One particular facet of these adaptive changes is the alteration of Ca2+ fluxes through the plasma membrane, as described in several studies. In this review, we summarize the current knowledge about the expression of different Ca2+ channels in the plasma membrane of melanoma cells and its impact on oncogenic Ca2+ signaling. In the last few years, new molecular components of Ca2+ influx pathways have been identified in melanoma cells. In addition, new links between Ca2+ homeostasis and specific cell processes important in melanoma tumor progression have been unveiled. Thus, not only do Ca2+ channels appear to have a potential as prognostic markers, but their pharmacological blockade or gene silencing is hinted as interesting therapeutic approaches.The remodeling of Ca The concentration of Ca2+ in the extracellular milieu is 1-2 mM whereas, at rest, intracellular Ca2+ is maintained at about 100 nM [2+-transporters and Ca2+-binding proteins are used by cells to extrude Ca2+ through the plasma membrane, transport Ca2+ into the intracellular reservoirs, and buffer cytosolic Ca2+ [2+ channels in the plasma membrane allowing Ca2+ entry into the cytosol. Ca2+ influx may cross-talk with Ca2+ channels present in the endoplasmic reticulum (ER), resulting in localized Ca2+ elevations that are decoded through a variety of Ca2+-dependent effectors [Ionized calcium Ca+ is a ublic Ca2+ . Conversffectors .2+ is needed to induce cell proliferation and cell cycle progression in mammalian cells [2+ influx to induce a G1/S-phase during the cell cycle process [2+-deficient media [It has been long known that external Caan cells . Some st process , 7. Howent media .2+ channels have been involved in cell cycle progression: transient receptor potential melastatin (TRPM), transient receptor potential vanilloid (TRPV), Transient Receptor Potential Canonical (TRPC), components of the store-operated calcium entry (SOCE) pathway such as Ca2+ influx channel (ORAI1) and endoplasmic Ca2+ depletion sensor (STIM1), and voltage-gated calcium channels (VGCCs) [ in vitro models, a role for TRPC1, ORAI1, or STIM1 in Ca2+ signaling changes associated with the proliferation of endothelial cells has been uncovered [2+ in nonexcitable cells that signal to favor mitotic progression through direct binding of Ca2+ to intracellular effectors such as calmodulin (CaM) [Several types of Ca (VGCCs) . Throughncovered , 10. In ncovered , 12. T-tin (CaM) .2+ influx also plays an important role in tumor growth. Commonly, cancer cells present alterations of Ca2+ fluxes across the plasma membrane that reflect changes in the expression, subcellular localization, and/or function of different types of Ca2+ channels [Cachannels , 14. Amochannels , and thechannels . Moreovechannels , 17.2+ from the ER may drive tumor growth by inducing Ca2+ influx through the plasma membrane, as the expression of the SOCE canonical components STIM1 and ORAI1 is augmented in various cancer types, including breast cancer, glioblastoma, melanoma, and esophageal carcinoma .2+ waves that favor cell cycle progression [v1.2 mRNA have been reported in colorectal cancer [v3.2 channels in breast, colon, prostate, ovarian, esophageal, and colon cancers and in glioblastoma, hepatoma, and melanoma [v3.1 isoform) occurs in different tumors including colon, pancreatic, and gastric cancer, suggesting that it acts as a tumor suppressor [VGCCs are also involved in cancer progression by generating oscillatory Cagression . Heightel cancer . Severalmelanoma . Howeverppressor .2+ influx too. Through cell migration, Ca2+ signaling is involved in the directional sensing of the cells, in the redistribution and traction force of the cytoskeleton and in the repositioning of new focal adhesions [2+ channel families involved in tumor growth have been implicated in cancer cell migration and metastasis, such as TRP channels [Cell physiology aspects other than proliferation are dependent on Cadhesions , 23. Celchannels –26, STIMchannels –30, and channels , 32. Forchannels , being achannels . Neverthchannels . The sigchannels , 30. Furchannels . Convers2+ influx and its molecular machinery. Both the pharmacological blockade and the siRNA-mediated silencing of TRPM8 channels have been shown to induce the apoptotic death of prostate cancer cells [2+ homeostasis maintenance. It has been suggested that TRPM8 could regulate either proliferation or apoptosis mechanism in prostate cells, depending on its intracellular localization [v3.1 and Cav3.2 T-type channel isoforms. Likewise, Valerie et al. found that inhibition of T-type channels by a selective antagonist or siRNA-mediated gene knockdown not only reduced glioma cell viability but also induced apoptosis. These effects were reached via inhibition of the mTORC2/Akt pathway followed by a reduction in the phosphorylation of antiapoptotic Bad [Finally, cell fate is also dependent on Caer cells , indicatlization . Moreovelization . VGCCs alization . Importaotic Bad .2+ channels expressed in the plasma membrane of melanoma cells, as well as the Ca2+ signaling pathways involved during tumorigenesis and tumor progression.Hereon, this review will discuss the current knowledge about the role of different Ca de novo or from dysplastic, congenital, or common nevi [Cutaneous melanoma is a malignant skin cancer that arises from transformed melanocytesmon nevi . MelanomTRP channels are known to regulate melanocyte physiology, particularly members of the TRPM subfamily . UntransTRPM1 was first discovered in B16 mouse melanoma cell lines as a result of a differential display analysis . This ch in vitro and in vivo during melanoma progression [The regulation of TRPM1 gene expression has been extensively investigated. It has been proposed that TRPM1 expression in melanocytes and melanoma cells is regulated by a promoter region of the gene that contains four microphthalmia transcription factor (MITF) binding sites. Several groups demonstrated that MITF directly regulates the expression of TRPM1gression , 53, 54.TRPM1 gene encodes both TRPM1 mRNA and miR-211 which is coded by the sixth intron of the gene. TRPM1 and miR-211 share the same promoter and are coregulated by MITF. Similar to TRPM1 protein, miR-211 is highly expressed in melanocytes and nevi and is reduced in melanoma cells , 56. Con2+ influx upon activation by menthol as agonist [2+ handling pathway and suggesting TRPM8 Ca2+ channels as novel targets of drug development for malignant melanoma , Cav2 , and Cav3 (T-type) families [v3.1 channels, whereas transcripts for T-type Cav3.2 and Cav3.3 channels were undetectable. We also found a correlation between the proliferation rate and the expression of specific T-type channels isoforms, such that the melanoma cell lines displaying a high proliferation rate expressed higher levels of Cav3.2 channels (v3.1 isoform. Interestingly, the expression of these two T-type channel isoforms was counterbalanced. Furthermore, the results attained in gene knockdown experiments showed that whereas both Cav3.1 and Cav3.2 isoforms promote the progression of melanoma cells, the expression of Cav3.1 is associated with slow cycling and it is induced under hypoxic conditions [v3.1 and Cav3.2 channels, thus allowing the identification of these T-type channels as novel targets to deregulate autophagy and induce cytotoxicity in melanoma cells [In 2012, our research group reported that both normal melanocytes and transformed melanoma cells express functional VGCCs, including members of the Cafamilies . Howeverchannels ; Table 1nditions . In a fonditions . An in-dma cells .2+ influx is involved in melanoma tumorigenesis and progression may provide new targeted therapies for melanoma treatment. For this to materialize, it is essential to further deepen the study of the role of plasma membrane Ca2+ channels in melanoma cell proliferation and also in other less explored areas, such as migration or survival. In light of current knowledge, the selective activation of certain TRP channel isoforms and/or the development of humanized inhibitory antibodies to extracellular domains of TRP channels arise as putative therapeutic strategies [2+ influx through the use of pharmacological SOCE blockers and/or raft-targeting agents appears as an interesting approach to tackle melanoma progression [2+ channels as novel prognosis markers and/or therapeutic targets in melanoma. In this regard, our studies have uncovered a dual role of T-type Ca2+ channels in controlling melanoma cell proliferation and Ca2+ homeostasis, with an impact on adaptive cancer cell mechanisms like ER stress and macroautophagy that are often associated with chemotherapeutic or radiotherapeutic resistance [2+ channel blockers may expand the pharmacological arsenal and become valuable partners for combined chemotherapies against melanoma.Recent advances in the understanding of how Carategies , 16, 51.gression , 48. Finsistance , 49. Thu


Cardiac hypertrophy is the most potent cardiovascular risk factor after age, and relative mortality risk linked with cardiac hypertrophy is greater in women. Ischemic heart disease is the most common form of cardiovascular pathology for both men and women, yet significant differences in incidence and outcomes exist between the sexes. Cardiac hypertrophy and ischemia are frequently occurring dual pathologies. Whether the cellular (cardiomyocyte) mechanisms underlying myocardial damage differ in women and men remains to be determined. In this study, utilizing an in vitro experimental approach, our goal was to examine the proposition that responses of male/female cardiomyocytes to ischemic (and adrenergic) stress may be differentially modulated by the presence of pre-existing cardiac hypertrophy.We used a novel normotensive custom-derived hypertrophic heart rat ). Cardiomyocyte morphologic and electromechanical functional studies were performed using microfluorimetric techniques involving simulated ischemia/reperfusion protocols.2+ responses to β-adrenergic challenge differed in hypertrophic male and female cardiomyocytes, with the accentuated response in males abrogated in females—even while contractile responses were similar. In simulated ischemia, a marked and selective elevation of end-ischemia Ca2+ in normal female myocytes was completely suppressed in hypertrophic female myocytes—even though all groups demonstrated similar shifts in myocyte contractile performance. After 30 min of simulated reperfusion, the Ca2+ desensitization characterizing the male response was distinctively absent in female cardiomyocytes.HHR females exhibited pronounced cardiac/cardiomyocyte enlargement, equivalent to males. Under basal conditions, a lower twitch amplitude in female myocytes was prominent in normal but not in hypertrophic myocytes. The cardiomyocyte Ca2+ operational status characteristic of female  cardiomyocytes comprising normal hearts is not exhibited by myocytes of hypertrophic hearts. After ischemia/reperfusion, availability of activator Ca2+ is suppressed in female hypertrophic myocytes, whereas sensitivity to Ca2+ is blunted in male hypertrophic myocytes. These findings demonstrate that selective intervention strategies should be pursued to optimize post-ischemic electromechanical support for male and female hypertrophic hearts.Our data demonstrate that cardiac hypertrophy produces dramatically different basal and stress-induced pathophenotypes in female- and male-origin cardiomyocytes. The lower CaThe online version of this article (doi:10.1186/s13293-016-0084-8) contains supplementary material, which is available to authorized users. For both women and men, cardiovascular disease is the leading cause of death and disability , 2. CardIschemic heart disease is the most common form of cardiovascular pathology for men and women . IschemiThe importance of achieving a more detailed understanding of basic mechanisms of sex difference in pathophysiologic processes, which have particular relevance to cardiovascular disease and health demography, has been emphasized . The valExperimentally, important insights have been gained in relation to sex-specific differentials in the impacts of cardiac hypertrophy and of exposure to ischemic insult—yet still, knowledge is lacking , 16. The2+ regulatory status. Whilst limited sex-specific information is available at the myocyte level, in vitro findings suggest differential female and male contractile/Ca2+ handling characteristics in simulated ischemia and also sex differences in the responsiveness of myocytes to manipulation of adrenergic activation [2+ transient amplitude (vs non-ischemic control) and that in reperfusion, female cardiomyocytes are hyper-contractile  and more likely to survive the ischemia/reperfusion challenge [2+ levels and internal Ca2+ store flux responses [Most fundamentally, protection against hypertrophy and ischemia-driven arrhythmogenesis and contractile dysfunction is determined by cardiomyocyte Cativation , 25. Spehallenge . Sex difesponses , 27.In this study, utilizing an in vitro experimental approach, our goal was to examine the proposition that responses of male and female cardiomyocytes to stress stimuli (β-adrenergic activation and simulated ischemia/reperfusion insult) may be differentially affected by the presence of pre-existing cardiac hypertrophy. Using a purpose-derived rat model of normotensive cardiac hypertrophy, the hypertrophic heart rat (HHR), a range of cardiomyocyte morphologic and electromechanical functional studies was performed using microfluorimetric techniques. We have previously reported premature mortality in the HHR ), with ex vivo HHR exhibiting normal systolic function and an increased vulnerability to ischemia/reperfusion dysfunction/injury , 28. TheAnimals were obtained from established colonies of the HHR and the control NHR, maintained at the Biological Research Facility at the University of Melbourne, Australia, and derived as reported . Briefly2+-free Krebs  at 73 mmHg for 3 min at 37 °C. Cardiomyocytes were then perfused with type II collagenase  to enable heart digestion. The heart was removed from the cannula, and the ventricular cardiomyocytes dispersed in Krebs/collagenase solution -1-piperazineethanesulfonic acid (HEPES), 11 d-glucose) supplemented with trypsin inhibitor  to inhibit residual collagenase activity. For each heart, 100 rod-shaped and regularly striated cardiomyocytes were selected at random for length and width measurement at ×400 magnification using an inverted light microscope and calibrated eye piece as previously described [−3 pl/μm2 [Ventricular myocytes were isolated from NHR and HHR rats, as described . Brieflyescribed . Cardiom3 pl/μm2 .2+ levels and shortening performance were measured [2+ fluorescent dye, fura2-AM . Myocytes were superfused with 2 mM Ca2+-HEPES buffer  and stimulated to establish steady state contractile performance at 4 Hz . Cardiomyocyte twitch properties were assessed by video-based edge detection . The indices used to describe twitch cycle were twitch amplitude normalized to diastolic cell length (twitch amplitude percentage) and maximal rate of shortening and lengthening . Myocyte Ca2+ signals were measured by microfluorimetry  [2+ transient were amplitude (F360:380) and the time constant of decay . Cardiomyocyte twitch and Ca2+ fluorescent ratio properties were measured off-line using IonWizard (IonOptix) and were determined by average of ten steady state transients for each myocyte. Ca2+-shortening phase loops were constructed for individual activation cycles by plotting Ca2+ level vs myocyte shortening throughout the shortening and lengthening phases of the cycle. During the relaxation (i.e. lengthening) phase, the Ca2+ level at half (50 %) relaxation may be interpreted as an index of myofilament Ca2+ sensitivity [Isolated myocyte Cameasured . Cell suMA, USA) . Fluoressitivity .2+-HEPES buffer were superfused with 10 nM isoproterenol for 5 min. Previous dose range and time-course studies confirmed this was an optimal time point to allow a maximal shortening and amplitude Ca2+ response to reach a new steady state [To assess the response of male and female NHR and HHR cardiomyocytes to an adrenergic challenge, myocytes stabilized for 5 min in 2 mM Cady state .2PO4-H2O, 1.1 MgSO4-7H2O, 2.0 CaCl2, 10 HEPES, 10 lactate, pH 6.8, 100 % N2 gas saturation) for 20 min to simulate conditions associated with ischemia, including hypoxia, hypercapnia, acidosis, substrate deprivation and lactate accumulation [2+-HEPES buffer for a further 30 min. Cardiomyocytes were electrically stimulated (4 Hz) throughout the ischemia/reperfusion protocol.To assess the response of male and female NHR and HHR cardiomyocytes to an ischemia and reperfusion challenge, cardiomyocytes were superfused with a modified HEPES buffer . Statistical annotation used throughout the study to designate significance level: #, strain effect; *, sex effect; †, sex-strain interaction. Post hoc analysis was performed on data in which a sex-strain interaction was evident, with significant differences in post hoc analysis represented in the figures  directly above the relevant figure bar or data point.Data are presented as mean ± SEM. Differences between groups were assessed by two-way ANOVA and with repeated measures as appropriate. Myocyte survival curves were assessed by log-rank Mantel-Cox test. Data were considered significant at p < 0.05; Fig. Female body and heart weights were significantly lower than strain and age-matched NHR and HHR values Table . Heart wp < 0.05). This lower female contractility was marked in normal female myocytes , though time to peak shortening was particularly slow in female HHR cardiomyocytes . Sex differences in the Ca2+ response to β-adrenoceptor stimulation were evident. The extent of isoproterenol-induced increase in Ca2+ transient amplitude was selectively blunted in female HHR myocytes compared with males . This sex-specific response was not observed in non-hypertrophic (NHR) myocytes. Diastolic Ca2+ levels were stable with isoproterenol challenge in all treatment groups . In contrast to males, female NHR cardiomyocytes exhibited a substantial increase in amplitude Ca2+ at the end of ischemia , which was not associated with a concomitant increase in shortening , the shortening responses between male and female HHR were not different . A sex-strain interaction was evident for diastolic Ca2+ during simulated ischemia . Male HHR cardiomyocytes demonstrated equivalent shortening to male NHR controls, yet amplitude Ca2+ was significantly increased. This directly contrasted with female HHR cardiomyocytes, where shortening was significantly increased compared with female NHR controls, yet amplitude Ca2+ was not different. Phase loop analyses were performed to assess individual myocyte Ca2+-shortening relationships . These data demonstrate that, in contrast to hypertrophic male myocytes, myofilament Ca2+ responsiveness in hypertrophic female cardiomyocytes was not characterized by ‘desensitization’. Thus, a major sex-specific stress response phenotype difference emerges between female and male hypertrophic cardiomyocytes when subjected to simulated ischemia and reperfusion challenge.During the simulated reperfusion phase of the protocol, myocytes of all groups most typically exhibited a rapid decrease in resting length (i.e. a diastolic contracture response) with decrement in twitch amplitude, followed by gradual return to a new steady state in the extent of myocyte shortening, albeit diastolic relaxation state was only partially regained Fig. . Myocyteips Fig. . Male, b2+ responses to β-adrenergic challenge differed in hypertrophic male and female cardiomyocytes, with the accentuated response in males abrogated in females—even while contractile responses were similar. In simulated ischemia, the Ca2+ transient responses of hypertrophic male and female myocytes were markedly different—though again, all groups demonstrated similar shifts in myocyte contractile performance. A prominent and selective elevation of end-ischemia Ca2+ in female NHR was completely suppressed in female hypertrophic myocytes. Similarly, after the 30-min simulated reperfusion, male and female cardiomyocytes evinced different Ca2+ response patterns indicating an absence of the Ca2+ desensitization characterizing the male response.This investigation provides new evidence that male and female hearts with an underlying pathological hypertrophy respond differently to cellular stresses. Using the novel HHR model , our findings document pronounced cardiac and cardiomyocyte enlargement in females—including cardiomyocyte dimensional increase in female HHR equivalent to male HHR. Under basal conditions, a lower twitch amplitude in female myocytes was prominent in normal (NHR) but not in hypertrophic (HHR) myocytes. The cardiomyocyte Ca2+ operational status characteristic of female  cardiomyocytes comprising normal hearts is not exhibited by myocytes of hypertrophic hearts. After ischemia/reperfusion, availability of activator Ca2+ is suppressed in female hypertrophic myocytes, whereas sensitivity to Ca2+ is blunted in male hypertrophic myocytes. These findings demonstrate that selective intervention strategies should be pursued to optimize post-ischemic electromechanical support for male and female hypertrophic hearts.Our data demonstrate that cardiac hypertrophy produces dramatically different basal and stress-induced pathophenotypes in female- and male-origin cardiomyocytes. The lower Ca2+ concentration) [2+ transporter function are undoubtedly implicated. While differences in protein expression levels of various Ca2+ transporters have been reported between males and females in some contexts [2+ handling effectors is likely to be of most functional importance [2+ transporter expression/activity have utilized a series of different species  and animal ages, which may explain variability in findings. We have recently shown that expression of the sarcoplasmic reticulum (SR) Ca2+ release channel (RyR2) does not differ in male/female Sprague Dawley (SpD) rats (12–16 weeks) [2+ release channel (RyR2) [2+ ATPase (SERCA2a) and phospholamban (PLB) expression in male/female SpD rats, consistent with previous reports [2+ transporter activity, and not the expression, that differs in males/females. In particular, we have shown that basal Ca2+/calmodulin-dependent kinase II (CaMKII) phosphorylation  is lower in females, a mediator which exerts major influence on numerous Ca2+ transporter properties [The present study assessed male/female cardiomyocyte function and stress responses in a novel pathological hypertrophic HHR strain (vs control NHR strain). Significantly lower body weight in female rats is consistent with previous reports , 37–39, tration) . The postration) , 39. Thicontexts , 30, oesportance , 44–48. 6 weeks) . This col (RyR2) , 50. We  reports . Directl2+ handling includes voltage-gated Ca2+ current suppression, internal Ca2+ store release modulation and myofilament Ca2+ sensitivity alteration [2+-dependent mechanism, but likely non-Ca2+ dependent estrogenic actions (cell energetics/gene expression) are involved [2+ transient amplitude differences.Estrogenic influence on cardiomyocyte Cateration , 51–59. involved  as the e2+ transients are smaller in female cardiomyocytes  treated with isoproterenol [Sex-specific aspects of altered β-adrenoceptor response in cardiac disease pathogenesis are not well characterized. Experimentally, evidence indicates that the β-adrenergic response is less pronounced in healthy cardiac tissues/myocytes of females compared with males under some circumstances. We and others have previously shown that Caoterenol , 61–63. oterenol , 65, andoterenol , 62.2+ transient amplitude in isoproterenol-treated NHR myocytes—suggesting that pacing under near-physiological conditions minimizes response difference [2+ transient amplitude observed in male HHR myocytes, even though both female and male HHR had similarly augmented contractile responses. This suggests a Ca2+-independent component determining adrenergic response selectively in females. The mechanism is unclear, but may be related to differential cellular metabolism profile—with hypertrophic hearts drawing on sex-selective energy reserves to effect an adrenergic-driven contractile response [We did not observe male/female differences in the extent of increase in twitch or Cafference . Notablyresponse , 66.In this investigation, distinct mechanisms underlying vulnerability to ischemia/reperfusion in female- and male-origin cardiomyocytes are identified. These findings build on our prior observations involving isolated ex vivo hearts where we established that the more robust functional recovery of female hearts to ischemic insult was abrogated in hypertrophy . In that2+ levels—suggesting that the interplay between Ca2+, Na+ and pH which is an important determinant of electromechanical status at end reperfusion operates differently in normal male and female myocytes. Related, but qualitatively different, observations have been previously reported. The sex differences in ionic homeostatic responses to simulated ischemia may depend on precise experimental conditions [During ischemia, female NHR showed a surprising tolerance for elevated cardiomyocyte Canditions . Sex-com2+ handling and response characteristic produced this similar net effect in HHR cell types. In the male HHR myocytes, a significant elevation of Ca2+ transient amplitude occurred with a major offsetting reduction in myofilament sensitivity to Ca2+. In the female HHR myocytes, this combination of Ca2+ adaptations was not apparent. In relation to adverse outcomes in early reperfusion, these findings may be extrapolated to suggest that in acute reperfusion, Ca2+ overload arrhythmogenic predisposition presents as a male myocyte vulnerability. In contrast, the blunted female myocyte Ca2+ transient response to β-adrenoceptor inotropic influence has the potential for compromised functional performance. Thus, ischemic recovery is similarly impaired in both males and females—but via different cellular mechanisms. Clinically, the importance of gender differences in arrhythmia substrate is increasingly recognized—and advancing the understanding of underlying cellular processes involved is essential [Cell shortening is typically reduced by approximately 50 % relative to basal in the first 5 min of simulated ischemia, though this reduction was attenuated in female HHR cardiomyocytes Fig. . This 50ssential .2+ loading levels [The present study, of young adult rodents at prime estrogenic life stage, demonstrates the absence of a female ‘protected’ state in the context of hypertrophy. The conventional view of female oestrogen cardioprotection may require refinement , 72. Oesg levels –22. New g levels –76, and 2+ sensitivity, there may be a case for review in selected at-risk female subgroups. This study provides a basis to support relevant focused pre-clinical research to pursue this possibility.In summary, this experimental investigation demonstrates that the responses of hypertrophic male and female cardiomyocytes to β-adrenoceptor stimulation and ischemia/reperfusion challenges are fundamentally different at the cellular level—and that hypertrophy in females confers a distinctive liability. Our findings indicate that therapeutic interventions targeted to the early reperfusion period may have differential sex-specific efficacy depending on the presence of an underlying comorbid hypertrophic state. Where clinical trials have previously failed to identify benefit in early reperfusion manipulation of cellular processes impacting on myofilament Ca2+/calmodulin-dependent kinase II; CWI, cardiac weight index; ERα, oestrogen receptor α; ERβ, oestrogen receptor β; GPER, G-protein-coupled oestrogen receptor; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HHR, hypertrophic heart rat; NHR, normal heart rat; RISK, reperfusion injury salvage kinase; SHR, spontaneously hypertensive ratANOVA, analysis of variance; CaMKII, Ca


In addition, we also observed that 21 Ca2+ signaling genes were significantly up-regulated in response to TLR3 priming of hMSCs by RNA sequencing analysis. Both poly(I:C) and LPS exposure enhanced cytokine release from hMSCs. The enhanced cytokine release vanished upon siRNA knockdown and chelation of intracellular Ca2+. These data demonstrate that TLR3- and TLR4-priming differentially enhance Ca2+ signaling and cytokine expression, and Ca2+ -dependently potentiates cytokine release in hMSCs.In human mesenchymal stem cells (hMSCs), toll-like receptor 3 (TLR3) and TLR4 act as key players in the tissue repair process by recognizing their ligands and stimulating downstream processes including cytokine release. The mechanisms of TLR3- and TLR4-mediated cytokine releases from hMSCs remain uncertain. Here, we show that exposure to the TLR3 agonist polyinosinic-polycytidylic acid (poly(I:C)) or incubation with the TLR4 agonist lipopolysaccharide (LPS) increased the mRNA expression levels of TLR3, TLR4 and cytokines in hMSCs. Poly(I:C) exposure rather than LPS incubation not only elevated inositol 1,4,5-triphosphate receptor (IP Human mesenchymal stem cells (hMSCs) are not only capable of self renewal and differentiation into osteoblasts, chondrocytes, adipocytes, myocytes and even neurons1345784101141113141531731718192+ entry through the plasma membrane and Ca2+ mobilization from intracellular stores leads to temporally and spatially distinct patterns of [Ca2+]i to generate a highly versatile intracellular signal that controls almost all known molecular and cellular events in germ cells, somatic cells and stem cells21222324252627212223242526272+ entry pathways, such as store-operated Ca2+ entry (SOCE), which is composed of Orai and STIM proteins or transient receptor potential (TRP) channels, as well as voltage-gated calcium channel (VGCC)-mediated Ca2+ influx operate in the plasma membrane29303132332+ and release these divalent ions through the ligand-gated calcium channels inositol triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs)2735362+-handling devices to control their [Ca2+]i responses and the corresponding downstream molecular and cellular events293031323334Extracellular Ca2+ signaling is crucial for both proliferation and differentiation in several types of stem cells3839402+ stores function as a key intracellular Ca2+ source to generate the complex oscillation, fast transient increase and sustained elevation of [Ca2+]i in various stem cells394243442+ channels IP3Rs and RyRs play an important role in the neuronal or cardiac differentiation of stem cells3943442+ transients to regulate both proliferation and differentiation in human neural progenitor cells2+ mobilization from the ER and Ca2+ influx through TRPM7 channels occur in hMSCs to generate [Ca2+]i oscillations upon application of mechanical traction on the plasma membrane2+ mobilization from the ER regulate numerous activities, including TLR-mediated cytokine production in immune cells31472+]i couples TLR-priming to cytokine release in hMSCs. The present work verifies that TLR3- and TLR4-priming differentially enhances Ca2+ signaling and cytokine expression and Ca2+ -dependently elevates cytokine release in hMSCs.CaMSC specific surface markers present on hMSCs were analyzed by flow cytometry . The cel2+]i in hMSCs. Real-time single-cell measurements of [Ca2+]i revealed two distinct patterns of basal [Ca2+]i in hMSCs  drastically increased [Ca2+]i in the absence and presence of extracellular Ca2+  did not cause a [Ca2+]i increase, but instead slightly decreased [Ca2+]i  (2+ entry was effectively abolished by application of the membrane permeable SOCE antagonist 2APB (50 μM)  . Moreove (50 μM) . It is cTo quantify the impact of the TLR3 agonist poly(I:C) and the TLR4 agonist LPS on the mRNA expression levels of TLR3, TLR4 and cytokines in hMSCs, we performed RT-PCR and real-time RT-PCR assays. RT-PCR analysis confirmed that control hMSCs expressed both TLR3 and TLR4 mRNAs. This analysis revealed that 4 h exposure to LPS and poly(I:C) elevated TLR4 and TLR3 mRNA expression in hMSCs in a concentration and time-dependent manner . QuantifInterestingly, real-time RT-PCR detection showed that incubation with 5 μg/ml poly(I:C) for 4 h preferably elevated IL4 mRNA levels. In contrast, 4 h treatment with LPS (10 ng/ml) preferentially up-regulated the mRNA expression levels of IL6, IL8 and IP10 . These f2+, we focused our attention on Ca2+ mobilization from IP3-sensitive stores, which is likely to be the only Ca2+ release mechanism in hMSCs (3R) expression and IP3R-mediated Ca2+ mobilization in hMSCs using RT-PCR analysis, [Ca2+]i measurements, confocal immunofluorescence microscopy and western blot analysis.To explore the possible signaling pathways of TLR3 and TLR4 that respond to the highly versatile intracellular signal Cain hMSCs . Therefo2+]i measurements showed that stimulation with 50 μM CCH evoked [Ca2+]i transients with somewhat different patterns in control cells bathed in extracellular solution without Ca2+ , ITPR2 (IP3R2) and ITPR3 (IP3R3) mRNAs, but very few RYR1 mRNAs and no RYR2 mRNAs (3R1), ITPR2 (IP3R2) and ITPR3 (IP3R3) mRNAs, whereas priming of TLR4 with LPS did not influence the mRNA expression of all three subtypes of IP3Rs , but not LPS, induced significant increases in the expression of the IP3R3  and the subsequent addition of extracellular Ca2+ evoked prominent [Ca2+]i transients in control (n = 18), LPS- (n = 17) and poly(I:C)-treated groups (n = 17) in Ca2+-free extracellular solution (2+]i reflected by the averaged delta F340/F380 ratios following CPA exposure was significantly higher in the poly(I:C) group than in the control group, whereas this parameter was similar between LPS-treated and control cells (2+]i induced by extracellular application of 4 mM Ca2+ following Ca2+ store depletion by CPA was significantly exaggerated in poly(I:C)-treated cells, but just marginally elevated in LPS-treated cells in comparison with that in control cells (2+]i was mirrored by the averaged F340/F380 ratios prior to application of CPA and was increased significantly in the poly(I:C) group, but was elevated only slightly in LPS the group compared with the control group  in comparison with control cells.Cytokine release is considered an important activity in TLR3- and TLR4-primed hMSCs. This led us to study whether the promotion of Ca2+ with BAPTA/AM (5 μM)  . Type I M (5 μM) . These repletion  affects epletion .2+]i, one exhibiting a stable resting [Ca2+]i and the other displaying spontaneous [Ca2+]i oscillations. It is plausible to postulate that one or more [Ca2+]i oscillation-generating devices, e.g., IP3R2, somehow become active under basal conditions to create this spontaneous [Ca2+]i oscillation in the latter population of hMSCs. This postulation is supported by the fact that the IP3R2 undergoes activation at the lowest concentration of IP3 among the three IP3R subtypes to generate [Ca2+]i oscillations3R subtypes are present in hMSCs (see below for details). The distinction in the basal [Ca2+]i profile may serve as a clue for the important aspects, such as cell cycle phase, differentiation fate, self-renewal capacity or immune-modulating features, of hMSCs. These two basal [Ca2+]i profiles deserve further investigation to clarify what exactly they signify.The present work confirms that two different populations of hMSCs in the same extracellular milieu show two distinct profiles of basal i oscillations to negatively regulates apoptosis in early differentiating embryonic stem cells493Rs are important signaling devices in hMSC development, although their exact roles remain to be explored. In addition, the present work also corroborates that hMSCs acquire Ca2+ from the extracellular environment through SOCE rather than VGCCs. These functional observations are in accordance with previous studies42The results presented herein demonstrate that hMSCs prefer to use IPhMSCs appropriately sense and efficiently respond to environmental challenges and adequately engraft in inflamed sites or lesion areas to accomplish repair missions31314151617183131415161741011142+]i. More importantly, we reveal that TLR3-priming produces not only a significant increase in IP3R-mediated Ca2+ mobilization but also a substantial elevation of the molecular expression of IP3Rs in hMSCs. In contrast, TLR4-priming has only marginal influences on these two parameters. Likewise, TLR3-priming significantly augments SOCE with a concomitant increase in basal [Ca2+]i and the molecular expression of candidate building blocks of SOCE, including two Orai subtypes and one STIM subtypes as well as TRPM4 and TRPC4 in hMSCs. However, TLR4-priming fails to do so. These findings demonstrate that TLR3-priming but not TLR4-priming exaggerates IP3R- and SOCE-mediated Ca2+ signaling. They also suggest that TLR3-priming does not allosterically modulate IP3R and SOCE activity, but instead increases their abundance via genomic mechanisms. In addition to these Ca2+ channels, K+ channels are also present in hMSCs53+ efflux causes a more negative membrane potential and thereby enhances Ca2+ influx due to the increased electric driving force for Ca2+ entry2+]i through the increased expression of these K+ channels. Therefore, we have quantified the mRNA expression of the large-conductance calcium-activated potassium channel gene MaxiK3Rs and SOCE.The present work has dissected the mechanisms linking TLR3 and TLR4 to i was measured as the fura-2 340/380 nm fluorescence ratio with a fluorescence microscope . The microscope was equipped with a xenon arc lamp, integrated shutter and cooled EM-CCD camera . The camera and shutter were controlled by MetaFluor software . Single cells were defined as regions of interest (ROIs)  138 NaCl, 5.6 KCl, 1 MgClt (ROIs) . The 16-To stain the hMSCs, the cells were harvested with trypsin, and then blocked with phosphate buffered saline (PBS) with 2% normal serum for 5 min. The cells were incubated with direct immunofluorescence using fluorescein isothiocyanate (FITC)-conjugated antibodies against CD105 , HLA-DR , CD29 , CD44 , and phycoerythrin (PE)-conjugated antibodies against CD34 , CD45 , CD31 , CD73 , and CD90  for 30 min. Control cells were prepared with FITC-, PE-mouse isotype antibodies . The stained cells were analyzed with a FACS Calibur A .The human mesenchymal stem cell functional identification kit  was employed for the differentiation of hMSCs into adipocytes and osteoblasts. Briefly, hMSCs were cultured in minimum essential medium  containing adipogenic and osteogenic supplements for 21 days to induce differentiation into adipocytes and osteoblasts respectively. The medium was replaced with fresh medium every 3–4 days. After 21 days, differentiated cells were fixed with 4% paraformaldehyde and incubated with a primary antibody against fatty acid binding protein 4  for adipocytes and osteocalcin  for osteoblasts. The cells were washed and incubated with fluorescein-labeled anti-rabbit IgG . Stained cells were observed using a microscope .3R3 , rabbit polyclonal anti-Orai1  or rabbit polyclonal anti-Orai2  at 4 °C overnight. A subsequent incubation of the samples with Goat anti-rabbit IgG conjugated to Alexa 488  was performed for 30 min at 37 °C. Finally, the samples were mounted in the mounting medium Vectashield  and visualized with a Zeiss LSM 710 confocal microscope .hMSCs were seeded onto coverslips in 4-well plates, cultured for a day and treated with LPS or poly(I:C). Subsequently, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with cold methanol for 5 min. Then, the samples were blocked with 3% bovine serum albumin for 1 h, incubated with rabbit polyclonal anti-IP2, 1% Triton X-100 and a complete protease inhibitor mixture tablet (Roche Applied Science). Equal amounts of protein (30–50 μg) were subjected to 6 or 10% SDS-PAGE and blotted onto a PVDF membrane. The membranes were then incubated with anti-IP3R3  or anti-Orai2  and anti-pancadherin  or anti-β-actin  for loading controls and signals were detected with ECL reagent.Cell lysates were prepared by sonication in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 5 mM MgCl4 cells/cm2 into 10 cm dish containing culture medium and cultivated for a day. Thereafter, the normal medium was replaced with the medium supplemented with TLR ligands followed by a 4 h incubation. Cytokine concentrations in the culture media were detected by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. Standard curves were established using mouse recombinant cytokines provided with the ELISA kit . The assay detection limit was 16 to 32 pg/ml.hMSCs were seeded at a density of 6 × 10Data are reported as mean ± SEM. Statistical analyses were performed using the Origin program . Statistical significance was determined by Student’s t test. The significance level was set at *p < 0.05 or **p < 0.005.How to cite this article: Park, K. S. et al. TLR3-/4-Priming Differentially Promotes Ca2+ Signaling and Cytokine Expression and Ca2+-Dependently Augments Cytokine Release in hMSCs. Sci. Rep.6, 23103; doi: 10.1038/srep23103 (2016).


Recently, it has been found using cell culture models that transient increases of intracellular Ca2+ induce cytoplasmic α-syn aggregates. Ca2+-dependent α-syn aggregation could be blocked by the Ca2+ buffering agent, BAPTA-AM, or by the Ca2+ channel blocker, Trimethadione. Furthermore, a greater proportion of cells positive for aggregates occurred when both raised Ca2+ and oxidative stress were combined, indicating that Ca2+ and oxidative stress cooperatively promote α-syn aggregation. Current on-going work using a unilateral mouse lesion model of Parkinson’s disease shows a greater proportion of calbindin-positive neurons survive the lesion, with intracellular α-syn aggregates almost exclusively occurring in calbindin-negative neurons. These and other recent findings are reviewed in the context of neurodegenerative pathologies and suggest an association between raised Ca2+, α-syn aggregation and neurotoxicity.In Parkinson’s disease and some atypical Parkinson’s syndromes, aggregation of the α-synuclein protein (α-syn) has been linked to neurodegeneration. Many triggers for pathological α-syn aggregation have been identified, including port-translational modifications, oxidative stress and raised metal ions, such as Ca In general, misfolded proteins that cannot be refolded are degraded by the ubiquitin proteasome system (UPS), or via lysosomal degradation. In disease, however, the misfolded protein may be resistant to degradation and undergo aggregate formation. Abnormal aggregation of the protein α-synuclein (α-syn) has been implicated in a number of neurological diseases, including Parkinson’s disease (PD), characterised by cytoplasmic aggregates in multiple cell types throughout the central nervous system. Collectively, these diseases are termed α-synucleinopathies and include PD and the atypical Parkinson’s syndrome, multiple system atrophy (MSA). In this report, key features of PD and MSA will be reviewed, two diseases that characterize the range of cell types affected by α-syn aggregates. Moreover, mechanisms of and factors influencing α-syn aggregation in the CNS will be discussed, with a special emphasis on the role of Caractions ,2,3.SNCA (PARK1/4), the gene responsible for the expression of α-syn. To date, five PD-linked point mutations in SNCA, have been identified [+) in comparison to cells expressing wild-type α-syn and control cells [PD has progressive clinical symptoms, including slight weakness, tremors, forward posture, sleep disturbance, constipation, the inability to walk unaided, speech impairment, difficulty swallowing, tremor, loss of urinary and gastrointestinal control and extreme exhaustion. The three main symptoms of PD are bradykinesia, or a slowing of voluntary controlled movement, rigidity, and tremor. Pathological examination of brain tissues from PD sufferers shows that there is a loss of dopaminergic neurons in the Substantia nigra (SN), a region of the brain that, through neural connections with the striatum, is responsible for controlled muscle movements. PD can be classified into two main groups: idiopathic and familial. Idiopathic PD, the larger of the two groups, accounts for roughly 85%–90% of all PD cases. Familial PD accounts for the remaining 10%–15% of cases and is due to mutations in a number of genes, including CA PARK1/, the genentified , G51D [7ol cells . Furtherol cells  and tripol cells  have alsEnvironmental factors, such as exposure to pesticides and insecticides, may play a role in the pathogenesis of idiopathic PD. Rotenone, 1-methyl-4-phenylpyridinium (MPP+) and Paraquat have all been linked to PD ,15. ThesA characteristic pathological feature of PD is a severe loss of the neuromelanin-positive dopaminergic neurons of the SN, located in the midbrain. Signalling between the SN and the striatum is involved in controlling muscle movements; therefore the resultant loss of nigro-striatal pathway signalling can explain the classical symptoms of PD. Of the neurons that remain, large protein aggregates called Lewy bodies (LB) are often observed. While LB’s are made up of numerous proteins, including proteasome components, lysosome components and chaperone proteins, α-syn immunoreactivity defined α-syn as a major component of the LB in PD and in dementia with Lewy bodies ,17.SNCA gene, have been identified with an increased risk of MSA [Multiple system atrophy (MSA) is also late onset, neurodegenerative, idiopathic and progressive . Howeverk of MSA . A numbek of MSA ,20.SNCA gene that is highly conserved in vertebrate species. Although the exact role of α-syn remains unclear, the protein is primarily expressed in the olfactory bulb, frontal cortex, striatum and the hippocampus with lower expression levels also observed in the hypothalamus, thalamus, midbrain, cerebellum and pons [2+ binding site [α-Syn is a 14 kDa protein encoded by the and pons  where itand pons ,23. It iand pons . Three ping site . The thiing site . The coning site ,28,29. αing site  through ing site  and thising site ,33. Deleing site  and bothing site .SNCA in autosomal dominant forms of PD suggests the importance of α-syn gene dosage and protein concentration in aggregation. Interestingly, mouse studies have shown that there is an age-dependent decline in both α-syn protein and mRNA levels [Gene triplication and duplication of A levels . WhereasA levels .2+ levels in SH-SY5Y cells [2+ was rapid, reaching a plateau around 200 s, and dependent on extracellular Ca2+, indicating a pore forming ability of α-syn oligomeric species. Treatment with α-syn oligomers resulted in an increased level of cleaved (active) caspase 3 indicating α-syn olimomers induced apoptosis. Outeiro et al. [in vitro and determined that the most favourable α-syn interaction was anti-parallel, resulting in cytotoxicity that could be suppressed by HSP70.Abnormal oligomeric α-syn species have been implicated in the pathogenesis of α-synucleinopathies ,41,42,43o et al.  used proOne possible mechanism for increased rate of α-syn oligomerization is an increase in mitochondrial mediated ROS production. Using PD cybrids, which have relatively high levels of oxidative stress and an enhancement in oligomer formation, treatment with CoQ10 and GSH antioxidants resulted in a decrease in oligomer formation . MoreovePhosphorylation of α-syn has been shown to be important in increasing the rate of aggregation  and has et al. [et al. [One factor contributing to the toxicity of oligomeric α-syn species is cell to cell spread of α-syn via exosomes . Exosomeet al.  demonstr [et al.  demonstr [et al. . Microgl [et al. . Moreove [et al. ,63.et al. [There is evidence that oxidative stress is increased in normal aged brain however the level of oxidative stress is greatly increased in patients with neurodegenerative diseases . The majet al.  showed tet al. . Moreoveet al. .2+ plays many important roles in normal cellular processes, such as apoptosis, metabolism, signal transduction, gene expression and cell death, and intracellular Ca2+ homeostasis is tightly regulated between the cytoplasm, intracellular Ca2+ stores, such as the endoplamic reticulum (ER), and between the intracellular environment and the extracellular environment. Resting intracellular Ca2+ is found not to be increased with age of the neuron, with studies indicating no difference between intracellular Ca2+ levels of young and aged neurons. However, the return time to resting levels after a stimulus is greatly reduced in aged neurons. Thus, the possible link between Ca2+ and α-syn aggregation in neurodegenerative diseases is a current focus of research [2+ influx [2+ and Co2+ binding have been shown to accelerate the formation of α-syn annular oligomeric species [2+ binding site has not yet been defined, truncation at residue 125 was found to abolish Ca2+ binding and Ca2+-dependent aggregation.Caresearch ,68. Inde+ influx . Further species . Althoug2+ include Ca2+ influx through ligand-gated glutamate receptors, such as N-methyl-d-aspartate receptor (NMDAR) or various voltage-dependent Ca2+ channels (VDCCs), as well as the release of Ca2+ from intracellular stores. Clearance of Ca2+ after stimulation is achieved either by intracellular Ca2+ binding, uptake into the ER and mitochondria or pumping into the extracellular space via plasma membrane Ca2+ ATPases, which have been shown to be impaired in aged neurons [2+ stimulus through uptake into the intracellular Ca2+ stores with a decline in sarcoplasmic ER Ca2+ ATPase Ca2+ function [2+ store and like the ER, the ability of this organelle to act as a reservoir for Ca2+ is also decreased with age [The major sources of intracellular Ca neurons ,71,72. Afunction . The mitwith age .2+ buffering in neurons. Bu et al. [et al. [et al. [Calbindin (CB), calretinin and parvalbumin are three cytosolic calcium binding proteins that are capable of Cau et al.  found a  [et al.  found th [et al. ,78. Furt [et al.  found re2+ concentration induced in cultured 1321N1 glioma cells by thapsigargin or Ca2+ ionophore (CI) chemical treatments caused a significant increase in the proportion of cells bearing microscopically-visible α-syn aggregates , also srization ). In thi2+ [2+ entry in differentiated SH-SY5Y [et al. [2+ dynamics in transgenic (tg) mice expressing human WT α-syn. α-Syn-tg mice exhibited augmented, long-lasting Ca2+ transients characterized by considerable deviation from the exponential decay. Furthermore control and α-syn KO groups demonstrated low percentages of neurons with Ca2+ abnormalities whereas the α-syn tg group showed Ca2+ response alteration, suggesting these alterations are related to α-syn expression.Elevated levels of intracellular α-syn have been shown to elevate levels of intracellular Ca2+ . Secrete SH-SY5Y . Reznich [et al.  investig2+ transients by enhancing ER-mitochondria interactions. Cali et al. [2+ in SH-SY5Y and HeLa cells. Additionally, treatment with naturally secreted α-syn increases Ca2+ entry in primary rat cortical neurons and induces mitochondrial Ca2+ uptake. Significantly higher levels of mitochondrial Ca2+ content in α-syn treated cells were observed compared to control cells.Other studies have shown that α-syn overexpression augments mitochondrial Cai et al.  demonstret al. [2+ in yeast that coincides with an increase in oxidative stress [2+ transient and then to oxidative stress. WT α-syn has also been shown to induce mitochondrial NO when it is associated with mitochondria [N-terminal kinase (JNK) pathway [Dryanovsky et al.  found the stress , suggestchondria . This in pathway .2+, to promote α-syn aggregation and neurodegeneration. The combination of oxidative stress and α-syn expression has been used to generate a model of MSA in mice, whereby the overexpression of α-syn in glial cells is combined with 3-nitropropionic acid [2+ binding. It was found that, when treated with Ca2+ ionophore (CI) or thapsigargin (TG) and H2O2 in combination, there was a dramatic increase in the number of protein aggregates per cell in 1321N1 cells (in vitro experiments that showed that the combination of Ca2+ treatment and oxidation of recombinant α-syn monomer caused the formation of stable, oligomeric α-syn aggregates, indicating a cooperative interaction between Ca2+ binding to α-syn and α-syn oxidation [2+ and oxidative stress work synergistically to induce α-syn aggregation. This may be extremely important in the pathogenic mechanisms behind α-syn aggregation and α-synopthathy disease progression as fibrillization of α-syn is highly dependent on nucleation centres, with pre-aggregated α-syn dramatically increasing the rate of α-syn aggregation. Furthermore, kinetic studies of α-syn aggregation by Nath et al. [2+/oxidation stabilized α-syn aggregates may serve to increase nucleation centres in disease. Indeed, the formation of di-tyrosine cross linked α-syn dimers have been found previously to be a rate-limiting step in the fibrillation process and the formation of nucleation centres [2+ influx into neurons can induce oxidative stress in mitochondria of mouse dopaminergic neurons [2+ influx was exacerbated in DJ-1 mutant mice.Oxidative stress is strongly implicated in α-synucleinopathy and may combine synergistically with other factors, such as α-syn expression and raised Canic acid . MoreoveN1 cells . This waxidation . Thus, th et al.  were con centres . Moreove neurons , as oxid2+ and oxidative stress. Recent studies have found that transient increases of intracellular Ca2+ induce cytoplasmic α-syn aggregates, that can be blocked by Ca2+ buffering or Ca2+ channel blocking agents. Furthermore, it has been shown that Ca2+ and oxidative stress cooperatively promote α-syn aggregation. These recent findings suggest an association between raised intracellular Ca2+, α-syn aggregation and neurotoxicity paving the way for the development of therapeutics that target raised Ca2+ [2+ lowering medications, such as anti-epileptic drugs, often have significant side-effects, successfully targeting raised intracellular free Ca2+ in the brain as a neuroprotective strategy will depend on the development of reliable genetic, imaging or biochemical tests. It is clear that in order to differentiate disease sub-types with strong Ca2+ involvement, multiple marker evaluation will be necessary.The link between PD and some atypical Parkinson’s syndromes and aggregation of α-syn makes this process a major target for the development of future neurodegenerative therapies. Many triggers for pathological α-syn aggregation have been identified, including raised Cased Ca2+ . Since C


Calmodulin is one of the most abundant presynaptic proteins and it binds Ca2+ faster than any other characterized endogenous neuronal Ca2+ buffer. Direct effects of calmodulin on fast presynaptic Ca2+ dynamics and vesicular release however have not been studied in detail. Using experimentally constrained three-dimensional diffusion modeling of Ca2+ influx–exocytosis coupling at small excitatory synapses we show that, at physiologically relevant concentrations, Ca2+ buffering by calmodulin plays a dominant role in inhibiting vesicular release and in modulating short-term synaptic plasticity. We also propose a novel and potentially powerful mechanism for short-term facilitation based on Ca2+-dependent dynamic dislocation of calmodulin molecules from the plasma membrane within the active zone.Action potential-dependent release of synaptic vesicles and short-term synaptic plasticity are dynamically regulated by the endogenous Ca CaM regulates synaptic transmission and synaptic plasticity via Ca2+-dependent binding to its target proteins in both the pre- and the post-synaptic compartments. These include protein kinases, adenylyl cyclases, calcineurin, Munc13s, and voltage-gated Ca2+ channels (VGCCs) , parvalbumin, and calretinin -evoked presynaptic Ca2+ dynamics and vesicular release have not been systematically studied.Calmodulin (CaM) is a major neuronal protein that acts as a key mediator of multiple Ca2+ dynamics and synaptic vesicle fusion in small excitatory synapses. We compared the relative contributions of Ca2+ buffering by CB and CaM to modulation of vesicular release probability and short-term synaptic plasticity. Our simulations demonstrate that, at physiologically relevant concentrations, fast Ca2+ binding to the N-lobe of CaM has a dominant effect in inhibiting AP-evoked vesicular release. We also show that the predicted effect of CaM Ca2+ buffering on short-term synaptic plasticity strongly depends on the location and mobility of CaM molecules.In this work we performed realistic, experimentally constrained model simulations of AP-evoked presynaptic Ca2+-dependent interaction of CaM with membrane-associated proteins that contain the isoleucine–glutamine (IQ) binding motif  . This in turn, leads to an increase of vesicular release probability during repeated APs. Thus, AP-evoked dislocation of CaM may provide a powerful mechanism for short-term facilitation of synaptic transmission.Finally, we propose a novel mechanism for a dynamic regulation of presynaptic strength based on Ca2+ influx, buffering, and diffusion, on a millisecond timescale, was performed in the Virtual Cell (VCell) simulation environment (http://vcell.org) using the fully implicit finite volume regular grid solver and a 10 nm mesh. In contrast to the simplified steady-state and/or non-stationary single compartment models that are normally used to approximate presynaptic Ca2+ dynamics on tens to hundreds of milliseconds timescale . The AZ containing VGCCs was modeled as a circle of radius AZR = 0.16 μm situated in the center of the truncated plane z = 0.25 μm. VGCCs were evenly distributed within a rectangular cluster (40 by 80 nm) which was placed in the center of the AZ. The cluster contained 7 P/Q-type, 8 N-type, and 1 R-type VGCCs ,Nonk = 770 μM−1 s−1, (T),Noffk = 1.6 × 105 s−1, (R),Nonk = 3.2 × 104 μM−1 s−1, (R),Noffk = 2.2 × 104 s−1, (T),Conk = 84 μM−1 s−1, (T),Coffk = 2.6 × 103 s−1, (R),Conk = 25 μM−1 s−1, (R),Coffk = 6.5 s−1. The total average CaM concentration was [CaM]tot = 100 μM . In the case of CaM associated with neuromodulin we assumed that (R),Coffk was increased 50-fold ,Coffk = 325 s−1).The model assumed Ca2+ binding sites (two fast and two slow)   was approximated by a first-order reaction: extrj = −extrk · ([Ca2+]−[Ca2+]rest)  = B · [ln(t/t0)]2], where A = 9.2246 × 10−4 pA s, B = 15.78, t0 = 8.036 × 10−4 s  for AP-evoked Ca2+ concentration profiles obtained in VCell simulations at each of the 10 × 10 × 10 nm voxels located immediately above the AZ plane  = 1 − ∑i Vi(t), where ∑i Vi(t) is the sum of occupancies of all six model states iV /dt. In this work we were specifically interested in dissecting the relative effects of CaM and CB on vesicular release and short-term facilitation. Therefore, we did not take into account any changes in the number of release-ready vesicles that occur during paired-pulse stimulation due to vesicle depletion and replenishment. We thus considered that at the onsets of both the first and second APs the vesicular release sensor was in Ca2+ unbound state Vt = 0ms = Vt = 20ms = . To account for sensitivity of AP-evoked release observed in small excitatory hippocampal and neocortical synapses to the slow endogenous buffer EGTA  with the AZ located at the truncated plane .We considered that in addition to ATP the presynaptic bouton contains two major presynaptic Cavp as a function of distance between the VGCC cluster and the vesicular release sensor  should correspond to an average coupling distance d within a 30–50 nm range  we performed simulations using different combinations of CB and CaM either absent or present at physiological concentrations  relative to control simulations without CB and CaM. CaM had even stronger inhibitory effect: ~80% reduction of vp at d = 40 nm (from 0.58 to 0.12). Consistent with the steep power relationship between vesicular release rate and [Ca2+] at the release sensors .To compare the relative contributions of CB and CaM to buffering of AP-evoked  transients at the AZ and vp  and calculated the dependencies of paired-pulse ratios (PPRs) on the coupling distance d both for peak [Ca2+] (PPR [Ca2+]peak = [Ca2+]AP2peak/[Ca2+]AP1peak) and for the vesicular release probability (vpPPR = pAP2v/pAP1v) .We first considered the case of mobile CaM Figure , 2. We m Figures . It shouPPR [Ca2+]peak and vpPPR  and in the rest of the bouton volume  . Ca2+ unbinding from the fully occupied C-lobe occurs on a longer timescale ,Coffk = 6.5 s−1) than the 20 ms inter-spike interval. Therefore, at a typical coupling distance d = 40 nm only 80% of Ca2+ binding sites on the C-lobe were free at the onset of the second AP ,Noffk = 2.2 × 104 s−1). Therefore, concentrations of the available N-lobe Ca2+ binding sites were similar at the onsets of the first and the second APs, which led to occlusion of the paired-pulse facilitation caused by saturation of CB and the C-lobe of CaM. In this set of simulations we used Ca2+ binding kinetics determined for free CaM  or decrease  Ca2+ affinity of CaM  in comparison to the case with evenly distributed CaM (100 μM). The detailed Ca2+ binding kinetics to CaM associated with neuromodulin remains unknown. However, binding of CaM to the post-synaptically expressed neurogranin (which contains a similar CaM-binding IQ motif) has been shown to decrease Ca2+ affinity of the CaM C-lobe because of ~ a fifty-fold acceleration of Ca2+ dissociation rate (R),Coffk ,Coffk 50-fold (from 6.5 to 325 s−1).We first considered a limiting case where all CaM molecules were irreversibly bound to neuromodulin molecules located in the bouton plasma membrane. In the VCell simulations we assumed that all CaM molecules were located within a single 10 nm layer adjacent to the plasma membrane Figure . This leThe simulations revealed that in the case of irreversible binding of CaM to membrane associated neuromodulin, the presence of CaM still partially occludes the short-term facilitation caused by saturation of CB Figure  to the d2+-dependent interaction between CaM and neuromodulin. Ca2+ binding by the C-lobe of CaM reduces its affinity to neuromodulin several fold which leads to dissociation of CaM—neuromodulin complex  all CaM molecules were bound to the plasma membrane via the interaction with neuromodulin. We assumed that upon binding of two Ca2+ ions by the C-lobe (independently of the Ca2+ occupancy of the N-lobe), a CaM molecule can irreversibly dissociate from the plasma membrane and freely diffuse in the cytosol (with CaMD = 20 μm2 s−1)  is unknown, but based on thermodynamics principles it is likely to be comparable to the effective Ca2+ dissociation rate. Therefore, we assumed that upon Ca2+ binding by the C-lobe there is a 50% chance of CaM dissociation from neuromodulin ,Coffk = 650 s−1).To test the feasibility of this hypothesis we modeled how Can Figure . As in S) Figure . The disCaM]tot in the AZ caused by Ca2+ influx during the first AP  on vesicular release probability and short-term synaptic plasticity. To constrain the model parameters we used recently published detailed kinetics of Ca2+ binding to CaM . Such an occlusion mechanism, and possible differences in concentration, location and mobility of CaM may explain why Ca2+ saturation of CB contributes to short-term facilitation only in certain types of synapses . This corresponds to ~25 CaM molecules located at an average sized AZ with an area AZS = 0.04 μm2 (Schikorski and Stevens, Ca2+]rest apoCaM molecules are also bound to the presynaptic VGCCs via a similar IQ-motif interaction (Ben-Johny and Yue, When modeling the effect of Ca2+ ions. Yet, the detailed kinetics of CaM and neuromodulin interaction in the presence and in the absence of Ca2+ remains largely unknown. Thus, further experimental and modeling work is required to obtain more realistic models of the complex kinetics of Ca2+-dependent interaction of CaM with its binding partners at the AZ. Furthermore, activity-dependent phosphorylation of neuromodulin and other IQ-motif containing proteins prevents their interaction with CaM (Xia and Storm, 2+ buffering capacity at the AZ and vp on a longer timescale. Our theoretical modeling study thus argues that Ca2+-dependent CaM dislocation from the plasma membrane could provide a powerful mechanism for dynamic modulation of vesicular release during physiological patterns of activity, and calls for direct experimental testing of this hypothesis.In this work we used a simplified model that did not take into account the mobility of VGCCs in the presynaptic membrane (Schneider et al., 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.


Mammalian target of rapamycin (mTOR) signaling is a critical pathway in the biology of acute myeloid leukemia (AML). Proviral integration site for moloney murine leukemia virus (PIM) serine/threonine kinase signaling takes part in various pathways exerting tumorigenic properties. We hypothesized that the combination of a PIM kinase inhibitor with an mTOR inhibitor might have complementary growth-inhibitory effects against AML. The simultaneous inhibition of the PIM kinase by pan-PIM inhibitor AZD1208 and of mTOR by selective mTORC1/2 dual inhibitor AZD2014 exerted anticancer properties in AML cell lines and in cells derived from primary AML samples with or without supportive stromal cell co-culture, leading to suppressed proliferation and increased apoptosis. The combination of AZD1208 and AZD2014 rapidly activated AMPKα, a negative regulator of translation machinery through mTORC1/2 signaling in AML cells; profoundly inhibited AKT and 4EBP1 activation; and suppressed polysome formation. Inhibition of both mTOR and PIM counteracted induction of heat-shock family proteins, uncovering the master negative regulation of heat shock factor 1 (HSF1), the dominant transcription factor controlling cellular stress responses. The novel combination of the dual mTOR inhibitor and pan-PIM inhibitor synergistically inhibited AML growth by effectively reducing protein synthesis through heat shock factor pathway suppression. FLT3) and KIT is a frequent event in AML pathogenesis, regulating various downstream pathways such as Ras/Raf/MEK/ERK, PI3K/PTEN/AKT/mTOR, and Jak/STAT and resulting in leukemogenesis [Acute myeloid leukemia (AML), the clonal expansion of hematopoietic progenitors characterized by acquired somatic mutations, represents one of the most common types of leukemia in adults, though it affects people of all ages . Activatogenesis . HoweverFLT3 signaling induces expression of the STAT5 gene, which subsequently induces expression of proviral integration site for moloney murine leukemia virus 1 (PIM1) in AML [FLT3 is the most frequently mutated gene in AML cells, and approximately 30% of AML patients harbor constitutively activating internal tandem duplication (ITD) mutations known to be associated with poor prognosis [Activated rognosis , 4-6.PI3K/AKT/mTOR is an evolutionarily conserved pathway that plays an important role in regulating cell growth and proliferation. Mammalian target of rapamycin (mTOR) has been an attractive target for treatment of various types of tumors for years, but the efficacy and clinical activity of the first-generation mTOR inhibitors, which selectively target mTORC1, have been disappointing , 5. InabAZD2014 is a second-generation dual mTORC1/2 inhibitor that has entered preclinical and clinical trials, showing highly specific activity against mTORC1/2 and thereby efficiently blocking the AKT/mTOR signal transduction pathway . AZD2014PIM proteins are considered “weak” oncoproteins because they require an accompanying oncoprotein to exert their tumorigenic properties. They are overexpressed in a broad range of tumors, including both hematological malignancies  and soliin vitro screening to identify the efficacy of a novel drug combination, pan-PIM inhibitor AZD1208 and dual-mTORC1/2 inhibitor AZD2014, and demonstrated that the combination of AZD2014 and AZD1208 synergistically inhibited growth of AML regardless of FLT3 mutation status through impaired polysome assembly and induction of apoptosis.Therefore, we hypothesized that these inhibitors used in combination might have complementary growth-inhibitory effects against AML, although no preclinical investigation of a regimen combining mTOR and PIM inhibitors against AML subtypes has been published. In this study, we used FLT3-ITD activation mutation leads to upregulation of PIM1 [FLT3 mutations are associated with high PIM1 expression levels in AML, we first determined the PIM1 mRNA expression levels in AML patient samples from 2 large studies (GSE14468 and GSE1159) using the Oncomine Platform . These analyses showed greater expression of PIM1 mRNA in the FLT3-mutant (FLT3-MT) samples than in the FLT3 wild-type (FLT3-WT) AML samples in both the GSE14468 series  and the GSE1159 series   revealed no association of PIM expression and FLT3 mutation status  further demonstrated no distinct relevance to FLT3-mutation status (FLT3 mutations might be associated with high levels of PIM1 mRNA but not protein expression in AML.It has been reported that  of PIM1 . To exam) Figure . Immunobs Figure . The PIMn status . These r. The sensitivity to PIM inhibitor AZD1208 varied among AML cell lines: MOLM-16 was extremely responsive to AZD1208, requiring the lowest concentration to induce cell growth inhibition and cell death, followed by OCI-AML3, MV4;11, and MOLM-13, which required progressively higher doses. To assess the interaction between PIM and mTOR inhibitors, we used Calcusyn software to analyze their effects on growth inhibition according to the Chou-Talalay method [To assess the effects of a combination of pan-PIM inhibitor AZD1208 and dual mTORC1/2 inhibitor AZD2014, we investigated the proliferation and survival of cultured AML cell lines treated with these agents. AML cells were treated with AZD1208, AZD2014, or their combination for 72 h, and the viable cells and Annexin V-positive cells were isolated and counted. As shown in Figure y method ; this anFLT3-WT or FLT3-ITD status , one of the key molecules in mTOR pathway-mediated CAP-dependent translation, is the known downstream target of AZD1208 in AML , 25. Phol Figure .The association of AKT phosphorylation with cell survival has been reported previously . AKT is CH10 (HSPE1). Metabolic pathway-related proteins such as fatty acid synthase also were significantly downregulated by the combination  analysis, which measures mature proteins as an approach to closely examining possible biological changes in cells. As shown in Table on Table .Using DAVID Bioinformatics Resources , 30 and The IPA platform highlighted repression of the transcription factor heat shock factor 1 (HSF1), a central upstream regulator, by the combination of AZD1208 and AZD2014 in both the MOLM-16 and OCI-AML3 cell lines . PreviouThe PI3K/AKT/mTOR signaling pathway plays an important role in polysome formation, which controls CAP-dependent translation for protein synthesis . We thercyclin D1 (CCND1) transcripts present in each sucrose gradient fraction relative to β-actin in MOLM-16 cells. In these cells, AZD2014 alone or in combination with AZD1208 strongly reduced the polysome association of CCND1 transcripts, whereas AZD1208 alone had weak influence on the CCND1-polysome association  differences in expression of multiple proteins in the same pathway [et al. [Our protein expression analysis and profiling of relevant pathways revealed suppression of Myc by the mTOR inhibitor, of Myb by the PIM inhibitor, and of HSF1 by either. Although the of 4EBP1 . Both Myof 4EBP1 . Notablyof 4EBP1 , 50, andof 4EBP1 . Global  pathway , indicat pathway . Inhibit pathway . We have pathway . While o [et al.  demonstr [et al. , 57. Imp [et al. .In summary, our findings provide the first evidence that the combination of dual mTOR inhibitor AZD2014 and pan-PIM inhibitor AZD1028 effectively reduces protein synthesis by simultaneous inhibition of the mTORC1/2 pathway and induces apoptosis in AML cells. These findings suggest that this combination is an attractive therapeutic strategy for AML and warrants further clinical investigation.FLT3-ITD mutant AML cell lines were used: MV4;11 [FLT3-WT AML cell lines OCI-AML3 [2.The following : MV4;11 , purchas: MV4;11 , provide: MV4;11 , provideOCI-AML3  and MOLM2 in minimum essential medium alpha supplemented with 20% FBS, 1% L-glutamine, and 1% penicillin-streptomycin as described elsewhere [5, with or without a layer of MSCs plated at a density of 0.2×105 cells/cm2. Co-cultured AML cells were separated from the MSC monolayer by careful pipetting with ice-cold phosphate-buffered saline solution (PBS), repeated twice. After the AML cells were collected, to rule out the possibility of contamination with MSCs, MSC monolayers were examined by microscopy (×100) to confirm that the monolayer was not damaged and that fewer than 10 leukemic cells per visual field remained attached. To verify lack of significant contamination in collected leukemic cells, they were analyzed by flow cytometry using CD45 as a discriminator between leukemic cells and MSCs [To represent the BM stroma, MSCs obtained from healthy BM donors and AML patients were cultured at a density of 5,000 to 6,000 cells/cmlsewhere . Passageand MSCs . Data wePeripheral blood and BM samples were obtained from patients with AML after informed consent was obtained in accordance with a protocol approved by The University of Texas MD Anderson Cancer Center Institutional Review Board regulations. Ficoll-Hypaque density gradient centrifugation was used to separate mononuclear cells .Cell viability, apoptosis, and specific cell death were assessed by the trypan blue exclusion cell count method and by Annexin V/7AAD staining positivity analyzed by a fluorescence-activated cell sorting FACS Array Bioanalyzer  as described previously .MOLM-16 and OCI-AML3 cells were treated for 6 h with AZD1208 (2 μM), AZD2014 (1 μM), or the combination. Cells were then fixed with 1.6% paraformaldehyde  and subjected to permeabilization in ice-cold methanol  for 20 min. After washing twice, cells were resuspended in 1% bovine serum albumin in PBS. Antibodies were added to the cell suspension and incubated for 30 min. Antibodies used were Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) mouse monoclonal antibody ; Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP rabbit monoclonal antibody ; Phospho-Akt (Ser473) rabbit monoclonal antibody ; and CXCR4 . After washing twice, cells were resuspended and analyzed by a Gallios flow cytometer (Beckman-Coulter).RealTimePrimers.com) and cyclin D1 .RNA (50-100 ng) from polysomal fractions isolated by using the RNeasy kit  was used to synthesize cDNA with the iScript Reverse Transcription Supermix . Real-time polymerase chain reactions (PCR) were then performed on a 7900 System  with a SYBR Green qPCR kit (Applied Biosystems) and gene-specific primers for β-actin . AZD1208 (1 or 3 μM), AZD2014 (0.25 or 0.5 μM), or the combination was added to the medium before plating. The cells were subjected to vortexing for 15 s and were then plated on 35×10-mm dishes with a 2×2-mm grid (NUNC) in triplicate and incubated in a humidified chamber at 37°C in 5% CO2 for 14 days. Colonies were scored by using a 1×-3 stereoscope .Mononuclear cells were seeded at 0.05-0.1×10Cells were solubilized in lysis buffer  and incubated for 30 min on ice. The lysates were then subjected to centrifugation for 10 min at 13,000 rpm at 4°C. Protein concentration was determined by the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories) according to the manufacturer's instructions. Total proteins (40 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories) and transferred to polyvinylidene-fluoride membranes , then probed with first and second antibodies according to the manufacturers’ protocols. For immunoblotting, the following antibodies were used: alpha-tubulin and beta-actin (Sigma-Aldrich), PIM1 , PIM3 , PIM2, p70 S6 Kinase, phospho-(p-) p70 S6 Kinase (Thr389), 4EBP1, phospho-(p-)4EBP1 (Thr37/46), AKT, p-AKT (Ser473), AMPK-α, pAMPK-α (Thr172), c-myc, GAPDH, and horseradish peroxidase-linked anti-mouse and anti-rabbit IgG .iTRAQ sample labeling, mass spectrometry analysis, and peptide identificationwww.qiagen.com/ingenuity).The iTRAQ chemical labeling mass spectrometry (MS) method was performed by following the manufacturer's protocol . Briefly2; 1.5 mM KCl) supplemented with cycloheximide (100 μg/mL), dithiothreitol , protease inhibitor , and RNase inhibitor . After the suspension was subjected to vortexing for 4 s, Triton ×100  and sodium deoxycholate  were added to the mix. After spinning at 12,000g for 5 min at 4°C, the supernatant was transferred to a new tube and snap-frozen in liquid nitrogen. Polysomal fractionation was carried out as previously described [Molm-16 and OCI-AML3 cells were treated for 6 h with 2 or 3 μM AZD1208, respectively, 1 μM AZD2014, or the combination. Cells were then washed twice with PBS supplemented with cycloheximide  and resuspended in hypotonic lysis buffer (5 mM Tris, pH 7.5; 2.5 mM MgClescribed .Synergism, additive effects, and antagonism were assessed by the Chou-Talalay method , utilizi