BBA - Molecular Cell Research (v.1813, #5)

Human malignant glioblastomas are highly invasive tumors. Increased cell motility and degradation of the surrounding extracellular matrix are essential for tumor invasion. PI3K/Akt signaling pathway emerges as a common pathway regulating cellular proliferation, migration and invasion; however, its contribution to particular process and downstream cascades remain poorly defined. We have previously demonstrated that Cyclosporin A (CsA) affects glioblastoma invasion in organotypic brain slices and tumorigenicity in mice. Here we show that CsA impairs migration and invasion of human glioblastoma cells by downregulation of Akt phosphorylation. Interference with PI-3K/Akt signaling was crucial for CsA effect on invasion, because overexpression of constitutively active myr-Akt antagonized drug action. Furthermore, the drug was not effective in T98G glioblastoma cells with constitutively high level of phosphorylated Akt. CsA, comparably to pharmacological inhibitors of PI3K/Akt signaling (LY294002, A443654), reduced motility of glioblastoma cells, diminished MMP-2 gelatinolytic activity and MMP-2 and MT1-MMP expression. The latter effect was mimicked by overexpression of dominant negative Akt mutants. We demonstrate that CsA and LY294002 reduced MMP transcription partly via modulation of IκB phosphorylation and NFκB transcriptional activity. Those effects were not mediated by inhibition of calcineurin, a classical CsA target. Additionally, CsA reduced phosphorylation and activity of focal adhesion kinase that was associated with rapid morphological alterations, rearrangement of lamellipodia and impairment of MT1-MMP translocation to membrane protrusions. Our results document novel, Akt-dependent mechanisms of interference with motility/invasion of human glioblastoma cells: through a rapid modulation of cell adhesion and MT1-MMP translocation to membrane protrusions and delayed, partly NFκB-dependent, downregulation of MMP-2 and MT1-MMP expression.► Cyclosporin A impairs phosporylation of Akt and FAK. ► Akt signaling regulates motility and invasiveness of malignant glioblastoma cells. ► PI-3K/Akt signaling regulates subcellular localisation of MT1-MMP. ► MT1-MMP locally influences MMP-2 activity and facilitates formation of invadopodia. ► Expression of MMP-2 and MT1-MMP is regulated in an Akt-dependent manner.
Keywords: PI3K/Akt signaling; Transcriptional regulation; MMPs; Migration; Invasion; Cyclosporin A;

Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number of transporters of heme and heme synthesis intermediates have been described. Here we review aspects of heme metabolism and discuss our current understanding of heme transporters, with emphasis on the function of the cell-surface heme exporter, FLVCR. Knockdown of Flvcr in mice leads to both defective erythropoiesis and disturbed systemic iron homeostasis, underscoring the critical role of heme transporters in mammalian physiology.► Review of our current understanding of heme catabolism and heme transporters. ► Focus is on the function of the cell-surface heme exporter, FLVCR. ► Knockdown of Flvcr in mice disturbs systemic iron homeostasis.
Keywords: Heme; Heme oxygenase; FLVCR; Membrane Transporter; Anemia; Erythrophagocytosis;

Characterization of sphere-propagating cells with stem-like properties from DU145 prostate cancer cells by Adrian P. Rybak; Lizhi He; Anil Kapoor; Jean-Claude Cutz; Damu Tang (683-694).
While accumulating evidence demonstrates the existence of prostate cancer stem cells (PCSCs), PCSCs have not been isolated and thoroughly characterized. We report here the enrichment and characterization of sphere-propagating cells with stem-like properties from DU145 PC cells in a defined serum-free medium (SFM). Approximately 1.25% of monolayer DU145 cells formed spheres in SFM and 26% of sphere cells formed secondary spheres. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins (34βE12 and CK18) and for cancer stem cell markers, including CD44, CD24, and integrin α2β1. Upon culturing spheres under differentiating media conditions in the presence of 10% serum, cells positive for CD44 and CD24 were substantially reduced. Furthermore, spheres could be generated from the sphere-derived adherent cell cultures and xenograft tumors, demonstrating the stemness of DU145 spheres. We have maintained spheres for more than 30 passages within 1.5 years without noticeable loss of their “stemness”. Sphere cells possess self-renewal capacity, display significant increases in proliferation potential, and initiate xenograft tumors with enhanced capacity compared to monolayer DU145 cells. While EGF promoted the generation and maintenance of these stem-like cells, bFGF inhibited these events. Sphere cells proliferate slowly with a significant reduction in the activation of the PI3K-AKT pathway compared to monolayer DU145 cells. While knockdown of PTEN enhanced AKT activation, this did not affect the generation of primary spheres and the propagation of secondary spheres. Consistent with this observation, we were able to demonstrate the generation and propagation of spheres without the addition of external growth factors. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.►We characterized prostate cancer stem cells (PCSCs) derived from DU145 cells. ►PCSCs can be long-term cultured as spheres in the presence of only EGF. ►bFGF reduces the generation and propagation of spheres. ►The PI3K-AKT pathway does not play a major role in PCSC culture.
Keywords: Prostate cancer stem cell; PI3K; AKT; PTEN;

Role of JNK/ATF-2 pathway in inhibition of thrombospondin-1 (TSP-1) expression and apoptosis mediated by doxorubicin and camptothecin in FTC-133 cells by Hassan El btaouri; Hamid Morjani; Yannick Greffe; Emmanuelle Charpentier; Laurent Martiny (695-703).
Our previous studies have shown that camptothecin and doxorubicin triggered ceramide accumulation via de novo synthesis pathway. De novo ceramide generation was responsible for the drug-induced apoptosis through a caspase-3-dependent pathway and a decrease of thrombospondin-1 expression in human thyroid carcinoma FTC-133 cells. Here, we demonstrate that Jun N-terminal kinases play a critical role in camptothecin- and doxorubicin-induced down-regulation of thrombospondin-1 expression: i) de novo ceramide synthesis pathway activates Jun N-terminal kinase 1/2 resulting in activating transcription factor 2 phosphorylation; ii) cell treatment by SP600125, a Jun N-terminal kinase specific inhibitor, strongly reduced activating transcription factor 2 phosphorylation and completely abolished camptothecin and doxorubicin effects; and iii) activating transcription factor 2 expression silencing greatly attenuated camptothecin- and doxorubicin-induced down-regulation of thrombospondin-1 expression and apoptosis. The set of our data established that camptothecin- and doxorubicin-induced activation of Jun N-terminal kinase/activating transcription factor 2 pathway via de novo ceramide synthesis down-regulates thrombospondin-1 expression and apoptosis in human thyroid carcinoma FTC-133 cells.► Camptothecin and doxorubicin stimulated de novo synthesis ceramide pathway. ► Produced ceramide activated JNK/ATF-2 pathway. ► JNK/ATF-2 decreased TSP-1 expression and induced apoptosis.
Keywords: Thrombospondin; Extracellular matrix; Cancer; JNK; ATF-2; Ceramide;

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells by Rita Santamaria; Filomena Fiorito; Carlo Irace; Luisa De Martino; Carmen Maffettone; Giovanna Elvira Granato; Antonio Di Pascale; Valentina Iovane; Ugo Pagnini; Alfredo Colonna (704-712).
Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin–Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes.►TCDD affects the maintenance of iron homeostasis. ►TCDD causes an opposite modulation of the iron regulatory proteins activity. ►TCDD determines an increase of TfR-1 expression and a decrease of ferritin content. ►TCDD causes an enlargement of the intracellular free iron pool. ►IRP2 becomes the effector of cellular iron homeostasis when TCDD exposure is prolonged.
Keywords: 2,3,7,8-Tetrachlorodibenzo-p-dioxin; Iron metabolism; Iron-related proteins; Iron regulatory proteins; Labile iron pool;

Ataxin-1 occupies the promoter region of E-cadherin in vivo and activates CtBP2-repressed promoter by Soyeon Lee; Sunghoi Hong; Sungsu Kim; Seongman Kang (713-722).
Ataxin-1 is a polyglutamine protein of unknown function that is encoded by the ATXN1 gene in humans. To gain insight into the function of ataxin-1, we sought to identify proteins that interact with ataxin-1 through yeast two-hybrid screening. In this study, transcriptional corepressor CtBP2 was identified as a protein that interacted with ataxin-1. CtBP2 and ataxin-1 colocalized in the nucleus of mammalian cells. Since the E-cadherin promoter is a target of CtBP-mediated repression, the relationship between ataxin-1 and the E-cadherin promoter was investigated. Chromatin immunoprecipitation assays showed that CtBP2 and ataxin-1 were recruited to the E-cadherin promoter in mammalian cells. Luciferase assays using E-cadherin promoter reporter constructs revealed that the luciferase activity was enhanced as the level of ataxin-1 protein expression increased. CtBP2 overexpression decreased E-cadherin expression, but expression of ataxin-1 inversely increased the mRNA and protein levels of endogenous E-cadherin. Interestingly, siRNA experiments showed that the transcriptional activation of ataxin-1 was associated with the presence of CtBP2. This study demonstrates that ataxin-1 occupies the promoter region of E-cadherin in vivo and that ataxin-1 activates the promoter in a CtBP2-mediated transcriptional regulation manner.► Ataxin-1 interacts and colocalizes with CtBP2. ► Ataxin-1 occupies the endogenous E-cadherin promoter in vivo. ► Overexpression of ataxin-1 enhances E-cadherin expression at the protein and mRNA levels. ► Ataxin-1 activates the E-cadherin gene promoter in a CtBP2-dependent manner.
Keywords: Ataxin-1; CtBP2; E-cadherin; Repression; Activation;

Regulation of IGF-1-dependent cyclin D1 and E expression by hEag1 channels in MCF-7 cells: The critical role of hEag1 channels in G1 phase progression by Anne-Sophie Borowiec; Frédéric Hague; Valérie Gouilleux-Gruart; Kaiss Lassoued; Halima Ouadid-Ahidouch (723-730).
Insulin-like Growth Factor-1 (IGF-1) plays a key role in breast cancer development and cell cycle regulation. It has been demonstrated that IGF-1 stimulates cyclin expression, thus regulating the G1 to S phase transition of the cell cycle. Potassium (K+) channels are involved in the G1 phase progression of the cell cycle induced by growth factors. However, mechanisms that allow growth factors to cooperate with K+ channels in order to modulate the G1 phase progression and cyclin expression remain unknown. Here, we focused on hEag1 K+ channels which are over-expressed in breast cancer and are involved in the G1 phase progression of breast cancer cells (MCF-7). As expected, IGF-1 increased cyclin D1 and E expression of MCF-7 cells in a cyclic manner, whereas the increase of CDK4 and 2 levels was sustained. IGF-1 stimulated p21WAF1/Cip1 expression with a kinetic similar to that of cyclin D1, however p27Kip1 expression was insensitive to IGF-1. Interestingly, astemizole, a blocker of hEag1 channels, but not E4031, a blocker of HERG channels, inhibited the expression of both cyclins after 6–8 h of co-stimulation with IGF-1. However, astemizole failed to modulate CDK4, CDK2, p21WAF1/Cip1 and p27Kip1 expression. The down-regulation of hEag1 by siRNA provoked a decrease in cyclin expression. This study is the first to demonstrate that K+ channels such as hEag1 are directly involved in the IGF-1-induced up-regulation of cyclin D1 and E expression in MCF-7 cells. By identifying more specifically the temporal position of the arrest site induced by the inhibition of hEag1 channels, we confirmed that hEag1 activity is predominantly upstream of the arrest site induced by serum-deprivation, prior to the up-regulation of both cyclins D1 and E.►IGF-1 regulates cyclin D1 and E expression in breast cancer. ►hEag1 is involved in the G1 phase progression in MCF-7 breast cancer cell line. ►hEag1 is a key actor of the IGF-1 pathway which modulates cyclin D1 and E expression. ►hEag1 activity occurs in G1 phase prior to the up-regulation of cyclins D1 and E.
Keywords: Cell cycle; hEag1; IGF-1; Cyclin expression regulation; G1/S transition;

Human T-cell leukemia virus type 1 Tax transactivates the matrix metalloproteinase 7 gene via JunD/AP-1 signaling by Sawako Nakachi; Tetsuro Nakazato; Chie Ishikawa; Ryuichiro Kimura; Derek A. Mann; Masachika Senba; Hiroaki Masuzaki; Naoki Mori (731-741).
Adult T-cell leukemia (ATL) is a T-cell malignancy associated with human T-cell leukemia virus type 1 (HTLV-1) and characterized by visceral invasion. Degradation of the extracellular matrix by matrix metalloproteinases (MMPs) is a crucial process in invasion of tumors and metastasis. MMP-7 (or matrilysin), is a “minimal domain MMP” with proteolytic activity against components of the extracellular matrix. To determine the involvement of MMP-7 in visceral spread in ATL, this study investigated MMP-7 expression in ATL. MMP-7 expression was identified in HTLV-1-infected T-cell lines, peripheral blood ATL cells and ATL cells in lymph nodes, but not in uninfected T-cell lines or normal peripheral blood mononuclear cells. MMP-7 expression was induced following infection of a human T-cell line with HTLV-1, and specifically by the viral protein Tax. Functionally, MMP-7 promoted cell migration of HTLV-1-infected T cells. The MMP-7 promoter activity was increased by Tax and reduced by deletion of the activator protein-1 (AP-1) binding site. Electrophoretic mobility shift assay showed high levels of AP-1 binding proteins, including JunD, in HTLV-1-infected T-cell lines and ATL cells, and Tax elicited JunD binding to the MMP-7 AP-1 element. Tax-induced MMP-7 activation was inhibited by dominant negative JunD and augmented by JunD/JunD homodimers. Short interfering RNA against JunD inhibited MMP-7 mRNA expression in HTLV-1-infected T-cell lines. These results suggest that the induction of MMP-7 by Tax is regulated by JunD and that MMP-7 could facilitate visceral invasion in ATL.Display Omitted► ATL is a T-cell malignancy associated with HTLV-1 and characterized by visceral invasion. ► MMP-7 is a “minimal domain MMP” with proteolytic activity against components of the extracellular matrix. ► Infection of a human T-cell line with HTLV-1 and induction of the viral protein Tax in a human T-cell line induced MMP-7 expression. ► The induction of MMP-7 by Tax was regulated by JunD/AP-1 signaling and MMP-7 could facilitate cell invasion in ATL.
Keywords: MMP-7; ATL; HTLV-1; Tax; AP-1; JunD;

The TGF-β co-receptor, CD109, promotes internalization and degradation of TGF-β receptors by Albane A. Bizet; Kai Liu; Nicolas Tran-Khanh; Anshuman Saksena; Joshua Vorstenbosch; Kenneth W. Finnson; Michael D. Buschmann; Anie Philip (742-753).
Transforming growth factor-β (TGF-β) is implicated in numerous pathological disorders, including cancer and mediates a broad range of biological responses by signaling through the type I and II TGF-β receptors. Internalization of these receptors via the clathrin-coated pits pathway facilitates SMAD-mediated signaling, whereas internalization via the caveolae pathway is associated with receptor degradation. Thus, molecules that modulate receptor endocytosis are likely to play a critical role in regulating TGF-β action. We previously identified CD109, a GPI-anchored protein, as a TGF-β co-receptor and a negative regulator of TGF-β signaling. Here, we demonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109 increases binding of TGF-β to its receptors and enhances their internalization via the caveolae. In addition, CD109 promotes localization of the TGF-β receptors into the caveolar compartment in the presence of ligand and facilitates TGF-β-receptor degradation. Thus, CD109 regulates TGF-β receptor endocytosis and degradation to inhibit TGF-β signaling.Display Omitted► CD109 associates with caveolin-1. ► CD109 facilitates TGF-β receptor localization and internalization into the caveolae. ► CD109's inhibition of TGF-β signaling requires caveolin-1. ► CD109 promotes TGF-β receptor degradation.
Keywords: TGF-β receptor; CD109; Internalization; Degradation; Caveolae;

Real-time cellular impedance measurements detect Ca2+ channel-dependent oscillations of morphology in human H295R adrenoma cells by Athanasios Denelavas; Franziska Weibel; Michael Prummer; Alexander Imbach; Roger G. Clerc; Christian M. Apfel; Cornelia Hertel (754-762).
Endocrine cells, such as H295R have been widely used to study secretion of steroid and other hormones. Exocytosis-dependent hormone release is accompanied by an increase in plasma membrane surface area and a decrease in vesicle content. Recovery of vesicles and decrease in plasma membrane area is achieved by endocytotic processes. These changes in the extent of the surface area lead to morphological changes which can be determined by label-free real-time impedance measurements. Exo- and endocytosis have been described to be triggered by activation of L-type Ca2+ channels. The present study demonstrates that activation of L-type calcium channels induces prolonged oscillating changes in cellular impedance. The data support the hypothesis that a tight regulation of the intracellular Ca2+ concentration is a prerequisite for the observed cellular impedance oscillations. Furthermore evidence is presented for a mechanism in which the oscillations depend on a Ca2+-triggered calmodulin-dependent cascade involving myosin light chain kinase, nonmuscle myosin II and ultimately actin polymerization, a known determinant for cell shape changes and exocytosis in secretory cells. The described assay provides a method to determine continuously prolonged changes in cellular morphology such as exo/endocytosis cycles.► L-type Ca channel opening induces oscillations of cellular impedance in H295R cells. ► Tight regulation of intracellular Ca is mandatory for impedance oscillations. ► Ca triggers calmodulin-dependent kinase cascade. ► Cellular impedance oscillations represent exo/endocytosis cycles.
Keywords: Impedance; BayK8644; Ca channel; Exocytosis; Endocytosis; Label-free technology;

Visfatin exerts angiogenic effects on human umbilical vein endothelial cells through the mTOR signaling pathway by Joo-Won Park; Won-Ho Kim; So-Hee Shin; Ji Yeon Kim; Mi Ran Yun; Keon Jae Park; Hyun-Young Park (763-771).
The biologically active factors known as adipocytokines are secreted primarily by adipose tissues and can act as modulators of angiogenesis. Visfatin, an adipocytokine that has recently been reported to have angiogenic properties, is upregulated in diabetes, cancer, and inflammatory diseases. Because maintenance of an angiogenic balance is critically important in the management of these diseases, understanding the molecular mechanism by which visfatin promotes angiogenesis is very important. In this report, we describe our findings demonstrating that visfatin stimulates the mammalian target of the rapamycin (mTOR) pathway, which plays important roles in angiogenesis. Visfatin induced the expression of hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF) in human endothelial cells. Inhibition of the mTOR pathway by rapamycin eliminated the angiogenic and proliferative effects of visfatin. The visfatin-induced increase in VEGF expression was also eliminated by RNA interference-mediated knockdown of the 70-kDa ribosomal protein S6 kinase (p70S6K), a downstream target of mTOR. Visfatin inactivated glycogen synthase kinase 3β (GSK3β) by phosphorylating it at Ser-9, leading to the nuclear translocation of β-catenin. Both rapamycin co-treatment and p70S6K knockdown inhibited visfatin-induced GSK3β phosphorylation at Ser-9 and nuclear translocation of β-catenin. Taken together, these results indicate that mTOR signaling is involved in visfatin-induced angiogenesis, and that this signaling leads to visfatin-induced VEGF expression and nuclear translocation of β-catenin.► Visfatin-induced angiogenesis requires mTOR and GSK3β signaling downstream of the PI3-kinase/Akt signaling pathway. ► Activation of mTOR inactivates GSK3β, which promotes the nuclear translocation of β-catenin, thereby increasing the expression of cyclin D1 and VEGF. ► β-Catenin knockdown attenuates visfatin-induced endothelial cell proliferation and capillary tube formation. ► Collectively, these results show that the activation of mTOR and the inactivation of GSK3β play key roles in visfatin-induced angiogenesis.
Keywords: Visfatin; Angiogenesis; Adipocytokine; mTOR; GSK3β;

ESeroS-GS modulates lipopolysaccharide-induced macrophage activation by impairing the assembly of TLR-4 complexes in lipid rafts by Wenjuan Duan; Juefei Zhou; Shen Zhang; Kai Zhao; Lijing Zhao; Kazumi Ogata; Takahiro Sakaue; Akitane Mori; Taotao Wei (772-783).
The binding of lipopolysaccharides (LPS) to macrophages results in inflammatory responses. In extreme cases it can lead to endotoxic shock, often resulting in death. A broad range of antioxidants, including tocopherols, can reduce LPS activity in vitro and in vivo. To elucidate the underlying mechanisms of their action, we investigated the effect of the sodium salt of γ-L-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-L-cysteinylglycine (ESeroS-GS), a novel α-tocopherol derivative, on LPS-induced inflammation in vitro and in vivo. ESeroS-GS reduced the transcription of TNF-α, IL-1β, IL-6 and iNOS genes in a dose-dependent manner in RAW264.7 macrophages, and inhibited the release of these inflammatory factors. In addition, ESeroS-GS inhibited LPS-induced mortality in a mouse sepsis model. Electrophoretic mobility shift assays (EMSA) and reporter gene assays revealed that ESeroS-GS down-regulated the transcriptional activity of NF-κB. By analyzing the partitioning of CD14 and Toll-like receptor 4 (TLR-4) in cell membrane microdomains, we found that ESeroS-GS attenuates the binding of LPS to RAW264.7 cells via interfering with the relocation of CD14 and TLR-4 to lipid rafts, blocking the activation of interleukin-1 receptor-associated kinase 1 (IRAK-1), and inhibiting the consequent phosphorylation of TAK1 and IKKα/β, which together account for the suppression of NF-κB activation. Taken together, our data suggest that ESeroS-GS can modulate LPS signaling in macrophages by impairing TLR-4 complex assembly via a lipid raft dependent mechanism.► ESeroS-GS is a novel antioxidant derived from vitamin E. ► LPS activates macrophages. ► ESeroS-GS inhibits the assembly of TLR-4 complex and regulates macrophage activation in vitro and in vivo through lipid raft dependent mechanism.
Keywords: ESeroS-GS; RAW264.7 macrophage; Lipopolysaccharide; Toll-like receptor-4; Lipid raft;

Hydrogen peroxide depletes phosphatidylinositol-3-phosphate from endosomes in a p38 MAPK-dependent manner and perturbs endocytosis by Fumi Kano; Tamaki Arai; Mariko Matsuto; Hanako Hayashi; Moritoshi Sato; Masayuki Murata (784-801).
Phosphatidylinositol-3-phosphate (PI3P) is a lipid that is enriched specifically in early endosomes. Given that early endosomes containing PI3P act as a microdomain to recruit proteins that contain a PI3P-binding domain (FYVE domain), the equilibrium between the production and degradation of PI3P influences a variety of processes, including endocytosis and signal transduction via endosomes. In the study reported herein, we have developed a novel analytical method to quantify the amount of PI3P in endosomes by introducing a GST-2xFYVE protein probe into semi-intact cells. The GST-2xFYVE probe was targeted specifically to intracellular PI3P-containing endosomes, which retained their small punctate structure, and allowed the semi-quantitative measurement of intracellular PI3P. Using the method, we found that treatment of HeLa cells with H2O2 decreased the amount of PI3P in endosomes in a p38 MAPK-dependent manner. In addition, H2O2 treatment delayed transport through various endocytic pathways, especially post-early endosome transport; the retrograde transport of cholera toxin was especially dependent on the amount of PI3P in endosomes.► Hydrogen peroxide depletes PI3P from early endosomes in a p38 MAPK-dependent manner. ► H2O2 delayed the endocytic pathway, especially post-early endosome transport. ► Endosomal PI3P is crucial for retrograde transport of cholera toxin to the Golgi.
Keywords: Oxidative stress; Endocytosis; Phosphatidylinositol-3-phosphate; p38 MAPK; Semi-intact cell; Visual assay;

Adiponectin (15–36) stimulates steroidogenic acute regulatory (StAR) protein expression and cortisol production in human adrenocortical cells: Role of AMPK and MAPK kinase pathways by Manjunath Ramanjaneya; Alex C. Conner; James E.P. Brown; Jing Chen; Janet E. Digby; Thomas M. Barber; Hendrik Lehnert; Harpal S. Randeva (802-809).
Adiponectin is an abundantly circulating adipokine, orchestrating its effects through two 7-transmembrane receptors (AdipoR1 and AdipoR2). Steroidogenesis is regulated by a variety of neuropeptides and adipokines. Earlier studies have reported adipokine mediated steroid production. A key rate-limiting step in steroidogenesis is cholesterol transportation across the mitochondrial membrane by steroidogenic acute regulatory protein (StAR). Several signalling pathways regulate StAR expression. The actions of adiponectin and its role in human adrenocortical steroid biosynthesis are not fully understood. The aim of this study was to investigate the effects of adiponectin on StAR protein expression, steroidogenic genes, and cortisol production and to dissect the signalling cascades involved in the activation of StAR expression. Using qRT-PCR, Western blot analysis and ELISA, we have demonstrated that stimulation of human adrenocortical H295R cells with adiponectin results in increased cortisol secretion. This effect is accompanied by increased expression of key steroidogenic pathway genes including StAR protein expression via ERK1/2 and AMPK-dependent pathways. This has implications for our understanding of adiponectin receptor activation and peripheral steroidogenesis. Finally, our study aims to emphasise the key role of adipokines in the integration of metabolic activity and energy balance partly via the regulation of adrenal steroid production.► Adiponectin receptors are expressed in human adrenocortical (H295R) cells. ► Adiponectin increases StAR protein expression and cortisol secretion. ► Adiponectin induces increase in expression of key steroidogenic genes. ► Adiponectin induces activation of ERK1/2 and AMPK kinase in H295R cells. ► AMPK and ERK inhibitions result in reduction of adiponectin-mediated StAR expression.
Keywords: StAR; Cortisol; Adiponectin; AMPK; MAPK; Steroidogenesis;

Mechanism of 1α,25-dihydroxyvitamin D3-dependent repression of interleukin-12B by Petra Gynther; Sari Toropainen; Juha M. Matilainen; Sabine Seuter; Carsten Carlberg; Sami Väisänen (810-818).
Interleukin 12 (IL-12) is a heterodimeric, pro-inflammatory cytokine that plays a central role in activation and differentiation of CD4+ T cells into interferon-γ secreting T-helper type 1 cells. IL-12B, a gene encoding the larger subunit of active IL-12, has been reported to be down-regulated by the nuclear hormone 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), but the mechanism of the regulation is unknown. In this study, we have examined the molecular mechanism of transcriptional regulation of the IL-12B gene by 1α,25(OH)2D3 in lipopolysaccharide (LPS)-treated human monocytes (THP-1). Quantitative RT-PCR showed that IL-12B mRNA displays a cyclical expression profile and is down-regulated 2.8-fold during the first 8 h and even 12.1-fold 24 h after exposure to 1α,25(OH)2D3. Gel shift and quantitative chromatin immunoprecipitation (ChIP) assays demonstrated vitamin D receptor (VDR) binding to genomic regions 480 and 6300 bp upstream of the IL-12B transcription start site (TSS). Quantitative ChIP assays also revealed that together with VDR and its partner RXR the above regions recruited the co-repressor NCOR2/SMRT and histone deacetylase 3 leading to a decreased histone 4 acetylation and increased histone 3 trimethylation at the IL-12B promoter and its TSS. We suggest that these repressive epigenetic changes eventually cause down-regulation of IL-12 expression.► 1a,25(OH)2D3 down-regulates the IL-12B gene both in cultured and primary monocytes. ► The repression of IL-12B by 1a,25(OH)2D3 is mediated via previously unknown VDREs. ► The repression of IL-12B by 1a,25(OH)2D3 involves repressive epigenetic changes. ► The repressive proteins associate cyclically to IL-12B promoter and TSS.
Keywords: Chromatin; IL-12B; VDR; Transcription; Inflammation;

Dimerization of Smac is crucial for its mitochondrial retention by XIAP subsequent to mitochondrial outer membrane permeabilization by Lorna Flanagan; Jordi Sebastia; Maria Eugenia Delgado; Jennifer C. Lennon; Markus Rehm (819-826).
Following the apoptotic permeabilization of the outer mitochondrial membrane, the inter-membrane space protein second mitochondria-derived activator of caspases (Smac) is released into the cytosol. Smac efficiently promotes apoptosis by antagonizing x-linked inhibitor of apoptosis protein (XIAP), an inhibitor of caspases-9, -3, and -7, via a short NH2-terminal inhibitor of apoptosis protein (IAP) binding motif (AVPI). Native Smac dimerizes to form a highly stable and inflexible elongated arch, however, a functional role for this outstretched structure so far remained unknown. Using time-lapse single-cell imaging of DLD-1 and HCT-116 colon cancer cells, we here demonstrate that upon mitochondrial outer membrane permeabilization physiological expression levels of XIAP are sufficient to selectively prolong the release of dimeric but not monomeric Smac. Elevating the expression of XIAP further extended the release duration of dimeric Smac and resulted in the mitochondrial retention of a significant proportion of the Smac pool. In contrast, monomeric Smac was always fully released and the release kinetics were not affected by altered XIAP expression. Our findings therefore indicate that the dimerization of Smac is critical for the XIAP-mediated retention of Smac at or inside the mitochondria.► We identified that physiological expression of XIAP selectively prolongs the release of dimeric Smac but not monomeric Smac. ► Dimeric Smac cannot efficiently leave the mitochondria if elevated amounts of XIAP are present. The release of monomeric Smac instead is not affected. ► In contrast to Smac dimerization, the dimerization of XIAP via its BIR1 region does not influence the kinetic or the efficiency of Smac release.
Keywords: Apoptosis; x-Linked inhibitor of apoptosis protein (XIAP); Second mitochondria-derived activator of caspases (Smac); Mitochondrial outer membrane permeabilization (MOMP); Time-lapse live cell imaging; Single-cell analysis;

Essential role of ERK activation in neurite outgrowth induced by α-lipoic acid by Xiaohui Wang; Zhuyao Wang; Yuzhen Yao; Jingjin Li; Xiaojin Zhang; Chuanfu Li; Yunlin Cheng; Guoxian Ding; Li Liu; Zhengnian Ding (827-838).
Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth.To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for β-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth.LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth.Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism.► Alpha-lipoic acid (LA) promotes neurite outgrowth. ► LA activates ERKs and Akt, increases ROS generation. ► Inhibition of MEK/ERK, but not PI3K/Akt, abolished LA-stimulated neurite growth. ► Inhibition of ROS abolished LA-induced neurite outgrowth and ERK activation.
Keywords: Alpha-lipoic acid; Neurite outgrowth; MEK/ERK signaling pathway; Reactive oxygen species;

DNA methylation restricts spontaneous multi-lineage differentiation of mesenchymal progenitor cells, but is stable during growth factor-induced terminal differentiation by Marlinda Hupkes; Eugene P. van Someren; Sjors H.A. Middelkamp; Ester Piek; Everardus J. van Zoelen; Koen J. Dechering (839-849).
The progressive restriction of differentiation potential from pluripotent embryonic stem cells, via multipotent progenitor cells to terminally differentiated, mature somatic cells, involves step-wise changes in transcription patterns that are tightly controlled by the coordinated action of key transcription factors and changes in epigenetic modifications. While previous studies have demonstrated tissue-specific differences in DNA methylation patterns that might function in lineage restriction, it is unclear at what exact developmental stage these differences arise. Here, we have studied whether terminal, multi-lineage differentiation of C2C12 myoblasts is accompanied by lineage-specific changes in DNA methylation patterns. Using bisulfite sequencing and genome-wide methylated DNA- and chromatin immunoprecipitation-on-chip techniques we show that in these cells, in general, myogenic genes are enriched for RNA polymerase II and hypomethylated, whereas osteogenic genes show lower polymerase occupancy and are hypermethylated. Removal of DNA methylation marks by 5-azacytidine (5AC) treatment alters the myogenic lineage commitment of these cells and induces spontaneous osteogenic and adipogenic differentiation. This is accompanied by upregulation of key lineage-specific transcription factors. We subsequently analyzed genome-wide changes in DNA methylation and polymerase II occupancy during BMP2-induced osteogenesis. Our data indicate that BMP2 is able to induce the transcriptional program underlying osteogenesis without changing the methylation status of the genome. We conclude that DNA methylation primes C2C12 cells for myogenesis and prevents spontaneous osteogenesis, but still permits induction of the osteogenic transcriptional program upon BMP2 stimulation. Based on these results, we propose that cell type-specific DNA methylation patterns are established prior to terminal differentiation of adult progenitor cells.► 5AC induces C2C12 osteogenic and adipogenic differentiation. ► 5AC induces promoter demethylation and mRNA upregulation of key osteogenic factors. ► BMP2-induced C2C12 osteogenesis occurs in the absence of DNA methylation changes. ► C2C12 myogenic differentiation occurs in the absence of DNA methylation changes.
Keywords: DNA methylation; Differentiation; Myoblast; Osteoblast; Bone morphogenetic protein 2; 5-Azacytidine;

Investigation of the Bcl-2 multimerisation process: Structural and functional implications by Alessia Camperchioli; Marisa Mariani; Silvia Bartollino; Lella Petrella; Marco Persico; Nausicaa Orteca; Giovanni Scambia; Shohreh Shahabi; Cristiano Ferlini; Caterina Fattorusso (850-857).
Bcl-2 plays a prominent role in regulating the function of mitochondria during respiration and in determining the threshold of apoptotic sensitivity. Despite its relevance, the mechanism through which these processes are achieved is still unknown. Using surface plasmon resonance technology to monitor Bcl-2 multimerisation we discovered that a simple dimeric model does not fit with experimental data. A molecular model of the experimentally observed Bcl-2 homomeric complex has been developed. Accordingly, using a panel of mutants we identified in the loop a critical region for the process of Bcl-2 multimerisation. Our results indicate that the Bcl-2 loop posttranscriptional changes can modulate its ability to make homo and hetero-complexes, ultimately leading to functional modulation, suggesting an intriguing relationship between the ability of Bcl-2 to form multimeric complexes and its multi-functional role as a membrane channel.Display Omitted► Bcl-2 forms a high-affinity homo-trimeric complex via its long loop. ► The formation of the Bcl-2 homo-trimer is related to its apoptotic function. ► The Bcl-2 homo-trimer shares structural similarity with some channels/transporters.
Keywords: Bcl-2 structure/function; Multimeric protein complexes; Apoptosis;

Single actomyosin motor interactions in skeletal muscle by Zeno Földes-Papp; Shih-Chu Jeff Liao; Ben Barbieri; Karol Gryczynski; Rafal Luchowski; Zygmunt Gryczynski; Ignacy Gryczynski; Julian Borejdo; Tiefeng You (858-866).
We present a study of intramuscular motion during contraction of skeletal muscle myofibrils. Myofibrillar actin was labeled with fluorescent dye so that the ratio of fluorescently labeled to unlabeled protein was 1:105. Such sparse labeling assured that there was on average only one actin-marker present in the focus at a given time. From the intensity signal in the two orthogonal detection channels, significant fluctuations, similar to fluorescent burst in diffusion-based single-molecule detection schemes, were identified via a threshold algorithm and analyzed with respect to their intensity and polarization. When only rigor complexes were formed, the fluctuations of polarized intensity were characterized by unimodal Gaussian photon distributions. During contraction, in contrast, bimodal Gaussian photon distributions were observed above the rigor background threshold. This suggests that the bimodal Gaussian photon distributions represent pre- and post-power stroke conformations. Clusters of polarized photons indicated an anisotropy decay of single actomyosin motors of ~ 9 s during muscle contraction.► Intensity fluctuations in two polarization selective detection channels. ► The ability to interact using different orientations of the reporter dipole on actin. ► During contraction, bimodal Gaussian photon distributions were observed above rigor noise. ► Clusters of polarized photons indicated an anisotropy decay of 9 s during contraction. ► The slow relaxation dynamics revealed molecular memory.
Keywords: Single actomyosin; Live skeletal muscle cell; Polarization dependent fluorescence fluctuations; Time-dependent photon distributions; Molecular orientations;

Abnormal actomyosin assembly in proliferating and differentiating myoblasts upon expression of a cytosolic DMPK isoform by Susan A.M. Mulders; Remco van Horssen; Lieke Gerrits; Miranda B. Bennink; Helma Pluk; Roelie T. de Boer-van Huizen; Huib J.E. Croes; Mietske Wijers; Fons A.J. van de Loo; Jack Fransen; Bé Wieringa; Derick G. Wansink (867-877).
DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine–threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast–myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells.► Expression of DMPK in the cytosol induces formation of stellar actomyosin stress fibers. ► Stellar stress fiber formation is accompanied by augmented phosphorylation of MLC2. ► Myoblasts expressing active cytosolic DMPK display altered cell morphology and motility. ► Early steps in myogenesis are delayed upon DMPK activity expression in the cytosol. ► High cytosolic DMPK activity during myogenesis supports formation of myosacs.
Keywords: Stress fiber; ROCK; Rho kinase; Myotonic dystrophy protein kinase; Actomyosin cytoskeleton; Myogenesis; Alternative splicing;

The pro- and anti-inflammatory properties of the cytokine interleukin-6 by Jürgen Scheller; Athena Chalaris; Dirk Schmidt-Arras; Stefan Rose-John (878-888).
Interleukin-6 is a cytokine not only involved in inflammation and infection responses but also in the regulation of metabolic, regenerative, and neural processes. In classic signaling, interleukin-6 stimulates target cells via a membrane bound interleukin-6 receptor, which upon ligand binding associates with the signaling receptor protein gp130. Gp130 dimerizes, leading to the activation of Janus kinases and subsequent phosphorylation of tyrosine residues within the cytoplasmic portion of gp130. This leads to the engagement of phosphatase Src homology domains containing tyrosin phosphatase-2 (SHP-2) and activation of the ras/raf/Mitogen-activated protein (MAP) kinase (MAPK) pathway. In addition, signal transducer and activator of transcription factors are recruited, which are phosphorylated, and consequently dimerize whereupon they translocate into the nucleus and activate target genes. Interestingly, only few cells express membrane bound interleukin-6 receptor whereas all cells display gp130 on the cell surface. While cells, which only express gp130, are not responsive to interleukin-6 alone, they can respond to a complex of interleukin-6 bound to a naturally occurring soluble form of the interleukin-6 receptor. Therefore, the generation of soluble form of the interleukin-6 receptor dramatically enlarges the spectrum of interleukin-6 target cells. This process has been named trans-signaling. Here, we review the involvement of both signaling modes in the biology of interleukin-6. It turns out that regenerative or anti-inflammatory activities of interleukin-6 are mediated by classic signaling whereas pro-inflammatory responses of interleukin-6 are rather mediated by trans-signaling. This is important since therapeutic blockade of interleukin-6 by the neutralizing anti-interleukin-6 receptor monoclonal antibody tocilizumab has recently been approved for the treatment of inflammatory diseases.Legend:Cells that express both gp130 and the membrane bound IL-6R are responsive to IL-6 (classic signaling). In contrast, cells that express gp130 but not IL-6R can only be activated by the complex of IL-6 and soluble IL-6R (sIL-6R). Activation of cells by the IL-6/sIL-6R complex is termed trans-signaling. The sIL-6R is generated by proteolytic cleavage of the membrane bound precursor by membrane bound metalloproteases of the ADAM family. The sIL-6R binds IL-6 with comparable affinity as the membrane bound form and mediates gp130 activation in an (1) autocrine or (2) paracrine manner.Display Omitted► The cytokine interleukin-6 (IL-6) has pro- and anti-inflammatory properties. ► Whereas only few cells express the IL-6 receptor and respond to IL-6 (classic signaling), all cells can be stimulated via a soluble IL-6 receptor (trans-signaling) since gp130 is ubiquitously expressed. ► Classic- and trans-signaling can be differentially inhibited. ► Apparently, IL-6 via classic signaling has regenerative and anti-inflammatory functions whereas trans-signaling is pro-inflammatory.
Keywords: IL-6 trans-signaling; sIL-6R; gp130; sgp130Fc; STAT3 activation; Inflammation associated cancer;

Gastrins, iron homeostasis and colorectal cancer by Suzana Kovac; Gregory J. Anderson; Graham S. Baldwin (889-895).
The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron deficiency anemia was recognized as a long-term consequence of partial gastrectomy. This review summarizes the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyze the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract.► Gastrins bind two ferric ions with micromolar affinity. ► Binding of iron to non-amidated gastrins is essential for biological activity. ► Gastrin interacts with, and may catalyse the loading of, transferrin with iron. ► Concentrations of circulating gastrins are increased in hemochromatosis. ► Transferrin saturation positively correlates with circulating gastrins.
Keywords: Gastrin; Ferric; Iron; Colon cancer; Colorectal cancer;

Molecular determinants and feedback circuits regulating type 2 CRH receptor signal integration by Danijela Markovic; Anu Punn; Hendrik Lehnert; Dimitris K. Grammatopoulos (896-907).
In most target tissues, the adenylyl cyclase/cAMP/PKA, the extracellular signal regulated kinase and the protein kinase B/Akt are the main pathways employed by the type 2 corticotropin-releasing hormone receptor to mediate the biological actions of urocortins (Ucns) and CRH. To decipher the molecular determinants of CRH-R2 signaling, we studied the signaling pathways in HEK293 cells overexpressing recombinant human CRH-R2β receptors. Use of specific kinase inhibitors showed that the CRH-R2β cognate agonist, Ucn 2, activated extracellular signal regulated kinase in a phosphoinositide 3-kinase and cyclic adenosine monophosphate/PKA-dependent manner with contribution from Epac activation. Ucn 2 also induced PKA-dependent association between AKAP250 and CRH-R2β that appeared to be necessary for extracellular signal regulated kinase activation. PKB/Akt activation was also mediated via pertussis toxin-sensitive G-proteins and PI3-K activation but did not require cAMP/PKA, Epac or protein kinase C for optimal activation. Potential feedback mechanisms that target the CRH-R2β itself and modulate receptor trafficking and endocytosis were also investigated. Indeed, our results suggested that inhibition of either PKA or extracellular signal regulated kinase pathway accelerates CRH-R2β endocytosis. Furthermore, Ucn 2-activated extracellular signal regulated kinase appeared to target β-arrestin1 and modulate, through phosphorylation at Ser412, β-arrestin1 translocation to the plasma membrane and CRH-R2β internalization kinetics. Loss of this “negative feedback” mechanism through inhibition of the extracellular signal regulated kinase activity resulted in significant attenuation of Ucn 2-induced cAMP response, whereas Akt phosphorylation was not affected by altered receptor endocytosis. These findings reveal a complex interplay between the signaling molecules that allow “fine-tuning” of CRH-R2β functional responses and regulate signal integration.► Intracellular signals and interactions with AKAP regulate CRH-R2β-ERK activation. ► CRH-R2β induces PKB/Akt activation via Gi/o-proteins-PI3-K activation. ► ERK phosphorylates β-arr1 and modulates its translocation to the plasma membrane. ► Inhibition of either PKA or ERK pathway accelerates CRH-R2β endocytosis. ► The Akt signaling is insensitive to changes in receptor endocytosis rate.
Keywords: CRH receptor; PKA; Ucn2; β-arrestin1; ERK1/2; AKAP;

The 11th Meeting of the European Calcium Society by Jacques Haiech; Claus W. Heizmann; Joachim Krebs (908).

Calcium Waves in Warszawa by Joachim Krebs (909-912).

The Ca2+-binding helix–loop–helix structural motif called “EF-hand” is a common building block of a large family of proteins that function as intracellular Ca2+-receptors. These proteins respond specifically to micromolar concentrations of Ca2+ in the presence of ~1000-fold excess of the chemically similar divalent cation Mg2+. The intracellular free Mg2+ concentration is tightly controlled in a narrow range of 0.5–1.0 mM, which at the resting Ca2+ levels is sufficient to fully or partially saturate the Ca2+-binding sites of many EF-hand proteins. Thus, to convey Ca2+ signals, EF-hand proteins must respond differently to Ca2+ than to Mg2+. In this review the structural aspects of Mg2+ binding to EF-hand proteins are considered and interpreted in light of the recently proposed two-step Ca2+-binding mechanism (Grabarek, Z., J. Mol. Biol., 2005, 346, 1351). It is proposed that, due to stereochemical constraints imposed by the two-EF-hand domain structure, the smaller Mg2+ ion cannot engage the ligands of an EF-hand in the same way as Ca2+ and defaults to stabilizing the apo-like conformation of the EF-hand. It is proposed that Mg2+ plays an active role in the Ca2+-dependent regulation of cellular processes by stabilizing the “off state” of some EF-hand proteins, thereby facilitating switching off their respective target enzymes at the resting Ca2+ levels. Therefore, some pathological conditions attributed to Mg2+ deficiency might be related to excessive activation of underlying Ca2+-regulated cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► “EF-hand” proteins convey intracellular Ca2+ signals in the presence of ~1000-fold excess of Mg2+. ► Due to stereochemical constraints Mg2+ ion cannot engage the ligands of an EF-hand in the same way as Ca2+. ► Mg2+ plays an active role in the Ca2+-dependent regulation of cellular processes by stabilizing the “off state” of some EF-hand proteins. ► Some pathological conditions attributed to Mg2+ deficiency might be related to excessive activation of underlying Ca2+-regulated cellular processes.
Keywords: EF-hand; Ca2+-binding; Calcium signaling; Mg2+-binding; Magnesium deficiency;

Atrial cardiomyocyte calcium signalling by Martin D. Bootman; Ioannis Smyrnias; Rüdiger Thul; Stephen Coombes; H. Llewelyn Roderick (922-934).
Whereas Ca2+ signalling in ventricular cardiomyocytes is well described, much less is known regarding the Ca2+ signals within atrial cells. This is surprising given that atrial cardiomyocytes make an important contribution to the refilling of ventricles with blood, which enhances the subsequent ejection of blood from the heart. The dependence of cardiac function on the contribution of atria becomes increasingly important with age and exercise. Disruption of the rhythmic beating of atrial cardiomyocytes can lead to life-threatening conditions such as atrial fibrillation. Atrial and ventricular myocytes have many structural and functional similarities. However, one key structural difference, the lack of transverse tubules (“T-tubules”) in atrial myocytes, make these two cell types display vastly different calcium patterns in response to electrical excitation. The lack of T-tubules in atrial myocytes means that depolarisation provokes calcium signals that originate around the periphery of the cells. Under resting conditions, such Ca2+ signals do not propagate towards the centre of the atrial cells and so do not fully engage the contractile machinery. Consequently, contraction of atrial myocytes under resting conditions is modest. However, when atrial myocytes are stimulated with a positive inotropic agonist, such as isoproterenol, the peripheral Ca2+ signals trigger a global wave of Ca2+ that propagates in a centripetal manner into the cells. Enhanced centripetal movement of Ca2+ in atrial myocytes leads to increased contraction and a more substantial contribution to blood pumping. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► Atrial cardiac myocytes make an important contribution to blood pumping. ► Dysregulation of atrial contraction can be debilitating or even fatal. ► Atrial myocytes have a different ultrastructure from ventricular myocytes. ► Calcium regulates heart contraction, and it has a unique pattern in atrial myocytes. ► Atrial myocytes change the pattern of calcium signalling to alter contraction strength.
Keywords: Cardiac; Calcium; Arrhythmia; Signalling; Atrial; Ryanodine; Inositol; Inotropy; Excitation–contraction coupling; T-tubules;

Annexin A6—Linking Ca2+ signaling with cholesterol transport by Carlos Enrich; Carles Rentero; Sandra Vilà de Muga; Meritxell Reverter; Vishwaroop Mulay; Peta Wood; Meryem Koese; Thomas Grewal (935-947).
Annexin A6 (AnxA6) belongs to a conserved family of Ca2+-dependent membrane-binding proteins. Like other annexins, the function of AnxA6 is linked to its ability to bind phospholipids in cellular membranes in a dynamic and reversible fashion, in particular during the regulation of endocytic and exocytic pathways. High amounts of AnxA6 sequester cholesterol in late endosomes, thereby lowering the levels of cholesterol in the Golgi and the plasma membrane. These AnxA6-dependent redistributions of cellular cholesterol pools give rise to reduced cytoplasmic phospholipase A2 (cPLA2) activity, retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. In addition to regulating cholesterol and caveolin distribution, AnxA6 acts as a scaffold/targeting protein for several signaling proteins, the best characterized being the Ca2+-dependent membrane targeting of p120GAP to downregulate Ras activity. AnxA6 also stimulates the Ca2+-inducible involvement of PKC in the regulation of HRas and possibly EGFR signal transduction pathways. The ability of AnxA6 to recruit regulators of the EGFR/Ras pathway is likely potentiated by AnxA6-induced actin remodeling. Accordingly, AnxA6 may function as an organizer of membrane domains (i) to modulate intracellular cholesterol homeostasis, (ii) to create a scaffold for the formation of multifactorial signaling complexes, and (iii) to regulate transient membrane–actin interactions during endocytic and exocytic transport. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.►Cholesterol modulates the membrane binding and intracellular distribution of AnxA6. ►AnxA6-induced alterations in cholesterol transport and caveolin export from the Golgi. ►AnxA6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity. ►AnxA6 inhibits Ras signaling in breast cancer cells. ►AnxA6 overexpression stabilizes cortical actin.
Keywords: Annexin A6; Actin; Caveolin-1; Caveolae; Cholesterol; cPLA2; PKCα; Ras/MAPK;

This review focuses on muscle disorders and diseases caused by defects in the Ca2+ release channels of the sarcoplasmic reticulum, the ryanodine receptors, and in the luminal, low affinity, high capacity Ca2+-binding proteins, calsequestrins. It provides a time line over the past half century of the highlights of research on malignant hyperthermia (MH), central core disease (CCD) and catecholaminergic polymorphic ventricular tachycardia (CPVT), that resulted in the identification of the ryanodine receptor (RYR), calsequestrin (CASQ) and dihydropyridine receptor (CACNA1S) genes as sites of disease-causing mutations. This is followed by a description of approaches to functional analysis of the effects of disease-causing mutations on protein function, focusing on studies of how mutations affect spontaneous (store overload-induced) Ca2+-release from the sarcoplasmic reticulum, the underlying cause of MH and CPVT. Subsequent sections describe results obtained by analysis of knockin mouse lines carrying MH- and CCD-causing mutations, including a Casq1 knockout. The review concludes with the presentation of two mechanistic models. The first shows how dysregulation of Ca2+ homeostasis can lead to muscle diseases involving both RyR and Casq proteins. The second describes a theory of central core formation wherein non-uniformity of Ca2+ release, resulting in non-uniformity of muscle contraction, is presented as an intrinsic property of the specific tertiary structure of mutant heterotetrameric ryanodine receptors and as the underlying cause of core formation in skeletal muscle. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► We provide an overview of the major advances in our understanding of the etiology of MH, CCD and CPVT. ► MH and CPVT are caused by mutations in the ryanodine receptor (RyR) or calsequestrin. ► We propose that store overload-induced Ca2+ release is the common causal factor in MH and CPVT. ► We propose that there is non-uniformity of Ca2+ release from heterogeneous Ca2+ release units. ► This causes non-uniformity of muscle contraction, leading to structural muscle damage for CCD.
Keywords: Calcium; Malignant hyperthermia; Central core disease; Catecholaminergic polymorphic ventricular tachycardia; Ryanodine receptor; Calsequestrin;

In this mini-review/opinion article we describe evidence that multiple cellular and molecular alterations in Alzheimer's disease (AD) pathogenesis involve perturbed cellular calcium regulation, and that alterations in synaptic calcium handling may be early and pivotal events in the disease process. With advancing age neurons encounter increased oxidative stress and impaired energy metabolism, which compromise the function of proteins that control membrane excitability and subcellular calcium dynamics. Altered proteolytic cleavage of the β-amyloid precursor protein (APP) in response to the aging process in combination with genetic and environmental factors results in the production and accumulation of neurotoxic forms of amyloid β-peptide (Aβ). Aβ undergoes a self-aggregation process and concomitantly generates reactive oxygen species that can trigger membrane-associated oxidative stress which, in turn, impairs the functions of ion-motive ATPases and glutamate and glucose transporters thereby rendering neurons vulnerable to excitotoxicity and apoptosis. Mutations in presenilin-1 that cause early-onset AD increase Aβ production, but also result in an abnormal increase in the size of endoplasmic reticulum calcium stores. Some of the events in the neurodegenerative cascade can be counteracted in animal models by manipulations that stabilize neuronal calcium homeostasis including dietary energy restriction, agonists of glucagon-like peptide 1 receptors and drugs that activate mitochondrial potassium channels. Emerging knowledge of the actions of calcium upstream and downstream of Aβ provides opportunities to develop novel preventative and therapeutic interventions for AD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► During aging brain cells are subjected to elevated levels of oxidative and metabolic stress which may perturb cellular calcium homeostasis in ways that render neurons vulnerable to synaptic dysfunction, degeneration and cell death. ► In Alzheimer’s disease, aberrant proteolytic processing of the β-amyloid precursor protein results in excessive production and self-aggregation of the amyloid β-peptide which can generate reactive oxygen species resulting in impairment of membrane ion-motive ATPases and glutamate and glucose transporters thereby contributing to cellular calcium overload and energy failure. ► Mutations in presenilin-1 that cause many cases of early-onset inherited Alzheimer’s disease alter calcium regulation in the endoplasmic reticulum, which may contribute to synaptic damage and neuronal degeneration. ► By activating adaptive cellular stress response pathways involving neurotrophic factors, protein chaperones and antioxidant systems, exercise, dietary energy restriction and cognitive enrichment may stabilize cellular calcium homeostasis and protect the brain against aging and Alzheimer’s disease. ► Pharmacological interventions that protect neurons against cellular calcium overload may prove beneficial in delaying the onset and slowing the progression of cognitive impairment in aging and Alzheimer’s disease.
Keywords: Amyloid; Calcium channels; Cognitive impairment; Endoplasmic reticulum; GLP-1; Mitochondria;

Local signals with global impacts and clinical implications: Lessons from the plasma membrane calcium pump (PMCA4) by Delvac Oceandy; Tamer M.A. Mohamed; Elizabeth J. Cartwright; Ludwig Neyses (974-978).
Calcium has been unequivocally regarded as a key signal messenger in almost every cell type. Calcium regulates a number of important cellular functions including cell growth, myofilament contraction, cell survival and apoptosis as well as gene transcription. A complex regulatory mechanism of cellular calcium is needed to fine tune the precise calcium concentration in each subcellular location and also to transmit the signals carried by the calcium pool to the correct end target. In this article we will review the recently emerging role of the plasma membrane calcium/calmodulin dependent ATPase isoform 4 (PMCA4) in regulating calcium signalling. We will then focus on the function of this molecule in cardiomyocytes, in which PMCA4 forms protein–protein interactions with several key signalling molecules. Recent evidence has shown in vivo physiological functionalities and possible clinical implications of the PMCA4 signalling complex. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► PMCA4 plays an important role in the regulation of intracellular calcium signalling. ► Protein interactions are key during calcium signal transmission. ► The PMCA4 signalling complex has physiological relevance and clinical implications.
Keywords: Calcium; Signalling; PMCA; Interaction; Hypertrophy; Contractility;

Calcium signaling in osteoclasts by Sung-Yong Hwang; James W. Putney (979-983).
It has long been known that many bone diseases, including osteoporosis, involve abnormalities in osteoclastic bone resorption. As a result, there has been intense study of the mechanisms that regulate both the differentiation and bone resorbing function of osteoclast cells. Calcium (Ca2+) signaling appears to play a critical role in the differentiation and functions of osteoclasts. Cytoplasmic Ca2+ oscillations occur during RANKL-mediated osteoclastogenesis. Ca2+ oscillations provide a digital Ca2+ signal that induces osteoclasts to up-regulate and autoamplify nuclear factor of activated T cells c1 (NFATc1), a Ca2+/calcineurin-dependent master regulator of osteoclastogenesis. Here we review previous studies on Ca2+ signaling in osteoclasts as well as recent breakthroughs in understanding the basis of RANKL-induced Ca2+ oscillations, and we discuss possible molecular players in this specialized Ca2+ response that appears pivotal for normal bone function. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► Calcium signaling plays a significant role in the process of osteoclastogenesis. ► The RANKL receptor utilizes calcium signaling and activation of NFAT to drive differentiation of osteoclasts. ► Recent studies demonstrate that a key component of osteoclast calcium signaling involves influx through plasma membrane channels, including members of the TRP channel superfamily, as well as store-operated channels.
Keywords: Osteoclasts; Calcium signaling; Calcium channels; Bone;

Ca2+ sources for the exocytotic release of glutamate from astrocytes by Vladimir Parpura; Vladimir Grubišić; Alexei Verkhratsky (984-991).
Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca2+ concentration is necessary and sufficient for this process. The predominant source of Ca2+ for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca2+ to the cytosol. The ER store is (re)filled by the store-specific Ca2+-ATPase. Ultimately, the depleted ER is replenished by Ca2+ which enters from the extracellular space to the cytosol via store-operated Ca2+ entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca2+ channels and plasma membrane Na+/Ca2+ exchangers are additional means for cytosolic Ca2+ entry. Cytosolic Ca2+ levels can be modulated by mitochondria, which can take up cytosolic Ca2+ via the Ca2+ uniporter and release Ca2+ into cytosol via the mitochondrial Na+/Ca2+ exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca2+ sources generates cytosolic Ca2+ dynamics that can drive Ca2+-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.►Astrocytes can exocytotically release the gliotransmitter glutamate. ►Various Ca2+ sources contribute to cytosolic Ca2+ dynamics that can drive exocytosis. ►The ER is the main source of Ca2+. ►There is also some Ca2+ entry from the extracellular space. ►Cytosolic Ca2+ levels can be modulated by mitochondria.
Keywords: Ca2+ signaling; Endoplasmic reticulum; Ca2+ channels; Ionotropic receptors; Store-operated Ca2+ entry; Mitochondria;

Ionotropic receptors in neuronal–astroglial signalling: What is the role of “excitable” molecules in non-excitable cells by Ulyana Lalo; Yuriy Pankratov; Vladimir Parpura; Alexei Verkhratsky (992-1002).
Astroglial cells were long considered to serve merely as the structural and metabolic supporting cast and scenery against which the shining neurones perform their illustrious duties. Relatively recent evidence, however, indicates that astrocytes are intimately involved in many of the brain's functions. Astrocytes possess a diverse assortment of ionotropic transmitter receptors, which enable these glial cells to respond to many of the same signals that act on neurones. Ionotropic receptors mediate neurone-driven signals to astroglial cells in various brain areas including neocortex, hippocampus and cerebellum. Activation of ionotropic receptors trigger rapid signalling events in astroglia; these events, represented by local Ca2+ or Na+ signals provide the mechanism for fast neuronal–glial signalling at the synaptic level. Since astrocytes can detect chemical transmitters that are released from neurones and can release their own extracellular signals, gliotransmitters, they are intricately involved in homocellular and heterocellular signalling mechanisms in the nervous system. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► Astrocytes possess a diverse assortment of ionotropic transmitter receptors. ► Using receptors astrocytes can detect transmitters released from neurones. ► Astrocytes can release their own gliotransmitters.
Keywords: Astrocyte; Glutamate; ATP; NMDA receptor; P2X purinoceptor; Calcium; Sodium; Synapse;

The IP3 receptor–mitochondria connection in apoptosis and autophagy by Jean-Paul Decuypere; Giovanni Monaco; Geert Bultynck; Ludwig Missiaen; Humbert De Smedt; Jan B. Parys (1003-1013).
The amount of Ca2+ taken up in the mitochondrial matrix is a crucial determinant of cell fate; it plays a decisive role in the choice of the cell between life and death. The Ca2+ ions mainly originate from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores of the endoplasmic reticulum (ER). The uptake of these Ca2+ ions in the mitochondria depends on the functional properties and the subcellular localization of the IP3 receptor (IP3R) in discrete domains near the mitochondria. To allow for an efficient transfer of the Ca2+ ions from the ER to the mitochondria, structural interactions between IP3Rs and mitochondria are needed. This review will focus on the key proteins involved in these interactions, how they are regulated, and what are their physiological roles in apoptosis, necrosis and autophagy. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► The Ca2+ in the mitochondria is crucial for cell fate. ► The localization of the IP3 receptors determines the Ca2+ uptake in the mitochondria. ► IP3 receptor function and localization is regulated by protein–protein interactions. ► IP3 receptors interact with mitochondrial Ca2+ uptake sites. ► The strength of the interactions affect the occurrence of apoptosis or autophagy.
Keywords: IP3 receptor; Ca2+ signaling; Mitochondria; Apoptosis; Autophagy; Endoplasmic reticulum;

Microglial calcium signal acts as a rapid sensor of single neuron damage in vivo by Gerhard Eichhoff; Bianca Brawek; Olga Garaschuk (1014-1024).
In the healthy adult brain microglia, the main immune-competent cells of the CNS, have a distinct (so-called resting or surveying) phenotype. Resting microglia can only be studied in vivo since any isolation of brain tissue inevitably triggers microglial activation. Here we used in vivo two-photon imaging to obtain a first insight into Ca2+ signaling in resting cortical microglia. The majority (80%) of microglial cells showed no spontaneous Ca2+ transients at rest and in conditions of strong neuronal activity. However, they reliably responded with large, generalized Ca2+ transients to damage of an individual neuron. These damage-induced responses had a short latency (0.4–4 s) and were localized to the immediate vicinity of the damaged neuron (< 50 μm cell body-to-cell body distance). They were occluded by the application of ATPγS as well as UDP and 2-MeSADP, the agonists of metabotropic P2Y receptors, and they required Ca2+ release from the intracellular Ca2+ stores. Thus, our in vivo data suggest that microglial Ca2+ signals occur mostly under pathological conditions and identify a Ca2+ store-operated signal, which represents a very sensitive, rapid, and highly localized response of microglial cells to brain damage. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► In vivo microglia rarely show Ca2+ transients at rest but reliably respond with rapid Ca2+ signals to damage of an individual neuron in their vicinity. ► Damage-induced Ca2+ transients (DICTs) have large amplitudes and are strictly localized to microglia dwelling within a 50-μm vicinity of a damaged neuron. ► DICTs require Ca2+ release from intracellular stores likely caused by an activation of metabotropic P2Y receptors.
Keywords: Two-photon microscopy; Calcium imaging; Resting microglia; ATP; Isolectin B4; CX 3 CR1;

Calmyrin1 binds to SCG10 protein (stathmin2) to modulate neurite outgrowth by Adam Sobczak; Katarzyna Debowska; Magdalena Blazejczyk; Michael R. Kreutz; Jacek Kuznicki; Urszula Wojda (1025-1037).
Calmyrin1 (CaMy1) is an EF-hand Ca2+-binding protein expressed in several cell types, including brain neurons. Using a yeast two-hybrid screen of a human fetal brain cDNA library, we identified SCG10 protein (stathmin2) as a CaMy1 partner. SCG10 is a microtubule-destabilizing factor involved in neuronal growth during brain development. We found increased mRNA and protein levels of CaMy1 during neuronal development, which paralleled the changes in SCG10 levels. In developing primary rat hippocampal neurons in culture, CaMy1 and SCG10 colocalized in cell soma, neurites, and growth cones. Pull-down, coimmunoprecipitation, and proximity ligation assays demonstrated that the interaction between CaMy1 and SCG10 is direct and Ca2+-dependent in vivo and requires the C-terminal domain of CaMy1 (residues 99–192) and the N-terminal domain of SCG10 (residues 1–35). CaMy1 did not interact with stathmin1, a protein that is homologous with SCG10 but lacks the N-terminal domain characteristic of SCG10. CaMy1 interfered with SCG10 inhibitory activity in a microtubule polymerization assay. Moreover, CaMy1 overexpression inhibited SCG10-mediated neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. This CaMy1 activity did not occur when an N-terminally truncated SCG10 mutant unable to interact with CaMy1 was expressed. Altogether, these data suggest that CaMy1 via SCG10 couples Ca2+ signals with the dynamics of microtubules during neuronal outgrowth in the developing brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► CaMy1 directly interacts with human brain SCG10 in Ca2+-dependent manner in vivo. ► CaMy1/SCG10 binding involves CaMy1 C-terminal domain and N-terminal SCG10 fragment. ► CaMy1 and SCG10 protein levels increase during early neural development. ► CaMy1 co-localizes with SCG10 in developing neurons. ► CaMy1 modulates SCG10-related neurite outgrowth and microtubule dynamics.
Keywords: Calmyrin; Calcium and integrin binding protein 1; SCG10; Calcium signaling; Microtubule; Neuronal development;

Weibel–Palade bodies (WPBs) are secretory organelles of endothelial cells that store the thrombogenic glycoprotein von Willebrand factor (vWF). Endothelial activation, e.g. by histamine and thrombin, triggers the Ca2+-dependent exocytosis of WPB that releases vWF into the vasculature and thereby initiates platelet capture and thrombus formation. Towards understanding the molecular mechanisms underlying this regulated WPB exocytosis, we here identify components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery associated with WPB. We show that vesicle-associated membrane protein (VAMP) 3 and VAMP8 are present on WPB and that VAMP3, but not VAMP8 forms a stable complex with syntaxin 4 and SNAP23, two plasma membrane-associated SNAREs in endothelial cells. By introducing mutant SNARE proteins into permeabilized endothelial cells we also show that soluble VAMP3 but not VAMP8 mutants comprising the cytoplasmic domain interfere with efficient vWF secretion. This indicates that endothelial cells specifically select VAMP 3 over VAMP8 to cooperate with syntaxin 4 and SNAP23 in the Ca2+-triggered fusion of WPB with the plasma membrane. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.►Endothelial VAMP3 and VAMP8 reside on WPB. ►VAMP3 forms a stable complex with syntaxin 4 and SNAP23 in endothelial cells. ►A cytosolic VAMP3-mutant interferes with regulated vWF secretion.
Keywords: Histamine; NSF; SNARE; Streptolysin O; vWF secretion;

Stress induced subcellular distribution of ALG-2, RBM22 and hSlu7 by Aleksandra Janowicz; Marek Michalak; Joachim Krebs (1045-1049).
ALG-2 is a highly conserved calcium binding protein in the cytoplasm which belongs to the family of penta-EF hand proteins. Recently, we showed that ALG-2 is interacting with RBM22, a highly conserved spliceosomal nuclear protein (Montaville et al. Biochim. Biophys. Acta 1763, 1335, 2006; Krebs, Biochim. Biophys. Acta 1793, 979, 2009). In NIH 3T3 cells expressing both proteins a significant amount of ALG-2mRFP is translocated to the nucleus due to the interaction with RBM22-EGFP. hSlu7, another spliceosomal nuclear protein, known to interact with RBM22 in yeast, has been shown to translocate to the cytoplasm under cellular stress conditions. Here we provide evidence that the 2 spliceosomal proteins differ significantly in their subcellular distributions under stress conditions, and that RBM22 enhances the cytoplasmic translocation of hSlu7 under stress, especially a stress induced by thapsigargin. On the other hand, in NIH 3T3 cells expressing RBM22-EGFP and ALG-2-mRFP, ALG-2 remains translocated into the nucleus under both stress conditions, i.e. heat shock or treatment with thapsigargin. We could further demonstrate that these stress conditions had a different influence on the splicing pattern of XBP-1, a marker for the unfolded protein response indicating that ER stress may play a role in stress-induced translocation of spliceosomal proteins. The article is part of a Special Issue entitled: 11th European Symposium on Calcium.► Influence of different stress conditions on the subcellular localization of ALG-2, RBM22, hSlu7. ► Different response of the spliceosomal proteins RBM22 and hSlu7 on cellular stress. ► Differences in the splicing pattern of XBP-1, a marker for the unfolded protein response, influenced by RBM22 and/or ALG-2 during ER stress.
Keywords: ALG-2; RBM22; hSlu7; ER stress response; Subcellular localization;

Sumoylation regulates nuclear localization of repressor DREAM by Malgorzata Palczewska; Iñigo Casafont; Kedar Ghimire; Ana M. Rojas; Alfonso Valencia; Miguel Lafarga; Britt Mellström; Jose R. Naranjo (1050-1058).
DREAM is a Ca2+-binding protein with specific functions in different cell compartments. In the nucleus, DREAM acts as a transcriptional repressor, although the mechanism that controls its nuclear localization is unknown. Yeast two-hybrid assay revealed the interaction between DREAM and the SUMO-conjugating enzyme Ubc9 and bioinformatic analysis identified four sumoylation-susceptible sites in the DREAM sequence. Single K-to-R mutations at positions K26 and K90 prevented in vitro sumoylation of recombinant DREAM. DREAM sumoylation mutants retained the ability to bind to the DRE sequence but showed reduced nuclear localization and failed to regulate DRE-dependent transcription. In PC12 cells, sumoylated DREAM is present exclusively in the nucleus and neuronal differentiation induced nuclear accumulation of sumoylated DREAM. In fully differentiated trigeminal neurons, DREAM and SUMO-1 colocalized in nuclear domains associated with transcription. Our results show that sumoylation regulates the nuclear localization of DREAM in differentiated neurons. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► DREAM is sumoylated at specific residues. ► In PC12, sumoylated DREAM is present exclusively in the nucleus. ► Neuronal differentiation increases the amount of sumoylated DREAM in the nucleus.
Keywords: DREAM; KChIPs; Nuclear localization; SUMO; Ubc9; Trigeminal neuron;

A general strategy to characterize calmodulin–calcium complexes involved in CaM–target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin–calcium complexes by Rania Dagher; Shan Peng; Sophie Gioria; Marie Fève; Maria Zeniou; Michael Zimmermann; Claire Pigault; Jacques Haiech; Marie-Claude Kilhoffer (1059-1067).
Calmodulin (CaM) is a ubiquitous Ca2+ sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four “EF hand” type Ca2+ binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca2+ ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM–Can (n = 0–4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► CaM interaction with its target peptides depends on the number of Ca2+ bound. ► CaM-Ca interacts with the unphosphorylated DAPK CaM binding domain. ► CaM-Ca3 interacts with the phosphorylated DAPK CaM binding domain peptide. ► CaM with four Ca2+ is required for interaction with the EGFR CaM binding domain.
Keywords: Calmodulin; Death associated protein kinase; EGFR; Fluorescence polarization; CaM binding domain;

Site-directed mutagenesis of the glycine-rich loop of death associated protein kinase (DAPK) identifies it as a key structure for catalytic activity by Laurie K. McNamara; Joseph S. Brunzelle; James P. Schavocky; D. Martin Watterson; Valerie Grum-Tokars (1068-1073).
Death associated protein kinase (DAPK) is a calmodulin (CaM)-regulated protein kinase that is a therapeutic target for central nervous system (CNS) disorders. We report here the results of studies that test the hypothesis of McNamara et al. (2009) that conformational selection in DAPK's glycine-rich region is key for catalytic activity. The hypothesis was tested by site-directed mutagenesis of glutamine-23 (Q23) in the middle of this loop. The glycine-rich loop exhibits localized differences in structure among DAPK conformations that correlate with different stages of the catalytic cycle. Changing the Q23 to a Valine (V23), found at the corresponding position in another CaM regulated protein kinase, results in a reduced catalytic efficiency. High resolution X-ray crystal structures of various conformations of the Q23V mutant DAPK and their superimposition with the corresponding conformations from wild type catalytic domain reveal localized changes in the glycine-rich region. The effect of the mutation on DAPK catalytic activity and the finding of only localized changes in the DAPK structure provide experimental evidence implicating conformational selection in this domain with activity. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► Glutamine-23 in Death associated protein kinase altered by point mutation. ► X-ray structures reveal localized changes in glycine-rich region. ► Reduced catalytic efficiency observed relative to wild type. > First reported structures with correlating activity of DAPK mutants.
Keywords: Death associated protein kinase; Calmodulin; Glycine-rich region; Neurodegeneration; Central nervous system; Phosphorylation; Protein kinase inhibitor;

Calmodulin binds HER2 and modulates HER2 signaling by Colin D. White; Zhigang Li; David B. Sacks (1074-1082).
Human epidermal growth factor receptor 2 (HER2), a member of the ErbB family of receptor tyrosine kinases, has defined roles in neoplastic transformation and tumor progression. Overexpression of HER2 is an adverse prognostic factor in several human neoplasms and, particularly in breast cancer, correlates strongly with a decrease in overall patient survival. HER2 stimulates breast tumorigenesis by forming protein–protein interactions with a diverse array of intracellular signaling molecules, and evidence suggests that manipulation of these associations holds therapeutic potential. To modulate specific HER2 interactions, the region(s) of HER2 to which each target binds must be accurately identified. Calmodulin (CaM), a ubiquitously expressed Ca2+ binding protein, interacts with multiple intracellular targets. Interestingly, CaM binds the juxtamembrane region of the epidermal growth factor receptor, a HER2 homolog. Here, we show that CaM interacts, in a Ca2+-regulated manner, with two distinct sites on the N-terminal portion of the HER2 intracellular domain. Deletion of residues 676–689 and 714–732 from HER2 prevented CaM–HER2 binding. Inhibition of CaM function or deletion of the CaM binding sites from HER2 significantly decreased both HER2 phosphorylation and HER2-stimulated cell growth. Collectively, these data suggest that inhibition of CaM–HER2 interaction may represent a rational therapeutic strategy for the treatment of patients with breast cancer. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► Human epidermal growth factor receptor 2 (HER2) stimulates tumorigenesis in human breast neoplasms by forming protein-protein interactions with a diverse array of intracellular signaling molecules. Manipulation of the associations between HER2 and selected binding partners therefore holds potential for anti-cancer therapy. To modulate specific HER2 binding interactions, the region(s) on HER2 to which each protein binds must be accurately identified. ► Here, we show that calmodulin binds, in a Ca2+-regulated manner, to two distinct sites on the N-terminal portion of the HER2 intracellular domain. Deletion of residues 676–689 and 714–732 from HER2 prevented calmodulin binding. Inhibition of calmodulin function or disruption of the association between HER2 and calmodulin significantly decreased both HER2 phosphorylation and HER2-stimulated cell growth. ► These data suggest that inhibition calmodulin-HER2 binding may represent a rational therapeutic strategy for the treatment of patients with breast cancer.
Keywords: Breast cancer; Calmodulin; HER2;

The crystal structures of human S100B in the zinc- and calcium-loaded state at three pH values reveal zinc ligand swapping by Thorsten Ostendorp; Joachim Diez; Claus W. Heizmann; Günter Fritz (1083-1091).
S100B is a homodimeric zinc-, copper-, and calcium-binding protein of the family of EF-hand S100 proteins. Zn2+ binding to S100B increases its affinity towards Ca2+ as well as towards target peptides and proteins. Cu2+ and Zn2+ bind presumably to the same site in S100B. We determined the structures of human Zn2+- and Ca2+-loaded S100B at pH 6.5, pH 9, and pH 10 by X-ray crystallography at 1.5, 1.4, and 1.65 Å resolution, respectively. Two Zn2+ ions are coordinated tetrahedrally at the dimer interface by His and Glu residues from both subunits. The crystal structures revealed that ligand swapping occurs for one of the four ligands in the Zn2+-binding sites. Whereas at pH 9, the Zn2+ ions are coordinated by His15, His25, His 85′, and His 90′, at pH 6.5 and pH 10, His90′ is replaced by Glu89′. The results document that the Zn2+-binding sites are flexible to accommodate other metal ions such as Cu2+. Moreover, we characterized the structural changes upon Zn2+ binding, which might lead to increased affinity towards Ca2+ as well as towards target proteins. We observed that in Zn2+–Ca2+-loaded S100B the C-termini of helix IV adopt a distinct conformation. Zn2+ binding induces a repositioning of residues Phe87 and Phe88, which are involved in target protein binding. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► Crystal structure of Ca2+ and Zn2+ bound human S100B. ► Structure determined at three different pH values. ► Ligand swapping for Zn2+, flexible Zn2+-binding site. ► Stabilization of C-terminus promotes target protein binding.
Keywords: S100 protein; S100B; EF-hand; Crystallography; Calcium; Zinc; Copper;

S100B protein activates IKKβ/NF-κB within myoblasts, thereby inhibiting the expression of MyoD and the MyoD-downstream effectors, myogenin and p21WAF1, and myoblast differentiation. Herein we show that myoblasts downregulate S100B expression once transferred from proliferation medium to differentiation medium via a p38 MAPK-driven transcriptional mechanism as well as a post-translational, proteasome-dependent mechanism, and that myoblasts that have not been committed to differentiation resume expressing S100B once transferred back to proliferation medium. Likewise, myoblasts downregulate S100B expression once transferred to quiescence medium, and interference with S100B downregulation as obtained by stable overexpression of the protein results in reduced acquisition of quiescence and a faster proliferation upon transfer of the cells from quiescence medium to proliferation medium, compared to controls. These latter effects are dependent on S100B-induced activation of JNK. Moreover, S100B reduces myoblast apoptosis in an MEK-ERK1/2, Akt, JNK, and NF-κB-dependent manner. However, myogenin+ myoblasts (i.e., myocytes) and myotubes abundantly express S100B likely induced by myogenin. Our results suggest that (1) a timely repression of S100B expression is required for efficient myogenic differentiation; (2) S100B plays an important role in the expansion of the activated (i.e., proliferating) myoblast population; (3) under conditions associated with enhanced expression of S100B, the transition from proliferation to quiescence and from quiescence to proliferation might be altered; and (4) S100B exerts different regulatory effects in myoblasts and myocytes/myotubes/myofibers. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► Differentiating myoblasts downregulate S100B expression via p38 MAPK. ► Serum mitogens upregulate S100B expression in myoblasts. ► Myoblasts induced to quiescence downregulate S100B expression. ► Downregulation of S100B is required for efficient myoblast differentiation. ► Downregulation of S100B is required for myoblast to attain quiescence in vitro.
Keywords: S100B; Myoblast; Differentiation; Quiescence;

Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membrane glycoprotein that localizes to myelin sheaths in the central nervous system. MOG has important implications in multiple sclerosis, as pathogenic anti-MOG antibodies have been detected in the sera of multiple sclerosis patients. As a membrane protein, MOG achieves its native structure in the endoplasmic reticulum where its folding is expected to be controlled by endoplasmic reticulum chaperones. Calnexin, calreticulin, and ERp57 are essential components of the endoplasmic reticulum quality control where they assist in the proper folding of newly synthesized glycoproteins. In this study, we show that expression of MOG is not affected by the absence of the endoplasmic reticulum quality control proteins calnexin, calreticulin, or ERp57. We also show that calnexin forms complexes with MOG and these interactions might be glycan-independent. Importantly, we show that cell surface targeting of MOG is not disrupted in the absence of the endoplasmic reticulum chaperones. This article is part of a special issue entitled: 11th European Symposium on Calcium.► Myelin oligodendrocyte glycoprotein (MOG) has important implication in multiple sclerosis, our data show that calnexin, an endoplasmic reticulum chaperone, forms complexes with MOG, suggesting that calnexin is a potential chaperone for this protein. ► Importantly we discover that cell surface trafficking of MOG in not disrupted in the absence of essential endoplasmic reticulum chaperones. ► Our results provide new insights into folding and processing of MOG—an important antigen involved in multiple sclerosis.
Keywords: Endoplasmic reticulum; Quality control; Chaperone; Calnexin; Calreticulin; ERp57;

Darier disease : A disease model of impaired calcium homeostasis in the skin by Magali Savignac; Anissa Edir; Marina Simon; Alain Hovnanian (1111-1117).
The importance of extracellular calcium in epidermal differentiation and intra-epidermal cohesion has been recognized for many years. Darier disease (DD) was the first genetic skin disease caused by abnormal epidermal calcium homeostasis to be identified. DD is characterized by loss of cell-to-cell adhesion and abnormal keratinization. DD is caused by genetic defects in ATP2A2 encoding the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2 (SERCA2). SERCA2 is a calcium pump of the endoplasmic reticulum (ER) transporting Ca2+ from the cytosol to the lumen of ER. ATP2A2 mutations lead to loss of Ca2+ transport by SERCA2 resulting in decreased ER Ca2+ concentration in Darier keratinocytes. Here, we review the role of SERCA2 pumps and calcium in normal epidermis, and we discuss the consequences of ATP2A2 mutations on Ca2+ signaling in DD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► Mutations in ATP2A2 encoding the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2 (SERCA2). ► Loss of Ca2+ transport from the cytosol to the lumen of the ER. ► Depleted ER Ca2+ stores ► Loss of cell-to-cell adhesion and abnormal keratinisation.
Keywords: SERCA2; Darier; Calcium; Keratinocytes; Adhesion; Differentiation;

Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases by Ilse Vandecaetsbeek; Søren Brøgger Christensen; Huizhen Liu; Paul P. Van Veldhoven; Etienne Waelkens; Jan Eggermont; Luc Raeymaekers; Jesper V. Møller; Poul Nissen; Frank Wuytack; Peter Vangheluwe (1118-1127).
The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca2+ ATPase (SERCA2b) and secretory-pathway Ca2+ ATPase (SPCA1a) belong both to the P2A-type ATPase subgroup of Ca2+ transporters and play a crucial role in the Ca2+ homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated carrying minor modifications in the Tg-binding site showing a lower affinity for Tg. In a second step, reactive dye chromatography was performed to further purify and concentrate the properly folded pumps and to exchange the detergent to one more suitable for crystallization. A similar strategy was successfully applied to purify WT SPCA1a. This study shows that it is possible to purify functionally active intracellular Ca2+ ATPases using successive thapsigargin and reactive dye affinity chromatography for future structural studies. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.► The ubiquitous Ca2+ ATPases SERCA2b and SPCA1a so far evaded purification. ► A new purification strategy was developed biasing towards properly folded protein. ► This method uses thapsigargin and reactive dye as ligands. ► Elution of WT hSERCA2b was hampered by its nM affinity for thapsigargin. ► Thapsigargin mutant hSERCA2b-I765A could be easily purified.
Keywords: SERCA2b; SPCA1a; Ca2+ pumps; Endoplasmic reticulum; Golgi; Chromatography;