Isoform-specific up-regulation of plasma membrane Ca2+ATPase expression during colon and gastric cancer cell differentiation

In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca²⁺ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na⁺/K⁺–ATPase or that of the sarco/endoplasmic reticulum Ca²⁺ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca²⁺ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells.Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype.     

Histone deacetylase inhibitor- and PMA-induced upregulation of PMCA4b enhances Ca clearance from MCF-7 breast cancer cells

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.     

Plasma membrane Ca2+-ATPase expression during colon cancer cell line differentiation

The differentiation of colon cancer cell lines is associated with changes in calcium homeostasis. Concomitantly there are changes in the expression of some calcium transporters and G-protein-coupled receptors, which are capable of altering cytosolic-free calcium levels. Recent studies associate alterations in calcium transporter expression with tumourigenesis, such as changes in specific isoforms of the plasma membrane calcium ATPase (PMCA) in breast cancer cell lines. In this study, we examined the expression of PMCA isoforms in the HT-29 colon cancer cell line using two methods of differentiation (sodium butyrate-mediated and spontaneous post-confluency induced differentiation). Our studies show that differentiation of HT-29 colon cancer cells is associated with the up-regulation of the PMCA isoform PMCA4 but no significant alteration in PMCA1. These results suggest that PMCA4 may be important and have a specific role in colon cells as well as being significant in colon cancer tumourigenesis.     

Plasma membrane calcium pumps and their emerging roles in cancer

Alterations in calcium signaling and/or the expression of calcium pumps and channels are an increasingly recognized property of some cancer cells. Alterations in the expression of plasma membrane calcium ATPase (PMCA) isoforms have been reported in a variety of cancer types, including those of breast and colon, with some studies of cancer cell line differentiation identifying specific PMCA isoforms, which may be altered in some cancers. Some studies have also begun to assess levels of PMCA isoforms in clinical tumor samples and to address mechanisms of altered PMCA expression in cancers. Both increases and decreases in PMCA expression have been reported in different cancer types and in many cases these alterations are isoform specific. In this review, we provide an overview of studies investigating the expression of PMCA in cancer and discuss how both the overexpression and reduced expression of a PMCA isoform in a cancer cell could bestow a growth advantage, through augmenting responses to proliferative stimuli or reducing sensitivity to apoptosis.     

Contribution of plasma membrane Ca ATPase to cerebellar synapse function

The cerebellum expresses one of the highest levels of the plasma membrane Ca(2+) ATPase, isoform 2 in the mammalian brain. This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex; i.e. the Purkinje neurons (PNs). Here we review recent evidence, including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2 (PMCA2) knockout mouse, to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour. These studies have also revealed that deletion of PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development, they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs.     

Evidence for a secretory pathway Ca2+-ATPase in sea urchin spermatozoa

Plasma membrane, sarco-endoplasmic reticulum and secretory pathway Ca2+-ATPases (designated PMCA, SERCA and SPCA) regulate intracellular Ca2+ in animal cells. The presence of PMCA, and the absence of SERCA, in sea urchin sperm is known. By using inhibitors of Ca2+-ATPases, we now show the presence of SPCA and Ca2+ store in sea urchin sperm, which refills by SPCA-type pumps. Immunofluorescence shows SPCA localizes to the mitochondrion. Ca2+ measurements reveal that approximately 75% of Ca2+ extrusion is by Ca2+ ATPases and 25% by Na+ dependent Ca2+ exchanger/s. Bisphenol, a Ca2+ ATPase inhibitor, completely blocks the acrosome reaction, indicating the importance of Ca2+-ATPases in fertilization.     

Expression,purification, and characterization of isoform 1 of the plasma membrane Ca2+ pump: focus on calpain sensitivity

The plasma membrane Ca2+ ATPase isoform 1(PMCA1) is ubiquitously distributed in tissues and cells, but only scarce information is available on its properties. The isoform was overexpressed in Sf9 cells, purified on calmodulin columns, and characterized functionally. The level of expression was very low, but sufficient amounts of the protein could be isolated for biochemical characterization. The affinity of PMCA1 for calmodulin was similar to that of PMCA4, the other ubiquitous PMCA isoform. The affinity of PMCA1 for ATP, evaluated by the formation of the phosphorylated intermediate, was higher than that of the PMCA4 pump. The recombinant PMCA1 pump was a much better substrate for the cAMP-dependent protein kinase than the PMCA2 and PMCA4 isoforms. Pulse and chase experiments on Sf9 cells overexpressing the PMCA pumps showed that PMCA1 was much less stable than the PMCA4 and PMCA2 isoforms, i.e. PMCA1 had a much higher sensitivity to degradation by calpain. The effect of calpain was not the result of a general higher susceptibility of the PMCA1 to proteolytic degradation, because the pattern of degradation by trypsin was the same in the three isoforms.     

Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32

Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.     

Expression of Na(+)/Ca(2+) exchanger isoforms (NCX1 and NCX3) and plasma membrane Ca(2+) ATPase during osteoblast differentiation

The ability to deliver calcium to the osteoid is critical to osteoblast function as a regulator of bone calcification. There are two known transmembrane proteins capable of translocating calcium out of the osteoblast, the Na(+)/Ca(2+) exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). In this study, we reveal the presence of the NCX3 isoform in primary osteoblasts and examine the expression of NCX1, NCX3, and PMCA1 during osteoblast differentiation. The predominant NCX isoform expressed by osteoblasts is NCX3. NCX1 also is expressed, but at low levels. Both NCX isoforms are expressed at nearly static levels throughout differentiation. In contrast, PMCA expression peaks at 8 days of culture, early in osteoblast differentiation, but declines thereafter. Immunocytochemical co-detection of NCX and PMCA reveal that NCX is positioned along surfaces of the osteoblast adjacent to osteoid, while PMCA is localized to plasma membrane sites distal to the osteoid. The expression pattern and spatial distribution of NCX support a role as a regulator of calcium efflux from osteoblasts required for calcification. The expression pattern and spatial distribution of PMCA makes its role in the mineralization process unlikely and suggests a role in calcium homeostasis following signaling events.     

Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines

The plasma membrane Ca²⁺ ATPase (PMCA) is an important regulator of free intracellular calcium, with dynamic regulation in the rat mammary gland during lactation. Recent studies suggest that Ca²⁺ plays a role in cellular proliferation. To determine if PMCA expression is altered in tumorigenesis, we compared relative levels of PMCA1 mRNA. We found that the relative expression of PMCA1 mRNA is increased, by approximately 270% and 170%, in MCF-7 and MDA-MB-231 human breast cancer cell lines deprived of serum for 72 h, respectively, compared to the similarly treated MCF-10A human mammary gland epithelial cell line. Characterization of PMCA mRNA isoforms revealed that PMCA1b and PMCA4 mRNA are expressed in MCF-7, MDA-MB-231, SK-BR-3, ZR-75-1 and BT-483 breast cancer cell lines. We also detected PMCA2 mRNA expression in all the breast cancer cell lines examined. However, PMCA3 mRNA was only detected in BT-483 cells. Our results suggest that PMCA expression may be altered in breast cancer cell lines, suggesting altered Ca²⁺ regulation in these cell lines. Our results also indicate that breast cancer cell lines can express mRNAs for a variety PMCA isoforms.     

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.     

Internalization of plasma membrane Ca2+-ATPase during Xenopus oocyte maturation

A transient increase in intracellular Ca²⁺ is the universal signal for egg activation at fertilization. Eggs acquire the ability to mount the specialized fertilization-specific Ca²⁺ signal during oocyte maturation. The first Ca²⁺ transient following sperm entry in vertebrate eggs has a slow rising phase followed by a sustained plateau. The molecular determinants of the sustained plateau are poorly understood. We have recently shown that a critical determinant of Ca²⁺ signaling differentiation during oocyte maturation is internalization of the plasma membrane calcium ATPase (PMCA). PMCA internalization is representative of endocytosis of several integral membrane proteins during oocyte maturation, a requisite process for early embryogenesis. Here we investigate the mechanisms regulating PMCA internalization. To track PMCA trafficking in live cells we cloned a full-length cDNA of Xenopus PMCA1, and show that GFP-tagged PMCA traffics in a similar fashion to endogenous PMCA. Functional data show that MPF activation during oocyte maturation is required for full PMCA internalization. Pharmacological and co-localization studies argue that PMCA is internalized through a lipid raft endocytic pathway. Deletion analysis reveal a requirement for the N-terminal cytoplasmic domain for efficient internalization. Together these studies define the mechanistic requirements for PMCA internalization during oocyte maturation.     

Plasma membrane calcium pump regulation by metabolic stress

The plasma membrane Ca(2+)-ATPase (PMCA) is an ATP-driven pump that is critical for the maintenance of low resting [Ca(2+)](i) in all eukaryotic cells. Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism, has the capacity to cause ATP depletion and thus inhibit PMCA activity. This has potentially fatal consequences, particularly for non-excitable cells in which the PMCA is the major Ca(2+) efflux pathway. This is because inhibition of the PMCA inevitably leads to cytosolic Ca(2+) overload and the consequent cell death. However, the relationship between metabolic stress, ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted. There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion. In particular, there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function. Moreover, membrane phospholipids, mitochondrial membrane potential, caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA. The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca(2+) overload and cytotoxicity.     

Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.     

Splicing of the Muscle-Specific Plasma Membrane Ca2+-ATPase Isoform PMCA1c Is Associated with Cell Fusion in C2 Myocytes

Abstract: The regulation of intracellular calcium is essential for proper muscle function. Muscle cells have several mechanisms for dealing with the rapid and large changes in cytosolic calcium level that occur during contraction. Among these is the plasma membrane Ca²⁺-ATPase (PMCA), which pumps calcium from the cytosol to the extracellular space. We have previously shown that in human fetal muscle the PMCA1 isoforms present are PMCA1a-d, with PMCA1b and c predominating. Alternative splicing of mRNAs encoding proteins involved in muscle contraction is common in developing muscle. Therefore, we examined the expression of muscle-specific PMCA mRNAs in pre- and postfusion mouse C2 myoblasts. The housekeeping form of the Ca²⁺-ATPase, PMCA1b, was found at all times and under all conditions. However, the other predominating isoform found in muscle, PMCA1c, was expressed on myotube formation. Simple cell-cell contact was not sufficient to induce PMCA1c expression, as cells plated at confluence but harvested before myotube formation did not express PMCA1c. The induction of this muscle-specific Ca²⁺-ATPase at myotube formation suggests that it may play an important role in muscle function.     

Expression and role of calcium-ATPase pump and sodium-calcium exchanger in differentiated trophoblasts from human term placenta

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are not completely elucidated. Especially, mechanisms of syncytiotrophoblast Ca(2+) extrusion into fetal circulation remain to be established. In the current study we have investigated the characteristics of Ca(2+) efflux in syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblasts isolated from human term placenta. Time-courses of Ca(2+) uptake by differentiated human trophoblasts displayed rapid initial entry (initial velocity (V(i)) of 8.82 +/- 0.86 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid decline of cell-associated (45)Ca(2+) with a V(i) of efflux (V(ie)) of 8.90 +/- 0.96 nmol/mg protein/min. Expression of membrane systems responsible for intracellular Ca(2+) extrusion from differentiated human trophoblast were investigated by RT-PCR. Messenger RNAs of four known isoforms of PMCA (PMCA 1-4) were detected. Messenger RNAs of two cloned human NCX isoforms (NCX1 and NCX3) were also revealed. More specifically, both splice variants NCX1.3 and NCX1.4 were amplified by PCR with total RNA of differentiated human trophoblast cells. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. However, erythrosine B (inhibitor of PMCA) time- and dose-dependently increased cell associated (45)Ca(2+) suggesting a principal role of plasma membrane Ca(2+)-ATPase (PMCA) in the intracellular Ca(2+) extrusion of syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblast cells isolated from human term placenta.     

Hypoxic up-regulation of triosephosphate isomerase expression in mouse brain capillary endothelial cells

A protein with a molecular mass of 27kDa was induced by hypoxia in a mouse brain capillary endothelial cell line and identified as triosephosphate isomerase (TPI) by amino-terminal sequencing. Hypoxia caused an elevation of the TPI protein level, concomitant with an increase of the TPI mRNA level. However, hypoxia resulted in an insufficient elevation of TPI activity level, compared to an increase of TPI protein level. When cells expressing the recombinant TPI protein with histidine tag were exposed to hypoxia and the TPI protein was affinity-purified, the catalytic activity (specific activity) of the TPI protein purified from hypoxic cells was substantially lower than that obtained from normoxic cells. In addition, three TPI isoforms with an electrophoretic multiplicity were found; two of the three isoforms were substantially increased in response to the hypoxia, but the level of the most acidic isoform was barely changed. The induction of TPI gene expression by hypoxia was suppressed by (1) a chelator of intracellular Ca(2+), (2) a blocker of non-selective cation channels, (3) a blocker of Na(+)/Ca(2+) exchangers, (4) an inhibitor of Ca(2+)/calmodulin-dependent protein kinases, and (5) an inhibitor of c-jun/AP-1 activation.