Plasma Membrane Calcium ATPase Isoform Research Articles

The PLEKHA7–PDZD11 complex regulates the localization of the calcium pump PMCA and calcium handling in cultured cells

The plasma membrane calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adapter protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here, we test this hypothesis using cultured cell model systems. We show using immunofluorescence microscopy and a surface biotinylation assay that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct epithelial cells results in increased accumulation of endogenous PMCA at lateral cell–cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, coexpression of PDZD11 reduces membrane accumulation of overexpressed PMCA4x/b, and analysis of cytosolic calcium transients shows that PDZD11 counteracts calcium extrusion activity of overexpressed PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mouse kidney collecting duct) cells increases the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7–PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA.

AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model

Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Müller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Müller cell-specific Crb1KOCrb2LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Müller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of dl-α-aminoadipate acid induced gliosis and retinal disorganization in Crb1KOCrb2LowMGC retinas but not in wild-type or Crb1-deficient retinas. Crb1KOCrb2LowMGC mice showed a substantial decrease in inner/outer photoreceptor segment length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 (hCRB2) in Müller cells of Crb1KOCrb2LowMGC mice subsequently exposed to low levels of dl-α-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (hCRB1) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that hCRB expression in Müller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-hCRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-hCRB1 in protection against loss of vision.

Evaluation of Single-Nucleotide Polymorphisms of Transcription Factor 7-Like 2 and ATP2B1 Genes as Cardiovascular Risk Predictors in Chronic Kidney Disease.

Introduction:Cardiovascular disease (CVD) is the primary cause of morbidity and premature mortality in chronic kidney disease (CKD). The transcription factor 7-like 2 (TCF7L2) gene product TCF4 is a transcription factor that acts as a downstream effector in the canonical Wnt signaling pathway and may be important in the development of both type 2 diabetes and renal development and disease. It is, therefore, plausible that mutations in this gene could manifest themselves in reduced kidney function or kidney disease through their effects on hyperglycemia as well as independent of this mechanism. The ATP2B1 gene encodes the plasma membrane calcium ATPase isoform 1, which removes bivalent calcium ions from eukaryotic cells against very large concentration gradients and is responsible for controlling the contraction and dilation of vascular smooth muscles.Aim and Objectives:The aims of this study are (1) to evaluate single-nucleotide polymorphisms (SNPs) of TCF7L2 gene as cardiovascular risk predictors in CKD and (2) to evaluate SNPs of ATP2B1 gene as cardiovascular risk predictors in CKD.Subjects and Methods:Fifty clinically diagnosed CKD patients in the age group between 20 and 60 years of both genders were selected as cases and fifty healthy participants from the master health checkup department were selected as controls. Genomic DNA was extracted based on the spin column kit method. The DNA samples were stored at −20°C until analysis. Genotyping for TCF7L2 gene rs7903146 (C/T) and ATP2B1 gene rs11105354 (A/G) was carried out through polymerase chain reaction.Results:T allele frequency was observed in 12 controls and 23 cases (odds ratio [OR] = 2.2, 95% confidence interval [CI]: 1.0–4.7). CC genotype was observed in 38 controls and 27 cases and CT genotype in 22 cases and 12 controls. A allele was found in 38 cases and 23 controls (OR = 2, 95% CI: 1.1–3.8). The mean values of cholesterol, low-density lipoprotein, triglycerides, glucose, insulin, urea, and creatinine were high in cases when compared to controls.Conclusion:T allele of TCF7L2 gene rs7903146 (C/T) and A allele of ATP2B1 (A/G) gene rs11105354 (A/G) are associated with CVD in CKD patients.

Contribution of Plasma Membrane Calcium ATPases to neuronal maladaptive responses: Focus on spinal nociceptive mechanisms and neurodegeneration.

Plasma membrane calcium ATPases (PMCAs) are ion pumps that expel Ca2+ from cells and maintain Ca2+ homeostasis. Four isoforms and multiple splice variants play important and non-overlapping roles in cellular function and integrity and have been implicated in diseases including disorders of the central nervous system (CNS). In particular, one of these isoforms, PMCA2, is critical for spinal cord (SC) neuronal function. PMCA2 expression is decreased in SC neurons at onset of symptoms in animal models of multiple sclerosis. Decreased PMCA2 expression affects the function and viability of SC neurons, with motor neurons being the most vulnerable population. Recent studies have also shown that PMCA2 could be an important contributor to pain processing in the dorsal horn (DH) of the SC. Pain sensitivity was altered in female, but not male, PMCA2+/- mice compared to PMCA2+/+ littermates in a modality-dependent manner. Changes in pain responsiveness in the female PMCA2+/- mice were paralleled by female-specific alterations in the expression of effectors, which have been implicated in the excitability of DH neurons, in mechanisms governing nociception and in the transmission of pain signals. Other PMCA isoforms and in particular, PMCA4, also contribute to the excitability of neurons in the dorsal root ganglia (DRG), which contain the first-order sensory neurons that convey nociceptive information from the periphery to the DH. These findings suggest that specific PMCA isoforms play specialized functions in neurons that mediate pain processing. Further investigations are necessary to unravel the precise contribution of PMCAs to mechanisms governing pathological pain in models of injury and disease.

156 Inducers of pulmonary arterial hypertension upregulate the expression of plasma membrane calcium atpase 1 in pulmonary artery smooth muscle cells

Pulmonary arterial hypertension (PAH) is a chronic and life-threatening disease with high morbidity and mortality in adult and paediatric patients. PAH is characterised by a progressive narrowing and occlusion of small pulmonary arteries leading to increased pulmonary resistance, right ventricular hypertrophy, and, finally, right ventricular failure. A large body of data has shown that proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) represent key events in the vascular remodelling of pulmonary arteries that occurs during PAH. Levels of cytoplasmic calcium are an important determinant of PASMC proliferation and migration, and failure in maintaining appropriate levels of intracellular calcium are associated with PAH. The plasma membrane calcium ATPase (PMCA) proteins extrude calcium from the cytosol to the extracellular medium, and in doing so, play a critical role in the modulation of intracellular calcium levels. In this work, we have investigated whether inducers of PAH trigger any changes in the expression of PMCA proteins in PASMCs. Analysis of RNA expression levels for PMCA genes has revealed that treatment of PASMCs with PDGF results in a significant increase in the level of the RNA encoding for the protein PMCA1. Interestingly, PMCA1 RNA levels were also elevated in lungs of rats with monocrotaline-induced PAH. No changes were observed in the RNA levels for PMCA4, the other major PMCA isoform expressed in PASMCs. Although previous studies on the regulation of PMCA1 gene expression have identified functional binding sites for the transcription factors NFAT in the PMCA1 promoter region, we show here that PDGF-mediated upregulation of PMCA1 transcriptional expression is independent of activation of the calcineurin/NFAT signalling pathway. Our results suggest the involvement of PMCA1 in PASMC deregulation during PAH, although determination of the link between increased expression of PMCA1 and PAH requires further investigation.

Abstract 2859: Chloroquine and vitamin D3 modulate proliferation in early stage breast cancer models

Abstract The defining feature of premalignant mammary lesions is proliferation of cells within the intraductal niche. Survival in this stressful duct microenvironment requires sophisticated mechanisms to avoid apoptosis caused by hypoxia, nutrient deprivation, metabolic acidosis, and intracellular calcium overload. Two major survival mechanisms of breast ductal carcinoma cells (DCIS) are 1) the upregulation of autophagy and 2) the enhanced export of calcium via plasma membrane calcium-ATPase isoform 2 (PMCA2) efflux pumps. We hypothesize that calcium efflux, through PMCA2, and activation of autophagic catabolism are critical interdependent mechanisms for the survival of pre-invasive human breast neoplastic cells. To assess the proteomic response to autophagy inhibition and/or elevated intracellular calcium, 41 signal transduction proteins/post-translationally modified proteins related to proliferation, autophagy, and calcium pathways were quantified by reverse phase protein microarrays (RPPA) in time course studies of T-47D metastatic ductal breast cancer cell line, and MCF-10A non-tumorigenic breast cell line, both of which express low basal levels of PMCA2, and in human primary DCIS organoid cultures. Cell lines were treated with chloroquine (50μM), or 1,25 dihydroxyvitamin D3 (Calcitriol) (50nM or 100nM), or chloroquine + Calcitriol, and harvested at periodic intervals over 197 hours. Stoichiometric time course studies (24 hours) were evaluated for Calcitriol as a single agent in both T-47D and MCF-10A cell lines. Cell morphology, adhesion, proliferation and autophagic flux in the DCIS organoids were assessed by Immunohistochemistry and RPPA. Calcitriol alone did not increase proliferation based on PCNA expression within DCIS ducts. Chloroquine markedly increased Cleaved PARP (Chi Sq p = 0.004) and CAMKinase II (p = 0.0068). Chloroquine (50μM) plus Calcitriol (100nM) increased E-Cadherin (p = 0.0379) and phosphoPAK1/2 (p = 0.0163), while PMCA2 levels were not significantly different between treatment groups. Autophagy inhibition and calcium efflux may be exploited therapeutically by co-administration of chloroquine and Calcitrol, to inhibit lysosomal catabolism and increase intracellular calcium levels, respectively, switching them from autophagic survival into apoptotic death. Citation Format: Virginia A. Espina, Solomon Yeon, Joshua N. VanHouten, John Wysolmerski, Lance A. Liotta. Chloroquine and vitamin D3 modulate proliferation in early stage breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2859.

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression.

Selective upregulation of the expression of plasma membrane calcium ATPase isoforms upon differentiation and 1,25(OH)2D3-vitamin treatment of colon cancer cells

We have previously presented co-expression of the plasma membrane calcium ATPase isoforms 4b (PMCA4b) and 1b (PMCA1b) in colon carcinoma cells, and selective upregulation of PMCA4b during differentiation initiated by short chain fatty acids or post-confluent growth. Here we show that the induction of PMCA4b expression is a characteristic feature of the post-confluency-induced differentiation of both enterocyte-type and goblet cell-type colon cancer cells. Vitamin D3 (1,25(OH)2D3) is a well-known regulator of intestinal Ca2+ absorption and of basic cell functions such as growth and differentiation in various cell types. As PMCA proteins are involved both in intestinal Ca2+ absorption and adenocarcinoma cell differentiation, we investigated the effect of 1,25(OH)2D3 on PMCA expression in enterocyte-like colon carcinoma cells, and monitored its effect on the expression of various differentiation markers. 1,25(OH)2D3 stimulated PMCA1b, but not PMCA4b expression without modulating the expression of the majority of the differentiation markers examined. Caco-2 cells differentiated in post-confluent cultures present normal enterocyte-like intestinal epithelial phenotype. To better understand the role of PMCA proteins in vectorial Ca2+ transport by enterocytes, we also studied their subcellular localization in mature polarized Caco-2 cells. Both PMCA isoforms were located to the basolateral membrane, and the PMCA-specific immunofluorescent signal was significantly higher in vitamin D3-treated cells, underlining the 1,25(OH)2D3-induced upregulation of PMCA (presumably 1b isoform) expression in differentiated Caco-2 cells. We suggest that while PMCA1b has a housekeeping function in colon cancer cells, PMCA4b participates in the reorganization of the Ca2+ signalling machinery during cell differentiation. The subcellular localization of PMCA1b and its selective 1,25(OH)2D3-dependent upregulation indicate that this isoform may have a specific role in 1,25(OH)2D3-stimulated intestinal Ca2+ absorption.

Plasma membrane calcium ATPases: From generic Ca2+ sump pumps to versatile systems for fine-tuning cellular Ca2+

The plasma membrane calcium ATPases (PMCAs) are ATP-driven primary ion pumps found in all eukaryotic cells. They are the major high-affinity calcium extrusion system for expulsion of Ca2+ ions from the cytosol and help restore the low resting levels of intracellular [Ca2+] following the temporary elevation of Ca2+ generated during Ca2+ signaling. Due to their essential role in the maintenance of cellular Ca2+ homeostasis they were initially thought to be “sump pumps” for Ca2+ removal needed by all cells to avoid eventual calcium overload. The discovery of multiple PMCA isoforms and alternatively spliced variants cast doubt on this simplistic assumption, and revealed instead that PMCAs are integral components of highly regulated multi-protein complexes fulfilling specific roles in calcium-dependent signaling originating at the plasma membrane. Biochemical, genetic, and physiological studies in gene-manipulated and mutant animals demonstrate the important role played by specific PMCAs in distinct diseases including those affecting the peripheral and central nervous system, cardiovascular disease, and osteoporosis. Human PMCA gene mutations and allelic variants associated with specific disorders continue to be discovered and underline the crucial role of different PMCAs in particular cells, tissues and organs.

Disrupted calcium homeostasis is involved in elevated zinc ion-induced photoreceptor cell death

Zinc (Zn), the second abundant trace element in living organisms, plays an important role in regulating cell metabolism, signaling, proliferation, gene expression and apoptosis. Meanwhile, the overload of Zn will disrupt the intracellular calcium homeostasis via impairing mitochondrial function. However, the specific molecular mechanism underlying zinc-induced calcium regulation remains poorly understood. In the present study, using zinc chloride (ZnCl2) as a stressor, we investigated the effect of exogenous Zn2+ in regulating murine photoreceptor cell viability, reactive oxygen species (ROS), cell cycle distribution and calcium homeostasis as well as plasma membrane calcium ATPase (PMCA) isoforms (PMCA1 and PMCA2, i.e., ATP2B1, ATP2B2) expression. We found that the exogenous Zn2+ in the exposure range (31.25–125.0μmol/L) results in the overgeneration of ROS, cell cycle arrest at G2/M phases, elevation of cytosolic [Ca2+], inactivation of Ca2+-ATPase and reduction of both PMCA1 and PMCA2 in 661Wcells, and thus induces cell death. In conclusion, ZnCl2 exposure can elevate the cytosolic [Ca2+], disrupt the intracellular calcium homeostasis, further initiate Ca2+-dependent signaling pathway in 661Wcells, and finally cause cell death. Our results will facilitate the understanding of cell death induced by the zinc ion-mediated calcium homeostasis disruption.

Impaired nitric oxide production and increased blood pressure in systemic heterozygous ATP2B1 null mice

In the 'Millennium Genome Project', we identified ATP2B1 as a gene responsible for hypertension through single-nucleotide polymorphism analysis. The ATP2B1 gene encodes the plasma membrane calcium ATPase isoform 1, which contributes to the maintenance of intracellular calcium homeostasis by removing calcium ions. Since ATP2B1 knockout mice are reported to be embryo-lethal, we generated systemic heterozygous ATP2B1 null (ATP2B1(+/-)) mice, and evaluated the implication of ATP2B1 in blood pressure. ATP2B1(+/-) mice revealed significantly higher SBP as measured by a radiotelemetric method. Phenylephrine-induced vasoconstriction was significantly increased in vascular rings from ATP2B1(+/-) mice, and the difference in this contraction disappeared in the presence of a nitric oxide synthase (NOS) inhibitor. Vasorelaxation to acetylcholine was significantly attenuated in vascular rings from ATP2B1(+/-) mice. In addition, cultured endothelial cells of ATP2B1(+/-) mice showed that the phosphorylation (Ser-1177) level of endothelial NOS protein was significantly lower, and nitric oxide production in endothelial cells and aorta was lower compared with those in control mice. In contrast, neural NOS expression in vascular smooth muscle cells from ATP2B1(+/-) mice and control mice were not significantly different. These results suggest that decreased ATP2B1 gene expression is associated with impaired endothelial NOS activity and nitric oxide production, and the ATP2B1 gene plays a crucial role in the regulation of blood pressure.

Enhanced Synaptic Inhibition in the Cerebellar Cortex of the Ataxic PMCA2−/− Knockout Mouse

Mice with genetic deletion of a calcium extrusion pump, the plasma membrane calcium ATPase isoform 2, PMCA2, exhibit overt cerebellar ataxia, but the cellular mechanisms are only partially understood. Here, we report an enhanced synaptic GABAergic inhibition within the molecular layer of cerebellar cortex slices from PMCA2 knockout (PMCA2(-/-)) mice, a finding that could contribute to the observed ataxia. Purkinje neurons from PMCA2(-/-) mice exhibited an increased frequency and amplitude of spontaneous inhibitory post-synaptic currents that was accompanied by an enhanced spontaneous firing frequency of molecular layer interneurons (both basket cells and stellate cells). The elevated inhibition was sufficient to reduce the frequency and regularity of spike firing by PMCA2(-/-) Purkinje neurons. Acute pharmacological inhibition of PMCA recapitulated some of these features in wild-type mice indicating that the changes were in part a direct result of PMCA2 removal. However, additional compensatory mechanisms within the PMCA2(-/-) mouse were also a major factor. Indeed, morphological studies revealed an abnormally large number of molecular layer interneurons (basket cells and stellate cells) and GABAergic synapses within the PMCA2(-/-) cerebellar cortex. We conclude that loss of PMCA2 adversely influences the function and organisation of Purkinje neuron synaptic inhibition as a major contributory mechanism to the ataxic phenotype of the PMCA2(-/-) mouse.

Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase

The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.

730 THE EFFECTS OF THE DISTAL TUBULES-SPECIFIC ATP2B1 DEFICIENCIES ON BLOOD PRESSURE AND URINARY OSMOLARITY IN MICE

Objectives: We have already demonstrated that ATP2B1 gene is associated with hypertension in Japanese populations. ATP2B1 encodes the plasma membrane calcium ATPase isoform 1(PMCA1), which is critical component in the regulation of cellular calcium homeostasis. Because PMCA1 is expressed ubiquitously, and it seems to be the major housekeeping enzyme, the systemic deletion of the ATP2B1 has been reported as embryonic lethal. Therefore, to determine the relationship between ATP2B1 and hypertension, we generated conditional knockout mice. We recently reported that vascular smooth muscle cell specific knockout of the ATP2B1 causes hypertension through alterations in calcium homeostasis (Hypertension, 2012). To evaluate the role of ATP2B1 in kidney, we used Cre-loxp technology to eliminate ATP2B1 genes from distal tubules. Methods: We generated mice with distal tubules specific knockout of the ATP2B1 by Cre-loxp technology using a kidney-specific cadherin promoter (Ksp). The male mice with homozygous for the floxed ATP2B1 and heterozygous for Ksp-Cre, were used in all studies. Blood pressure was measured by tail-cuff method and telemetry method. Results: The birth ratios were not different between KO and control mice. KO mice grow and increase their body weight as with control mice. There were no significant differences in blood pressure between KO and controls mice measured by the tail-cuff method. However, KO mice increased the urine volume for three times. Similarly, the urine osmolarity was decreased to one-third in KO mice. Conclusion: The tubular knockout of ATP2B1 gene affects the urinary osmolality. The effects of ATP2B1 gene deletion in tubular cells on blood pressure might be limited.

Distinct Regulation of Cytoplasmic Calcium Signals and Cell Death Pathways by Different Plasma Membrane Calcium ATPase Isoforms in MDA-MB-231 Breast Cancer Cells

Plasma membrane calcium ATPases (PMCAs) actively extrude Ca(2+) from the cell and are essential components in maintaining intracellular Ca(2+) homeostasis. There are four PMCA isoforms (PMCA1-4), and alternative splicing of the PMCA genes creates a suite of calcium efflux pumps. The role of these different PMCA isoforms in the control of calcium-regulated cell death pathways and the significance of the expression of multiple isoforms of PMCA in the same cell type are not well understood. In these studies, we assessed the impact of PMCA1 and PMCA4 silencing on cytoplasmic free Ca(2+) signals and cell viability in MDA-MB-231 breast cancer cells. The PMCA1 isoform was the predominant regulator of global Ca(2+) signals in MDA-MB-231 cells. PMCA4 played only a minor role in the regulation of bulk cytosolic Ca(2+), which was more evident at higher Ca(2+) loads. Although PMCA1 or PMCA4 knockdown alone had no effect on MDA-MB-231 cell viability, silencing of these isoforms had distinct consequences on caspase-independent (ionomycin) and -dependent (ABT-263) cell death. PMCA1 knockdown augmented necrosis mediated by the Ca(2+) ionophore ionomycin, whereas apoptosis mediated by the Bcl-2 inhibitor ABT-263 was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4 silencing in promoting ABT-263-induced cell death. This study demonstrates distinct roles for PMCA1 and PMCA4 in the regulation of calcium signaling and cell death pathways despite the widespread distribution of these two isoforms. The targeting of some PMCA isoforms may enhance the effectiveness of therapies that act through the promotion of cell death pathways in cancer cells.

Hair cells, plasma membrane Ca2+ ATPase and deafness

Hearing relies on the ability of the inner ear to convert sound waves into electrical signals. The main actors in this process are hair cells. Their stereocilia contain a number of specific proteins and a scaffold of actin molecules. They are organized in bundles by tip-link filaments composed of cadherin 23 and protocadherin 15. The bundle is deflected by sound waves leading to the opening of mechano-transduction channels and to the influx of K+ and Ca2+ into the stereocilia. Cadherin 23 and the plasma membrane calcium ATPase isoform 2 (PMCA2) are defective in human and murine cases of deafness. While the involvement of cadherin 23 in deafness/hearing could be expected due to its structural role in the tip-links, that of PMCA2 has been discovered only recently. This review will summarize the structural and functional characteristics of hair cells, focusing on the proteins whose mutations may lead to a deafness phenotype.

Mice Lacking Hypertension Candidate Gene ATP2B1 in Vascular Smooth Muscle Cells Show Significant Blood Pressure Elevation

We reported previously that ATP2B1 was one of the genes for hypertension receptivity in a large-scale Japanese population, which has been replicated recently in Europeans and Koreans. ATP2B1 encodes the plasma membrane calcium ATPase isoform 1, which plays a critical role in intracellular calcium homeostasis. In addition, it is suggested that ATP2B1 plays a major role in vascular smooth muscle contraction. Because the ATP2B1 knockout (KO) mouse is embryo-lethal, we generated mice with vascular smooth muscle cell-specific KO of ATP2B1 using the Cre-loxP system to clarify the relationship between ATP2B1 and hypertension. The KO mice expressed significantly lower levels of ATP2B1 mRNA and protein in the aorta compared with control mice. KO mice showed significantly higher systolic blood pressure as measured by tail-cuff method and radiotelemetric method. Similar to ATP2B1, the expression of the Na(+)-Ca(2+) exchanger isoform 1 mRNA was decreased in vascular smooth muscle cells of KO mice. However, ATP2B4 expression was increased in KO mice. The cultured vascular smooth muscle cells of KO mice showed increased intracellular calcium concentration not only in basal condition but also in phenylephrine-stimulated condition. Furthermore, phenylephrine-induced vasoconstriction was significantly increased in vascular rings of the femoral artery of KO mice. These results suggest that ATP2B1 plays important roles in the regulation of blood pressure through alteration of calcium handling and vasoconstriction in vascular smooth muscle cells.

Abstract 9368: Plasma Membrane Calcium ATPase Isoform 1 Protects the Heart from Cardiomyopathy, Arrhythmias and Hypertension

With 1 in 3 people in the USA dying as a result of cardiovascular disease, and the incidence of these diseases rapidly increasing in the developing world, it is essential that we continue to provide mechanistic insights into their development and progression. Several recent human genome wide association studies have identified single nucleotide polymorphisms in the plasma membrane calcium ATPase isoform 1 (PMCA1) gene as the strongest association with hypertension and blood pressure variance; hypertension along with cardiomyopathy and arrhythmias are central features of heart failure. In this study we provide evidence that PMCA1, which is a transmembrane calcium transporter known to eject calcium from the cell, is involved in all of these key features of heart failure. To elucidate the role of PMCA1 in cardiac function we generated mice carrying a cardiomyocyte-specific gene deletion of PMCA1 (PMCA1 cko ). In response to pressure overload hypertrophic stress induced by transverse aortic constriction (TAC) PMCA1 cko mice displayed accelerated cardiac dysfunction shown by significantly reduced fractional shortening compared to controls (FS; PMCA1 cko /TAC: 21±2%, PMCA1 f/f /TAC 32±3%, PMCA1 cko /Sham and [alpha]-MHC-Cre/TAC 35±2%, p <0.05, n=6). This was accompanied by increased lung weight/body weight in PMCA1 cko /TAC (11.5±1 vs 7.8±0.8 mg/g in PMCA1 f/f /TAC, p <0.05, n=6), greater cardiomyocyte cross-sectional area and significantly elevated interstitial fibrosis. Our data shows that in the myocardium PMCA1 has an additional role in stabilising cardiac rhythm. At 3 months of age PMCA1 cko displayed episodes of spontaneous ventricular tachycardia whilst exhibiting an 80% downregulation in the expression of the voltage gated sodium channel Na v 1.5 and a significant reduction in the corresponding current. To investigate the relationship between PMCA1 and blood pressure we generated PMCA1 heterozygous knockout mice (PMCA1 Ht ) in which both systolic and diastolic carotid pressure was significantly increased in PMCA1 Ht in comparison to wild type littermates. In conclusion, this data addresses the mechanistic basis for the association of PMCA1 with hypertension, cardiomyopathy and arrhythmias which are key contributors to heart failure.

Abstract 9943: Prevention and Reversal of Hypertrophy Through a Novel Drug-Like Compound That Inhibits Local Calcium Oscillations in a Pmca4/calcineurin Microdomain

In pathological hypertrophy, the calcium/calmodulin dependent phosphatase calcineurin has been shown to transmit upstream calcium signals through dephosphorylation of the transcription factor NFAT which subsequently translocates to the nucleus and initiates a hypertrophic gene programme. We have previously shown that plasma membrane calcium ATPase isoform 4 (PMCA4) binds to calcineurin when overexpressed in HEK293 cells. We here investigated whether targeting PMCA4 by genetic or pharmacological strategies provides novel opportunities for the reduction of cardiac hypertrophy through modulation of calcineurin. Germline PMCA4 ablation resulted in the attenuation of pathological hypertrophy in response to pressure overload (Heart weight /tibia length ratio after transverse aortic constriction (TAC), PMCA4-/-: 6.74 ± 0.33 mg/mm vs WT: 8.34 ± 0.88 mg/mm, P10). Mechanistically, PMCA4 ablation led to a significant reduction in calcineurin at the sarcolemma as shown by Western blot and electron microscopy. We then developed a specific pharmacological inhibitor of PMCA4, which had not previously been available. Using a modified colorimetric ATPase assay we screened a library of medically optimized drug-like molecules and identified AP2 which has an IC50 of 150 nM for PMCA4. Injection of AP2 in mice (5 mg/kg body weight/day ip) significantly reduced cardiac hypertrophy following TAC for 2 weeks by 50% (n=10 each group, p<0.01). Moreover, AP2 reversed pre-established TAC-induced hypertrophy (HW/TL (mg/mm): sham, 5.7+0.3, TAC+vehicle, 9.1+0.7, TAC+AP2, 7.6+0.6, n=4-6 each group, P<0.05). Using a novel fluorescent Ca2+ sensor (GCaMP2) attached to the N-terminus of PMCA4 we found that AP2 reduced calcium oscillations in the microdomain around PMCA4 by 45% leading to reduced calcineurin activation. Furthermore, AP2 inhibited the NFAT pathway as shown by expression of the bona fide calcineurin target RCAN 1.4 and NFAT phosphorylation levels. Overall, our results demonstrated that specific inhibition of PMCA4 prevents and reverses pressure-overload hypertrophy, likely through regulation of local calcium oscillations around calcineurin making the plasma membrane calcium pump a potential target for the treatment of cardiac hypertrophy.

Abstract A30: Inhibition of PMCA4 as a strategy to increase the effectiveness of Bcl-2 inhibitor-mediated cell death in breast cancer cells.

Abstract Intracellular calcium is a vital cell signaling molecule that can influence a myriad of important cellular functions, including those deregulated in the cancer cell. Consequently Ca2+ levels within the cell must be precisely controlled. Amongst the suite of proteins controlling calcium homeostasis are the plasma membrane calcium ATPases (PMCAs), which belong to a family of druggable proteins. PMCAs are pumps that reside on the plasma membrane and are responsible for the efflux of intracellular Ca2+ and thus are important in maintaining basal cellular calcium levels. Sustained increases in intracellular Ca2+ can be a trigger for cell death and agents that induce alterations in calcium signaling could be a strategy to restore the sensitivity of a cancer cell to death stimuli. The aim of this study was to explore the effectiveness of inhibiting two PMCA isoforms, PMCA1 and PMCA4, on the augmentation of cell death initiated by a Ca2+ionophore (ionomycin) and a Bcl-2 inhibitor (ABT-263) in MDA-MB-231 breast cancer cells. Ionomycin represents a caspase-independent mechanism of cell death and ABT-263 enhances cell death via a caspase-dependent mechanism. PMCA1 and PMCA4 were inhibited using siRNA and the effect on cell death was measured by staining with Hoechst 33342 and propidium iodide using high content imaging (ImageXpress). Intracellular Ca2+ was determined using a FLIPRTETRA with cells loaded with the calcium sensor Fluo4-AM. Inhibition of PMCA1 was associated with significant changes in global cytosolic free Ca2+ induced by purinergic receptor activation, whereas the same effect was not seen with PMCA4 inhibition. Consistent with effects on global calcium levels, ionomycin (3 μM)-induced cell death was significantly augmented by PMCA1 but not PMCA4 siRNA. However, ABT-263 (3 μM)-induced cell death was augmented only by inhibition of PMCA4, suggesting that PMCA4 siRNA-mediated inhibition of ABT-263 apoptosis is mediated through effects on local and/or specific calcium signaling processes. PMCA4 can alter the activity of calcium-dependent transcription factors in cardiac myocytes independent of global cytosolic calcium. In MDA-MB-231 cells, the effects of PMCA4 inhibition appear to be associated with alterations in the activity of NFkappaB. These results highlight the specificity of different isoforms of PMCA in the modulation of cell death pathways in MDA-MB-231 breast cancer cells. Inhibition of PMCA4 may be a viable adjuvant therapy when combined with Bcl-2 family inhibitors for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A30.