CD95 triggers Orai1-mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) β2, and prevents death-inducing signaling complex formation.
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ABSTRACT:
SUBMITTER: Khadra N
PROVIDER: S-EPMC3223456 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The uppermost thin layer on the surface of the skin, called the epidermis, is responsible for the barrier function of the skin. The epidermis has a multilayered structure in which each layer consists of keratinocytes (KCs) of different differentiation status. The integrity of KC differentiation is crucial for the function of skin and its loss causes or is accompanied by skin diseases. Intracellular and extracellular Ca(2+) is known to play important roles in KC differentiation. However, the molecular mechanisms underlying Ca(2+) regulation of KC differentiation are still largely unknown. Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most non-excitable cells. SOCE is evoked in response to a fall in Ca(2+) concentration in the endoplasmic reticulum. Two proteins have been identified as essential components of SOCE: STIM1, a Ca(2+) sensor in the ER, and Orai1, a subunit of Ca(2+) channels in the plasma membrane. In this study, we analyzed the contribution of SOCE to KC growth and differentiation using RNAi knockdown of STIM1 and Orai1 in the human keratinocyte cell line, HaCaT. KC differentiation was induced by a switch in extracellular Ca(2+) concentration from low (0.03 mM; undifferentiated KCs) to high (1.8 mM; differentiated KCs). This Ca(2+) switch triggers phospholipase-C-mediated intracellular Ca(2+) signals (Ca(2+)-switch-induced Ca(2+) response), which would probably involve the activation of SOCE. Knockdown of either STIM1 or Orai1 strongly suppressed SOCE and almost completely abolished the Ca(2+)-switch-induced Ca(2+) responses, resulting in impaired expression of keratin1, an early KC differentiation marker. Furthermore, loss of either STIM1 or Orai1 suppressed normal growth of HaCaT cells in low Ca(2+) and inhibited the growth arrest in response to a Ca(2+) switch. These results demonstrate that SOCE plays multiple crucial roles in KC differentiation and function.
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Project description:Exercise promotes the formation of intracellular junctions in skeletal muscle between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of transverse-tubules (TT) that increase co-localization of proteins required for store-operated Ca2+ entry (SOCE). Here, we report that SOCE, peak Ca2+ transient amplitude and muscle force production during repetitive stimulation are increased after exercise in parallel with the time course of TT association with SR-stacks. Unexpectedly, exercise also activated constitutive Ca2+ entry coincident with a modest decrease in total releasable Ca2+ store content. Importantly, this decrease in releasable Ca2+ store content observed after exercise was reversed by repetitive high-frequency stimulation, consistent with enhanced SOCE. The functional benefits of exercise on SOCE, constitutive Ca2+ entry and muscle force production were lost in mice with muscle-specific loss of Orai1 function. These results indicate that TT association with SR-stacks enhances Orai1-dependent SOCE to optimize Ca2+ dynamics and muscle contractile function during acute exercise.
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Project description:Endothelial barrier function is critical for tissue fluid homeostasis, and its disruption contributes to various pathologies, including inflammation and sepsis. Thrombin is an endogenous agonist that impairs endothelial barrier function. We showed that the thrombin-induced decrease in transendothelial electric resistance of cultured human endothelial cells required the endoplasmic reticulum-localized, calcium-sensing protein stromal interacting molecule 1 (STIM1), but was independent of Ca2+ entry across the plasma membrane and the Ca2+ release-activated Ca2+ channel protein Orai1, which is the target of STIM1 in the store-operated calcium entry pathway. We found that STIM1 coupled the thrombin receptor to activation of the guanosine triphosphatase RhoA, stimulation of myosin light chain phosphorylation, formation of actin stress fibers, and loss of cell-cell adhesion. Thus, STIM1 functions in pathways that are dependent on and independent of Ca2+ entry.
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Project description:Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ?15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ?15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.
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Project description:Early embryo development and endometrial differentiation are initially independent processes, and synchronization, imposed by a limited window of implantation, is critical for reproductive success. A putative negative regulator of endometrial receptivity is LEFTY2, a member of the transforming growth factor (TGF)-? family. LEFTY2 is highly expressed in decidualizing human endometrial stromal cells (HESCs) during the late luteal phase of the menstrual cycle, coinciding with the closure of the window of implantation. Here, we show that flushing of the uterine lumen in mice with recombinant LEFTY2 inhibits the expression of key receptivity genes, including Cox2, Bmp2, and Wnt4, and blocks embryo implantation. In Ishikawa cells, a human endometrial epithelial cell line, LEFTY2 downregulated the expression of calcium release-activated calcium channel protein 1, encoded by ORAI1, and inhibited store-operated Ca2+ entry (SOCE). Furthermore, LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, as well as YM-58483, inhibited, whereas the Ca2+ ionophore, ionomycin, strongly upregulated COX2, BMP2 and WNT4 expression in decidualizing HESCs. These findings suggest that LEFTY2 closes the implantation window, at least in part, by downregulating Orai1, which in turn limits SOCE and antagonizes expression of Ca2+-sensitive receptivity genes. KEY MESSAGES:•Endometrial receptivity is negatively regulated by LEFTY2. •LEFTY2 inhibits the expression of key murine receptivity genes, including Cox2, Bmp2 and Wnt4, and blocks embryo implantation. •LEFTY2 downregulates the expression of Orai1 and inhibits SOCE. •LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, and YM-58483 inhibit COX2, BMP2, and WNT4 expression in endometrial cells. •Targeting LEFTY2 and Orai1 may represent a novel approach for treating unexplained infertility.
| S-EPMC5778154 | biostudies-literature
Project description:BACKGROUND:Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ channels is an essential signaling pathway in many cell types. Ca2+ release-activated Ca2+ channels are formed by ORAI1, ORAI2, and ORAI3 proteins and activated by stromal interaction molecule (STIM) 1 and STIM2. Mutations in the ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and nonimmunologic symptoms. OBJECTIVE:We performed molecular and immunologic analysis of patients with CID, anhidrosis, and ectodermal dysplasia of unknown etiology. METHODS:We performed DNA sequencing of the ORAI1 gene, modeling of mutations on ORAI1 crystal structure, analysis of ORAI1 mRNA and protein expression, SOCE measurements, immunologic analysis of peripheral blood lymphocyte populations by using flow cytometry, and histologic and ultrastructural analysis of patient tissues. RESULTS:We identified 3 novel autosomal recessive mutations in ORAI1 in unrelated kindreds with CID, autoimmunity, ectodermal dysplasia with anhidrosis, and muscular dysplasia. The patients were homozygous for p.V181SfsX8, p.L194P, and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. In addition to impaired T-cell cytokine production, ORAI1 mutations were associated with strongly reduced numbers of invariant natural killer T and regulatory T (Treg) cells and altered composition of γδ T-cell and natural killer cell subsets. CONCLUSION:ORAI1 null mutations are associated with reduced numbers of invariant natural killer T and Treg cells that likely contribute to the patients' immunodeficiency and autoimmunity. ORAI1-deficient patients have dental enamel defects and anhidrosis, representing a new form of anhidrotic ectodermal dysplasia with immunodeficiency that is distinct from previously reported patients with anhidrotic ectodermal dysplasia with immunodeficiency caused by mutations in the nuclear factor κB signaling pathway (IKBKG and NFKBIA).
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Project description:RATIONALE:Orai1 and the associated calcium release-activated calcium (CRAC) channel were discovered in the immune system. Existence also in endothelial cells has been suggested, but the relevance to endothelial biology is mostly unknown. OBJECTIVE:The aim of this study was to investigate the relevance of Orai1 and CRAC channels to vascular endothelial growth factor (VEGF) and endothelial tube formation. METHODS AND RESULTS:In human umbilical vein endothelial cells, Orai1 disruption by short-interfering RNA or dominant-negative mutant Orai1 inhibited calcium release-activated (store-operated) calcium entry, VEGF-evoked calcium entry, cell migration, and in vitro tube formation. Expression of exogenous wild-type Orai1 rescued the tube formation. VEGF receptor-2 and Orai1 partially colocalized. Orai1 disruption also inhibited calcium entry and tube formation in endothelial progenitor cells from human blood. A known blocker of the immune cell CRAC channel (3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide) was a strong blocker of store-operated calcium entry in endothelial cells and inhibited calcium entry evoked by VEGF in 3 types of human endothelial cell. The compound lacked effect on VEGF-evoked calcium-release, STIM1 clustering, and 2 types of transient receptor potential channels, TRPC6 and TRPV4. Without effect on cell viability, the compound inhibited human endothelial cell migration and tube formation in vitro and suppressed angiogenesis in vivo in the chick chorioallantoic membrane. The compound showed 100-fold greater potency for endothelial compared with immune cell calcium entry. CONCLUSIONS:The data suggest positive roles for Orai1 and CRAC channels in VEGF-evoked calcium entry and new opportunity for chemical modulation of angiogenesis.
| S-EPMC3512576 | biostudies-literature
Project description:An unconventional interaction between SPCA2, an isoform of the Golgi secretory pathway Ca(2+)-ATPase, and the Ca(2+) influx channel Orai1, has previously been shown to contribute to elevated Ca(2+) influx in breast cancer derived cells. In order to investigate the physiological role of this interaction, we examined expression and localization of SPCA2 and Orai1 in mouse lactating mammary glands. We observed co-induction and co-immunoprecipitation of both proteins, and isoform-specific differences in the localization of SPCA1 and SPCA2. Three-dimensional cultures of normal mouse mammary epithelial cells were established using lactogenic hormones and basement membrane. The mammospheres displayed elevated Ca(2+) influx by store independent mechanisms, consistent with upregulation of both SPCA2 and Orai1. Knockdown of either SPCA2 or Orai1 severely depleted Ca(2+) influx and interfered with mammosphere differentiation. We show that SPCA2 is required for plasma membrane trafficking of Orai1 in mouse mammary epithelial cells and that this function can be replaced, at least in part, by a membrane-anchored C-terminal domain of SPCA2. These findings clearly show that SPCA2 and Orai1 function together to regulate Store-independent Ca(2+) entry (SICE), which mediates the massive basolateral Ca(2+) influx into mammary epithelia to support the large calcium transport requirements for milk secretion.
| S-EPMC3696057 | biostudies-literature
Project description:Sigma1 receptors (?1Rs) are expressed widely; they bind diverse ligands, including psychotropic drugs and steroids, regulate many ion channels, and are implicated in cancer and addiction. It is not known how ?1Rs exert such varied effects. We demonstrate that ?1Rs inhibit store-operated Ca(2+)entry (SOCE), a major Ca(2+)influx pathway, and reduce the Ca(2+)content of the intracellular stores. SOCE was inhibited by expression of ?1R or an agonist of ?1R and enhanced by loss of ?1R or an antagonist. Within the endoplasmic reticulum (ER), ?1R associated with STIM1, the ER Ca(2+)sensor that regulates SOCE. This interaction was modulated by ?1R ligands. After depletion of Ca(2+)stores, ?1R accompanied STIM1 to ER-plasma membrane (PM) junctions where STIM1 stimulated opening of the Ca(2+)channel, Orai1. The association of STIM1 with ?1R slowed the recruitment of STIM1 to ER-PM junctions and reduced binding of STIM1 to PM Orai1. We conclude that ?1R attenuates STIM1 coupling to Orai1 and thereby inhibits SOCE.
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Project description:Increasing evidence supports that activation of store-operated Ca2+ entry (SOCE) is implicated in the chemoresistance of cancer cells subjected to chemotherapy. However, the molecular mechanisms underlying chemoresistance are not well understood. In this study, we aim to investigate whether 5-FU induces hepatocarcinoma cell death through regulating Ca2+ -dependent autophagy. [Ca2+ ]i was measured using fura2/AM dye. Protein expression was determined by Western blotting and immunohistochemistry. We found that 5-fluorouracil (5-FU) induced autophagic cell death in HepG2 hepatocarcinoma cells by inhibiting PI3K/AKT/mTOR pathway. Orai1 expression was obviously elevated in hepatocarcinoma tissues. 5-FU treatment decreased SOCE and Orai1 expressions, but had no effects on Stim1 and TRPC1 expressions. Knockdown of Orai1 or pharmacological inhibition of SOCE enhanced 5-FU-induced inhibition of PI3K/AKT/mTOR pathway and potentiated 5-FU-activated autophagic cell death. On the contrary, ectopic overexpression of Orai1 antagonizes 5-FU-induced autophagy and cell death. Our findings provide convincing evidence to show that Orai1 expression is increased in hepatocarcinoma tissues. 5-FU can induce autophagic cell death in HepG2 hepatocarcinoma cells through inhibition of SOCE via decreasing Orai1 expression. These findings suggest that Orai1 expression is a predictor of 5-FU sensitivity for hepatocarcinoma treatment and blockade of Orai1-mediated Ca2+ entry may be a promising strategy to sensitize hepatocarcinoma cells to 5-FU treatment.
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