Phosphatase Calcineurin Research Articles

Regulation of dual leucine zipper kinase activity through its interaction with calcineurin

Hyperglycemia enhancing the intracellular levels of reactive oxygen species (ROS) contributes to dysfunction and progressive loss of beta cells and thereby to diabetes mellitus. The oxidation sensitive calcium/calmodulin dependent phosphatase calcineurin promotes pancreatic beta cell function and survival whereas the dual leucine zipper kinase (DLK) induces apoptosis. Therefore, it was studied whether calcineurin interferes with DLK action. In a beta cell line similar concentrations of H2O2 decreased calcineurin activity and activated DLK. DLK interacted via its φLxVP motif (aa 362–365) with the interface of the calcineurin subunits A and B. Mutation of the Val prevented this protein protein interaction, hinting at a distinct φLxVP motif. Indeed, mutational analysis revealed an ordered structure of DLK's φLxVP motif whereby Val mediates the interaction with calcineurin and Leu maintains an enzymatically active conformation. Overexpression of DLK wild-type but not the DLK mutant unable to bind calcineurin diminished calcineurin-induced nuclear localisation of the nuclear factor of activated T-cells (NFAT), suggesting that both, DLK and NFAT compete for the substrate binding site of calcineurin. The calcineurin binding-deficient DLK mutant exhibited increased DLK activity measured as phosphorylation of the downstream c-Jun N-terminal kinase, inhibition of CRE-dependent gene transcription and induction of apoptosis. These findings show that calcineurin interacts with DLK; and inhibition of calcineurin increases DLK activity. Hence, this study demonstrates a novel mechanism regulating DLK action. These findings suggest that ROS through inhibition of calcineurin enhance DLK activity and thereby lead to beta cell dysfunction and loss and ultimately diabetes mellitus.

BCL11B Regulates Arterial Stiffness and Related Target Organ Damage.

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A role for the endocannabinoid enzymes monoacylglycerol and diacylglycerol lipases in cue-induced cocaine craving following prolonged abstinence.

Following exposure to drugs of abuse, long-term neuroadaptations underlie persistent risk to relapse. Endocannabinoid signaling has been associated with drug-induced neuroadaptations, but the role of lipases that mediate endocannabinoid biosynthesis and metabolism in regulating relapse behaviors following prolonged periods of drug abstinence has not been examined. Here, we investigated how pharmacological manipulation of lipases involved in regulating the expression of the endocannabinoid 2-AG in the nucleus accumbens (NAc) influence cocaine relapse via discrete neuroadaptations. At prolonged abstinence (30days) from cocaine self-administration, there is an increase in the NAc levels of diacylglycerol lipase (DAGL), the enzyme responsible for the synthesis of the endocannabinoid 2-AG, along with decreased levels of monoacylglycerol lipase (MAGL), which hydrolyzes 2-AG. Since endocannabinoid-mediated behavioral plasticity involves phosphatase dysregulation, we examined the phosphatase calcineurin after 30days of abstinence and found decreased expression in the NAc, which we demonstrate is regulated through the transcription factor EGR1. Intra-NAc pharmacological manipulation of DAGL and MAGL with inhibitors DO-34 and URB-602, respectively, bidirectionally regulated cue-induced cocaine seeking and altered the phosphostatus of translational initiation factor, eIF2α. Finally, we found that cocaine seeking 30days after abstinence leads to decreased phosphorylation of eIF2α and reduced expression of its downstream target NPAS4, a protein involved in experience-dependent neuronal plasticity. Together, our findings demonstrate that lipases that regulate 2-AG expression influence transcriptional and translational changes in the NAc related to drug relapse vulnerability.

Calcineurin: A star is reborn

The protein phosphatase calcineurin has long been familiar to the calcium community, but the definition of its physiological substrates has been far from complete. A new study rectifies this deficiency and sets the stage for new insights into the role of calcineurin in diverse cellular processes.

Genetic contribution to high temperature tolerance in Cryptococcus neoformans.

The human fungal pathogen Cryptococcus neoformans relies on a complex signaling network for the adaptation and survival at the host temperature. Protein phosphatase calcineurin is central to proliferation at 37°C but its exact contributions remain ill-defined. To better define genetic contributions to the C. neoformans temperature tolerance, 4031 gene knockouts were screened for genes essential at 37°C and under conditions that keep calcineurin inactive. Identified 83 candidate strains, potentially sensitive to 37°C, were subsequently subject to technologically simple yet robust assay, in which cells are exposed to a temperature gradient. This has resulted in identification of 46 genes contributing to the maximum temperature at which C. neoformans can proliferate (Tmax). The 46 mutants, characterized by a range of Tmax on drug-free media, were further assessed for Tmax under conditions that inhibit calcineurin, which led to identification of several previously uncharacterized knockouts exhibiting synthetic interaction with the inhibition of calcineurin. A mutant that lacked septin Cdc11 was among those with the lowest Tmax and failed to proliferate in the absence of calcineurin activity. To further define connections with calcineurin and the role for septins in high temperature growth, the 46 mutants were tested for cell morphology at 37°C and growth in the presence of agents disrupting cell wall and cell membrane. Mutants sensitive to calcineurin inhibition were tested for synthetic lethal interaction with deletion of the septin-encoding CDC12 and the localization of the septin Cdc3-mCherry. The analysis described here pointed to previously uncharacterized genes that were missed in standard growth assays indicating that the temperature gradient assay is a valuable complementary tool for elucidating the genetic basis of temperature range at which microorganisms proliferate.

Novel small chemical compound with NFAT modulatory properties alleviates synaptic dysfunction and improves cognition in mouse models of amyloid pathology

AbstractBackgroundHyperactivity of the protein phosphatase calcineurin (CN) in brain directly corresponds to key neuropathological and clinical features of Alzheimer’s disease (AD). While inhibition of CN ameliorates pathophysiologic and cognitive changes in mice with AD‐like pathology, concerns over adverse effects have slowed the transition of common CN‐inhibiting drugs to the clinic for treatment of AD and AD‐related disorders. Targeting substrates of CN, like the nuclear factor of activated T‐cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that modulates NFAT signaling, without inhibiting CN activity.Method Cell culture studies — Rat cortical astrocyte cultures were treated with a variety of pathogenic factors in the presence of Q134R or the CN inhibitor, cyclosporine. NFAT‐dependent luciferase activity and dephosphorylation of non‐NFAT substrates were assessed. Intact mouse studies — Q134R was delivered (via oral gavage) to APP/PS1 mice at different ages, or to wild type (WT) mice infused with oligomeric amyloid‐beta peptides. Cognition was assessed on a Y maze along with several postmortem measures including NFAT4 activity, glial activation (GFAP and Iba‐1 expression), amyloid deposition, long‐term survival, and synaptic function and plasticity.ResultIn primary astrocytes, Q134R significantly inhibited NFAT activation in a concentration‐dependent manner, but did not inhibit the CN‐dependent dephosphorylation of non‐NFAT substrates in vivo or in vitro. Short‐term treatment of APP/PS1 mice with Q134R inhibited the nuclear translocation of NFAT4 in hippocampal astrocytes while causing a significant drop in GFAP volume, a mild reduction in Iba1, and no change in amyloid pathology. Short‐term treatment with Q134R also improved cognitive function in APP/PS1 mice and amyloid‐infused WT mice. Long‐term oral delivery of Q134R over many months promoted survival in WT mice and improved synaptic function and plasticity in APP/PS1 mice.ConclusionQ134R has novel CN‐independent, NFAT modulatory properties and improves key neurologic function parameters in mouse models of AD‐like pathology. The results suggest that Q134R is a promising drug for treating AD and AD‐related disorders.

Functional Characterization of Calcineurin-Responsive Transcription Factors Fg01341 and Fg01350 in Fusarium graminearum.

Ca2 +/calmodulin-dependent phosphatase calcineurin is one of the important regulators of intracellular calcium homeostasis and has been investigated extensively in Saccharomyces cerevisiae. However, only a few reports have explored the function of the Crz1 homolog in filamentous fungi, especially in Fusarium graminearum. In this study, we identified Fg01341 as a potential ortholog of yeast Crz1. Fg01341 could interact with calcineurin and initiate nuclear transport in a calcineurin-dependent manner. The ΔFg01341 mutant exhibited normal hyphal growth on basic medium and conidia formation, but sexual reproduction was partially blocked. Pathogenicity assays showed that the virulence of the ΔFg01341 mutant in flowering wheat heads and corn silks dramatically decreased and was thus consistent with the reduction in deoxynivalenol production. Unexpectedly, the sensitivity to osmotic stress of the deletion mutant and that of the wild-type strain did not present any differences. The deletion mutant showed higher sensitivity to tebuconazole than the wild-type strain. Results also showed that the transcription factor Fg01350 might be the calcineurin target and was independent of Crz1. Furthermore, ΔFg01350 showed defects in hyphal growth, sexual production, virulence, and deoxynivalenol production. Collectively, the results indicate that these two proteins functionally redundant and that the calcineurin–Crz1-independent pathway is particularly important in F. graminearum.

Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion.

Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin pathogenesis based on studies with gB and gH hyperfusogenic VZV mutants. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Two structurally related non-antibiotic macrolides, tacrolimus and pimecrolimus, both significantly increased VZV gB/gH-gL mediated cell fusion. These compounds form a drug-protein complex with FKBP1A, which binds to calcineurin and specifically inhibits calcineurin phosphatase activity. Inhibition of calcineurin phosphatase activity also enhanced both herpes simplex virus-1 fusion complex and syncytin-1 mediated cell fusion, indicating a broad role of calcineurin in modulating this process. To characterize the role of calcineurin phosphatase activity in VZV gB/gH-gL mediated fusion, a series of biochemical, biological and infectivity assays was performed. Pimecrolimus-induced, enhanced cell fusion was significantly reduced by shRNA knockdown of FKBP1A, further supporting the role of calcineurin phosphatase activity in fusion regulation. Importantly, inhibition of calcineurin phosphatase activity during VZV infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with the previously reported phenotypes of gB and gH hyperfusogenic VZV mutants. Seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited were identified as potential downstream factors involved in fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis.

The establishment of a novel high-throughput screening system using RNA-guided genome editing to identify chemicals that suppress aldosterone synthase expression

Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca2+) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca2+ signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter into CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA.

PDE5 inhibition improves cardiac morphology and function in SHR by reducing NHE1 activity: Repurposing Sildenafil for the treatment of hypertensive cardiac hypertrophy

Previously, we have shown that an increased cGMP-activated protein Kinase (PKG) activity after phosphodiesterase 5 (PDE5) inhibition by Sildenafil (SIL), leads to myocardial Na+/H+ exchanger (NHE1) inhibition preserving its basal homeostatic function. Since NHE1 is hyperactive in the hypertrophied myocardium of spontaneous hypertensive rats (SHR), while its inhibition was shown to prevent and revert this pathology, the current study was aimed to evaluate the potential antihypertrophic effect of SIL on adult SHR myocardium. We initially tested the inhibitory capability of SIL on NHE1 in isolated cardiomyocytes of SHR by comparing H+ efflux during the recovery from an acid load. After confirmed that effect, eight-month-old SHR were chronically treated for one month with SIL through drinking water. Compared to their littermate controls, SIL-treated rats presented a decreased NHE1 activity, which correlated with a reduction in its phosphorylation level assigned to activation of a PKG-p38 MAP kinase-PP2A signaling pathway. Moreover, treated animals showed a decreased oxidative stress that appears to be a consequence of a decreased mitochondrial NHE1 phosphorylation. Treated SHR showed a significant reduction in the pro-hypertrophic phosphatase calcineurin, despite slight tendency to decrease hypertrophy was detected. When SIL treatment was prolonged to three months, a significant decrease in myocardial hypertrophy and interstitial fibrosis that correlated with a lower myocardial stiffness was observed. In conclusion, the current study provides evidence concerning the ability of SIL to revert established cardiac hypertrophy in SHR, a clinically relevant animal model that resembles human essential hypertension.

Calcineurin Regulates Conidiation, Chlamydospore Formation and Virulence in Fusarium oxysporum f. sp. lycopersici.

Fusarium wilt of tomato caused by the ascomycetous fungus Fusarium oxysporum f. sp. lycopersici (Fol) is widespread in most tomato planting areas. Calcineurin is a heterodimeric calcium/calmodulin-dependent protein phosphatase comprised of catalytic (Cna1) and regulatory (Cnb1) subunits. Calcineurin has been studied extensively in human fungal pathogens, but less is known about its roles in plant fungal pathogens. It is known that calcineurin regulates fungal calcium signaling, growth, drug tolerance, and virulence. However, the roles of calcineurin in Fol have not yet been characterized. In this study, we deleted calcineurin CNA1 and CNB1 genes to characterize their roles in conidiation, chlamydospore formation and virulence in Fol. Our results revealed that both cna1 and cnb1 mutants show defects in calcineurin phosphatase activity, vegetative growth and conidiation as compared to the wild type. Furthermore, calcineurin mutants exhibited blunted and swollen hyphae as observed by scanning electron microscopy. Interestingly, we found that Fol calcineurin is critical for chlamydospore formation, a function of calcineurin previously undocumented in the fungal kingdom. According to transcriptome analysis, the expression of 323 and 414 genes was up- and down-regulated, respectively, in both cna1 and cnb1 mutants. Based on the pathogen infection assay, tomato plants inoculated with cna1 or cnb1 mutant have a dramatic reduction in disease severity, indicating that calcineurin has a vital role in Fol virulence. In conclusion, our findings suggest that Fol calcineurin is required, at least in part, for phosphatase activity, vegetative growth, conidiation, chlamydospore formation, and virulence.

A Peptidyl Inhibitor that Blocks Calcineurin-NFAT Interaction and Prevents Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease with a high morbidity and mortality rate, for which no pharmacologic treatment is currently available. Our previous studies discovered that a pivotal step in the disease process is the activation of the nuclear factor of activated T cells (NFAT) c3 in lung macrophages, suggesting that inhibitors against the upstream protein phosphatase calcineurin should be effective for prevention/treatment of ARDS. Herein, we report the development of a highly potent, cell-permeable, and metabolically stable peptidyl inhibitor, CNI103, which selectively blocks the interaction between calcineurin and NFATc3, through computational and medicinal chemistry. CNI103 specifically inhibited calcineurin signaling in vitro and in vivo and exhibited a favorable pharmacokinetic profile, broad tissue distribution following different routes of administration, and minimal toxicity. Our data indicate that CNI103 is a promising novel treatment for ARDS and other inflammatory diseases.

Cyclosporin A but not FK506 activates the integrated stress response in human cells

Cyclosporin A (CsA) and tacrolimus (FK506) are valuable immunosuppressants for a range of clinical settings, including (but not limited to) organ transplantation and the treatment of autoimmune diseases. They function by inhibiting the activity of the Ca2+/calmodulin-dependent phosphatase calcineurin toward nuclear factor of activated T-cells (NF-AT) in T-lymphocytes. However, use of CsA is associated with more serious side effects and worse clinical outcomes than FK506. Here we show that CsA, but not FK506, causes activation of the integrated stress response (ISR), an event which is normally an acute reaction to various types of intracellular insults, such as nutrient deficiency or endoplasmic reticulum stress. These effects of CsA involve at least two of the stress-activated protein kinases (GCN2 and PERK) that act on the translational machinery to slow down protein synthesis via phosphorylation of the eukaryotic initiation factor (eIF) 2α and thereby induce the ISR. These actions of CsA likely contribute to the adverse effects associated with its clinical application.

Calreticulin regulates Src kinase in osteogenic differentiation from embryonic stem cells.

Calreticulin, the major Ca2+ buffer of the endoplasmic reticulum plays an important role in the choice of fate by embryonic stem cells. Using the embryoid body method of organogenesis, we showed impaired osteogenesis in crt-/- cells vis-à-vis calreticulin-containing osteogenic WT cells. In the non-osteogenic crt-/- cells, c-Src- a non-receptor tyrosine kinase- was activated and its inhibition rescued osteogenesis. Most importantly, we demonstrated that calreticulin-containing cells had lower c-Src kinase activity, and this was accomplished via the Ca2+-homeostatic function of calreticulin. Specifically, lowering cytosolic [Ca2+] in calreticulin-containing osteogenic WT cells with BAPTA-AM, activated c-Src and impaired osteogenic differentiation. Conversely, increasing cytosolic [Ca2+] in crt-/- cells with ionomycin deactivated c-Src kinase and restored osteogenesis. The immediate effector of calreticulin, the Ser/Thr phosphatase calcineurin, was less active in crt-/- cells, however, its activity was rescued upon inhibition of c-Src activity by small molecule inhibitors. Finally, we showed that higher activity of calcineurin correlated with increased level of nuclear Runx2, a transcription factor that is the master regulator of osteogenesis. Collectively, our work has identified a novel pathway involving calreticulin regulated Ca2+ signalling via c-Src in osteogenic differentiation of embryonic stem cells.

Transcription factor EB and TFE3: new metabolic coordinators mediating adaptive responses to exercise in skeletal muscle?

In response to the increased energy demands of contractions, skeletal muscle adapts remarkably well through acutely regulating metabolic pathways to maintain energy balance and in the longer term by regulating metabolic reprogramming, such as remodeling and expanding the mitochondrial network. This long-term adaptive response involves modulation of gene expression at least partly through the regulation of specific transcription factors and transcriptional coactivators. The AMPK-peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) pathway has long been known to orchestrate contraction-mediated adaptive responses, although AMPK- and PGC1α-independent pathways have also been proposed. Transcription factor EB (TFEB) and TFE3, known as important regulators of lysosomal biogenesis and autophagic processes, have emerged as new metabolic coordinators. The activity of TFEB/TFE3 is regulated through posttranslational modifications (i.e., phosphorylation) and spatial organization. Under nutrient and energy stress, TFEB and TFE3 are dephosphorylated and translocate to the nucleus, where they activate transcription of their target genes. It has recently been reported that exercise promotes nuclear translocation and activation of TFEB/TFE3 in mouse skeletal muscle through the Ca2+-stimulated protein phosphatase calcineurin. Skeletal muscle-specific ablation of TFEB exhibits impaired glucose homeostasis and mitochondrial biogenesis with reduced metabolic flexibility during exercise, and global TFE3 depletion results in diminished endurance and abolished exercise-induced metabolic benefits. Transcriptomic analysis of the muscle-specific TFEB-null mice has demonstrated that TFEB regulates the expression of genes involved in glucose metabolism and mitochondrial homeostasis. This review aims to summarize and discuss emerging roles for TFEB/TFE3 in metabolic and adaptive responses to exercise and contractile activity in skeletal muscle.

Influence of electrostatic forces on the association kinetics and conformational ensemble of an intrinsically disordered protein.

Recent work has revealed that the association of a disordered region of a protein with a folded binding partner can occur as rapidly as association between two folded proteins. This is the case for the phosphatase calcineurin (CaN) and its association with its activator calmodulin. Calmodulin binds to the intrinsically disordered regulatory domain of CaN. Previous studies have shown that electrostatic steering can accelerate the binding of folded proteins with disordered ligands. Given that electrostatic forces are strong determinants of disordered protein ensembles, the relationship between electrostatics, conformational ensembles, and quaternary interactions is unclear. Here, we employ experimental approaches to explore the impact of electrostatic interactions on the association of calmodulin with the disordered regulatory region of CaN. We find that estimated association rate constants of calmodulin with our chosen calmodulin-substrates are within the diffusion-limited regime. The association rates are dependent on the ionic strength, indicating that favorable electrostatic forces increase the rate of association. Further, we show that charged amino acids outside the calmodulin-binding site modulate the binding rate. Conformational ensembles obtained from computer simulations suggest that electrostatic interactions within the regulatory domain might bias the conformational ensemble such that the calmodulin binding region is readily accessible. Given the prevalence of charged residues in disordered protein chains, our findings are likely relevant to many protein-protein interactions.

3077 – THE ROLE OF CALCIUM IN HEMATOPOIETIC ONTOGENY

Hematopoietic Stem Cells (HSCs) reside in the bone marrow and generate blood cells throughout life. Our lab recently discovered that HSCs from adult bone marrow (ABM HSCs) are endowed with low intracellular calcium and maintained in low calcium environments both in vitro and in vivo (Luchsinger et al., Cell Stem Cell, 2019). ABM HSCs derive from fetal liver (FL) HSCs and while both populations perform multilineage hematopoiesis they differ in key aspects such as cell cycle status, self-renewal capacity, and transcription factor expression, raising the question how HSCs at earlier developmental stages handle calcium. We employed single-cell perfusion live imaging of calcium dyes and flow cytometry of genetically-encoded calcium indicators to show that FL HSCs possess approximately two-fold increased intracellular calcium from ABM HSCs. Perfusion live imaging also showed increased store-operated calcium entry (SOCE) and decreased calcium efflux through Plasma Membrane Calcium ATPases (PMCAs) from fetal to adult stages. Consistent with these changes, FL HSCs displayed decreased expression of the PMCA gene Atp2b4 and decreased pan-PMCA protein expression. FL HSCs also showed increased nuclear localization of the transcription factor NFAT1, which translocates to the nucleus upon dephosphorylation by the calcium-dependent phosphatase Calcineurin, consistent with increased calcium of FL HSCs. Finally, we show that NFAT1 localization is controlled in part by cytosolic calcium levels, and only ABM HSCs respond to low extracellular calcium to decrease nuclear NFAT localization. These data indicate profound, developmentally regulated changes in calcium physiology between FL and ABM HSCs, which may potentially explain other functional differences between these populations.

Abstract MP161: A Calcineurin-hoxb13 Axis Regulates Growth Mode of Mammalian Cardiomyocytes

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. Our group recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2. We recently also showed that Meis1, a TALE family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a Meis1 cofactor in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can both extend the postnatal window of cardiomyocyte proliferation and reactivate cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1/Hoxb13 double knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and an improvement in left ventricular systolic function following myocardial infarction both by echocardiography and MRI. ChIP-seq analysis demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204 (S204), resulting in its nuclear localization and cell cycle arrest. Collectively, these results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

Calcineurin Aβ-Specific Anchoring Confers Isoform-Specific Compartmentation and Function in Pathological Cardiac Myocyte Hypertrophy.

The Ca2+/calmodulin-dependent phosphatase calcineurin is a key regulator of cardiac myocyte hypertrophy in disease. An unexplained paradox is how the β isoform of the calcineurin catalytic A-subunit (CaNAβ) is required for induction of pathological myocyte hypertrophy, despite calcineurin Aα expression in the same cells. It is unclear how the pleiotropic second messenger Ca2+ drives excitation-contraction coupling while not stimulating hypertrophy by calcineurin in the normal heart. Elucidation of the mechanisms conferring this selectivity in calcineurin signaling should reveal new strategies for targeting the phosphatase in disease. Primary adult rat ventricular myocytes were studied for morphology and intracellular signaling. New Förster resonance energy transfer reporters were used to assay Ca2+ and calcineurin activity in living cells. Conditional gene deletion and adeno-associated virus-mediated gene delivery in the mouse were used to study calcineurin signaling after transverse aortic constriction in vivo. CIP4 (Cdc42-interacting protein 4)/TRIP10 (thyroid hormone receptor interactor 10) was identified as a new polyproline domain-dependent scaffold for CaNAβ2 by yeast 2-hybrid screen. Cardiac myocyte-specific CIP4 gene deletion in mice attenuated pressure overload-induced pathological cardiac remodeling and heart failure. Blockade of CaNAβ polyproline-dependent anchoring using a competing peptide inhibited concentric hypertrophy in cultured myocytes; disruption of anchoring in vivo using an adeno-associated virus gene therapy vector inhibited cardiac hypertrophy and improved systolic function after pressure overload. Live cell Förster resonance energy transfer biosensor imaging of cultured myocytes revealed that Ca2+ levels and calcineurin activity associated with the CIP4 compartment were increased by neurohormonal stimulation, but minimally by pacing. Conversely, Ca2+ levels and calcineurin activity detected by nonlocalized Förster resonance energy transfer sensors were induced by pacing and minimally by neurohormonal stimulation, providing functional evidence for differential intracellular compartmentation of Ca2+ and calcineurin signal transduction. These results support a structural model for Ca2+ and CaNAβ compartmentation in cells based on an isoform-specific mechanism for calcineurin protein-protein interaction and localization. This mechanism provides an explanation for the specific role of CaNAβ in hypertrophy and its selective activation under conditions of pathologic stress. Disruption of CaNAβ polyproline-dependent anchoring constitutes a rational strategy for therapeutic targeting of CaNAβ-specific signaling responsible for pathological cardiac remodeling in cardiovascular disease deserving of further preclinical investigation.

The nonselective cation channel TRPV4 inhibits angiotensin II receptors

G-protein-coupled receptors (GPCRs) are a ubiquitously expressed family of receptor proteins that regulate many physiological functions and other proteins. They act through two dissociable signaling pathways: the exchange of GDP to GTP by linked G-proteins and the recruitment of β-arrestins. GPCRs modulate several members of the transient receptor potential (TRP) channel family of nonselective cation channels. How TRP channels reciprocally regulate GPCR signaling is less well-explored. Here, using an array of biochemical approaches, including immunoprecipitation and fluorescence, calcium imaging, phosphate radiolabeling, and a β-arrestin-dependent luciferase assay, we characterize a GPCR-TRP channel pair, angiotensin II receptor type 1 (AT1R), and transient receptor potential vanilloid 4 (TRPV4), in primary murine choroid plexus epithelial cells and immortalized cell lines. We found that AT1R and TRPV4 are binding partners and that activation of AT1R by angiotensin II (ANGII) elicits β-arrestin-dependent inhibition and internalization of TRPV4. Activating TRPV4 with endogenous and synthetic agonists inhibited angiotensin II-mediated G-protein-associated second messenger accumulation, AT1R receptor phosphorylation, and β-arrestin recruitment. We also noted that TRPV4 inhibits AT1R phosphorylation by activating the calcium-activated phosphatase calcineurin in a Ca2+/calmodulin-dependent manner, preventing β-arrestin recruitment and receptor internalization. These findings suggest that when TRP channels and GPCRs are co-expressed in the same tissues, many of these channels can inhibit GPCR desensitization.