However, recent studies demonstrate a significant contribution of Ca2+ influx to spontaneous and evoked Ca2+ signaling in astrocytes, suggesting that Ca2+ influx might account for astrocytic Ca2+ signaling to a greater extent than previously thought. Here, we investigated AMPA-evoked Ca2+ influx into olfactory bulb astrocytes in mouse brain slices using Fluo-4 and GCaMP6s, respectively. Bath application of AMPA evoked Ca2+ transients in periglomerular astrocytes that persisted after neuronal transmitter release was inhibited by tetrodotoxin and bafilomycin A1. Withdrawal of external Ca2+ suppressed AMPA-evoked Ca2+ transients, whereas depletion of Ca2+ stores had no effect. Both Ca2+ transients and inward currents induced by AMPA receptor activation were partly reduced by Naspm, a blocker of Ca2+-permeable AMPA receptors lacking the GluA2 subunit. Antibody staining revealed a strong expression of GluA1 and GluA4 and a weak expression of GluA2 in periglomerular astrocytes. Our results indicate that Naspm-sensitive, Ca2+-permeable AMPA receptors contribute to Ca2+ signaling in periglomerular astrocytes in the olfactory bulb.Ca Recent studies, however, challenge this notion and demonstrate pivotal roles of Ca2+ influx in spontaneous Ca2+ signaling and in astrocyte function such as neurovascular coupling62+-permeable ion channels such as ionotropic neurotransmitter receptors, transient receptor potential channels and store-operated Ca2+ channels that mediate Ca2+ influx from the extracellular space3562+ permeability82+-impermeable and results in retraction of glial processes from synapses of Purkinje cells and abnormal synaptic currents2+-permeable AMPA receptors in neuron-glia interactions2+ permeability131415It has become increasingly evident during the past decade that astrocytes are far more than supportive cells in the brain, but rather take active part in information processing such as synaptic transmission and synaptic plasticity12+ transients182+ signaling in periglomerular astrocytes by mGluR5 and P2Y1 receptors202A receptors2+ increases in olfactory bulb astrocytes have been reported to release ATP from astrocytes and to trigger vasoresponses in blood vessels contacted by astrocytic end feet2022232+ responses to neurotransmitters measured in olfactory bulb astrocytes were mediated by Ca2+ release from internal stores, while Ca2+ influx from the extracellular space has not been demonstrated so far2+-permeable AMPA receptors and whether these receptors are activated by glutamate released from olfactory receptor neurons. We studied Ca2+ responses and membrane currents in olfactory bulb astrocytes to application of AMPA and kainate, using Ca2+ imaging in brain slices and whole-cell patch-clamp recordings in acutely isolated astrocytes. Kainate-evoked membrane currents as well as Ca2+ transients induced by AMPA and electrical stimulation of olfactory receptor axons were partly reduced by Naspm (N-[3-[[4-[(3-aminopropyl) amino] butyl] amino] propyl]-1-naphthaleneacetamide trihydrochloride), an AMPA receptor blocker selective for GluA2-lacking, Ca2+-permeable AMPA receptors. Immunohistological staining revealed expression of GluA1, GluA2 and GluA4 in olfactory bulb astrocytes. The results indicate that olfactory bulb astrocytes possess both GluA2-containing and GluA2-lacking AMPA receptors, the latter being blocked by Naspm.In the olfactory bulb, activation of neurotransmitter receptors in astrocytes has been shown to result in Ca2+ signaling. We used application of ATP to test the viability of the cells and to identify astrocytes in the glomerular layer. Olfactory bulb astrocytes express P2Y1 receptors and respond to bath application of ATP and ADP with Ca2+ transients, whereas olfactory bulb neurons do not2+-dependent increase in Fluo-4 fluorescence by 1.34 ± 0.08 ΔF/F0 (n = 53) in periglomerular astrocytes, while in neurons, ATP did not evoke Ca2+ signaling (2+ increase was 1.18 ± 0.09 ΔF/F0 (n = 53) in AMPA-responding astrocytes and 1.62 ± 0.14 ΔF/F0 (n = 59) in neurons. To test whether AMPA evoked Ca2+ transients in periglomerular astrocytes directly or via activation of neurons and subsequent neurotransmitter release, we suppressed vesicular neurotransmitter release by incubation of the brain slices with 10 μM bafilomycin A1 (duration 40 min) and added 1 μM tetrodotoxin (TTX) to the artificial cerebrospinal fluid (ACSF) (2+ transients in astrocytes are expected to be suppressed by TTX/bafilomycin2+ of 0.97 ± 0.07 ΔF/F0 (n = 53) under control conditions, while the increase was significantly reduced to 0.32 ± 0.03 ΔF/F0  by treatment with TTX/bafilomycin  and 1.02 ± 0.13 ΔF/F0 . The results indicate that ATP and AMPA directly induced Ca2+ transients in astrocytes. Therefore, we used bafilomycin A1 and TTX in all following Ca2+ imaging experiments  to isolate the direct response in astrocytes.We were interested whether olfactory bulb astrocytes respond to bath application of AMPA with Caignaling . 59.3% oignaling . The mead (ACSF) . We usedilomycin . In contilomycin . The amp2+ influx through the receptor channel itself. In addition, AMPA/kainate receptors have been shown to stimulate G protein-coupled pathways, including Ca2+ release from intracellular stores26272+ is released from internal stores, we applied AMPA (25 μM) after Ca2+ stores were depleted by incubation with 20 μM cyclopiazonic acid (CPA). As shown in 2+ response in periglomerular astrocytes was only slightly reduced after Ca2+ store depletion. In Ca2+-free, EGTA-buffered ACSF, however, the Ca2+ response was entirely blocked , indicating that AMPA induced Ca2+ influx from the extracellular space.AMPA can activate AMPA receptors as well as kainate receptors2+ influx could be mediated by Ca2+-permeable AMPA receptors or by AMPA-evoked depolarization and subsequent activation of voltage-gated Ca2+ channels. To test whether AMPA evoked Ca2+ influx through Ca2+-permeable AMPA receptors we employed Naspm, a highly selective antagonist of GluA2-lacking, Ca2+-permeable AMPA receptors292+ transients were significantly reduced by 68.0 ± 2.2%  , leading to the conclusion that Ca2+ influx through voltage-gated Ca2+ channels is negligible , confirming the involvement of Ca2+-permeable AMPA receptors in Ca2+ signaling in olfactory bulb astrocytes (2+ transients with an amplitude of 2.50 ± 0.50 ΔF/F0 (n = 31), which were reduced to 52.7 ± 5.3% by Naspm.The AMPA-induced Ca< 0.001) . In addigligible . Hence, trocytes  and e. O+ influx that plugs K+ channels322 was applied. Kainate application evoked an inward current of 427.8 ± 190.3 pA (n = 15) (at −70 mV) corresponding to a current density of 16.0 ± 5.1 pA/pF (n = 15) (2+ into periglomerular astrocytes that in the majority (75%) of cells is Naspm-sensitive and thus mediated by Ca2+-permeable AMPA receptors.We aimed to verify these results in electrophysiological experiments on acutely dissociated astrocytes from the olfactory bulb glomerular layer, using hGFAP-eGFP reporter mice to identify astrocytes . We appl12(n = 15) . 100 μM (n = 15)  and i. I(n = 15)  and i. T2+ permeability of AMPA receptors critically depends on the relative abundance of GluA2 within the channel complex14352+ imaging data suggest Ca2+-permeable AMPA receptors in periglomerular astrocytes, we investigated the cellular distribution of GluA2 in the olfactory bulb. GluA2 immunoreactivity was mainly found rather homogeneously in the external plexiform layer and in somata of periglomerular cells, whereas GluA2 immunoreactivity was much weaker in the synaptic neuropil in the core of the glomeruli  in periglomerular astrocytes. We aimed to isolate the putative Ca2+ response induced by activation of Ca2+-permeable AMPA receptors in astrocytes by applying a mix of receptor blockers antagonizing metabotropic neurotransmitter receptors known to induce Ca2+ transients in olfactory bulb astrocytes. In addition, we reduced glutamatergic activation of neurons and hence indirect effects with D-APV . This blocker mix significantly reduced the stimulation-induced Ca2+ response in periglomerular astrocytes to 56.4 ± 3.0% of the control  (2+ transients to 24.4 ± 2.2% of the control (n = 51). Ca2+ transients in the presence of Naspm were significantly smaller as compared to Ca2+ transients in the presence of the mix of blockers without Naspm (p < 0.001), indicating that glutamate release from OSN axons triggers Ca2+ signaling in periglomerular astrocytes via Ca2+-permeable AMPA receptors.Olfactory receptor neurons (ORN) release glutamate from their axon terminals in the glomeruli, which evokes Caes18202+ . We cros< 0.001) . Additio2+ signaling in periglomerular astrocytes of the olfactory bulb. Our results show that axonal stimulation activates AMPA receptor-mediated Ca2+ influx into periglomerular astrocytes. This Ca2+ influx was largely reduced by Naspm, a blocker of GluA2-lacking, Ca2+-permeable AMPA receptors29In the present study, we have investigated the role of AMPA receptors in Ca2+-permeable and Naspm-sensitive3536Abundant expression of GluA1, GluA2 and GluA4 in the rodent olfactory bulb has been published before. GluA1, e.g., is mainly found in the external plexiform and the glomerular layers and is expressed by periglomerular neurons and mitral/tufted cells394344382+ influx. Firstly, AMPA-induced Ca2+ transients upon agonist bath application were not reduced by suppressing potential indirect effects  with the sodium channel blocker TTX and bafilomycin, an inhibitor of vesicular H+ pumps required for filling synaptic vesicles with neurotransmitter molecules+-evoked Ca2+ transients in astrocytes, in contrast, were greatly reduced by TTX/bafilomycin, in line with the notion that olfactory astrocytes do not significantly express voltage-gated Ca2+ channels and NMDA receptors and hence high-K+-evoked and NMDA-evoked Ca2+ signaling was mainly due to neuronal transmitter release. Whether the remaining, TTX/bafilomycin-insensitive part of NMDA-evoked Ca2+ transients reflects insufficient efficacy of TTX/bafilomycin or expression of NMDA receptors in olfactory bulb astrocytes, as demonstrated for cortical astrocytes and for oligodendrocytes, remains to be shown4849505152+ in neurons and subsequent activation of astrocytic receptors might also contribute to NMDA-evoked Ca2+-transients in astrocytes. Such TTX/bafilomycin-insensitive neurotransmitter release could contribute to astrocytic Ca2+ signalling not only upon application of NMDA, but also AMPA. However, it is very unlikely that this has a major contribution to the AMPA-evoked Ca2+ signaling, since the same mechanism is activated during NMDA and high K+ application, which produced only small Ca2+ transients in the presence of TTX/bafilomycin.Several results indicate that in periglomerular astrocytes, AMPA directly gates Ca2+ release from internal stores, but induced Ca2+ transients that mainly depended on the presence of Ca2+ in the bath solution, indicating Ca2+ influx. Besides Ca2+ influx through AMPA receptors, an increase in Ca2+ due to inhibition or reversal of Na+/Ca2+ exchanger (NCX) upon the AMPA-evoked Na+ increase in astrocytes might contribute to the Ca2+ transients triggered by AMPA application, as shown before for Na+ increases evoked by GABA transport into olfactory bulb and hippocampal astrocytes222+ increases were small yet sufficient to trigger Ca2+-induced Ca2+ release, which was abolished upon store depletion with CPA2+ transients in the present study, in contrast, were only weakly affected by store depletion, indicating that they were not mainly mediated by NCX-dependent Ca2+-induced Ca2+ release.Secondly, AMPA receptor activation did only weakly evoke Ca2+ permeability of the receptors. It should be noted that during our patch-clamp experiments, AMPA receptor-evoked currents were isolated by blocking potassium conductances with Ba2+ and quinine, which affect membrane properties of astrocytes55+-dependent block of K+ channels32+ channel blockers, AMPA receptor-evoked effects on membrane currents might be more complex than shown in our experiments. Importantly, we have demonstrated that Ca2+-permeable AMPA receptors of periglomerular astrocytes are activated through electrical stimulation of ORN axons. ORN axons release glutamate and ATP1819212+ signaling in periglomerular astrocytes by activation of mGluR5, P2Y1 and A2A receptors2058592+ response in periglomerular astrocytes remaining in this blocker cocktail was sensitive to Naspm, indicating that this form of axon-glia interaction activates Ca2+-permeable AMPA receptors. Olfactory bulb astrocytes have been shown to mediate neurovascular coupling2022242+ influx through their AMPA receptors, thereby modulating gliotransmitter release and adapting local circulation and energy supply to the actual metabolic requirements. In addition, astrocytes are involved in the development of the glomeruli, and AMPA receptor-mediated astrocyte Ca2+ signaling might affect neurite growth and synaptogenesis62Thirdly, our findings were confirmed with acutely dissociated cells excluding indirect effects through neuronal activation. The observation that AMPA receptor currents were sensitive to Naspm further substantiated a Ca2+ imaging experiments, NMRI, GCaMP6sfl/fl and GLAST-CreETR2 mice of both genders at postnatal days 14 to 40 were used64fl/fl mice were raised in the animal facility at the University of Hamburg (Germany). hGFAP-eGFP miceFor Ca2PO4 × 2H2O, 26 NaHCO3, 2.8 D-(+)-glucose, 1 MgCl2, 2 CaCl2; Ca2+-free ACSF: 120 NaCl, 2.5 KCl, 1 NaH2PO4 × 2H2O, 26 NaHCO3, 2.8 D-(+)-glucose, 3 MgCl2, 0.5 EGTA; preparation solution for Ca2+ imaging: 83 NaCl, 1 NaH2PO4 × 2H2O, 26.2 NaHCO3, 2.5 KCl, 70 Sucrose, 20 D-(+)-glucose, 2.5 MgSO4 × 7 H2O; preparation solution for electrophysiology: 87 NaCl, 1.25 NaH2PO4 × 2H2O, 25 NaHCO3, 2.5 KCl, 7 MgCl2 × 6 H2O, 0.5 CaCl2 × 6 H2O, 60 Sucrose, 25 D-(+)-glucose (325 mOsm). ACSF, preparation solutions and Ca2+-free ACSF were continuously gassed with carbogen  to buffer the pH at 7.4 and to supply oxygen. Patch clamp-recording of isolated cells was performed in bath solution containing (in mM): 150 NaCl, 5 KCl, 2 MgCl2 × 6 H2O, 2 CaCl2 × 6 H2O, 10 D-(+)-glucose, 10 HEPES, pH 7.4, gassed with oxygen. The compounds amino-3-hydroxy-5-methyl-4-isoxazolephosphonic acid (AMPA), cyclothiazide, (E)-ethyl 1,1a,7,7a-tetrahydro-7-(hydroxyimino)cyclopropa[b]chromene-1a-carboxylate (CPCCOEt), 2-methyl-6-(phenylethynyl)pyridine (MPEP), 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate (MRS2179), 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolotriazolopyrimidin-5-amine (SCH442416) and (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) were obtained from Abcam . Adenosine 5′-triphosphate (ATP), kainate, N-methyl-D-aspartic acid (NMDA) and papain was purchased from Sigma Aldrich , GYKI 53655 from Tocris . Bafilomycin A1 and CPA were acquired from Enzo Life Sciences . The reagents D-APV, Naspm and TTX were received from Alomone labs . All reagents were stored as stock solutions corresponding to the manufacturer’s instructions and added to ACSF directly before the experiment.The following solutions were employed (molarity in mM), ACSF: 120 NaCl, 2.5 KCl, 1 NaH2+ indicator Fluo-4  made from a 4-mM stock solution (dissolved in DMSO and 20% pluronic acid) for 40 min. In most of the experiments, bafilomycin A1 (10 μM) was added to the Fluo-4-AM solution. In some experiments, GCaMP6s fluorescence was used as an indicator of Ca2+ concentration. Changes in intracellular Ca2+ levels in periglomerular astrocytes were recorded by confocal microscopy . An excitation laser wavelength of 488 nm and a frame rate of 0.3–0.5 fps were used. Drugs were administered via the perfusion system except for AMPA. The application solution containing AMPA was applied directly into the perfusion stream in the bath with a custom made application system to allow for a semi-fast application (within 3–5 s). The flow rate of the application system equaled the flow rate of the perfusion system, resulting in a 1:2 dilution of the agonist concentration as adjusted in the application solution.Tissue slices were placed in a recording chamber and fixed with a platinum frame fitted with nylon strings. Slices were incubated with the membrane-permeable form of the CaFor electrical stimulation of axons in tissue slices, a glass pipette with a resistance of 2–2.5 MΩ filled with ACSF was used. The pipette was placed on the surface of the olfactory nerve layer (ONL) comprising axons of olfactory receptor neurons and 250-μA stimuli were applied for 2 s at 10 Hz.2/5% CO2) gassed ACSF supplemented with sucrose (preparation solution) using a vibratome . Slices were transferred for 15 min to warm (35 °C) preparation solution and then to standard ACSF . Cells were isolated using an enzymatic/mechanical approach2+ imaging experiments, astrocytes were isolated from NMRI mice, seeded on concanavalin A-coated cover slipes, loaded with Fluo-4-AM (2 μM in ACSF) for 30–45 min and imaged as discribed for brain slices.For tissue preparation, 300 μm thick sagittal slices from the olfactory bulb were prepared from hGFAP-eGFP mice  in ice cold, carbogen  and BaCl2 (100 μM)Experiments on isolated cells employed a customized concentration clamp device connected to an EPC-7 amplifier and TIDA software  as described elsewhere2+ level in astrocytes, cell somata were marked as regions of interests (ROIs) using EZC1 Viewer software (Nikon). Cells located in the glomerular layer that showed a Ca2+ response to ATP were identified as periglomerular astrocytes2+ levels over time, Fluo-4 and GCaMP6s fluorescence intensity (F), respectively, was recorded throughout the experiment and normalized to the basal fluorescence intensity in absence of stimuli (F0). Changes in Ca2+ are given by ΔF/F0. All values are given as mean values ± standard error of the mean. Data for every set of experiment was acquired from at least three different animals. The assessment of statistical significance by comparing two means was done by Student’s t-test or, if applicable, one-way ANOVA with Fisher’s post-hoc test at an error probability p .To analyze changes of the Ca2HPO4, 3 NaH2PO4. Slices were cut with a vibratome  and incubated with the primary antibodies anti-GluA1 , anti-GluA2 , anti-GluA4 , anti-GFAP , anti-GFAP  and anti-GFP . Antibodies were diluted in 1% NGS, 0.05% TritonX100 in PBS. Subsequently, slices were incubated with secondary antibodies over night at room temperature. Secondary antibodies (1:1000 in PBS) used were: goat anti-rabbit Alexa Fluor 488 , goat anti-rabbit Alexa Fluor 555 (Invitrogen Thermo Fisher), goat anti-chicken Alexa Fluor 555 and goat anti-chicken Alexa 488 (Abcam), CF488A donkey anti-guinea pig (Sigma-Aldrich). Hoechst 33342  was used for nuclear staining. Stacks of confocal images were acquired  using a 40x/NA 1.3 oil immersion lens. The axial step size was 150 nm. Image stacks of GluA staining were deconvolved using Huygen’s software . Projections were made using Image J  and adjusted for brightness and contrast.Immunohistological staining was performed on 100-μm thick sagittal slices of olfactory bulbs of NMRI and hGFAP-eGFP mice. For anti-GluA staining, cerebella of the same animals were used as a positive control to verify specific antibody staining. After dissection, olfactory bulbs and cerebella were stored and refrigerated in paraformaldehyde solution  in phosphate buffered solution (PBS) containing (in mM): 130 NaCl, 7 NaHow to cite this article: Droste, D. et al. Ca2+-permeable AMPA receptors in mouse olfactory bulb astrocytes. Sci. Rep.7, 44817; doi: 10.1038/srep44817 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Studies on the role of mitochondrial Ca2+ signaling in pathophysiology have been favored by the identification of the genes encoding the mitochondrial calcium uniporter (MCU) and its regulatory subunits. Thus, research carried on in the last years on one hand has determined the structure of the MCU complex and its regulation, on the other has uncovered the consequences of dysregulated mitochondrial Ca2+ signaling in cell and tissue homeostasis. Whether mitochondrial Ca2+ uptake can be exploited as a weapon to counteract cancer progression is debated. In this review, we summarize recent research on the molecular structure of the MCU, the regulatory mechanisms that control its activity and its relevance in pathophysiology, focusing in particular on its role in cancer progression.Mitochondrial Ca Warburg proposed that mitochondrial dysfunction was the primary cause of preferential aerobic glycolysis. In addition, only cells able to increase glycolytic ATP production during periods of respiratory failure would progress toward cancer. Thus, cancer would be a consequence of progressive and irreversible defect in oxidative metabolism. However, experimental evidence revealed the weaknesses of this model. First, in some tumors, cells have normal respiratory capacity and/or are able to reactivate oxidative phosphorylation in case of impaired glycolysis. Second, in alternative to glucose metabolism, certain cancers utilize glutamine as an energy source  and release of proapoptotic factors which eventually lead to cell death , the highly selective channel responsible for mitochondrial Caescribed , the genescribed . Therefo2+ overload in triggering cell death. Thus, the old paradigm of excessive Ca2+ uptake being permissive for apoptotic cell death still holds true. However, other studies highlighted novel and still incompletely defined aspects of MCU in cancer, indicating that mitochondrial Ca2+ uptake plays multiple and complex roles in cancer biology. This review will summarize the molecular structure of MCU, the regulatory mechanisms underlying its activity, and will uncover its role in pathophysiology, with particular attention to cancer biology.Genetic modulation of MCU expression confirmed the previously recognized role of mitochondrial Ca2+ transport from the cytosol to the intermembrane space  are the most abundant proteins of the outer mitochondrial membrane. VDAC1, VDAC2, and VDAC3 are permeable to <5 kDa metabolites and are characterized by a weak anion selectivity in the open channel conformation, while they show strong cation preference in the closed states . However, despite this huge driving force, resting mitochondrial [Ca2+] is in the nanomolar range, indicating that the affinity of the channel for Ca2+ is low. In addition, the mitochondrial Na+/Ca2+ exchanger ensures efficient extrusion of Ca2+ from mitochondria , but also in the presence of too high [Ca2+] at which the inhibitory effect of Ca2+ on InsP3R prevails. Thus, efficient mitochondrial Ca2+ uptake is warranted by the prompt removal of Ca2+ from microdomains exerted by mitochondria.A 480 kDa inner mitochondrial membrane (IMM) multimeric complex, i.e., the MCU, comprising channel pore-forming subunits and regulatory elements, ensures Caa Figure . Long bechondria . Upon stly close , 14. The2+-selective mitochondrial channel whose current had the properties of that mediated by MCU  domains separated by a short hydrophilic loop enriched in acidic amino acids  and electron microscopy. Differently from the quaternary complex proposed for mammalian MCU  in mushroom body neurons during pupation caused memory impairment associated with a decrease in synaptic vesicles and an increased length in the axons  injury, although a difference was apparent in the effects of the mPTP inhibitor cyclosporin A (CsA). In contrast to wild-type hearts, MCU−/− hearts were not protected from I/R injury by CsA treatment. These data indicate that, besides mPTP opening by mitochondrial Ca2+ overload, alternative damaging pathways are activate by I/R which are not counteracted by MCU deletion  protease, is essential for correct assembly of the gatekeeping subunits  , particie effect . MICU1 ae effect . Howeverown mice . The phyown mice . Patron + uptake . This wo+ uptake . In addi+ uptake . However tissues .2+ uptake, which exceed those triggered by MICU1. While MICU2 overexpression reduces MICU1-induced mitochondrial Ca2+ uptake . CamKII would positively regulate MCU activity, and CamKII inhibition protects cardiomyocytes from I/R injury  promptly respond to InsP3-generating stimuli with high mitochondrial Ca2+ peaks. Clearly, efficient mitochondrial Ca2+ uptake in these cells is not primarily utilized to stimulate TCA cycle and oxidative phosphorylation and should have other functions, if any. Partial answers derive from studies of mitochondria metabolism that have unraveled important roles for mROS production, regulation of NADH/NADPH, and utilization of metabolic intermediates in cancer.The idea that mitochondrial Caic death . In recef cancer , includiin situ. Finally, MCU expression correlates with lymph node invasion  . During per se did not affect at all cell proliferation and viability. Also, when cells were treated with the BCL-2 inhibitor ABT-263, caspase-dependent cell death was unaffected. However, caspase-independent death triggered by treatment with the Ca2+ ionophore ionomycin was potentiated by MCU knockdown, suggesting that MCU inhibition could in principle be viewed as a therapeutical strategy against TNBC.In a particular cancer model, i.e., triple negative breast cancer (TNBC), the role of MCU has been extensively investigated both in terms of cell viability and on migration/invasion properties Table . Based o2+ uptake in cancer cell survival depends on cancer type and on stress stimuli, not to mention the role that cancer niche and extracellular environment exert. In this context, the role of MCU is still obscure.A similar hypothesis was tested by Hall et al. . As repo2+ entry (SOCE) which, in turn, was required for proper cell migration. Tosatto et al. confirmed the data that MCU is required for MDA-MB-231 migration and extended this observation to other triple-negative breast cancer cell lines. However, despite the consistent role on cell motility, the effect of MCU silencing on SOCE was cell line dependent, indicating that different mechanisms are involved, likely directly dependent on the strong reduction in mitochondrial Ca2+ uptake, rather than on a secondary effect on global Ca2+ homeostasis. MCU inhibition caused a strong reduction in mROS production and HIF-1α expression. In addition, restoring HIF-1α levels was sufficient to overcome the decrease in motility. The essential role of MCU in TNBC progression was demonstrated by in vivo experiments in which MDA-MB-231 cells were injected into the fat pad of immune-deficient SCID mice. Deletion of MCU markedly retarded primary tumor formation. In addition, at equal primary tumor size, absence of MCU impaired lung and lymph node colonization , MCU−/− and wild-type genotypes display similar decline in ejection fraction, fractional shortening, hypertrophy and fibrosis.On this issue, one of the first studies after the identification of MCU was carried out in neonatal cardiomyocytes ectively . HoweverR injury . Interes− strain , consist− strain . However of life . Upon ph2+ accumulation  cells. MCU inhibition impaired fight or flight heart rate by decreasing ATP levels, and dialysis of ATP was sufficient to rescue the phenotype in cardiac pacemaking cells . MCU gene deletion was induced in adult mice, and cardiac function was evaluated , 83. Sim−/− mice although at very mild levels. Skeletal muscle mitochondrial matrix [Ca2+] was reduced compared to wild-type controls, although not completely abolished. A decrease maximal oxygen consumption rate was detected. In addition, PDH activity was impaired in agreement with the pivotal role of mitochondrial Ca2+ accumulation in the control of ATP production. Finally, MCU−/− animals show defective maximal power output generation. Subsequently, to discriminate the compensatory effects occurring during embryonic development, MCU modulation was achieved after birth by means of adeno-associated viral vectors for both overexpression and silencing  the identity and role of uniporter channel components and interacting subunits, and their mutual regulation; (b) the transcriptional and posttranscriptional mechanisms that contribute to channel activity control; and (c) the pathophysiological consequences of dysregulated mitochondrial Ca2+ signaling. For any of these topics, impressive progresses have been achieved in a relatively short time. However, many questions are still unanswered. First, work is still needed to better clarify how the different channel subunits and regulatory components functionally interact with each other and to determine cell-type specificity of these mechanisms. Second, the regulation of uniporter components expression and activity by transcriptional and posttranscriptional events is a still largely unexplored field. Third, the pathophysiological role of MCU in different tissues is still unclear. Specifically on tumor development and progression, the role and significance of mitochondrial Ca2+ signaling has been studied only in few cancer types, and nothing is known on the effects of MCU activity in the tumor niche. In addition, although patient-derived samples have been analyzed for the expression levels of MCU components and correlation data between expression and prognosis have been obtained, in vivo tumorigenesis experiments aimed to determine the significance of aberrant mitochondrial Ca2+ uptake at different cancer stages have been rarely performed. Research in the next years will uncover these and others aspects of mitochondrial Ca2+ signaling.The seminal discoveries of CM, GG, and RR wrote the manuscript. GG prepared figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Its cytoplasmic tail contains six tyrosine residues , including three (Y2/5/6) lying within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that serve to recruit the protein tyrosine phosphatase SHP-1 after BCR activation-induced phosphorylation. The mechanism of inhibiting Ca2+i by CD22 has been poorly understood. Previous study demonstrated that CD22 associated with plasma membrane calcium-ATPase (PMCA) and enhanced its activity . The association is dependent on BCR activation-induced cytoplasmic tyrosine phosphorylation, because CD22 with either all six tyrosines mutated to phenylalanines or cytoplasmic tail truncated loses its ability to associate with PMCA. However, which individual or a group of tyrosine residues determine the association and how CD22 and PMCA interacts, are still unclear. In this study, by using a series of CD22 tyrosine mutants, we found that ITIM Y2/5/6 accounts for 34.3~37.1% Ca2+i inhibition but is irrelevant for CD22/PMCA association. Non-ITIM Y4 and its YEND motif contribute to the remaining 69.4~71.7% Ca2+i inhibition and is the binding site for PMCA-associated Grb2. Grb2, independently of BCR cross-linking, is constitutively associated with and directly binds to PMCA in both chicken and human B cells. Knockout of Grb2 by CRISPR/Cas9 completely disrupted the CD22/PMCA association. Thus, our results demonstrate for the first time that in addition to previously-identified ITIM/SHP-1-dependent pathway, CD22 holds a major pathway of negative regulation of Ca2+i signal, which is ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent.CD22 is a surface immunoglobulin implicated in negative regulation of B cell receptor (BCR) signaling; particularly inhibiting intracellular Ca Ca2+i B cells -8. Thus,as lupus -11. On tas lupus , 13.2+i signals and mitogen-activated protein kinase (MAPK) cascades through dephosphorylation of various signaling molecules and regulation of PLC-γ2 activity i i . The ass2+i signal, Ca2+i efflux, and CD22/PMCA interaction. Here we present data showing: Mutation of any two of the three ITIM tyrosines (Y2/5/6) has no impact on [Ca2+]i. Mutation of all three Y2/5/6 contributes to 34.3~37.1% of the [Ca2+]i attenuation. Surprisingly, all three ITIM tyrosines do not mediate the interaction with PMCA. Non-ITIM Y4 and Y4-affiliated YEND motif contributes to 69.4~71.7% of the [Ca2+]i attenuation and is the PMCA interacting site. Knockout of Grb2 completely disrupts the CD22/PMCA association and most of the CD22-dependent [Ca2+]i attenuation. Interestingly, we found that Grb2 is constitutively associated with PMCA in both avian and human B cells, independently of BCR cross-linking. In vitro pull-down experiments demonstrate a direct interaction between Grb2 and PMCA. These results indicate CD22 recruits PMCA through Grb2. Thus, our results identify for the first time that in addition to the classic ITIM/SHP-1-dependent pathway, CD22 exerts its negative regulation of Ca2+i signal through an ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent pathway.In this study, we introduced serial point mutations on the six tyrosine residues in the cytoplasmic tail of CD22 and have analyzed the contributions of each tyrosine residue to Ca2+i and its association with PMCA . Fura-2 buffer was also used as extracellular medium for measuring [Ca2+]i. The Ca2+-free buffer consisted of Fura-2 buffer with CaCl2 replaced by 1mM EGTA. Fluorescence from cells in suspension was monitored with a QuantaMaster Series fluorometer  at excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm. Anti-chicken IgM M4 or anti-human IgM (1~10 μg/ml) was used to activate BCR on B cells [2+]i  / [Empty vector's AUC - CD22-WT's AUC] x100%) by computer-assisted planimetry. Of the five, Δpeak and %ΔAUC are two key parameters.Cells . Cells (3×106) were suspended in 2 ml efflux buffer and [Ca2+]o brought to ~160nM with EGTA (pH 8.0). Efflux buffer contains 135mM NaCl, 1mM MgCl2, 5mM KCl, 10mM glucose, 10mM HEPES, 3mM EGTA, 0.1% BSA, pH 7.4. Similarly, the amplitude of [Ca2+]o elevation in response to BCR activation was calculated using the percentage of increase of F340/F380 with reference to F340/F380 at the resting state. The percentage at 300 seconds after BCR stimulation was set as a point for comparison between groups.[Cad method . In brieTM XRS+ (Bio-Rad).Cells were solubilized in 1% NP-40 lysis buffer  with a proteinase inhibitor mixture . Lysates were incubated sequentially with appropriate antibodies and protein A/G-agarose (Roche). The immunoprecipitants were washed four times with lysis buffer, boiled in SDS-sample buffer, and resolved by electrophoresis in a 7~12% SDS-PAGE, followed by blotting onto polyvinylidene difluoride membranes (Millipore). After overnight incubation at 4°C with primary antibodies , blots were washed 4 times in PBS containing 0.05% Tween-20 and then incubated with HRP-conjugated secondary antibodies (1:1000 dilution) for 1 hour at room temperature before extensive washes. The blots were visualized using an ECL detection kit (Thermo-Pierce) on the Chemi Dochttp://www.e-crisp.org) [The Grb2-specific guide RNA (gRNA) sequence preceding the PAM motif for CRISPR/Cas9 interference were designed from the region of the exon-1 of the chicken Grb2 gene Figure  and wereisp.org) , 57. The5 cells/ml in a 6-well plate were transfected with 2.5 μg of plasmid pVK-Grb2 by electroporation. Twelve hours post-transfection, media was refreshed. At 72 hours, the cells were re-seeded into a 10 cm dish with 1 μg/mL puromycin. The media with antibiotics was changed every 2 days. Upon complete cell death in the controls (untransfected cells with single puromycin selection) after a week of selection, cells were plated in two 96-well plates at a density of 0.3 cells per well to isolate single cell derived clones. Wells were monitored for single clone expansion. Upon observation, the expanded clone was divided into two parts, half for freezing and half for genomic DNA extraction.DT40 cells at a density of 2×10Western blotting.Genomic DNA from transfected and control DT40 cells was extracted using a genome isolation kit (Promega). The genomic fragment was obtained by PCR using KOD-FX-neo and primers (Grb2-Exon1-F: 5′-ACTTCCCCTAATGGCACGGC-3′ and Grb2-Exon1-R: 5′-CCAACATTTGACAGCCTGATG-3′) under the following conditions: 94°C for 2min; 35 cycles . The fragments from both transfected and control cells were purified, mixed in equal amount, and subjected to re-annealing reaction. Re-annealed PCR products were incubated for 30 min at 37 °C in the presence of 10 U of mismatch-sensitive T7 endonuclease I (New England Biolabs) and analyzed by agarose gel electrophoresis. Monoallelic deletion clones were defined as having both the non-deletion band and deletion band. Biallelic deletion clones were defined as having the deletion band and absence of the non-deletion band. Having the non-deletion band and absence of the deletion band were defined as non-deletion clones Figure . By usint test. P < 0.05 was taken as a significant difference between data sets.All data are given as the mean±S.E. Data were analyzed by using a paired or unpaired Student's


This review focuses on MCU and its regulators in cell migration during physiological and pathophysiological processes including development and cancer. We also present hypotheses to explain the molecular mechanisms by which MCU may regulate mitochondrial dynamics and motility to drive cell migration.Mitochondria are dynamic organelles involved in numerous physiological functions. Beyond their function in ATP production, mitochondria regulate cell death, reactive oxygen species (ROS) generation, immunity and metabolism. Mitochondria also play a key role in the buffering of cytosolic calcium, and calcium transported into the matrix regulates mitochondrial metabolism. Recently, the identification of the mitochondrial calcium uniporter (MCU) and associated regulators has allowed the characterization of new physiological roles for calcium in both mitochondrial and cellular homeostasis. Indeed, recent work has highlighted the importance of mitochondrial calcium homeostasis in regulating cell migration. Cell migration is a property common to all metazoans and is critical to embryogenesis, cancer progression, wound-healing and immune surveillance. Previous work has established that cytoplasmic calcium is a key regulator of cell migration, as oscillations in cytosolic calcium activate cytoskeletal remodelling, actin contraction and focal adhesion (FA) turnover necessary for cell movement. Recent work using animal models and  •Mitochondrial calcium dynamics control cell migration.•MCU and regulators are involved in development and cancer invasion.•MCU regulates cytoskeletal dynamics.•MCUM modulates ATP and ROS production, and intracellular calcium signalling. Mitochondrial plasticity allows their trafficking along the microtubules resulting in their strategic partitioning within the cell, which is crucial to ensure specialized functions such as immunity 2+ in cell migration is well established, the function of mitochondrial Ca2+ and dynamics has only emerged recently. Indeed, many studies have shown that the mechanisms regulating cell migration are deregulated during metastasis, and Ca2+ signalling dysfunction is correlated with increased metastatic invasion and poor prognosis 2+ uniporter (MCU) and its main regulators, the role of mitochondrial Ca2+ homeostasis in cell migration can be directly interrogated.Cell migration is a natural process, essential for a number of physiological functions including embryonic development, immunity and wound-healing. This process is controlled by different regulatory effectors, which orchestrate the remodelling of the cytoskeleton architecture 2+ flux in cell migration. We will discuss the intimate connection between mitochondrial Ca2+ homeostasis and mitochondria dynamics/motility during this process.In this review, we will describe recent evidence highlighting the role of mitochondrial Ca1.12+ signals are regulated by Ca2+ influx through the plasma membrane (PM) (extracellular [Ca2+] ≈ 1 mM) and Ca2+ release from intracellular stores, in particular from the Golgi ([Ca2+] ≈ 300 μM) and the ER ([Ca2+] ≈ 200–650 μM). To maintain the optimal cytosolic Ca2+ concentration (resting cytosolic [Ca2+] ≈ 100 nM), intracellular Ca2+ stores are constantly refilled while cytosolic Ca2+ is extruded from the cell by the plasma membrane Ca2+ ATPase (PMCA) pump. Intracellular Ca2+ is mainly stored in the ER lumen that is constantly refilled by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump 2+ release from the ER. ER Ca2+ can be released directly into the cytosol, or into juxtaposed organelles including mitochondria , so the basal Ca2+ concentration in the cytosol is not sufficient to allow an efficient mitochondrial uptake 2+ concentrations are needed to activate MCU activity. It is now well established that one of the main functions of mitochondria-ER contact sites 2+ microdomains facilitating Ca2+ transport into mitochondria 2+ first passes the outer mitochondrial membrane (OMM) through the voltage-dependent anion channel (VDAC) 2+ uptake has been associated with energized mitochondria, where depletion of mitochondrial membrane potential abrogates mitochondrial Ca2+ uptake and defects in the respiratory chain have been associated with a decreased ability of mitochondria to pump Ca2+The affinity of MCU for Ca2+ uptake has been associated with numerous physiological functions such as cell death, autophagy, skeletal muscle trophism, immunity, cardiomyocyte contraction and heart rate 2+ regulation and to investigate its role in intracellular Ca2+ signalling.Well before the discovery and the characterization of the uniporter, mitochondrial Ca1.22+ uptake in different animal species and cell types 2+ entry Saccharomyces cerevisiae, which lacks the mitochondrial uniporter, showed that expression of the human MCU alone was not sufficient to allow mitochondrial Ca2+ pumping activity 2+ concentration through its C-terminus end, facing the mitochondrial matrix, and modulate MCU activity 2+ uptake protein 1 (MICU1) 2+ concentration. MICU1 is considered as the MCU “gatekeeper”. At low extramitochondrial Ca2+ concentration, MICU1 stabilizes MCU in a close state inhibiting Ca2+ uptake; whereas at high Ca2+ concentration, the EF-hand motif of MICU1 binds Ca2+ leading to MICU1 conformational change and subsequently the opening of the channel and Ca2+ entry 2+ entry that probably needs to be finely regulated in different tissues  ensured by the calmodulin Ca2+-dependent kinase MLCK 2+-dependent proteases 2+ gradient with low Ca2+ concentration at the leading edge 2+2+ concentration allows the different components of the cell migration machinery to respond to local pulses of intracellular Ca2+ changes. Recently, transient and localized microdomains of high Ca2+ concentrations have been shown to be more active at the front of the migrating cells  disassembly 2+ are dependent on the store-operated Ca2+ entry (SOCE) Directional cell migration involves three important steps: triggering and maintaining cell polarity, remodelling cytoskeleton to activate linear locomotion and modifying the direction of movement in response to gradients of environmental variations. Cell movement begins with the formation of protrusions of the cell membrane, followed by the establishment of new focal adhesions (FA) at the leading edge to anchor the cytoskeleton to the extracellular matrix . Tractiong cells . These “1.42+ entry in non-excitable cells is the SOCE allowing entry in the cell of extracellular Ca2+ through the PM-localized Ca2+-activated Ca2+-release channel ORAI1 2+ concentration and the Ca2+ sensor ER-resident Stromal Interacting Molecule 1 (STIM1). Upon ER Ca2+-depletion, induced for example by sustained IP3 stimulation, cytosolic Ca2+ is extruded by PMCA and the ER refilled by SERCA. However, if the ER Ca2+ concentration remains too low, SOCE is activated by the STIM1/ORAI1 pathway in order to refill it 2+ concentration, Ca2+ dissociates from the STIM1-EF hand that senses the lumenal ER Ca2+, leading to the oligomerization of the protein. Cytoskeletal remodelling then promotes STIM1 relocalization specifically at ER-PM contact points where it interacts with ORAI1 and maintains the channel open 2+ influx required to maintain, for example, prolonged stimulation during cell migration.The main path for Ca2+ uptake. It has been proposed that mitochondria relocalize to the PM to directly buffer Ca2+ entry to inhibit the Ca2+-dependent inactivation of the ORAI1 channels  was associated with a deregulation of cell directionality and a decrease in actin polymerization dynamics leading to cell migration defects in vivo models have highlighted physiological functions of MCU. An elegant study showed that loss of the nematode orthologue of MCU (MCU-1) suppressed mitochondrial Ca2+ uptake and impaired wound healing 2+ sensitive GCaMP3 fluorescent probes, that a mitochondrial Ca2+ wave, induced by the cytosolic Ca2+ wave occurs after wounding. This wave of mitochondrial Ca2+ was totally inhibited in MCU-1 knockout preventing cytoskeleton remodelling during the healing process 2+ at wound sites to locally recruit polymerized actin. In fact, it was described that wounding induced Ca2+ waves in epithelial cells that were crucial to increase cell motility rate 2+ signalling linked to the regulation of cytoskeleton remodelling.Increasing evidence support an active role for mitochondrial Ca2+ entry 2+ level and a decreased capacity for mitochondria to uptake Ca2+ at high concentration (>15 μM). However, one study showed that MICU1-KO in C57BL/6 J background was lethal a few hours after birth due to failure in basic vital functions Surprisingly, the total MCU-KO in a mixed genetic mice background (outbred CD1 strain) exhibits only a discrete phenotype with a reduced exercise tolerance and skeletal muscle respiration correlating to a defect in PDH phosphorylation 2+ homeostasis can lead to an alteration of cell migration via defects in actin dynamics or premature embryonic death.Taken together, these studies indicate that deregulation of mitochondrial Ca2.22+ uptake resulted in enhanced cell resistance to apoptosis 2+ up-regulation can greatly enhance metastatic behavior. Recently, clinical data analysis of breast cancer patients has associated overexpression of MCU and downregulation of MICU1 to poor prognosis 2+ uptake accelerates cancer dissemination. Indeed, multiple in vitro studies using triple negative breast cancer (TNBC) and other breast cancer cell models have shown that depletion of MCU led to a drastic cell migration decrease, independent of cell proliferation in vivo tumor growth and metastasis progression in mice 2+ uptake by the Ruthenium 360, a potent inhibitor of MCU, also led to decreased cell migration capacity 2+ signalling from the ER and cytosol.The regulation of cell migration plays a major role in tumor metastasis allowing the movement of cancer cells to the periphery and the circulation. In prostate and colon cancers, it has been shown that overexpressed microRNA specifically downregulating MCU and dampening mitochondrial Ca2+ overload .It is now generally accepted that MICU1 serves as a molecular gatekeeper preventing mitochondria Ca2+ dynamics are important in cell migration. The challenge is to determine whether these roles are direct and/or indirect, and identify the molecular mechanisms that couple these dynamics to the signalling pathways that drive the migration process.Evidence is therefore emerging that mitochondrial Ca33.12+ on TCA enzyme activities, a deficiency of MCUM could potentially alter mitochondrial ATP production and subsequently global or local cytoskeleton remodelling. It has been put forward that a deficiency of MCU affects ATP production by decreasing resting mitochondrial Ca2+ levels. However, numerous studies reported that different cell lines with MCU deficiency did not exhibit respiration defect in basal conditions. Indeed, no detectable difference in oxygen consumption was noted in MCU-KO derived fibroblasts d-glucose treatment failed to induce an increase of ATP production 2+-induced ATP raise, stimulated by the cytokine cl-CD95L and impaired cell migration Cytoskeleton dynamics is an active process that is directly dependent on ATP levels. Given the regulatory role of mitochondrial Ca2+ in MCU deficient cells, the MICU1 deficiency that resulted in migration defects was associated with a consistent increase of resting mitochondrial Ca2+ levels. This rise in mitochondrial Ca2+ has been correlated to a better response to increased ATP demand 2+ load, variation in ATP levels is not likely to play a major role in the migratory phenotypes of MCUM deficient cells; rather the dynamics of the Ca2+ wave regulation may be more crucial.In contrast to the loss of mitochondrial Ca3.2in vivo tumor growth and metastasis progression in vitro and in vivo2+ concentration with increased mtROS production In 2016, Rizzuto's group proposed a mechanism involving mitochondrial MCU-regulated ROS production during cell migration in triple negative breast cancer (TNBC) cell lines 2+, ROS and cell migration have also been made with the Bcl-2 family of proteins. Beyond their role as key modulators of apoptosis 2+ uptake 2+ homeostasis via their direct interaction with VDAC1/3 2+ channeling through VDAC. The exact mechanism of ROS action is still controversial, however pioneering work showed that ROS produced within migrating cells promotes cell movement and are necessary for chemotaxis 202), which is believed to induce cellular changes by reversibly oxidizing the thiol group of cysteine residues of specific proteins 202 levels are regulated by peroxiredoxins, which have been shown to regulate ROS-mediated signal transduction in mammalian cells; for example, peroxiredoxin-2 overexpression has been shown to suppress chemotactic migration and adhesion induced by platelet-derived growth factor (PDGF) in MEFs in vivo promoted the growth and migration of smooth muscle cells during vascular remodelling C.elegans, it has been shown that wound closure by F-actin accumulation at wound site, requires the Cdc42 small GTPase and Arp2/3-dependent actin polymerization and is negatively regulated by RHO-1 and non-muscle myosin c.elegans model of MCU-1 KO leading to wound-healing defects, Xu et al. demonstrated that mitochondrial Ca2+ was totally inhibited in MCU-1 knockout and the mtROS production necessary to remodel the cytoskeleton during healing was prevented. The authors proposed that mtROS inhibited RHO-1 activity by oxidizing its redox sensitive motif to promote wound closure Links between mitochondrial Ca2+ regulates this release, Booth et al. recently demonstrated the interdependence of mitochondrial Ca2+ uptake and H202 release at the mitochondria-ER interface 2O2 accumulating in the mitochondrial cristae are compressed and released at mitochondria-ER contact sites upon Ca2+ signal propagation to the mitochondria, likely due to concomitant K+ and water influx to the matrix. Transient release of H2O2 in turn, would sensitize ER Ca2+ release to maintain Ca2+ oscillations 2+ at membrane contact sites is crucial for the generation and release of mtROS required for proper cell migration.Collectively, these studies show that mtROS production are involved in cell migration and their release in the cytoplasm requires a functional MCU. However, in MICU1 deficient EDV cells where cell migration was decreased, the authors observed a large increase of mtROS 3.32+ buffering 2+ pulses but also to directly control local extracellular Ca2+ entry at the PM during SOCE. Early work has provided solid evidence demonstrating that mitochondria can play a role in controlling the opening of the Ca2+ release-activated channels (CRAC) that regulate cellular Ca2+ entry through SOCE regulation 2+ homeostasis in regulating transient fluxes of cytosolic Ca2+. For example, it has been shown that MCU regulated leukotriene-induced physiological oscillations of cytoplasmic Ca2+ in a rat basophile cell line (RBL-1) 2+, which were inhibited by mitochondrial membrane depolarisation or MCU knockdown. Moreover knockdown of MCU induced a reduction in cytosolic Ca2+ oscillations indicating a proactive role of mitochondria in the propagation of physiological cytosolic Ca2+ signalling 2+ uptake to ensure IP3-induced STIM1 oligomerization in HeLa cells Mitochondria are scattered throughout the cytoplasm but their distribution can vary depending on local high-energy demands. For example, human ovarian adenocarcinoma cells increase local AMP-activated protein kinase (AMPK) activated mitochondria in cellular protrusions to respond to metabolic demands 2+ entry after ER-store depletion upon thapsigargin treatment. In these studies, inhibition of the SOCE by pharmaceutical approaches mcu-loss. These data suggest that mitochondrial Ca2+ homeostasis may control cytoskeleton dynamics and cell migration via the SOCE regulation. On the other hand, it has been reported that MCU silencing did not affect SOCE in three different breast cancer cell lines, attributing cell migration defects to a decrease in ROS production and HIF1 signalling 2+ entry during SOCE 2+-dependent inactivation of the CRAC channels, mitochondria would need to be closely located to the PM to influence this process (d < 30 nm). It has been shown that in contrast to ER Ca2+ release, Ca2+ entry via SOCE did not generate Ca2+ hotspots at the OMM. This suggests that mitochondria could not relocalize at ER-PM contact, where SOCE occurs, due to steric hindrance 2+ generated during SOCE by acting directly at the ER  have been identified and shown to be most active at the leading edge of migrating cells 2+ flicker activity develops across the lamella and promotes the turning of the cells towards the chemo-attractant 2+ pulses restricted to the leading edge were generated by IP3-stimulated local depletion of ER Ca2+, and local activation of STIM1, supporting pulsatile front retraction and adhesion of the cell 2+ buffer capacity of the mitochondria in this phenomenon. It is tempting to hypothesize that MCU may also regulate the intensity and the duration of those flickers. MCU may act on these Ca2+ flickers in two different ways: by its potential ability to regulate SOCE, or by direct Ca2+ buffering at the ER. Therefore, it will be important to design new experiments in migrating cells to elucidate the role of MCUM in this phenomenon.Recently, in human lung fibroblasts and umbilical vein EC (HUVEC), local high Ca3.42+ uptake may also impact cell migration via its capacity to regulate this process. So far, the role of MCU in mitochondrial motility and dynamics has not been fully investigated. Mitochondrial motility along microtubules is ensured by mitochondrial adaptor, motor proteins and cytoskeleton components, which are regulated by cytosolic Ca2+2+ binding domains and plays an important role in mitochondrial motility along microtubules. High cytoplasmic Ca2+ levels halt mitochondrial movement by binding to miro1 EF-hand domains 2+ can also play a critical role in mitochondrial transport along the axons 2+ content was inversely proportional to the speed of mitochondrial movement. Thus, by regulating intracellular Ca2+ signal, MCU may indirectly control mitochondrial motility.As described previously, mitochondria need to relocalize to the leading edge of the migrating cells to ensure their function . Mitocho2+2+ levels, MCUM is likely to affect mitochondrial dynamics and distribution, which may impact cell migration. In mouse embryonic fibroblasts derived from MCU-KO, the mitochondrial shape was not altered by MCU loss 2+ pool harbored fragmented mitochondria 2+ influx into mitochondria that drove mitochondrial fragmentation 2+ was required for mitochondrial fragmentation by modulating Drp1 level 2+ uptake in mitochondrial dynamics.Mitochondrial motility is also regulated by mitochondrial shape and it has been shown that fragmented and smaller mitochondria move faster along microtubules. Increasing evidence in multiple studies has shown that mitochondrial dynamics are involved in cell migration and cancer invasiveness Thus, it will be interesting to further investigate the specific role of MCUM in mitochondrial motility/dynamics during cell migration.42+ homeostasis in immune cell polarization and chemotaxis Recent evidence has shed a light on the crucial role of MCU and its regulators in cell migration. Independently of the role of MCU in cancer cell migration, different studies have highlighted the role of mitochondrial Ca2+ signal regulation have been investigated. To fully elucidate these mechanisms, it will be important to decipher the potential role of the MCUM on Ca2+ flickers formation/intensity but also the impact of MCUM on mitochondrial dynamics at the leading edge during cell migration.If MCU is involved in cytoskeleton remodelling, the full mechanism of its impact on cell migration remains to be discovered. So far, the non-exclusive connections between ATP and ROS production and cytosolic Ca2+ uptake as a potential therapeutic target in highly proliferative cancers.Finally, the fact that MCU is overexpressed in breast cancer patients and the clear evidence linking MCU to cancer invasion and growth, points to mitochondrial Ca


The purpose of this study was to determine the actions of LXA4 on cultured human conjunctival goblet cell mucin secretion and increase in intracellular [Ca2+] ([Ca2+]i) and on histamine-stimulated responses. LXA4 increased mucin secretion and [Ca2+]i, and activated ERK1/2 in human goblet cells. Addition of LXA4 before resolvin D1 (RvD1) decreased RvD1 responses though RvD1 did not block LXA4 responses. LXA4 inhibited histamine-stimulated increases in mucin secretion, [Ca2+]i, and ERK1/2 activation through activation of β-adrenergic receptor kinase 1. We conclude that conjunctival goblet cells respond to LXA4 through the ALX/FPR2 receptor to maintain homeostasis of the ocular surface and regulate histamine responses and could provide a new therapeutic approach for allergic conjunctivitis and dry eye diseases.Conjunctival goblet cells synthesize and secrete mucins which play an important role in protecting the ocular surface. Pro-resolution mediators, such as lipoxin A Conjunctival goblet cells are specialized cells that span the thickness of the conjunctiva from the ocular surface to the stroma. These cells synthesize and secrete mucins which include the gel forming mucin MUC5AC in humans and in rats13In the context of the ocular surface, the types of inflammation observed include seasonal allergic conjunctivitis, and dry eye syndrome5789101 and CysLT2, are expressed in goblet cells2+] ([Ca2+]i) and high molecular weight glycoconjugate secretion including MUC5AC15During the allergic response, mast cells are recruited to the ocular surface, degranulate, and release histamine and leukotrienes (LT)1315162+]i, activate extracellular regulated kinase (ERK) 1/2, and stimulate mucin secretion and (2) block LTD4- and histamine-stimulated increase in [Ca2+]i and mucin secretion19Termination of inflammation occurs with the biosynthesis of the specialized pro-resolution mediators (SPMs) resolvins (Rvs), lipoxins (LX), maresins, and protectins from omega-3 and -6 essential fatty acids192021224 and lipoxin B4 (LXB4) are both biosynthesized from arachidonic acid. LXA4 binds to the ALX/FPR2 receptor causing a conformational change leading to stimulation of pro-resolution pathways4, LXB4, and several stable analogs of LXA4 alone increased [Ca2+]i in conjunctival goblet cells from rats. LXA4 also increased mucin secretion utilizing the signaling pathways of phospholipase C, -D, and A22+]i stimulated by LXA4 was directly linked to mucin secretion as chelation of intracellular Ca2+ blocked secretion4 with cultured human conjunctival goblet cells, as well as the impact of LXA4 on histamine-stimulated increase in [Ca2+]i, mucin secretion, and ERK 1/2 activation in rat and human goblet cells. In human goblet cells, LXA4 binds to the ALX/FPR2 and GPR32 receptors while RvD1 binds to GPR32 receptors. In rat, LXA4 and RvD1 preferentially bind to the ALX/FPR2 receptor. In addition, we report that LXA4 utilizes β-adrenergic receptor kinase (βARK) 1 to counter-regulate the H1 histamine receptor.LXA4 stimulated an increase in [Ca2+]i and mucin secretion in rat goblet cells4 on goblet cells grown from human conjunctiva, cells were stimulated with LXA4 from 10−11 M–10−9 M and the increase in [Ca2+]i was measured. Pseudo colored images of goblet cells treated with increasing concentrations of LXA4 are shown in Fig. 1A. LXA4 increased [Ca2+]i in a concentration dependent manner with increases in peak [Ca2+]i of 97.6 ± 31.2, 762.0 ± 259.4, and 50.0 ± 14.4 nM at 10−11, 10−10, and 10−9 M, respectively .We previously demonstrated that LXAectively . The inc4 has been shown to bind to the ALX/FPR2 receptor in goblet cells isolated from rat conjunctiva4 acts via ALX/FPR2 receptors in human goblet cells, goblet cells were pretreated with the ALX/FPR2 antagonist BOC-2 (10−4 M) for 30 min, prior to addition of LXA4 (10−10 M). Similar to rat goblet cells, in cultured human cells BOC-2 inhibited LXA4-stimulated increase in [Ca2+]i at 10−10 M by 98.5 ± 1.1% to 13.3 ± 11.7 nM (p=0.05) (LXA(p=0.05) .4 on glycoprotein secretion from cultured human goblet cells was determined. Goblet cells were serum-starved for 2 h and LXA4 was added (10−10–10−8 M) for 2 h and glycoconjugate secretion measured. LXA4 (10−9 M) increased secretion 2.6 ± 0.1 fold above basal  prior to stimulation with RvD1 (10−8 M) and [Ca2+]i was measured. In the absence of inhibitor, RvD1 increased [Ca2+]i by 242.8 ± 70.8 nM  first caused a peak increase in [Ca2+]i of 256.2 ± 62.5 nM .In a second set of experiments, the following experimental paradigm was used: addition of first agonist was followed 5 minutes later by addition of second agonist and the increase in peak i of 576.5 ± 149.8 nM (4 resulted in a decrease from the first peak and was 20.4 ± 13.8 nM (p = 0.04). If RvD1 is given first, the addition of LXA4 resulted in change in peak [Ca2+]i of 391.7 ± 53.7 nM. This was not different from the result obtained with LXA4 added first  for 30 min and stimulated with histamine (10−5 M) for 2 h. In untreated rat cells, histamine increased mucin secretion 1.9 ± 0.1 fold increase above basal (p=0.002). LXA4 blocked histamine-stimulated secretion by 75.8 ± 8.8 and 90.8 ± 4.0% at 3 × 10−10 and 10−9 M, respectively  increased [Ca2+]i. with a peak of 587.1 ± 92.3 nM  decreased histamine-stimulated increase in [Ca2+]i at 10−9 and 10−8 M with maximum inhibition occurring at 10−9 M LXA4 which decreased the histamine response by 64.1 ± 14.1% to 185.6 ± 52.1 nM  stimulated an increase in peak [Ca2+]i 1152.5 ± 173.9 nM  decreased histamine-stimulated increase in [Ca2+]i at all concentrations with maximum inhibition occurring at 10−10 M LXA4 which decreased the histamine response by 66.1 ± 7.3% to 378.6 ± 92.6 nM  for 15 min prior to addition of LXA4 (10−9 M) for 30 min. The [Ca2+]i was then measured in response to histamine (10−5 M). In the absence of BOC-2 and LXA4, the change in peak [Ca2+]i in response to histamine was 586.8 ± 173.1 nM . Preincubation with BOC-2 reversed the inhibition by LXA4 on the histamine response and increased [Ca2+]i by 495.0 ± 76.6 nM  for 30 min prior to incubation with histamine (10−6 M) for 5 min and ERK 1/2 activity was measured. Histamine increased ERK 1/2 activity 1.3 ± 0.1 fold increase above basal , showed that this receptor has consensus sequences for β-adrenergic receptor kinase 1 (βARK1), also known as G-protein coupled receptor kinase (GRK)-2 and protein kinase C (PKC). We previously demonstrated that RvD1 binding to GPR32 activates both these kinases to counter-regulate the H1 histamine receptor to block the increase in [Ca2+]i4 also use βARK1 and/or PKC to counter regulate histamine H1 receptor, rat goblet cells were pretreated with either LXA4 or LXA4 plus inhibitors to βARK1 and PKC. The increase in [Ca2+]i in response to the specific H1 receptor agonist, histamine dimaleate was measured in cultured rat goblet cells. Pretreatment with LXA4 decreased the histamine dimaleate stimulated increase in [Ca2+]i from 823.7 ± 154.1 nM above basal in the absence of LXA4 to 140.9 ± 68.7 nM  alone did not have an effect of the histamine dimaleate . In these experiments, histamine dimaleate increased [Ca2+]i by 751.7  C, PLD, and PLA2 signaling pathways  and RvD1 was purchased from Cayman Chemical, Ann Arbor, MI). Both compounds were dissolved in ethanol as supplied by the manufacturer and were stored at −80 °C with minimal exposure to light. Immediately prior to use, the SPMs were diluted in with Krebs-Ringer bicarbonate buffer with HEPES  to the desired concentrations and added to the cells. The cells were then incubated at 37 °C in the dark. Daily working stock dilutions were discarded following each experiment. N-BOC-Phe-Leu-Phe-Leu-Phe (BOC-2) was purchased from Genescript .Synthetic LXAHuman conjunctiva was obtained from Eversight . Tissue was placed in Optisol media within 18 h after death.Male Sprague-Dawley rats  weighing between 125 and 150 g were anesthetized with CO2 for 1 min, decapitated, and the bulbar and forniceal conjunctival membranes removed from both eyes. All experiments were approved by the Schepens Eye Research Institute Animal Care and Use Committee and carried out in accordance to the protocols approved by this committee.Goblet cells from human and rat conjunctiva were grown in organ culture as described and extensively characterized previously4161740412+]i with time or as the change in peak [Ca2+]i. Change in peak [Ca2+]i was calculated by subtracting the average of the basal value (no added agonist) from the peak [Ca2+]i. Although data are not shown, the plateau [Ca2+]i was affected similarly to the peak [Ca2+]i.Goblet cells were incubated for 1 h at 37 °C with KRB-HEPES with 0.5% BSA containing 0.5 μM fura-2/AM , 8 μM pluronic acid F127, and 250 μM sulfinpyrazone followed by washing in KRB-HEPES containing sulfinpyrazone. Inhibitors were added for the last 30 min of the fura-2 incubation. Calcium measurements were made with a ratio imaging system  using wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. At least 10 cells were selected in each experimental condition. Data were collected in real time and are presented as the actual [Ca4 or histamine in serum-free RPMI 1640 supplemented with 0.5% BSA for 2 h. Inhibitors were added 30 min prior to stimulation. Goblet cell secretion was measured using an enzyme-linked lectin assay (ELLA) with the lectin UEA-I. UEA-1 detects high molecular weight glycoconjugates containing L-fucose including mucin MUC5AC produced by goblet cellsCultured goblet cells were serum starved for 2 h before use and then stimulated with either LXACultured human goblet cells were homogenized in TRIzol and total RNA was isolated. One microgram of purified total RNA was used for complementary DNA (cDNA) synthesis using the Superscript First-Strand Synthesis system for RT-PCR . The cDNA was amplified by the polymerase chain reaction (PCR) using primers specific to human ALX/FPR2 receptor using the Jumpstart REDTaq Readymix Reaction Mix  in a thermal cycler . The primers were from published sequences4 was added 30 min prior to histamine (10−6 M) for 5 min. The antibody against phosphorylated (active) ERK 1/2 was used diluted 1:200 and total ERK 1/2  was diluted 1:500. Immunoreactive bands were visualized by the enhanced chemiluminescence method. The films were analyzed with Image J software (http://rsbweb.nih.gov/ij/). Values for phosphorylated ERK 1/2 were normalized to total ERK 1/2. Control value was set as 1.Cultured goblet cells were homogenized in RIPA buffer  containing a protease inhibitor cocktail . The lysate was centrifuged at 2000 g for 30 min at 4 °C. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and processed for western blotting. The antibody against the ALX/FPR2 receptor  was diluted 1:1000. To measure activation of ERK 1/2, LXA2HPO4, and 2.7 mM NaH2PO4 (pH 7.2)) for 4 hours at 4 °C. The coverslips were rinsed for 5 minutes in PBS, and nonspecific sites were blocked by incubation with 1% bovine serum albumin, and 0.2% Triton X-100 in PBS for 45 minutes at room temperature. ALX/FPR2 receptor antibody (Novus Biologics) was used at 1:100 dilution overnight at 4 °C. UEA-1 directly conjugated to FITC  was used at a dilution of 1:300 to identify goblet cells. Secondary antibodies were conjugated to Cy 3  was used at a dilution of 1:150 for 1 h at room temperature. Negative control experiments included incubation with the isotype control antibody. The cells were viewed by fluorescence microscopy  and micrographs were taken with a digital camera .First passage cells were grown on glass cover slips and were fixed in 4% formaldehyde diluted in phosphate buffered saline .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


However, the low aqueous solubility and easy photodegradation impede the topical application of hyperforin for therapeutic purposes. We designed a compound hydroxypropyl-β-cyclodextrin- (HP-β-CD-) tetracapped hyperforin, which had increased aqueous solubility and improved photoprotection. We assessed the physiological effects of hyperforin/HP-β-CD on wound healing in HaCaT keratinocytes using live imaging to observe the ATP release and the intracellular Ca2+ increase. In response to stretching (20%), ATP was released only from the foremost cells at the wound edge; it then diffused to the cells behind the wound edge and activated the P2Y receptors, which caused propagating Ca2+ waves via TRPC6. This process might facilitate wound closure, because the Ca2+ response and wound healing were inhibited in parallel by various inhibitors of ATP-Ca2+ signaling. We also applied hyperforin/HP-β-CD on an ex vivo skin model of atopic dermatitis and found that hyperforin/HP-β-CD treatment for 24 h improved the stretch-induced Ca2+ responses and oscillations which failed in atopic skin.Cutaneous wound healing is accelerated by mechanical stretching, and treatment with hyperforin, a major component of a traditional herbal medicine and a known TRPC6 activator, further enhances the acceleration. We recently revealed that this was due to the enhancement of ATP-Ca During the wound healing process, these exogenous stimuli and the endogenous stimuli, such as the tension and traction forces generated between the migration of the foremost cells and the cells that are located behind them, may affect the rate of wound closure. Our earlier study demonstrated that mechanical stretching facilitated wound closure in bovine aortic endothelial cells . We receout mice .Hypericum perforatum L.) extract, which is widely used in traditional herbal medicines, to promote wound healing [β- and γ-cyclodextrin (CD) was reported to enhance the photoprotection and solubility of hyperforin in aqueous solutions [β-cyclodextrin (HP-β-CD) to improve its aqueous solubility and photostability, because HP-β-CD has been shown to possess the highest solubility not only in water but also in ethanol among several of the CD compounds that are commonly used. We also assessed the effects of the compound on the wound healing and ATP-Ca2+ signaling in HaCaT keratinocytes.Hyperforin is a major active constituent of St. John's wort  and hydroxypropyl-β-cyclodextrin  as described below. The other chemicals and reagents were as follows: carbenoxolone disodium salt (CBX), apyrase (from potato), GdCl3, U73122, and Cremophor EL ; ionomycin ; suramin hexasodium ; GsMTx-4 ; diC8-PIP2 ; dispase ; Fluo-8 AM ; Cellmatrix type IA ; Lipofectamine ; DME/F12 ; FBS .Hyperfolin/hydroxypropyl-−4 M hyperforin (in 1 mL methanol) and 1.86 × 10−3 M HP-β-CD (in 1 mL ethanol) were mixed and then stirred for 30 min. The solvents were then removed in vacuo with a centrifugal evaporator  at ambient temperature. The obtained white solid was dissolved in Milli-Q water by ultrasonication for at least 10 min. The resulting aqueous solution of hyperforin/HP-β-CD was syringe-filtered with a 0.20 μm pore size and kept in a freezer until use. All of the procedures were performed under light-shielded conditions.Solutions of 4.66 × 10β-CD was spectroscopically determined. The concentration of hyperforin in these studies was 4.66 × 10−4 M whereas the HP-β-CD concentration was used in the range of 0–8.0 equivalents. The UV spectra of hyperforin were recorded using a UV/VIS scanning spectrophotometer . The changes in the absorbance of hyperforin following the addition of various concentrations of the HP-β-CD complexing agent were measured at λmax 281 ± 7 nm.Stoichiometry of the reaction between hyperforin and HP-μL were taken every 30 min for the analysis. All of the quantitative measurements were conducted using a Hitachi LaChrom Elite HPLC system  equipped with a quaternary pump (L-2130), an autosampler (L-2200), a column oven (L-2300), and a diode array detector (DAD/L-2450). Separation was performed using a TSKgel ODS-100Z reversed phase column  with a mobile phase composed of acetonitrile-water-methanol-trifluoroacetic acid . The flow rate was 1.6 mL/min. The UV detector was set at 270 nm. Curve fitting was performed using Excel (MS Office 2013) to minimize the R2 value.An irradiation test was performed using a 6-watt LED light bulb  that was placed 14 cm above the samples. Irradiation was conducted in a dark room under temperature control (25°C). Aliquots of 40 2+) supplemented with 2% FBS at 37°C in a humidified atmosphere of 5% CO2. The growth medium was prepared from DME/F12  by adding 0.07 mM Ca2+, 365 mg/L L-glutamine, 59.05 mg/L L-leucine, 91.25 mg/L L-lysine·HCl, 61.2 mg/L MgCl2·6H2O, 48.84 mg/L MgSO4 (anhydrous), 17.2 mg/L L-methionine, and 1.2 g/L NaHCO3 and was adjusted to pH 7.4 with 1 mM NaOH. For the experiments, the cells were seeded on a collagen-coated (Cellmatrix type IA) silicone stretch chamber (see the following) or 15 mm round glass coverslips  and cultured in DME/F12 (1.05 mM Ca2+) supplemented with 10% FBS to allow cell attachment. After 1 day, the medium was replaced with DME/F12 (0.07 mM Ca2+) supplemented with 2% FBS, and the cells were further incubated for 1 day to achieve confluence.HaCaT human keratinocyte cells  at passaThe physiological experiments were performed as described previously . A brief2+ imaging or an upright microscope  for extracellular ATP imaging. HaCaT cells were seeded on collagen-coated silicone stretch chambers or 15 mm round glass coverslips at 3 × 105 cells/cm2 and grown to confluence. A narrow cell-free gap (about 250 μm) was created in a fully confluent monolayer by removing a silicone strip that was attached to the bottom of the stretch chamber during cell seeding. The wound closure process was monitored every 3 h after making the scratch using an inverted microscope (IX-70 Olympus) with a 4x  objective. The wound closure speed was defined as the percentage of the wound closure area, which was calculated from the ratio of the final migrated area to the initial cell-free area.The cells were cultured in a stretch chamber molded out of Silpot 184 W/C silicone elastomers . A chamber with cultured cells was attached to a stretching machine  mounted on the stage of an inverted microscope  for intracellular CaμM Fluo-8 AM using 0.1–0.2% of Cremophor EL (Sigma-Aldrich) for 40–60 min in an incubator at 37°C. After washing away the dye with DME/F12 containing 2.0 mM Ca2+, the chamber with the cells was attached to the stretching device on the stage of an inverted microscope with 4x  or 10x (UPlanFL 0.30) objectives. Time-lapse Fluo-8 fluorescence images were acquired at 0.5 s intervals using MetaMorph software .At 3 h after making a scratch, the HaCaT cells in the stretch chamber were loaded with 1 2+ and 10 mM HEPES, pH 7.4) containing high-sensitivity luciferin-luciferase solution . Images were acquired using the MetaMorph software with a stream acquisition mode (exposure time 100 ms).The stretch-induced release of ATP was measured in real-time using the imaging system, as described previously . BrieflyTRPC6 shRNA plasmids that coexpressed RFP  were used. The shTRPC6 targeting sequence was 59-AAGCAGGACATCTCAAGTCTCCGCTATGA-39. A scrambled noneffective plasmid with the same nucleotide content was used as a negative control. Each shRNA at a concentration of 45 nM was transfected into HaCaT cells using Lipofectamine reagent, according to the manufacturer's instructions.β-CD at 4°C, the epidermis was peeled off from the dermis with forceps. The detached pieces of epidermis were fixed with intradermal needles on an elastic silicone chamber and were further incubated at 37°C in a humidified 5% CO2 atmosphere for 12 h. Epidermis tissue was loaded with 1 μM Fluo-8 AM using 0.2% of Cremophor EL in culture media for 1 h at 37°C. After washing away the dye with DME/F12 containing 2.0 mM Ca2+, the chamber containing the cells was attached to a pulse-motor-driven stretching machine  mounted on the stage of an inverted laser scanning confocal microscope . Time-lapse Fluo-8 fluorescence and Nomarski differential interference contrast images were acquired at 1 s intervals. The Ca2+ imaging experiments were performed at room temperature (24 ± 2°C).Biopsies were taken from the outer forearm of a volunteer with atopic skin. Written informed consent was obtained from the volunteer. The study was approved by the Pixy Central Institute Ethics Committee, 2009. The sample of skin tissue was placed in PBS prior to treatment with dispase. After overnight digestion with 500 U/mL dispase in serum-free F12/DME with or without hyperforin/HP-β-cyclodextrin (HP-β-CD) at different molar ratios using the solvent evaporation method. The hyperforin/HP-β-CD complexes were investigated with UV/Vis spectroscopy in aqueous solution. The molar ratio method was used to determine the stoichiometry of the inclusion complex formed by hyperforin and HP-β-CD. ΔA, the difference in the absorbance of hyperforin with and without HP-β-CD, was plotted against the molar ratio of HP-β-CD to hyperforin at 280 nm  in the curve for hyperforin/HP-β-CD, suggesting the existence of a nondegraded (photoprotected) form in the complex  treatment further enhanced the effect of stretching, as shown previously. Hyperforin/HP-β-CD (1 μM) was equally (or more) effective in facilitating wound closure.We previously demonstrated that the wound closure of keratinocytes was accelerated by stretching and that hyperforin treatment further enhanced the effect  Figure . In the 2+ waves in hyperforin-treated HaCaT cells, which were initiated from the leading cells on the wound edge and that this occurred due to the release of ATP from the leading cells and the activation of TRPC6 on the cells behind the leading edge through the activation of P2Y with the spread ATP [β-CD also has the same effects on HaCaT keratinocytes. At 3 h after making a narrow scar on the confluent monolayer of hyperforin/HP-β-CD-treated cells, stretching  induced an increase in the intracellular Ca2+ in almost all of the leading cells on the wound edge and the Ca2+ increase propagated towards the rear cells behind the edge in a wave-like pattern (https://doi.org/10.1155/2017/8701801). The Ca2+ waves occurred due to the release of ATP from the leading cells and its diffusion to the surrounding cells behind the edge, as shown in 2+ signaling and wound healing in HaCaT cells [It was reported that stretch stimulation induced intercellular Caβ-CD was that the effects on the stretch-induced ATP and Ca2+ signaling were more reproducible than those obtained simple hyperforin. This may be attributed to the improvement of photostability and the aqueous solubility of hyperforin/HP-β-CD.One notable advantage of hyperforin/HP-2+response, the effects of various inhibitors on the Ca2+response were evaluated in hyperforin/HP-β-CD-treated HaCaT cells. The time course of the intracellular Ca2+response induced by a 20% stretch was measured at different distances (0–240 μm) from the scar (β-CD-treated cells). At 0 μm (wound edge), the Ca2+response was evoked immediately after stretching and it was prolonged by several min. When the distance from the edge was increased, a longer time lag was found before the onset of the activity; however, the amplitudes of the plateau phase were nearly the same. These results were consistent with the idea that Ca2+ waves caused by the simple diffusion of ATP released from the leading cells at wound edge and the activation of P2Y in the surrounding cells behind the wound edge. When Gd3+ (10 μM), an inhibitor of the stretch-activated channel, was applied, the Ca2+ response in the peak was reduced and the rate of decay was obviously faster, especially at the distant regions , apyrase , and CBX  suggested the contribution of ATP signaling in this process. The reduction by each treatment with U73122  and diC8-PIP2  suggested that the P2Y receptor-Gq-PLC-DAG-mediated signaling cascade was involved in the activation of TRPC6. These results were the same as those obtained by treatment with hyperforin (nonencapsulate) and stretch stimulation [2+ via TRPC6 through the Gq-PLC-DAG cascade.To analyze the characteristics of the stretch-induced Ca regions . The Ca2istances . The supβ-CD facilitates wound closure by amplifying ATP-Ca2+ signaling, we assessed the effects of the various inhibitors that were used above on the wound closure during sustained stretching. The wound gap was almost closed at approximately 6 h , CBX (100 μM), apyrase (20 U/mL), suramin (100 μM), Gd3+(10 μM), GsMTx-4 (5 μM), and shTRPC6 treatment delayed wound closure  and subsequent Ca2+ waves with the long-lasting elevation of Ca2+ , which mimicked the extracellular Ca2+ environment for barrier-perturbed epidermis, such as the environment that would result from skin stripping or the use of surfactants. Atopic dermatitis is also a skin barrier dysfunction. Topical medication of hyperforin-rich St. John's wort cream has been shown to be effective in patients with atopic dermatitis [2+dynamics of atopic skin behave under mechanical environments such as wound healing and reepithelialization. We observed the Ca2+ dynamics induced by the stretching of epidermis ex vivo using a confocal microscope , which possessed increased aqueous solubility and improved photoprotection. Treatment with hyperforin/HP-β-CD enhanced the mechanically induced ATP-Ca2+ signaling and accelerated wound closure in HaCaT keratinocytes with equal (or greater) efficacy to hyperforin. We also applied hyperforin/HP-β-CD on atopic skin ex vivo and found that hyperforin/HP-β-CD treatment for 24 h improved the stretch-induced Ca2+ responses and oscillations, which reduced in atopic skin. The data suggest that hyperforin/HP-β-CD is a potent targeted therapeutic agent that can be used to promote epidermal wound healing and to treat atopic dermatitis.Cutaneous wound healing is accelerated by mechanical stress both exogenously and endogenously, and treatment with hyperforin enhances the acceleration through the facilitation of ATP-CaSupplemental Movies2+ responses and the propagation of Ca2+ waves from the wound edge. S1. The stretch-induced CaS2. The stretch-induced release of ATP from the leading cells of the wound edge . 2+ oscillations and the Ca2+ response to stretching in the epidermis of normal skin ex vivo. S3. The Ca2+ response to stretching in the epidermis of atopic skin ex vivo. S4. The reduced Ca2+ oscillations and Ca2+ response to stretching in the epidermis of atopic skin ex vivo. S5. The hyperforin/HP-β-CD treatment-enhanced Ca


Besides, transient receptor potential channels also participate in the process of calcium inwards. In this review, we summarize the recent researches on the spatial-temporal distribution of store-operated calcium entry components and transient receptor potential channels. Questions about how these channels play function for calcium influx and what impacts these channels have on oocytes are discussed. At the time of sperm-egg fusion, sperm-specific factor(s) diffuse and enable eggs to mount intracellular calcium oscillations. In this review, we also focus on the basic knowledge and the modes of action of the potential sperm factor phospholipase C zeta, as well as the downstream receptor, type 1 inositol 1,4,5-trisphosphate receptor. From the achievement in the previous several decades, it is easy to find that there are too many doubtful points in the field that need researchers take into consideration and take action in the future.Under the guidance and regulation of hormone signaling, large majority of mammalian oocytes go through twice cell cycle arrest-resumption prior to the fertilized egg splits: oocyte maturation and egg activation. Cytosolic free calcium elevations and endoplasmic reticulum calcium store alternations are actively involved in triggering the complex machineries and events during oogenesis. Among these, calcium influx had been implicated in the replenishment of endoplasmic reticulum store during oocyte maturation and calcium oscillation during egg activation. This process also drove successful fertilization and early embryo development. Store-operated Ca Close attention had been paid to the molecular signaling basis of arrest exit downstream calcium (Ca2+) oscillation. The cytostatic factor, maturation-promoting factor (MPF), is composed of catalytic subunit cyclin-dependent kinase 1 (Cdk1)/cell division cycle protein 2 (cdc2) and regulatory subunit cyclin B. The inactivated MPF enforces Ca2+ command to call for cell cycle resumption. Two multiple interacting programs are responsible for the inactivation of MPF. On the one hand, the phosphorylation state of Cdk1/Cdc2 is coordinated by kinases and phosphatases. On the other hand, the synthesis and degradation of cyclin B are balanced. Reactivation and down-regulation of Wee1B exercised a great influence on Cdk1/Cdc2 kinase activity and pronucleus forming, and Wee1B inhibited Cdk1/Cdc2 activity by phosphorylating tyrosine 15 i), known as “Ca2+ oscillation”, triggered by specific sperm factor. The successful cell cycle arrest and punctual exit are orchestrated by well-balanced [Ca2+]i concentrations and complicated regulators/pathways, deeper issues needs to be solved in the future. The Ca2+ oscillation in mammalian eggs is featured by a train of long-lasting repetitive intracellular Ca2+ transients i was broadened and the interval between spikes was widened, indicating the active participation of PMCA in [Ca2+]i oscillations shaping [2+ reservoir” and is indispensable for Ca2+ oscillations. Replenishment and discharge of [Ca2+]ER require sarco-endoplasmic reticulum Ca2+ ATPases (SERCA) and type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) function during [Ca2+]i oscillations. Addition of SERCA inhibitors (thapsigargin or cyclopiazonic acid (CPA)) could perturb Ca2+ oscillations by reducing basal [Ca2+]ER levels and holding the recovery back ER content increased, which showed the gradual inactivation of Ca2+ entry in mouse i oscillations in mouse [2+]i without affecting IP3R1 i was broadened and the interval between spikes was widened, indicating the active participation of PMCA in [Ca2+]i oscillations shaping [2+ reservoir” and is indispensable for Ca2+ oscillations. Replenishment and discharge of [Ca2+]ER require sarco-endoplasmic reticulum Ca2+ ATPases (SERCA) and type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) function during [Ca2+]i oscillations. Addition of SERCA inhibitors (thapsigargin or cyclopiazonic acid (CPA)) could perturb Ca2+ oscillations by reducing basal [Ca2+]ER levels and holding the recovery back i vibration, blocked ER refilling of Ca2+ and thus enhanced [Ca2+]i [2+]ER refilling and normal oscillations maintaining, but there are other possibilities, including, just conceivably, it may uptake Ca2+ into the matrix i , 69. ATPe matrix .2+ oscillation during oocyte maturation and egg activation is achieved by the coordination of various types of channels. However, recent studies only rest on the enumeration of the phenomenon and the expression levels of different channels. All of this means it is imperative that more research should focus on the signal interactions and mechanisms involved in Ca2+ handling in the future.Above all, the accomplishment of well-balanced Ca2+ releasing function was found to depend on IP3R to produce IP3, and therewith, Ca2+-sensitive PLC became the most suspicion i increases associated with fertilization [2+ releasing from ER [2+]i oscillatory ability at the MII stage, lost over half of the receptors in pronucleus stage, corresponding to the sperm-initiated oscillations subsiding [2+]i oscillations [2+ can also act as a co-agonist [2+ release in mouse and porcine eggs [2+ release [Diacylglycerol (DAG) and IP3 are two products of PLCζ hydrolysis of PIP2, IP3 then identifies and binds with IP3 receptors (IP3R), subsequently release the first Calization , 24. Suclization , 98. IP3 from ER , 100. Coubsiding , 103. Pellations . In addi-agonist . On the ine eggs , 109. Th release . The aboPAWP, postacrosomal sheath WW domain-binding protein, locates in the post-acrosomal sheath region (PAS) of the perinuclear theca (PT) in elongating spermatids. It is composed of WW domain binding protein 2-similar homology in the N-terminal, PPXY consensus binding site for group-IWW domain-containing proteins, and numerous unique repeating motifs, YGXPPXG, in the C-terminal . The per2+ allocation is debatable, the battle of true identity of PAWP never ceased since its discovery. In 2015, two opposite research teams stated their points of view respectively in the same journal “Asian Journal of Andrology”, referring to “Is PAWP the “real” sperm factor?” and “Re: Is PAWP the ‘real’ sperm factor?” [2+ dynamics was further uncovered. Once again, the dispensable role of PAWP was supported by no indistinguishable changes in Ca2+ oscillations or in subsequent embryo development following gross depletion of PAWP [While the effectiveness of PAWP to Cafactor?” , 112. Fa of PAWP . The eni2+ homeostasis play a significant role in these two processes. The regulation of intracellular Ca2+ homeostasis involves Ca2+ in-out of extracellular and intracellular stores. Amidst all of this, Ca2+ influx not only maintains Ca2+ oscillations by replenishing Ca2+ stores during oocyte maturation, but also provides an important spatially restricted Ca2+ signal required for complete egg activation at fertilization [2+ influx. In this review, we analyzed the channel composition and molecular effectors, especially the basic knowledge and spatial-temporal changes of SOCE and TRP family proteins P2 residing distinct vesicular structures into IP3 and DAG. As tetrameric Ca2+ channels located on the membrane of ER, IP3R1s take in IP3 and transform it into Ca2+ surge. Confronted with the intricate and multivariate factors in the action pathways, we went deep into and summarized the structural and modificatory regulation of PLCζ-induced Ca2+ oscillation (Figure 2+ oscillations need to justified. More efforts should be redoubled to gain insight into the mechanism and molecular effectors that mediate the optimization of Ca2+ adjustment.In most mammal species, Ca