Elevated [Ca2+]i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca2+ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca2+ influx and increase in [Ca2+]i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca2+]i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca2+/NFAT pathway.Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca2) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca2+]i was measured with a dynamic digital Ca2+ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.Human PASMC were cultured under hypoxia (3% O2+]i and Ca2+ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca2+]i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.Hypoxia induced PASMC proliferation with increases in basal [Ca2+/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.The SOC/Ca Pulmonary arterial hypertension (PAH) is a progressive disease characterized by a sustained increase in pulmonary arterial pressure and vascular remodeling. A few molecular mechanisms such as prostacyclin, nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) and endothelin pathways have been shown of pathological importance and involved in the abnormal proliferation and contraction of pulmonary artery smooth muscle cells (PASMC) in PAH patients. Therapies developed towards these targets, such as prostacyclin analogs, endothelin-1 receptor antagonists and phosphodiesterase type-5 (PDE5) inhibitors . Long-te2+ signal and other signaling pathways through induction of a specific genetic program, and it has been proposed to be involved in PAH pathogenesis. The Ca2+/NFAT pathway plays an important part in the cell proliferation including osteoblasts i. Ca2+ influx is the important determinant of NFAT activity in skeletal muscle cells and smooth muscle cells i. Subsequently, the depletion of intracellular Ca2+ stores triggers a sustained Ca2+ flux called capacitive calcium entry (CCE). Ca2+ entry via SOC (SOCE) in the membrane caused by ER depletion is the dominated component of CCE [2+ influx via SOC appears to be a determinant in maintaining a sustained increase in [Ca2+]i and regulation of vascular tone and arterial wall structure i. The promoted [Ca2+]i leads to increased calcineurin phosphatase activity, which induces nuclear translocation of NFAT. NFAT activation in PASMC could regulate multiple gene transcriptions including TRPC1 gene which positively reinforce NFAT activation and cell proliferation. The SOC/Ca2+/NFAT pathway may be a downstream mediator for the anti-proliferative effect of sildenafil.Thus, we hypothesized that hypoxia-induced PASMC proliferation involves up-regulation of TRPC1 expression, which in turn resulted in the enhancement of SOCE and elevation of i in a single cell was measured using a Ca2+-sensitive fluorescent indicator fura 2-AM . Cells were loaded with 3 μM fura 2-AM for 30 min in the dark at room temperature. Fura 2-AM loaded cells were transferred to glass-bottomed culture dishes , fixed on a microscope stage, and perfused with physiological salt solution (PSS) for 30 min to remove extracellular fura 2-AM and to activate intracellular fura 2-AM into fura 2. The [Ca2+]i was measured using an xenon lamp  equipped with a Nikon's Epi-fluorescence microscope  and band-pass filters for wavelengths of 340 nm and 380 nm. [Ca2+]i was based on the equation, [Ca2+]i = Kd × (Sf2/Sb2) × (R-Rmin)/(Rmax-R) [2+]i, cyclopiazonic acid -induced ER Ca2+ release and SOCE upon changing perfusion from Ca2+-free PSS to 1.8 mM Ca2+ PSS were measured in different groups. In most experiments, 5-10 cells were imaged in a single field, and a selected peripheral cytosolic area from each cell used for analysis.[Call bound]. RestingTotal RNA was isolated from PASMC by using TRIzol reagent  according to manufacturer's instructions. RNA was reverse-transcribed to synthesize first-strand cDNA. The specific primers were designed from coding regions of human TRPC1 . The primers of β-actin  were used as control for RNA integrity. PCR was done using an Icycler Thermal cycler  under conditions described below. The PCR reaction mixture was denatured at 94°C (0.5 min), annealed at 55°C (0.5 min), and extended at 72°C (0.5 min) for 30 cycles. This was followed by a final extension at 72°C (5 min) to ensure complete product extension. Amplified products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. PCR product bands were visualized by ultraviolet light . Intensity values were measured by densitometric analysis with Quantitative One software , and normalized to the intensity values of β-actin for quantitative comparisons. PCR products were sequenced. The amplified production of TRPC1 and β-actin were 372 bp and 539 bp respectively. The ratio of normoxia group was regarded as 100%.TRPC1 protein was detected using a standard Western blotting protocol. Briefly, adherent PASMC were harvested and 40 μg proteins from each sample of different groups separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 0.5 h, and at 120 V for 1.5 h. They were transferred onto a nitrocellulose membrane  at 100 V for 1.5 h at 4°C onto Western blotting apparatus . The blocked membrane was incubated with primary antibody of TRPC1  and β-actin  overnight at 4°C. After incubation with horseradish peroxidase-conjugated secondary antibody  for 1 h at room temperature, immunoblotting signals were visualized using Western Luminescent kit . Results were quantified by densitometry, and the densities of immunoblotting were analyzed by scanning X-ray film with Quantitative One software. The value of the relative density of the TRPC1 band was normalized to the density of the β-actin band to represent the amount of TRPC1 protein. The ratio of normoxia group was regarded as 100%.The human PASMC after 24 h starvation were cultured in 2% FBS under normoxia, hypoxia or hypoxia plus sildenafil or other drugs for 72 h respectively. After treatment, cells were fixed for 30 min at room temperature in 4% formaldehyde in Dulbecco's Phosphate-Buffered Saline (D-PBS), blocked with blocking solution (2% BSA in D-PBS) for 15 min and incubated with 0.2% Triton X-100 in blocking buffer for 30 min at room temperature. Cells were incubated with primary antibodies  for 1 h at room temperature and then fluorescent-conjugated secondary antibodies  for 30 min at room temperature. The nucleus was stained with Hoechest33258 . Fluorescence was examined using a Leica laser scanning confocal microscope .2, 1.2 MgCl2, 10 HEPES, and 10 glucose, (pH 7.4). For Ca2+-free PSS, CaCl2 was replaced by equimolar MgCl2 and 1 mM EGTA added to chelate residual Ca2+ i in PASMC. Solvents (DMSO and ethanol) had no obvious effect on cell growth (data not shown).Firstly, the mitogenic effect of hypoxia on human PASMC was tested. Cell proliferation was quantified by MTT assay. Hypoxia  triggered a transient rise in [Ca2+]i in human PASMC  induced a rise in [Ca2+]i that was obviously due to SOCE , the CPA-induced [Ca2+]i transient rise due to Ca2+ release from the SR  and the peak in [Ca2+]i due to SOCE  in human PASMC compared with normoxia group . These results gave evidence that sildenafil may exert its anti-proliferative effect by inhibiting the activated SOC/[Ca2+]i pathway under hypoxia exposure.Hypoxia-induced PASMC proliferation is associated with extracellular CaSMC Fig.  due to lOCE Fig. . Hypoxiaoup Fig. . Sildena2+ influx [TRPC-encoded proteins may be involved in the molecular identity of SOC . Inhibit+ influx .2+ influx may be the potential pathway mechanism involved in the anti-proliferative effect of sildenafil.We examined if the anti-proliferation effect of sildenafil is related to the SOC expression. Sildenafil significantly inhibited the up-regulated mRNA and protein expression level of TRPC1 by hypoxia stimulus Fig. . These d2+]i activates calcineurin which dephosphorylates cytoplasmic NFAT, allowing its entry to the nucleus where it forms complexes with other transcription factors and regulates gene transcriptions [2+]i was significantly increased in hypoxic PASMC. We assessed if this hypoxia-induced [Ca2+]i increase through SOC could mediate NFAT nuclear translocation. The results show that hypoxia induced significant nuclear translocation of NFATc3  and an indirect inhibitor of NFAT (Cyclosporin A) on hypoxia-induced TRPC1 up-regulation and human PASMC proliferation were examined. As shown in Fig. 2+ influx and increase in [Ca2+]i are involved in hypoxia-induced human PASMC proliferation. (b) Potentiation of [Ca2+]i resulting from enhancement of SOC leads to nuclear translocation of NFATc3. (c) NFATc3 nuclear translocation is involved in hypoxia-induced human PASMC proliferation; (d) Inhibiting NFAT nuclear translocation reduces TRPC1 expression in human PASMC. (e) Anti-proliferative effects of sildenafil is related to the SOC/Ca2+/NFAT pathway. PAH is a disease of progressive vascular remodeling of the small pulmonary arteries (<500 μM in diameter), which results in a progressive increase in pulmonary vascular resistance and, eventually, right ventricular failure and death i and SOC-mediated Ca2+ influx i is reported higher in the PASMC of PAH patients. Hypoxia-induced PASMC proliferation was accompanied by a significant increase in resting [Ca2+]i, calcium release from the ER, and SOC-mediated Ca2+ influx in cytoplasm  agonists such as ET-1, angiotensin-II, and phenylephrine [2+]i, stimulate vasoconstriction, and promote cell growth. We demonstrated that hypoxia promoted TRPC1 expression on the genetic and protein level which might be responsible for the enhancement of SOC-mediated Ca2+ influx.Intracellular Caferation . [Ca2+]iasm Fig. . Chelatiion Fig. . TRPC1 hlephrine . Up-regu2+ is its role in regulation of gene expression. One mechanism by which the Ca2+ signal can be translated into a change in gene activity is the calcineurin-mediated activation of NFAT i. It has been showed that the TRPC1 promoter has one binding sequence with NFAT. Conceivably, once activated, NFAT might stimulate TRPC1 expression through a positive feedback mechanism. The results in this experiment showed that blocking NFAT nuclear translocation inhibited hypoxia-induced TRPC1 expression. These results suggested that the Ca2+/calcineurin/NFAT pathway especially this positive feedback mechanism could feasibly stimulate the development of hypoxia-induced PASMC proliferation.The effect of Caertrophy ,46. A mo program . Given t program  demonstr program . The pre2+]i, but also inhibited NFAT nuclear translocation. The results indicated that therapeutic effects (vasodilation/antiproliferation) of sildenafil was involved in inhibition of the SOC/Ca2+/NFAT pathway. Additionally, activation PKG pathway by sildenafil and 8-brom-cGMP inhibited NFAT nuclear translocation. We speculate that the sildenafil effect in PAH therapy may act through multiple downstream signaling pathways and target the progression of pulmonary vascular remodeling in PAH. Novel strategies involving NFAT inhibition can be useful for PAH treatment.Our experiments demonstrated that sildenafil not only attenuated hypoxia-induced elevation of TRPC1 expression, enhancement of SOC function and increase in [CaThe authors declare that they have no competing interests.2+ imaging and the design of the study. JFL, JL, JW and YXW carried out immunofluorescence and RT-PCR as well as MTT experiment. LZ, JW and CW prepared the manuscript. All authors read and approved the final manuscript.CW carried out Western blotting, Ca


The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.−/− mice to monitor the Ca2+ dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca2+ channels, or Ca2+ stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca2+-homeostasis in these neurons by influencing the activity of the plasma membrane Na+/Ca2+-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca2+ elevation by stimulating the reverse mode of NCX in both WT and OMP−/− mice. The resulting Ca2+ responses indicate that OMP facilitates NCX activity and allows rapid Ca2+ extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).We used intact olfactory epithelium obtained from WT and OMPSince CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions. Interestingly, Bex protein, an interacting partner of OMP 2+-dependent manner Despite a recent report suggesting that OMP plays a modulatory role in cAMP formation 2+ homeostasis, we imaged Ca2+ dynamics of single olfactory knobs in wild-type and OMP−/− mice. Ca2+responses were induced by activating and blocking various pathways involved in Ca2+ influx, sequestration, and extrusion. We present here evidence that OMP facilitates NCX activity and allows rapid Ca2+ extrusion from OSN knobs. Furthermore, our protein interaction data suggest that the influence of OMP on NCX activity may be mediated through its interactions with Bex protein and calmodulin.To investigate the possibility that OMP participates in modulating Ca2+ signaling and to determine where in the signal transduction cascade OMP might be acting, we compared the response kinetics of the Ca2+ transients elicited in OSN knobs of WT and OMP−/− mice by confocal Ca2+ imaging. All elements required during olfactory signal transduction have been reported to be present in both the cilia and knob 2+ entry during the olfactory signal transduction cascade are the cyclic-nucleotide gated (CNG) channels in OSN cilia 2+ increases seen in this OSN compartment too 2+ dynamics in the mouse olfactory knob could be achieved at excellent spatial and temporal resolution using confocal imaging  observed in both the transmission , were captured  to 0.6 µM (low Ca2+)  , demonst2+ signaling differs in knobs of OMP−/−vs. WT OSNs, we first compared the Ca2+ transients evoked by a 1-s pulse of IBMX (100 µM) . By contrast, the recovery kinetics of the Ca2+ signal back to baseline were significantly altered between WT and OMP−/− (p<0.0001). Fits of the single-exponential decay yielded a time constant (τ) that gave a measure of the Ca2+ recovery rate. On average, the decay time constant of an IBMX-evoked Ca2+ transient was approximately 2.5-fold slower in OMP−/−vs. WT knobs , therefore, the deficit in Ca2+ recovery kinetics is attributed to a compromised ability of events subsequent to cAMP generation, e.g. inefficient adaptation of CNG channels allowing Ca2+ entry for a longer period of time or a defect in the Ca2+ clearance processes.To determine whether Ca(100 µM) . AnalysiWT knobs . Hence, 2+ influx through CNG channels is an important factor in determining the overall Ca2+ flux, we next asked whether there could be altered feedback inhibition of CNG channels due to elevated resting Ca2+i in the OMP−/− mouse. Resting endogenous Ca2+i levels in OSNs were measured using calibration procedures with either fluo-4 or fura-2. From single wavelength calibration with fluo-4, the free Ca2+i at rest was estimated to be 76.3±3.3 nM  in WT knobs and 79.4±4.3 nM  in OMP−/− knobs. Based on ratiometric calibration with fura-2, resting levels of Ca2+i were calculated to be 83.0±13.7 nM in WT  and 87.1±15.6 nM  in OMP−/− knobs. In neither measurement was there any significant difference in Ca2+i resting levels between WT and OMP−/− mice, arguing against the possibility of altered inhibition of CNG channels due to elevated Ca2+i in OMP−/− mice. To determine whether the reduced Ca2+ recovery rate in OMP−/− mice was dependent on the source of Ca2+-entry, we tested the effects of raising Ca2+i by membrane depolarisation which leads to the opening of voltage-operated Ca2+ channels, or by releasing Ca2+ from intracellular stores 2+ transients evoked by application of KCl or caffeine were monitored in subsequent experiments. We found that Ca2+ transients elicited by a 1-s pulse of KCl (80 mM) showed an approximately 1.5-fold slower recovery time constant in OMP−/− knobs compared to WT controls (p<0.001) , a condition under which IBMX failed to induce a measurable Ca2+ rise  external Ca2+  . Thus, aa2+ rise . This shnal Ca2+ . Togethe2+i transients of OMP−/− mice indicates that there is a defect in the Ca2+ clearance mechanism in the OSN knobs. In mammalian cells, elevated Ca2+i can be reduced by sequestering it into Ca2+ stores through sarco-endoplasmic-reticulum Ca2+-ATPase (SERCA) pumps or by expelling Ca2+ through the plasma membrane by a Na+/Ca2+ exchanger (NCX) and/or plasma membrane Ca2+-ATPases (PMCA). Using electrophysiological criteria it has been reported that NCX is the major means of Ca2+ extrusion following odor-evoked stimulation of the apical region of amphibian and mouse OSNs 2+ imaging of mouse OSN knobs. Recent reports suggests that PMCA may also play a significant role in Ca2+ removal in the rat 2+ clearance mechanism to the removal of elevated Ca2+i.The observed delay in restitution kinetics of the Ca2+ stores, we next evaluated whether the SERCA pumps could play a significant role in Ca2+ removal in the WT OSNs. To test this, the decay rate constants of the depolarization-induced Ca2+ transients were characterized both before and after inhibition of the SERCA pumps with 200 nM thapsigargin (Tg)  following treatment with Tg  confirming that SERCA pumps do not affect the recovery kinetics of Ca2+ responses of the OMP−/− mice. These results suggest that the elimination of Ca2+i from OSN knobs should be mediated mostly by plasma membrane-dependent Ca2+ extrusion mechanisms.Since mouse OSN knobs contain accessible Cagin (Tg) . After t with Tg , confirm2+-ATPase and the Na+/Ca2+ exchanger to Ca2+i removal. To achieve this, decay kinetics of Ca2+ transients were evaluated under conditions where each was selectively inhibited. In the presence of carboxyeosin , a potent PMCA inhibitor 2+ transients showed, on average, less than 1.5-fold reduction in the recovery rate to basal level  on WT knobs. In the presence of DCB, the recovery rate of an IBMX-induced Ca2+ transient was considerably altered, increasing by approximately 3.8-fold (2+i recovery were restored close to those observed before inhibition (data not shown), demonstrating both the efficacy and reversibility of the DCB effect. Conversely, DCB has been reported to block CNG channels 2+ influx. We observed a slight decrease in maximal IBMX-induced fluorescence intensity during DCB treatment , however this decrease was not significant (t-test: p = 0.16) excluding a potential involvement of CNG channels. These results suggest, therefore, that OSN knobs use NCX as their primary Ca2+i extrusion mechanism in response to stimulus-induced Ca2+i elevation  2+ rise , whereas 5 µM KB-R7943 was ineffective  treatment, the magnitude of the Ca2+ signal was significantly enhanced, by as much as 50–100%, confirming the Na+i-dependent nature of the Ca2+ influx through NCX  compared to WT mice , indicating a reduced activity of the Ca2+ entry mode of NCX in the OMP−/− knobs. When the low Na+ pulse was turned off, the modality of NCX reversed to once again extrude the elevated Ca2+i. The decay rate measured in the Ca2+ efflux mode . These results suggest that NCX activity in the OMP−/− mice is reduced in the Ca2+ efflux mode as well as in the reverse Ca2+ entry mode. To partially decrease the adaptive behavior of the NCX activity in the WT response, 24 mM Na+o was also applied for 10 s. The decay rate was again almost 2-fold slower in the OMP−/− mice , suggesting that the decay kinetics were not affected by adaptation. Taken together, our data demonstrate that in the absence of OMP a kinetic deficit exists in transporting Ca2+ in both directions through NCX, suggesting that OMP serves as a modulator of NCX function in OSN knobs.To test if a deficit in NCX function is responsible for the delayed recovery of elevated Cahe pulse . In conthe pulse . The timlux mode  was apprIn situ hybridization indicates that mRNAs for multiple NCX genes are present in OSNs −/− mouse (not shown). These observations provide supporting evidence that the compromised ability in reducing the elevated [Ca2+]i in OMP−/− mice could rather be attributed to a functional deficit of NCX activity than to a deficit in NCX protein expression.NCX protein has been reported on the apical regions of OSNs 2+-dependent manner 2+-dependent manner. Using the 1∶1 Langmuir binding model, the Kd value for XIP peptide binding to CaM was calculated to be 20±6 nM.A possible mechanism by which OMP could influence NCX activity is through a dynamic interaction of OMP and Bex1. Bex1 protein was recently identified as an interacting protein partner of OMP 2+-dependent interaction of Bex1 peptide with CaM by SPR analysis. CaM interacted with immobilized Bex1 (residues 50–75) rapidly and reversibly  and OMP and Bex1 peptide (Kd = 90±12 µM) were Ca2+-independent (not shown).We also confirmed Caversibly , consistTo further assess the interactions of Bex and XIP with CaM, we evaluated the ability of these peptides to influence the CaM-dependent activation of cAMP-PDE activity 2+ imaging of the dendritic knobs of OSNs from OMP−/− mice shows that the lack of OMP compromises rapid extrusion of elevated Ca2+i generated in response to activation of the prototypic cAMP signalling pathway that is used in canonical olfactory sensory neurons for signal transduction. (2) The findings indicate that this effect depends on altered regulation of NCX activity in the OSN dendritic knobs of OMP−/− mice. (3) Molecular analysis indicates that proper Ca2+ extrusion might depend on a series of protein-protein interactions among OMP, Bex1, Ca2+/CaM and NCX, and that these interactions could be altered in OSNs from OMP−/− mice. (4) Together, these findings provide new insight into the mechanisms underlying Ca2+ regulation in dendritic knobs of mouse OSNs.Several major results emerge from this study. (1) Ca−/− mice 2+ transients was significantly slower in the OMP−/− mouse illustrates that OMP plays an important role in the processes of Ca2+ removal in the dendritic knob as distinct from the various mechanisms of Ca2+ entry through CNG channels, voltage-gated Ca2+ channels, or release from Ca2+ stores  as confirmed by our SPR experiments  similar to that reported for OMP−/− mice Thus the interaction of the XIP site of NCX with CaM and with OMP provides a linkage between OMP and NCX through the interactions of OMP, XIP, Bex1 and CaM. Consistent with this postulate is our recent observation that odor-evoked field potential responses (EOG) of the Bex12+i in OSN dendritic knobs. The persistently delayed restitution of Ca2+ transients in the OSN knobs of OMP−/− mice, demonstrates that OMP participates in the regulation of NCX activity. In summary, we propose a model, and provide evidence, to explain the involvement of OMP in the regulation of NCX activity that involves the interaction of OMP, Bex and CaM to regulate NCX activity. Further, we suggest that the dynamic interactions among these proteins provides a mechanism to help explain the complex phenotype of the OMP-KO mouse based on OMP modulation of the efficacy of CaM at multiple steps of the olfactory chemo-transduction pathway. This provides the first mechanistic insight to the elusive role of OMP and provides a framework for further investigation of its function.In conclusion, we have demonstrated that OMP plays an important role in olfactory signal transduction by influencing the rate of removal of elevated Ca−/− mice or the parental strain  were used. The generation and genotyping of the OMP-null mice has been described previously 2/5% CO2) containing (in mM) 120 NaCl, 25 NaHCO3, 5 KCl, 5 BES, 1 MgSO4, 1 CaCl2, and 10 mM glucose. The epithelial preparation was loaded with the Ca2+ indicator fluo-4/AM 2+ concentration were imaged in single knobs by using a confocal laser system n) and mice (N) indicated. Unless otherwise stated, Ca2+ transients shown are averaged traces from 5 representative knobs. Statistical significance between two groups was evaluated using the Student's unpaired t-test. Probability values (p) of <0.05 were considered statistically significant.Mice (3–6 month old) of homozygous OMP2+ solution contained (in mM): 120 NaCl, 25 NaHCO3, 5 KCl, 4.25 CaCl2, 5 EGTA, 1 MgCl2, 5 BES, and 6 glucose at pH = 7.3 and 300 mOsm, giving a free Ca2+ concentration of approximately 0.6 µM. Low external Na+ solution contained (mM): 125 LiCl, 20 NaCl, 5 KCl, 1 CaCl2, 1 Mg Cl2, 10 Hepes, 4 NaOH, pH = 7.3, 300 mOsm (glucose). 3-isobutyl-1-methylxanthine (IBMX), KB-R7943 , 3,4-dichlorobenzamil hydrochloride, and 5,6-carboxyeosin diacetate, succinimidyl ester  were prepared in DMSO. Dilutions were made freshly with final DMSO concentrations of <0.1% (v/v). Unless otherwise stated, chemicals were obtained from Sigma .Stimuli were diluted in extracellular solution  immediately before use and focally ejected onto the olfactory knobs by air pressure and using multi-barrelled stimulation pipettes. Low external CaCarboxyeosin (CE) stock solution, initially made in DMSO, was added to the extracellular solution (for composition see above) to give a concentration of 10 µM. The tissue was preincubated for 15 minutes and then superfused with CE-free solution for an additional 10 minutes to allow de-esterification of CE and to wash off the residual compound in the bath. The concentration of 10 µM CE is 500-fold higher than the reported IC50 value KB-R7943 and DCB  were both first dissolved in DMSO and then further diluted in extracellular solution immediately before use to yield a concentration of 5–10 µM and 10 µM, respectively. Both KB-R7943 and DCB were directly applied to the knobs 5 minutes prior and during additional measurements. DCB was also tested at a concentration of 20 µM. The effect of 10 or 20 µM DCB was indistinguishable, except at the higher concentration where the inhibitory effect of DCB was irreversible. The general inhibitory working concentration of DCB on NCX used by several authors is 10–40 µM in various systems e.g. .Thapsigargin (Tg) was initially dissolved in DMSO to give a 2 mM stock solution. The agent was diluted to the final concentration of 200 nM immediately before use, sonicated, and applied to the extracellular solution for 15 minutes. In some experiments recordings were made from the same knob before and after loading with Tg.2+ solution containing papain (1.4 mg/ml) and DNase . The tissue was then gently extruded in normal oxygenated external solution and centrifuged for 10 min at 600 rpm. The pellet was resuspended in external solution and the released single cells were plated onto a glass cover slip previously coated with 0.01% poly-L-lysine and 0.01% laminin. Resting Ca2+concentrations were determined by imaging olfactory knobs using the in situ preparation or freshly dissociated OSNs. Single-wavelength calibration (fluo-4/AM) was performed as described by Kao et al. 2+i in fura-2/AM loaded OSNs:d is the Ca2+-fura-2 dissociation constant, 224 nM. R is (w1−w)/(w2−w) where w1 and w2 are the fluorescence intensity at excitation wavelength of 340 and 380 nm, respectively, and w equals autofluorescence after cell lysis with digitonin (50 µM). Some of the values were predetermined by Dr. Kao for this specific imaging system: Rmin = 0.358±0.0585; Rmax = to4.69±0.678; Sf2/Sb2 = 4.072±0.533.For a few selected experiments, we used freshly dissociated mouse OSNs by adapting previously described procedures Olfactory neuroepithelial tissue was obtained from postnatal day 7 (P7) WT mice. Mice were anesthetized with 60 mg/kg nembutal and perfused transcardially with 20 ml of ice cold PBS followed by 30 ml of 4% freshly prepared phosphate buffered paraformaldehyde. Tissue was dissected and postfixed for two hours in cold fixative and cryoprotected overnight in 30% sucrose at 4°C. Tissues were embedded in OCT  and snap-frozen in a dry ice/isopentane bath. Coronal cryostat sections of olfactory tissue (12 µm) were attached to Superfrost-plus microscope slides , dried at 37°C for 15 min and stored at −80°C until needed. For single and double-labeled immunofluorescence staining goat anti-OMP antibody  under a continuous flow of 5 µl/min for 2 minutes, unless otherwise stated. Flow cells were regenerated by flushing with running buffer without CaCl2. Binding data were fitted and analyzed using Biacor evaluation version 4.1 software (Biacore). Data are representative of three separate experiments.The Biacore 3000  was used to study peptide-protein interactions by SPR. Bovine calmodulin was purchased from Sigma, recombinant rat OMP was expressed and purified as previously described


In rhizobia it is not yet known whether Ca2+ can act as a transducer of symbiotic signals.During the interaction between rhizobia and leguminous plants the two partners engage in a molecular conversation that leads to reciprocal recognition and ensures the beginning of a successful symbiotic integration. In host plants, intracellular Ca2+ probe aequorin was introduced into Mesorhizobium loti USDA 3147T strain to investigate whether a Ca2+ response is activated in rhizobia upon perception of plant root exudates. We find that M. loti cells respond to environmental and symbiotic cues through transient elevations in intracellular free Ca2+ concentration. Only root exudates from the homologous host Lotus japonicus induce Ca2+ signalling and downstream activation of nodulation genes. The extracellular Ca2+ chelator EGTA inhibits both transient intracellular Ca2+ increase and inducible nod gene expression, while not affecting the expression of other genes, either constitutively expressed or inducible.A plasmid encoding the bioluminescent CaThese findings indicate a newly described early event in the molecular dialogue between plants and rhizobia and highlight the use of aequorin-expressing bacterial strains as a promising novel approach for research in legume symbiosis. When lview see .nod) genes which are required for the synthesis of rhizobial signal molecules of lipo-chitooligosaccharide nature, the Nod factors. In the host plant, the generation of intracellular Ca2+ oscillations triggered by Nod factors has been firmly established as one of the earliest crucial events in symbiosis signalling; these oscillations are transduced into downstream physiological and developmental responses i) tightly is a prerequisite for a Ca2+-based signalling system, and is known to be present in bacteria i and its changes in live cells. This has proven difficult because of problems in loading fluorescent Ca2+ indicator dyes, such as fura-2, into bacterial cells. However, the recombinant expression of the Ca2+-sensitive photoprotein aequorin, which has been demonstrated to be a suitable method to monitor [Ca2+]i changes accurately in eukaryotes [E.coli [Anabaena sp. PCC7120 [2+ ]i with specific Ca2+ signatures.In order to establish precisely when and how Cakaryotes -12, has  i) residing in the submicromolar range (around 500 nM) , confirming that the recorded signal was due only to Ca2+-dependent light emission from aequorin.The occurrence in 2+]i changes in both plants i in both eukaryotic i) in resting M. loti cells grown to mid-exponential phase.Click here for file


The aim of this study was to determine whether Ca2+ is necessary and sufficient for inducing long-term memory formation. A suitable model to address this question is the Pavlovian appetitive conditioning of the proboscis extension reflex in the honeybee Apis mellifera, in which animals learn to associate an odor with a sucrose reward.Synaptic plasticity associated with an important wave of gene transcription and protein synthesis underlies long-term memory processes. Calcium (Ca2+ concentration ([Ca2+]i) in the brain, we show that: (i) blocking [Ca2+]i increase during multiple-trial conditioning selectively impairs long-term memory performance; (ii) conversely, increasing [Ca2+]i during single-trial conditioning triggers long-term memory formation; and finally, (iii) as was the case for long-term memory produced by multiple-trial conditioning, enhancement of long-term memory performance induced by a [Ca2+]i increase depends on de novo protein synthesis.By modulating the intracellular Ca2+ is both a necessary and a sufficient signal for the formation of protein-dependent long-term memory. Ca2+ therefore appears to act as a switch between short- and long-term storage of learned information.Altogether our data suggest that during olfactory conditioning Ca Moreover, most studies suggested that Ca2+ is necessary for LTM formation, but they did not show that it may be sufficient; that is, that in association with a learning procedure producing only short-term memory, Ca2+ may induce LTM formation. The aim of our work is to demonstrate the direct role of Ca2+ in memory formation and to show that it is both necessary and sufficient during conditioning to induce LTM formation. Our hypothesis is that an increase in the [Ca2+]i during multiple-trial learning could be a key for triggering long-lasting gene expression-dependent phenomena involved in LTM formation.One possible candidate for this role is Caes is AC ,13,15,16in (CaM) ,18 and crlie LTM ,20. Seveocessing -25. In tApis mellifera is a well-established model to study the molecular basis of memory formation i during learning, we demonstrate that Ca2+ is both necessary and sufficient during acquisition for LTM formation.To investigate whether Caex (PER) , in whic or more . Shortlyructures ,32. Acco2+ is necessary for LTM formation, we first evaluated the effect of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), a membrane permeant Ca2+ chelator, injected 1 h before conditioning on memory performance at 72 h. The results in Figure Three conditioning trials with 10-min inter-trial intervals normally lead to high LTM performance at 72 h. To test whether Ca2+]i increase during conditioning selectively impaired LTM performance, indicating that Ca2+ increase during learning is necessary for LTM formation.Comparison of CS-specific responses (SR)  between groups confirmed the significant reduction of LTM performance after treatment with BAPTA-AM, relative to controls Figure . We also2+ is necessary to establish LTM, but is it sufficient? To answer this question we increased [Ca2+]i during one-trial conditioning, a training protocol normally inefficient to produce LTM [2+ from ryanodine-sensitive stores [2+]i in the honeybee brain. As a prerequisite for this experiment, we confirmed with Ca2+ imaging experiments that an injection of caffeine (20 mM) induces an increase of [Ca2+]i. The Ca2+ signal was recorded during 60 min in three different olfactory structures . After 10 min of recording, caffeine solution or saline was injected. As shown for the antennal lobe -N,N,N',N'-tetraacetoxymethyl ester (NP-EGTA-AM) was used to confirm the promnesic effect of Ca2+. As shown in Figure 2+ release by uncaging of NP-EGTA-AM 5 min before one-trial conditioning also induced a specific LTM increase at 72 h. This increase was not found at 3 h or at 24 h. Altogether, these data indicate that increasing [Ca2+]i during one-trial conditioning leads to a specific olfactory LTM enhancement. Therefore, associated with one-trial, Ca2+ is sufficient for LTM formation.As caffeine has broader effects than just acting on Ca+ levels -37, a sp2+]i is dependent upon protein synthesis, we replicated the previous experiment in the presence of the transcription inhibitor, actinomycin-D (ACT-D). Bees were subjected to one-trial conditioning associated with the injection of caffeine (20 mM) or saline solution, followed 3 h later either by an injection of ACT-D  or saline solution (control group). These groups were then tested at 72 h. As shown in Figure de novo protein synthesis during consolidation.LTM is dependent on a new wave of protein synthesis, required for the functional and structural synaptic modifications involved in long-term storage of information ,30. To t2+]i increase during multiple-trial conditioning selectively impairs LTM performance; (ii) conversely, treatments that increase [Ca2+]i during one-trial conditioning greatly enhance LTM performance; and finally, (iii) as was the case for LTM produced by multiple-trial conditioning, the increased memory performance at long-term induced by an increase of [Ca2+]i depends on de novo protein synthesis. Altogether our data suggest that Ca2+ is both a necessary and a sufficient signal during olfactory conditioning for the formation of protein-dependent LTM.Here we show that: (i) treatment that inhibits i at the moment of a conditioning trial can act as the trigger for biochemical pathways that will lead to de novo protein synthesis, and to LTM.The repetition of associative CS-US trials is thought to be the key for the formation of an odor-specific protein-dependent LTM. Therefore, additional conditioning trials act as confirmations of the information gained during the first conditioning trial. A question that was left unanswered until now was which intracellular messengers were keeping track of the first associative trial? Our present results suggest that Ca2+]i during learning affects specifically LTM while leaving learning and short-term memory (STM) intact. Similar results have been observed in rats in which blocking of the L-type voltage-gated Ca2+ channel with the antagonist verapamil also impaired LTM selectively and showed no effect on learning or STM [2+]i level has to be reached during learning to trigger downstream pathways leading to de novo gene transcription required for LTM formation. It does not, however, mean that learning and STM do not rely at all on Ca2+. In fact, this might indicate that they require different ranges of [Ca2+]i than LTM. In any case, the memory formed before 24 h in honeybees is not dependent on de novo gene transcription i increase were applied to the whole animal and likely affected all brain structures, yet the enhanced memory was odor specific. This is because odor specificity is ensured by mechanisms within the subset of neurons activated during the single CS-US trial, in particular the neurons that represent the CS in the olfactory network. This could correspond to subsets of antennal lobe neurons  or mushroom body neurons (Kenyon cells), which receive input from the US pathway  i i . Thus, te stores  and sevee stores ,46 and pe stores ,48. Othe+ stores .2+]i during the first conditioning trial determines the fate of olfactory cells when the second trial is applied, inducing the cascades leading to LTM. This second trial can take place 10 min or even later after the first one. Disruption of Ca2+ homeostasis over such a long period of time could have detrimental effects on the survival of the cells. Therefore, we favor the hypothesis that an early and transient Ca2+ signal is sufficient to prime olfactory cells for the formation of LTM, and that a relay bridges the gap between associative trials. One possible hypothesis is that Ca2+-dependent signaling pathways could be this relay. For instance, the involvement of CaMKII, a Ca2+/CaM-activated kinase, in LTM formation has been demonstrated in several studies i: intracellular calcium concentration; Ca2+/CaMKII: calcium/calmodulin-dependent kinase II; CaM: calmodulin; cAMP: cyclic adenosine monophosphate; CR: conditioned responses; CREB: cyclic adenosine monophosphate responsive element binding protein; CS: conditioned stimulus; DMSO: dimethylsulfoxyde; LTM: long-term memory; NO: nitric oxide; NP-EGTA-AM: O-Nitrophenyl-ethyleneglycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester; PER: proboscis extension reflex; PKA: protein kinase A; SR: specific responses; US: unconditioned stimulus; UV: ultra violet; VUMmx1: ventral unpaired median neuron of the maxillary neuromere 1.AC: adenylate cyclase; ACT-D: actinomycin-D; BAPTA-AM: 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester; CaEP carried out most of the experiments, performed the statistical analysis and participated in its design, and contributed to the draft of the manuscript. VR, IN, CL, MM and JCS conceived the study, participated in its design and coordination and helped to draft the manuscript. YM carried out part of the behavioral experiments. JCS helped to perform the statistical analysis. All authors read and approved the final manuscript.Control injections of BAPTA-AM and caffeine after conditioning. Effect of drug injection on long-term memory (LTM) performance (at 72 h), plotted as the difference (ΔSR) of the percentage of specific response  between treated and control groups. A negative value indicates a memory disruption while a positive value indicates a memory improvement. Thus, bees injected with a Ca2+ chelator, BAPTA-AM 1 h before a three-trial conditioning, show a strong and significant memory disruption . Conversely, bees injected with caffeine, allowing a release of Ca2+ from internal stores, 20 min before a single conditioning trial, show improved LTM performance . However, injection 1 h after the conditioning trial does not show any memory increase  : nearly significant, NS: non-significant).Click here for fileControl for the conditioned stimulus-unconditioned stimulus association needed for the promnesic caffeine effect. Retention performances at 72 h, as the percentage of specific response  and not to the new odor), between controls and bees injected with caffeine 20 min, before one conditioning trial (CS + unconditioned stimulus (US)) or a CS-alone trial. The results of the CS + US group were presented in details in Figure 2 = 0, P = 0.97; Control: n = 39; Caffeine: n = 40). Caffeine therefore has no effect without a full CS-US conditioning trial. .Click here for file


Specific gene silencing was obtained by siRNAs transfection. A murine BT474 xenograft cancer model was used to assess the effect of lapatinib on gene expression in vivo. We found that lapatinib induces upregulation of Grb7, an adaptor protein involved in receptor tyrosine kinase signaling and promoting cell survival and cell migration. Grb7 upregulation induced by lapatinib was found to occur in cancer cells in vitro and in vivo. We demonstrate that Grb7 upregulation is recreated by PI3K inhibitors while being prevented by constitutively active Akt. Thus, Grb7 is repressed by PI3K signaling and lapatinib-mediated Akt inhibition is responsible for Grb7 de-repression. Finally, we show that Grb7 removal by RNA-interference reduces breast cancer cell viability and increases the activity of lapatinib. In conclusion, Grb7 upregulation is a potentially adverse consequence of HER2 signaling inhibition. Preventing Grb7 accumulation and/or its interaction with receptor tyrosine kinases may increase the benefit of HER2-targeting drugs.The efficacy of anti-HER2 therapeutics, such as lapatinib and trastuzumab, is limited by primary and acquired resistance. Cellular adaptations that allow breast cancer cell to survive prolonged HER2 inhibition include de-repression of the transcription factor FOXO3A with consequent estrogen receptor activation, and/or increased HER3 signaling. Here, we used low-density arrays, quantitative PCR, and western blotting to determine how HER2 signaling inhibition with lapatinib or PI3K inhibitors affects the expression of genes involved in breast cancer metastatic spread and overall prognosis. Retroviral transgenesis was used to express constitutively active forms of Akt in the HER2 HER2/ERBB2/neu is amplified in 20–30% breast cancers where it acts as a driving oncogene conferring propensity to metastasize and adverse prognosis + breast cancer HER2-targeting therapeutics, such as the monoclonal antibody trastuzumab (Herceptin) and the dual HER2/EGFR tyrosine kinase inhibitor lapatinib (Tykerb), are successfully applied to the treatment of patients with HER2Chronic exposure to inhibitors of HER-family proteins results in cellular adaptations with the potential of reducing the efficacy of these drugs. Sergina at al. described a feedback mechanism controlled by Akt whereby Akt inhibition in response to HER2 tyrosine kinase inhibitors leads to HER3 redistribution to the cell membrane and to sustained HER3 phosphorylation Here, we used low-density arrays (LDAs) to assess how HER2 tyrosine kinase inhibition by lapatinib would affect the expression of a panel of genes involved in breast cancer biology and metastatic behavior. We show that lapatinib causes rapid upregulation of Grb7, an adaptor protein that is typically co-amplified with HER2, participates in HER2 signaling, and promotes cell survival and cell migration. We identify a negative feedback loop mediated by Akt as responsible for repressing Grb7. Finally, we show that preventing Grb7 accumulation by RNA-interference potentiates the activity of lapatinib.SKBR3, BT474, MCF7, MDA-MB231, and Phoenix cells were all obtained from ATCC. Cell were grown in RPMI medium , supplemented with 10%FBS, L-glutamine and antibiotics. Lapatinib was generously provided by GlaxoSmithKline. LY294002, wortmannin, puromycin, G418, and protamine sulfate, were purchased from Sigma-Aldrich .3 cells/well were seeded in 96 well plates in medium containing 1% FBS. Cells were allowed to adhere for 24 h and were subsequently incubated with the indicated drug concentrations. Each condition was tested in triplicate wells. Viability was assessed 120 h later by Celltiter96 Aqueous1  using a standard ELISA reader. Specific death was calculated with the following formula: 100-. Cell lysates were boiled at 100°C for 10 minutes and stored at −20°C for subsequent use. Proteins were separated on a SDS-polyacrylamide gel and electroblotted to a polyvinylidene fluoride membrane . Proteins were visualized by probing the membranes with the following antibodies: anti-phospho Akt (Ser473), anti-Akt, anti-HER2 , anti-Grb7 , anti-FOXO3a , and γ-tubulin (Sigma Aldrich). Standard enhanced chemiluminescence was used for protein bands detection.6) were washed twice with ice-cold PBS and then scraped off the plates in cell-lysis buffer  containing protease inhibitor cocktail and phosphatase inhibitor cocktail I & II (Sigma-Aldrich). Lysates were prepared after 15 min of incubation on ice by repeated pipetting and centrifugation in a microfuge at 12,000 g for 10 min at 4°C. For coimmunoprecipitations, a anti-HER2 antibody  was covalently coupled to protein G Dynabeads (2 µg of antibodies per 30 µl beads) using BS3  according to the manufacturer's specification, and then incubated with 1 mg of cell extract supernatant for 3 h at 4°C. The immunoprecipitates were washed five times with lysis buffer, eluted with 25 µl of electrophoresis sample buffer, and heated at 95°C for 5 min. Thereafter, proteins were separated by immunoblotting, transferred to PVDF membranes and revealed using appropriate antibodies and standard enhanced chemiluminescence.Cells . 50 mg/kg body weight lapatinib was given daily for three days by oral gavage in 0.5% hydroxypropylmethycellulose and 0.1% Tween 80. Thereafter, mice were anesthetized and killed by cervical dislocation. Tumors were divided in two parts and stored in RNAlater  for subsequent RNA extraction, and in formalin for histological examination (hematoxylin-eosin) and HER2 immunohistochemistry .This study was performed in compliance with the US Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and approved by the Internal Review Board of the Advanced Biotechnology Center (ABC) in Genoa, Italy. Six- to eight-week-old female BALB/c athymic mice  were acquired from Charles Rivers Laboratories. A 17 β-estradiol pellet (Innovative Research of America) was inserted s.c. to each mouse 1 d before injection with BT474 cells. 2×10Promoter analysis of the Grb7 gene was performed by Genomatix software .t-tests were performed to evaluate the significance of the results.Each experiment was performed at least three times with similar results. Unpaired Vice versa, in MCF7 and MDA-MB-231 cells lapatinib showed no effect on cell cycle and viability for concentrations up to 1 µM  or overexpress a WT Akt allele Akt is responsible for inhibiting the forkhead box-O (FOXO) transcription factors by phosphorylating them on several residues and thereby inducing their sequestration in the cytoplasm by 14-3-3 proteins in vivo, we made use of a BT474 murine xenograft model . On the other hand, similar to what observed with lapatinib, Grb7 removal decreased TFRC/CD71 expression, in line with a role for Grb7 in the HER2-Akt-mTOR pathway . FinallyIn this study, we identify a functional interplay between HER2 and its interactor Grb7 whereby HER2 signaling represses Grb7 using the PI3K-Akt arm of its downstream signaling cascades. Inhibition of HER2 tyrosine kinase activity or of PI3K/Akt de-represses Grb7 causing its rapid upregulation . NoticeaOur study reinforces the concept that adaptations involving gene de-repression and/or protein relocalization/posttranslational modification (as in the case of HER3) occur as a consequence of RTK inhibition and have the potential to reduce the benefit of RTK-targeting therapeutics Grb7 acts as a pro-survival factor in breast cancer cells as shown by the fact that RNAi-mediated removal of this protein reduces cell viability. The mechanisms whereby Grb7 promotes cell survival are still unclear. Our data indicate a role for Grb7 in the HER2-Akt-mTOR pathway. Grb7 silencing reduces Akt activation and leads to TFRC/CD71 downregulation In line with its biological properties, Grb7 belongs to a group of genes conferring adverse prognosis in node-negative breast cancer per se does not seem to be sufficient to restore Akt phosphorylation  in the presence of lapatinib (at least in the incubation times tested in our experiments) regardless of a persistent interaction with HER2. Thus, Grb7 silencing is unlikely to cooperate with lapatinib by eliminating a residual Akt activity. Vice versa, it appears likelier that Grb7 reduction  affect other signaling pathways/intracellular processes whose obstruction increase susceptibility to HER2 inhibition.Our observation that Grb7 silencing increases lapatinib activity provides the proof of principle that interfering with this adaptor protein may be beneficial, although the mechanism(s) underlying this synergism is (are) not entirely clear. Grb7 upregulation RNAi-based therapeutics are eventually being developed and Grb7 siRNAs may thus potentially be coupled to anti-HER2 drugs In conclusion, Grb7 upregulation is a potentially adverse molecular side effect of HER2 signaling inhibition. Interfering with Grb7 accumulation may be advisable given its oncogenic activity and its capacity to increase the metastatic potential of a cell.Table S1Gene expression modulation by lapatinib in breast cancer cell lines. Supporting material. Gene list and LDA results.(0.16 MB DOC)Click here for additional data file.Figure S1FOXO3A does not affect Grb7 expression in SKBR3 cells. SKBR3 cells were transfected with plasmids encoding WT FOXO3A, FOXO3A AAA or the empty vector (pcDNA3) as a control. Cells were selected for 2 weeks using G418 before being used for cell lysates preparation. FOXO3A, Grb7, and γ-tubulin expression were detected by immunoblotting.(0.70 MB TIF)Click here for additional data file.


To reveal the spatiotemporal properties of the propagation of an excitatory signal with action potentials in the primary visual cortical circuit, we conducted a Ca2+ imaging study on slices of the mouse visual cortex. Electrical stimulation of layer 4 evoked [Ca2+]i transients around the stimulus electrode. Subsequently, the high [Ca2+]i region mainly propagated perpendicular to the cortical layer , with horizontal propagation being restricted. When the excitatory synaptic transmission was blocked, only weak and concentric [Ca2+]i transients were observed. When the action potential was blocked, the [Ca2+]i transients disappeared almost completely. These results suggested that the action potential contributed to the induction of the [Ca2+]i transients, and that excitatory synaptic connections were involved in the propagation of the high [Ca2+]i region in the primary visual cortical circuit. To elucidate the involvement of inhibitory synaptic connections in signal propagation in the primary visual cortex, the GABAA receptor inhibitor bicuculline was applied. In this case, the evoked signal propagated from layer 4 to the entire field of view, and the prolonged [Ca2+]i transients were observed compared with the control condition. Our results suggest that excitatory neurons are widely connected to each other over the entire primary visual cortex with recurrent synapses, and inhibitory neurons play a fundamental role in the organization of functional sub-networks by restricting the propagation of excitation signals.The calcium ion (Ca The calcium ion . Due to these functions, Ca2+ can affect synaptic transmissions, gene expression, and morphological changes of cellular processes 2+]i transient allows the detection of the excitability of a neuron and helps to reveal the plastic changes of the synaptic transmission.Ca2+]i changes evoked by layer 4 stimulation in primary visual cortical slice preparations by means of Ca2+ imaging to study the propagation of action potentials in the primary visual cortical neuronal networks. These results also provide quantitative data to study the plasticity of the neuronal circuits, which is highly dependent to [Ca2+]i alterations.Here, we demonstrate the spatiotemporal properties of the [CaSome of our preliminary results have been previously reported at meetings 2+]i transients induced by electrical stimulation at layer 4 in the cell body regions in each of the cortical layers  near the stimulus position and within 20 ms in the other regions. The time-to-peak values were 40–50 ms in each neuron. The time courses of the [Ca2+]i transients obtained from the cell body region and from the binned area were nearly identical. This observation indicates that the fluorescent signal in the cell body region may actually include out-of-focus areas just above and below the cells. To avoid the incorporation of any misleading information, 16×16-pixel binned data were used for quantitative data analyses  20–30 ms after stimulation, and the [Ca2+]i transients in layer 2/3 increased in size up to 40 ms after stimulation. In contrast, the horizontal spread of the high [Ca2+]i region was restricted to approximately 200 µm in layers 4 and 2/3. The high [Ca2+]i region gradually shrank, starting approximately 60 ms after stimulation, and the [Ca2+]i level returned to a near-basal level approximately 200 ms after stimulation. When the 80 µA stimulus was applied, the width of a high [Ca2+]i region, in which the amplitude of [Ca2+]i transient exceeded 30% of that observed at the stimulus position under the control condition, were 204±29 µm, 125±18 µm, 107±16 µm, and 109±14 µm in the dorsal, ventral, lateral, and medial directions, respectively . Blocking the excitatory synaptic transmission by the application of CNQX and AP5 reduced the [Ca2+]i transients around the stimulus position, . Anisotropic signal propagation could not be detected . No [Ca2+]i transients were observed after TTX administration . CNQX and AP5 also reduced the amplitudes at layer 2/3 . These results, combined with the results of the response widths, suggested that excitatory synaptic transmission contributed to the propagation of the high [Ca2+]i region , and 4.9±4.9% and 10.6±10.6% in the case of the 200 µA stimuli . These results indicated that action potentials involved in the induction of the [Ca2+]i transients . There was no horizontal restriction of signal propagation under the administration of 5 µM bicuculline, and the evoked signal propagated over the entire observation area.As mentioned above, layer 4 stimulation evoked the [Caondition . The amp2+]i transients did not differ between the control conditions and those under the administration of 5 µM bicuculline (2+]i transients over the entire observation area (n = 9 slices) and 415±113 ms (n = 4 slices), respectively  (The onset of the [Cauculline  and S4. ion area . The tim t-test) .2+]i transients obtained from the peak amplitudes of the 16×16-pixel binned ΔF/F data in the regions indicated in 2+]i transients increased in a dose-dependent manner. In particular, the signal evoked in layer 4 under the administration of 5 and 10 µM bicuculline propagated over the entire observation area  B receptors are known to be involved in slow inhibitory postsynaptic potentials (slow IPSPs) B receptors to signal propagation in the visual cortex was also tested by the application of 1–30 µM CGP55845, a GABAB receptor antagonist. No significant changes in the properties of the signal propagation were observed . These results strongly suggest that synaptic inhibition using GABAA receptors plays a crucial role in confining the action potentials within a functional sub-network in the visual cortical circuit.GABA2+]i dynamics evoked by electrical stimulation in primary visual cortical slice preparations by means of Ca2+ imaging. The spatio-temporal distribution of the high [Ca2+]i regions revealed the propagation properties of the action potentials in the primary visual cortical neuronal circuit. The evoked [Ca2+]i transients in the cell somata are thought to be caused by the generation of action potentials 2+]i transients were observed in the case of 80 µA stimulation, and the [Ca2+]i transients almost completely disappeared in the case of 200 µA stimulation  from the stimulus position, with restricted horizontal signal propagation . The onset of the [Ca2+]i transients were slower at the layer 2/3 above the stimulus position compared with that at stimulus position. In the case of 80 µA stimuli, the average onset time was 19±4 ms at layer 2/3 . When excitatory synaptic transmission was blocked by the application of CNQX and AP5, the amplitudes of the [Ca2+]i transients around the stimulus position and at layer 2/3 were reduced, the response widths shrank, and anisotropic nature  of the high [Ca2+]i region disappeared  and were conducted in accordance with the guidelines established by the Ministry of Education, Culture, Sports, Science and Technology, Japan.2–5% CO2. The composition of normal ACSF was as follows (in mM): 137 NaCl, 2.5 KCl, 0.58 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 21 NaHCO3, and 10 glucose. Coronal slices of the visual cortex  were prepared using a vibratome tissue slicer . The slices were incubated at room temperature in a submerged chamber containing gassed ACSF for at least 60 min prior to the experiments.Standard slice preparation protocols were used 2+]i in the slices was measured using the membrane-permeant acetoxymethyl (AM) ester of Oregon Green 488 BAPTA-1  dissolved in dimethylsulfoxide . The visual cortical slices were placed in a small plastic Petri dish containing 100 µl ACSF with 10 µM OGB1-AM and 0.02% Cremophor EL . The dish was incubated at 35°C for 30 min in a small chamber, which was humidified and continuously aerated with 95% O2–5% CO2, and then washed with 100 µl ACSF at 35°C for 15 min. OGB1-loaded slices were transferred to a continuously superfused (2–2.5 ml/min) chamber on the stage of a BX51WI epifluorescent upright microscope . The excitation light source  was coupled to the epifluorescent port of the microscope equipped with the filter cube  for excitation and emission. The fluorescence signals were imaged with a 20×, NA 0.95 water-immersion objective (Olympus) at 29–31°C and were captured using a cooled-CCD NeuroCCDsm256 camera  at a frame rate of 100 Hz (100 fps). The actual imaging area was 640×640 µm. The [Ca2+]i transients were evoked by stimulation with a 200 µs, 40 to 240 µA biphasic current from a glass microelectrode  placed in layer 4 of the visual cortical slice. The stimulation with each current under each condition was repeated 3–10 times, and the responses were averaged. Because a current stimulator generally has internal impedance, the actual amount of the stimulus current is likely different from the command current pulse. To confirm this issue, we measured the actual stimulus current. The current wave forms, when various intensities of 100 µs negative and 100 µs positive square command pulses were applied to the stimulus electrode in saline, are shown in [Ca2+]i were quantified as ΔF/F, where F indicates the average fluorescence intensity before stimulation, and ΔF is the change in fluorescence intensity from F. For quantitative data analyses, the 256×256 fluorescence images were binned by 16×16 pixels  followed by an application of the Hanning filter to the time series of the binned images and the calculation of ΔF/F (Images were recorded using NeuroPlex software (Redshirt Imaging) and processed using custom-made MATLAB  programs. For noise reduction, the captured fluorescence images were spatially filtered with a 5×5 Gaussian Kernel, and the time series of the images were temporally filtered with a Hanning filter . The relative changes in i transients evoked by layer 4 stimulation. The evoked [Ca2+]i transients obtained from the 16 × 16-pixel binned area under the control condition (black line), under the condition of 10 μM CNQX + 50 μM AP5 administration (red line), and under the condition of 1 μM TTX administration (blue line) in the same slice shown in Time courses of the [Ca(0.40 MB PDF)Click here for additional data file.Figure S42+]i transients evoked by layer 4 stimulation under bicuculline administration. The [Ca2+]i transients evoked by the application of an 80 μA stimulus to layer 4 obtained from the 16 × 16-pixel binned area under the control condition (black line), under the condition of 2 μM bicuculline administration (red line), and under the condition of 5 μM bicuculline administration (blue line) in the same slice shown in Time courses of the [Ca(0.49 MB PDF)Click here for additional data file.Figure S52+]i transients under the control condition and under the administration of 5 μM bicuculline. (A) The values of time-to-peak were obtained from the 16 × 16-pixel binned data in the three red regions  on the pseudocolor [Ca2+]i transient images. The vertical line is drawn perpendicular to the cortical layers and passes through the stimulus position. The L2/3 horizontal line is drawn along layer 2/3, and its vertical distance from the stimulus position is 200 μm. The L4 horizontal line is drawn along layer 4 and passes through the stimulus position. Scale bar  =  100 μm. The distributions of the time-to-peaks (B) of the [Ca2+]i transients under the control condition (filled circles) and under the administration of 5 μM bicuculline (open circles) along with the vertical, L2/3 horizontal, and L4 horizontal lines. In the vertical panel, the distance indicates the displacement from the stimulus position, with negative corresponding to the dorsal direction and positive to the ventral direction. In the L2/3 horizontal and L4 horizontal line panels, the distance indicates the horizontal displacement from the stimulus position, with negative corresponding to the lateral direction and positive to the medial direction. Error bars are represented as SEM.Time-to-peaks of the [Ca(0.44 MB PDF)Click here for additional data file.Figure S62+]i transients evoked by the application of the 120 μA stimulus in layer 4 under the control condition (A) and under the condition of 10 μM picrotoxin administration (B). The illustrations are the same as those in Blockade of inhibitory synaptic transmission by picrotoxin also enhanced the propagation of the evoked signal. Time-lapse pseudocolor images of the [Ca(1.25 MB PDF)Click here for additional data file.


Nine human tumour cell lines  were studied as to whether cellular radiosensitivity is related to the number of initial or residual double-strand breaks (dsb). Cellular sensitivity was measured by colony assay and dsb by means of constant- and graded-field gel electrophoresis . The nine tumour cell lines showed a broad variation in cellular sensitivity (SF2 0.17–0.63). The number of initial dsb as measured by GFGE ranged between 14 and 27 dsb/Gy/diploid DNA content. In contrast, normal fibroblasts raised from skin biopsies of seven individuals showed only a marginal variation with 18–20 dsb/Gy/diploid DNA content. For eight of the nine tumour cell lines, there was a significant correlation between the number of initial dsb and the cellular radiosensitivity. The tumour cells showed a broad variation in the amount of dsb measured 24 h after irradiation by CFGE, which, however, was not correlated with the cellular sensitivity. This residual damage was found to be influenced not only by the actual number of residual dsb, but also by apoptosis and cell cycle progression which had impact on CFGE measurements. Some cell line strains were able to proliferate even after exposure to 150 Gy while others were found to degrade their DNA. Our results suggest that for tumour cells, in contrast to normal cells, the variation in sensitivity is mainly determined by differences in the initial number of dsb induced. GFGE opHere we applied the GFGE to determine the number of dsb induced in nine tumour cell lines and for control in seven normal fibroblast lines and compared it with the respective data obtained by CFGE. We further measured the kinetics of dsb repair and the residual damage 24 h after irradiation in order to clarify, whether dsb critically determine tumour cell survival.2.The LNCaP and DU145 cell lines were originally isolated from metastatic lesions of patients suffering from prostate cancer  and pipetted into 180 μl plug moulds to solidify . Lysis was started on ice for 30 min and continued at 37°C overnight. The plugs were washed thrice with Tris-EDTA buffer and sliced into pieces containing about 105 cells, which were inserted into a 14 × 20 cm 0.8% agarose gel . The gel was then covered with a thin overlayer of 0.8% agarose to avoid light fraction artefacts upon optical imaging. Electrophoresis was performed at 0.6 V cm−1 for 30 h in 0.5 × TBE buffer  in a conventional apparatus . The gel was then stained overnight (0.5 μg ml−1 ethidium bromide), destained overnight in ddH2O and CCD camera (Sony XC-75CE) equipped with an image analysis system  was used to quantify the FDR.Subconfluent cells were suspended .Cells were treated as before but electrophoresis was performed differently. Running conditions were now 0.6 V cmeviously  the preseviously . In prinusly −1, . The cur sigmoid  and coulCell cycle distribution of PI-stained cells was determined by flow cytometry in an FACScan .Each experiment was repeated three times and the data were given as a mean with its standard error (±s.e.m.). Statistical analysis, data fitting and graphics were performed by means of the Prism 3.1 computer program .α and β and the mean inactivation dose, Dbar (Table 1Dbar of the most radiosensitive cell line (LNCaP) was 2.9 times lower as compared to the most radioresistant strain (RT112).The nine tumour cell lines showed a wide range of radiosensitivity . The comparison of all strains gave by fitting the size of the fragments in each band. According to these fits band 1 contained DNA fragments of 5.25–11.0 Mbp and band 2 of less than 5.25 Mbp. Theses values are slightly higher than previously reported (−12 dsb/Gy/Da for MCF7-Bus (−12 dsb/Gy/Da). These values are equivalent to a variation of 14–27 dsb/Gy/diploid DNA content for tumour cells and 18–20 dsb/Gy/diploid DNA content for normal fibroblasts.For each strain, the FDR in bands 1 and 2 were simultaneously fitted as function of dose using a model previously described in detail . Those freported . Now, 16reported . The numMCF7-Bus . The resfret, depends on cell type, proliferation and presumably other effects. It needs to be known for calculation of the exact number of dsb. For the tumour cells the retention factor fret ranged between 0.11 (HeLa) and 0.4 (MCF7-Bus)  , which iτfast=3–4 min and τslow=90–100 min). These data illustrate that tumour cells are unlikely to process dsb with different kinetics compared to normal cells, which was also observed previously for mouse and rat tumour cells  was found to be significantly correlated with the number of induced dsb . LNCaP cells were excluded, since the data point was exterior to the range of 2 s.d. However, the correlation was even significant when LNCaP was included . The data indicate that at least for eight out of nine tumour cells, the variation in sensitivity could result from differences in the initial damage.Figure 7The relationship between residual dsb and sensitivity gave a nonsignificant trend. This trend, however, was presumably not meaningful, since an increase in the number of residual dsb was associated with a decrease in sensitivity. In contrast to the initial damage, residual dsb as determined by CFGE appeared not to be an appropriate indicator of the clonogenic radiosensitivity.The present study was aimed to define the role of initial and residual dsb on the radiosensitivity of human tumour cells.−12 dsb/Gy/Da  of the FDR curve of CFGE. However, this does not mean that the initial slope can reliably taken as an indicator for the amount of initial damage. In many cases, the variation in the initial slopes of FDR curves may not be large enough to reveal significant differences between the cell lines. The initial slope further depend on S-phase cells and on the retention factor −12 dsb/Gy/Da. (−12 dsb/Gy/Da) based on PFGE, from which a number of dsb can be calculated when it is combined with either 125I-decay, analysis of fragment size distribution after high doses, or restriction digest (−12/Gy/Da.The mean dsb induction frequency of all 16 cell lines was 8.1 × 10b/Gy/Da. . This van digest , 2003. OThe amount of nonrepaired damage showed a broad variation among the nine tumour cell lines. However, it was shown that apoptosis (γ-H2AX phosphorylation (γ-H2AX will be an advantage over conventional gel electrophoresis for monitoring residual damage in tumour cells.The recently introduced technique to visualise sites of histone rylation  and may rylation . HoweverIn contrast to tumour cells, for normal human and rodent fibroblasts, the variation in cellular sensitivity did not result from differences in the induction, but the repair of dsb . These rper se but also on the cell cycle progression and in some cases on DNA degradation, presumably due to apoptosis.Tumour cells vary considerably in their amount of induced damage. These differences are most likely due to variations of chromatin structure and they may largely account for tumour cell survival. The number of residual damage was also different among the cell lines studied, but we could now show that those measurements do not only depend on the repair capacity


Exploration into the roles of P100 and CTF in SOCE inhibition reveal that P100, when expressed in Xenopus laevis oocytes, directly inhibits the SOCE currents but CTF does not, nor does P100 when containing the disease causing R4227X mutation. Interestingly, we also found that in PC1 expressing MDCK cells, translocation of the ER Ca2+ sensor protein STIM1 to the cell periphery was significantly altered. In addition, P100 Co-immunoprecipitates with STIM1 but CTF does not. The expression of P100 in CHO cells recapitulates the STIM1 translocation inhibition seen with FL PC1. These data describe a novel polycystin-1 cleavage product, P100, which functions to reduce SOCE via direct inhibition of STIM1 translocation; a function with consequences for ADPKD.Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder resulting in large kidney cysts and eventual kidney failure. Mutations in either the PKD1 or PKD2/TRPP2 genes and their respective protein products, polycystin-1 (PC1) and polycystin-2 (PC2) result in ADPKD. PC2 is known to function as a non-selective cation channel, but PC1's function and the function of PC1 cleavage products are not well understood. Here we identify an endogenous PC1 cleavage product, P100, a 100 kDa fragment found in both wild type and epitope tagged PKD1 knock-in mice. Expression of full length human PC1 (FL PC1) and the resulting P100 and C-Terminal Fragment (CTF) cleavage products in both MDCK and CHO cells significantly reduces the store operated Ca PC1's function in Ca2+ homeostasis is far less clear.Ca2+ channel 2+, where a disruption of any Ca2+ regulator can lead to cyst formation. Normal PC2 expression and the resulting lower ER Ca2+ levels should increase Ca2+ influx and lead to apoptosis and increased cell proliferation 2+ influx. PC1 may also play an important role in regulating cellular Ca2+ responses to stimuli, as PC1 has been reported to alter ATP activated intracellular Ca2+ increases by influencing the rate of Ca2+ reuptake in the ER Tubular cysts result from a dysfunction in either PC1 or PC2, suggesting a common functional pathway. This idea is supported by evidence that PC1 must bind PC2 in order for PC2 to function as a Ca2+ store depletion is sensed by proteins of the stromal interaction molecule (STIM) family, specifically, STIM1 2+ molecules 2+ channels (SOC) 2+ sensor that directly activates the SOC channels to replenish ER Ca2+ levels.ER Ca2+ homeostasis either at the ER or plasma membranes is further complicated by the cleavage of the PC1 protein and a lack of understanding about PC1 cleavage product localization. The first cleavage occurs constitutively and partially at the juxtamembrane GPS domain, generating a stable N-terminal fragment (NTF), and a C-terminal fragment (CTF) that coexist with uncleaved full-length PC1 Deciphering the role PC1 plays in Ca2+ influx at the plasma membrane, and we provide evidence that PC1 interacts with STIM1, preventing its re-localization after store depletion. Our data support the hypothesis that PC1 and P100 regulate store operated calcium entry by interfering with STIM1's ability to activate SOC channels on the plasma membrane.Here we describe a novel PC1 cleavage product, P100. We show that PC1, through its P100 product, inhibits Cav/v mice where PC1 cleavage at GPS is disrupted and CTF is thus absent (HA/HA), which express fully functional PC1 tagged with a 3xHA epitope at the C-terminus HA/HA mouse by anti-HA after immunoprecipitation or directly in total lysate  directly at the cell surface. However, in both MDCK and CHO cells, PC1 was cleaved into CTF and P100. Could the cleavage products themselves be responsible for modulating the SOCE currents?Next we began an investigation into the possible functions of the PC1 cleavage products on Calar Ca2+  and thislar Ca2+ . Our resXenopus laevis oocytes have well characterized Ca2+ activated Cl- currents (CaCC) 2+ channels and store operated Ca2+ currents Xenopus oocytes, when subjected to a pretreatment with a zero Ca2+ solution containing thapsigargin, displayed large inward currents with complex kinetics , or affecting both the SOCE and the CaCC directly.  current  signific current . In contXenopus oocytes did not significantly reduce the transient peak (p<0.50) or steady state currents (p<0.44)(Xenopus oocytes. AESW expressing oocytes displayed an inhibited transient peak (p<0.01) and steady state amplitudes (p<0.01) as compared to controls. Similarly, AESW transient over expression inhibited extracellular Ca2+ entry in thapsigargin treated CHO cells . The impHO cells . These r2+ ringers (5 mM) after 15 minutes exposure to 4 µM thapsigargin , suggesting that STIM1 did not translocate after ER store depletion. In the control cells with no mPC1 expression, thapsigargin induced a significant change in the STIM1 ratio (p<0.001). Interestingly, the expression of PC1 also limited the overall STIM1 fluorescence in the high Ca2+ ringer (p<0.001) in addition to the translocation of STIM1.We hypothesized that PC1, through the actions of its P100 product, may exert its inhibitory effect on SOCE by interfering with STIM1 in the ER, retarding STIM1's ability to transduce a Ca2+ depletion signal to the SOCE channels of the plasma membrane. Functionally, we sought to address this by measuring the amount of STIM1 translocation to the periphery with or without the expression of PC1. We chose to use a MDCK cell line stably expressing mouse PC1, which we compared to empty vector control cells. The MDCK (with or without mPC1) cells were transfected with a YFP-STIM1 construct and photographed in high Casigargin . Similar2+ ringers and again 10 minutes after exposure to 8 µM thapsigargin 2+ levels and shorten/reduce cytosolic Ca2+ transients e.g. ATP) act through the PLC/IP3 pathway to increase the release of Ca2+ form ER stores and raise cytosolic Ca2+. PC1, therefore, may attenuate the extent of net intracellular Ca2+ increases upon exposure to physiological stimuli. Li et al (2009)2+ release from ER stores upon IP3 stimulation, an action that results in attenuated responses to exogenous ATP application in MDCK cells 2+ responses to physiological stimuli. Interestingly, a number of studies using the C-terminal tail of PC1 over expressed as a fusion protein have found the inverse; the expression of C-terminal tail leads to increases in intracellular Ca2+ and SOCE We have also found that PC1 expression, and specifically P100, leads to an inhibition SOCE, an occurrence hereto unreported. PC1 mediated inhibition of SOCE could lower ER CaXenopus oocytes. These results suggest dysfunctional SOCE regulation . To increase the conductance of endogenous SOC currents, oocytes were pretreated with 4 µM thapsigargin  for up to 2 hours in the zero Ca2+ bath solution; generally, recordings were begun 3 minutes after the Ca2+ containing ND-96 bath solution was re-introduced to the oocytes. A holding potential of −40 mV was used on all oocytes.Whole cell voltage clamp recordings from the Stably transfected Madin-Darby canine kidney (MDCK) cells were a generous gift from Gregory G. Germino  and cultured with selection agents hygromycin (100 µg/ml) and blasticidin (5 µg/ml).The human P100, the P100 R4227X, and the human PC1 fragment AESW constructs were also described previously under the names QIF38, R4227X, and AESW respectively 2+ solution . Images were acquired once every 5 seconds using IPLab Software . Thapsigargin  was used to deplete ER stores and high Ca2+ ringers  was secondarily applied to observe the store depletion activated Ca2+ entry. For STIM1 translocation assays, CHO cells expressing either YFP-STIM1 alone or with the CTF-100 construct began in high Ca2+ ringer then the bath was replaced with the zero Ca2+ ringer and 8 µM thapsigargin. For live images of STIM1 translocation in MDCK cells, MDCK cells stably transfected with either murine PC1 or the blank vector in addition to the YFP-STIM1 construct. 4 µM thapsigargin was used for store depletion. STIM1 translocation analysis was done as described by Luik et al (2008)CHO cells were rinsed with non-supplemented media then loaded with fura 2 . The cover slips were then bathed in a zero CaMethods S1Detailed methods.(0.03 MB DOC)Click here for additional data file.Figure S1Myc and PKD1ΔCMyc are 5xMyc-tagged Pkd1 knock-in alleles that produce fully functional PC1 protein. The domains in polycystin-1 are shown. The Pkd1ΔCmyc allele produces 5xMyc-tagged truncated polycystin-1 protein with deletion of C-terminal 257 amino acids myc/+ and Pkd1ΔCmyc/+ and their wild-type littermate embryo (E17) using anti-Myc antibody after immunoprecipitation. The truncated CTFΔCmyc/+ and P100ΔCmyc/+ are detected in the Pkd1ΔCmyc/+ embryo.Biochemical characterization of PC-1 product, P100 in Myc-tagged knock in mouse model. (A) PKD1 alleles and schematic diagram of their corresponding polycystin-1 proteins. PKD1(1.73 MB EPS)Click here for additional data file.Figure S2Xenopus laevis oocytes. (A) Currents elicited from a representative H2O injected control Xenopus laevis oocytes 3–5 days after injection in a ND96 bath solution subjected to a +20mV step protocol from −100 mV to +60 mV from an initial holding potential of −40 mV. (B) Currents elicited from a representative H2O injected control oocyte pretreated with 4 µM thapsigargin in a zero Ca2+ ND96. (C) Summary steady state current/voltage relation from H2O injected control oocytes with  or without  thapsigargin/zero Ca2+ ND96 pretreatment. ±SEM; (**p<0.01). (D) Averaged currents in H2O inject control oocytes elicited by a 10 second voltage step to −120 mV from a −40 mV holding potential. For each cell (n = 4) currents were first recorded in zero Ca2+ ND96 (blue trace), then in the normal ND96 (1.8 mM Ca2+)(black trace) and finally in ND96 with 100 µM La3+ (red trace). Inset: mean current amplitudes at the transient peak (1.5 sec from beginning of record) and at the steady state (10 sec from beginning of record). ±SEM; (**p<0.01)(*p<0.05). (E) Averaged currents in H2O inject control oocytes elicited by a 10 second voltage step to −120 mV from a −40 mV holding potential. For each cell (n = 10) currents were recorded before (black trace) and after (red trace) replacing the bath with a low Cl- bath containing 100 µM Niflumic acid (NFA). Currents were recorded from a subset of cells (n = 4) further treated with 100 µM La3+ (blue trace). Subtraction of currents before and after treatment with NFA indicates the waveform of the NFA sensitive, probable Cl- currents (black dotted black trace). Inset: mean current amplitudes at the transient peak and steady state. ±SEM; (**p<0.01)(*p<0.05). (F) Measurements of transient peak current reversal potentials. Current/voltage relation of tail current amplitudes after initial −120 mV step in normal ND96 bath , low Cl-/100 µM NFA bath , or low Cl-/100 µM NFA/100 µM La3+ bath . Inset: representative tail current measurements using reversal potential paradigm.SOC and CaCC currents in (3.58 MB EPS)Click here for additional data file.Figure S32+ influx. Fura 2- AM measurements in CHO cells expressing PC1 cleavage products. Recordings began after cells reached steady state in a zero Ca2+, 4 µM thapsigargin ringers. The bath was then replaced with a high Ca2+ bath and Ca2+ influx through SOC channels is measured. (A) CHO cells transiently transfected with CTF  or empty plasmid control . (B) CHO cells transiently transfected with AESW  or empty plasmid control . ±SEM; (*p<0.05).Expression of Polycystin-1 cleavage products have differing effects on store operated Ca(0.82 MB EPS)Click here for additional data file.


Damage elicits both repair and death responses; intriguingly, in those responses Ca2+ also participates as second messenger. This delineates a dual role for Ca2+ in cell stress, making difficult to separate the different and multiple mechanisms required for Ca2+-mediated control of cell survival and apoptosis. Here we attempt to disentangle the two scenarios, examining on the one side, the events implicated in deregulated Ca2+ toxicity and the mechanisms through which this elicits reparative or death pathways; on the other, reviewing the role of Ca2+ as a messenger in the transduction of these same signaling events.Ca For many decades the research on ion  roles in cell physiopathology was hampered by intrinsic technical difficulties. A big impulse came with the development of Ca2+-sensitive fluorescent probes c) low and stable. These include active pumping against gradient by Ca2+ ATPases, enzymes present on the cytosolic side of plasma membrane and endoplasmic reticulum (ER) performing the high energy-expensive task of pumping Ca2+ out of the cytosol against gradient, or by ion exchangers  , which is rapidly extinguished by cytosolic buffering proteins, by the Ca2+-ATPases and by mitochondria, thus being in fact Ca2+ transients. To restore ER homeostasis, the partially emptied ER is replenished by Ca2+ entry from the extracellular space through controlled opening of plasma membrane channels  channels c) for cell homeostasis is that it must be maintained low against gradient , since excess or deregulated [Ca2+]c pose cells in such a dramatic asset to be in fact cell-toxic. To this purpose, a wide range of mechanisms are displayed, including high capacity binding proteins, pumps and exchangers, and mitochondria, which possess a low affinity Ca2+ uniporter that sequesters cytosolic Ca2+ when it reaches the dangerous threshold of 500 nM, thus being a major detoxifying mechanism against Ca2+ overload mt mitochondria stress or collapse c and depletes [Ca2+]er. Moreover, oxidative stress impairs the buffering capacity of mitochondria, lowering the internal Ca2+ threshold level of PTP opening c increases over 500 nM [2+ signals [2+]mt increase modulates mitochondrial activity  c was indeed occurring in some examples of apoptosis, such as the paradigmatic model of rat thymocytes treated with corticosteroids c, it was later shown that in many instances intracellular Ca2+ buffering did not abrogate, but even increased, THG-induced apoptosis, demonstrating that also ER Ca2+ depletion was an apoptogenic event c rises in apoptosis are not rough event, but precise signaling steps, such as, for example, the Ca2+-dependent proteases calpains mt, favoring PTP opening by lowering the Ca2+ threshold required for ANT-VDAC interaction c is involved in the stimulation of the autophagic response through the activation of calcium/calmodulin-dependent kinase-b that inhibits mTOR c and impairing Ca2+-mediated cell signaling. Many pieces of evidence from the literature show that cells with partial Ca2+-depleted ER cannot initiate apoptosis c is reduced [2O2-induced apoptosis begins [2+ signaling is required for stress-induced apoptosis [The decision between cell repair or demise of damaged cells is a choice between the risk of mal-repair, leading to stabilize mutations and potentially preserve precancerous cells, versus loss of viable cells performing useful functions, which must be expensively replaced. Even though the former risk is definitely worse than the latter, mechanisms aiming at avoiding unnecessary loss of precious cells have evolved. To this purpose, it is important that damaged cells do not initiate apoptosis before attempting to repair the damage: this is actively achieved by damaged cells via the set-up of reversible standby scenarios, during which apoptotic signaling is transiently kept at bay. One of such standby mechanisms implies that potentially apoptogenic stress conditions such as H (GAPDH) ; this inytic ATP , thus depoptosis –132, andpoptosis . Startin resumes , ER Ca2+ reduced ; only ths begins ; 2+ and cell death has a long and complex story. It was a reasonably simple task when the goal was describing how strongly deregulated intracellular Ca2+ may cause the passive cell death by necrosis. The scenario became very much complex when the increasing information of the mechanisms of Ca2+-mediated cell signaling in general, and apoptotic signaling in particular, begun to merge.2+ alterations, as an intrinsic stressor, play in the survival or death of damaged cells, aiming at separating Ca2+ deregulation from pro-apoptotic Ca2+ signaling. Perhaps the hottest topic to-date in this field is the role that ER, and the Ca2+ messages it exchanges with mitochondria, plays in the amplification of the apoptotic signal, ending up with the promotion of MOMP and the trigger of the commitment phase of the intrinsic apoptotic signaling. The amplification loops created by the concerted action of ER, Bax, IP3 channels and cytochrome c with Ca2+ signals, spanning ER and mitochondria via cytosol, as depicted in The relationship between Ca2+-dependent pre-commitment apoptotic phase would place Ca2+-related events among the earliest of apoptosis, which would make the closing of a circle that begun almost 20 years ago, when Ca2+ as an intrinsic stressor was considered as “the” mediator of apoptosis.The acknowledgement of a Ca