Intracellular Alkalinization Induces Cytosolic Ca2+ Increases by Inhibiting Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA)

Intracellular pH (pHi) and Ca2+ regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca2+. The sources of the Ca2+ increase are from the endoplasmic reticulum (ER) Ca2+ pools as well as from Ca2+ influx. The store-mobilization component of the Ca2+ increase induced by the pHi rise was not sensitive to antagonists for either IP3-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), leading to depletion of the ER Ca2+ store. We further showed that the physiological consequence of depletion of the ER Ca2+ store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca2+ influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca2+ leak from ER pools followed by Ca2+ influx via SOCs

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

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Regulatory and functional studies of store-operated calcium entry

Ca2+ signaling is essential for a wide variety of cellular activities, ranging from short term activities, such as synaptic and muscle contraction, to long term processes, such as proliferation and differentiation. Store-operated Ca2+ entry (SOCE), an important Ca2+ influx pathway in non-excitable cells, well coordinates Ca2+ release from ER and Ca2+ influx through plasma membrane. STIM1 and Orai1, serving as ER Ca2+ sensor and pore forming subunit, respectively, are the two essential components of SOCE machinery. In addition to activate Orai1 channel, studies have shown that STIM1 regulates other plasma membrane Ca2+ channels and senses a variety of cellular stresses to regulate SOCE. Therefore, it is of great interests to investigate the mechanisms and physiological functions of STIM1 and Orai1 mediated SOCE. Here, we performed tandem affinity purification to identify STIM1 associated proteins in Hela cells stably expressing STIM1-His6-3×Flag. Four candidate proteins, including GRP78, HSP70, IQGAP1, and Actin, were identified by mass spectrometry analyses. Surprisingly, IQGAP1 failed to affect the activity of SOCE. Interestingly, GRP78 knockdown only affected the inactivation phase while exerted no effect on the activation phase of SOCE. In addition, GRP78 knockdown markedly induced cell apoptosis and dramatically increased the ER Ca2+ concentration. Moreover, GRP78 was involved in the regulation of SOCE by the ER stress. These data indicate that GRP78 is an important regulator of SOCE to prevent Ca2+ overload in cells. HSP70, however, significantly reduced the activity of SOCE by inhibiting STIM1 translocation to ER-PM junctions. Future studies will explore the mechanism of GRP78 and HSP70 in regulating SOCE by confocal and TIRF imaging. Embryonic stem (ES) cells proliferate unlimitedly and can differentiate into all fetal and adult cell types. This property endows ES cells to be the promising candidates in the therapy of neurodegenerative diseases. Thus, it is important to identify novel signaling molecules or events that could play a role in the neural commitment of ES cells. Accumulated evidences have documented the role of STIM1 and Orai1 mediated SOCE in neuronal activities. Yet, the role of SOCE in early neural development remains to be determined. Here we examined the role of STIM1 and Orai1 during neural differentiation of mouse ES cells. Both of STIM1 and Orai1 were expressed and functionally active in ES cells, and expressions of STIM1 and Orai1 were dynamically regulated during neural differentiation of mouse ES cells. STIM1 knockdown inhibited the differentiation of mouse ES cells into neural progenitors, neurons, and astrocytes. In addition, STIM1 knockdown caused severe cell death and markedly suppressed the proliferation of neural progenitors. Surprisingly, Orai1 knockdown had little effect on neural differentiation of mouse ES cells, but the neurons derived from Orai1 knockdown ES cells, like those from STIM1 knockdown cells, had defective SOCE. Taken together, our data indicate that STIM1 is required for neural differentiation of mouse ES cells independent of Orai1-mediated SOCE.published_or_final_versionPhysiologyDoctoralDoctor of Philosoph

TRPC3 is required for the survival, pluripotency and neural differentiation of mouse embryonic stem cells (mESCs)

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Application of Antibodies to Neuronally Expressed Nogo-A Increases Neuronal Survival and Neurite Outgrowth

Nogo-A, a glycoprotein expressed in oligodendrocytes and central nervous system myelin, inhibits regeneration after injury. Antibodies against Nogo-A neutralize this inhibitory activity, improve locomotor recovery in spinal cord-injured adult mammals, and promote regrowth/sprouting/saving of damaged axons beyond the lesion site. Nogo-A is also expressed by neurons. Complete ablation of Nogo-A in all cell types expressing it has been found to lead to recovery in some studies but not in others. Neuronal ablation of Nogo-A reduces axonal regrowth after injury. In view of these findings, we hypothesized that, in addition to neutralizing Nogo-A in oligodendrocytes and myelin, Nogo-A antibodies may act directly on neuronal Nogo-A to trigger neurite outgrowth and neuronal survival. Here, we show that polyclonal and monoclonal antibodies against Nogo-A enhance neurite growth and survival of cultured cerebellar granule neurons and increase expression of the neurite outgrowth-promoting L1 cell adhesion molecule and polysialic acid. Application of inhibitors of signal transducing molecules, such as c-src, c-fyn, protein kinase A, and casein kinase II reduce antibody-triggered neurite outgrowth. These observations indicate that the recovery-promoting functions of antibodies against Nogo-A may not only be due to neutralizing Nogo-A in oligodendrocytes and myelin, but also to their interactions with Nogo-A on neurons

Intracellular alkalinization induces Stim1 and Orai1 colocalization in HeLa cells.

<p>HeLa cells, co-transfected with plasmids, Stim1-mCherry and Orai1-EGFP, were incubated in Ca²⁺ free HBSS for 15 min as control or treated with thapsigargin (10 M) or DIEA.HBr (4 mM) for 15 min in Ca²⁺ free HBSS. Confocal imaging of both mCherry and EGFP were taken. The graphs represent data from three independent experiments. Scale bar: 5 µm.</p

Intracellular alkalinization induces extracellular Ca²⁺ influx through SOCs in NIH 3T3 cells.

<p>(A) DIEA.HBr (4 mM) induced-Ca²⁺ influx was inhibited by La³⁺ (100 µM), a SOC blocker, treatment in Fura-2 loaded NIH3T3 cells incubated in regular HBSS. (B) Immunoblot analysis of Stim1-knockdown in NIH3T3 cells. MEK1 immunoblot was used as the internal control. (C) Quantitative real-time RT-PCR analysis of Orai1-knockdown in NIH3T3 cells. GAPDH was used as the internal control. Data are expressed as means ± S.D., n = 3. (D) and (E) Stim1 or Orai1 knockdown abolished the sustained Ca²⁺ influx induced by thapsigargin (10 µM) (D) and by DIEA.HBr (4 mM) (E) in Fura-2 loaded NIH3T3 cells incubated in regular HBSS. (F) Stim1 and Orai1 knockdown diminished the Ca²⁺ influx induced by DIEA.HBr (4 mM) in Fura-2 loaded NIH3T3 cells. Cells were initially treated with thapsigargin (1 µM) in Ca²⁺-free HBSS to deplete ER Ca²⁺ pool, followed by 2 mM Ca²⁺ addition. All graphs represent data from three independent experiments. Data quantification in (A), (D), (E), and (F) are presented as mean ± S.E., n = 30–50 cells. The * symbols indicate the results of <i>t</i> Test analysis, <i>p</i><0.05.</p

Intracellular alkalinization induces cytosolic Ca²⁺ increases in HeLa cells.

<p>(A) DIEA.HBr, similar to NH₄Cl, induced cytosolic Ca²⁺ increases in a dose-dependent manner in HeLa cells as measured by the Ca²⁺-indicator, Fura-2 AM. (B) Intracellular alkalinization induced by DIEA.HBr (10 mM) and NH₄Cl (10 mM) were inhibited by sodium acetate (40 mM) as measured by the pH-indicator, BCECF AM. (C) Cytosolic Ca²⁺ increases induced by DIEA.HBr (10 mM) and NH₄Cl (10 mM) were markedly inhibited by sodium acetate (40 mM). The graphs represent data from three independent experiments. Data quantifications of the time to reach pH_i peak (B) or [Ca²⁺]_i peak (C) after drug treatment were expressed as mean ± S.E., n = 30–50 cells.</p

Intracellular alkalinization releases Ca²⁺ from ER pools in HeLa cells and PC12 cells.

<p>(A) DIEA.HBr (4 mM)-induced Ca²⁺ increase in Fura-2 loaded HeLa cells was abolished by thapsigargin (1 µM) pretreatment. This Ca²⁺ increase was inhibited by removal of external Ca²⁺ (Ca²⁺-free HBSS with 4 mM EGTA). (B) Pretreatment of Fura-2 loaded HeLa cells with either <i>Xestospongin C (XeC)</i> (10 µM) or U73122 (10 µM) did not inhibit the DIEA.HBr-induced Ca²⁺ increase compared with untreated cells. The graphs represent data from three independent experiments. (C) Pretreatment of Fura-2 loaded PC12 cells with ryanodine (20 µM) or 8-Br-cADPR (100 µM) did not inhibit the DIEA.HBr-induced Ca²⁺ increase compared with untreated cells. The graphs represent data from three independent experiments. (D) Pretreatment of Fura-2 loaded HeLa cells with glycyl-l-phenylalanine 2-naphthylamide (GPN) (50 µM) did not inhibit the DIEA.HBr-induced Ca²⁺ increase compared with untreated cells while completely blocked GPN or bafilomycin A1 (0.5 µM)-induced Ca²⁺ increase. The graphs represent data from three independent experiments.</p