Erythroid Differentiation Regulator 1 Strengthens TCR Signaling by Enhancing PLCγ1 Signal Transduction Pathway

Erythroid differentiation regulator 1 (Erdr1) has previously been reported to control thymocyte selection via TCR signal regulation, but the effect of Erdr1 as a TCR signaling modulator was not studied in peripheral T cells. In this report, it was determined whether Erdr1 affected TCR signaling strength in CD4 T cells. Results revealed that Erdr1 significantly enhanced the anti-TCR antibody-mediated activation and proliferation of T cells while failing to activate T cells in the absence of TCR stimulation. In addition, Erdr1 amplified Ca²⁺ influx and the phosphorylation of PLCγ1 in CD4 T cells with the TCR stimuli. Furthermore, NFAT1 translocation into nuclei in CD4 T cells was also significantly promoted by Erdr1 in the presence of TCR stimulation. Taken together, our results indicate that Erdr1 positively modulates TCR signaling strength via enhancing the PLCγ1/Ca²⁺/NFAT1 signal transduction pathway.     

Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells

Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy), mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER) and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca²⁺ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1), as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT) and the IP3-receptors (IP3Rs) as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca²⁺ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca²⁺ uniporter (MCU) primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca²⁺ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca²⁺ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis.     

Enhanced Ca2+ Entry Sustains the Activation of Akt in Glucose Deprived SH-SY5Y Cells

The two crucial cellular insults that take place during cerebral ischemia are the loss of oxygen and loss of glucose, which can both activate a cascade of events leading to neuronal death. In addition, the toxic overactivation of neuronal excitatory receptors, leading to Ca²⁺ overload, may contribute to ischemic neuronal injury. Brain ischemia can be simulated in vitro by oxygen/glucose deprivation, which can be reversible by the re-establishment of physiological conditions. Accordingly, we examined the effects of glucose deprivation on the PI3K/Akt survival signaling pathway and its crosstalk with HIF-1α and Ca²⁺ homeostasis in SH-SY5Y human neuroblastoma cells. It was found that glucose withdrawal decreased HIF-1α protein levels even in the presence of the ischemia-mimicking CoCl_2. On the contrary, and despite neuronal death, we identified a strong activation of the master pro-survival kinase Akt, a finding that was also confirmed by the increased phosphorylation of GSK3, a direct target of p-Akt. Remarkably, the elevated Ca²⁺ influx recorded was found to promptly trigger the activation of Akt, while a re-addition of glucose resulted in rapid restoration of both Ca²⁺ entry and p-Akt levels, highlighting the plasticity of neurons to respond to ischemic challenges and the important role of glucose homeostasis for multiple neurological disorders.     

CFH Loss in Human RPE Cells Leads to Inflammation and Complement System Dysregulation via the NF-κB Pathway

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease, and chronic inflammatory processes may be involved. Besides aging and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism of CFH dysregulation confers such a great risk for AMD and its role in RPE cell homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources, or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g., IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g., C3, CFB upregulation, and C5 downregulation) that are known to play a role in AMD. Moreover, our results identify the NF-κB pathway as the major pathway involved in regulating these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-κB pathway work in synergy to maintain inflammatory and complement balance, and in case either one of them is dysregulated, the RPE microenvironment changes towards a proinflammatory AMD-like phenotype.     

Ultrasensitive Imaging of Ca2+ Dynamics in Pancreatic Acinar Cells of Yellow Cameleon-Nano Transgenic Mice

Yellow Cameleons are genetically encoded Ca²⁺ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Förster) resonance energy transfer Ca²⁺-sensor probe. To achieve ultrasensitive Ca²⁺ imaging for low resting Ca²⁺ or small Ca²⁺ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca²⁺ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca²⁺ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca²⁺ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca²⁺ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca²⁺ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from R_max and R_min values under in vivo condition. The mice will be useful for ultrasensitive Ca²⁺ imaging in vivo.     

The Regulatory Roles of Mitochondrial Calcium and the Mitochondrial Calcium Uniporter in Tumor Cells

Mitochondria, as the main site of cellular energy metabolism and the generation of oxygen free radicals, are the key switch for mitochondria-mediated endogenous apoptosis. Ca²⁺ is not only an important messenger for cell proliferation, but it is also an indispensable signal for cell death. Ca²⁺ participates in and plays a crucial role in the energy metabolism, physiology, and pathology of mitochondria. Mitochondria control the uptake and release of Ca²⁺ through channels/transporters, such as the mitochondrial calcium uniporter (MCU), and influence the concentration of Ca²⁺ in both mitochondria and cytoplasm, thereby regulating cellular Ca²⁺ homeostasis. Mitochondrial Ca²⁺ transport-related processes are involved in important biological processes of tumor cells including proliferation, metabolism, and apoptosis. In particular, MCU and its regulatory proteins represent a new era in the study of MCU-mediated mitochondrial Ca²⁺ homeostasis in tumors. Through an in-depth analysis of the close correlation between mitochondrial Ca²⁺ and energy metabolism, autophagy, and apoptosis of tumor cells, we can provide a valuable reference for further understanding of how mitochondrial Ca²⁺ regulation helps diagnosis and therapy.     

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKβ/AMPK Signaling Pathway in Human Endothelial Cells

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca²⁺ concentration and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and Ca²⁺/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca²⁺ concentration and phosphorylation of CaMKII, CaMKKβ, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKβ inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca²⁺/CaMKII and CaM/CaMKKβ/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.     

IRAG2 Interacts with IP3-Receptor Types 1, 2, and 3 and Regulates Intracellular Ca2+ in Murine Pancreatic Acinar Cells

The inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is also known as Jaw1 or lymphoid-restricted membrane protein (LRMP) and shares homology with the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1). IRAG1 interacts with inositol trisphosphate receptors (IP₃ receptors /IP₃R) via its coiled-coil domain and modulates Ca²⁺ release from intracellular stores. Due to the homology of IRAG1 and IRAG2, especially in its coiled-coil domain, it is possible that IRAG2 has similar interaction partners like IRAG1 and that IRAG2 also modulates intracellular Ca²⁺ signaling. In our study, we localized IRAG2 in pancreatic acinar cells of the exocrine pancreas, and we investigated the interaction of IRAG2 with IP₃ receptors and its impact on intracellular Ca²⁺ signaling and exocrine pancreatic function, like amylase secretion. We detected the interaction of IRAG2 with different subtypes of IP₃R and altered Ca²⁺ release in pancreatic acinar cells from mice lacking IRAG2. IRAG2 deficiency decreased basal levels of intracellular Ca²⁺, suggesting that IRAG2 leads to activation of IP₃R under unstimulated basal conditions. Moreover, we observed that loss of IRAG2 impacts the secretion of amylase. Our data, therefore, suggest that IRAG2 modulates intracellular Ca²⁺ signaling, which regulates exocrine pancreatic function.     

Muscarinic Receptors and BK Channels Are Affected by Lipid Raft Disruption of Salivary Gland Cells

Activity-dependent fluid secretion is the most important physiological function of salivary glands and is regulated via muscarinic receptor signaling. Lipid rafts are important for G-protein coupled receptor (GPCR) signaling and ion channels in plasma membranes. However, it is not well understood whether lipid raft disruption affects all membrane events or only specific functions in muscarinic receptor-mediated water secretion in salivary gland cells. We investigated the effects of lipid raft disruption on the major membrane events of muscarinic transcellular water movement in human salivary gland (HSG) cells. We found that incubation with methyl-β-cyclodextrin (MβCD), which depletes lipid rafts, inhibited muscarinic receptor-mediated Ca²⁺ signaling in HSG cells and isolated mouse submandibular acinar cells. However, MβCD did not inhibit a Ca²⁺ increase induced by thapsigargin, which activates store-operated Ca²⁺ entry (SOCE). Interestingly, MβCD increased the activity of the large-conductance Ca²⁺-activated K⁺ channel (BK channel). Finally, we found that MβCD did not directly affect the translocation of aquaporin-5 (AQP5) into the plasma membrane. Our results suggest that lipid rafts maintain muscarinic Ca²⁺ signaling at the receptor level without directly affecting the activation of SOCE induced by intracellular Ca²⁺ pool depletion or the translocation of AQP5 into the plasma membrane.     

Size-Dependent Cytoprotective Effects of Selenium Nanoparticles during Oxygen-Glucose Deprivation in Brain Cortical Cells

It is known that selenium nanoparticles (SeNPs) obtained on their basis have a pleiotropic effect, inducing the process of apoptosis in tumor cells, on the one hand, and protecting healthy tissue cells from death under stress, on the other hand. It has been established that SeNPs protect brain cells from ischemia/reoxygenation through activation of the Ca²⁺ signaling system of astrocytes and reactive astrogliosis. At the same time, for a number of particles, the limitations of their use, associated with their size, are shown. The use of nanoparticles with a diameter of less than 10 nm leads to their short life-time in the bloodstream and rapid removal by the liver. Nanoparticles larger than 200 nm activate the complement system and are also quickly removed from the blood. The effects of different-sized SeNPs on brain cells have hardly been studied. Using the laser ablation method, we obtained SeNPs of various diameters: 50 nm, 100 nm, and 400 nm. Using fluorescence microscopy, vitality tests, PCR analysis, and immunocytochemistry, it was shown that all three types of the different-sized SeNPs have a cytoprotective effect on brain cortex cells under conditions of oxygen-glucose deprivation (OGD) and reoxygenation (R), suppressing the processes of necrotic death and inhibiting different efficiency processes of apoptosis. All of the studied SeNPs activate the Ca²⁺ signaling system of astrocytes, while simultaneously inducing different types of Ca²⁺ signals. SeNPs sized at 50 nm- induce Ca²⁺ responses of astrocytes in the form of a gradual irreversible increase in the concentration of cytosolic Ca²⁺ ([Ca²⁺]_i), 100 nm-sized SeNPs induce stable Ca²⁺ oscillations without increasing the base level of [Ca²⁺]_i, and 400 nm-sized SeNPs cause mixed patterns of Ca²⁺ signals. Such differences in the level of astrocyte Ca²⁺ signaling can explain the different cytoprotective efficacy of SeNPs, which is expressed in the expression of protective proteins and the activation of reactive astrogliosis. In terms of the cytoprotective efficiency under OGD/R conditions, different-sized SeNPs can be arranged in descending order: 100 nm-sized > 400 nm-sized > 50 nm-sized.     

Targeting Lysosomes to Reverse Hydroquinone-Induced Autophagy Defects and Oxidative Damage in Human Retinal Pigment Epithelial Cells

In age-related macular degeneration (AMD), hydroquinone (HQ)-induced oxidative damage in retinal pigment epithelium (RPE) is believed to be an early event contributing to dysregulation of inflammatory cytokines and vascular endothelial growth factor (VEGF) homeostasis. However, the roles of antioxidant mechanisms, such as autophagy and the ubiquitin-proteasome system, in modulating HQ-induced oxidative damage in RPE is not well-understood. This study utilized an in-vitro AMD model involving the incubation of human RPE cells (ARPE-19) with HQ. In comparison to hydrogen peroxide (H₂O₂), HQ induced fewer reactive oxygen species (ROS) but more oxidative damage as characterized by protein carbonyl levels, mitochondrial dysfunction, and the loss of cell viability. HQ blocked the autophagy flux and increased proteasome activity, whereas H₂O₂ did the opposite. Moreover, the lysosomal membrane-stabilizing protein LAMP2 and cathepsin D levels declined with HQ exposure, suggesting loss of lysosomal membrane integrity and function. Accordingly, HQ induced lysosomal alkalization, thereby compromising the acidic pH needed for optimal lysosomal degradation. Pretreatment with MG132, a proteasome inhibitor and lysosomal stabilizer, upregulated LAMP2 and autophagy and prevented HQ-induced oxidative damage in wildtype RPE cells but not cells transfected with shRNA against ATG5. This study demonstrated that lysosomal dysfunction underlies autophagy defects and oxidative damage induced by HQ in human RPE cells and supports lysosomal stabilization with the proteasome inhibitor MG132 as a potential remedy for oxidative damage in RPE and AMD.     

Linalool Activates Oxidative and Calcium Burst and CAM3-ACA8 Participates in Calcium Recovery in Arabidopsis Leaves

Plants produce linalool to respond to biotic stress, but the linalool-induced early signal remains unclear. In wild-type Arabidopsis, plant resistance to diamondback moth (Plutella xylostella) increased more strongly in a linalool-treated group than in an untreated control group. H₂O₂ and Ca²⁺, two important early signals that participated in biotic stress, burst after being treated with linalool in Arabidopsis mesophyll cells. Linalool treatment increased H₂O₂ and intracellular calcium concentrations in mesophyll cells, observed using a confocal microscope with laser scanning, and H₂O₂ signaling functions upstream of Ca²⁺ signaling by using inhibitors and mutants. Ca²⁺ efflux was detected using non-invasive micro-test technology (NMT), and Ca²⁺ efflux was also inhibited by NADPH oxidase inhibitor DPI (diphenyleneiodonium chloride) and in cells of the NADPH oxidase mutant rbohd. To restore intracellular calcium levels, Ca²⁺-ATPase was activated, and calmodulin 3 (CAM3) participated in Ca²⁺-ATPase activation. This result is consistent with the interaction between CAM7 and Ca²⁺-ATPase isoform 8 (ACA8). In addition, a yeast two-hybrid assay, firefly luciferase complementation imaging assay, and an in vitro pulldown assay showed that CAM3 interacts with the N-terminus of ACA8, and qRT-PCR showed that some JA-related genes and defense genes expressions were enhanced when treated with linalool in Arabidopsis leaves. This study reveals that linalool enhances H₂O₂ and intracellular calcium concentrations in Arabidopsis mesophyll cells; CAM3-ACA8 reduces intracellular calcium concentrations, allowing cells to resume their resting state. Additionally, JA-related genes and defense genes’ expression may enhance plants’ defense when treated with linalool.     

DOT1L Methyltransferase Regulates Calcium Influx in Erythroid Progenitor Cells in Response to Erythropoietin

Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of murine hematopoietic progenitor cells (HPCs). An important downstream response of EPO signaling is calcium (Ca²⁺) influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca²⁺ influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca²⁺ influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in KIT-positive erythroid progenitor cells was regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development including early erythropoiesis. We previously reported that Dot1l knockout (Dot1l^KO) HPCs in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we detected a marked downregulation of Trpc6 gene expression in Dot1l^KO progenitor cells in the yolk sac compared to the wild type (WT). The promoter and the proximal regions of the Trpc6 gene locus exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. However, the expression of Trpc2, the positive regulator of Ca²⁺ influx, remained unchanged, resulting in an increased TRPC2/TRPC6 ratio. As the loss of DOT1L decreased TRPC6, which inhibited Ca²⁺ influx by TRPC2, Dot1l^KO HPCs in the yolk sac exhibited accelerated and sustained elevated levels of Ca²⁺ influx. Such heightened Ca²⁺ levels might have detrimental effects on the growth and proliferation of HPCs in response to EPO.