RCAN1 ( Regulator of calcineurin 1; DSCR1, Down syndrome critical region gene 1; Dscr1; ADAPT78; MCIP1, Modulatory calcineurin-interacting protein1; Calcipressin 1, Csp1, CALP1; RCN-1, RCN1, Rcn1p; CBP1, calcineurin binding protein1; Nebula, nla; Sarah, sra); RCAN2 (Regulator of calcineurin 2; Dscr1l1, Dscr1-like 1; MCIP2, Modulatory calcineurin-interacting protein 2; Calcipressin 2; Thyroid Hormone-Responsive Protein ZAKI-4); RCAN3 ( Regulator of calcineurin 3; DSCR1L2, DSCR1-like 2; Dscr1l2; Calcipressin 3)
The protein family of the regulators of calcineurin (RCANs), previously termed as DSCR, MCIP, or calcipressins among other names, has been shown to function as regulators of the calcium-calmodulin regulated phosphatase calcineurin. The gene encoding for the first RCAN protein was cloned more than 20 years ago and named DSCR1, as it was identified in the Down syndrome critical region, which encoded a proline-rich protein highly expressed in the human fetal brain and heart (Fuentes et al. 1995). Although the identification and characterization of new members of the RCAN family in humans and several homolog proteins in other species (Saccharomyces cerevisiae, Caenorhabditis elegans, Cryptococcus neoformans, Drosophila melanogaster, and Mus musculus) was initially focused in the nervous system, RCAN proteins have also been shown to inhibit calcineurin activity in other systems and cell types (Serrano-Candelas et al. 2014). The fact that calcineurin is a serine-threonine phosphatase that plays important roles in different key biological processes, and RCAN proteins regulate the activity of calcineurin under multiple physiological and pathological conditions, makes members of this family involved in the regulation of a multitude of programs of development and differentiation in many cell types and tissues. This chapter reviews the current understanding of the mechanisms that regulate the activity of RCAN proteins and the functions that these proteins have in different cell types and systems.
RCAN Members and Structure
RCAN1: The RCAN1 gene consists of seven exons. Exons 1 to 4 can be spliced yielding different mRNA isoforms, which in turn encode two proteins, RCAN1-1 of 197 or 252 amino acids and RCAN1-4 of 197 amino acids. RCAN1-1 and RCAN 1-4 begin with exon 1 and 4, respectively, and share exons 5, 6, and 7. Interestingly, exon 1 can produce two isoforms due to the fact that it has two different ATG start codons, which generate the long 252-amino acid protein known as RCAN1-1L or the short 197-amino acid protein known as RCAN1-1S (Genesca et al. 2003).
RCAN2: Similar to RCAN1, the RCAN2 gene contains seven exons that encode two proteins, RCAN2-3 and RCAN2-4. Interestingly the RCAN2-3 protein product is generated from either of the two alternatively spliced transcripts, RCAN2-1 or RCAN2-2, due to the fact that the first initiation codon lies within the common exon 3 (Davies et al. 2007).
RCAN3: The RCAN3 gene is also composed of seven exons (where the first three exons are mutually exclusive and non-coding) and produces up to 21 distinct transcripts, out of which so far only 10 have been accepted as complete mRNA transcripts in the NCBI’s RefSeq database. These 10 transcripts generate six final protein products: From these, RCAN3-4, of 241 amino acids, is the only RCAN3 protein detected in human and mice at the endogenous level and it can come from any of these mRNAs: 3-1, 3-2, 3-2a, or 3-3. The remaining protein products are RCAN3-4,5,6a,7; RCAN3-4,5,7; RCAN3-4,7; RCAN3-4,6,7; and finally RCAN3-6,7, which is translated from any of the following mRNAs: 3-2a,4,6,7; 3-2,5,6,7 or 3-1,5,6,7 (Facchin et al. 2011; Serrano-Candelas et al. 2014).
The docking motif is essential for the inhibition of calcineurin and is conserved across all RCANs. Additionally RCANs contain motifs that are specifically required for their stimulatory or inhibitory effects. In vertebrates, all RCAN proteins are remarkably conserved in their central calcineurin-inhibitor RCAN (CIC) and C-terminal PxIIxT regions and diverge in their amino-terminal region. The last three exons are common to all RCAN family members, and their functional differences arise from using alternative transcription start sites (TSS). The first three or four exons of each RCAN gene are mutually exclusive and regulated by different proximal promoter regions.
RRM: The N-terminal domain folds into a conformation similar to that of an RNA recognition motif (RRM) domain.
L, S and E: This includes an LxxP motif (L) and the highly conserved SP domain (a repeat from NFAT) with a site for phosphorylation by GSK-3 (S) and an ExxP motif (E).
CIC: The calcineurin-inhibitor RCAN domain, a PxIxIT-like motif (consensus sequence [PG]x[IV]x[IVL][EDNHT]) responsible for docking substrates to calcineurin and required to inhibit its function.
- 4.P/T: The C-terminal domain contains two overlapping regions: first a PxIIxT motif at exon 7, only present in mammalian RCAN1, that efficiently blocks calcineurin activity by binding to its surface with the help of the LxxP motif. Secondly, there is a TxxP motif, required for the stimulatory effects on calcineurin, which is universally conserved in fungal and animal RCANs with a few exceptions; three budding yeast (Candida albicans, Candida dubliniensis, and Candida tropicalis), two fission yeast (Schizosaccharomyces pombe and Schizosaccharomyces japonicus), and a protist (Dictyostelium discoideum).
Regulation of Calcineurin by RCANs
Specific Functions in Different Systems
RCAN proteins develop their functions in several pathologies as natural inhibitors of the calcium-calcineurin-NFAT signaling pathway. These proteins must be expressed in several mammalian tissues where calcineurin is found such as brain, adipose tissue, adrenal cells, heart, osteoclasts, kidney, liver, T-lymphocytes, lung, pancreas, placenta, platelets, eye, skeletal and smooth muscles, thymus, thyroid, testis, sperm. Their extensive distribution confers RCAN family member-specific functions that act in a cell-dependent manner. Such is the case of RCAN1 in cardiovascular system, where it has a dual role in cardiac hypertrophy depending on the nature of the hypertrophic stimulus (Minami 2014). The absence of calcineurin inhibition caused by RCAN1 deletion leads to drastic cardiac hypertrophy, whereas in response to pressure overload or chronich adrenergic stimulation, cardiac hypertrophy is reduced when RCAN1 is absent. Furthermore, expression of RCAN1 in vascular endothelial cells reduces blood vessel formation and acts as an anti-angiogenic and anti-inflammatory molecule, inhibiting the eNOS (nitric oxide endothelial synthase) production induced by calcineurin (Minami 2014). Thus, RCAN inhibits indirectly the release of nitric oxide among other inflammatory molecules that may increase the mesenteric vasoconstriction. In atherosclerosis progression, bone marrow-derived macrophages in the atherosclerotic plaques upregulate RCAN1 gene expression, whereas RCAN1 expressed in endothelial cells inhibits endothelial cell activation, monocytes cell adhesion, and vessel growth mediated by the endothelial cell-specific mitogen known as vascular endothelial growth factor (VEGF) (Minami 2014) (Fig. 2). RCAN2 has been less studied than the others members, and its initial role was identified in human skin fibroblasts in response to regulation by thyroid hormones (Miyazaki et al. 1996; Davies et al. 2007). Later on, it was shown to be expressed in brain, heart, skeletal muscle, and kidney, having also a role in modulating calcineurin activity in endothelial cells and during angiogenesis (Cao et al. 2002). RCAN3 also plays a role in angiogenesis (Serrano-Candelas et al. 2014; Martinez-Hoyer et al. 2015).
It has been well established that RCAN proteins interact with many other proteins in addition to its main known substrate, calcineurin, thereby influencing other signaling pathways. Recent research has shown interaction of RCANs with proteins such as Tollip (positively modulating interleukin-1 receptor-mediated signaling), NF-kappaB-inducing kinase (which phosphorylates and blocks the degradation of RCAN1), or cardiac troponin I (TNNI3) (Serrano-Candelas et al. 2015). RCAN3 has been reported to bind to cardiac troponin I (TNNI3), the heart-specific inhibitory subunit of the troponin complex, a central component of the contractile apparatus, indicating a possible implication of this protein in cardiac contraction (Facchin et al. 2011; Serrano-Candelas et al. 2014).
RCAN proteins have been associated to other pathologies such as cancer, where overexpression of RCAN1 and RCAN3 member reduces significantly blood vessel formation in tumors and therefore tumor growth (Serrano-Candelas et al. 2014; Martinez-Hoyer et al. 2015). These proteins have different roles in cell migration in several cell types. For example, their overexpression in different cancer lines and endothelial cells reduces motility through calcineurin activity inhibition; however, in smooth muscle cells, they enhance cell migration (Ryeom et al. 2008; Minami 2014). RCAN1 is essential to avoid metastasis of melanoma or lung carcinoma, and in leukemia cells it has been shown to induce apoptosis through calcineurin inhibition, in response to Glucocorticoids (Serrano-Candelas et al. 2014; Minami 2014). RCAN3 is a potent tumor suppressor in human breast cancer models, inhibiting tumor growth and tumor angiogenesis. In addition, an RCAN3-derived peptide can reproduce all the antitumor effects of RCAN3 full-length protein (Martinez-Hoyer et al. 2015). Interestingly, studies with tumor xenografts in Down syndrome mice models suggest that angiogenesis suppression by RCAN1 is in part responsible of the cancer protection found in Down syndrome. The expression of this RCAN family member, which is upregulated by reactive oxygen species generation and the activation of the MAP kinases signaling pathway, alters NFAT regulation through calcineurin inactivation, promoting multiple features of Down syndrome (Baek et al. 2009). Although all RCAN family members can be involved in neurodegeneration, given their tissue distribution mainly in brain, RCAN1 is well known for its role in neurodegeneration (Cao et al. 2002; Facchin et al. 2011; Minami 2014). RCAN1, expressed predominantly in neurons rather than in astrocytes or microglia, has been shown to upregulate in response to multiple stresses such as traumatic injury o brain damage by psychosocial/emotional stress. These stresses are associated to neurodegeneration, where overexpression of RCAN1 is identified as a key factor that contributes, at least in part, to Alzheimer disease. RCAN1 is also overexpressed in Down syndrome and, interestingly an aggressive form of Alzheimer disease takes place at early-onset in some Down syndrome patients. Elevated levels of Tau protein in the cerebro-espinal fluid predict incipient Alzehimer’s and other tautophaties, due to impaired degradation of this protein (Ermak et al. 2009; Ermak et al. 2011). Inhibition of calcineurin by RCAN may lead to the generation of aberrant forms of Tau protein that are not degraded and, consequently, might enhance neurodegenerative disease. In addition, it has been suggested that impaired degradation of RCAN1 through alterations of the proteosome and the lysosome in neurodegeneration (Fig. 2) might be the reason why RCAN1 is overexpressed in Alzheimer‘s disease (Liu et al. 2009). Conversely, expression of one isoform of RCAN1 in brain appears to be deficient in Hungtinton’s disease, and its overexpression has been found to protect against mutant Huntingtin toxicity in vitro (Ermak et al. 2009).
RCAN proteins have been shown to participate in the regulation of the adaptive immune response. The expression of all the murine isoforms of RCAN has been detected in varying quantities in all lymphoid tissues. In humans, all isoforms have also been detected in thymus and liver among other tissues (Serrano-Candelas et al. 2015). RCAN members play an important role inhibiting calcium-calcineurin signaling pathway and thus blocking gene expression that is dependent of NFAT, including cytokine genes (Mulero et al. 2007). In addition, they have been described as negative regulators of T cell activation in response to oxidative radicals (ROS) released upon TCR engagement, modulating the maintenance of an efficient T cell function (Valdor et al. 2014). Recently, RCAN1 and RCAN3 have been shown to be expressed in lymphoid tissues and modulate T cell development. Thymic positive selection is facilitated by the interaction of RCANs with RAF kinases and calcineurin; however, in this case calcineurin activity is not affected (Serrano-Candelas et al. 2015).
Expression of RCANs is highly induced by calcineurin-dependent transcription factors in yeast, nematodes, mammals, and humans. Mice lacking the Rcan1 gene but retaining full function of the other two, RCAN2 and RCAN3, show elevated calcineurin signaling in some tissues. Interestingly, not only the presence or absence of the protein but the level of expression and the phosphorylation state can influence its activity, which has been associated with several human pathologies. For example, high RCAN1 expression reduces calcineurin activity and blocks proliferation of endothelial cells, whereas low RCAN1 expression may hyperactivate calcineurin activity and trigger apoptosis (Ryeom et al. 2008). Furthermore, the expression of RCAN1 is increased in the brains of Down syndrome patients. However, reduced expression in flies has been shown to induce the same characteristic pathologies associated with Down syndrome (Ermak et al. 2009, 2011), suggesting that altered expression of RCAN1 contributes to the neurological defects. Another example of the importance of the regulation of these proteins in neuronal pathologies is the abnormally increased expression of RCAN1 seen in the cerebral cortex of Alzheimer’s disease patients. In this case, a specific isoform, RCAN1-1L, is upregulated in neurons. Not surprisingly RCAN1 transcription is upregulated by the aggregated amyloid Ab peptide in these patients (Ermak et al. 2011). For simplicity, only human RCAN gene regulation will be discussed in the reminder of this section.
The human RCAN1 gene is located on chromosome 21 (HSA 21q22.12) and both of its isoforms, RCAN1–1 and RCAN1–4, are ubiquitously expressed. Basal levels of RCAN1-1 isoform are constitutively expressed, although they can be upregulated by vascular endothelial growth factor (VEGF) and glucocorticoids. In addition, RCAN1-1 has been shown to be downregulated by the Notch signaling pathway. Differently, the RCAN1-4 expression is controlled, among other stimuli, by estrogen hormones, intracellular increase of calcium concentration via multiple NFATc and C/EBPb binding sites in its promoter region, and by osmotic and oxidative stress. The transcription of this isoform is regulated via a negative feedback loop (Fig. 2). In the presence of calcium, NFAT is activated by the phosphatase activity of calcineurin to enhance the expression of RCAN1. Excessive expression of RCAN1 inhibits calcineurin activity and the NFAT signaling pathway, functioning as a negative feedback loop that controls its own expression (Ryeom et al. 2008; Serrano-Candelas et al. 2014). The RCAN2 gene is located on chromosome 6 (HSA 6p12.3). The RCAN2-3 isoform is ubiquitously expressed in various tissues such as brain, heart, muscle, and liver, while the other isoform, RCAN2-4, has only been detected in brain, and it has been shown to be upregulated by the thyroid hormone via an AKT/PKB-dependent signaling pathway (Cao et al. 2002; Davies et al. 2007). RCAN3, located on chromosome 1 (HSA 1p35.3-p33), is constitutively expressed in all tissues, but it is highly abundant in heart, brain, small intestine, lung, testis, prostate, and peripheral blood leukocytes. It has been demonstrated that unlike the other two forms of RCAN, RCAN3 expression is not affected by intracellular calcium concentrations (Mulero et al. 2007). Some studies on the promoter of the gene revealed that RCAN3, like the RCAN1 promoter, contains CpG islands regions, which are not methylated and thus correlate with the transcriptional activity. At least, RCAN3-associated CpG island has been identified to be functional. Furthermore, identification of antisense transcripts in the genes of the three members of RCAN family reveals a high sequence identity among mammals, indicating that they could affect or regulate RCAN gene expression (Facchin et al. 2011; Serrano-candelas et al. 2014).
RCAN proteins have been shown to be also regulated at protein level by degradation. Initially, phosphorylation of RCAN proteins in a specific motif was demonstrated to reduce half-life of these proteins in the cytosol after inhibition of calcineurin (Genesca et al. 2003). Later on, RCAN1 was shown to be overexpressed in neurons, promoting inhibition of the calcineurin-NFAT activity, when the ubiquitin proteosome pathway and chaperone-mediated autophagy (CMA) lysosomes were blocked. Two specific motifs for CMA were founded in RCAN1, indicating that these proteins might be regulated by CMA and proteosome activity depending on the cell stimulus (Liu et al. 2009). Recently, the degradation of RCAN1 by CMA has been also reported in CD4+ T cells. It is well established that T cell activation requires tight regulation of signaling pathways downstream of the T cell receptor (TCR). In this context, autophagy plays an important role in modulating the abundance of negative signaling molecules in the cell. Here RCAN1 acts as a negative regulator of TCR signaling, and its degradation through CMA allows the generation of effective T cell responses. Interestingly, the expression of RCAN1 increases following engagement of the TCR. Thus, in response to ROS production, NFAT activates RCAN1 expression in a feedback loop that regulates NFAT activation by controlling calcineurin activity. CMA participates in this loop by regulating the degradation of RCAN1 and preventing its accumulation in the cytosol, which otherwise could allow for uncontrolled inhibition of calcineurin activity and blocking of efficient T cell activation. It is possible that post-translational modifications additionally account for the degradation of RCAN1 by CMA either by creating new CMA motifs or by promoting conformational changes that expose existing hidden CMA motifs (Valdor et al. 2014).
In vertebrates, the RCAN family is formed by three members, RCAN1, RCAN2, and RCAN3, whereas only RCAN1 is present in the rest of Eukarya. Members of RCAN family have been established as key regulators in cellular programs of development, differentiation, and activation in many cell types and tissues, such as the immune response or muscle fiber remodeling and memory. RCAN proteins bind specifically through defined domains to calcineurin, inhibiting calcineurin activity and therefore modulating the calcineurin-NFATc signaling pathway. The endogenous modulation of this signaling pathway implicates RCAN in many pathological processes, such as cardiac hypertrophy, neurodegenerative diseases, and cancer. Given the wide range of tissue expression of RCAN proteins, it is clear that novel functions of these proteins still remain to be discovered. The tight regulation at both gene and protein levels of RCAN ensures specific regulation of distinct program of gene expression. Specific functions for individual RCAN family members and the characterization of the expression distribution of specific RCAN isoforms during unique cell programs should also lead to a better understanding on how specific programs of development are integrated and modulated by RCANs. Studies into the molecular mechanisms underlying RCAN regulation should also provide new insight for the development of future therapies. The large amount of evidence that clearly involves RCAN function in several pathologies makes these proteins good targets for new therapeutic approaches, for instance, to suppress immune responses regulated by calcineurin-NFAT signaling pathway, but also to design new drugs for the treatment of neurodegenerative diseases, cancer, and other pathologies controlled by this family of proteins.
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