Calcineurin-dependent Signaling Research Articles

Rcn1, the fission yeast homolog of human DSCR1, regulates arsenite tolerance independently from calcineurin.

Calcineurin (CN) is a conserved Ca2+/calmodulin-dependent phosphoprotein phosphatase that plays a key role in Ca2+ signaling. Regulator of calcineurin 1 (RCAN1), also known as Down syndrome critical region gene 1 (DSCR1), interacts with calcineurin and inhibits calcineurin-dependent signaling in various organisms. Ppb1, the fission yeast calcineurin regulates Cl--homeostasis, and Ppb1 deletion induces MgCl2 hypersensitivity. Here, we characterize the conserved and novel roles of the fission yeast RCAN1 homolog rcn1+. Consistent with its role as an endogenous calcineurin inhibitor, Rcn1 overproduction reproduced the calcineurin-null phenotypes, including MgCl2 hypersensitivity and inhibition of calcineurin signaling upon extracellular Ca2+ stimuli as evaluated by the nuclear translocation and transcriptional activation of the calcineurin substrate Prz1. Notably, overexpression of rcn1+ causes hypersensitivity to arsenite, whereas calcineurin deletion induces arsenite tolerance, showing a phenotypic discrepancy between Rcn1 overexpression and calcineurin deletion. Importantly, although Rcn1 deletion induces modest sensitivities to arsenite and MgCl2 in wild-type cells, the arsenite tolerance, but not MgCl2 sensitivity, associated with Ppb1 deletion was markedly suppressed by Rcn1 deletion. Collectively, our findings reveal a previously unrecognized functional collaboration between Rcn1 and calcineurin, wherein Rcn1 not only negatively regulates calcineurin in the Cl- homeostasis, but also Rcn1 mediates calcineurin signaling to modulate arsenite cytotoxicity.

Calcineurin mediates the inhibitory effects of TNF on NKCC2 phosphorylation

We previously showed that TNF inhibits NKCC2 activity and phosphorylation along the TAL, however, the mechanism has not yet been defined. As calcineurin (CN) activity in synoviocytes was increased by proinflammatory cytokines such as IL-1 and TNF, we tested the hypothesis that induction of CN activity and expression of CN isoforms is part of a pathway by which TNF inhibits NKCC2 phosphorylation. In this study, we found that CN activity increased by approximately 2-fold in primary cultures of medullary thick ascending limb (mTAL) cells that were maintained at 37oC/5%CO2 then challenged with mouse recombinant TNF (1 nM) for 60 min. Similarly, TNF induced a 3-fold increase in CN activity when mTAL cells were exposed to hypertonic media (400 mOsm/Kg H2O), an effect that was prevented by cyclosporine A (CsA). In contrast, silencing TNF production in mTAL cells using lentivirus (U6-TNF-ex4) significantly reduced CN activity by approximately 56% in cells exposed to 400 mOsm/Kg H2O for 1 h. CN activity also was inhibited in cells pretreated with CsA for 30 min followed by incubation in 400 mOsm/Kg H2O. Our data indicate that NKCC2 phosphorylation was significantly decreased in cultured mTAL cells challenged with TNF (1 nM) for 4 hr while inhibition of CN activity with CsA increased pNKCC2 expression. Analysis of qRT-PCR experiments showed that mTAL cells express the calcineurin A subunit (CNA) isoforms a (Sequence ID: NM_001293622.1) and b (sequence ID: NM_001310426.1). However, only the mRNA abundance of the CNAb isoform increased when mTAL cells were incubated with TNF (1 nM) for 4 hr under NS or HS (400 mOsm/Kg H2O) conditions as TNF had no effect on the a isoform of CNA. In vivo, both TNF and CNAb expression increased in outer medulla (OM) from mice given 1% NaCl in the drinking water (HS) for 7 days while intrarenal silencing of TNF with the U6-TNF-ex4 lentivirus reduced expression of CNAb under NS or HS conditions. Similarly, intrarenal injection of a lentivirus construct that specifically silenced CNAb in the kidney (U6-CNAb-ex6) reduced expression of CNAb in mice ingesting HS for 7 days. Interestingly, co-administration of U6-CNAb-ex6 with U6-TNF-ex4 increased pNKCC2 expression by approximately 63% in the OM from mice compared with silencing of TNF alone. Collectively, these data are the first to demonstrate that TNF increases CN activity as well as specific isoform expression in the kidney. Since NKCC2 has been identified as a target of the CNAb isoform, which is increased by TNF, these data demonstrate that a calcineurin-dependent signaling pathway is part of the mechanism by which TNF inhibits the phosphorylation of NKCC2. NIH grants R01 HL133077 and HL153525. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cyclophilin BcCyp2 Regulates Infection-Related Development to Facilitate Virulence of the Gray Mold Fungus Botrytis cinerea.

Cyclophilin (Cyp) and Ca2+/calcineurin proteins are cellular components related to fungal morphogenesis and virulence; however, their roles in mediating the pathogenesis of Botrytis cinerea, the causative agent of gray mold on over 1000 plant species, remain largely unexplored. Here, we show that disruption of cyclophilin gene BcCYP2 did not impair the pathogen mycelial growth, osmotic and oxidative stress adaptation as well as cell wall integrity, but delayed conidial germination and germling development, altered conidial and sclerotial morphology, reduced infection cushion (IC) formation, sclerotial production and virulence. Exogenous cyclic adenosine monophosphate (cAMP) rescued the deficiency of IC formation of the ∆Bccyp2 mutants, and exogenous cyclosporine A (CsA), an inhibitor targeting cyclophilins, altered hyphal morphology and prevented host-cell penetration in the BcCYP2 harboring strains. Moreover, calcineurin-dependent (CND) genes are differentially expressed in strains losing BcCYP2 in the presence of CsA, suggesting that BcCyp2 functions in the upstream of cAMP- and Ca2+/calcineurin-dependent signaling pathways. Interestingly, during IC formation, expression of BcCYP2 is downregulated in a mutant losing BcJAR1, a gene encoding histone 3 lysine 4 (H3K4) demethylase that regulates fungal development and pathogenesis, in B. cinerea, implying that BcCyp2 functions under the control of BcJar1. Collectively, our findings provide new insights into cyclophilins mediating the pathogenesis of B. cinerea and potential targets for drug intervention for fungal diseases.

Long-Term Decreases in the Expression of Calcineurin and GABAA Receptors Induced by Early Maternal Separation Are Associated with Increased Anxiety-Like Behavior in Adult Male Rats

Introduction: Early life stress is a well-described risk factor of anxiety disorders in adulthood. Dysfunction in GABA/glutamate receptors and their functional regulator, calcineurin, is linked to anxiety disorders. Here, we investigated the effect of early life stress, such as repeated maternal separation (MS; 3 h per day from postnatal day [P] 2 to 11), on changes in the expression of calcineurin as well as the ionotropic glutamatergic and GABAergic receptors including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and GABA_A receptors in the hippocampus and prefrontal cortex (PFC) of adolescent (P35) and adult (P62) male Wistar rats and their correlations with anxiety-like behavior in adulthood. Methods: The protein levels were assessed by Western blot analysis. Anxiety-like behavior was measured in the elevated plus maze (EPM) and open field (OF) tests. Results: MS induced a regional transient decrease of glutamate receptors expression at P35, with decreased NMDA and AMPA receptor levels, respectively, in the hippocampus and PFC, suggesting a possible decrease in excitatory synaptic strength. In contrast to glutamate receptors, MS had long-lasting influence on GABA_A receptor and calcineurin levels, with reduced expression of GABA_A receptor and calcineurin in both brain regions at P35 that continued into adulthood. These results were accompanied by increased anxiety behavior in adulthood, shown by lower percentage of number of total entries and time spent in the open arms of the EPM, and by lower time spent and number of entries in the OF central area. Conclusions: Together, our study suggests that GABA_A receptors via calcineurin-dependent signaling pathways may play an important role in the expression of stress-induced anxiety-like behavior.

Canonical Transient Potential Receptor-3 Channels in Normal and Diseased Airway Smooth Muscle Cells.

All seven canonical transient potential receptor (TRPC1-7) channel members are expressed in mammalian airway smooth muscle cells (ASMCs). Among this family, TRPC3 channel plays an important role in the control of the resting [Ca2+]i and agonist-induced increase in [Ca2+]i. This channel is significantly upregulated in molecular expression and functional activity in airway diseases. The upregulated channel significantly augments the resting [Ca2+]i and agonist-induced increase in [Ca2+]i, thereby exerting a direct and essential effect in airway hyperresponsiveness. The increased TRPC3 channel-mediated Ca2+ signaling also results in the transcription factor nuclear factor-κB (NF-κB) activation via protein kinase C-α (PKCα)-dependent inhibitor of NFκB-α (IκBα) and calcineurin-dependent IκBβ signaling pathways, which upregulates cyclin-D1 expression and causes cell proliferation, leading to airway remodeling. TRPC3 channel may further interact with intracellular release Ca2+ channels, Orai channels and Ca2+-sensing stromal interaction molecules, mediating important cellular responses in ASMCs and the development of airway diseases.

Testin protects against cardiac hypertrophy by targeting a calcineurin-dependent signalling pathway.

Multiple organs express testin (TES), including the heart. Nevertheless, current understanding of the influence of TES on cardiovascular diseases, especially on cardiac hypertrophy and its etiology, is insufficient. This study investigated the influence of TES on cardiac hypertrophy and its etiology. Murine models with excessive TES expression specific to the heart were constructed with an adeno‐associated virus expression system. Cardiac hypertrophy was stimulated through aortic banding (AB). The severity of cardiac hypertrophy was evaluated through molecular, echocardiographic, pathological, and hemodynamic examination. The findings of our study revealed that TES expression was remarkably suppressed not only in failing human hearts but also in mouse hearts with cardiac hypertrophy. It was discovered that excessive TES expression driven by an adeno‐associated viral vector noticeably inhibited hypertrophy triggered by angiotensin II (Ang II) in cultivated cardiomyocytes from newborn rats. It was also revealed that TES knockdown via AdshTES caused the reverse phenotype in cardiomyocytes. Furthermore, it was proved that excessive TES expression attenuated the ventricular dilation, cardiac hypertrophy, dysfunction, and fibrosis triggered by AB in mice. It was discovered that TES directly interacted with calcineurin and suppressed its downstream signalling pathway. Moreover, the inactivation of calcineurin with cyclosporin A greatly offset the exacerbated hypertrophic response triggered by AB in TES knockdown mice. Overall, the findings of our study suggest that TES serves as a crucial regulator of the hypertrophic reaction by hindering the calcineurin‐dependent pathway in the heart.

Cardiac apoptosis induced under high glucose condition involves activation of IGF2R signaling in H9c2 cardiomyoblasts and streptozotocin-induced diabetic rat hearts

The insulin-like growth factor type 2 receptor (IGF2R) overexpression has been implicated in heart disease progression. Unregulated IGF2R signaling triggers cardiac hypertrophy, apoptosis, and cardiomyopathies. The present study investigated the role of IGF2R in cardiomyocyte apoptosis under high glucose (HG) levels and in streptozotocin (STZ) induced diabetic rat hearts. We found that IGF2 and IGF2R protein expression were highly upregulated under high glucose condition in H9c2 cells as well as in STZ induced diabetic rat hearts. Using immunoblotting and TUNEL assay, we found that elevated glucose condition induced IGF2R expression leads to activation of Gαq mediated calcineurin-dependent signaling pathway, which further leads to downstream activation and expression of cardiac hypertrophy related proteins, ANP and BNP. Further, we found that glucose-induced IGF2R expression downregulated survival protein p-Akt, p-Bad (Ser 155) and enhanced the expression of apoptosis-inducing proteins cytochrome c and cleaved Caspase-3. Our results suggested that hyperglycemic condition leads to cellular cardiomyocyte apoptosis both in vitro and in vivo models, via abnormally increased activation of the IGF2R signaling pathway.

The novel cardiac z-disc protein CEFIP regulates cardiomyocyte hypertrophy by modulating calcineurin signaling

The z-disc is a structural component at the lateral borders of the sarcomere and is important for mechanical stability and contractility of both cardiac and skeletal muscles. Of note, the sarcomeric z-disc also represents a nodal point in cardiomyocyte function and signaling. Mutations of numerous z-disc proteins are associated with cardiomyopathies and muscle diseases. To identify additional z-disc proteins that might contribute to cardiac disease, we employed an in silico screen for cardiac-enriched cDNAs. This screen yielded a previously uncharacterized protein named cardiac-enriched FHL2-interacting protein (CEFIP), which exhibited a heart- and skeletal muscle-specific expression profile. Importantly, CEFIP was located at the z-disc and was up-regulated in several models of cardiomyopathy. We also found that CEFIP overexpression induced the fetal gene program and cardiomyocyte hypertrophy. Yeast two-hybrid screens revealed that CEFIP interacts with the calcineurin-binding protein four and a half LIM domains 2 (FHL2). Because FHL2 binds calcineurin, a phosphatase controlling hypertrophic signaling, we examined the effects of CEFIP on the calcineurin/nuclear factor of activated T-cell (NFAT) pathway. These experiments revealed that CEFIP overexpression further enhances calcineurin-dependent hypertrophic signal transduction, and its knockdown repressed hypertrophy and calcineurin/NFAT activity. In summary, we report on a previously uncharacterized protein CEFIP that modulates calcineurin/NFAT signaling in cardiomyocytes, a finding with possible implications for the pathogenesis of cardiomyopathy.

Pancreatic β-Cell-Derived IP-10/CXCL10 Isletokine Mediates Early Loss of Graft Function in Islet Cell Transplantation.

Pancreatic islets produce and secrete cytokines and chemokines in response to inflammatory and metabolic stress. The physiological role of these “isletokines” in health and disease is largely unknown. We observed that islets release multiple inflammatory mediators in patients undergoing islet transplants within hours of infusion. The proinflammatory cytokine interferon-γ–induced protein 10 (IP-10/CXCL10) was among the highest released, and high levels correlated with poor islet transplant outcomes. Transgenic mouse studies confirmed that donor islet–specific expression of IP-10 contributed to islet inflammation and loss of β-cell function in islet grafts. The effects of islet-derived IP-10 could be blocked by treatment of donor islets and recipient mice with anti–IP-10 neutralizing monoclonal antibody. In vitro studies showed induction of the IP-10 gene was mediated by calcineurin-dependent NFAT signaling in pancreatic β-cells in response to oxidative or inflammatory stress. Sustained association of NFAT and p300 histone acetyltransferase with the IP-10 gene required p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) activity, which differentially regulated IP-10 expression and subsequent protein release. Overall, these findings elucidate an NFAT-MAPK signaling paradigm for induction of isletokine expression in β-cells and reveal IP-10 as a primary therapeutic target to prevent β-cell–induced inflammatory loss of graft function after islet cell transplantation.

The impact of calcineurin inhibitors on neutrophil gelatinase-associated lipocalin and fibroblast growth factor 23 in long-term kidney transplant patients.

Neutrophil gelatinase-associated lipocalin (NGAL), a protein with bacteriostatic functions rapidly excreted from stimulated or damaged epithelial cells, is elevated in acute and chronic kidney disease. A calcineurin dependent signaling pathway for fibroblast growth factor 23 (FGF23) has been revealed, but the effect of calcineurin inhibitors (CNIs) on the levels of NGAL and markers of mineral metabolism in long-term kidney transplant patients has not been explored. In a cross-sectional study, 39 patients who received a first kidney transplant more than 10years ago were split into two groups based on whether (n=28) or not (n=11) they used CNIs. Only patients with well-functioning grafts defined as an estimated glomerular filtration rate ≥45mL/min per 1.73m2 were included. The median levels of NGAL, intact parathyroid hormone (iPTH), and iFGF23 were significantly higher in CNI users vs CNI nonusers, 167.0 (134.0-235.0) ng/mL vs 105.0 (91.3-117.0) ng/mL, P<.001, 13.8 (10.0-17.3)pmol/L vs 8.4 (6.4-9.9) pmol/L, P=.003, and 81.6 (56.4-116.5) pg/mL vs 61.8 (43.3-72.1) pg/mL, P=.04 respectively. The median levels of iFGF23 were higher in CNI users compared to CNI nonusers giving support to the notion of a CNI induced FGF23 resistance in the parathyroid. The net result of CNIs side effects needs to be further explored.

Analysis of Taurine's Anti-Down Syndrome Potential in Caenorhabditis elegans.

Down syndrome (DS) patients overexpress human DS critical region gene 1 (hDSCR-1), whose translational product inhibits calcineurin-dependent signaling pathways of genetic transcription. Compared to hDSCR-1, C. elegans rcn-1 has 40% sequence similarity and its proteins share an analogous function with hDSCR-1 in regulating calcineurin. Taurine has had a positive effect on DS patients. According to animal research studies, taurine reduces the expression of MCIP1, a calcineurin inhibitory protein, on C2C12 myotubes and fibroblast in mouse. This study utilizes two C. elegans models for DS: rcn-1 overexpression model, displaying a calcineurin-deficient phenotype, and calcineurin loss-of function mutants. C. elegans larvae were treated with taurine to characterize its effect and mechanism in helping DS patients. RCN-1 expression and behavioral changes were examined in rcn-1 overexpression and calcineurin-deficient models at different concentrations of taurine. When treated with taurine, transgenic worms harboring an rcn-1 reporter (RCN-1::GFP) showed a reduced level of rcn-1 mRNA expression and improved behaviors that were comparable to those in the wild type. These results indicate that taurine exerts a down-regulating effect on the expression of rcn-1 and, consequently, a positive effect on the expression of calcineurins. In summary, taurine may improve the DS symptoms by prompting a positive interaction between RCN-1 and calcineurin. Furthermore, these results suggest that novel mechanisms may regulate interactions among taurine, RCN-1 and calcineurin.

What can FGF23 do without Klotho?

What can FGF23 do without Klotho?

Cisd2 modulates the differentiation and functioning of adipocytes by regulating intracellular Ca2+ homeostasis

CISD2 is a causative gene associated with Wolfram syndrome (WFS). However, it remains a mystery as to how the loss of CISD2 causes metabolic defects in patients with WFS. Investigation on the role played by Cisd2 in specific cell types may help us to resolve these underlying mechanisms. White adipose tissue (WAT) is central to the maintenance of energy metabolism and glucose homeostasis in humans. In this study, adipocyte-specific Cisd2 knockout (KO) mice showed impairment in the development of epididymal WAT (eWAT) in the cell autonomous manner. A lack of Cisd2 caused defects in the biogenesis and function of mitochondria during differentiation of adipocytes in vitro. Insulin-stimulated glucose uptake and secretion of adiponectin by the Cisd2 KO adipocytes were decreased. Moreover, Cisd2 deficiency increased the cytosolic level of Ca(2+) and induced Ca(2+)-calcineurin-dependent signaling that inhibited adipogenesis. Importantly, Cisd2 was found to interact with Gimap5 on the mitochondrial and ER membranes and thereby modulate mitochondrial Ca(2+) uptake associated with the maintenance of intracellular Ca(2+) homeostasis in adipocytes. Thus, it would seem that Cisd2 plays an important role in intracellular Ca(2+) homeostasis, which is required for the differentiation and functioning of adipocytes as well as the regulation of glucose homeostasis in mice.

Parathyroid-Specific Deletion of Klotho Unravels a Novel Calcineurin-Dependent FGF23 Signaling Pathway That Regulates PTH Secretion

Klotho acts as a co-receptor for and dictates tissue specificity of circulating FGF23. FGF23 inhibits PTH secretion, and reduced Klotho abundance is considered a pathogenic factor in renal secondary hyperparathyroidism. To dissect the role of parathyroid gland resident Klotho in health and disease, we generated mice with a parathyroid-specific Klotho deletion (PTH-KL−/−). PTH-KL−/− mice had a normal gross phenotype and survival; normal serum PTH and calcium; unaltered expression of the PTH gene in parathyroid tissue; and preserved PTH response and sensitivity to acute changes in serum calcium. Their PTH response to intravenous FGF23 delivery or renal failure did not differ compared to their wild-type littermates despite disrupted FGF23-induced activation of the MAPK/ERK pathway. Importantly, calcineurin-NFAT signaling, defined by increased MCIP1 level and nuclear localization of NFATC2, was constitutively activated in PTH-KL−/− mice. Treatment with the calcineurin-inhibitor cyclosporine A abolished FGF23-mediated PTH suppression in PTH-KL−/− mice whereas wild-type mice remained responsive. Similar results were observed in thyro-parathyroid explants ex vivo. Collectively, we present genetic and functional evidence for a novel, Klotho-independent, calcineurin-mediated FGF23 signaling pathway in parathyroid glands that mediates suppression of PTH. The presence of Klotho-independent FGF23 effects in a Klotho-expressing target organ represents a paradigm shift in the conceptualization of FGF23 endocrine action.

α-Actinin-3 deficiency alters muscle adaptation in response to denervation and immobilization

Homozygosity for a common null polymorphism (R577X) in the ACTN3 gene results in the absence of the fast fibre-specific protein, α-actinin-3 in ∼16% of humans worldwide. α-Actinin-3 deficiency is detrimental to optimal sprint performance and benefits endurance performance in elite athletes. In the general population, α-actinin-3 deficiency is associated with reduced muscle mass, strength and fast muscle fibre area, and poorer muscle function with age. The Actn3 knock-out (KO) mouse model mimics the human phenotype, with fast fibres showing a shift towards slow/oxidative metabolism without a change in myosin heavy chain (MyHC) isoform. We have recently shown that these changes are attributable to increased activity of the calcineurin-dependent signalling pathway in α-actinin-3 deficient muscle, resulting in enhanced response to exercise training. This led us to hypothesize that the Actn3 genotype influences muscle adaptation to disuse, irrespective of neural innervation. Separate cohorts of KO and wild-type mice underwent 2 weeks immobilization and 2 and 8 weeks of denervation. Absence of α-actinin-3 resulted in reduced atrophic response and altered adaptation to disuse, as measured by a change in MyHC isoform. KO mice had a lower threshold to switch from the predominantly fast to a slower muscle phenotype (in response to immobilization) and a higher threshold to switch to a faster muscle phenotype (in response to denervation). We propose that this change is mediated through baseline alterations in the calcineurin signalling pathway of Actn3 KO muscle. Our findings have important implications for understanding individual responses to muscle disuse/disease and training in the general population.

Weighted Gene Coexpression Network Analysis of Human Left Atrial Tissue Identifies Gene Modules Associated With Atrial Fibrillation

Genetic mechanisms of atrial fibrillation (AF) remain incompletely understood. Previous differential expression studies in AF were limited by small sample size and provided limited understanding of global gene networks, prompting the need for larger-scale, network-based analyses. Left atrial tissues from Cleveland Clinic patients who underwent cardiac surgery were assayed using Illumina Human HT-12 mRNA microarrays. The data set included 3 groups based on cardiovascular comorbidities: mitral valve (MV) disease without coronary artery disease (n=64), coronary artery disease without MV disease (n=57), and lone AF (n=35). Weighted gene coexpression network analysis was performed in the MV group to detect modules of correlated genes. Module preservation was assessed in the other 2 groups. Module eigengenes were regressed on AF severity or atrial rhythm at surgery. Modules whose eigengenes correlated with either AF phenotype were analyzed for gene content. A total of 14 modules were detected in the MV group; all were preserved in the other 2 groups. One module (124 genes) was associated with AF severity and atrial rhythm across all groups. Its top hub gene, RCAN1, is implicated in calcineurin-dependent signaling and cardiac hypertrophy. Another module (679 genes) was associated with atrial rhythm in the MV and coronary artery disease groups. It was enriched with cell signaling genes and contained cardiovascular developmental genes including TBX5. Our network-based approach found 2 modules strongly associated with AF. Further analysis of these modules may yield insight into AF pathogenesis by providing novel targets for functional studies.

Skin cancer in organ transplant recipients: effects of immunosuppressive medications on DNA repair

UV-induced skin cancers comprise a major problem in organ transplant recipients (OTRs). Cyclosporin A, a calcineurin inhibitor, is used as a standard immunosuppressant and clearly increases the skin cancer risk. Azathioprine does not appear to result in such an increase in skin cancer risk, and mTOR inhibitors are associated with an even lesser skin cancer risk. The underlying molecular mechanisms of these clinically important differences among immunosuppressants are still unclear and may relate to other than immunological effects. Insights may be gained by the multistep skin cancer theory and xeroderma pigmentosum, where defective nucleotide excision repair (NER) results in a cellular mutator phenotype and cutaneous carcinogenesis. This viewpoint assay summarizes current knowledge about the influence of the most commonly used immunosuppressive drugs in OTRs on DNA repair. Calcineurin inhibition results in a 200-fold increased skin cancer risk compared with the normal population and inhibits NER. The skin cancer risk under azathioprine is threefold less compared with calcineurin inhibitors, which may relate to inhibition of only the last step of NER, i.e. gap filling. mTOR inhibitors do not reduce NER in the global genome and can inhibit the growth of already initiated tumors, which may account for the markedly reduced skin cancer risk compared with calcineurin inhibitors. We conclude that OTRs may benefit from treatment regimens other than calcineurin inhibitors and speculate that a targeted modulation of calcineurin-dependent signalling may prevent UV-induced tumor formation by enhancing NER not only in OTRs but also in the general population, at least in part.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Cyclosporin A inhibits nucleotide excision repair via downregulation of the xeroderma pigmentosum group A and G proteins, which is mediated by calcineurin inhibition

Cyclosporin A (CsA) inhibits nucleotide excision repair (NER) in human cells, a process that contributes to the skin cancer proneness in organ transplant patients. We investigated the mechanisms of CsA-induced NER reduction by assessing all xeroderma pigmentosum (XP) genes (XPA-XPG). Western blot analyses revealed that XPA and XPG protein expression was reduced in normal human GM00637 fibroblasts exposed to 0.1 and 0.5 μm CsA. Interestingly, the CsA treatment reduced XPG, but not XPA, mRNA expression. Calcineurin knockdown in GM00637 fibroblasts using RNAi led to similar results suggesting that calcineurin-dependent signalling is involved in XPA and XPG protein regulation. CsA-induced reduction in NER could be complemented by the overexpression of either XPA or XPG protein. Likewise, XPA-deficient fibroblasts with stable overexpression of XPA (XP2OS-pCAH19WS) did not show the inhibitory effect of CsA on NER. In contrast, XPC-deficient fibroblasts overexpressing XPC showed CsA-reduced NER. Our data indicate that the CsA-induced inhibition of NER is a result of downregulation of XPA and XPG protein in a calcineurin-dependent manner.

Sustained Hemodynamic Stress Disrupts Normal Circadian Rhythms in Calcineurin-Dependent Signaling and Protein Phosphorylation in the Heart

Despite overwhelming evidence of the importance of circadian rhythms in cardiovascular health and disease, little is known regarding the circadian regulation of intracellular signaling pathways controlling cardiac function and remodeling. To assess circadian changes in processes dependent on the protein phosphatase calcineurin, relative to changes in phosphorylation of cardiac proteins, in normal, hypertrophic, and failing hearts. We found evidence of large circadian oscillations in calcineurin-dependent activities in the left ventricle of healthy C57BL/6 mice. Calcineurin-dependent transcript levels and nuclear occupancy of the NFAT (nuclear factor of activated T cells) regularly fluctuated as much as 20-fold over the course of a day, peaking in the morning when mice enter a period of rest. Phosphorylation of the protein phosphatase 1 inhibitor 1 (I-1), a direct calcineurin substrate, and phospholamban, an indirect target, oscillated directly out of phase with calcineurin-dependent signaling. Using a surgical model of cardiac pressure overload, we found that although calcineurin-dependent activities were markedly elevated, the circadian pattern of activation was maintained, whereas, oscillations in phospholamban and I-1 phosphorylation were lost. Changes in the expression of fetal gene markers of heart failure did not mirror the rhythm in calcineurin/NFAT activation, suggesting that these may not be direct transcriptional target genes. Cardiac function in mice subjected to pressure overload was significantly lower in the morning than in the evening when assessed by echocardiography. Normal, opposing circadian oscillations in calcineurin-dependent activities and phosphorylation of proteins that regulate contractility are disrupted in heart failure.