RESUMEN
BACKGROUND: STIM- and Orai-mediated store operated calcium entry (SOCE) is a ubiquitous Ca2+ signaling process, crucial for the proper function of immune, muscle and neuronal systems. To treat SOCE-related disorder or diseases of these systems, and to mechanistically dissect activation and function of SOCE, specific SOCE inhibitors are needed. However, strategies for developing new SOCE modifiers are still limited.
Methodology: In this study, we identified a novel SOCE inhibitor named 2PHDO from a small pool of Chinese herbal extracts used for treating psoriasis. It could block SOCE and SOCE-mediated NFAT translocation in multiple types of cells with a half inhibitory concentration around 1 µM. At this concentration, 2PHDO was specific for SOCE. Mechanistically, 2PHDO didn't affect the activation of STIM1 or its physical coupling with Orai1. Rather, 2PHDO inhibited SOCE via its actions on Orai1. Results: 2PHDO may serve as a good template for developing new medicines aiming to treat SOCE related diseases. Conclusion: Overall, we proved the feasibility of screening and identification of novel SOCE inhibitors from active monomers of Chinese herbal medicine.RESUMEN
During sepsis, acute lung injury (ALI) results from activation of innate immune cells and endothelial cells by endotoxins, leading to systemic inflammation through proinflammatory cytokine overproduction, oxidative stress, and intracellular Ca2+ overload. Despite considerable investigation, the underlying molecular mechanism(s) leading to LPS-induced ALI remain elusive. To determine whether stromal interaction molecule 1-dependent (STIM1-dependent) signaling drives endothelial dysfunction in response to LPS, we investigated oxidative and STIM1 signaling of EC-specific Stim1-knockout mice. Here we report that LPS-mediated Ca2+ oscillations are ablated in ECs deficient in Nox2, Stim1, and type II inositol triphosphate receptor (Itpr2). LPS-induced nuclear factor of activated T cells (NFAT) nuclear accumulation was abrogated by either antioxidant supplementation or Ca2+ chelation. Moreover, ECs lacking either Nox2 or Stim1 failed to trigger store-operated Ca2+ entry (SOCe) and NFAT nuclear accumulation. LPS-induced vascular permeability changes were reduced in EC-specific Stim1-/- mice, despite elevation of systemic cytokine levels. Additionally, inhibition of STIM1 signaling prevented receptor-interacting protein 3-dependent (RIP3-dependent) EC death. Remarkably, BTP2, a small-molecule calcium release-activated calcium (CRAC) channel blocker administered after insult, halted LPS-induced vascular leakage and pulmonary edema. These results indicate that ROS-driven Ca2+ signaling promotes vascular barrier dysfunction and that the SOCe machinery may provide crucial therapeutic targets to limit sepsis-induced ALI.
Asunto(s)
Lesión Pulmonar Aguda/prevención & control , Glicoproteínas de Membrana/antagonistas & inhibidores , NADPH Oxidasas/antagonistas & inhibidores , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Anilidas/farmacología , Animales , Canales de Calcio , Señalización del Calcio , Células Cultivadas , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Técnicas de Silenciamiento del Gen , Receptores de Inositol 1,4,5-Trifosfato/deficiencia , Receptores de Inositol 1,4,5-Trifosfato/genética , Lipopolisacáridos/toxicidad , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , NADPH Oxidasa 2 , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Factores de Transcripción NFATC/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sepsis/complicaciones , Transducción de Señal , Molécula de Interacción Estromal 1 , Tiadiazoles/farmacologíaRESUMEN
Many ion channels are regulated by lipids, but prominent motifs for lipid binding have not been identified in most ion channels. Recently, we reported that phospholipase Cgamma1 (PLC-gamma1) binds to and regulates TRPC3 channels, components of agonist-induced Ca2+ entry into cells. This interaction requires a domain in PLC-gamma1 that includes a partial pleckstrin homology (PH) domain-a consensus lipid-binding and protein-binding sequence. We have developed a gestalt algorithm to detect hitherto 'invisible' PH and PH-like domains, and now report that the partial PH domain of PLC-gamma1 interacts with a complementary partial PH-like domain in TRPC3 to elicit lipid binding and cell-surface expression of TRPC3. Our findings imply a far greater abundance of PH domains than previously appreciated, and suggest that intermolecular PH-like domains represent a widespread signalling mode.