STIM1Redistribution And Involvement In Nucleoplasmic Ca2+Signaling In HUVEC

Part ITranslocation of STIM1after calcium store depleting and it’s involvement of [Ca2+]N in HUVECsBackgroundStore-operated Ca2+(SOC) influx was an important process in cellular physiology. It manipulated such diverse functions as refilling of intracellular Ca2+stores, activation of enzymatic activity, gene transcription, and release of cytokines. The molecular components of the underlying channels and the mechanisms controlling them are still unclear. STIM1was first concerned to people due to its tumor inhibition, it played an important role in the immune deficiency disease and many types of tumors, until2005, Jack Roos team found, STIM1was essential to SOC channels in mammals. The single transmembrane protein STIM1was present in the endoplasmic reticulum, which contained several conserved domains:the EF-hand located in the ER lumen and the sterile a the motif (SAM), sequencing, and EF-hand and SAM was conserved domains from worms to butterflies to the human; coiled-coil domains, located in the cytoplasm, Pro/Ser-rich region, and Lys-rich region.STIM1N-terminus of the EF-hand was a calcium sensor, it sensed Ca2+「by an EF hand Ca2+-binding site in the lumen of the ER:when ER was filled, Ca2+-binding EF-hand formed a conventional helix-loop-helix EF motif; when ER was depleted, STIM1formed dimers and oligomers. And other domains, such as SAM, coiled-coil domains, Pro/Ser-rich region, and Lys-rich region played crucial roles in the STIM1translocation and activation of SOC channels.ER uptake and stored calcium by calcium pump ATPase. TG as the calcium pump ATPase inhibitors, it made calcium stores can not be refilled, so as to achieve the purpose of depleting calcium stores. When refilling the ER calcium stores, STIM1was mainly located in the cytoplasm, including the endoplasmic reticulum within the N-terminal EF-Hand and endoplasmic reticulum Ca2+binding. After calcium store depletion by TG, the EF-Hand and Ca2+dissociated, STIM1translocated to the PM, the cytoplasmic C-terminal and Oraill binding uncovered another gated channel, CRAC activation domain (CRAC activation domain (CAD), also known as SOAR or OASF), and Ca2+influx, the calcium store refilled, then the STIM1got back to the resting structure.STIM1caused extracellular calcium influx are summarized as follows:(1) dissociation of Ca2+;(2) rapid oligomerization;(3) spatially restricted translocation to nearby ER-PM junctions;(4) activation of PM Ca2+「Channels.But, we found STIM1translocated to nuclear membrane after TG in the HUVEC cells.(see Hu Yali’s thesis). Did such translocation to the nuclear membrane have a special physiological significance? For example, it may participate in the regulation of nuclear calcium signaling? Sowe explore it in this study.ObjectiveTo explore the role of STIM1involved in nuclear calcium signalings after STIM1knockdown.MethodIn this study we transfected STIM1knockdown plasmid to knockdown STIM1expression, and readded2mM Ca2+「after TG-evoked store depletion. Then measured the [Ca2+]C and [Ca2+]N to analyze whether STIM1involved in nuclear calcium regulation. Result1, After stable transfecting of STIM1shRNA in HUVECs, STIM1knockdown group was declined (73.4±7.2)%,p<0.01. While the vector group (98.5±8.6)%and the nonspecial group (99.1±5.9)%were no differences to the control group.2, After TG-evoked store depletion(Rl), readded2mM Ca2+(R2), the STIM1knockdown group cytoplasmic R2/R1was0.31±0.03, it was declined (73.4±3.7)%compared to control group, p<0.01, nucleoplasmic R2/R1was0.13±0.006, n=54, it was declined (89.6±5.6)%compared to control group, p<0.01, and the deduction of nucleoplasmic R2/R1was more noticeable than cytoplasmic, p<0.01; the vector group cytoplasmic R2/R1was1.36±0.05, nucleoplasmic R2/R1was1.28±0.05, the nonspecial group cytoplasmic R2/R1was, nucleoplasmic R2/R1was1.28±0.05, n=94; the control group cytoplasmic R2/R1was1.23±0.06, nucleoplasmic R2/R1was1.30±0.10, n=80, there were no differences among the other three groups both cytoplasmic and nucleoplasmic R2/R1,p>0.05.Conclusions1, SOCs influx was supressed when STIM1was knockdown2, nucleoplasma calcium was supressed when STIM1was knockdown The results suggest that STIM1was essential to SOCs opening, and it participated in nucleoplasma calcium regulation. Part ⅡTranslation of mutated STIM1and it’s involvment of [Ca2+]N in HUVECsBackgroundIn our laboratory we had found STIM1translocated to NE after TG-induced ER depletion in HUVECs. and it participated in both the cytoplasmic and nucleoplasmic calcium regulation. What are the reasons for such differences? We speculate it may be due to STIM1mutations in HUVECs which cause its function change. Then three sites of point mutations were found in HUVECs, so we constructed mutant STIM1plasmids for the further study. What’s more, we found cytoplasmic independent nucleoplasmic calcium regulation(see Yan Yan’s thesis). Readding the extracellular calcium after TG-induced calcium stores depletion in the HUVECs, different concentrations of extracellular calcium induced different rise of cytoplasmic and nuclear calcium signaling. Cytoplasmic calcium signal decreased with declining of extracellular calcium, but when given less than0.1mM extracellular calcium the nucleoplasmic calcium signal is not decreased any more. So we believe in HUVECs the nuclei had its own independent calcium signal regulation system, and we study cytoplasmic independent nucleoplasmic calcium signaling by0.1mM extracellular calcium.Objective1, To explore the correlation of wild type and mutated STIM1to cytoplasmic and nucleoplasmic calcium. 2, To survey the tanslocation of wild type and mutated STIM1after ER depletion.3, To clarify if wild type and mutated STIM1participated in nucleoplasmic calcium regulation.4, To observed if wild type and mutated STIM1regulated the nucleoplasmic calcium in HEK293.Method1, quantitated the expression of wild type and mutated STIM1by Realtime PCR, and analyze its correlation to cytoplasmic and nucleoplasmic calcium by linear regression analysis.2, observed the translocation of wild type and mutated STIM1after TG-treated when over-expressed wild type and mutated STIM1.3, investigate what role the wild type and mutated STIM1played in nucleoplasmic calcium regulation in HUVECs by over-expressing wild type and mutated STIM1and knockdown STIM1expression.4, investigate what role the wild type and mutated STIM1played in nucleoplasmic calcium regulation in HEK293by over-expressing wild type and mutated STIM1and deducing STIM1expression via gene interference.Results1, HUVECs were isolated from three different donors. The relative abundance of wild type and mutated STIM1mRNA as well as [Ca2+]C and [Ca2+]N alterations stimulated by histamine in the presence of2.0mM Ca2+were monitored. Regression analysis revealed a good correlation between [Ca2+]N amplitude and mutated STIM1abundance (mutated STIM1mRNA=1.00:1.33:2.44, R=0.636, p<0.01), whereas no correlation between [Ca2+]N and wild type STIM1abundance (wild STIM1mRNA=0.60:1.00:1.07, p>0.01). These results provide an additional support for the mutated STIM1in regulating [Ca2+]N. There is no correlation between [Ca2+]c amplitude and wild STIM1abundance (p>0.01), indicating that the difference of wild STIM1abundance among HUVECs from the donors is not sufficient to affect [Ca2+]C or the effect of wild STIM1among HUVECs from the donors is possibly saturated. While the difference of mutated STIM1abundance does not affect [Ca2+]C amplitude either (p>0.01), further suggesting the specific role of mutated STIM1in regulating [Ca2+]N signaling.2, we constructed and transfected a vector expressing humanized recombinant GFP fused with STIM1into HUVEC on coverslips using established techniques. A time-series of images were taken using a laser-scanning confocal microscope system at various time periods up to10min after intracellular Ca2+store(s) depletion. Intracellular Ca2+store(s) depletion stimulated a rapid translocation of mutated STIM1-GFP from the cytoplasm to nuclear membrane and the subsequent sliding along nuclear membrane in HUVEC. By contrast, translocation from cytoplasm to nuclear membrane was not observed in wild type STIM1-GFP expressed HUVEC after intracellular Ca2+store(s) depletion. The translocation of STIM1-GFP from the cytoplasm to cytoplasmic membrane was noted in both mutated and wild type STIMl-expressing HUVEC after intracellular Ca2+store(s) depletion. The real time and continuous monitoring of fluorescence intensities in one dotted area in nuclear membrane revealed interval or repetitive GFP translocation after intracellular Ca2+store(s) depletion in HUVEC expressing mutated, not wild type STIM1-GFP or in a time control HUVEC without intracellular Ca2+store(s) depletion. 3,The overexpression of wild STIM1slightly increased [Ca2+]c and [Ca2+]N. By contrast, the overexpression of mutated STIM1greatly increased [Ca2+]c and [Ca2+]N including both [Ca2+]c-independent [Ca2+]N (in the presence of0.1mM Ca2+) and [Ca2+]c-dependent [Ca2+]N amplitude (in the presence of2.0mM Ca2+). Importantly, the overexpression of mutated STIM1increased [Ca2+]c-independent [Ca2+]N more profoundly than [Ca2+]c (changed by～600%for [Ca2+]N amplitude, p<0.01vs.～150%for [Ca2+]c amplitude). This result provides evidence that mutated STIM1plays a role in regulating nucleoplasmic Ca2+signal. Of note, the overexpression of mutated STIMl also increased [Ca2+]N amplitude in the presence of2.0mM Ca2+, which is probably due to the role of mutated STIM1in regulating nucleoplasmic Ca2+itself and the increased cytoplasmic Ca2+signal under this condition. The altered cytoplasmic Ca2+signal by the manipulation of mutated STIM1expression suggests that mutated STIM1can also provide an additional role in store-operated Ca2+entry into cytoplasma, although this is much weaker than its role in regulating nucleoplasmic Ca2+. While the expression of STIM1is knockdown by shRNA, both [Ca2+]C-independent [Ca2+]N and [Ca2+]C-dependent [Ca2+]N as well as [Ca2+]C itself were inhibited. The more profound inhibition of [Ca2+]N compared with [Ca2+]c in the presence of2.0mM Ca2+is probably due to the decreased levels of endogenous wild and mutated STIM1. The inhibition of [Ca2+]C-independent [Ca2+]N by STIM1knockdown provides an additional evidence for the role of STIM1in nucleoplasmic Ca2+signaling.Conclusions1, STIM1translocated to NE was due to STIM1mutated in HUVECs.2, STIM1participated in nucleoplasmic calcium regulation by mutated STIM1. Part IIIThe importance of STIM1in [Ca2+]N-regulated transcription and gene expression in HUVECBackgroundIt was also reported that nucleoplasmic and cytoplasmic Ca2+distinctly control gene expression and nuclear calcium plays a key role in neuroprotection which is acquired by synaptic activity via activating the cAMP response element binding protein (CREB). In mouse hippocampal neurons, CREB can derectly activate the transcription factor ATF3(activating transcription factor3). Induction of ATF3expression by CREB in hippocampal neurons was initiated by calcium entry through synaptic NMDA receptors and required nuclear calcium transients and calcium/calmodulin-dependent protein kinase IV activity. Overexpression of cAMP-response element (CRE)-binding protein (CREB) and activating transcription factor (ATF)1contributes to melanoma progression and metastasis at least in part by promoting tumor cell survival and stimulating matrix metalloproteinase(MMP)2expression. To explore any functional significance of STIM1-associated nucleoplasmic Ca2+signaling in HUVEC, we performed molecular assay to measure CREB activity and gene expression of MMP2which has been shown to be regulated by nucleoplasmic Ca2+-activated CREB pathway in previous studies.Objective 1, To characterize the biological significance of STIM1-participated [Ca2+]N signaling in HUVECs, agonist-stimulated cytoplasmic and [Ca2+]N oscillations, CREB activity.2, and CREB-dependent matrix metalloproteinase-2(MMP2) gene expressionMethodWe have two groups, both include the follow groups:control, vector, wild type STIM1, mutated STIM1, STIM1knockdown, vehicle and CREB inhibitor HUVECs. Histamine stimulated both [Ca2+]c and [Ca2+]N oscillations, the former ones ceased at-30min after stimulation, whereas the later ones continued during the experimental period (～60min). Measured the activtiy of CREB by ELISA, and expression of MMP2by Realtime PCR.ResultHistamine stimulated [Ca2+]c oscillations-independent [Ca2+]N oscillations in～55%mutated STIM1-overexpressed HUVECs, in～21-26%control HUVECs, vector, wild STIM1-overexpressed HUVECs, vehicle and CREB inhibitor-pretreated HUVECs, and in only～12%STIM1-knockdown HUVECs (in38,28,19,34,17,22and17cells from at least3separate experiments with [Ca2+]c oscillations in response to histamine stimulation for mutated STIM1-, control-, vector-, wild type STIM1-, vehicle-or CREB inhibitor-HUVECs). The frequency and spike duration of [Ca2+]c oscillations-independent [Ca2+]N oscillations were not affected by the manipulation of STIM1expression or the treatment of KG501. However, the overexpression of mutated STIM1, not wild STIM1significantly increased, whereas STIM1knockdown obviously attenuated the amplitude of histamine-stimulated [Ca2+]c oscillations-independent [Ca2+]N oscillations p<0.05). HUVECs with the above manipulated STIM1expressions were stimulated by1M histamine in the presence of2.0mM extracellular Ca2+for30and60min, then endogenous CREB activity and MMP-2mRNA expression were measured.Conclusions1, Mutated STIM1regulated nucleoplasmic Ca2+in control of CREB activity.2, Mutated STIM1regulated nucleoplasmic Ca2+in control of gene expression.