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1.
Adv Exp Med Biol ; 1131: 93-129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646508

RESUMO

Plasma membrane Ca2+ transport ATPases (PMCA1-4, ATP2B1-4) are responsible for removing excess Ca2+ from the cell in order to keep the cytosolic Ca2+ ion concentration at the low level essential for normal cell function. While these pumps take care of cellular Ca2+ homeostasis they also change the duration and amplitude of the Ca2+ signal and can create Ca2+ gradients across the cell. This is accomplished by generating more than twenty PMCA variants each having the character - fast or slow response, long or short memory, distinct interaction partners and localization signals - that meets the specific needs of the particular cell-type in which they are expressed. It has become apparent that these pumps are essential to normal tissue development and their malfunctioning can be linked to different pathological conditions such as certain types of neurodegenerative and heart diseases, hearing loss and cancer. In this chapter we summarize the complexity of PMCA regulation and function under normal and pathological conditions with particular attention to recent developments of the field.


Assuntos
Membrana Celular , ATPases Transportadoras de Cálcio da Membrana Plasmática , Animais , Membrana Celular/enzimologia , Membrana Celular/patologia , Citosol/metabolismo , Homeostase/fisiologia , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo
2.
BMC Cancer ; 18(1): 1029, 2018 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-30352569

RESUMO

BACKGROUND: Remodeling of Ca2+ signaling is an important step in cancer progression, and altered expression of members of the Ca2+ signaling toolkit including the plasma membrane Ca2+ ATPases (PMCA proteins encoded by ATP2B genes) is common in tumors. METHODS: In this study PMCAs were examined in breast cancer datasets and in a variety of breast cancer cell lines representing different subtypes. We investigated how estrogen receptor alpha (ER-α) and histone deacetylase (HDAC) inhibitors regulate the expression of these pumps. RESULTS: Three distinct datasets displayed significantly lower ATP2B4 mRNA expression in invasive breast cancer tissue samples compared to normal breast tissue, whereas the expression of ATP2B1 and ATP2B2 was not altered. Studying the protein expression profiles of Ca2+ pumps in a variety of breast cancer cell lines revealed low PMCA4b expression in the ER-α positive cells, and its marked upregulation upon HDAC inhibitor treatments. PMCA4b expression was also positively regulated by the ER-α pathway in MCF-7 cells that led to enhanced Ca2+ extrusion capacity in response to 17ß-estradiol (E2) treatment. E2-induced PMCA4b expression was further augmented by HDAC inhibitors. Surprisingly, E2 did not affect the expression of PMCA4b in other ER-α positive cells ZR-75-1, T-47D and BT-474. These findings were in good accordance with ChIP-seq data analysis that revealed an ER-α binding site in the ATP2B4 gene in MCF-7 cells but not in other ER-α positive tumor cells. In the triple negative cells PMCA4b expression was relatively high, and the effect of HDAC inhibitor treatment was less pronounced as compared to that of the ER-α positive cells. Although, the expression of PMCA4b was relatively high in the triple negative cells, a fraction of the protein was found in intracellular compartments that could interfere with the cellular function of the protein. CONCLUSIONS: Our results suggest that the expression of Ca2+ pumps is highly regulated in breast cancer cells in a subtype specific manner. Our results suggest that hormonal imbalances, epigenetic modifications and impaired protein trafficking could interfere with the expression and cellular function of PMCA4b in the course of breast cancer progression.

3.
Front Oncol ; 7: 95, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28596940

RESUMO

Several new therapeutic options emerged recently to treat metastatic melanoma; however, the high frequency of intrinsic and acquired resistance among patients shows a need for new therapeutic options. Previously, we identified the plasma membrane Ca2+ ATPase 4b (PMCA4b) as a metastasis suppressor in BRAF-mutant melanomas and found that mutant BRAF inhibition increased the expression of the pump, which then inhibited the migratory and metastatic capability of the cells. Earlier it was also demonstrated that histone deacetylase inhibitors (HDACis) upregulated PMCA4b expression in gastric, colon, and breast cancer cells. In this study, we treated one BRAF wild-type and two BRAF-mutant melanoma cell lines with the HDACis, SAHA and valproic acid, either alone, or in combination with the BRAF inhibitor, vemurafenib. We found that HDACi treatment strongly increased the expression of PMCA4b in all cell lines irrespective of their BRAF mutational status, and this effect was independent of ERK activity. Furthermore, HDAC inhibition also enhanced the abundance of the housekeeping isoform PMCA1. Combination of HDACis with vemurafenib, however, did not have any additive effects on either PMCA isoform. We demonstrated that the HDACi-induced increase in PMCA abundance was coupled to an enhanced [Ca2+]i clearance rate and also strongly inhibited both the random and directional movements of A375 cells. The primary role of PMCA4b in these characteristic changes was demonstrated by treatment with the PMCA4-specific inhibitor, caloxin 1c2, which was able to restore the slower Ca2+ clearance rate and higher motility of the cells. While HDAC treatment inhibited cell motility, it decreased only modestly the ratio of proliferative cells and cell viability. Our results show that in melanoma cells the expression of both PMCA4b and PMCA1 is under epigenetic control and the elevation of PMCA4b expression either by HDACi treatment or by the decreased activation of the BRAF-MEK-ERK pathway can inhibit the migratory capacity of the highly motile A375 cells.

4.
Int J Cancer ; 140(12): 2758-2770, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27813079

RESUMO

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca2+ signaling is a well-known regulator of tumor progression, the crosstalk between Ca2+ signaling and the Ras-BRAF-MEK-ERK pathway is much less explored. Here we show that in BRAF mutant melanoma cells the abundance of the plasma membrane Ca2+ ATPase isoform 4b (PMCA4b, ATP2B4) is low at baseline but markedly elevated by treatment with the mutant BRAF specific inhibitor vemurafenib. In line with these findings gene expression microarray data also shows decreased PMCA4b expression in cutaneous melanoma when compared to benign nevi. The MEK inhibitor selumetinib-similarly to that of the BRAF-specific inhibitor-also increases PMCA4b levels in both BRAF and NRAS mutant melanoma cells suggesting that the MAPK pathway is involved in the regulation of PMCA4b expression. The increased abundance of PMCA4b in the plasma membrane enhances [Ca2+ ]i clearance from cells after Ca2+ entry. Moreover we show that both vemurafenib treatment and PMCA4b overexpression induce marked inhibition of migration of BRAF mutant melanoma cells. Importantly, reduced migration of PMCA4b expressing BRAF mutant cells is associated with a marked decrease in their metastatic potential in vivo. Taken together, our data reveal an important crosstalk between Ca2+ signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor.


Assuntos
Movimento Celular/genética , Melanoma/genética , Mutação , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Animais , Western Blotting , Cálcio/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/metabolismo , Melanoma/patologia , Camundongos SCID , Microscopia Confocal , Metástase Neoplásica , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Sulfonamidas/farmacologia , Transplante Heterólogo , Vemurafenib
5.
Methods Mol Biol ; 1307: 141-7, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-24482125

RESUMO

Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well.


Assuntos
Bioquímica/métodos , Sinalização do Cálcio , Células-Tronco Embrionárias Humanas/metabolismo , Espaço Intracelular/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Estatística como Assunto
6.
Biochim Biophys Acta ; 1863(6 Pt B): 1351-63, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26707182

RESUMO

Plasma membrane Ca(2+) ATPases (PMCAs) are intimately involved in the control of intracellular Ca(2+) concentration. They reduce Ca(2+) in the cytosol not only by direct ejection, but also by controlling the formation of inositol-1,4,5-trisphosphate and decreasing Ca(2+) release from the endoplasmic reticulum Ca(2+) pool. In mammals four genes (PMCA1-4) are expressed, and alternative RNA splicing generates more than twenty variants. The variants differ in their regulatory characteristics. They localize into highly specialized membrane compartments and respond to the incoming Ca(2+) with distinct temporal resolution. The expression pattern of variants depends on cell type; a change in this pattern can result in perturbed Ca(2+) homeostasis and thus altered cell function. Indeed, PMCAs undergo remarkable changes in their expression pattern during tumorigenesis that might significantly contribute to the unbalanced Ca(2+) homeostasis of cancer cells. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Membrana Celular/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Homeostase , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética
7.
Sci Signal ; 8(364): ra19, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25690014

RESUMO

Calcium (Ca(2+)) is a critical cofactor and signaling mediator in cells, and the concentration of cytosolic Ca(2+) is regulated by multiple proteins, including the plasma membrane Ca(2+)-ATPases (adenosine triphosphatases) (PMCAs), which use ATP to transport Ca(2+) out of cells. PMCA isoforms exhibit different kinetic and regulatory properties; thus, the presence and relative abundance of individual isoforms may help shape Ca(2+) transients and cellular responses. We studied the effects of three PMCA isoforms (PMCA4a, PMCA4b, and PMCA2b) on Ca(2+) transients elicited by conditions that trigger store-operated Ca(2+) entry (SOCE) and that blocked Ca(2+) uptake into the endoplasmic reticulum in HeLa cells, human embryonic kidney (HEK) 293 cells, or primary endothelial cell isolated from human umbilical cord veins (HUVECs). The slowly activating PMCA4b isoform produced long-lasting Ca(2+) oscillations in response to SOCE. The fast-activating isoforms PMCA2b and PMCA4a produced different effects. PMCA2b resulted in rapid and highly PMCA abundance-sensitive clearance of SOCE-mediated Ca(2+) transients, whereas PMCA4a reduced cytosolic Ca(2+), resulting in the establishment of a higher than baseline cytosolic Ca(2+) concentration. Mathematical modeling showed that slow activation was critical to the sustained oscillation induced by the "slow" PMCA4b pump. The modeling and experimental results indicated that the distinct properties of PMCA isoforms differentially regulate the pattern of SOCE-mediated Ca(2+) transients, which would thus affect the activation of downstream signaling pathways.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Membrana Celular/enzimologia , Modelos Biológicos , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Isoformas de Proteínas/metabolismo , Transdução de Sinais
8.
Cell Calcium ; 55(2): 78-92, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24439526

RESUMO

The expression of the plasma membrane Ca2+ ATPase (PMCA) isoforms is altered in several types of cancer cells suggesting that they are involved in cancer progression. In this study we induced differentiation of MCF-7 breast cancer cells by histone deacetylase inhibitors (HDACis) such as short chain fatty acids (SCFAs) or suberoylanilide hydroxamic acid (SAHA), and by phorbol 12-myristate 13-acetate (PMA) and found strong upregulation of PMCA4b protein expression in response to these treatments. Furthermore, combination of HDACis with PMA augmented cell differentiation and further enhanced PMCA4b expression both at mRNA and protein levels. Immunocytochemical analysis revealed that the upregulated protein was located mostly in the plasma membrane. To examine the functional consequences of elevated PMCA4b expression, the characteristics of intracellular Ca2+ signals were investigated before and after differentiation inducing treatments, and also in cells overexpressing PMCA4b. The increased PMCA4b expression - either by treatment or overexpression - led to enhanced Ca2+ clearance from the stimulated cells. We found pronounced PMCA4 protein expression in normal breast tissue samples highlighting the importance of this pump for the maintenance of mammary epithelial Ca2+ homeostasis. These results suggest that modulation of Ca2+ signaling by enhanced PMCA4b expression may contribute to normal development of breast epithelium and may be lost in cancer.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ésteres de Forbol/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Regulação para Cima/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Células MCF-7 , Maleimidas/farmacologia , Ácidos Pentanoicos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Vorinostat
9.
J Cell Sci ; 127(Pt 1): 72-84, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24198396

RESUMO

Plasma membrane Ca(2+) ATPases (PMCAs, also known as ATP2B1-ATP2B4) are known targets of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], but if and how they control the PtdIns(4,5)P2 pool has not been considered. We demonstrate here that PMCAs protect PtdIns(4,5)P2 in the plasma membrane from hydrolysis by phospholipase C (PLC). Comparison of active and inactive PMCAs indicates that the protection operates by two mechanisms; one requiring active PMCAs, the other not. It appears that the mechanism requiring activity is the removal of the Ca(2+) required for sustained PLC activity, whereas the mechanism not requiring activity is PtdIns(4,5)P2 binding. We show that in PMCA overexpressing cells, PtdIns(4,5)P2 binding can lead to less inositol 1,4,5-triphosphate (InsP3) and diminished Ca(2+) release from intracellular Ca(2+) pools. Inspection of a homology model of PMCA suggests that PMCAs have a conserved cluster of basic residues forming a 'blue collar' at the interface between the membrane core and the cytoplasmic domains. By molecular dynamics simulation, we found that the blue collar forms four binding pockets for the phosphorylated inositol head group of PtdIns(4,5)P2; these pockets bind PtdIns(4,5)P2 strongly and frequently. Our studies suggest that by having the ability to bind PtdIns(4,5)P2, PMCAs can control the accessibility of PtdIns(4,5)P2 for PLC and other PtdIns(4,5)P2-mediated processes.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Sinalização do Cálcio , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Membrana Celular/química , Expressão Gênica , Regulação da Expressão Gênica , Células HeLa , Humanos , Hidrólise , Inositol 1,4,5-Trifosfato/química , Transporte de Íons , Simulação de Dinâmica Molecular , Fosfatidilinositol 4,5-Difosfato/química , Ligação Proteica , Coelhos , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fosfolipases Tipo C/química , Fosfolipases Tipo C/genética
10.
Biochim Biophys Acta ; 1833(12): 2561-2572, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23830917

RESUMO

Recent evidences show that the localization of different plasma membrane Ca(2+) ATPases (PMCAs) is regulated in various complex, cell type-specific ways. Here we show that in low-density epithelial and endothelial cells PMCA4b localized mostly in intracellular compartments and its plasma membrane localization was enhanced upon increasing density of cells. In good correlation with the enhanced plasma membrane localization a significantly more efficient Ca(2+) clearance was observed in confluent versus non-confluent HeLa cell cultures expressing mCherry-PMCA4b. We analyzed the subcellular localization and function of various C-terminally truncated PMCA4b variants and found that a truncated mutant PMCA4b-ct24 was mostly intracellular while another mutant, PMCA4b-ct48, localized more to the plasma membrane, indicating that a protein sequence corresponding to amino acid residues 1158-1181 contained a signal responsible for the intracellular retention of PMCA4b in non-confluent cultures. Alteration of three leucines to alanines at positions 1167-1169 resulted in enhanced cell surface expression and an appropriate Ca(2+) transport activity of both wild type and truncated pumps, suggesting that the di-leucine-like motif (1167)LLL was crucial in targeting PMCA4b. Furthermore, upon loss of cell-cell contact by extracellular Ca(2+) removal, the wild-type pump was translocated to the early endosomal compartment. Targeting PMCA4b to early endosomes was diminished by the L(1167-69)A mutation, and the mutant pump accumulated in long tubular cytosolic structures. In summary, we report a di-leucine-like internalization signal at the C-tail of PMCA4b and suggest an internalization-mediated loss of function of the pump upon low degree of cell-cell contact.


Assuntos
Membrana Celular/enzimologia , Leucina/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Asparagina/metabolismo , Cálcio/metabolismo , Compartimento Celular , Contagem de Células , Cães , Dinaminas/metabolismo , Endocitose , Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Lisina/metabolismo , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Sinais Direcionadores de Proteínas , Transporte Proteico , Alinhamento de Sequência , Relação Estrutura-Atividade , Frações Subcelulares/metabolismo
11.
Cell Signal ; 25(4): 752-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23305950

RESUMO

Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca(2+) indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca(2+) oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca(2+) signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging.


Assuntos
Cálcio/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Compostos de Anilina/química , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Histamina/farmacologia , Humanos , Microscopia Confocal , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Trombina/farmacologia , Xantenos/química , Ácido gama-Aminobutírico/farmacologia
12.
FEBS J ; 280(21): 5408-18, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23157274

RESUMO

Cellular calcium signaling plays important roles in several signal transduction pathways that control proliferation, differentiation and apoptosis. In epithelial cells calcium signaling is initiated mainly by calcium release from endoplasmic-reticulum-associated intracellular calcium pools. Because calcium is accumulated in the endoplasmic reticulum by sarco/endoplasmic reticulum calcium ATPases (SERCA), these enzymes play a critical role in the control of calcium-dependent cell activation, growth and survival. We investigated the modulation of SERCA expression and function in human lung adenocarcinoma cells. In addition to the ubiquitous SERCA2 enzyme, the SERCA3 isoform was also expressed at variable levels. SERCA3 expression was selectively enhanced during cell differentiation in lung cancer cells, and marked SERCA3 expression was found in fully differentiated normal bronchial epithelium. As studied by using a recombinant fluorescent calcium probe, induction of the expression of SERCA3, a lower calcium affinity pump, was associated with decreased intracellular calcium storage, whereas the amplitude of capacitative calcium influx remained unchanged. Our observations indicate that the calcium homeostasis of the endoplasmic reticulum in lung adenocarcinoma cells presents a functional defect due to decreased SERCA3 expression that is corrected during pharmacologically induced differentiation. The data presented in this work show, for the first time, that endoplasmic reticulum calcium storage is anomalous in lung cancer cells, and suggest that SERCA3 may serve as a useful new phenotypic marker for the study of lung epithelial differentiation.


Assuntos
Adenocarcinoma/patologia , Sinalização do Cálcio , Diferenciação Celular , Retículo Endoplasmático/enzimologia , Neoplasias Pulmonares/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Adenocarcinoma/metabolismo , Western Blotting , Proliferação de Células , Homeostase , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/metabolismo
13.
Cell Calcium ; 51(2): 171-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22252018

RESUMO

Local Ca(2+) signaling requires proper targeting of the Ca(2+) signaling toolkit to specific cellular locales. Different isoforms of the plasma membrane Ca(2+) pump (PMCA) are responsible for Ca(2+) extrusion at the apical and basolateral membrane of polarized epithelial cells, but the mechanisms and signals for differential targeting of the PMCAs are not well understood. Recent work demonstrated that the alternatively spliced w-insert in PMCA2 directs this pump to the apical membrane. We now show that inserting the w-insert into the corresponding location of the PMCA4 isoform confers apical targeting to this normally basolateral pump. Mutation of a di-leucine motif in the C-tail thought to be important for basolateral targeting did not enhance apical localization of the chimeric PMCA4(2w)/b. In contrast, replacing the C-terminal Val residue by Leu to optimize the PDZ ligand site for interaction with the scaffolding protein NHERF2 enhanced the apical localization of PMCA4(2w)/b, but not of PMCA4x/b. Functional studies showed that both apical PMCA4(2w)/b and basolateral PMCA4x/b handled ATP-induced Ca(2+) signals with similar kinetics, suggesting that isoform-specific functional characteristics are retained irrespective of membrane targeting. Our results demonstrate that the alternatively spliced w-insert provides autonomous apical targeting information in the PMCA without altering its functional characteristics.


Assuntos
Processamento Alternativo/fisiologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/enzimologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/biossíntese , Animais , Linhagem Celular , Membrana Celular/genética , Cães , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética
14.
Mol Cell Endocrinol ; 353(1-2): 57-67, 2012 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-21945604

RESUMO

Pluripotent stem cells represent a new source of biological material allowing the exploration of signaling phenomena during normal cell development and differentiation. Still, the calcium signaling pathways and intracellular calcium responses to various ligands or stress conditions have not been sufficiently explored as yet in embryonic or induced pluripotent stem cells and in their differentiated offspring. This is partly due to the special culturing conditions of these cell types, the rapid morphological and functional changes in heterogeneous cell populations during early differentiation, and methodological problems in cellular calcium measurements. In this paper, we review the currently available data in the literature on calcium signaling in pluripotent stem cells and discuss the potential shortcomings of these studies. Various assay methods are surveyed for obtaining reliable data both in undifferentiated embryonic stem cells and in specific, stem cell-derived human tissues. In this paper, we present the modulation of calcium signaling in human embryonic stem cells (hESC) and in their derivates; mesenchymal stem cell like (MSCl) cells and cardiac tissues using the fluorescent calcium indicator Fluo-4 and confocal microscopy. LPA, trypsin and angiotensin II were effective in inducing calcium signals both in HUES9 and MSCl cells. Histamine and thrombin induced calcium signal exclusively in the MSCl cells, while ATP was effective only in HUES9 cells. There was no calcium signal evoked by GABA, even at relatively high concentrations. In stem cell-derived cardiomyocytes a rapid increase in the beating rate and an increase of the calcium signal peaks could be observed after the addition of adrenaline, while verapamil led to a strong decrease in cellular calcium and stopped spontaneous contractions in a relaxed state.


Assuntos
Sinalização do Cálcio/fisiologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/citologia , Histamina/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/citologia , Trombina/metabolismo
15.
Biochem Biophys Res Commun ; 410(2): 322-7, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21672522

RESUMO

The "w" splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.


Assuntos
Membrana Celular/enzimologia , Polaridade Celular , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Linhagem Celular , Cães , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Transfecção
16.
J Biol Chem ; 285(41): 31704-12, 2010 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-20663896

RESUMO

The membrane localization of the plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.


Assuntos
Processamento Alternativo/fisiologia , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Fosfoproteínas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Actinas/metabolismo , Processamento Alternativo/efeitos dos fármacos , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citocalasina D/farmacologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Cães , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosfoproteínas/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Trocadores de Sódio-Hidrogênio/genética , Tiazolidinas/farmacologia
17.
Biochim Biophys Acta ; 1793(6): 1023-32, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19073225

RESUMO

Besides the control of global calcium changes, specific plasma membrane calcium ATPase (PMCA) isoforms are involved in the regulation of local calcium signals. Although local calcium signaling requires the confinement of signaling molecules into microdomains, little is known about the specific organization of PMCA molecules within the plasma membrane. Here we show that co-expression with the postsynaptic density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4b and redistributed the pump into clusters. The clustering of PMCA4b was fully dependent on the presence of its PDZ-binding sequence. Using the fluorescence recovery after photobleaching (FRAP) technique, we show that the lateral membrane mobility of the clustered PMCA4b is significantly lower than that of the non-clustered molecules. Disruption of the actin-based cytoskeleton by cytochalasin D resulted in increased cluster size. Our results suggest that PSD-95 promotes the formation of high-density PMCA4b microdomains in the plasma membrane and that the membrane cytoskeleton plays an important role in the regulation of this process.


Assuntos
Membrana Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
18.
Ann N Y Acad Sci ; 1099: 440-50, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17446484

RESUMO

Maintenance of Ca2+ homeostasis is essential for normal cellular function and survival. Recent evidences suggest that Ca2+ is also an important player of apoptosis. We demonstrated that the plasma membrane Ca2+ ATPase (PMCA) isoform 4b, a key element of cellular Ca2+ homeostasis, was cleaved by caspase-3 during the course of apoptosis. This cleavage of PMCA removed the entire regulatory region from the C terminus, leaving behind a 120-kDa catalytic fragment. Since loss of PMCA activity could lead to intracellular Ca2+ overload and consequently necrotic cell death, an important question is whether the apoptotic fragment of PMCA retains full activity or it is inactivated. To address this question, we constructed a C-terminally truncated mutant that corresponded to the caspase-3 fragment of PMCA4b and showed that it was fully and constitutively active. This mutant was targeted properly to the plasma membrane when it was expressed stably or transiently in several different cell lines. We followed truncation of PMCA during apoptosis induced by mitochondrial or receptor-mediated pathways and found that a similar fragment of 120 kDa was formed and remained intact for several hours after treatment. We have also demonstrated that the caspase-3 cleavage site is an important structural element of PMCA and found that the accessibility of the caspase-3 site depended strongly on the conformational state of the protein.


Assuntos
Apoptose , ATPases Transportadoras de Cálcio/metabolismo , Animais , Caspase 3/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Humanos , Imuno-Histoquímica , Proteínas Recombinantes/metabolismo
19.
Cell Signal ; 18(3): 294-306, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16099142

RESUMO

Sphingomyelinase (SMase)-mediated release of ceramide in the plasma membrane of T-lymphocytes induced by different stimuli such as ligation of Fas/CD95, irradiation, stress, inflammation or anticancer drugs primarily involves mitochondrial apoptosis signaling, but under specific conditions non-apoptotic Fas-signaling was also reported. Here we investigated, using a quantitative simulation model with exogenous C2-ceramide (and SMase), the dependence of activation and fate of T-cells on the strength and duration of ceramide accumulation. A murine, influenza virus hemagglutinin-specific T-helper cell (IP12-7) alone or together with interacting antigen presenting B-cells (APC) was used. C2-ceramide induced apoptosis of TH cells above a 'threshold' stimulus (>25 microM in 'strength' or >30 min in duration), while below the threshold C2-ceramide was non-apoptotic, as confirmed by early and late apoptotic markers (PS-translocation, mitochondrial depolarization, caspase-3 activation, DNA-fragmentation). The modest ceramide stimuli strongly suppressed the calcium response and inhibited several downstream signal events (e.g. ERK1/2-, JNK-phosphorylation, CD69 expression or IL-2 production) in TH cells during both anti-CD3 induced and APC-triggered activation. Ceramide moderately affected the Ca2+ -release from internal stores upon antigen-specific engagement of TCR in immunological synapses, while the influx phase was remarkably reduced in both amplitude and rate, suggesting that the major target(s) of ceramide-effects are membrane-proximal. Ceramide inhibited Kv1.3 potassium channels, store operated Ca2+ -entry (SOC) and depolarized the plasma membrane to which contribution of spontaneously formed ceramide channels is possible. The impaired function of these transporters may be coupled to the quantitative, membrane raft-remodeling effect of ceramide and responsible, in a concerted action, for the suppressed activation. Our results suggest that non-apoptotic Fas stimuli, received from previously activated, FasL+ interacting lymphocytes in the lymph nodes, may negatively regulate subsequent antigen-specific T-cell activation and thus modulate the antigen-specific T-cell response.


Assuntos
Apoptose , Sobrevivência Celular , Ativação Linfocitária , Transdução de Sinais , Esfingosina/análogos & derivados , Linfócitos T Auxiliares-Indutores/fisiologia , Animais , Linfócitos B/fisiologia , Caspase 3 , Caspases/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Fragmentação do DNA , Humanos , Interleucina-2/metabolismo , Canal de Potássio Kv1.3/fisiologia , Potenciais da Membrana/fisiologia , Camundongos , Receptores de Antígenos de Linfócitos T/fisiologia , Esfingomielina Fosfodiesterase/metabolismo , Esfingosina/metabolismo , Esfingosina/farmacologia , Linfócitos T Auxiliares-Indutores/imunologia , Fatores de Tempo , Receptor fas/metabolismo
20.
Biochem J ; 391(Pt 3): 687-92, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16080782

RESUMO

The calmodulin-activated transporter hPMCA4 (human plasma membrane Ca2+-ATPase isoform 4) is a target for cleavage by caspase-3 during apoptosis. We have demonstrated that caspase-3 generates a 120 kDa fragment of this pump which lacks the complete autoinhibitory sequence [Paszty, Verma, Padanyi, Filoteo, Penniston and Enyedi (2002) J. Biol. Chem. 277, 6822-6829]. In the present study we analysed further the characteristics of the fragment of hPMCA4b produced by caspase-3. We did this by overexpressing the caspase-3 cleavage product of hPMCA4b in COS-7 and MDCKII (Madin-Darby canine kidney II) cells. This technique made it possible to clearly define the properties of this fragment, and we showed that it is constitutively active, as it forms a phosphoenzyme intermediate and has high Ca2+ transport activity in the absence of calmodulin. When this fragment of hPMCA4b was stably expressed in MDCKII cell clones, it was targeted without degradation to the basolateral plasma membrane. In summary, our studies emphasize that the caspase-3 cleavage product of hPMCA4b is constitutively active, and that the C-terminus is not required for proper targeting of hPMCA4b to the plasma membrane. Also, for the first time, we have generated cell clones that stably express a constitutively active PMCA.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Caspases/metabolismo , Membrana Celular/metabolismo , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/química , Calmodulina/metabolismo , Caspase 3 , Linhagem Celular , Membrana Celular/enzimologia , Cercopithecus aethiops , Cães , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática , Transporte Proteico , Especificidade por Substrato
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