Calcium signaling plays an important role in B lymphocyte survival and activation, and is critically dependent on the inositol-1,4,5-tris-phosphate-induced release of calcium stored in the endoplasmic reticulum (ER). Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes), and therefore these enzymes play an important role in ER calcium homeostasis and in the control of B of cell activation. Because Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis, in this work the effects of the virus on SERCA-type calcium pump expression and calcium accumulation in the endoplasmic reticulum of B cells was investigated.
Two Sarco-Endoplasmic Reticulum Calcium transport ATPase isoforms, the low Ca2+-affinity SERCA3, and the high Ca2+-affinity SERCA2 enzymes are simultaneously expressed in B cells. Latency type III infection of Burkitt's lymphoma cell lines with immortalization-competent virus expressing the full set of latency genes selectively decreased the expression of SERCA3 protein, whereas infection with immortalization-deficient virus that does not express the EBNA2 or LMP-1 viral genes was without effect. Down-modulation of SERCA3 expression could be observed upon LMP-1, but not EBNA2 expression in cells carrying inducible transgenes, and LMP-1 expression was associated with enhanced resting cytosolic calcium levels and increased calcium storage in the endoplasmic reticulum. Similarly to virus-induced B cell immortalisation, SERCA3 expression was also decreased in normal B cells undergoing activation and blastic transformation in germinal centers of lymph node follicles.
The data presented in this work indicate that EBV-induced immortalization leads to the remodelling of ER calcium homeostasis of B cells by LMP-1 that copies a previously unknown normal phenomenon taking place during antigen driven B cell activation. The functional remodelling of ER calcium homeostasis by down-regulation of SERCA3 expression constitutes a previously unknown mechanism involved in EBV-induced B cell immortalisation.
Rationale: Modulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca2+ homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown.
Objectives: This study provides a comprehensive investigation of expression and activity of SERCA in CF airway epithelium. A detailed study of the mechanisms underlying SERCA changes and its consequences was also undertaken.
Methods: Lung tissue samples (bronchus and bronchiole) from subjects with and without CF were evaluated by immunohistochemistry. Protein and mRNA expression in primary non-CF and CF cells was determined by Western and Northern blots.
Measurements and Main Results: SERCA2 expression was decreased in bronchial and bronchiolar epithelia of subjects with CF. SERCA2 expression in lysates of polarized tracheobronchial epithelial cells from subjects with CF was decreased by 67% as compared with those from subjects without CF. Several non-CF and CF airway epithelial cell lines were also probed. SERCA2 expression and activity were consistently decreased in CF cell lines. Adenoviral expression of mutant F508 cystic fibrosis transmembrane regulator gene (CFTR), inhibition of CFTR function pharmacologically (CFTRinh172), or stable expression of antisense oligonucleotides to inhibit CFTR expression caused decreased SERCA2 expression. In CF cells, SERCA2 interacted with Bcl-2, leading to its displacement from caveolae-related domains of endoplasmic reticulum membranes, as demonstrated in sucrose density gradient centrifugation and immunoprecipitation studies. Knockdown of SERCA2 using siRNA enhanced epithelial cell death due to ozone, hydrogen peroxide, and TNF-α.
Conclusions: Reduced SERCA2 expression may alter calcium signaling and apoptosis in CF. These findings decrease the likelihood of therapeutic benefit of SERCA inhibition in CF.
cystic fibrosis; SERCA2; pulmonary epithelium; ER
The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) plays a critical role in Ca2+ homeostasis via sequestration of this ion into the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in ageing tissues and cardiovascular disease. We have shown previously that the myeloperoxidase- (MPO) derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca2+ levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to pre-formed or enzymatically-generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrently with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca2+, to HOSCN or HOCl, resulted in a time- and concentration-dependent increase in intracellular Ca2+ under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca2+ pumps/channels, completely attenuated the increase in intracellular Ca2+ consistent with a critical role for SERCA in maintaining endothelial cell Ca2+ homeostasis. Angiotensin II pre-treatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca2+ levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers due to their higher SCN− levels.
Myeloperoxidase; calcium; oxidation; thiols; sarco/endoplasmic reticulum Ca2+-ATPase (SERCA); hypochlorous acid; hypothiocyanous acid
The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca2+ signaling. However, whether the changes in Ca2+ signaling and Ca2+ signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known.
Materials and Methods
In SERCA2+/- mouse parotid gland acinar cells, Ca2+ signaling, expression levels of Ca2+ signaling proteins, and amylase secretion were investigated.
SERCA2+/- mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca2+ ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP3Rs), but the localization and activities of IP3Rs were not altered. In SERCA2+/- mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice.
These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca2+ signaling proteins in the parotid gland acini, however, overall Ca2+ signaling is unchanged.
sarco/endoplasmic reticulum Ca2+-ATPase; Ca2+ signaling proteins; parotid gland acinar cells
The sarco/endoplasmic reticulum calcium ATPase (SERCA) is essential for the control of intracellular free Ca2+ levels. Recently, SERCA has been identified as an important effector of nitric oxide (NO) action in vascular cells. NO stimulates the uptake of cytosolic Ca2+ via SERCA by adducting glutathione to the reactive cysteine-674 thiol. Mutation of this single amino acid prevents the stimulation of Ca2+ uptake by NO, as well as its ability to decrease Ca2+ and cell migration. NO function is impaired in a variety of cardiovascular diseases, including diabetes and atherosclerosis, which are associated with irreversible oxidation of SERCA cysteine-674. Targeting the sources of oxidants in vascular diseases to prevent SERCA oxidation and/or increasing the expression of SERCA may impede vascular disease.
The loss of calcium homeostasis in the lens of the eye appears to be a factor contributing to lens opacity. In the human lens, calcium homeostasis depends on the Ca2+-ATPase pumps found only in the epithelium. A plasma membrane calcium pump, PMCA2 is upregulated in human cataractous lenses. To determine if oxidation caused the plasma membrane Ca2+-ATPases (PMCA) or sarcoplasmic/endoplasmic Ca2+-ATPases (SERCA) to become upregulated, we cultured a human lens epithelial cell line, in the presence of hydrogen peroxide. We observed an increase in PMCA1, PMCA2 SERCA2b and SERCA3 mRNA levels and protein expression with increasing hydrogen peroxide concentrations and treatment times. Hydrogen peroxide caused a rise in the intracellular calcium which could be an initiating factor in the concerted upregulation of PMCA1 and SERCA3. Our data support the idea that oxidative stress could contribute to a selective rise in PMCA/SERCA expression in human cataractous lenses.
In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca2+ is maintained at low levels by pumping Ca2+ into stores in the endoplasmic reticulum (ER) via the sarco ER Ca2+-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca2+, whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca2+ clearance and exaggerating inositol 1,4,5-trisphosphate–mediated Ca2+ liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid β production. Our results point to a physiological role for the presenilins in Ca2+ signaling via regulation of the SERCA pump.
The sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) is a key intracellular calcium transporter, which regulates cellular calcium concentration [Ca2+] by transporting Ca2+ ions from the cytosol into the endoplasmic reticulum. SERCA-mediated Ca2+ sequestration controls proper folding of newly synthesized proteins within the ER as well as the timing and spatial patterning of depolarization-evoked Ca2+ responses in the cytoplasm. We studied expression and distribution of all three SERCA isoforms in the mouse retina using isoform-specific antibodies. No immunostaining was observed with the SERCA1 antibody. SERCA2 was expressed in photoreceptor inner segments, amacrine and ganglion cells of the mouse retina. Similar SERCA2 localization was observed in adult rat, macaque and ground squirrel retinas. Analysis of distribution of SERCA2 immunofluorescence in the developing mouse retina revealed prominent SERCA2 signals throughout postnatal development. The antibody raised against the SERCA3 isoform labeled inner segments of photoreceptors and cell bodies in the inner nuclear layer of the mouse retina. The SERCA3 signal was detected in the inner plexiform layer of the early postnatal retina, but moved by P10 to the outer retina where it was concentrated in outer segments of cones. These results indicate that SERCA2 represents the dominant SERCA isoform in the mammalian retina. SERCA3 may contribute to calcium regulation in photoreceptors and bipolar cells.
calcium store; photoreceptor; endoplasmic reticulum; mouse; squirrel; rat
By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca2+ accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.
SERCA1; endoplasmic reticulum; calcium; apoptosis; splice variants
Intracellular calcium controls several crucial cellular events in apicomplexan parasites, including protein secretion, motility, and invasion into and egress from host cells. The plant compound thapsigargin inhibits the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), resulting in elevated calcium and induction of protein secretion in Toxoplasma gondii. Artemisinins are natural products that show potent and selective activity against parasites, making them useful for the treatment of malaria. While the mechanism of action is uncertain, previous studies have suggested that artemisinin may inhibit SERCA, thus disrupting calcium homeostasis. We cloned the single-copy gene encoding SERCA in T. gondii (TgSERCA) and demonstrate that the protein localizes to the endoplasmic reticulum in the parasite. In extracellular parasites, TgSERCA partially relocalized to the apical pole, a highly active site for regulated secretion of micronemes. TgSERCA complemented a calcium ATPase-defective yeast mutant, and this activity was inhibited by either thapsigargin or artemisinin. Treatment of T. gondii with artemisinin triggered calcium-dependent secretion of microneme proteins, similar to the SERCA inhibitor thapsigargin. Artemisinin treatment also altered intracellular calcium in parasites by increasing the periodicity of calcium oscillations and inducing recurrent, strong calcium spikes, as imaged using Fluo-4 labeling. Collectively, these results demonstrate that artemisinin perturbs calcium homeostasis in T. gondii, supporting the idea that Ca2+-ATPases are potential drug targets in parasites.
The calcium pump SERCA2a (sarcoplasmic reticulum calcium ATPase 2a), which plays a central role in cardiac contraction, shows decreased expression in heart failure (HF). Increasing SERCA2a expression in HF models improves cardiac function. We used direct cardiac delivery of adeno-associated virus encoding human SERCA2a (AAV6-hSERCA2a) in HF and normal canine models to study safety, efficacy, and the effects of immunosuppression. Tachycardic-paced dogs received left ventricle (LV) wall injection of AAV6-hSERCA2a or solvent. Pacing continued postinjection for 2 or 6 weeks, until euthanasia. Tissue/serum samples were analyzed for hSERCA2a expression (Western blot) and immune responses (histology and AAV6-neutralizing antibodies). Nonpaced dogs received AAV6-hSERCA2a and were analyzed at 12 weeks; a parallel cohort received AAV-hSERCA2a and immunosuppression. AAV-mediated cardiac expression of hSERCA2a peaked at 2 weeks and then declined (to ∼50%; p<0.03, 6 vs. 2 weeks). LV end diastolic and end systolic diameters decreased in 6-week dogs treated with AAV6-hSERCA2a (p<0.05) whereas LV diameters increased in control dogs. Dogs receiving AAV6-hSERCA2a developed neutralizing antibodies (titer ≥1:120) and cardiac cellular infiltration. Immunosuppression dramatically reduced immune responses (reduced inflammation and neutralizing antibody titers <1:20), and maintained hSERCA2a expression. Thus cardiac injection of AAV6-hSERCA2a promotes local hSERCA2a expression and improves cardiac function. However, the hSERCA2a protein level is reduced by host immune responses. Immunosuppression alleviates immune responses and sustains transgene expression, and may be an important adjuvant for clinical gene therapy trials.
Zhu and colleagues employ direct cardiac delivery of adeno-associated virus encoding the human calcium pump SERCA2a (AAV6-hSERCA2a) in heart failure and normal canine models in order to study the safety and efficacy of the approach as well as the effects of concomitant immunosuppressant treatment. Tachycardic-paced dogs injected with AAV6-hSERCA2a via the left ventricle wall displayed hSERCA2a expression and improved cardiac function, although hSERCA2a protein levels were reduced by host immune responses. Immunosuppression dramatically reduced inflammation and neutralizing antibody titers while maintaining hSERCA2a expression.
The cardiac isoform of the sarco/endoplasmic reticulum Ca2+ATPase (SERCA2a) plays a major role in controlling excitation/contraction coupling. In both experimental and clinical heart failure (HF), SERCA2a expression is significantly reduced which leads to abnormal Ca2+ handling and deficient contractility. A large number of studies in isolated cardiac myocytes, in small and large animal models of HF showed that restoring SERCA2a expression by gene transfer corrects the contractile abnormalities and improves energetics and electrical remodeling. Following a long line of investigation, a clinical trial is underway to restore SERCA2a expression in patients with HF using adeno-associated virus type 1.
This review addresses the following issues regarding HF gene therapy: 1) new insights on calcium regulation by SERCA2a; 2) SERCA2a as a gene therapy target in animal models of HF; 3) advances in the development of viral vectors and gene delivery; 4) clinical trials of HF using SERCA2a
This review focuses on the new advances in SERCA2a- targeted gene therapy made in the last three years.
SERCA2a is an important therapeutic target in various cardiovascular disorders. Ongoing clinical gene therapy trials will provide answers on its safety and applicability.
Sacroplasmic reticulum Ca2+ ATPase; heart failure; gene therapy
We analyze a recently published model of calcium handling in cardiac myocytes in order to find conditions for the presence of instabilities in the resting state of the model. Such instabilities can create calcium waves which in turn may be able to initiate cardiac arrhythmias. The model was developed by Swietach, Spitzer and Vaughan-Jones  in order to study the effect, on calcium waves, of varying ryanodine receptor (RyR)-permeability, sarco/endoplasmic reticulum calcium ATPase (SERCA) and calcium diffusion. We study the model using the extracellular calcium concentration ce and the maximal velocity of the SERCA-pump vSERCA as control parameters. In the (ce, vSERCA)-domain we derive an explicit function v*
= v*(ce), and we claim that any resting state based on parameters that lie above the curve (i.e. any pair (ce, vSERCA) such that with vSERCA > v*(ce)) is unstable in the sense that small perturbations will grow and can eventually turn into a calcium wave. And conversely; any pair (ce, vSERCA) below the curve is stable in the sense that small perturbations to the resting state will decay to rest. This claim is supported by analyzing the stability of the system in terms of computing the eigenmodes of the linearized model. Furthermore, the claim is supported by direct simulations based on the non-linear model.
Since the curve separating stable from unstable states is given as an explicit function, we can show how stability depends on other parameters of the model.
Sarco-endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and SERCA3 pump Ca2+ in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca2+ concentration ([Ca2+]ER) and the role of SERCA3 in the control of insulin secretion and ER stress.
RESEARCH DESIGN AND METHODS
β-Cell [Ca2+]ER of SERCA3+/+ and SERCA3−/− mice was monitored with an adenovirus encoding the low Ca2+-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca2+ concentration ([Ca2+]c) and [Ca2+]ER were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied.
Glucose elicited synchronized [Ca2+]ER and [Ca2+]c oscillations. [Ca2+]ER oscillations were smaller in SERCA3−/− than in SERCA3+/+ β-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [Ca2+]ER rise in SERCA3+/+ and SERCA3−/− β-cells. In a Ca2+-free medium, glucose moderately raised [Ca2+]ER from a highly buffered cytosolic Ca2+ pool. Increasing [Ca2+]c with high [K] elicited a [Ca2+]ER rise that was larger but more transient in SERCA3+/+ than SERCA3−/− β-cells because of the activation of a Ca2+ release from the ER in SERCA3+/+ β-cells. Glucose-induced insulin release was larger in SERCA3−/− than SERCA3+/+ islets. SERCA3 ablation did not induce ER stress.
[Ca2+]c and [Ca2+]ER oscillate in phase in response to glucose. Upon [Ca2+]c increase, Ca2+ is taken up by SERCA2b and SERCA3. Strong Ca2+ influx triggers a Ca2+ release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca2+ uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress.
Coronary restenosis, a major complication of percutaneous balloon angioplasty, results from neointimal proliferation of vascular smooth muscle cells (VSMCs). The sarco/endoplasmic reticulum calcium ATPase isoform 2a (SERCA2a), specific to contractile VSMCs, has been reported previously to be involved in the control of the Ca2+-signaling pathways governing proliferation and migration. Moreover, SERCA2a gene transfer was reported to inhibit in vitro VSMC proliferation and to prevent neointimal thickening in a rat carotid injury model.
The aim of this study was to evaluate the potential therapeutic interest of SERCA2a gene transfer for prevention of in-stent restenosis using a human ex vivo model of left internal mammary artery (hIMA) intimal thickening.
Left hIMAs, obtained at the time of aorto-coronary bypass surgeries, were subjected to balloon dilatation followed by infection for 30 min with adenoviruses encoding either human SERCA2 and GFP or control gene (beta-galactosidase) and GFP. Proliferation of subendothelial VSMCs and neointimal thickening were observed in balloon-injured hIMA maintained 14 days in organ culture under constant pressure and perfusion. SERCA2a gene transfer prevented vascular remodeling and significantly (p<0.01, n=5) reduced neointimal thickening in injured arteries (intima/media ratio was 0.07 ± 0.01 vs 0.40 ± 0.03 in βGal-infected arteries).
These findings could have potential implications for treatment of pathological in stent-restenosis.
Restenosis; SERCA2a; Gene Transfer; Internal Mammary Artery
AIM: To investigate the effect of Chaiqinchengqi decoction (CQCQD) on sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) mRNA expression of pancreatic tissues in acute pancreatitis (AP) rats.
METHODS: Thirty Sprague-Dawley (SD) rats were randomized into control group, AP group and CQCQD group (n = 3 × 10). The rats in the CQCQD group were intragastrically administered with CQCQD (10 mL/kg every 2 h) after induction of AP by intraperitoneal injection of caerulein (50 μg/kg.h × 5) within 4 h. At 6 h after the induction of AP model, pancreatic tissues were collected for the pathological observation, mRNA extraction for determination of SERCA1 and SERCA2 mRNA expression or pancreatic acinar cell isolation for measurement of fluorescence intensity (FI) of intracellular calcium ion concentration [Ca2+]i.
RESULTS: There was no expression of pancreatic SERCA1 mRNA in the control group and the AP group. The expression of pancreatic SERCA2 mRNA in the AP group was down-regulated (expression ratio = 0.536; P = 0.001) compared with the control group, while that in the CQCQD group was up-regulated (expression ratio = 2.00; P = 0.012) compared with AP group. The FI of intracellular [Ca2+] of pancreatic acinar cells in the AP group (138.2 ± 23.1) was higher than the C group (111.0 ± 18.4) and the CQCQD group (118.7 ± 15.2 ) (P < 0.05) and the pancreatic pathological score in the CQCQD group was lower than that in the AP group (5.7 ± 1.9 vs 9.2 ± 2.7, P < 0.05).
CONCLUSION: CQCQD can up-regulate the expression of SERCA2 mRNA of pancreatic tissues, reduce intracellular calcium overload and relieve pancreatic tissue lesions.
Chaiqinchengqi decoction; Pancreatitis; Calcium overload; Sarco/endoplasmic reticulum Ca2 +-ATPase
The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The “2-color” SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.
Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na+/K+ transporters, H+/K+ transporters, and plasma membrane Ca2+ pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endo)plasmic reticulum calcium ATPase (SERCA), which maintains calcium homeostasis in the cell by actively pumping Ca2+ into the sarco(endo)plasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain.
In blood vessels, tone is maintained by agonist-induced cytosolic Ca2+ oscillations of quiescent/contractile vascular smooth muscle cells (VSMCs). However, in synthetic/proliferative VSMCs, Gq/phosphoinositide receptor-coupled agonists trigger a steady-state increase in cytosolic Ca2+ followed by a Store Operated Calcium Entry (SOCE) which translates into activation of the proliferation-associated transcription factor NFAT. Here, we report that in human coronary artery smooth muscle cells (hCASMCs), the sarco/endoplasmic reticulum calcium ATPase type 2a (SERCA2a) expressed in the contractile form of the hCASMCs, controls the nature of the agonist-induced Ca2+ transient and the resulting down-stream signaling pathway. Indeed, restoring SERCA2a expression by gene transfer in synthetic hCASMCs 1) increased Ca2+ storage capacity; 2) modified agonist-induced IP3R Ca2+ release from steady-state to oscillatory mode (the frequency of agonist-induced IP3R Ca2+ signal was 11.66 ± 1.40/100 sec in SERCA2a-expressing cells (n=39) vs 1.37 ± 0.20/100 sec in control cell (n=45), p<0.01); 3) suppressed SOCE by preventing interactions between SR calcium sensor STIM1 and pore forming unit ORAI1; 4) inhibited calcium regulated transcription factor NFAT and its down-stream physiological function such as proliferation and migration.
This study provides evidence for the first time that oscillatory and steady-state patterns of Ca2+ transients have different effects on calcium-dependent physiological functions in smooth muscle cells.
SERCA; NFAT; proliferation; signal transduction; calcium oscillations; store-operated calcium entry
In neonatal ventricular cardiomyocytes (NVCM), decreased contractile activity stimulates sarco-endoplasmic reticulum Ca2+-ATPase2a (SERCA2a), analogous to reduced myocardial load in vivo. This study investigated in contracting NVCM the role of load-dependent RhoA-ROCK signaling in SERCA2a regulation. Contractile arrest of NVCM resulted in low peri-nuclear localized RhoA levels relative to contracting NVCM. In arrested NVCM, ROCK activity was decreased (59%) and paralleled a loss in F-actin levels. Y-27632-induced ROCK inhibition in contracting NVCM increased SERCA2a messenger RNA expression by 150%. This stimulation was transcriptional, as evident from transfections with the SERCA2a promoter. A reciprocal effect of Y-27632 treatment on the promoter activity of atrial natriuretic factor was observed. SERCA2a transcription was not altered by co-transfection of the RhoA-ROCK-dependent serum response factor (SRF) alone or in combination with myocardin. Furthermore, GATA4, another ROCK-dependent transcription factor, induced rather than repressed SERCA2a transcription. This study shows that contractile activity suppresses SERCA2a gene expression via RhoA-ROCK-dependent transcription modulation. This modulation is likely to be accomplished by a transcription factor other than SRF, myocardin, or GATA4.
Transcription regulation; Signal transduction; RhoA; ROCK; SRF; GATA4; SERCA2a
Malignant gliomas have low survival expectations regardless of current treatments. Nonsteroidal anti-inflammatory drugs (NSAIDs) prevent cell transformation and slow cancer cell growth by mechanisms independent of cyclooxygenase (COX) inhibition. Certain NSAIDs trigger the endoplasmic reticulum stress response (ERSR), as revealed by upregulation of molecular chaperones such as GRP78 and C/EBP homologous protein (CHOP). Although celecoxib (CELE) inhibits the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA), an effect known to induce ERSR, sulindac sulfide (SS) has not been reported to affect SERCA. Here, we investigated these two drugs for their effects on Ca2+ homeostasis, ERSR, and glioma cell survival. Our findings indicate that SS is a reversible inhibitor of SERCA and that both SS and CELE bind SERCA at its cyclopiazonic acid binding site. Furthermore, CELE releases additional Ca2+ from the mitochondria. In glioma cells, both NSAIDS upregulate GRP78 and activate ER-associated caspase-4 and caspase-3. Although only CELE upregulates the expression of CHOP, it appears that CHOP induction could be associated with mitochondrial poisoning. In addition, CHOP induction appears to be uncorrelated with the gliotoxicity of these NSAIDS in our experiments. Our data suggest that activation of ERSR is primarily responsible for the gliotoxic effect of these NSAIDS. Because SS has good brain bioavailability, has lower COX-2 inhibition, and has no mitochondrial effects, it represents a more appealing molecular candidate than CELE to achieve gliotoxicity via activation of ERSR.
GRP78; gliotoxicity; Ca2+; NSAIDs
We have previously reported on calcium transport mechanisms in American lobster, Homarus americanus, using 45Ca2+ coupled with vesicle preparations of hepatopancreatic endoplasmic reticulum. The active transport of calcium across membranes bordering calcium-sequestering stores such as sarcoplasmic or endoplasmic reticulum is catalyzed by membrane-spanning proteins, the sarco-endoplasmic Ca2+-ATPases (SERCAs). In the study described here we used advanced bioinformatics and molecular techniques to clone SERCA from the economically important Caribbean spiny lobster, Panulirus argus. We report the complete cloning of a full-length SERCA from P. argus antenna cDNA (GenBank accession number AY702617). This cDNA has a 1020-amino acid residue open reading frame which is 90% identical to published sequences of other crustacean SERCA proteins. Our data support the hypothesis that one crustacean and three vertebrate genes controlling calcium transport were derived from a common ancestral gene.
Spiny lobster; Cloning; Polymerase chain reaction; Rapid amplification of cDNA ends; Sequencing; Real time PCR
Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular bulk degradation pathway that plays critical roles in eliminating intracellular pathogens, presenting endogenous antigens, and regulating T lymphocyte survival and proliferation. In this study, we have investigated the role of autophagy in regulating the ER compartment in T lymphocytes. We found that ER content is expanded in mature Atg7-deficient T lymphocytes. Atg7-deficient T cells stimulated through the T cell receptor display impaired influx, but not efflux, of calcium, and ER calcium stores are increased in Atg7-deficient T cells. Treatment with the ER sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor thapsigargin rescues the calcium influx defect in Atg7-deficient T lymphocytes, suggesting that this impairment is caused by an intrinsic defect in ER. Furthermore, we found that the stimulation-induced redistribution of STIM-1, a critical event for the store-operated Ca2+-release activated Ca2+ (CRAC) channel opening, is impaired in Atg7-deficient T cells. Together, these findings indicate that the expanded ER compartment in Atg7-deficient T cells contains increased calcium stores, and the inability of these stores to be depleted causes defective calcium influx in these cells. Our results demonstrate that autophagy plays an important role in maintaining ER and calcium homeostasis in T lymphocytes.
Autophagy; Atg7; ER homeostasis; calcium influx; T lymphocytes
Traditional analyses of calcium homeostasis have separately quantified either calcium accumulation or release mechanisms. To define the system as a whole, however, requires multiple experimental techniques to examine both accumulation and release. Here we describe a technique that couples the simultaneous quantification of radio-labeled calcium accumulation in endoplasmic reticulum (ER) microsomes with the release of inorganic phosphate (Pi) by the hydrolytic activity of sarco-endoplasmic reticulum calcium ATPase (SERCA) all in the convenience of a 96-well format.
Calcium; SERCA activity; Microsomes; Inorganic phosphate; Malachite green
Cardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3'-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI.
The protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed.
Combing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.
Human myocardial infarction; Expression; SERCA2; miRNA; Bioinformatics