Forchlorfenuron and Novel Analogs Cause Cytotoxic Effects in Untreated and Cisplatin-Resistant Malignant Mesothelioma-Derived Cells.

Malignant mesothelioma (MM) is a currently incurable, aggressive cancer derived from mesothelial cells, most often resulting from asbestos exposure. The current first-line treatment in unresectable MM is cisplatin/pemetrexed, which shows very little long-term effectiveness, necessitating research for novel therapeutic interventions. The existing chemotherapies often act on the cytoskeleton, including actin filaments and microtubules, but recent advances indicate the 'fourth' form consisting of the family of septins, representing a novel target. The septin inhibitor forchlorfenuron (FCF) and FCF analogs inhibit MM cell growth in vitro, but at concentrations which are too high for clinical applications. Based on the reported requirement of the chloride group in the 2-position of the pyridine ring of FCF for MM cell growth inhibition and cytotoxicity, we systematically investigated the importance (cell growth-inhibiting capacity) of the halogen atoms fluorine, chlorine, bromine and iodine in the 2- or 3-position of the pyridine ring. The MM cell lines ZL55, MSTO-211H, and SPC212, and-as a control-immortalized Met-5A mesothelial cells were used. The potency of the various halogen substitutions in FCF was mostly correlated with the atom size (covalent radius); the small fluoride analogs showed the least effect, while the largest one (iodide) most strongly decreased the MTT signals, in particular in MM cells derived from epithelioid MM. In the latter, the strongest effects in vitro were exerted by the 2-iodo and, unexpectedly, the 2-trifluoromethyl (2-CF3) FCF analogs, which were further tested in vivo in mice. However, FCF-2-I and, more strongly, FCF-2-CF3 caused rapidly occurring strong symptoms of systemic toxicity at doses lower than those previously obtained with FCF. Thus, we investigated the effectiveness of FCF (and selected analogs) in vitro in MM cells which were first exposed to cisplatin. The slowly appearing population of cisplatin-resistant cells was still susceptible to the growth-inhibiting/cytotoxic effect of FCF and its analogs, indicating that cisplatin and FCF target non-converging pathways in MM cells. Thus, a combination therapy of cisplatin and FCF (analogs) might represent a new avenue for the treatment of repopulating chemo-resistant MM cells in this currently untreatable cancer.

Amyloid pathology-produced unexpected modifications of calcium homeostasis in hippocampal subicular dendrites.

INTRODUCTION: Alzheimer's disease (AD) is linked to neuronal calcium dyshomeostasis, which is associated with network hyperexcitability. Decreased expression of the calcium-binding protein cal- bindin-D28K (CB) might be a susceptibility factor for AD. The subiculum is affected early in AD, for unknown reasons. METHODS: In AD, CB knock-out and control mice fluorescence Ca2+ imaging combined with patch clamp were used to characterize Ca2+ dynamics, resting Ca2+ , and Ca2+ -buffering capacity in subicular neurons. CB expression levels in wild-type and AD mice were also analyzed. RESULTS: The subiculum and dentate gyrus of wild-type mice showed age-related decline in CB expression not observed in AD mice. Resting Ca2+ and Ca2+ -buffering capacity was increased in aged AD mice subicular dendrites. Modeling suggests that AD calcium changes can be explained by alterations of Ca2+ extrusion pumps rather than by buffers. DISCUSSION: Overall, abnormal Ca2+ homeostasis in AD has an age dependency that comprises multiple mechanisms, including compensatory processes.

Biological noise and positional effects influence cell stemness.

Biological (or cellular) noise is the random quantitative variability of proteins and other molecules in individual, genetically identical cells. As the result of biological noise in the levels of some transcription factors that determine a cell's differentiation status, differentiated cells may dedifferentiate to a stem cell state given a sufficiently long time period. Here, to provide direct evidence supporting this hypothesis, we used a live-cell monitoring system based on enhanced green fluorescent protein (eGFP) expression to continuously assess the "stemness" of individual human and murine malignant mesothelioma cells over a period of up to 3 months. Re-expression of the transcription factors, the top hierarchical stemness markers Sox2 (SRY-box 2) and Oct4 (octamer-binding transcription factor), monitored as cell eGFP expression was observed in a subpopulation of differentiated eGFP(-) malignant mesothelioma cells. However, we found that this transition was extremely rare. Of note, when it did occur, neighboring cells that were not direct descendants of a newly emerged eGFP(+) stem cell were more likely than non-neighboring cells to also become an eGFP(+) stem cell. This observation suggested a positional effect and led to a clustered "mosaic" reappearance of eGFP(+) stem cells. Moreover, stem cells reappeared even in cell cultures derived from one single differentiated eGFP(-) cell. On the basis of our experimental in vitro and in vivo findings, we developed a tumor growth model to predict the clustered localization of cancer stem cells within a tumor mass.

Inducible and reversible silencing of the Pvalb gene in mice: An in vitro and in vivo study.

Inducible and reversible regulation of gene expression is a powerful approach for unraveling gene functions. Here, we describe the generation of a system to efficiently downregulate in a reversible and inducible manner the Pvalb gene coding for the calcium-binding protein parvalbumin (PV) in mice. We made use of an IPTG-inducible short hairpin RNA to activate Pvalb transcript knockdown and subsequently downregulate PV. The downregulation was rapidly reversed after withdrawal of IPTG. In vitro and in vivo experiments revealed a decrease in PV expression of ≥50% in the presence of IPTG and full reversibility after IPTG removal. We foresee that the tightly regulated and reversible PV downregulation in mice in vivo will provide a new tool for the control of Pvalb transcript expression in a temporal manner. Because PV protein and PVALB transcript levels were found to be lower in the brain of patients with autism spectrum disorder and schizophrenia, the novel transgenic mouse line might serve as a model to investigate the putative role of PV in these neurodevelopmental disorders.

Extending the Mathematical Palette for Developmental Pattern Formation: Piebaldism.

Here, we present a theoretical investigation with potential insights on developmental mechanisms. Three biological factors, consisting of two diffusing factors and a cell-autonomous immobile transcription factor are combined with different feedback mechanisms. This results in four different situations or fur patterns. Two of them reproduce classical Turing patterns: (1) regularly spaced spots, (2) labyrinth patterns or straight lines with an initial slope in the activation of the transcription factor. The third situation does not lead to patterns, but results in different homogeneous color tones. Finally, the fourth one sheds new light on the possible mechanisms leading to the formation of piebald patterns exemplified by the random patterns on the fur of some cows' strains and Dalmatian dogs. Piebaldism is usually manifested as white areas of fur, hair, or skin due to the absence of pigment-producing cells in those regions. The distribution of the white and colored zones does not reflect the classical Turing patterns. We demonstrate that these piebald patterns are of transient nature, developing from random initial conditions and relying on a system's bistability. We show numerically that the presence of a cell-autonomous factor not only expands the range of reaction diffusion parameters in which a pattern may arise, but also extends the pattern-forming abilities of the reaction-diffusion equations.

Parvalbumin alters mitochondrial dynamics and affects cell morphology.

The Ca2+-binding protein parvalbumin (PV) and mitochondria play important roles in Ca2+ signaling, buffering and sequestration. Antagonistic regulation of PV and mitochondrial volume is observed in in vitro and in vivo model systems. Changes in mitochondrial morphology, mitochondrial volume and dynamics (fusion, fission, mitophagy) resulting from modulation of PV were investigated in MDCK epithelial cells with stable overexpression/downregulation of PV. Increased PV levels resulted in smaller, roundish cells and shorter mitochondria, the latter phenomenon related to reduced fusion rates and decreased expression of genes involved in mitochondrial fusion. PV-overexpressing cells displayed increased mitophagy, a likely cause for the decreased mitochondrial volumes and the smaller overall cell size. Cells showed lower mobility in vitro, paralleled by reduced protrusions. Constitutive PV down-regulation in PV-overexpressing cells reverted mitochondrial morphology and fractional volume to the state present in control MDCK cells, resulting from increased mitochondrial movement and augmented fusion rates. PV-modulated, bi-directional and reversible mitochondrial dynamics are key to regulation of mitochondrial volume.

Modulation of Calretinin Expression in Human Mesothelioma Cells Reveals the Implication of the FAK and Wnt Signaling Pathways in Conferring Chemoresistance towards Cisplatin.

Malignant mesothelioma (MM) is an aggressive asbestos-linked neoplasm, characterized by dysregulation of signaling pathways. Due to intrinsic or acquired chemoresistance, MM treatment options remain limited. Calretinin is a Ca2+-binding protein expressed during MM tumorigenesis that activates the FAK signaling pathway, promoting invasion and epithelial-to-mesenchymal transition. Constitutive calretinin downregulation decreases MM cells' growth and survival, and impairs tumor formation in vivo. In order to evaluate early molecular events occurring during calretinin downregulation, we generated a tightly controlled IPTG-inducible expression system to modulate calretinin levels in vitro. Calretinin downregulation significantly reduced viability and proliferation of MM cells, attenuated FAK signaling and reduced the invasive phenotype of surviving cells. Importantly, surviving cells showed a higher resistance to cisplatin due to increased Wnt signaling. This resistance was abrogated by the Wnt signaling pathway inhibitor 3289-8625. In various MM cell lines and regardless of calretinin expression levels, blocking of FAK signaling activated the Wnt signaling pathway and vice versa. Thus, blocking both pathways had the strongest impact on MM cell proliferation and survival. Chemoresistance mechanisms in MM cells have resulted in a failure of single-agent therapies. Targeting of multiple components of key signaling pathways, including Wnt signaling, might be the future method-of-choice to treat MM.

Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter.

BACKGROUND: The calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood. METHODS: We searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments. RESULTS: Septin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7. CONCLUSIONS: Our results demonstrate septin 7 not only serving as a "cytoskeletal" protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.

Putative cancer stem cells may be the key target to inhibit cancer cell repopulation between the intervals of chemoradiation in murine mesothelioma.

BACKGROUND: Cancer cell repopulation during chemotherapy or radiotherapy is a major factor limiting the efficacy of treatment. Cancer stem cells (CSC) may play critical roles during this process. We aim to demonstrate the role of mesothelioma stem cells (MSC) in treatment failure and eventually to design specific target therapies against MSC to improve the efficacy of treatment in malignant mesothelioma. METHODS: Murine mesothelioma AB12 and RN5 cells were used to compare tumorigenicity in mice. The expression of CSC-associated genes was evaluated by quantitative real-time PCR in both cell lines treated with chemo-radiation. Stemness properties of MSC-enriched RN5-EOS-Puro2 cells were characterized with flow cytometry and immunostaining. A MSC-specific gene profile was screened by microarray assay and confirmed thereafter. Gene Ontology analysis of the selected genes was performed by GOMiner. RESULTS: Tumor growth delay of murine mesothelioma AB12 cells was achieved after each cycle of cisplatin treatment, however, tumors grew back rapidly due to cancer cell repopulation between courses of chemotherapy. Strikingly, a 10-times lower number of irradiated cells in both cell lines led to a similar tumor incidence and growth rate as with untreated cells. The expression of CSC-associated genes such as CD24, CD133, CD90 and uPAR was dramatically up-regulated, while others did not change significantly after chemoradiation. Highly enriched MSC after selection with puromycin displayed an increasing GFP-positive population and showed typical properties of stemness. Comparatively, the proportion of MSC significantly increased after RN5-EOS parental cells were treated with either chemotherapy, γ-ray radiation, or a combination of the two, while MSC showed more resistance to the above treatments. A group of identified genes are most likely MSC-specific, and major pathways related to regulation of cell growth or apoptosis are involved. Upregulation of the gene transcripts Tnfsf18, Serpinb9b, Ly6a, and Nppb were confirmed. CONCLUSION: Putative MSC possess the property of stemness showing more resistance to chemoradiation, suggesting that MSC may play critical roles in cancer cell repopulation. Further identification of selected genes may be used to design novel target therapies against MSC, so as to eliminate cancer cell repopulation in mesothelioma.

EF-hand protein Ca2+ buffers regulate Ca2+ influx and exocytosis in sensory hair cells.

EF-hand Ca(2+)-binding proteins are thought to shape the spatiotemporal properties of cellular Ca(2+) signaling and are prominently expressed in sensory hair cells in the ear. Here, we combined genetic disruption of parvalbumin-α, calbindin-D28k, and calretinin in mice with patch-clamp recording, in vivo physiology, and mathematical modeling to study their role in Ca(2+) signaling, exocytosis, and sound encoding at the synapses of inner hair cells (IHCs). IHCs lacking all three proteins showed excessive exocytosis during prolonged depolarizations, despite enhanced Ca(2+)-dependent inactivation of their Ca(2+) current. Exocytosis of readily releasable vesicles remained unchanged, in accordance with the estimated tight spatial coupling of Ca(2+) channels and release sites (effective "coupling distance" of 17 nm). Substitution experiments with synthetic Ca(2+) chelators indicated the presence of endogenous Ca(2+) buffers equivalent to 1 mM synthetic Ca(2+)-binding sites, approximately half of them with kinetics as fast as 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Synaptic sound encoding was largely unaltered, suggesting that excess exocytosis occurs extrasynaptically. We conclude that EF-hand Ca(2+) buffers regulate presynaptic IHC function for metabolically efficient sound coding.

Calretinin Functions in Malignant Mesothelioma Cells Cannot Be Replaced by the Closely Related Ca2+-Binding Proteins Calbindin-D28k and Parvalbumin.

Calretinin (CR; CALB2) belonging to the family of EF-hand Ca2+-binding proteins (CaBP) is widely used as a positive marker for the identification of human malignant mesothelioma (MM) and functionally was suggested to play a critical role during carcinogenesis of this highly aggressive asbestos-associated neoplasm. Increasing evidence suggests that CR not only acts as a prototypical Ca2+ buffer protein, i.e., limiting the amplitude of Ca2+ signals but also as a Ca2+ sensor. No studies have yet investigated whether other closely related CaBPs might serve as substitutes for CR's functions(s) in MM cells. Genetically modified MM cell lines with medium (MSTO-211H and ZL5) or low (SPC111) endogenous CR expression levels were generated that overexpress either CR's closest homologue calbindin-D28k (CB) or parvalbumin (PV), the latter considered as a "pure" Ca2+ buffer protein. After lentiviral shCALB2-mediated CR downregulation, in both MSTO-211H and ZL5 cells expressing CB or PV, the CR deficiency-mediated increase in cell death was not prevented by CB or PV. With respect to proliferation and cell morphology of SPC111 cells, CB was able to substitute for CR, but not for CR's other functions to promote cell migration or invasion. In conclusion, CR has a likely unique role in MM that cannot be substituted by "similar" CaBPs.

Endogenous TRPV1 stimulation leads to the activation of the inositol phospholipid pathway necessary for sustained Ca2+ oscillations.

Sensory neuron subpopulations as well as breast and prostate cancer cells express functional transient receptor potential vanilloid type 1 (TRPV1) ion channels; however little is known how TRPV1 activation leads to biological responses. Agonist-induced activation of TRPV1 resulted in specific spatiotemporal patterns of cytoplasmic Ca2+ signals in breast and prostate cancer-derived cells. Capsaicin (CAPS; 50µM) evoked intracellular Ca2+ oscillations and/or intercellular Ca2+ waves in all cell lines. As evidenced in prostate cancer Du 145 cells, oscillations were largely dependent on the expression of functional TRPV1 channels in the plasma membrane, phospholipase C activation and on the presence of extracellular Ca2+ ions. Concomitant oscillations of the mitochondrial matrix Ca2+ concentration resulted in mitochondria energization evidenced by increased ATP production. CAPS-induced Ca2+ oscillations also occurred in a subset of sensory neurons, yet already at lower CAPS concentrations (1µM). Stimulation of ectopically expressed TRPV1 channels in CAPS-insensitive NIH-3T3 cells didn't provoke CAPS-triggered Ca2+ oscillations; rather it resulted in low-magnitude, long-lasting elevations of the cytosolic Ca2+ concentration. This indicates that sole TRPV1 activation is not sufficient to generate Ca2+ oscillations. Instead the initial TRPV1-mediated signal leads to the activation of the inositol phospholipid pathway. This in turn suffices to generate a biologically relevant frequency-modulated Ca2+ signal.

Activation of endogenous TRPV1 fails to induce overstimulation-based cytotoxicity in breast and prostate cancer cells but not in pain-sensing neurons.

Vanilloids including capsaicin and resiniferatoxin are potent transient receptor potential vanilloid type 1 (TRPV1) agonists. TRPV1 overstimulation selectively ablates capsaicin-sensitive sensory neurons in animal models in vivo. The cytotoxic mechanisms are based on strong Na(+) and Ca(2+) influx via TRPV1 channels, which leads to mitochondrial Ca(2+) accumulation and necrotic cell swelling. Increased TRPV1 expression levels are also observed in breast and prostate cancer and derived cell lines. Here, we examined whether potent agonist-induced overstimulation mediated by TRPV1 might represent a means for the eradication of prostate carcinoma (PC-3, Du 145, LNCaP) and breast cancer (MCF7, MDA-MB-231, BT-474) cells in vitro. While rat sensory neurons were highly vanilloid-sensitive, normal rat prostate epithelial cells were resistant in vivo. We found TRPV1 to be expressed in all cancer cell lines at mRNA and protein levels, yet protein expression levels were significantly lower compared to sensory neurons. Treatment of all human carcinoma cell lines with capsaicin didn't lead to overstimulation cytotoxicity in vitro. We assume that the low vanilloid-sensitivity of prostate and breast cancer cells is associated with low expression levels of TRPV1, since ectopic TRPV1 expression rendered them susceptible to the cytotoxic effect of vanilloids evidenced by plateau-type Ca(2+) signals, mitochondrial Ca(2+) accumulation and Na(+)- and Ca(2+)-dependent membrane disorganization. Moreover, long-term monitoring revealed that merely the ectopic expression of TRPV1 stopped cell proliferation and often induced apoptotic processes via strong activation of caspase-3 activity. Our results indicate that specific targeting of TRPV1 function remains a putative strategy for cancer treatment.

The Effect of Gap Junctional Coupling on the Spatiotemporal Patterns of Ca2+ Signals and the Harmonization of Ca2+-Related Cellular Responses.

Calcium ions (Ca2+) are important mediators of a great variety of cellular activities e.g. in response to an agonist activation of a receptor. The magnitude of a cellular response is often encoded by frequency modulation of Ca2+ oscillations and correlated with the stimulation intensity. The stimulation intensity highly depends on the sensitivity of a cell to a certain agonist. In some cases, it is essential that neighboring cells produce a similar and synchronized response to an agonist despite their different sensitivity. In order to decipher the presumed function of Ca2+ waves spreading among connecting cells, a mathematical model was developed. This model allows to numerically modifying the connectivity probability between neighboring cells, the permeability of gap junctions and the individual sensitivity of cells to an agonist. Here, we show numerically that strong gap junctional coupling between neighbors ensures an equilibrated response to agonist stimulation via formation of Ca2+ phase waves, i.e. a less sensitive neighbor will produce the same or similar Ca2+ signal as its highly sensitive neighbor. The most sensitive cells within an ensemble are the wave initiator cells. The Ca2+ wave in the cytoplasm is driven by a sensitization wave front in the endoplasmic reticulum. The wave velocity is proportional to the cellular sensitivity and to the strength of the coupling. The waves can form different patterns including circular rings and spirals. The observed pattern depends on the strength of noise, gap junctional permeability and the connectivity probability between neighboring cells. Our simulations reveal that one highly sensitive region gradually takes the lead within the entire noisy system by generating directed circular phase waves originating from this region.

Routes of Ca2+ Shuttling during Ca2+ Oscillations: FOCUS ON THE ROLE OF MITOCHONDRIAL Ca2+ HANDLING AND CYTOSOLIC Ca2+ BUFFERS.

In some cell types, Ca(2+) oscillations are strictly dependent on Ca(2+) influx across the plasma membrane, whereas in others, oscillations also persist in the absence of Ca(2+) influx. We observed that, in primary mesothelial cells, the plasmalemmal Ca(2+) influx played a pivotal role. However, when the Ca(2+) transport across the plasma membrane by the "lanthanum insulation method" was blocked prior to the induction of the serum-induced Ca(2+) oscillations, mitochondrial Ca(2+) transport was found to be able to substitute for the plasmalemmal Ca(2+) exchange function, thus rendering the oscillations independent of extracellular Ca(2+). However, in a physiological situation, the Ca(2+)-buffering capacity of mitochondria was found not to be essential for Ca(2+) oscillations. Moreover, brief spontaneous Ca(2+) changes were observed in the mitochondrial Ca(2+) concentration without apparent changes in the cytosolic Ca(2+) concentration, indicating the presence of a mitochondrial autonomous Ca(2+) signaling mechanism. In the presence of calretinin, a Ca(2+)-buffering protein, the amplitude of cytosolic spikes during oscillations was decreased, and the amount of Ca(2+) ions taken up by mitochondria was reduced. Thus, the increased calretinin expression observed in mesothelioma cells and in certain colon cancer might be correlated to the increased resistance of these tumor cells to proapoptotic/pronecrotic signals. We identified and characterized (experimentally and by modeling) three Ca(2+) shuttling pathways in primary mesothelial cells during Ca(2+) oscillations: Ca(2+) shuttled between (i) the endoplasmic reticulum (ER) and mitochondria, (ii) the ER and the extracellular space, and (iii) the ER and cytoplasmic Ca(2+) buffers.

Characterization and modeling of Ca2+ oscillations in mouse primary mesothelial cells.

Brief changes in the cytosolic and intra-organellar Ca2+ concentration serve as specific signals for various physiological processes. In mesothelial cells lining the surface of internal organs and the walls of body cavities, a re-entry in the cell cycle (G0-G1 transition) evoked by serum re-administration induces long-lasting Ca2+ oscillations with a slowly decreasing frequency. Individual mesothelial cells show a wide range of different oscillatory patterns within a single, supposedly homogenous cell population. Changes in the cytoplasmic Ca2+ concentration (ccyt) show baseline oscillatory patterns i.e., discrete Ca2+ transients starting from a constant basal ccyt level. The ER Ca2+ concentration (cER) displays a sawtooth wave at a semi-depleted ER state; the minimum level is reached just briefly after the maximal value for ccyt. These oscillations depend on plasmalemmal Ca2+ influx and on the inositol trisphosphate concentration [InsP3]; the Ca2+ influx is a crucial determinant of the oscillation frequency. Partial blocking of SERCA pumps modifies the oscillation frequency in both directions, i.e. increasing it in some cells and lowering it in others. Current mathematical models for Ca2+ oscillations mostly fail to reproduce two experimentally observed phenomena: the broad range of interspike intervals and constant basal ccyt levels between two Ca2+ spikes. Here we developed a new model based on--and fitted to--Ca2+ recordings of ccyt and cER recorded in primary mouse mesothelial cells. The model allowed for explaining many features of experimentally observed Ca2+ oscillations. We consider this model to be suitable to simulate various types of InsP3 receptor-based baseline Ca2+ oscillations.

Overexpression or absence of calretinin in mouse primary mesothelial cells inversely affects proliferation and cell migration.

BACKGROUND: The Ca(2+)-binding protein calretinin is currently used as a positive marker for identifying epithelioid malignant mesothelioma (MM) and reactive mesothelium, but calretinin's likely role in mesotheliomagenesis remains unclear. Calretinin protects immortalized mesothelial cells in vitro from asbestos-induced cytotoxicity and thus might be implicated in mesothelioma formation. To further investigate calretinin's putative role in the early steps of MM generation, primary mesothelial cells from calretinin knockout (CR-/-) and wildtype (WT) mice were compared. METHODS: Primary mouse mesothelial cells from WT and CR-/- mice were investigated with respect to morphology, marker proteins, proliferation, cell cycle parameters and mobility in vitro. Overexpression of calretinin or a nuclear-targeted variant was achieved by a lentiviral expression system. RESULTS: CR-/- mice have a normal mesothelium and no striking morphological abnormalities compared to WT animals were noted. Primary mouse mesothelial cells from both genotypes show a typical "cobblestone-like" morphology and express mesothelial markers including mesothelin. In cells from CR-/- mice in vitro, we observed more giant cells and a significantly decreased proliferation rate. Up-regulation of calretinin in mesothelial cells of both genotypes increases the proliferation rate and induces a cobblestone-like epithelial morphology. The length of the S/G2/M phase is unchanged, however the G1 phase is clearly prolonged in CR-/- cells. They are also much slower to close a scratch in a confluent cell layer (2D-wound assay). In addition to a change in cell morphology, an increase in proliferation and mobility is observed, if calretinin overexpression is targeted to the nucleus. Thus, both calretinin and nuclear-targeted calretinin increase mesothelial cell proliferation and consequently, speed up the scratch-closure time. The increased rate of scratch closure in WT cells is the result of two processes: an increased proliferation rate and augmented cell mobility of the border cells migrating towards the empty space. CONCLUSIONS: We hypothesize that the differences in proliferation and mobility between WT and CR-/- mesothelial cells are the likely result from differences in their developmental trajectories. The mechanistic understanding of the function of calretinin and its putative implication in signaling pathways in normal mesothelial cells may help understanding its role during the processes that lead to mesothelioma formation and could possibly open new avenues for mesothelioma therapy, either by directly targeting calretinin expression or indirectly by targeting calretinin-mediated downstream signaling.

Mechanism of capsaicin receptor TRPV1-mediated toxicity in pain-sensing neurons focusing on the effects of Na(+)/Ca(2+) fluxes and the Ca(2+)-binding protein calretinin.

Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a pain-sensing, ligand-gated, non-selective cation channel expressed in peripheral sensory neurons. Prolonged activation of TRPV1 by capsaicin leads to cell swelling and formation of membrane blebs in rat dorsal root ganglion (DRG) neurons. Similar results were obtained in NIH3T3 fibroblast cells stably expressing TRPV1. Here, we assessed the contribution of Ca(2+) and Na(+) ions to TRPV1-mediated changes. Cell swelling was caused by a substantial influx of extracellular Na(+) via TRPV1 channels, causing concomitant transport of water. In the absence of extracellular Na(+), the membrane blebbing was completely inhibited, but Ca(2+) influx did not change under these conditions. Na(+) influx was modulated by the intracellular Ca(2+) concentration ([Ca(2+)]i). Elevation of [Ca(2+)]i by ionomycin sensitized/activated TRPV1 channels causing cell swelling in TRPV1-positive cells. In the absence of extracellular Ca(2+), capsaicin caused only little increase in [Ca(2+)]i indicating that the increase in [Ca(2+)]i observed after capsaicin application is derived essentially from extracellular Ca(2+) and not from internal Ca(2+) stores. In the absence of extracellular Ca(2+) also the process of cell swelling was considerably slower. Calretinin is a Ca(2+) buffer protein, which is expressed in a subset of TRPV1-positive neurons. Calretinin decreased the amplitude, but slowed down the decay of Ca(2+) signals evoked by ionomycin. Cells co-expressing TRPV1 and calretinin were less sensitive to TRPV1-mediated, capsaicin-induced volume increases. In TRPV1-expressing NIH3T3 cells, calretinin decreased the capsaicin-induced Ca(2+) and Na(+) influx. Swelling and formation of membrane blebs resulted in impaired plasma membrane integrity finally leading to cell death. Our results hint towards a mechanistic explanation for the apoptosis-independent capsaicin-evoked neuronal loss and additionally reveal a protective effect of calretinin; we propose that the Ca(2+)-buffering capacity of calretinin reduces the susceptibility of calretinin-expressing DRG neurons against cell swelling/death caused by overstimulation of TRPV1 channels. This article is part of a Special Issue entitled:12th European Symposium on Calcium.

Parvalbumin tunes spike-timing and efferent short-term plasticity in striatal fast spiking interneurons.

Striatal fast spiking interneurons (FSIs) modulate output of the striatum by synchronizing medium-sized spiny neurons (MSNs). Recent studies have broadened our understanding of FSIs, showing that they are implicated in severe motor disorders such as parkinsonism, dystonia and Tourette syndrome. FSIs are the only striatal neurons to express the calcium-binding protein parvalbumin (PV). This selective expression of PV raises questions about the functional role of this Ca(2+) buffer in controlling FSI Ca(2+) dynamics and, consequently, FSI spiking mode and neurotransmission. To study the functional involvement of FSIs in striatal microcircuit activity and the role of PV in FSI function, we performed perforated patch recordings on enhanced green fluorescent protein-expressing FSIs in brain slices from control and PV-/- mice. Our results revealed that PV-/- FSIs fired more regularly and were more excitable than control FSIs by a mechanism in which Ca(2+) buffering is linked to spiking activity as a result of the activation of small conductance Ca(2+)-dependent K(+) channels. A modelling approach of striatal FSIs supports our experimental results. Furthermore, PV deletion modified frequency-specific short-term plasticity at inhibitory FSI to MSN synapses. Our results therefore reinforce the hypothesis that in FSIs, PV is crucial for fine-tuning of the temporal responses of the FSI network and for the orchestration of MSN populations. This, in turn, may play a direct role in the generation and pathology-related worsening of motor rhythms.

Restricted diffusion of calretinin in cerebellar granule cell dendrites implies Ca²âº-dependent interactions via its EF-hand 5 domain.

Ca²âº-binding proteins (CaBPs) are important regulators of neuronal Ca²âº signalling, acting either as buffers that shape Ca²âº transients and Ca²âº diffusion and/or as Ca²âº sensors. The diffusional mobility represents a crucial functional parameter of CaBPs, describing their range-of-action and possible interactions with binding partners. Calretinin (CR) is a CaBP widely expressed in the nervous system with strong expression in cerebellar granule cells. It is involved in regulating excitability and synaptic transmission of granule cells, and its absence leads to impaired motor control. We quantified the diffusional mobility of dye-labelled CR in mouse granule cells using two-photon fluorescence recovery after photobleaching. We found that movement of macromolecules in granule cell dendrites was not well described by free Brownian diffusion and that CR diffused unexpectedly slow compared to fluorescein dextrans of comparable size. During bursts of action potentials, which were associated with dendritic Ca²âº transients, the mobility of CR was further reduced. Diffusion was significantly accelerated by a peptide embracing EF-hand 5 of CR. Our results suggest long-lasting, Ca²âº-dependent interactions of CR with large and/or immobile binding partners. These interactions render CR a poorly mobile Ca²âº buffer and point towards a Ca²âº sensor function of CR.