Calcium channel Orai1 promotes lymphocyte IL-17 expression and progressive kidney injury

We hypothesized that the store-operated calcium entry (SOCE) channel, Orai1, participates in the activation of Th17 cells and influences renal injury. In rats, following renal ischemia/reperfusion (I/R), there was a rapid and sustained influx of Orai1+ CD4 T cells and IL-17 expression was restricted to Orai1+ cells. When kidney CD4+ cells of post-acute kidney injury (post-AKI) rats were stimulated with angiotensin II and elevated Na+ (10-7 M/170 mM) in vitro, there was an enhanced response in intracellular Ca2+ and IL-17 expression, which was blocked by SOCE inhibitors 2APB, YM58483/BTP2, or AnCoA4. In vivo, YM58483/BTP2 (1 mg/kg) attenuated IL-17+ cell activation, inflammation, and severity of AKI following either I/R or intramuscular glycerol injection. Rats treated with high-salt diet (5-9 weeks after I/R) manifested progressive disease indicated by enhanced inflammation, fibrosis, and impaired renal function. These responses were significantly attenuated by YM58483/BTP2. In peripheral blood of critically ill patients, Orai1+ cells were significantly elevated by approximately 10-fold and Th17 cells were elevated by approximately 4-fold in AKI versus non-AKI patients. Further, in vitro stimulation of CD4+ cells from AKI patients increased IL-17, which was blocked by SOCE inhibitors. These data suggest that Orai1 SOCE is a potential therapeutic target in AKI and CKD progression

Regional differences in store-operated Ca2+ entry in the epithelium of the intact human lens

An elevated level of Ca2+ is an important factor in cataract, yet precisely how Ca2+ enters the lens is unknown. Lens epithelial cells contain a range of G-protein–coupled receptors and receptor tyrosine kinases that induce increases in intracellular Ca2+. Receptor-associated Ca2+ influx is, therefore, likely to be an important route for Ca2+ influx to the lens. The authors investigated stimulated and passive Ca2+ influx in in situ human lens epithelium. Ca2+ changes in equatorial (E) and central anterior (CA) epithelial cells were monitored with the use of a Ca2+ indicator (Fluo4) and confocal microscopy. Gene expression was monitored by RT-PCR and immunoblotting. Adenosine triphosphate (ATP) induced Ca2+ responses that were smaller in CA than E. Ca2+ store depletion, using ATP (100 µM) or thapsigargin (1 µM), revealed greater relative store capacity and Ca2+ influx in E. Ca2+ influx was blocked by La3+ (0.5 µM) in both regions. Unstimulated Ca2+ influx was greater in E than CA. Greater expression of Orai1 and STIM1 was detected in E than in CA. Greater Ca2+ store capacity and Ca2+ influx in E compared with CA reflects underlying differences in proliferation and differentiation between the regions. The relatively small resting Ca2+ influx in CA epithelium suggests that store-operated Ca2+ entry (SOCE) is the main route of Ca2+ influx in these cells. Greater resting influx and SOCE in E cells suggests that these are a major route for Ca2+ influx into the lens. Increased expression of Orai1 and STIM1 in E could account for the differences in Ca2+ entry. Receptor activation will modulate Ca2+ influx, and inappropriate activity may contribute to cortical cataract

Hyperbolicity Measures "Democracy" in Real-World Networks

We analyze the hyperbolicity of real-world networks, a geometric quantity that measures if a space is negatively curved. In our interpretation, a network with small hyperbolicity is "aristocratic", because it contains a small set of vertices involved in many shortest paths, so that few elements "connect" the systems, while a network with large hyperbolicity has a more "democratic" structure with a larger number of crucial elements. We prove mathematically the soundness of this interpretation, and we derive its consequences by analyzing a large dataset of real-world networks. We confirm and improve previous results on hyperbolicity, and we analyze them in the light of our interpretation. Moreover, we study (for the first time in our knowledge) the hyperbolicity of the neighborhood of a given vertex. This allows to define an "influence area" for the vertices in the graph. We show that the influence area of the highest degree vertex is small in what we define "local" networks, like most social or peer-to-peer networks. On the other hand, if the network is built in order to reach a "global" goal, as in metabolic networks or autonomous system networks, the influence area is much larger, and it can contain up to half the vertices in the graph. In conclusion, our newly introduced approach allows to distinguish the topology and the structure of various complex networks

The prion protein constitutively controls neuronal store-operated ca2+ entry through Fyn Kinase

The prion protein (PrPC) is a cell surface glycoprotein mainly expressed in neurons, whose misfolded isoforms generate the prion responsible for incurable neurodegenerative disorders. Whereas PrPC involvement in prion propagation is well established, PrPC physiological function is still enigmatic despite suggestions that it could act in cell signal transduction by modulating phosphorylation cascades and Ca2+ homeostasis. Because PrPC binds neurotoxic protein aggregates with high-affinity, it has also been proposed that PrPC acts as receptor for amyloid-\u3b2 (A\u3b2) oligomers associated with Alzheimer\u2019s disease (AD), and that PrPC-A\u3b2 binding mediates AD-related synaptic dysfunctions following activation of the tyrosine kinase Fyn. Here, use of gene-encoded Ca2+ probes targeting different cell domains in primary cerebellar granule neurons (CGN) expressing, or not, PrPC, allowed us to investigate whether PrPC regulates store-operated Ca2+ entry (SOCE) and the implication of Fyn in this control. Our findings show that PrPC attenuates SOCE, and Ca2+ accumulation in the cytosol and mitochondria, by constitutively restraining Fyn activation and tyrosine phosphorylation of STIM1, a key molecular component of SOCE. This data establishes the existence of a PrPC-Fyn-SOCE triad in neurons. We also demonstrate that treating cerebellar granule and cortical neurons with soluble A\u3b2(1\u201342) oligomers abrogates the control of PrPC over Fyn and SOCE, suggesting a PrPC-dependent mechanizm for A\u3b2-induced neuronal Ca2+ dyshomeostasis

STIM1, PKC-δ and RasGRP set a threshold for proapoptotic Erk signaling during B cell development.

Clonal deletion of autoreactive B cells is crucial for the prevention of autoimmunity, but the signaling mechanisms that regulate this checkpoint remain undefined. Here we characterize a previously unrecognized Ca(2+)-driven pathway for activation of the kinase Erk, which was proapoptotic and biochemically distinct from Erk activation induced by diacylglycerol (DAG). This pathway required protein kinase C-δ (PKC-δ) and the guanine nucleotide-exchange factor RasGRP and depended on the concentration of the Ca(2+) sensor STIM1, which controls the magnitude of Ca(2+) entry. Developmental regulation of these proteins was associated with selective activation of the pathway in B cells prone to negative selection. This checkpoint was impaired in PKC-δ-deficient mice, which developed B cell autoimmunity. Conversely, overexpression of STIM1 conferred a competitive disadvantage to developing B cells. Our findings establish Ca(2+)-dependent Erk signaling as a critical proapoptotic pathway that mediates the negative selection of B cells

Role of Dicer Enzyme in the Regulation of Store Operated Calcium Entry (SOCE) in CD4+ T Cells

Background/Aims: Activation of T cell receptors (TCRs) in CD4+ T cells leads to a cascade of signalling reactions including increase of intracellular calcium (Ca2+) levels with subsequent Ca2+ dependent stimulation of gene expression, proliferation, cell motility and cytokine release. The increase of cytosolic Ca2+ results from intracellular Ca2+ release with subsequent activation of store-operated Ca2+ entry (SOCE). Previous studies suggested miRNAs are required for the development and functions of CD4+ T cells. An enzyme called Dicer is required during the process of manufacturing mature miRNAs from the precursor miRNAs. In this study, we explored whether loss of Dicer in CD4+ T cells affects SOCE and thus Ca2+ dependent regulation of cellular functions. Methods: We tested the expression of Orai1 by q-RT-PCR and flow cytometry. Further, we measured SOCE by an inverted phase-contrast microscope with the Incident-light fluorescence illumination system using Fura-2. Intracellular Ca2+ was also measured by flow cytometry using Ca2+ sensitive dye Fluo-4. Results: We found that in Dicer deficient (DicerΔ/Δ) mice Orai1 was downregulated at mRNA and protein level in CD4+ T cells. Further, SOCE was significantly smaller in DicerΔ/Δ CD4+ T cells than in CD4+ T cells isolated from wild-type (Dicerfl/fl) mice. Conclusion: Our data suggest that miRNAs are required for adequate Ca2+ entry into CD4+ T cells and thus triggering of Ca2+ sensitive immune functions

RASSF4: Regulator of plasma membrane PI(4,5)P2.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a negatively charged phospholipid that plays a major role in recruiting and regulating proteins at the plasma membrane-cytosol interface. In this issue, Chen et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201606047) demonstrate that RAS association domain family 4 (RASSF4) positively influences PI(4,5)P2 synthesis through ARF6-dependent regulation of PIP5K

Transient receptor potential canonical type 3 channels control the vascular contractility of mouse mesenteric arteries

Transient receptor potential canonical type 3 (TRPC3) channels are non-selective cation channels and regulate intracellular Ca2+ concentration. We examined the role of TRPC3 channels in agonist-, membrane depolarization (high K+)-, and mechanical (pressure)-induced vasoconstriction and vasorelaxation in mouse mesenteric arteries. Vasoconstriction and vasorelaxation of endothelial cells intact mesenteric arteries were measured in TRPC3 wild-type (WT) and knockout (KO) mice. Calcium concentration ([Ca2+]) was measured in isolated arteries from TRPC3 WT and KO mice as well as in the mouse endothelial cell line bEnd.3. Nitric oxide (NO) production and nitrate/nitrite concentrations were also measured in TRPC3 WT and KO mice. Phenylephrine-induced vasoconstriction was reduced in TRPC3 KO mice when compared to that of WT mice, but neither high K+- nor pressure-induced vasoconstriction was altered in TRPC3 KO mice. Acetylcholine-induced vasorelaxation was inhibited in TRPC3 KO mice and by the selective TRPC3 blocker pyrazole-3. Acetylcholine blocked the phenylephrine-induced increase in Ca2+ ratio and then relaxation in TRPC3 WT mice but had little effect on those outcomes in KO mice. Acetylcholine evoked a Ca2+ increase in endothelial cells, which was inhibited by pyrazole-3. Acetylcholine induced increased NO release in TRPC3 WT mice, but not in KO mice. Acetylcholine also increased the nitrate/nitrite concentration in TRPC3 WT mice, but not in KO mice. The present study directly demonstrated that the TRPC3 channel is involved in agonist-induced vasoconstriction and plays important role in NO-mediated vasorelaxation of intact mesenteric arteries.Fil: Yeon, Soo-In. Yonsei University College of Medicine; Corea del SurFil: Kim, Joo Young. Yonsei University College Of Medicine; . Yonsei University College of Medicine; Corea del SurFil: Yeon, Dong-Soo. Kwandong University College of Medicine; Corea del SurFil: Abramowitz, Joel. National Institute of Environmental Health Sciences; Estados UnidosFil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. National Institute of Environmental Health Sciences; Estados UnidosFil: Muallem, Shmuel. National Institutes of Health; Estados UnidosFil: Lee, Young-Ho. Yonsei University College of Medicine; Corea del Su

RPGR protein complex regulates proteasome activity and mediates store-operated calcium entry

Ciliopathies are a group of genetically heterogeneous disorders, characterized by defects in cilia genesis or maintenance. Mutations in the RPGR gene and its interacting partners, RPGRIP1 and RPGRIP1L, cause ciliopathies, but the function of their proteins remains unclear. Here we show that knockdown (KD) of RPGR, RPGRIP1 or RPGRIP1L in hTERT-RPE1 cells results in abnormal actin cytoskeleton organization. The actin cytoskeleton rearrangement is regulated by the small GTPase RhoA via the planar cell polarity (PCP) pathway. RhoA activity was upregulated in the absence of RPGR, RPGRIP1 or RPGRIP1L proteins. In RPGR, RPGRIP1 or RPGRIP1L KD cells, we observed increased levels of DVl2 and DVl3 proteins, the core components of the PCP pathway, due to impaired proteasomal activity. RPGR, RPGRIP1 or RPGRIP1L KD cells treated with thapsigargin (TG), an inhibitor of sarcoendoplasmic reticulum Ca2+ - ATPases, showed impaired store-operated Ca2+ entry (SOCE), which is mediated by STIM1 and Orai1 proteins. STIM1 was not localized to the ER-PM junction upon ER store depletion in RPGR, RPGRIP1 or RPGRIP1L KD cells. Our results demonstrate that the RPGR protein complex is required for regulating proteasomal activity and for modulating SOCE, which may contribute to the ciliopathy phenotype