Regulator Of Calcineurin Research Articles

Regulation of vascular function by RCAN1 ( ADAPT78)

RCAN1 (Adapt78) functions mainly, if not exclusively, as a regulator of calcineurin, a phosphatase that mediates many cellular responses to calcium. Identification of this regulatory activity has led to a surge of interest in RCAN1, since calcineurin is involved in many cellular and tissue functions, and its abnormal expression is associated with multiple pathologies. Recent studies have implicated RCAN1 as a regulator of angiogenesis. To more fully investigate the role of RCAN1 in vascular function, we first extended previous studies by assessing RCAN1 response in cultured endothelial cells to various vascular agonists. Strong induction of isoform 4 but not isoform 1 was observed in human umbilical vein- and bovine pulmonary aortic-endothelial cells in response to VEGF, thrombin, and ATP but not other agonists. Inductions were both calcium and calcineurin dependent, with the relative effect of each agonist cell-type dependent. Ectopic RCAN1 expression also inhibited calcineurin signaling in the HUVEC cells. Based on these strong RCAN1 responses and a lack of RCAN1-associated vascular studies beyond angiogenesis, we investigated the potential role of RCAN1 in vascular tone using whole mounted mesenteric artery. RCAN1 knockout mice exhibited an attenuated mesenteric vasoconstriction to phenylephrine as compared with wild-type. Overall contractility was unaffected, suggesting that this component of smooth muscle action is similar in the two mouse strains. Constriction in the knockout artery appeared to be potentiated by the addition of the nitric oxide synthase (NOS) inhibitor l-NAME, suggesting that elevated nitric oxide (NO) production occurs in the knockout vasculature and contributes to the weakened vasoconstriction. Our results reveal a newly identified vascular role for RCAN1, and a potential new target for treating vascular- and calcineurin-related disorders.

The Down Syndrome Critical Region Protein RCAN1 Regulates Long-Term Potentiation and Memory via Inhibition of Phosphatase Signaling

Regulator of calcineurin 1 (RCAN1/MCIP1/DSCR1) regulates the calmodulin-dependent phosphatase calcineurin. Because it is located on human chromosome 21, RCAN1 has been postulated to contribute to mental retardation in Down syndrome and has been reported to be associated with neuronal degeneration in Alzheimer's disease. The studies herein are the first to assess the role of RCAN1 in memory and synaptic plasticity by examining the behavioral and electrophysiological properties of RCAN1 knock-out mice. These mice exhibit deficits in spatial learning and memory, reduced associative cued memory, and impaired late-phase long-term potentiation (L-LTP), phenotypes similar to those of transgenic mice with increased calcineurin activity. Consistent with this, the RCAN1 knock-out mice display increased enzymatic calcineurin activity, increased abundance of a cleaved calcineurin fragment, and decreased phosphorylation of the calcineurin substrate dopamine and cAMP-regulated phosphoprotein-32. We propose a model in which RCAN1 plays a positive role in L-LTP and memory by constraining phosphatase signaling.

Identification and analysis of human RCAN3 ( DSCR1L2) mRNA and protein isoforms

Human RCAN3 (Regulator of calcineurin 3; previously known as DSCR1L2, Down syndrome critical region gene 1-like 2) is a five-exon gene mapped on chromosome 1 and belongs to the human RCAN gene family which also includes RCAN1 and RCAN2. The novel denomination RCAN for genes and proteins, instead of DSCR1L (Down syndrome critical region gene 1-like) has recently been widely discussed. The aim of the present work was to perform a multiple approach analysis of five RCAN3 mRNA and encoded protein isoforms, two of which have been identified for the first time in this research. The two new RCAN3 mRNA isoforms, RCAN3-2,4,5, which lacks exon 3, and RCAN3-2,3,5, which lacks exon 4, were identified during RCAN3 RT-PCR (reverse transcription-polymerase chain reaction) cloning, the product of which unexpectedly revealed the presence of five isoforms as opposed to the three previously known. In order to analyze the expression pattern of the five RCAN3 mRNA isoforms in seven different human tissues, a quantitative relative RT-PCR was performed: interestingly, all isoforms are present in all tissues investigated, with a statistically significant constant prevalence of RCAN3 isoform (the most complete, “reference” isoform). The RCAN3 locus expression level was comparable in all seven tissues analyzed, considering all isoforms, which indicates a ubiquitous expression of this human RCAN family member. To date two possible interactors have been described for this protein: human cardiac troponin I (TNNI3) and calcineurin. Here we report the interaction between the new RCAN3 variants and TNNI3, demonstrated by both yeast cotransformation and by the GST (glutathione-sepharose transferase) fusion protein assay, as was to be expected from the presence of exon 2 whose product has been seen to be sufficient for binding to TNNI3.

RCAN1 (DSCR1) increases neuronal susceptibility to oxidative stress: a potential pathogenic process in neurodegeneration

Oxidative stress (OS) underlies neuronal dysfunction in many neurodegenerative disorders. Regulator of Calcineurin 1 (RCAN1 or DSCR1) is a dose-sensitive gene whose overexpression has been linked to Down syndrome (DS) and Alzheimer's disease (AD) neuropathology and to the response of cells to stress stimuli. Here, we show that RCAN1 mRNA and protein expression are sensitive to OS in primary neurons, and we evaluate the involvement of RCAN1 dosage in neuronal death induced by OS. We find that Rcan1(-/-) neurons display an increased resistance to damage by H(2)O(2), which can be reverted by RCAN1 overexpression or by exogenous inhibitors of calcineurin. Although increased intracellular Ca(2+) concentration is an important factor in OS-mediated cell death, our results show that Ca(2+) loading after exposure to H(2)O(2) was similar in Rcan1(+/+) and Rcan1(-/-) neurons. Our data further suggest that CaN and NFAT signaling protect against OS in both Rcan1(+/+) and Rcan1(-/-) neurons. To explain the observed differential vulnerability, we therefore propose a mechanism downstream of H(2)O(2)-mediated Ca(2+) entry, involving calcineurin-NFAT signaling. These findings highlight the importance of RCAN1 gene dosage in the modulation of cell survival and death pathways and suggest that changes in the amount of RCAN1 could represent an important mechanism for regulating susceptibility to neurodegeneration, especially in DS and AD.

The Drosophila Calcipressin Sarah Is Required for Several Aspects of Egg Activation

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.

The Calcineurin Regulator Sra Plays an Essential Role in Female Meiosis in Drosophila

Modulatory calcineurin-interacting proteins (MCIPs)--also termed regulators of calcineurin (RCNs), calcipressins, or DSCR1 (Down's syndrome critical region 1)--are highly conserved regulators of calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase . Although overexpression experiments in several organisms have revealed that MCIPs inhibit calcineurin activity , their in vivo functions remain unclear. Here, we show that the Drosophila MCIP sarah (sra) is essential for meiotic progression in oocytes. Eggs from sra null mothers are arrested at anaphase of meiosis I. This phenotype was due to loss of function of sra specifically in the female germline. Sra is physically associated with the catalytic subunit of calcineurin, and its overexpression suppresses the phenotypes caused by constitutively activated calcineurin, such as rough eye or loss of wing veins. Hyperactivation of calcineurin signaling in the germline cells resulted in a meiotic-arrest phenotype, which can also be suppressed by overexpression of Sra. All these results support the hypothesis that Sra regulates female meiosis by controlling calcineurin activity in the germline. To our knowledge, this is the first unambiguous demonstration that the regulation of calcineurin signaling by MCIPs plays a critical role in a defined biological process.

Crystal structure of TM1367 from Thermotoga maritima at 1.90 Å resolution reveals an atypical member of the cyclophilin (peptidylprolyl isomerase) fold

Crystal structure of TM1367 from <i>Thermotoga maritima</i> at 1.90 Å resolution reveals an atypical member of the cyclophilin (peptidylprolyl isomerase) fold

Calreticulin is an upstream regulator of calcineurin

Ca 2+ is a signalling molecule involved in virtually every aspect of cell function. The endoplasmic reticulum (ER) is an important and dynamic organelle responsible for storage of the majority of intracellular Ca 2+. Within the ER lumen are proteins that function as Ca 2+ buffers and/or molecular chaperones including calreticulin, a multifunctional Ca 2+-binding protein. Calreticulin-deficiency is lethal in utero due to impaired cardiac development. In the absence of calreticulin Ca 2+ storage capacity in the ER and InsP 3 receptor mediated Ca 2+ release from ER are compromised. Remarkably, over-expression of constitutively active calcineurin in the hearts of calreticulin deficient mice rescues them from embryonic lethality and produces live calreticulin deficient animals. These observations provide first evidence that calreticulin is a key upstream regulator of calcineurin in the Ca 2+-signalling cascade and they highlight the importance of ER during early stages of cellular commitment and tissue development during organogenesis.

Anticoagulants in the treatment of small cell carcinoma of the bronchus.

The object of this study was to see if the addition of anticoagulants to a regimen of cytotoxic drugs would improve the prognosis in patients with small cell carcinoma of the bronchus. Twenty-four patients were randomly allocated to receive chemotherapy or chemotherapy plus anticoagulants. The median survival in the group receiving the anticoagulants was not improved.