Sarcoendoplasmic Reticulum Calcium Transport ATPase Research Articles

PDX1, a transcription factor essential for organ differentiation, regulates SERCA-dependent Ca2+ homeostasis in sensory neurons

Pancreatic and duodenal homeobox 1 (PDX1) is a transcription factor required for the development and differentiation of the pancreas. Previous studies indicated that PDX1 expression was restricted to the gastrointestinal tract. Using a cre-dependent reporter, we observed PDX1-dependent expression of tdtomato (PDX1-tom) in a subpopulation of sensory nerves. Many of these PDX1-tom afferents expressed the neurofilament 200 protein and projected to the skin. Tdtomato-labeled terminals were associated with hair follicles in the form of longitudinal and circumferential lanceolate endings suggesting a role in tactile and proprioceptive perception. To begin to examine the functional significance of PDX1 in afferents, we used Fura-2 imaging to examine calcium (Ca2+) handling under naïve and nerve injury conditions. Neuropathic injury is associated with increased intracellular Ca2+ signaling that in part results from dysregulation of the sarco/endoplasmic reticulum calcium transport ATPase (SERCA). Here we demonstrate that under naïve conditions, PDX1 regulates expression of the SERCA2B isoform in sensory neurons. In response to infraorbital nerve injury, a significant reduction of PDX1 and SERCA2B expression and dysregulation of Ca2+ handling occurs in PDX1-tom trigeminal ganglia neurons. The identification of PDX1 expression in the somatosensory system and its regulation of SERCA2B and Ca2+ handling provide a new mechanism to explain pathological changes in primary afferents that may contribute to pain associated with nerve injury.

Knockout of ACE-N facilitates improved cardiac function after myocardial infarction.

Angiotensin-converting enzyme (ACE) hydrolyzes N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) into inactive fragments through its N-terminal site (ACE-N). We previously showed that Ac-SDKP mediates ACE inhibitors' cardiac effects. Whether increased bioavailability of endogenous Ac-SDKP caused by knocking out ACE-N also improves cardiac function in myocardial infarction (MI)-induced heart failure (HF) is unknown. Wild-type (WT) and ACE-N knockout (ACE-NKO) mice were subjected to MI by ligating the left anterior descending artery and treated with vehicle or Ac-SDKP (1.6 mg/kg/day, s.c.) for 5 weeks, after which echocardiography was performed and left ventricles (LV) were harvested for histology and molecular biology studies. ACE-NKO mice showed increased plasma Ac-SDKP concentrations in both sham and MI group compared to WT. Exogenous Ac-SDKP further increased its circulating concentrations in WT and ACE-NKO. Shortening (SF) and ejection (EF) fractions were significantly decreased in both WT and ACE-NKO mice post-MI, but ACE-NKO mice exhibited significantly lesser decrease. Exogenous Ac-SDKP ameliorated cardiac function post-MI only in WT but failed to show any additive improvement in ACE-NKO mice. Sarcoendoplasmic reticulum calcium transport ATPase (SERCA2), a marker of cardiac function and calcium homeostasis, was significantly decreased in WT post-MI but rescued with Ac-SDKP, whereas ACE-NKO mice displayed less loss of SERCA2 expression. Our study demonstrates that gene deletion of ACE-N resulted in improved LV cardiac function in mice post-MI, which is likely mediated by increased circulating Ac-SDKP and minimally reduced expression of SERCA2. Thus, future development of specific and selective inhibitors for ACE-N could represent a novel approach to increase endogenous Ac-SDKP toward protecting the heart from post-MI remodeling.

MP08-17 ALTERED CALCIUM SIGNALING AND SERCA DYSFUNCTION CONTRIBUTE TO BLADDER DYSFUNCTION IN METABOLIC SYNDROME

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology (MP08)1 Sep 2021MP08-17 ALTERED CALCIUM SIGNALING AND SERCA DYSFUNCTION CONTRIBUTE TO BLADDER DYSFUNCTION IN METABOLIC SYNDROME Leo Song, John Strobel, James Byrd, Mouhamad Alloosh, Michael Sturek, and CR Powell Leo SongLeo Song More articles by this author , John StrobelJohn Strobel More articles by this author , James ByrdJames Byrd More articles by this author , Mouhamad AllooshMouhamad Alloosh More articles by this author , Michael SturekMichael Sturek More articles by this author , and CR PowellCR Powell More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001981.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Bladder dysfunction in metabolic syndrome (MetS) remains poorly understood despite being one of its most common complications. We hypothesize that bladder dysfunction in chronic, late stage MetS results in underactive bladder driven in part by sarcoendoplasmic reticulum calcium transport ATPase (SERCA) dysfunction. METHODS: Age matched Ossabaw pig models of MetS were fed a hypercaloric, atherogenic diet for 12 months to induce MetS and compared with control pigs. Urodynamic parameters were obtained in-vivo. Detrusor smooth muscle (DSM) cells were enzymatically isolated and intracellular Ca2+ handling was interrogated with fura-2. RESULTS: MetS was confirmed by obesity, hypertension, serum glucose, and increased total and LDL cholesterol (n=2). MetS exhibited inhibited SERCA activity based on less rapid and less vigorous “undershoot” during recovery of the intracellular Ca2+ response to caffeine (5.6±5 vs -10.8±4, p=0.03). As SERCA is the primary driver for Ca2+ recovery, impaired SERCA is a potential cause of subsequent bladder dysfunction. MetS also exhibited a trend toward increased response to carbachol 10µM but not 1µM (72.5±26 vs 47.8±16 cmH2O, p=0.08) and no difference in bladder pressure at capacity compared to controls (n=2). CONCLUSIONS: This pilot study indicates that DSM from MetS pigs demonstrates decreased SERCA activity, highlighting a potential therapeutic target for bladder dysfunction in MetS. This is the first time SERCA dysfunction has been demonstrated in MetS DSM. Source of Funding: This project was supported by a Project Development Team within the ICTSI NIH/NCRR Grant Number RR025761 (Powell, PI) and NIDDK DiaComp Pilot & Feasibility project, sub-award DK076169 (Powell, PI) and NIH grant HL062552 (Sturek, PI), NIH UL1 RR025761 (Sturek, CO-I), NIH P30 DK097512 (Sturek Co-I), Indiana University MedSTAR program (Song) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e157-e157 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Leo Song More articles by this author John Strobel More articles by this author James Byrd More articles by this author Mouhamad Alloosh More articles by this author Michael Sturek More articles by this author CR Powell More articles by this author Expand All Advertisement Loading ...

Abstract MP43: Knockout Of ACE-N Facilitates Improved Cardiac Function After Myocardial Infarction

Angiotensin-converting enzyme (ACE) hydrolyzes N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) into inactive fragments through its N-terminal domain (ACE-N). We reported that cardioprotective effects of ACE inhibitor in angiotensin II-induced hypertension were partially mediated through increased Ac-SDKP bioavailability. Whether increased endogenous Ac-SDKP by knocking out ACE-N leads to improved cardiac function in myocardial infarction (MI) is unknown. Wild-type (WT) and ACE-N knockout (ACE-N -/- ) mice were subjected to MI induced by ligating the left anterior descending artery and treated with either vehicle or Ac-SDKP (1.6 mg/kg/day, s.c.) for 5 weeks. Echocardiography was performed on awake mice at the end of experiment and left ventricles (LV) were harvested for histology and molecular biology studies. ACE-N-/- mice showed increased plasma Ac-SDKP level in Sham or MI group compared to WT. Exogenous Ac-SDKP further increased Ac-SDKP level in both WT and ACE-N-/-. SF and EF were significantly decreased in both WT and ACE-N-/- mice post-MI, . Exogenous Ac-SDKP further increased EF and SF post-MI only in WT, but not in ACE-N-/- mice. Sarcoendoplasmic reticulum calcium transport ATPase 2 (SERCA2), a marker of cardiac calcium homeostatsis, significantly decreased in WT post-MI was rescued with Ac-SDKP, whereas ACE-N-/- mice displayed less reduction in SERCA2 expression. These results demonstrate that gene deletion of ACE-N improves cardiac function in mice post-MI, which was associated with increased Ac-SDKP level and minimally reduced expression of SERCA2.Therefore,this study illustrates that endogenous Ac-SDKP results in cardioprotective role in MI.

Hyperglycaemia-Induced Contractile Dysfunction and Apoptosis in Cardiomyocyte-Like Pulsatile Cells Derived from Mouse Embryonic Stem Cells.

Hyperglycaemia, a key metabolic abnormality in diabetes mellitus, is implicated in pathological cardiogenesis during embryological development. However, the underlying mechanisms and potential therapeutic targets remain unknown. We, therefore, studied the effect of hyperglycaemia on mouse embryonic stem cell (mESC) cardiac differentiation. The mESCs were differentiated via embryoid body (EB) formation and cultured under conditions with baseline (25 mM) or high (50 mM) glucose. Time-lapse microscopy images of pulsatile mESCs and Ca2+ transients were recorded. Biomarkers of cellular changes were detected using immunocytochemistry, terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and Western blot analyses. Differentiated, spontaneously beating mESCs stained positive for cardiac troponin T, α-actinin 2, myosin heavy chain, and connexin 43. Hyperglycaemia decreased the EB diameter and number of beating EBs as well as the cellular amplitude of contraction, the Ca2+ transient, and the contractile response to caffeine (1 mM), but had no effect on the expression of the sarco-endoplasmic reticulum calcium transport ATPase 2 (SERCA 2). Furthermore, hyperglycaemia decreased the expression of B cell lymphoma 2 (Bcl-2) and increased the expression of cytoplasmic cytochrome c and the number of TUNEL-positive cells, but had no effect on the expression of one of the mitochondrial fusion regulatory proteins, optic atrophy protein 1 (OPA1). Overall, hyperglycaemia suppressed the mESC cardiomyocyte-like differentiation and induced contractile dysfunction. The results are consistent with mechanisms involving abnormal Ca2+ handling and mitochondrial-dependent apoptosis, factors which represent potential therapeutic targets in developmental diabetic cardiac disease.

The p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.

Lemnalol Modulates the Electrophysiological Characteristics and Calcium Homeostasis of Atrial Myocytes.

Sepsis, an inflammatory response to infection provoked by lipopolysaccharide (LPS), is associated with high mortality, as well as ischemic stroke and new-onset atrial arrhythmia. Severe bacterial infections causing sepsis always result in profound physiological changes, including fever, hypotension, arrhythmia, necrosis of tissue, systemic multi-organ dysfunction and finally death. LPS challenge-induced inflammatory responses during sepsis may increase the likelihood of the arrhythmogenesis. Lemnalol is known to possess potent anti-inflammatory effects. This study examined whether Lemnalol (0.1 μM) could modulate the electrophysiological characteristics and calcium homeostasis of atrial myocytes under the influence of LPS (1μg/mL). Under challenge with LPS, Lemnalol-treated LA myocytes, had a longer AP duration at 20%, 50% and 90% repolarization of the amplitude, compared to the LPS-treated cells. LPS-challenged LA myocytes showed increased late sodium current, Na+-Ca2+ exchanger current, transient outward current, rapid component of delayed rectifier potassium current, tumor necrosis factor-α, NF-κB and increased phosphorylation of ryanodine receptor (RyR), but a lower L-type Ca2+ current than the control LA myocytes. Exposure to Lemnalol reversed the LPS-induced effects. The LPS-treated and control groups of LA myocytes, with or without the existence of Lemnalol. showed no apparent alterations in the sodium current amplitude or Cav1.2 expression. The expression of sarcoendoplasmic reticulum calcium transport ATPase (SERCA2) was reduced by LPS treatment, while Lemnalol ameliorated the LPS-induced alterations. The phosphorylation of RyR was enhanced by LPS treatment, while Lemnalol attenuated the LPS-induced alterations. In conclusion, Lemnalol modulates LPS-induced alterations of LA calcium homeostasis and blocks the NF-κB pathways, which may contribute to the attenuation of LPS-induced arrhythmogenesis.

Astragalin augments basal calcium influx and insulin secretion in rat pancreatic islets

Astragalin is a flavonol glycoside with several biological activities, including antidiabetic properties. The objective of this study was to investigate the effects of astragalin on glycaemia and insulin secretion, in vivo, and on calcium influx and insulin secretion in isolated rat pancreatic islets, ex vivo. Astragalin (1 and 10 mg / kg) was administered by oral gavage to fasted Wistar rats and serum glucose and plasma insulin were measured. Isolated pancreatic islets were used to measure basal insulin secretion and calcium influx. Astragalin (10 mg/ kg) decreased glycaemia and increased insulin secretion significantly at 15–180 min, respectively, in the glucose tolerance test. In isolated pancreatic cells, astragalin (100 μM) stimulated calcium influx through a mechanism involving ATP-dependent potassium channels, L-type voltage-dependent calcium channels, the sarcoendoplasmic reticulum calcium transport ATPase (SERCA), PKC and PKA. These findings highlight the dietary coadjuvant, astragalin, as a potential insulin secretagogue that may contribute to glucose homeostasis.

Calneuron 1 Increased Ca2+ in the Endoplasmic Reticulum and Aldosterone Production in Aldosterone-Producing Adenoma.

Aldosterone production is initiated by angiotensin II stimulation and activation of intracellular Ca2+ signaling. In aldosterone-producing adenoma (APA) cells, the activation of intracellular Ca2+ signaling is independent of the renin-angiotensin-aldosterone systems. The purpose of our study was to clarify molecular mechanisms of aldosterone production related to Ca2+ signaling. Transcriptome analysis revealed that the CALN1 gene encoding calneuron 1 had the strongest correlation with CYP11B2 (aldosterone synthase) among genes encoding Ca2+-binding proteins in APA. CALN1 modulation and synthetic or fluorescent compounds were used for functional studies in human adrenocortical carcinoma (HAC15) cells. CALN1 expression was 4.4-fold higher in APAs than nonfunctioning adrenocortical adenomas. CALN1 expression colocalized with CYP11B2 expression as investigated using immunohistochemistry in APA and zona glomerulosa of male rats fed by a low-salt diet. CALN1 expression was detected in the endoplasmic reticulum (ER) by using GFP-fused CALN1, CellLight ER-RFP, and the corresponding antibodies. CALN1-overexpressing HAC15 cells showed increased Ca2+ in the ER and cytosol fluorescence-based studies. Aldosterone production was potentiated in HAC15 cells by CALN1 expression, and dose-responsive inhibition with TMB-8 showed that CALN1-mediated Ca2+ storage in ER involved sarcoendoplasmic reticulum calcium transport ATPase. The silencing of CALN1 decreased Ca2+ in ER, and abrogated angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells. Increased CALN1 expression in APA was associated with elevated Ca2+ storage in ER and aldosterone overproduction. Suppression of CALN1 expression prevented angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells, suggesting that CALN1 is a potential therapeutic target for excess aldosterone production.

Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes.

Key points Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart.Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur.We report alterations to the atrial systolic Ca2+ transient that have implications for the function of the atrial in the elderly.We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca2+ in the old atria.The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops. Many cardiovascular diseases, including those affecting the atria, are associated with advancing age. Arrhythmias, including those in the atria, can arise as a result of electrical remodelling or alterations in Ca2+ homeostasis. In the atria, age‐associated changes in the action potential have been documented. However, little is known about remodelling of intracellular Ca2+ homeostasis in the healthy aged atria. Using single atrial myocytes from young and old Welsh Mountain sheep, we show the free Ca2+ transient amplitude and rate of decay of systolic Ca2+ decrease with age, whereas sarcoplasmic reticulum (SR) Ca content increases. An increase in intracellular Ca buffering explains both the decrease in Ca2+ transient amplitude and decay kinetics in the absence of any change in sarcoendoplasmic reticulum calcium transport ATPase function. Ageing maintained the integrated Ca2+ influx via I Ca‐L but decreased peak I Ca‐L. Decreased peak I Ca‐L was found to be responsible for the age‐associated increase in SR Ca content but not the decrease in Ca2+ transient amplitude. Instead, decreased peak I Ca‐L offsets increased SR load such that Ca2+ release from the SR was maintained during ageing. The results of the present study highlight a novel mechanism by which increased Ca buffering decreases systolic Ca2+ in old atria. Furthermore, for the first time, we have shown that SR Ca content is increased in old atrial myocytes.

The Energy-Transduction Domain of the SERCA Calcium Pump is a Prime Therapeutic Target in Heart Failure and Obesity

We have used a comprehensive array of techniques to characterize the energy-transduction domain of the sarco/endoplasmic reticulum calcium transport ATPase (SERCA), including x-ray crystallography, solution biochemistry, fluorescence spectroscopy, molecular dynamics simulation, and small-molecule activation. The energy-transduction domain of SERCA mediates long-range allosteric coupling of ATP hydrolysis in the cytosolic headpiece to Ca2+ transport in the transmembrane domain. In heart failure, decreased SERCA activity correlates with disease progression in patients; increasing SERCA activity by drug or gene therapy reverses heart failure in animal models. Here we solved the x-ray crystal structure of SERCA in complex with a novel small-molecule activator, thus identifying a unique binding site on the energy-transduction domain. Conformation-specific proteolytic cleavage and intramolecular glutaraldehyde cross-linking was used to determine activator effect on SERCA headpiece structure. Fluorescence spectroscopy was used to identify kinetic transitions accelerated by the activator. We propose a structural and biochemical mechanism for small-molecule activation of SERCA. Interestingly, the activator site is located vicinal to the binding site of sarcolipin (SLN), a muscle phosphoprotein that regulates SERCA energy expenditure and muscle metabolism to provide resistance against diet-induced obesity and extreme cold through non-shivering thermogenesis. SLN uncoupling of ATP hydrolysis from Ca2+ transport by SERCA was examined using microsecond MD simulation, finding that the cytosolic domain of SLN induces a salt bridge-mediated structural rearrangement of the energy-transduction domain, which allosterically uncouples SERCA by disrupting Ca2+ occlusion at residue E309 in transport site II, and thereby facilitating futile Ca2+ backflux in muscle. Our results demonstrate that the energy-transduction domain is a key control element that provides a structural motif for activation of Ca2+ transport by cardiac SERCA2a in heart failure and for uncoupling ATP hydrolysis by muscle SERCA1a in obesity.

A Report of Two Patients with Darier Disease and Hidradenitis Suppurativa

We report two unique cases of patients with Darier disease and hidradenitis suppurativa. Although it is unknown whether there is a connection between the two diseases, it is possible that an interaction between sarcoendoplasmic reticulum calcium transport ATPase and Notch homolog 1, translocation-associated (Drosophila) caused both diseases in these individuals.

A homozygous STIM1 mutation impairs store-operated calcium entry and natural killer cell effector function without clinical immunodeficiency

A homozygous STIM1 mutation impairs store-operated calcium entry and natural killer cell effector function without clinical immunodeficiency

Vitamin D Receptor Ablation and Vitamin D Deficiency Result in Reduced Grip Strength, Altered Muscle Fibers, and Increased Myostatin in Mice.

Vitamin D deficiency is associated with muscle weakness, pain, and atrophy. Serum vitamin D predicts muscle strength and age-related muscle changes. However, precise mechanisms by which vitamin D affects skeletal muscle are unclear. To address this question, this study characterizes the muscle phenotype and gene expression of mice with deletion of vitamin D receptor (VDRKO) or diet-induced vitamin D deficiency. VDRKO and vitamin D-deficient mice had significantly weaker grip strength than their controls. Weakness progressed with age and duration of vitamin D deficiency, respectively. Histological assessment showed that VDRKO mice had muscle fibers that were significantly smaller in size and displayed hyper-nuclearity. Real-time PCR also indicated muscle developmental changes in VDRKO mice with dysregulation of myogenic regulatory factors (MRFs) and increased myostatin in quadriceps muscle (>2-fold). Vitamin D-deficient mice also showed increases in myostatin and the atrophy marker E3-ubiqutin ligase MuRF1. As a potential explanation for grip strength weakness, both groups of mice had down-regulation of genes encoding calcium-handling and sarco-endoplasmic reticulum calcium transport ATPase (Serca) channels. This is the first report of reduced strength, morphological, and gene expression changes in VDRKO and vitamin D-deficient mice where confounding by calcium, magnesium, and phosphate have been excluded by direct testing. Although suggested in earlier in vitro work, this study is the first to report an in vivo association between vitamin D, myostatin, and the regulation of muscle mass. These findings support a direct role for vitamin D in muscle function and corroborate earlier work on the presence of VDR in this tissue.

The tyrosine kinase inhibitor bafetinib inhibits PAR2-induced activation of TRPV4 channels in vitro and pain in vivo.

Protease-activated receptor 2 (PAR2) is expressed on nociceptive neurons, and can sensitize transient receptor potential (TRP) ion channels to amplify neurogenic inflammation and pain. The mechanisms by which this occurs are not fully understood. PAR2 causes receptor-operated activation of TRPV4 channels and TRPV4 null mice have attenuated PAR2-stimulated neurogenic inflammation and mechanical hyperalgesia. Here we investigate the intracellular signalling mechanisms underlying PAR2-induced TRPV4 channel activation and pain. Responses of non-transfected and TRPV4-transfected HEK293 cells to agonists of PAR2 (trypsin and SLIGRL) and TRPV4 channels (GSK1016790A) were determined using calcium imaging. Inhibitors of TRPV4 channels (HC067047), sarcoendoplasmic reticulum calcium transport ATPase (thapsigargin), Gαq (UBO-QIC), tyrosine kinases (bafetinib and dasatinib) or PI3 kinases (wortmannin and LY294002) were used to investigate signalling mechanisms. In vivo effects of tyrosine kinase inhibitors on PAR2 -induced mechanical hyperalgesia were assessed in mice. In non-transfected HEK293 cells, PAR2 activation transiently increased intracellular calcium ([Ca(2+) ]i ). Functional expression of TRPV4 channels caused a sustained increase of [Ca(2+) ]i , inhibited by HC067047, bafetinib and wortmannin; but not by thapsigargin, UBO-QIC, dasatinib or LY294002. Bafetinib but not dasatinib inhibited PAR2-induced mechanical hyperalgesia in vivo. This study supports a role for tyrosine kinases in PAR2-mediated receptor-operated gating of TRPV4 channels, independent of Gαq stimulation. The ability of a tyrosine kinase inhibitor to diminish PAR2-induced activation of TRPV4 channels and consequent mechanical hyperalgesia identifies bafetinib (which is in development in oncology) as a potential novel analgesic therapy.

Xestospongin C induces monocytic differentiation of HL60 cells through activation of the ERK pathway

Xestospongin C (XC), which is a group of macrocyclic bis-1-oxaquinolizidines, is a potent inhibitor of sarcoendoplasmic reticulum calcium transport ATPase and IP3 receptor. Nevertheless, very less information is available regarding whether XC induces AML differentiation. We investigated the potential role of XC in the differentiation of human leukemia HL60 cells and mechanisms underlying XC actin. XC treatment inhibited proliferation by inducing G1-phase cell cycle arrest in the HL60 cells. In addition, XC induced differentiation of HL60 cells into the CD14(+) monocytic lineage, which was indicated by morphological changes, nitroblue tetrazolium reduction assay, and expressions of CD11b and CD14 surface antigens. Our results also showed that XC promotes phagocytic activity and granularity in HL60 cells, suggesting that the cells are functionally activated. Furthermore, XC enhanced tumor necrosis factor (TNF)-α-mediated cytotoxic effect by increasing the numbers of TNF receptors. Moreover, we showed that XC activates extracellular signal-regulated kinase (ERK) pathway in the differentiation stages. Inhibition of ERK activation using PD98059 significantly decreased NBT+HL60 cells induced by XC treatment. Taken together, the results show that XC promotes monocytic differentiation of HL60 cells via ERK pathway activation, suggesting that XC could be a candidate for use as a differentiation-inducing agent for AML treatment.

Excess of glucocorticoid induces myocardial remodeling and alteration of calcium signaling in cardiomyocytes

Ventricular dysfunction is one of the important side effects of the anti-inflammatory agent, glucocorticoid (GC). The present study was undertaken to examine whether abnormal calcium signaling is responsible for cardiac dysfunction due to an excess of GC hormone. The synthetic GC drug, dexamethasone (DEX), significantly (P<0.001, n=20) increased heart weight to body weight ratio, left ventricular remodeling, and fibrosis. The microarray analysis showed altered expression of several genes encoding calcium cycling/ion channel proteins in DEX-treated rat heart. The altered expression of some of the genes was validated by real-time PCR and western blotting analyses. The expression of the L-type calcium channels and calsequestrin was increased, whereas sarcoendoplasmic reticulum calcium transport ATPase 2a (SERCA2a) and junctin mRNAs were significantly reduced in DEX-treated rat left ventricular tissues. In neonatal rat ventricular cardiomyocytes, DEX also increased the level of mRNAs of atrial- and brain natriuretic peptides, L-type calcium channels, and calsequestrin after 24 h of treatment, which were mostly restored by mifepristone. The caffeine-induced calcium release was prolonged by DEX compared to the sharp release in control cardiomyocytes. Taken together, these data show that impaired calcium kinetics may be responsible for cardiac malfunction by DEX. The results are important in understanding the pathophysiology of the heart in patients treated with excess GC.

Modulation of B-cell endoplasmic reticulum calcium homeostasis by Epstein-Barr virus Latent Membrane Protein-1

BackgroundCalcium 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.ResultsTwo 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.ConclusionThe 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.

Lineage-Specific Modulation of Calcium Pump Expression During Myeloid Differentiation

Calcium is accumulated from the cytosol into the endoplasmic reticulum by sarco-endoplasmic reticulum calcium transport ATPase (SERCA) enzymes. Because calcium stored in the endoplasmic reticulum is essential for cell growth, differentiation, calcium signaling, and apoptosis and because different SERCA enzymes possess distinct functional characteristics, in the present report we explored SERCA expression during in vitro differentiation of the human myeloid/promyelocytic cell lines HL-60 and NB4 and of freshly isolated acute promyelocytic leukemia cells. Two SERCA species have been found to be coexpressed in these cells: SERCA 2b and another isoform, SERCAPLIM, which is recognized by the PLIM430 monoclonal antibody. Induction of differentiation along the neutrophil granulocytic lineage by all-trans retinoic acid or cyclic AMP analogs led to an increased expression of SERCAPLIM, whereas the expression of the SERCA 2b isoform was decreased. The modulation of SERCA expression was manifest also on the mRNA level. Experiments with retinoic acid receptor isoform-specific retinoids indicated that SERCA expression is modulated by retinoic acid receptor alpha-dependent signaling. SERCA expression of retinoic acid-resistant cell variants was refractory to treatment. Differentiation along the monocyte/macrophage lineage by phorbol ester resulted in an increased expression of both SERCA isoforms. In addition, when cells were treated by phorbol ester in the presence of the glucocorticoid dexamethasone, a known inhibitor of monocyte differentiation, a selective blockage of the induction of SERCAPLIM was observed. Altered SERCA expression modified the functional characteristics of calcium transport into the endoplasmic reticulum. These observations show for the first time that the modulation of calcium pump expression is an integral component of the differentiation program of myeloid precursors and indicate that a lineage-specific remodelling of the endoplasmic reticulum occurs during cell maturation. In addition, these data show that SERCA isoforms may serve as useful markers for the study of myeloid differentiation.

Lineage-Specific Modulation of Calcium Pump Expression During Myeloid Differentiation

AbstractCalcium is accumulated from the cytosol into the endoplasmic reticulum by sarco-endoplasmic reticulum calcium transport ATPase (SERCA) enzymes. Because calcium stored in the endoplasmic reticulum is essential for cell growth, differentiation, calcium signaling, and apoptosis and because different SERCA enzymes possess distinct functional characteristics, in the present report we explored SERCA expression during in vitro differentiation of the human myeloid/promyelocytic cell lines HL-60 and NB4 and of freshly isolated acute promyelocytic leukemia cells. Two SERCA species have been found to be coexpressed in these cells: SERCA 2b and another isoform, SERCAPLIM, which is recognized by the PLIM430 monoclonal antibody. Induction of differentiation along the neutrophil granulocytic lineage by all-trans retinoic acid or cyclic AMP analogs led to an increased expression of SERCAPLIM, whereas the expression of the SERCA 2b isoform was decreased. The modulation of SERCA expression was manifest also on the mRNA level. Experiments with retinoic acid receptor isoform-specific retinoids indicated that SERCA expression is modulated by retinoic acid receptor α-dependent signaling. SERCA expression of retinoic acid-resistant cell variants was refractory to treatment. Differentiation along the monocyte/macrophage lineage by phorbol ester resulted in an increased expression of both SERCA isoforms. In addition, when cells were treated by phorbol ester in the presence of the glucocorticoid dexamethasone, a known inhibitor of monocyte differentiation, a selective blockage of the induction of SERCAPLIM was observed. Altered SERCA expression modified the functional characteristics of calcium transport into the endoplasmic reticulum. These observations show for the first time that the modulation of calcium pump expression is an integral component of the differentiation program of myeloid precursors and indicate that a lineage-specific remodelling of the endoplasmic reticulum occurs during cell maturation. In addition, these data show that SERCA isoforms may serve as useful markers for the study of myeloid differentiation.