Synergistic effect of cerium chloride and calcium chloride alters calcium signaling in keratinocytes to promote epidermal differentiation.

Epidermal keratinocytes undergo morphological and functional changes during differentiation, eventually being enucleated to become corneocytes. Calcium has been shown to be involved in various cellular functions of epidermal cells, including proliferation, differentiation, and apoptosis. Cerium is a lanthanide-series element and rare earth metal. For skin, cerium oxide has been investigated for use in absorbing UV and promoting wound healing. However, the functions and physiological effects of inorganic cerium on the skin have rarely been investigated. Here, we focused on cerium's function in epidermal keratinocytes and its interaction with calcium by investigating their effects on cell differentiation and intracellular calcium concentration. This study showed that applying cerium chloride to epidermal keratinocytes altered calcium signaling. It also suggested that cerium and calcium induced an increase in intracellular calcium concentration and promoted keratinocyte differentiation.

Changes of Trigeminal Ganglion Neurons Innervating the Temporomandibular Joint in Chronic Pain Rat Model.

Background: Phenotype alterations of nociceptive neurons have been shown to be a key step in the pathogenesis of many pain-related diseases. However, it is unclear if the characteristic changes of temporomandibular joint (TMJ) primary afferent neurons are related to the pathogenesis of temporomandibular joint osteoarthritis (TMJOA) chronic pain. This study aimed to determine the morphological and neurochemical changes in trigeminal ganglion (TG) neurons innervating the TMJ in TMJOA chronic pain rats. Materials and Methods: Monosodium iodoacetate (MIA)-induced TMJOA chronic pain rat model was established (n = 6), and saline was injected in rats of the control group (n = 6). TMJ primary afferent neurons were labeled with retrograde tracing (Dil). The spatial distribution and the expression of calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), and neurofilament 200 (NF200) of TMJ primary afferent neurons in TG were investigated using immunofluorescence. Intracellular calcium signaling was recorded by calcium imaging (n = 20). Results: TMJ primary afferent neurons were located only in the V3 region of the TG from both saline- and MIA-injected rats. The number of TG neurons innervating the TMJ was increased in MIA-injected rats. Elevated number and intracellular calcium concentration of small- and medium-sized instead of large-sized Dil+ TG neurons were found in MIA-injected rats. The upregulated expression of CGRP and IB4, but not NF200, in TG neurons innervating the rat TMJs was accompanied by TMJOA chronic pain. Conclusion: This study suggests that sensitization of small- to medium-sized Dil+ TG neurons and CGRP- and IB4-positive Dil+ TG neurons might contribute to the development of TMJOA chronic pain in rats. This will provide valuable information for more efficient control of TMJOA chronic pain.

Purinergic signaling in liver disease: calcium signaling and induction of inflammation.

Purinergic signaling regulates many metabolic functions and is implicated in liver physiology and pathophysiology. Liver functionality is modulated by ionotropic P2X and metabotropic P2Y receptors, specifically P2Y1, P2Y2, and P2Y6 subtypes, which physiologically exert their influence through calcium signaling, a key second messenger controlling glucose and fat metabolism in hepatocytes. Purinergic receptors, acting through calcium signaling, play an important role in a range of liver diseases. Ionotropic P2X receptors, such as the P2X7 subtype, and certain metabotropic P2Y receptors can induce aberrant intracellular calcium transients that impact normal hepatocyte function and initiate the activation of other liver cell types, including Kupffer and stellate cells. These P2Y- and P2X-dependent intracellular calcium increases are particularly relevant in hepatic disease states, where stellate and Kupffer cells respond with innate immune reactions to challenges, such as excess fat accumulation, chronic alcohol abuse, or infections, and can eventually lead to liver fibrosis. This review explores the consequences of excessive extracellular ATP accumulation, triggering calcium influx through P2X4 and P2X7 receptors, inflammasome activation, and programmed cell death. In addition, P2Y2 receptors contribute to hepatic steatosis and insulin resistance, while inhibiting the expression of P2Y6 receptors can alleviate alcoholic liver steatosis. Adenosine receptors may also contribute to fibrosis through extracellular matrix production by fibroblasts. Thus, pharmacological modulation of P1 and P2 receptors and downstream calcium signaling may open novel therapeutic avenues.

Corydaline attenuates osteolysis in rheumatoid arthritis via mitigating reactive oxygen species production and suppressing calcineurin-Nfatc1 signaling.

AIM OF THE STUDY: Osteolysis in Rheumatoid arthritis (RA) is principally provoked by osteoclast hyperactivity. This study aims to employ Corydaline (Cory), a plant extract, as an osteoclast inhibitor in treating RA-inflicted osteolysis while unveiling the corresponding mechanism. MATERIALS AND METHODS: Osteoclasts were derived from mouse bone marrow-derived monocytes (BMMs) stimulated with M-CSF and RANKL. Subsequently, utilizing network pharmacology, we performed a thorough analysis of Cory's molecular structure and discerned its preliminary therapeutic potential. Subsequently, LPS was used to simulate and establish an in vitro model of RA, and the biological effect of Cory on osteoclast behaviors was evaluated through various staining methods, RT-qPCR, and Western blot. In addition, a collagen-induced arthritis (CIA) mouse model was developed to evaluate the therapeutic effects of Cory in vivo. RESULTS: The results from network pharmacology indicated a significant correlation between Cory, oxidative stress, and calcium signaling. Subsequent in vitro experiments demonstrated Cory's capacity to inhibit the formation and function of osteoclast under inflammatory stimuli, thereby protecting against abnormal bone resorption. This effect is achieved by activating the Nrf2 signaling pathway, mitigating the generation of reactive oxygen species (ROS), and modulating the calcineurin-Nfatc1 signaling. Furthermore, this therapeutic effect of Cory on RA-associated osteolysis was proved in CIA mice models. CONCLUSIONS: Cory demonstrates the potential to activate the Nrf2 signaling pathway, effectively countering oxidative stress, and simultaneously inhibit the calcineurin-Nfatc1 signaling pathway to regulate the terminals of calcium signaling. These dual effects collectively reduce osteoclast activity, ultimately contributing to a therapeutic role in RA osteolysis. Therefore, our study presents Cory as a novel pharmaceutical candidate for the prevention and treatment of RA.

Expanded ATXN1 alters transcription and calcium signaling in SCA1 human motor neurons differentiated from induced pluripotent stem cells.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited and lethal neurodegenerative disease caused by the abnormal expansion of CAG repeats in the ATAXIN-1 (ATXN1) gene. Pathological studies identified dysfunction and loss of motor neurons (MNs) in the brain stem and spinal cord, which are thought to contribute to premature lethality by affecting the swallowing and breathing of SCA1 patients. However, the molecular and cellular mechanisms of MN pathogenesis remain unknown. To study SCA1 pathogenesis in human MNs, we differentiated induced pluripotent stem cells (iPSCs) derived from SCA1 patients and their unaffected siblings into MNs. We examined proliferation of progenitor cells, neurite outgrowth, spontaneous and glutamate-induced calcium activity of SCA1 MNs to investigate cellular mechanisms of pathogenesis. RNA sequencing was then used to identify transcriptional alterations in iPSC-derived MN progenitors (pMNs) and MNs which could underlie functional changes in SCA1 MNs. We found significantly decreased spontaneous and evoked calcium activity and identified dysregulation of genes regulating calcium signaling in SCA1 MNs. These results indicate that expanded ATXN1 causes dysfunctional calcium signaling in human MNs.

The effect of CGRP and SP and the cell signaling dialogue between sensory neurons and endothelial cells.

Increasing evidences demonstrate the role of sensory innervation in bone metabolism, remodeling and repair, however neurovascular coupling in bone is rarely studied. Using microfluidic devices as an indirect co-culture model to mimic in vitro the physiological scenario of innervation, our group demonstrated that sensory neurons (SNs) were able to regulate the extracellular matrix remodeling by endothelial cells (ECs), in particular through sensory neuropeptides, i.e. calcitonin gene-related peptide (CGRP) and substance P (SP). Nonetheless, still little is known about the cell signaling pathways and mechanism of action in neurovascular coupling. Here, in order to characterize the communication between SNs and ECs at molecular level, we evaluated the effect of SNs and the neuropeptides CGRP and SP on ECs. We focused on different pathways known to play a role on endothelial functions: calcium signaling, p38 and Erk1/2; the control of signal propagation through Cx43; and endothelial functions through the production of nitric oxide (NO). The effect of SNs was evaluated on ECs Ca2+ influx, the expression of Cx43, endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production, p38, ERK1/2 as well as their phosphorylated forms. In addition, the role of CGRP and SP were either analyzed using respective antagonists in the co-culture model, or by adding directly on the ECs monocultures. We show that capsaicin-stimulated SNs induce increased Ca2+ influx in ECs. SNs stimulate the increase of NO production in ECs, probably involving a decrease in the inhibitory eNOS T495 phosphorylation site. The neuropeptide CGRP, produced by SNs, seems to be one of the mediators of this effect in ECs since NO production is decreased in the presence of CGRP antagonist in the co-culture of ECs and SNs, and increased when ECs are stimulated with synthetic CGRP. Taken together, our results suggest that SNs play an important role in the control of the endothelial cell functions through CGRP production and NO signaling pathway.

A comparison of basal and activity-dependent exon splicing in cortical-patterned neurons of human and mouse origin.

Rodent studies have shown that alternative splicing in neurons plays important roles in development and maturity, and is regulatable by signals such as electrical activity. However, rodent-human similarities are less well explored. We compared basal and activity-dependent exon splicing in cortical-patterned human ESC-derived neurons with that in cortical mouse ESC-derived neurons, primary mouse cortical neurons at two developmental stages, and mouse hippocampal neurons, focussing on conserved orthologous exons. Both basal exon inclusion levels and activity-dependent changes in splicing showed human-mouse correlation. Conserved activity regulated exons are enriched in RBFOX, SAM68, NOVA and PTBP targets, and centered on cytoskeletal organization, mRNA processing, and synaptic signaling genes. However, human-mouse correlations were weaker than inter-mouse comparisons of neurons from different brain regions, developmental stages and origin (ESC vs. primary), suggestive of some inter-species divergence. The set of genes where activity-dependent splicing was observed only in human neurons were dominated by those involved in lipid biosynthesis, signaling and trafficking. Study of human exon splicing in mouse Tc1 neurons carrying human chromosome-21 showed that neuronal basal exon inclusion was influenced by cis-acting sequences, although may not be sufficient to confer activity-responsiveness in an allospecific environment. Overall, these comparisons suggest that neuronal alternative splicing should be confirmed in a human-relevant system even when exon structure is evolutionarily conserved.

CaMKK2: bridging the gap between Ca2+ signaling and energy-sensing.

Calcium (Ca2+) ions are ubiquitous and indispensable signaling messengers that regulate virtually every cell function. The unique ability of Ca2+ to regulate so many different processes yet cause stimulus specific changes in cell function requires sensing and decoding of Ca2+ signals. Ca2+-sensing proteins, such as calmodulin, decode Ca2+ signals by binding and modifying the function of a diverse range of effector proteins. These effectors include the Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) enzyme, which is the core component of a signaling cascade that plays a key role in important physiological and pathophysiological processes, including brain function and cancer. In addition to its role as a Ca2+ signal decoder, CaMKK2 also serves as an important junction point that connects Ca2+ signaling with energy metabolism. By activating the metabolic regulator AMP-activated protein kinase (AMPK), CaMKK2 integrates Ca2+ signals with cellular energy status, enabling the synchronization of cellular activities regulated by Ca2+ with energy availability. Here, we review the structure, regulation, and function of CaMKK2 and discuss its potential as a treatment target for neurological disorders, metabolic disease, and cancer.

Forskolin improves experimental autoimmune encephalomyelitis in mice probably by inhibiting the calcium and the IL-17-STEAP4 signaling pathway.

Multiple sclerosis (MS) is a chronic autoimmune disease in the central nervous system. Forskolin (FSK) is a plant-derived diterpene with excellent immunomodulatory properties and has not been systematically reported for treating MS. This study investigated the therapeutic effects of FSK on cellular and animal MS models and preliminarily explored related mechanisms. The results showed that FSK suppressed the inflammatory response, reduced the expression of STEAP4, and relieved iron deposition in BV-2 cells pretreated by LPS at the cellular level. Meanwhile, at the animal level, FSK treatment halted the progression of experimental autoimmune encephalomyelitis (EAE), alleviated the damage at the lesion sites, reduced the concentration of proinflammatory factors in peripheral blood, and inhibited the immune response of peripheral immune organs in EAE mice. Besides, FSK treatment decreased the expression of STEAP4 in the spinal cord and effectively restored the iron balance in the brain, spinal cord, and serum of EAE mice. Further investigation showed that FSK can reduce IL-17 expression, prevent the differentiation of TH17 cells, and inhibit the calcium signaling pathway. Thus, these results demonstrate that FSK may have the potential to treat MS clinically.

Activin A plays an essential role in migration and proliferation of hepatic stellate cells via Smad3 and calcium signaling.

Activin A and hepatic stellate cells (HSCs) are involved in tissue repair and fibrosis in liver injury. This study investigated the impact of activin A on HSC activation and migration. A microfluidic D4-chip was used for examining the cell migration of mouse hepatic stellate cell line MHSteC. The analysis of differentially expressed genes revealed that activin ßA (Inhba), activin receptor type 1A (Acvr1a) and type 2A (Acvr2a) mRNAs were more significantly expressed in human HSCs than in the hepatocytes. Moreover, activin A promoted MHSteC proliferation and induced MHSteC migration. Furthermore, the MHSteCs treated with activin A exhibited increased levels of migration-related proteins, N-cadherin, Vimentin, α-SMA, MMP2 and MMP9, but a decreased level of E-cadherin. Additionally, activin A treatment significantly increased the p-Smad3 levels and p-Smad3/Smad3 ratio in the MHSteCs, and the Smad3 inhibitor SIS3 attenuated activin A-induced MHSteC proliferation and migration. Simultaneously, activin A increased the calcium levels in the MHSteCs, and the migratory effects of activin A on MHSteCs were weakened by the intracellular calcium ion-chelating agent BAPTA-AM. These data indicate that activin A can promote MHSteC activation and migration through the canonical Smad3 signaling and calcium signaling.

Systematic mapping of mitochondrial calcium uniporter channel (MCUC)-mediated calcium signaling networks.

The mitochondrial calcium uniporter channel (MCUC) mediates mitochondrial calcium entry, regulating energy metabolism and cell death. Although several MCUC components have been identified, the molecular basis of mitochondrial calcium signaling networks and their remodeling upon changes in uniporter activity have not been assessed. Here, we map the MCUC interactome under resting conditions and upon chronic loss or gain of mitochondrial calcium uptake. We identify 89 high-confidence interactors that link MCUC to several mitochondrial complexes and pathways, half of which are associated with human disease. As a proof-of-concept, we validate the mitochondrial intermembrane space protein EFHD1 as a binding partner of the MCUC subunits MCU, EMRE, and MCUB. We further show a MICU1-dependent inhibitory effect of EFHD1 on calcium uptake. Next, we systematically survey compensatory mechanisms and functional consequences of mitochondrial calcium dyshomeostasis by analyzing the MCU interactome upon EMRE, MCUB, MICU1, or MICU2 knockdown. While silencing EMRE reduces MCU interconnectivity, MCUB loss-of-function leads to a wider interaction network. Our study provides a comprehensive and high-confidence resource to gain insights into players and mechanisms regulating mitochondrial calcium signaling and their relevance in human diseases.

An L-type calcium channel blocker nimodipine exerts anti-fibrotic effects by attenuating TGF-ß1 induced calcium response in an in vitro model of thyroid eye disease.

BACKGROUND: Thyroid eye disease (TED) is a vision-threatening autoimmune disorder. Orbital tissue fibrosis leading to intractable complications remains a troublesome issue in TED management. Exploration of novel therapeutic targets and agents to ameliorate tissue fibrosis is crucial for TED. Recent work suggests that Ca2+ signaling participates in tissue fibrosis. However, whether an alteration of Ca2+ signaling has a role in fibrogenesis during TED remains unclear. In this study, we aimed to investigate the role of Ca2+ signaling in the fibrogenesis process during TED and the potential therapeutic effects of a highly selective inhibitor of the L-type calcium channel (LTCC), nimodipine, through a TGF-ß1 induced in vitro TED model. METHODS: Primary culture of orbital fibroblasts (OFs) were established from orbital adipose connective tissues of patients with TED and healthy control donors. Real-time quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing were used to assess the genes expression associated with LTCC in OFs. Flow cytometry, RT-qPCR, 5-ethynyl-2'-deoxyuridine (EdU) proliferation assay, wound healing assay and Western blot (WB) were used to assess the intracellular Ca2+ response on TGF-ß1 stimulation, and to evaluate the potential therapeutic effects of nimodipine in the TGF-ß1 induced in vitro TED model. The roles of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 1 (STAT1) in fibrogenesis during TED were determined by immunohistochemistry, WB, flow cytometry and co-immunoprecipitation assay. Selective inhibitors were used to explore the downstream signaling pathways. RESULTS: LTCC inhibitor nimodipine blocked the TGF-ß1 induced intracellular Ca2+ response and further reduced the expression of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (Col1A1) and collagen type I alpha 2 (Col1A2) in OFs. Besides, nimodipine inhibited cell proliferation and migration of OFs. Moreover, our results provided evidence that activation of the CaMKII/STAT1 signaling pathway was involved in fibrogenesis during TED, and nimodipine inhibited the pro-fibrotic functions of OFs by down-regulating the CaMKII/STAT1 signaling pathway. CONCLUSIONS: TGF-ß1 induces an LTCC-mediated Ca2+ response, followed by activation of CaMKII/STAT1 signaling pathway, which promotes the pro-fibrotic functions of OFs and participates in fibrogenesis during TED. Nimodipine exerts potent anti-fibrotic benefits in vitro by suppressing the CaMKII/STAT1 signaling pathway. Our work deepens our understanding of the fibrogenesis process during TED and provides potential therapeutic targets and alternative candidate for TED.

Endoplasmic reticulum-mitochondria lockdown in Wolfram syndrome.

Wolfram syndrome (WS) is an incurable autosomal recessive disorder originally described as a mitochondriopathy. In a recent work, Liiv and colleagues found that an impaired endoplasmic reticulum (ER)-to-mitochondria calcium shuttling underlies mitochondrial dysfunction in WS models.

Effect of Evogliptin on the Progression of Aortic Valvular Calcification.

BACKGROUND: Medical therapy for aortic stenosis (AS) remains an elusive goal. OBJECTIVES: This study sought to establish whether evogliptin, a dipeptidyl peptidase-4 inhibitor, could reduce AS progression. METHODS: A total of 228 patients (age 67 ± 11 years; 33% women) with AS were randomly assigned to receive placebo (n = 75), evogliptin 5 mg (n = 77), or evogliptin 10 mg (n = 76). The primary endpoint was the 96-week change in aortic valve calcium volume (AVCV) on computed tomography. Secondary endpoints included the 48-week change in active calcification volume measured using 18F-sodium fluoride positron emission tomography (18F-NaF PET). RESULTS: There were no significant differences in the 96-week changes in AVCV between evogliptin 5 mg and placebo (-5.27; 95% CI: -55.36 to 44.82; P = 0.84) or evogliptin 10 mg and placebo (-18.83; 95% CI: -32.43 to 70.10; P = 0.47). In the placebo group, the increase in AVCV between 48 weeks and 96 weeks was higher than that between baseline and 48 weeks (136 mm3; 95% CI: 108-163 vs 102 mm3; 95% CI: 75-129; P = 0.0485). This increasing trend in the second half of the study was suppressed in both evogliptin groups. The 48-week change in active calcification volume on 18F-NaF PET was significantly lower in both the evogliptin 5 mg (-1,325.6; 95% CI: -2,285.9 to -365.4; P = 0.008) and 10-mg groups (-1,582.2; 95% CI: -2,610.8 to -553.5; P = 0.0038) compared with the placebo group. CONCLUSIONS: This exploratory study did not demonstrate the protective effect of evogliptin on AV calcification. Favorable 18F-NaF PET results and possible suppression of aortic valve calcification with longer medication use in the evogliptin groups suggest the need for larger confirmatory trials. (A Multicenter, Double-blind, Placebo-controlled, Stratified-randomized, Parallel, Therapeutic Exploratory Clinical Study to Evaluate the Efficacy and Safety of DA-1229 in Patients With Calcific Aortic Valve Disease; NCT04055883).

Resting human trabecular meshwork cells experience tonic cation influx.

The trabecular meshwork (TM) regulates intraocular pressure (IOP) by converting biochemical and biomechanical stimuli into intracellular signals. Recent electrophysiological studies demonstrated that this process is mediated by pressure sensing ion channels in the TM plasma membrane while the molecular and functional properties of channels that underpin ionic homeostasis in resting cells remain largely unknown. Here, we demonstrate that the TM resting potential is subserved by a powerful cationic conductance that disappears following Na+ removal and substitution with choline or NMDG+. Its insensitivity to TTX, verapamil, phenamil methanesulfonate and amiloride indicates it does not involve voltage-operated Na+, Ca2+ and epithelial Na+ (ENaC) channels or Na+/H+ exchange while a modest hyperpolarization induced by SEA-0440 indicates residual contribution from reversed Na+/Ca2+ exchange. Tonic cationic influx was inhibited by Gd3+ and Ruthenium Red but not GsMTx4, indicating involvement of TRP-like but not Piezo channels. Transcriptional analysis detected expression of most TRP genes, with the canonical transcriptome pool dominated by TRPC1 followed by the expression ofTRPV1, TRPC3 and TRPC5. TRPC3 antagonist Pyr3 and TRPC1,4,5 antagonist Pico1,4,5 did not affect the standing current, whereas the TRPC blocker SKF96365 promoted rather than suppressed, Na+ influx. TM cells thus maintain the resting membrane potential, control Na+ homeostasis, and balance K+ efflux through a novel constitutive monovalent cation leak current with properties not unlike those of TRP channels. Yet to be identified at the molecular level, this novel channel sets the homeostatic steady-state and controls the magnitude of pressure-induced transmembrane signals.

Resting trabecular meshwork cells experience constitutive cation influx.

A quintessential sentinel of cell health, the membrane potential in nonexcitable cells integrates biochemical and biomechanical inputs, determines the driving force for ionic currents activated by input signals and plays critical functions in cellular differentiation, signaling, and pathology. The identity and properties of ion channels that subserve the resting potential in trabecular meshwork (TM) cells is poorly understood, which impairs our understanding of intraocular pressure regulation in healthy and diseased eyes. Here, we identified a powerful cationic conductance that subserves the TM resting potential. It disappears following Na+ removal or substitution with choline or NMDG+, is insensitive to TTX, verapamil, phenamil methanesulfonate, amiloride and GsMTx4, is substituted by Li+ and Cs+, and inhibited by Gd3+ and Ruthenium Red. Constitutive cation influx is thus not mediated by voltage-operated Na+, Ca2+, epithelial Na+ (ENaC) channels, Piezo channels or Na+/H+ exchange but may involve TRP-like channels. Transcriptional analysis detected expression of many TRP genes, with the transcriptome pool dominated by TRPC1 followed by expression of TRPV1, TRPC3, TRPV4 and TRPC5. Pyr3 and Pico1,4,5 did not affect the standing current whereas SKF96365 promoted rather than suppressed, Na+ influx. SEA-0400 induced a modest hyperpolarization, indicating residual contribution from Na+/Ca2+ exchange. The resting membrane potential in human TM cells is thus maintained by a constitutive monovalent cation leak current with properties not unlike those of TRP channels. This conductance is likely to influence conventional outflow by setting the homeostatic steady-state and by regulating the magnitude of pressure-induced currents in normotensive and hypertensive eyes.

Melatonin protects RPE cells from necroptosis and NLRP3 activation via promoting SERCA2-related intracellular Ca2+ homeostasis.

BACKGROUND: Melatonin is an antioxidant that also has anti-inflammatory effects. It has been reported to delay the progression of age-related macular degeneration (AMD), however, the mechanism has not been fully recognized. PURPOSE: The aim of the present study was to investigate the effects of melatonin on sodium iodate (SI)-induced retinal degeneration and elucidate the specific mechanisms, then, provide novel targets in AMD treatment. METHODS: Retinal degeneration mouse model and in vitro retinal pigment epithelium (RPE) death model were established by SI treatment. Melatonin was administrated intraperitoneally at a concentration of 20, 40 or 80 mg/kg for in vivo study or treated at 48 h before SI treatment. To confirm the therapeutic effects of melatonin on mouse, the retinal structure and visual function were evaluated. The specific cell death rates were determined by CCK-8 assay, PI staining and protein level of RIPK3. The cytosolic or mitochondrial calcium levels were determined by Fluo-4AM or Rhod-2AM staining. Mitochondrial functions including mitochondrial dynamics, mitochondrial membrane potential, or mitochondrial permeability pore opening were evaluated. The proteins involved in endoplasmic reticulum (ER) stress were measured by western blot assay while the genes expression in calcium signaling pathway were measured by RT-qPCR. RESULTS: We show that melatonin protects RPE cells from necroptosis and NLRP3 inflammasome activation induced by SI. Mechanistically, melatonin suppresses ER stress and intracellular calcium overload triggered by SI through restoring the function of SERCA2. Silencing of SERCA2 or blocking of melatonin receptors inhibit the protective effects of melatonin. Melatonin reduces mitochondrial Ca2+ levels and restores mitochondrial membrane potential. Constant mitochondrial Ca2+ overload directly promote cell necroptosis through mitochondrial fission. Inhibition of mitochondrial fission by Mdivi-1 prevent necroptosis induced by SI without altering the level of mitochondrial Ca2+. CONCLUSIONS: The results confirmed that melatonin protects RPE cells from SI-induced injury by regulates MT2/SERCA2/Ca2+ axis. This study highlighted the potential of melatonin in the treatment of AMD and elucidated the mechanism and signaling pathway that mediate the protective effects.

ELD607 specifically traffics Orai1 to the lysosome leading to inhibition of store operated calcium entry.

Orai1 is a plasma membrane Ca2+ channel involved in store operated calcium entry (SOCE). SOCE can regulate cell growth, exocytosis, gene expression and inflammation. We previously found that short palate lung and nasal epithelial clone 1's (SPLUNC1) sixth α-helix (α6) bound Orai1 to inhibit SOCE. SPLUNC1 was not proteolytically stable, so we developed ELD607, an 11 amino acid peptide based on SPLUNC1's α6 region which was more stable and more potent than SPLUNC1/α6. Here, we studied ELD607's mechanism of action. We overexpressed either Orai1-HA or Orai1-YFP in HEK293T cells to probe ELD607-Orai1 interactions by confocal microscopy. We also measured changes in Fluo-4 fluorescence in a multiplate reader as a marker of cytoplasmic Ca2+ levels. ELD607 internalized Orai1 independently of STIM1. Both 15 min and 3 h exposure to ELD607 similarly depleted Orai1 in the plasma membrane. However, 3 h exposure to ELD607 yielded greater inhibition of SOCE. ELD607 continued to colocalize with Orai1 after internalization and this process was dependent on the presence of the ubiquitin ligase NEDD4.2. Similarly, ELD607 increased the colocalization between Orai1 and ubiquitin. ELD607 also increased the colocalization between Orai1 and Rab5 and 7, but not Rab11, suggesting that Orai1 trafficked through early and late but not recycling endosomes. Finally, ELD607 caused Orai1, but not Orai2, Orai3, or STIM1 to traffic to lysosomes. We conclude that ELD607 rapidly binds to Orai1 and works in an identical fashion as full length SPLUNC1 by internalizing Orai1 and sending it to lysosomes, leading to a decrease in SOCE.

Mitochondrial Ca2+-coupled generation of reactive oxygen species, peroxynitrite formation, and endothelial dysfunction in Cantú syndrome.

Cantú syndrome is a multisystem disorder caused by gain-of-function (GOF) mutations in KCNJ8 and ABCC9, the genes encoding the pore-forming inward rectifier Kir6.1 and regulatory sulfonylurea receptor SUR2B subunits, respectively, of vascular ATP-sensitive K+ (KATP) channels. In this study, we investigated changes in the vascular endothelium in mice in which Cantú syndrome-associated Kcnj8 or Abcc9 mutations were knocked in to the endogenous loci. We found that endothelium-dependent dilation was impaired in small mesenteric arteries from Cantú mice. Loss of endothelium-dependent vasodilation led to increased vasoconstriction in response to intraluminal pressure or treatment with the adrenergic receptor agonist phenylephrine. We also found that either KATP GOF or acute activation of KATP channels with pinacidil increased the amplitude and frequency of wave-like Ca2+ events generated in the endothelium in response to the vasodilator agonist carbachol. Increased cytosolic Ca2+ signaling activity in arterial endothelial cells from Cantú mice was associated with elevated mitochondrial [Ca2+] and enhanced reactive oxygen species (ROS) and peroxynitrite levels. Scavenging intracellular or mitochondrial ROS restored endothelium-dependent vasodilation in the arteries of mice with KATP GOF mutations. We conclude that mitochondrial Ca2+ overload and ROS generation, which subsequently leads to nitric oxide consumption and peroxynitrite formation, cause endothelial dysfunction in mice with Cantú syndrome.

Calcium ion delivery by microbubble-assisted sonoporation stimulates cell death in human gastrointestinal cancer cells.

Ultrasound-mediated cell membrane permeabilization - sonoporation, enhances drug delivery directly to tumor sites while reducing systemic side effects. The potential of ultrasound to augment intracellular calcium uptake - a critical regulator of cell death and proliferation - offers innovative alternative to conventional chemotherapy. However, calcium therapeutic applications remain underexplored in sonoporation studies. This research provides a comprehensive analysis of calcium sonoporation (CaSP), which combines ultrasound treatment with calcium ions and SonoVue microbubbles, on gastrointestinal cancer cells LoVo and HPAF-II. Initially, optimal sonoporation parameters were determined: an acoustic wave of 1 MHz frequency with a 50 % duty cycle at intensity of 2 W/cm2. Subsequently, various cellular bioeffects, such as viability, oxidative stress, metabolism, mitochondrial function, proliferation, and cell death, were assessed following CaSP treatment. CaSP significantly impaired cancer cell function by inducing oxidative and metabolic stress, evidenced by increased mitochondrial depolarization, decreased ATP levels, and elevated glucose uptake in a Ca2+ dose-dependent manner, leading to activation of the intrinsic apoptotic pathway. Cellular response to CaSP depended on the TP53 gene's mutational status: colon cancer cells were more susceptible to CaSP-induced apoptosis and G1 phase cell cycle arrest, whereas pancreatic cancer cells showed a higher necrotic response and G2 cell cycle arrest. These promising results encourage future research to optimize sonoporation parameters for clinical use, investigate synergistic effects with existing treatments, and assess long-term safety and efficacy in vivo. Our study highlights CaSP's clinical potential for improved safety and efficacy in cancer therapy, offering significant implications for the pharmaceutical and biomedical fields.