylation of BAD by CaN, BAD dissociates from its scaffolding proteins and translocates from the cytosol to the mitochondria. In the mitochondrial outer cell membrane, BAD forms a dimer with another pro-apoptotic protein, Bcl-2/Bcl-xL, triggering the release of cytochrome c, which contributes to the activation of the post mitochondrial caspase apoptotic cascade^46,129 (Fig. 4B). Recently, a study reported that prion protein increases CaN activity, resulting in decreased AMPK phosphorylation at threonine residue 172 and increased autophagy activation, which induces neuronal cell death; wherease FK506 may prevent this effect.¹⁰⁷ Taken together, inhibition of CaN may represent a novel therapeutic approach for preventing neurodegenerative diseases.

Cancer

In recent years, emerging evidence has shown that CaN may play an important role in the development and progression of human cancers.¹³⁰ Indeed, activation of CaN and its downstream targets has been defined as having oncogenic potential in colorectal, breast, prostate, ovarian, pancreatic, and liver cancers, as well as glioblastoma, lung cancer, and leukemia.^77,131, 132, 133, 134, 135, 136, 137, 138 Specifically, activated CaN reportedly regulates cancer stem cell survival and proliferation, cell migration, invasion, and metastasis in response to hypoxic conditions, inflammation, and vascular endothelial growth factor signaling.

The molecular mechanisms mediating these effects are based on the ability of CaN to dephosphorylate and activate NFAT and other target genes. Importantly, NFAT is constitutively activated or overexpressed in numerous cancers and can contribute to cancer development and progression.¹³⁰ Meanwhile, the CaN/NFATc1 signaling pathway is critically involved in the pathogenesis of solid tumors. Recently, nuclear NFATc1 was identified in human colon cancer specimens (stage II and stage III), as well as in human colon cancer cell lines, while being described as being strongly associated with poor survival rates. Studies have also shown that this transcription factor could promote migration capacity of colorectal cancer cells (CRC) via modulating runt-related transcription factor 2 (RUNX2) and gelsolin (GSN).⁵² Meanwhile, nuclear NFATc1 was detected in 50.6% of triple negative breast tumors, in 69.5% of pancreatic carcinomas and activated in hepatocellular carcinoma cells.^133,139 NFATc1 enhances proliferation and migration of breast cancer, pancreatic cancer, and hepatocellular carcinoma cells through regulating certain oncogenes such as c-myc. Loss-of-function studies show that NFATc1 silencing in 4T1cells (breast cancer cells) inhibits their migratoty and proliferative capacity. Besides, treatment with CsA in pancreatic carcinoma cell lines inhibits the nuclear localization of transcriptionally active NFATc1 and cell cycle progression. In addition, Xu and colleagues found that NFATc1 is significantly higher in ovarian cancer tissues than in paired normal control tissues and activates the extracellular regulated protein kinases1/2 (ERK1/2)/p38/MAPK signal pathway,¹⁴⁰ which leads to promotion of cell growth and tumorigenesis. Meanwhile, the involvement of CaN and NFATc1 has also been reported in hematologic malignancies. In fact, nuclear localization of NFATc1 was detectable in 72% of Burkitt's lymphoma (BL) cases and 28% of diffuse large B cell lymphoma (DLBCL) cases.¹⁴¹ Nuclear accumulation of NFATc1 was also discovered in aggressive T cell lymphoma.¹⁴¹

Additional CaN targets include cyclin D1, glycogen synthase kinase-3b (GSK-3b), nuclear factor I (NFI), kinase suppressor of ras 2 (KSR2), and c-Jun, which have all been shown to have pro-tumorigenic roles.^137,142, 143, 144, 145 For example, CaN dephosphorylates cyclin D1 at residue T286, inhibiting its degradation, thereby facilitating cell cycle progression and robust cell growth in invasive breast cancer cells.¹⁴² Similarly, CaN enhances transcription of nuclear factor I (NFI) via dephosphorylation, altering the migratory properties of malignant glioma (MG) cells.¹³⁷ Additionally, CaN stabilizes c-Jun by dephosphorylating it at Ser-243 to enhance its tumorigenic ability in COS-7 cells.¹⁴⁵

Activation of CaN and its dephosphorylated substrates regulate various genes critical for proliferation, apoptosis, migration, and survival in both solid tumors and lymphoid malignancies. Hence, efficient inhibition of CaN and its critical effectors, may be useful as a therapeutic strategy for cancer. In fact, a report showed that pancreatic cancer cells treated with CsA or FK506 exhibited a dramatic time- and dose-dependent reduction in proliferation.¹³⁹ Moreover, FK506 treatment of a breast cancer mouse model decreased tumor growth and angiogenesis in vivo and reduced migration of breast cancer cells in vitro.¹⁴⁶ Both CsA and FK506 treatment markedly increase the number of apoptotic cells in lymphoma and leukemia cell lines, and induces regression of T-cell acute lymphoblastic leukemia (T-ALL) in mice, prolonging their survival.¹⁴⁷ It appears that combination therapy comprising CNIs and other anti-cancer drugs may represent a promising approach for targeted treatment. A recent study revealed that when non-small cell lung cancer (NSCLC) cells were treated with crizotinib and CsA, apoptosis was promoted, and G2/M arrest was induced compared with crizotinib-only treatment.¹⁴⁸ In contrast, CsA and FK506 increased the risk of cancers in organ-transplant patients due to suppression of tumor immunosurveillance mechanisms.⁴⁹ For example, CsA treatment promotes the rapid growth and survival of renal cancer cells via activating Ras and inducing the expression of cytoprotective molecule heme oxygenase-1 (HO-1).¹⁴⁹ CsA increases expression of activating transcription factor 3 (ATF3) which enhances keratinocyte tumor formation and suppresses cancer cell senescence.¹⁵⁰ Therefore, further investigations are required to explore the downstream effectors of activated CaN in different malignancies to develop more specific inhibitors targeting these effectors for the treatment of cancers, which might overcome limitations linked to direct inhibition of CaN activity.

Concluding remarks and future perspectives

CaN plays a central role in a number of physiological and pathological processes, owing to its ability to control a network of transcriptional regulators coupled to posttranscriptional and posttranslational modifications that amplify the initial signals. CaN is a versatile protein able to modulate several fundamental pathways within the cell including activation, apoptosis, cycle, proliferation, migration, and invasion, as well as stem cell generation, transformation and fate. Inhibiting CaN activity may serve as a promising therapeutic strategy for several diseases. CNIs such as CsA and FK506 have been tested as therapeutic drugs in mouse models of disease and are currently used in clinical settings.¹²⁴ For example, CsA and FK506 have been widely used to prevent organ rejection in transplant patients, as well as for the treatment of aggressive forms of RA and SLE.^4,13 However, their use is associated with certain side effects, including hypertension, nephrotoxicity, neurotoxicity and metabolic disorders.⁴ It is therefore important that further research be conducted to investigate newly discovered molecular mechanisms of CaN activity and regulation, to enable more specific targeting of signaling pathways downstream of CaN activation. Specifically, new inhibitors should be assessed for their ability to interact with specific substrates or sets of substrates to inhibit CaN signal transmission. Thus, more efficient therapies may be designed to prevent excessive intracellular Ca²⁺ and correctly regulate CaN signaling. Importantly, drugs designed to affect CaN function can be applied to many prevalent diseases and pathological processes, with important social, medical and economic impacts.

Author contributions

Writing and original draft preparation: Lei Chen; review and editing: Chunyan Yao; Min Song. All authors have read and agreed to the published version of the manuscript.

Conflict of interests

The authors declare no conflict of interests.

Abbreviations

CaN

calcineurin

Ca²⁺

calcium

CaM

calmodulin

NFAT

nuclear factor of activated T-cells

IL-2

interleukin 2

IL-4

interleukin 4

Drp1

dynamin-related protein 1

NHE1

Na⁺/H⁺-exchanger 1

TRESK

TWIK-related spinal cord K⁺ channel

CRZ1

calcineurin response zinc finger

KSR2

kinase suppressor of ras 2

CNIs

CaN inhibitors

CsA

cyclosporine A

FK506

tacrolimus

CnA

catalytic A subunit

CnB

regulatory B subunit

EF hands

EF-hand motifs

ER

endoplasmic reticulum

Th2

T helper type 2

NZ

nephrogenic zone

IL-17

interleukin 17

IFNγ

interferon-γ

TNF-α

tumor necrosis factor-α

RA

rheumatoid arthritis

SLE

systemic lupus erythematosus

DC

dendritic cell

FKBP12

FK-binding protein 12

DCs

dendritic cells

PRRs

pattern recognition receptors

CLR

C-type lectin receptors

Syk

spleen tyrosine kinase

PLC

phospholipase

BMDCs

bone marrow-derived dendritic cells

COX-2

cyclooxygenase-2

ECM

extracellular matrix

AngII

angiotensin II

AT1-AA

angiotensin II type I receptor agonistic autoantibody

TRPC6

transient receptor potential channel 6

RANK

receptor activator of NF-κB

SYNPO

synaptopodin

uPAR-ITGB3

urokinase plasminogen activator receptor-integrin β3

RAC1

Rac family small GTPase 1

CDC42

cell division cycle 42

CH

cardiac hypertrophy

MEF2

myocyte enhancer factor2

GATA4

GATA binding protein4

NF-κB

nuclear factor-κ-gene binding

PKC

protein kinase C

MAPK

mitogen-activated protein kinases

CaMKII

calmodulin-dependent protein kinase II

PI3–K

phosphatidylinositol 3-kinase

RCAN1

regulator of calcineurin 1

LTP

long-term potentiation

AD

Alzheimer's disease

PD

Parkinson's disease

HD

Huntington's disease

TSEs

transmissible spongiform encephalopathies

ALS

amyotrophic lateral sclerosis

NMDA-R

N-methyl-d-aspartate receptor

GABAARs

γ-aminobutyric acid type A receptors

CREB

cAMP response element-binding

AMPA

amino-3-hydroxy-5-methyl-4-isoxazole propionic acid

pCREB

phosphorylated CREB

BDNF

brain-derived neurotrophic factor

trkB

tropomyosin-related kinase B

CNS

central nervous system

Aβ

amyloid β-protein

MCI

mild cognitive impairment

PPARγ

peroxisome proliferator-activated receptor γ

FOXO3

forkhead box O3

BAD

Bcl2-associated death protein

CRC

colorectal cancer cells

GSN

gelsolin

ERK1/2

extracellular regulated protein kinases1/2

BL

Burkitt's lymphoma

DLBCL

diffuse large B cell lymphoma

GSK-3b

glycogen synthase kinase-3b

NFI

nuclear factor I

KSR2

kinase suppressor of ras 2

MG

malignant glioma

T-ALL

T-cell acute lymphoblastic leukemia

NSCLC

non-small cell lung cancer

SCC

skin squamous cell carcinoma

HO-1

heme oxygenase-1

ATF3

activating transcription factor 3

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Figure 1.

Calcineurin dysfunctions result in many common disorders.