Regulator of Calcineurin 1 (RCAN1)
Regulator of calcineurin 1 (RCAN1) was first isolated by Fuentes et al. in 1995 during a search for genes associated with clinical features of Down syndrome (e.g., mental retardation and congenital heart disease) (Fuentes et al. 1995). Coding sequences of RCAN1 were identified from the 21q22.1–q22.2 regions of human chromosome 21 by an Alu-splice PCR method. It was initially thought that RCAN1 was significantly associated with the Down syndrome phenotype (Fuentes et al. 1997b); thus RCAN1 was first designated Down syndrome critical region 1 (DSCR1). Future studies showed that the DSCR1 gene product is a calcineurin regulator, and the new name “regulator of calcineurin (RCAN)” was adopted to describe the gene function. Although RCAN1 is still referred to as DSCR1, ADAPT78, MCIP1, Calpressin1, RCN-1, Nebular, Sarah, or CBP1, both the HUGO Gene Nomenclature Committee (HGNC) and the Mouse Genomic Nomenclature Committee (MGNC) (Davies et al. 2007) have adopted the RCAN1 nomenclature.
RCAN1 is an endogenous inhibitor of the serine phosphatase calcineurin (Fuentes et al. 2000; Gorlach et al. 2000; Kingsbury and Cunningham 2000). Calcineurin is a heterodimer that is composed of the catalytic subunit calcineurin A (CnA) and regulatory subunit calcineurin B (CnB). RCAN1 directly binds to CnA and inhibits the catalytic activity of calcineurin. Calcineurin controls the phosphorylation/dephosphorylation of several transcription factors, such as the nuclear factor of activated T-cells (NFAT) (Crabtree and Olson 2002), CREB (Liu and Graybiel 1996), and the MEF2 transcription factor families (Mao and Wiedmann 1999). Dephosphorylation of NFAT by activated calcineurin promotes translocation of NFAT to the nucleus, where the transcription factor binds to DNA and activates gene transcription (Crabtree and Olson 2002). A wide variety of physiological processes, such as lymphocyte activation (Fruman et al. 1995), neurite outgrowth (Chang et al. 1995), aging (Mair et al. 2011), heart development (Yang et al. 2000), skeletal muscle fiber type differentiation (Olson and Williams 2000), and cardiac function (Ryeom et al. 2003) are regulated by calcineurin-activated transcription factors. Thus, RCAN1, which controls calcineurin activity, regulates various physiological functions.
Domains and Motifs of RCAN1
The PxIxIT-like and LxxP motifs appear to be important for the stimulation of calcineurin signals at low RCAN1 expression level in addition to their roles in inhibition at high expression level. The TxxP motif, which is adjacent to the PxIIxT motif at exon 7, is only required for the stimulatory effects (Mehta et al. 2009). Together with the FLISPPxSPP motif phosphorylation site (see Inhibitory and Stimulatory Effects of RCAN1), these motifs are important for RCAN1-mediated stimulation of calcineurin signaling (Fig. 2).
RCAN1 is highly expressed in the human fetal brain and adult heart. Lower expression levels have been detected in the adult brain, lung, liver, skeletal muscle, kidney, and pancreas. The fetal lung, liver, kidney, and placenta also have low RCAN1 expression (Fuentes et al. 1995). RCAN1 expression in rat and mice is similar to the associated human tissues, and there is high expression in the rodent brain and heart (Fuentes et al. 1997a; Fuentes et al. 1995). In the rat brain, the in situ hybridization signal for RCAN1 expression was high in the olfactory bulb, the piriform cortex, the dentate granule cell layer, the pyramidal cell layer of the hippocampus, the striatum, and the cerebellar cortex. No signal for RCAN1 expression was detected in the white matter (Fuentes et al. 1995). The brains of 2- to 7-day-old neonatal rats had higher RCAN1 expression in the neocortex and the hypothalamus compared to adult rats older than 16 days (Fuentes et al. 1995).
RCAN1 expression is diverse throughout many different cell and tissue types and is regulated by various stimuli. Vascular endothelial growth factor (VEGF) (Minami et al. 2004; Yao and Duh 2004), thrombin (Minami et al. 2004), oxidative stress (Crawford et al. 1997), Ca2+-mediated stress (Cano et al. 2005), ß-amyloid fragments (Ermak et al. 2001), TNFα (Minami et al. 2004; Yao and Duh 2004), and endoplasmic reticulum stress (Zhao et al. 2008) have been reported to induce RCAN1 expression. Oxidative stress in hamster HA-1 cells induced RCAN1 expression as early as 90 min after peroxide exposure, indicating that the transcriptional response of RCAN1 is rapid and robust. A previous study indicated that the maximal increase in RCAN1 expression was 7.8-fold after 5 h of initial exposure (Crawford et al. 1997). Similarly, rapid and robust RCAN1 expression occurs with other types of stimuli, such as VEGF and thrombin. After 1 h of treatment, VEGF and thrombin stimulation increased RCAN1 expression in human endothelial cells by 22.3-fold and 17.7-fold, respectively (Minami et al. 2004).
RCAN1 transcription is regulated via a negative feedback loop with RCAN1-4, which is an isoform that is abundant in the fetal kidney, adult heart, placenta, and skeletal muscle (Ermak et al. 2002; Fuentes et al. 1997a, 2000). RCAN1 expression is induced by the transcription factor NFAT, and NFAT activation and nuclear translocation are regulated by calcineurin phosphatase activity. Therefore, an excessive amount of RCAN1 inhibits calcineurin and attenuates the calcineurin-NFAT signaling pathway, thereby establishing a negative feedback loop that suppresses its own expression. Calcineurin/NFAT signal-dependent RCAN1-4 transcription is controlled by a promoter region that is located upstream of exon 4 (between nucleotides −350 and −166); this promoter region also contains putative NFAT and AP-1 binding sites (Cano et al. 2005; Yang et al. 2000; Zhao et al. 2008). In contrast, RCAN1-1L, which is the isoform that is predominantly expressed in the fetal and adult brains (Ermak et al. 2002; Fuentes et al. 1997a, 2000), is controlled by a conserved muscle-specific CAT (M-CAT) site located 1426 bp upstream of exon 1 (Liu et al. 2008). Transcription enhancer factor 3 directly interacts with the M-CAT site in the promoter and is required for RCAN1-1L expression (Liu et al. 2008).
Inhibitory and Stimulatory Effects of RCAN1
RCAN1 is an endogenous inhibitor of calcineurin; RCAN1 overexpression inhibits NFAT-mediated calcineurin signaling by directly binding to the catalytic subunit of calcineurin (Fuentes et al. 2000). However, RCAN1 gene disruption also inhibits calcineurin-NFAT signaling instead of stimulating the signaling. In yeast, disruption of the RCAN1 orthologous gene (RCN1) results in significantly decreased calcineurin-NFAT signaling (Kingsbury and Cunningham 2000). In RCAN1−/− mice, calcineurin activity is decreased in response to cardiac hypertrophy induced by pressure overload (Vega et al. 2003). These conflicting results suggest reciprocal effect of RCAN1 to the calcineurin activities, as high RCAN1 expression is associated with calcineurin inhibition, while physiological levels of RCAN1 expression are associated with calcineurin stimulation.
A number of reports have indicated that phosphorylation of RCAN1 is important for RCAN1-mediated biphasic effects on calcineurin. Using both human and yeast RCAN1, Hilioti et al. showed that RCAN1-mediated calcineurin stimulation requires phosphorylation at the conserved FLISPPxSPP motif when RCAN1 is expressed at low concentrations (Hilioti et al. 2004). Moreover, Liu et al. also showed that phosphorylation of low concentrations of RCAN1 activates calcineurin-NFAT signaling (Liu et al. 2009). Because the phosphorylated FLISPPxSPP RCAN1 motif is rapidly degraded by the SCFCdc4 ubiquitin ligase complex (Genesca et al. 2003; Kishi et al. 2007), it has been hypothesized that phosphorylated RCAN1 rapidly degrades and releases calcineurin from the inactive complex (Kishi et al. 2007). RCAN1-mediated stimulation of calcineurin activity is observed only at physiological RCAN1 expression levels because the net balance of phosphorylated RCAN1 is much higher. On the other hand, when RCAN1 is overexpressed, a majority of the protein is not phosphorylated; this stable RCAN1 binds to calcineurin, which covers the catalytic domain and inhibits phosphatase activity. Overexpressed RCAN1 also increases calcineurin proteolysis (Lee et al. 2009), which also contributes to RCAN1-mediated inhibition. Collectively, RCAN1 inhibits calcineurin signaling by binding to calcineurin and interfering with substrate binding; phosphorylated RCAN1 has a shorter half-life and appears to be stimulatory when expressed at low levels.
Role of RCAN1 in Angiogenesis
Patients with Down syndrome have an extremely low incidence of solid tumors, which indicates that RCAN1 plays an important role in both angiogenesis and tumor development (Hasle 2001). RCAN1 gain-of-function studies show that constitutive expression of RCAN1 in endothelial cells impairs NFAT nuclear localization, proliferation, and tube formation. RCAN1 also reduces vascular density in Matrigel plugs and melanoma tumor growth in mice (Minami et al. 2004). Another study with transgenic mice with three copies of RCAN1 showed that these mice have significantly suppressed growth of Lewis lung carcinoma and B16F10 melanoma cells in vivo. Moreover, a study by Baek et al. showed that a modest increase in RCAN1 expression (2.4-fold increase in mRNA relative to littermate controls) is sufficient for tumor growth suppression (Baek et al. 2009). In Xenopus laevis, overexpression of RCAN1 decreased the number of branching points that sprouted from intersomitic vessels and decreased the vascular density of the microvessels (Fujiwara et al. 2011).
Consistent with the previous loss-of-function studies, studies in RCAN1−/− mice have shown that RCAN1 inhibits angiogenesis. RCAN1 deletion suppressed subcutaneous and metastatic tumor growth, and endothelial cells isolated from these knockout mice showed decreased VEGF-induced proliferation (Ryeom et al. 2008). Specific knockdown of RCAN1 expression by antisense oligonucleotides also inhibited VEGF-stimulated migration of endothelial cells (Iizuka et al. 2004). Researchers have hypothesized that the appropriate RCAN1 expression level, as well as the phosphorylation state, influences RCAN1 regulation of calcineurin activity. High RCAN1 expression may reduce calcineurin activity and block proliferation of endothelial cells, whereas low RCAN1 expression may hyperactivate calcineurin activity and trigger apoptosis (Ryeom et al. 2008).
RCAN1 and Down Syndrome
Because RCAN1 is highly expressed in the brains of Down syndrome patients, it is thought to be associated with the Down syndrome phenotype (Fuentes et al. 1995). Overexpression of the Drosophila ortholog of RCAN1 (nebula) causes neuronal defects that are similar to Down syndrome, such as impaired synaptic development, synaptic terminal structure, vesicle recycling, and locomotor activity (Chang and Min 2009). Additionally, reduction or overexpression of nebula impairs mitochondrial enzyme activity, the number and size of mitochondria, and accumulation of toxic reactive oxygen species (ROS) in the fly brains, which are all characteristic of pathologies associated with Down syndrome (Chang and Min 2005). These results strongly suggest that altered expression of RCAN1 contributes to the neurological defects in Down syndrome. Furthermore, cardiac defects, which are another common feature associated with Down syndrome, have been reported in RCAN1/DYRK1A double transgenic mice. A craniofacial defect was also reported in Nfatc2−/−/Nfatc4−/− double-knockout mice, which have impaired RCAN1-calcineurin-NFAT signaling (Arron et al. 2006). These results indicate that RCAN1 is an important gene that is associated with the Down syndrome phenotype.
RCAN1 and Alzheimer’s Disease
Overexpression of RCAN1 has been observed in Alzheimer’s disease patients’ brains, where RCAN1 expression was 2-fold higher in the cerebral cortex and 3-fold higher in the hippocampus (Ermak et al. 2001). Of the RCAN isoforms that are expressed in the brain, RCAN1-1L is upregulated in the neurons of Alzheimer’s disease patients (Harris et al. 2007). Overexpressed RCAN1 colocalizes with the neurodegenerative disease-associated proteins huntingtin (Q148) and ataxia-3 (Q84) in cultured primary neurons (Ma et al. 2004). Additionally, RCAN1 expression is directly stimulated by the aggregated amyloid Aß peptide, which is a peptide that plays a role in neuronal degeneration in Alzheimer’s disease and human neuroblastoma cell lines (Ermak et al. 2001).
Functional analyses of RCAN1 further indicate that it is closely associated with Alzheimer’s disease. Hypomorphic fly mutants with RCAN1 overexpression have decreased long-term memory compared to control D. melanogaster, which display 40% memory retention 24 hr after training. Correspondingly, calcineurin activity is 40% higher in these RCAN1 mutants (Chang et al. 2003). Inside the cell, RCAN1 regulates the number of vesicles undergoing exocytosis and the speed of vesicle fusion, which opens and closes the pore (Keating et al. 2008). These data indicate that RCAN1 is highly associated with neuronal memory and learning and is thus a strong candidate for Alzheimer’s disease neuropathology.
Other Unique Functions of RCAN1
Interestingly, female-specific RCAN1 functions have been reported in non-vertebrates, such as C. elegans and D. melanogaster. In C. elegans, the RCAN1 ortholog (RCN-1) is expressed in the vulva epithelial and muscle cells, and overexpression of RCN-1 results in egg retention (Lee et al. 2003). Calcineurin null mutants, which carry a large deletion in the calcineurin B regulatory subunit gene, have similar defects in fertility and egg-laying. In D. melanogaster, the RCAN1 ortholog (sarah) is expressed in the oocytes and nurse cells of normal flies and is critical for ovulation and female courtship behavior. Inhibition of sarah expression decreases the number of eggs laid, and a majority of the eggs arrest at metaphase I of meiosis (Ejima et al. 2004). Moreover, misexpression affects female courtship behavior, and D. melanogaster virgin mutant females frequently display extrusion behavior (Ejima et al. 2004). These studies indicate that RCAN1 may exert female-specific effects via regulation of calcineurin signaling.
Breakthrough in understanding the RCAN1 function is found in mitochondria (Chang and Min 2005). A Drosophila homolog of RCAN1, nebular, is found to be localized in the mitochondria as well as to cytoplasm and nucleus. Consistent with this finding, defects in mitochondrial respiration, generation of reactive oxygen species (ROS), and the number and size of mitochondria are shown in nebular hypomorphic mutant and transgenic flies. The role of nebular in mitochondria is calcineurin independent, and its signaling pathway was different from the conventional pathway. Furthermore, a new binding partner, mitochondrial adenine nucleotide translocator (ANT), has also been identified. ANT is the main component of the mitochondrial permeability transition pore (mtPTP) and regulates opening of mtPTP. Since opening of mtPTP initiates mitochondrial degradation and cell death (Javadov and Karmazyn 2007), nebular could exert its cytotoxic effects by regulating ANT-mtPTP. These mitochondria-related results in Drosophila suggest a critical role of RCAN1 in cell survival.
Other reports from human and rat neuronal cells also indicate similar effects of RCAN1 in mitochondria (Ermak et al. 2012). First, a significant decrease in mitochondrial number is found in RCAN1-1L overexpressed mammal neuronal cells. Second, mtPTP opening is observed in cells expressing RCAN1. Third, mtPTP opening causes mitochondrial autophagy, which eventually reduces the cell survival. These data in mammals confirm the role of RCAN1 in mitochondria.
RCAN1 plays an important role in the cell by regulating the multifunctional phosphatase calcineurin. RCAN1 function remains an area of ongoing investigation, although a number of reports have identified several RCAN1 molecular mechanisms. However, there is little information regarding the functional difference(s) between the RCAN1 isoforms, which are differentially expressed by various human tissues. Previous reports have indicated that RCAN1-1L overexpression activates the transcription factor NFAT and promotes pathologic angiogenesis in human endothelial cells, while RCAN1-4 inhibits angiogenesis (Qin et al. 2006). The mechanism for these opposing effects of the two isoforms remains unknown because this data does not agree with the current model of dual RCAN1 function. Moreover, additional analyses of the nonconserved N-terminal is needed. The N-terminus of RCAN1 contains an aggregation-prone domain, and overexpression of RCAN1 results in the formation of aggresome-like aggregates in cultured primary neurons, and the number of synapses is reduced in these neurons (Ma et al. 2004). Therefore, studies of the RCAN1 N-terminus may be informative for neurodegenerative diseases, such as Down syndrome or Alzheimer’s disease. In conclusion, RCAN1, which is an endogenous inhibitor of calcineurin, has a wide variety of physiological functions; future studies are necessary to identify the molecular mechanisms of RCAN1.
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