High glucose-induced apoptosis through store-operated calcium entry and calcineurin in human umbilical vein endothelial cells

Diabetes mellitus causes multiple cardiovascular complications. Previous studies have shown that prolonged exposure (96 h) of human umbilical vein endothelial cells (HUVECs) to hyperglycemia causes a significant increase in apoptosis. We report here that this increase in apoptosis is associated with an increase in Ca(2+) current (whole cell patch-clamp recorded) resulting from Ca(2+) entry mediated by store-operated channels (SOCs). The number of apoptotic cells after prolonged high glucose (HG, 30 mmol/L) exposure was significantly reduced in the presence of the SOC inhibitor 2-APB or of La(3+). A marked increase (approximately 80%) in Ca(2+)-dependent calcineurin (CN-A) phosphatase activity also occurred after prolonged HG exposure. Prolonged HG exposure-induced increase in CN-A activity was prevented by 2-APB, and selective CN-A phosphatase inhibition by FK506 or calmodulin inhibition by calmidazolium decreased HG-induced apoptosis. Blocking hydrogen peroxide production using catalase or inhibiting the tyrosine kinase pp60(src) during prolonged exposure to HG, resulted in a marked decrease in apoptosis and was further associated with a significant reduction in CN-A phosphatase activity. The results demonstrate a significant role for Ca(2+) entry in HG-induced apoptosis in HUVECs, and suggest that this role is mediated via H(2)O(2) generation and the action of the Ca(2+)-activated protein phosphatase calcineurin.     

Temporal modulation of calcium sensing in hematopoietic stem cells is crucial for proper stem cell expansion and engraftment

Hematopoietic stem cells (HSCs) are known to reside in a bone marrow (BM) niche, which is associated with relatively higher calcium content. HSCs sense and respond to calcium changes. However, how calcium-sensing components modulate HSC function and expansion is largely unknown. We investigated temporal modulation of calcium sensing and Ca²⁺ homeostasis during ex vivo HSC culture and in vivo. Murine BM-HSCs, human BM, and umbilical cord blood (UCB) mononuclear cells (MNCs) were treated with store-operated calcium entry (SOCE) inhibitors SKF 96365 hydrochloride (abbreviated as SKF) and 2-aminoethoxydiphenyl borate (2-APB). Besides, K⁺ channel inhibitor TEA chloride (abbreviated as TEA) was used to compare the relationship between calcium-activated potassium channel activities. Seven days of SKF treatment induced mouse and human ex vivo BM-HSC expansion as well as UCB-derived primitive HSC expansion. SKF treatment induced the surface expression of CaSR, CXCR4, and adhesion molecules on human hematopoietic stem and progenitor cells. HSCs expanded with SKF successfully differentiated into blood lineages in recipient animals and demonstrated a higher repopulation capability. Furthermore, modulation of SOCE in the BM-induced HSC content and differentially altered niche-related gene expression profile in vivo. Intriguingly, treatments with SOCE inhibitors SKF and 2-APB boosted the mouse BM mesenchymal stem cell (MSC) and human adipose-derived MSCs proliferation, whereas they did not affect the endothelial cell proliferation. These findings suggest that temporal modulation of calcium sensing is crucial in expansion and maintenance of murine HSCs, human HSCs, and mouse BM-MSCs function.     

Complex actions of 2-aminoethyldiphenyl borate on store-operated calcium entry

Store-operated Ca2+ entry (SOCE) is likely the most common mode of regulated influx of Ca2+ into cells. However, only a limited number of pharmacological agents have been shown to modulate this process. 2-Aminoethyldiphenyl borate (2-APB) is a widely used experimental tool that activates and then inhibits SOCE and the underlying calcium release-activated Ca2+ current (I CRAC). The mechanism by which depleted stores activates SOCE involves complex cellular movements of an endoplasmic reticulum Ca2+ sensor, STIM1, which redistributes to puncta near the plasma membrane and, in some manner, activates plasma membrane channels comprising Orai1, -2, and -3 subunits. We show here that 2-APB blocks puncta formation of fluorescently tagged STIM1 in HEK293 cells. Accordingly, 2-APB also inhibited SOCE and I(CRAC)-like currents in cells co-expressing STIM1 with the CRAC channel subunit, Orai1, with similar potency. However, 2-APB inhibited STIM1 puncta formation less well in cells co-expressing Orai1, indicating that the inhibitory effects of 2-APB are not solely dependent upon STIM1 reversal. Further, 2-APB only partially inhibited SOCE and current in cells co-expressing STIM1 and Orai2 and activated sustained currents in HEK293 cells expressing Orai3 and STIM1. Interestingly, the Orai3-dependent currents activated by 2-APB showed large outward currents at potentials greater than +50 mV. Finally, Orai3, and to a lesser extent Orai1, could be directly activated by 2-APB, independently of internal Ca2+ stores and STIM1. These data reveal novel and complex actions of 2-APB effects on SOCE that can be attributed to effects on both STIM1 as well as Orai channel subunits.     

Synthesis of bisboron compounds and their strong inhibitory activity on store-operated calcium entry

Store-operated calcium entry (SOCE) is an important mechanism for replenishing intracellular calcium stores and for sustaining calcium signaling. We developed a method for synthesis of bisboron compounds that have two borinic acids or their esters in one molecule. These compounds are analogues of 2-APB, which is widely used as a membrane-permeable SOCE inhibitor. Further, we examined the effect of the newly synthesized bisboron compounds on SOCE in Jurkat T cells. All the bisboron compounds showed strong inhibitory activity on SOCE, with IC₅₀ values of less than 1 μM, which were 20–45 times lower than observed with 2-APB.     

Overexpression of Bax induces down-regulation of store-operated calcium entry in prostate cancer cells

Store-operated Ca2+ channels control homeostasis between extracellular Ca2+ reservoir and intracellular Ca2+ storage and play important roles in apoptosis in a wide variety of cells, including prostate epithelia. Recent studies have shown that the acquired apoptosis-resistant nature of androgen-independent prostate cancer is associated with reduced function of store-operated Ca2+ entry (SOCE). This study investigates the functional interaction between Bax and SOCE in the apoptosis signaling cascade in prostate cancer. Our previous findings show that NRP-154, an androgen-independent prostate cancer cell line, could sustain overexpression of exogenous Bax without undergoing apoptosis. Here we show that sustained overexpression of Bax in NRP-154 cells leads to down-regulation of SOCE and reduced Ca2+ storage inside the endoplasmic reticulum. While reduced SOCE may represent an adaptive mechanism for cell survival, increased levels of Bax in the latent state enhances the sensitivity of NRP-154 cells to TGF-beta and thapsigargin-induced apoptosis. This enhanced apoptosis can be reduced by 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of SOCE, or reversed under conditions where SOCE is only partially activated. Our results demonstrate a functional interaction between Bax and SOCE in apoptosis of prostate cancer, and support the concept that improving this interaction has therapeutic implications for prostate cancer.     

Estrogen controls embryonic stem cell proliferation via store-operated calcium entry and the nuclear factor of activated T-cells (NFAT)

Embryonic stem cells (ESCs) can self-renew indefinitely and differentiate into all cell lineages. Calcium is a universal second messenger which regulates a number of cellular pathways. Previous studies showed that store-operated calcium channels (SOCCs) but not voltage-operated calcium channels are present in mouse ESCs (mESCs). In this study, store-operated calcium entry (SOCE) was found to exist in mESCs using confocal microscopy. SOCC blockers lanthanum, 2-aminoethoxydiphenyl borate (2-APB) and SKF-96365 reduced mESC proliferation in a concentration-dependent manner, suggesting that SOCE is important for ESC proliferation. Pluripotent markers, Sox-2, Klf-4, and Nanog, were down-regulated by 2-APB, suggesting that self-renewal property of mESCs relies on SOCE. 17β-estradiol (E2) enhanced mESC proliferation. This enhanced proliferation was associated with an increment of SOCE. Both stimulated proliferation and increased SOCE could be reversed by SOCC blockers suggesting that E2 mediates its stimulatory effect on proliferation via enhancing SOCE. Also, cyclosporin A and INCA-6, inhibitors of calcineurin [phosphatase that de-phosphorylates and activates nuclear factor of activated T-cells (NFAT)], reversed the proliferative effect of E2, indicating that NFAT is involved in E2-stimulated proliferation. Interestingly, E2 caused the nuclear translocation of NFATc4, and this could be reversed by 2-APB. These results suggested that NFATc4 is the downstream target of E2-induced SOCE. The present investigation provides the first line of evidence that SOCE and NFAT are crucial for ESCs to maintain their unique characteristics. In addition, the present investigation also provides novel information on the mechanisms of how E2, an important female sex hormone, affects ESC proliferation.     

Differential roles for STIM1 and STIM2 in store-operated calcium entry in rat neurons

The interaction between Ca(2+) sensors STIM1 and STIM2 and Ca(2+) channel-forming protein ORAI1 is a crucial element of store-operated calcium entry (SOCE) in non-excitable cells. However, the molecular mechanism of SOCE in neurons remains unclear. We addressed this issue by establishing the presence and function of STIM proteins. Real-time polymerase chain reaction from cortical neurons showed that these cells contain significant amounts of Stim1 and Stim2 mRNA. Thapsigargin (TG) treatment increased the amount of both endogenous STIM proteins in neuronal membrane fractions. The number of YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1 complexes was also enhanced by such treatment. The differences observed in the number of STIM1 and STIM2 complexes under SOCE conditions and the differential sensitivity to SOCE inhibitors suggest their distinct roles. Endoplasmic reticulum (ER) store depletion by TG enhanced intracellular Ca(2+) levels in loaded with Fura-2 neurons transfected with YFP-STIM1 and ORAI1, but not with YFP-STIM2 and ORAI1, which correlated well with the number of complexes formed. Moreover, the SOCE inhibitors ML-9 and 2-APB reduced Ca(2+) influx in neurons expressing YFP-STIM1/ORAI1 but produced no effect in cells transfected with YFP-STIM2/ORAI1. Moreover, in neurons transfected with YFP-STIM2/ORAI1, the increase in constitutive calcium entry was greater than with YFP-STIM1/ORAI1. Our data indicate that both STIM proteins are involved in calcium homeostasis in neurons. STIM1 mainly activates SOCE, whereas STIM2 regulates resting Ca(2+) levels in the ER and Ca(2+) leakage with the additional involvement of STIM1.     

Cytoskeletal reorganization internalizes multiple transient receptor potential channels and blocks calcium entry into human neutrophils

Store-operated calcium entry (SOCE) is required for polymorphonuclear neutrophil (PMN) activation in response to G protein-coupled agonists. Some immunocytes express proteins homologous to the Drosophila transient receptor potential gene (trp) calcium channel. TRP proteins assemble into heterotetrameric ion channels and are known to support SOCE in overexpression systems, but the evidence that TRP proteins support SOCE and are functionally important in wild-type cells remains indirect. We therefore studied the expression and function of TRP proteins in primary human PMN. TRPC1, TRPC3, TRPC4, and TRPC6 were all expressed as mRNA as well as membrane proteins. Immunofluorescence microscopy demonstrated localization of TRPC1, TRPC3, and TRPC4 to the PMN cell membrane and their internalization after cytoskeletal reorganization by calyculin A (CalyA). Either TRPC internalization by CalyA or treatment with the inositol triphosphate receptor inhibitor 2-aminoethoxydiphenyl borane resulted in the loss of PMN SOCE. Cytochalasin D (CytoD) disrupts actin filaments, thus preventing cytoskeletal reorganization, and pretreatment with CytoD rescued PMN SOCE from inhibition by CalyA. Comparative studies of CytoD and 2-aminoethoxydiphenyl borane inhibition of PMN cationic entry after thapsigargin or platelet-activating factor suggested that SOCE occurs through both calcium-specific and nonspecific pathways. Taken together, these studies suggest that the multiple TRPC proteins expressed by human PMN participate in the formation of at least two store-operated calcium channels that have differing ionic permeabilities and regulatory characteristics.     

Functional interactions among STIM1, Orai1 and TRPC1 on the activation of SOCs in HL-7702 cells

STIM1, Orai1 and TRPC1 are all reported to be important for store-operated Ca²⁺ entry (SOCE) in diverse cells. However, there is no evidence for the functional interaction of the three proteins in SOCE in human liver cells. The objective of this study is to determine whether they are involved in SOCE in normal human liver cells. Liposomal transfection method was used to increase expression levels of the three proteins in HL-7702 cells, a normal human liver cell line. Western blot and single cell RT–PCR were applied to evaluate transfection effectiveness. Changes in store-operated current (I_SOC) and SOCE were investigated using whole-cell patch-clamp recording and calcium imaging. I_SOC is detected in HL-7702 cells and it is inhibited either by 2-Aminoethoxydiphenyl borate (2-APB) or La³⁺. Overexpression of STIM1 or Orai1 alone did not induce any change in I_SOC. TRPC1-transfection, however, caused approximate 2.5-fold increase in I_SOC. A large increase (>10-fold) in I_SOC emerged when both STIM1 and Orai1 were co-transfected into HL-7702 cells. Co-overexpression of STIM1 + TRPC1 also caused >10-fold increase in I_SOC, and addition of Orai1 did not cause any further increase. In HL-7702 cells, TRPC1 and Orai1 take part in SOCE independently of each other. Functional interactions of STIM1 and Orai1 or TRPC1 contribute to I_SOC activation.     

Phosphatase inhibition reveals a calcium entry pathway dependent on protein kinase A in thyroid FRTL-5 cells: comparison with store-operated calcium entry

Calcium entry through store-operated calcium channels is an important entry mechanism. In the present report we have described a novel calcium entry pathway that is independent of depletion of intracellular calcium stores. Treatment of the cells with the phosphatase inhibitor calyculin A (caly A), which blocked thapsigargin-evoked store-operated calcium entry (SOCE), induced a potent concentration-dependent calcium entry. In a calcium-free buffer, acute addition of caly A evoked a very modest increase in cytosolic free calcium ([Ca(2+)](i)). This increase was not from the agonist-mobilizable calcium stores, as the thapsigargin-evoked increase in [Ca(2+)](i) was unaltered in caly A-treated cells. The caly A-evoked calcium entry was not blocked by Gd(3+) or 2-APB, whereas SOCE was. Caly A enhanced the entry of barium, indicating that the increase in intracellular calcium was not the result of a decreased extrusion of calcium from the cytosol. Jasplakinolide and cytochalasin D had only marginal effects on calcium entry. The protein kinase A (PKA) inhibitor H-89 and an inhibitory peptide for PKA abolished the caly A-evoked entry of both calcium and barium. The SOCE was, however, enhanced in cells treated with H-89. In cells grown in the absence of thyrotropin (TSH), the caly A-evoked entry of calcium was smaller compared with cells grown in TSH-containing buffer. Stimulation of cells grown without TSH with forskolin or TSH restored the calyculin A-evoked calcium entry to that seen in cells grown in TSH-containing buffer. SOCE was decreased in these cells. Our results thus suggest that TSH, through the production of cAMP and activation of PKA, regulates a calcium entry pathway in thyroid cells. The pathway is distinctly different from the SOCE. As TSH is the main regulator of thyroid cells, we suggest that the novel calcium entry pathway participates in the regulation of basal calcium levels in thyroid cells.     

Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels

Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca(2+) sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca(2+) entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca(2+) entry and activated typical I(CRAC) current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, I(SOC), that was blocked by 1 microm Gd(3+) and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and I(SOC) in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and I(SOC), whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE.     

3,3'-Diindolylmethane induces gastric cancer cells death via STIM1 mediated store-operated calcium entry

3,3''-Diindolylmethane (DIM), a natural phytochemicals isolated from cruciferous vegetables, has been reported to inhibit human gastric cancer cells proliferation and induce cells apoptosis as well as autophagy, but its mechanisms are still unclear. Store-operated calcium entry (SOCE) is a main Ca²⁺ influx pathway in various of cancers, which is activated by the depletion of endoplasmic reticulum (ER) Ca²⁺ store. Stromal interaction molecular 1 (STIM1) is the necessary component of SOCE. In this study, we focus on to examine the regulatory mechanism of SOCE on DIM-induced death in gastric cancer. After treating the human BGC-823 and SGC-7901 gastric cancer cells with DIM, cellular proliferation was determined by MTT, apoptosis and autophagy were detected by flow cytometry or Hoechst 33342 staining. The expression levels of related proteins were evaluated by Western blotting. Free cytosolilc Ca²⁺ level was assessed by fluorescence monitoring under a laser scanning confocal microscope. The data have shown that DIM could significantly inhibit proliferation and induce apoptosis as well as autophagy in two gastric cancer cell lines. After DIM treatment, the STIM1-mediated SOCE was activated by upregulating STIM1 and decreasing ER Ca²⁺ level. Knockdown STIM1 with siRNA or pharmacological inhibition of SOCE attenuated DIM induced apoptosis and autophagy by inhibiting p-AMPK mediated ER stress pathway. Our data highlighted that the potential of SOCE as a promising target for treating cancers. Developing effective and selective activators targeting STIM1-mediated SOCE pathway will facilitate better therapeutic sensitivity of phytochemicals acting on SOCE in gastric cancer. Moreover, more research should be performed to validate the efficacy of combination chemotherapy of anti-cancer drugs targeting SOCE for clinical application.     

Slow and persistent increase of [Ca2+]c in response to ligation of surface IgM in WEHI-231 cells

WEHI-231 and Bal 17 B cell lines are representative models for immature and mature B cells, respectively. Their regulation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) was compared using fura-2 fluorescence ratiometry. The ligation of B cell antigen receptor (BCR) by anti-IgM antibody induced a slow but large increase of [Ca(2+)](c) in WEHI-231 cells while not in Bal 17 cells. The thapsigargin-induced store-operated Ca(2+) entry (SOCE) of Bal 17 cells reached a steady state which was blocked by 2-aminoethoxydiphenyl borate (2-APB). On the contrary, the thapsigargin-induced SOCE of WEHI-231 cells increased continuously, which was accelerated by 2-APB. The increase of [Ca(2+)](c) by BCR ligation was also enhanced by 2-APB in WEHI-231 cells while blocked in Bal 17 cells. The Mn(2+) quenching study showed that the thapsigargin-, or the BCR ligation-induced Ca(2+) influx pathway of WEHI-231 was hardly permeable to Mn(2+). The intractable increase of [Ca(2+)](c) may explain the mechanism of BCR-driven apoptosis of WEHI-231 cells, a well-known model of clonal deletion of autoreactive immature B cells.     

The heterogeneity of store-operated calcium entry in melanoma

Calcium is a key regulator of many physiological processes that are perturbed in cancer, such as migration, proliferation and apoptosis. The proteins STIM and Orai mediate store-operated calcium entry (SOCE), the main pathway for calcium entry in non-excitable cells. Changes in the expression and function of STIM and Orai have been found in a range of cancer types and thus implicated in disease progression. Here we discuss the role of STIM, Orai and the SOCE pathway in the progression of melanoma and explore how the heterogeneous nature of melanoma may explain the lack of consensus in the field regarding the role of SOCE in the progression of this disease.     

S1P activates store-operated calcium entry via receptor- and non-receptor-mediated pathways in vascular smooth muscle cells

Sphingosine-1-phosphate (S1P) has been shown to modulate intracellular Ca(2+) through both G protein-coupled receptors and intracellular second messenger pathways. The precise mechanism by which S1P activates store-operated calcium entry (SOCE) in vascular smooth muscle cells (VSMCs) has not been fully characterized. Because sphingolipids and Ca(2+) modulate proliferation and constriction in VSMCs, characterizing the connection between S1P and SOCE may provide novel therapeutic targets for vascular diseases. We found that S1P triggered STIM1 puncta formation and SOCE in VSMCs. S1P-activated SOCE was inhibited by 2-aminoethoxydiphenyl borate (2-APB), diethylstilbestrol (DES), and gadolinium (Gd(3+)). SOCE was observed in VSMCs lacking either S1P(2) or S1P(3) receptors, suggesting that S1P acts via multiple signaling pathways. Indeed, both extracellular and intracellular S1P application increased the total internal reflection fluorescence signal in VSMCs cells transfected with STIM1-yellow fluorescent protein in a 2-APB-sensitive manner. These data, and the fact that 2-APB, DES, and Gd(3+) all inhibited S1P-induced cerebral artery constriction, suggest that SOCE modulates S1P-induced vasoconstriction in vivo. Finally, S1P-induced SOCE was larger in proliferative than in contractile VSMCs, correlating with increases in STIM1, Orai1, S1P(1), and S1P(3) receptor mRNA. These data demonstrate that S1P can act through both receptors and a novel intracellular pathway to activate SOCE. Because S1P-induced SOCE contributes to vessel constriction and is increased in proliferative VSMCs, it is likely that S1P/SOCE signaling in proliferative VSMCs may play a role in vascular dysfunction such as atherosclerosis and diabetes.     

Requirement for store-operated calcium entry in sodium butyrate-induced apoptosis in human colon cancer cells

The SOCE (store-operated Ca2+ entry) pathway plays a key role in both normal cells and cancerous cells. However, its molecular mechanism remains a long-lasting puzzle of Ca2+ signalling. In this paper, we provide evidence that butyric acid, a dietary fibre-derived short-chain fatty acid, induces apoptosis of colon cancer cells via SOCE signalling networks. We found that sodium butyrate (NaB) induces Ca2+ release from endoplasmic reticulum, which in turn causes extracellular Ca2+ influx in HCT-116 cells. The Ca2+ release and influx are important, because the addition of chelators, EGTA or BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] respectively blocked NaB-induced apoptosis. Furthermore, down-regulation of STIM1 (stromal interaction molecule 1) by RNA interference or pharmacological blockade of the SOCC (store-operated Ca2+ channel) by 2-APB (2-aminoethoxydiphenyl borate) or SKF-96365 inhibited NaB-induced extracellular Ca2+ influx and apoptosis in HCT-116 cells. Thus we conclude that NaB triggers colon cancer cell apoptosis in an SOCE-dependent manner. This finding provides new insights into how butyric acid suppresses colon carcinogenesis.     

Store-operated calcium entry in human oocytes and sensitivity to oxidative stress

Calcium signaling is a cellular event that plays a key role at many steps of fertilization and early development. However, little is known regarding the contribution of extracellular Ca(2+) influx into the cell to this signaling in gametes and early embryos. To better know the significance of calcium entry on oocyte physiology, we have evaluated the mechanism of store-operated calcium entry (SOCE) in human metaphase II (MII) oocytes and its sensitivity to oxidative stress, one of the major factors implicated in the outcome of in vitro fertilization (IVF) techniques. We show that depletion of intracellular Ca(2+) stores through inhibition of sarco(endo)plasmic Ca(2+)-ATPase with thapsigargin triggers Ca(2+) entry in resting human oocytes. Ba(2+) and Mn(2+) influx was also stimulated following inhibition, and Ca(2+) entry was sensitive to pharmacological inhibition because the SOCE blocker 2-aminoethoxydiphenylborate (2-APB) reduced calcium and barium entry. These results support the conclusion that there is a plasma membrane mechanism responsible for the capacitative divalent cation entry in human oocytes. Moreover, the Ca(2+) entry mechanism described in MII oocytes was found to be highly sensitive to oxidative stress. Hydrogen peroxide, at micromolar concentrations that could mimic culture conditions in IVF, elicited an increase of [Ca(2+)](i) that was dependent on the presence of extracellular Ca(2+). This rise was preventable by 2-APB, indicating that it was mainly due to the enhanced influx through store-operated calcium channels. In sum, our results demonstrate the occurrence of SOCE in human MII oocytes and the modification of this pathway due to oxidative stress, with possible consequences in IVF.     

Downregulation of BRD4 attenuates high glucose-induced damage of trophoblast cells by inhibiting activation of AKT/mTOR pathway

It was elucidated that bromodomain-containing protein 4 (BRD4) has involvement with diabetic complication. However, the role and molecular mechanism of BRD4 in gestational diabetes mellitus (GDM) are still unclear. In this study, the mRNA and protein contents of BRD4 in placenta tissues of GDM patients and high glucose (HG)-induced HTR8/SVneo cells were detected by qRT-PCR and western blot assay. CCK-8, EdU staining, flow cytometry as well as western blot were applied for the appraisement of cell viability and apoptosis. Wound healing assay and transwell assay were conducted for the assessment of cell migration and invasion. Oxidative stress and inflammatory factors were detected. Additionally, the contents of AKT/mTOR pathway-related proteins were estimated applying western blot. It was discovered that BRD4 expression was ascended in tissues and HG-induced HTR8/SVneo cells. BRD4 downregulation cut down the contents of p-AKT and p-mTOR but had no effects on the total protein levels of AKT or mTOR in HG-induced HTR8/SVneo cells. BRD4 depletion promoted cell viability, enhanced proliferative capability, and reduced cell apoptotic level. Moreover, BRD4 depletion facilitated cell migrative and invasive capabilities, and repressed the oxidative stress as well as inflammatory damage in HG-induced HTR8/SVneo cells. The activation of Akt reversed the protective impacts of BRD4 depletion on HG-induced HTR8/SVneo cells. To sum up, BRD4 silencing may alleviate HG-induced HTR8/SVneo cell damage through the inhibition of the AKT/mTOR pathway.     

Characterization of novel store-operated calcium entry effectors

2-Aminoethyl diphenylborinate (2-APB) is a well-known effector of the store-operated Ca^2 + entry of several cell types such as immune cells, platelets and smooth muscle cells. 2-APB has a dual effect: potentiation at 1–5 μM and inhibition at > 30 μM. Unfortunately, it is also able to modify the activity of other Ca^2 + transporters and, thus, cannot be used as a therapeutic tool to control the leukocyte activity in diseases like inflammation. Previously, we have shown that SOCE potentiation by 2-APB depends on the presence of the central boron-oxygen core (BOC) and that the phenyl groups determine the sensitivity of the molecule to inhibit and/or potentiate the SOCE.     

Reversible translocation of EYFP-tagged STIM1 is coupled to calcium influx in insulin secreting β-cells

Calcium (Ca²⁺) signaling regulates insulin secretion in pancreatic β-cells. STIM1 has been proposed to function as an endoplasmic reticulum (ER) Ca²⁺ sensor regulating store-operated Ca²⁺ entry (SOCE). Here we studied the translocation of EYFP-STIM1 in response to ER calcium depletion in mouse insulinoma MIN6 cells by fluorescent microscopy. While in resting cells EYFP-STIM1 is co-localized with an ER marker, in thapsigargin (Tg)-stimulated cells it occupied highly defined areas of the peri-PM space in punctae adjacent to, but not entirely coincident with the ER. Co-staining with fluorescent phalloidin revealed that EYFP-STIM1 punctae was located in actin-poor areas. Use of the SOCE blocker in MIN6 cells, 2-aminoethoxy diphenylborate (2-APB), prevented store depletion-dependent translocation of EYFP-STIM1 to the PM in a concentration-dependent (3.75–100 μM) and reversible manner. TIRF microscopy revealed that 2-APB treatment led to the reversible disappearance of peri-PM EYFP-STIM1 punctae, while the ER structure in this compartment remained grossly unaffected. We conclude from this data that in these cells EYFP-STIM1 is delivered to a peri-PM location from the ER upon store depletion and this trafficking is reversibly blocked by 2-APB.