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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.
J Exp Clin Cancer Res ; 36(1): 87, 2017 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-28651627

RESUMO

BACKGROUND: Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. METHODS: In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. RESULTS: We show that E2A-PBX1+ leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. CONCLUSION: These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.


Assuntos
Expressão Gênica , Leucemia de Células B/genética , Leucemia de Células B/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Cálcio/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Leucemia de Células B/metabolismo , Gradação de Tumores , Estadiamento de Neoplasias , Proteína Quinase C/genética
4.
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.

5.
Cell Calcium ; 65: 73-79, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28216081

RESUMO

Plasma membrane Ca2+-ATPases are key calcium exporter proteins in most tissues, and PMCA4b is the main calcium transporter in the human red blood cells (RBCs). In order to assess the expression level of PMCA4b, we have developed a flow cytometry and specific antibody binding method to quantitatively detect this protein in the erythrocyte membrane. Interestingly, we found several healthy volunteers showing significantly reduced expression of RBC-PMCA4b. Western blot analysis of isolated RBC membranes confirmed this observation, and indicated that there are no compensatory alterations in other PMCA isoforms. In addition, reduced PMCA4b levels correlated with a lower calcium extrusion capacity in these erythrocytes. When exploring the potential genetic background of the reduced PMCA4b levels, we found no missense mutations in the ATP2B4 coding regions, while a formerly unrecognized minor haplotype in the predicted second promoter region closely correlated with lower erythrocyte PMCA4b protein levels. In recent GWA studies, SNPs in this ATP2B4 haplotype have been linked to reduced mean corpuscular hemoglobin concentrations (MCHC), and to protection against malaria infection. Our data suggest that an altered regulation of gene expression is responsible for the reduced RBC-PMCA4b levels that is probably linked to the development of human disease-related phenotypes.


Assuntos
Eritrócitos/metabolismo , Regulação da Expressão Gênica , Haplótipos , ATPases Transportadoras de Cálcio da Membrana Plasmática , Polimorfismo de Nucleotídeo Único , Feminino , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Masculino , ATPases Transportadoras de Cálcio da Membrana Plasmática/biossíntese , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética
6.
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
7.
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
8.
Sci Rep ; 5: 12645, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26234466

RESUMO

In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na(+)/Ca(2+) exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Ratos Transgênicos/genética , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Homozigoto , Masculino , Mefloquina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , Ratos Sprague-Dawley , Ratos Wistar , Tioureia/análogos & derivados , Tioureia/farmacologia
9.
Biochem Biophys Res Commun ; 464(1): 189-94, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26116539

RESUMO

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 Ca(2+) absorption and of basic cell functions such as growth and differentiation in various cell types. As PMCA proteins are involved both in intestinal Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) absorption.


Assuntos
Calcitriol/farmacologia , Cálcio/metabolismo , Regulação Neoplásica da Expressão Gênica , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Células CACO-2 , Sinalização do Cálcio , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Polaridade Celular , Ativação Enzimática/efeitos dos fármacos , Células HT29 , Humanos , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Ativação Transcricional/efeitos dos fármacos
10.
J Am Soc Nephrol ; 26(11): 2731-40, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25788535

RESUMO

Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin-based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules. The transposon-based method used allowed the generation of homozygous transgenic rats containing one copy of the transgene per allele with a defined insertion pattern, without genetic or phenotypic alterations. We applied in vitro confocal and in vivo two-photon microscopy to examine basal calcium levels and ligand- and drug-induced alterations in these levels in proximal tubular epithelial cells. Notably, renal ischemia induced a transient increase in cellular calcium, and reperfusion resulted in a secondary calcium load, which was significantly decreased by systemic administration of specific blockers of the angiotensin receptor and the Na-Ca exchanger. The parallel examination of in vivo cellular calcium dynamics and renal circulation by fluorescent probes opens new possibilities for physiologic and pharmacologic investigations.


Assuntos
Cálcio/metabolismo , Túbulos Renais Proximais/metabolismo , Microscopia Confocal , Transgenes , Animais , Animais Geneticamente Modificados , Citoplasma/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Homozigoto , Hipóxia/patologia , Isquemia/patologia , Rim/metabolismo , Rim/patologia , Córtex Renal/metabolismo , Nefropatias/patologia , Túbulos Renais/metabolismo , Túbulos Renais Proximais/patologia , Ligantes , Ratos , Traumatismo por Reperfusão , Trocador de Sódio e Cálcio/metabolismo
11.
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
12.
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
13.
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
14.
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
15.
J Leukoc Biol ; 93(4): 537-47, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23362305

RESUMO

B cells acquire membrane-bound cognate antigens from the surface of the APCs by forming an IS, similar to that seen in T cells. Recognition of membrane-bound antigens on the APCs initiates adhesion of B lymphocytes to the antigen-tethered surface, which is followed by the formation of radial lamellipodia-like structures, a process known as B cell spreading. The spreading response requires the rearrangement of the submembrane actin cytoskeleton and is regulated mainly via signals transmitted by the BCR. Here, we show that cytoplasmic calcium is a regulator of actin cytoskeleton dynamics in B lymphocytes. We find that BCR-induced calcium mobilization is indispensible for adhesion and spreading of B cells and that PLCγ and CRAC-mediated calcium mobilization are critical regulators of these processes. Measuring calcium and actin dynamics in live cells, we found that a generation of actin-based membrane protrusion is strongly linked to the dynamics of a cytoplasmic-free calcium level. Finally, we demonstrate that PLCγ and CRAC channels regulate the activity of actin-severing protein cofilin, linking BCR-induced calcium signaling to the actin dynamics.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/imunologia , Actinas/genética , Actinas/imunologia , Animais , Apresentação do Antígeno , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/imunologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Cofilina 1/genética , Cofilina 1/imunologia , Cofilina 1/metabolismo , Regulação da Expressão Gênica/imunologia , Vetores Genéticos , Lentivirus/genética , Camundongos , Fosfolipase C gama/genética , Fosfolipase C gama/imunologia , Fosfolipase C gama/metabolismo , Pseudópodes/imunologia , Pseudópodes/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais , Transdução Genética
16.
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
17.
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
18.
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
19.
Commun Integr Biol ; 4(3): 340-3, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21980575

RESUMO

The localization of plasma membrane calcium ATPase (PMCA) isoforms in specified membrane compartments is crucial for their function in local Ca(2+) handling. PMCA2w/b is present in the apical membrane whereas alternative splice variants PMCA2x/b and 2z/b reside in the basolateral membrane in polarized epithelial cells. Here we found that the apical scaffolding protein NHERF2 greatly enhances the apical concentration of PMCA2w/b by tethering the pump to the underlying actin cytoskeleton. The interaction requires the C-terminal PDZ binding sequence in PMCA2b and results in increased membrane retention and decreased lateral mobility of the pump. In contrast, PMCA2x/b remains exclusively basolateral even when NHERF2 is overexpressed. Our results suggest that the alternatively spliced intracellular loop in PMCA2 imposes dominant membrane targeting information. NHERF2-mediated recruitment may be an effective means for polarized cells to regulate the abundance of PMCA2w/b in the apical membrane to meet an increased demand for local Ca(2+) extrusion.

20.
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
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