Apolipoprotein C3 induces inflammation and organ damage by alternative inflammasome activation.

NLRP3-inflammasome-driven inflammation is involved in the pathogenesis of a variety of diseases. Identification of endogenous inflammasome activators is essential for the development of new anti-inflammatory treatment strategies. Here, we identified that apolipoprotein C3 (ApoC3) activates the NLRP3 inflammasome in human monocytes by inducing an alternative NLRP3 inflammasome via caspase-8 and dimerization of Toll-like receptors 2 and 4. Alternative inflammasome activation in human monocytes is mediated by the Toll-like receptor adapter protein SCIMP. This triggers Lyn/Syk-dependent calcium entry and the production of reactive oxygen species, leading to activation of caspase-8. In humanized mouse models, ApoC3 activated human monocytes in vivo to impede endothelial regeneration and promote kidney injury in an NLRP3- and caspase-8-dependent manner. These data provide new insights into the regulation of the NLRP3 inflammasome and the pathophysiological role of triglyceride-rich lipoproteins containing ApoC3. Targeting ApoC3 might prevent organ damage and provide an anti-inflammatory treatment for vascular and kidney diseases.

A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP-modulated NFAT signaling.

Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca2+ entry (SOCE) triggering activation of transcription factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astrocytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and ICRAC, facilitating sequence-specific fast calcium-dependent inactivation and destabilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteomics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regulate the NFAT signalosome and cAMP-SOCE crosstalk.

Redox signals at the ER-mitochondria interface control melanoma progression.

Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)-mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER-mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial- and NOX4-derived ROS. The TMX-knockdown-induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1-positive and NFAT1-negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial- and redox-related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox-controlled ER-mitochondria-NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease.

Store-operated calcium entry is reduced in spastin-linked hereditary spastic paraplegia.

Pathogenic variants in SPAST, the gene coding for spastin, are the single most common cause of hereditary spastic paraplegia, a progressive motor neuron disease. Spastin regulates key cellular functions, including microtubule-severing and endoplasmic reticulum-morphogenesis. However, it remains unclear how alterations in these cellular functions due to SPAST pathogenic variants result in motor neuron dysfunction. Since spastin influences both microtubule network and endoplasmic reticulum structure, we hypothesized that spastin is necessary for the regulation of Ca2+ homeostasis via store-operated calcium entry. Here, we show that the lack of spastin enlarges the endoplasmic reticulum and reduces store-operated calcium entry. In addition, elevated levels of different spastin variants induced clustering of STIM1 within the endoplasmic reticulum, altered the transport of STIM1 to the plasma membrane and reduced store-operated calcium entry, which could be rescued by exogenous expression of STIM1. Importantly, store-operated calcium entry was strongly reduced in induced pluripotent stem cell-derived neurons from hereditary spastic paraplegia patients with pathogenic variants in SPAST resulting in spastin haploinsufficiency. These neurons developed axonal swellings in response to lack of spastin. We were able to rescue both store-operated calcium entry and axonal swellings in SPAST patient neurons by restoring spastin levels, using CRISPR/Cas9 to correct the pathogenic variants in SPAST. These findings demonstrate that proper amounts of spastin are a key regulatory component for store-operated calcium entry mediated Ca2+ homeostasis and suggest store-operated calcium entry as a disease relevant mechanism of spastin-linked motor neuron disease.

Acute Downregulation but Not Genetic Ablation of Murine MCU Impairs Suppressive Capacity of Regulatory CD4 T Cells.

By virtue of mitochondrial control of energy production, reactive oxygen species (ROS) generation, and maintenance of Ca2+ homeostasis, mitochondria play an essential role in modulating T cell function. The mitochondrial Ca2+ uniporter (MCU) is the pore-forming unit in the main protein complex mediating mitochondrial Ca2+ uptake. Recently, MCU has been shown to modulate Ca2+ signals at subcellular organellar interfaces, thus fine-tuning NFAT translocation and T cell activation. The mechanisms underlying this modulation and whether MCU has additional T cell subpopulation-specific effects remain elusive. However, mice with germline or tissue-specific ablation of Mcu did not show impaired T cell responses in vitro or in vivo, indicating that 'chronic' loss of MCU can be functionally compensated in lymphocytes. The current work aimed to specifically investigate whether and how MCU influences the suppressive potential of regulatory CD4 T cells (Treg). We show that, in contrast to genetic ablation, acute siRNA-mediated downregulation of Mcu in murine Tregs results in a significant reduction both in mitochondrial Ca2+ uptake and in the suppressive capacity of Tregs, while the ratios of Treg subpopulations and the expression of hallmark transcription factors were not affected. These findings suggest that permanent genetic inactivation of MCU may result in compensatory adaptive mechanisms, masking the effects on the suppressive capacity of Tregs.

Interleukin-1α Is a Central Regulator of Leukocyte-Endothelial Adhesion in Myocardial Infarction and in Chronic Kidney Disease.

BACKGROUND: Cardiovascular diseases and chronic kidney disease (CKD) are highly prevalent, aggravate each other, and account for substantial mortality. Both conditions are characterized by activation of the innate immune system. The alarmin interleukin-1α (IL-1α) is expressed in a variety of cell types promoting (sterile) systemic inflammation. The aim of the present study was to examine the role of IL-1α in mediating inflammation in the setting of acute myocardial infarction (AMI) and CKD. METHODS: We assessed the expression of IL-1α on the surface of monocytes from patients with AMI and patients with CKD and determined its association with atherosclerotic cardiovascular disease events during follow-up in an explorative clinical study. Furthermore, we assessed the inflammatory effects of IL-1α in several organ injury models in Il1a-/- and Il1b-/- mice and investigated the underlying mechanisms in vitro in monocytes and endothelial cells. RESULTS: IL-1α is strongly expressed on the surface of monocytes from patients with AMI and CKD compared with healthy controls. Higher IL-1α surface expression on monocytes from patients with AMI and CKD was associated with a higher risk for atherosclerotic cardiovascular disease events, which underlines the clinical relevance of IL-1α. In mice, IL-1α, but not IL-1ß, mediates leukocyte-endothelial adhesion as determined by intravital microscopy. IL-1α promotes accumulation of macrophages and neutrophils in inflamed tissue in vivo. Furthermore, IL-1α on monocytes stimulates their homing at sites of vascular injury. A variety of stimuli such as free fatty acids or oxalate crystals induce IL-1α surface expression and release by monocytes, which then mediates their adhesion to the endothelium via IL-1 receptor-1. IL-1α also promotes expression of the VCAM-1 (vascular cell adhesion molecule-1) on endothelial cells, thereby fostering the adhesion of circulating leukocytes. IL-1α induces inflammatory injury after experimental AMI, and abrogation of IL-1α prevents the development of CKD in oxalate or adenine-fed mice. CONCLUSIONS: IL-1α represents a key mediator of leukocyte-endothelial adhesion and inflammation in AMI and CKD. Inhibition of IL-1α may serve as a novel anti-inflammatory treatment strategy.

Detecting single ORAI1 proteins within the plasma membrane reveals higher-order channel complexes.

ORAI1 proteins form highly selective Ca2+ channels in the plasma membrane. Crystallographic data point towards a hexameric stoichiometry of ORAI1 channels, whereas optical methods postulated ORAI1 channels to reside as dimers at rest, and other data suggests that they have a tetrameric configuration. Here, liquid-phase scanning transmission electron microscopy (STEM) and quantum dot (QD) labeling was utilized to study the conformation of ORAI1 proteins at rest. To address the question of whether ORAI1 was present as a dimer, experiments were designed using single ORAI1 monomers and covalently linked ORAI1 dimers with either one or two label-binding positions. The microscopic data was statistically analyzed via the pair correlation function. Label pairs were found in all cases, even for concatenated dimers with one label-binding position, which is only possible if a significant fraction of ORAI1 was assembled in larger order oligomers than dimers, binding at least two QDs. This interpretation of the data was consistent with Blue Native PAGE analysis showing that ORAI1 is mainly present as a complex of an apparent molecular mass larger than that calculated for a dimer.

High glucose distinctively regulates Ca2+ influx in cytotoxic T lymphocytes upon target recognition and thapsigargin stimulation.

In CTLs: High glucose-culture enhances thapsigargin-induced SOCE but decreases target recognition-induced Ca2+ influx. High glucose-culture regulates expression of ORAIs and STIMs without affecting glucose uptake. More high glucose-cultured CTLs are prone to necrosis after execution of killing.

Cross-linking of Orai1 channels by STIM proteins.

The transmembrane docking of endoplasmic reticulum (ER) Ca2+-sensing STIM proteins with plasma membrane (PM) Orai Ca2+ channels is a critical but poorly understood step in Ca2+ signal generation. STIM1 protein dimers unfold to expose a discrete STIM-Orai activating region (SOAR1) that tethers and activates Orai1 channels within discrete ER-PM junctions. We reveal that each monomer within the SOAR dimer interacts independently with single Orai1 subunits to mediate cross-linking between Orai1 channels. Superresolution imaging and mobility measured by fluorescence recovery after photobleaching reveal that SOAR dimer cross-linking leads to substantial Orai1 channel clustering, resulting in increased efficacy and cooperativity of Orai1 channel function. A concatenated SOAR1 heterodimer containing one monomer point mutated at its critical Orai1 binding residue (F394H), although fully activating Orai channels, is completely defective in cross-linking Orai1 channels. Importantly, the naturally occurring STIM2 variant, STIM2.1, has an eight-amino acid insert in its SOAR unit that renders it functionally identical to the F394H mutant in SOAR1. Contrary to earlier predictions, the SOAR1-SOAR2.1 heterodimer fully activates Orai1 channels but prevents cross-linking and clustering of channels. Interestingly, combined expression of full-length STIM1 with STIM2.1 in a 5:1 ratio causes suppression of sustained agonist-induced Ca2+ oscillations and protects cells from Ca2+ overload, resulting from high agonist-induced Ca2+ release. Thus, STIM2.1 exerts a powerful regulatory effect on signal generation likely through preventing Orai1 channel cross-linking. Overall, STIM-mediated cross-linking of Orai1 channels is a hitherto unrecognized functional paradigm that likely provides an organizational microenvironment within ER-PM junctions with important functional impact on Ca2+ signal generation.

Supra-Molecular Assemblies of ORAI1 at Rest Precede Local Accumulation into Puncta after Activation.

The Ca2+ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca2+ entry (SOCE). Using liquid phase electron microscopy (LPEM), the distribution of ORAI1 proteins was examined at rest and after SOCE-activation at nanoscale resolution. The analysis of over seven hundred thousand ORAI1 positions revealed a number of ORAI1 channels had formed STIM-independent distinct supra-molecular clusters. Upon SOCE activation and in the presence of STIM proteins, a fraction of ORAI1 assembled in micron-sized two-dimensional structures, such as the known puncta at the ER plasma membrane contact zones, but also in divergent structures such as strands, and ring-like shapes. Our results thus question the hypothesis that stochastically migrating single ORAI1 channels are trapped at regions containing activated STIM, and we propose instead that supra-molecular ORAI1 clusters fulfill an amplifying function for creating dense ORAI1 accumulations upon SOCE-activation.

Expression of the LRRC52 γ subunit (γ2) may provide Ca2+-independent activation of BK currents in mouse inner hair cells.

Mammalian inner hair cells (IHCs) transduce sound into depolarization and transmitter release. Big conductance and voltage- and Ca2+-activated K+ (BK) channels are responsible for fast membrane repolarization and small time constants of mature IHCs. For unknown reasons, they activate at around -75 mV with a voltage of half-maximum activation (Vhalf) of -50 mV although being largely insensitive to Ca2+ influx. Ca2+-independent activation of BK channels was observed by others in heterologous expression systems if γ subunits leucine-rich repeat-containing protein (LRRC)26 (γ1) and LRRC52 (γ2) were coexpressed with the pore-forming BKα subunit, which shifted Vhalf by -140 and -100 mV, respectively. Using nested PCR, we consistently detected transcripts for LRRC52 but not for LRRC26 in IHCs of 3-wk-old mice. Confocal immunohistochemistry showed synchronous up-regulation of LRRC52 protein with BKα at the onset of hearing. Colocalization of LRRC52 protein and BKα at the IHC neck within ≤40 nm was specified using an insitu proximity ligation assay. Mice deficient for the voltage-gated Cav1.3 Ca2+ channel encoded by Cacna1d do not express BKα protein. LRRC52 protein was neither expressed in IHCs of BKα nor in IHCs of Cav1.3 knockout mice. Together, LRRC52 is a γ2 subunit of BK channel complexes and is a strong candidate for causing the Ca2+-independent activation of BK currents at negative membrane potentials in mouse IHCs.-Lang, I., Jung, M., Niemeyer, B. A., Ruth, P., Engel, J. Expression of the LRRC52 γ subunit (γ2) may provide Ca2+-independent activation of BK currents in mouse inner hair cells.

Excitable T cells: Ca(v)1.4 channel contributions and controversies.

Store-operated CRAC channels encoded by the Orai genes mediate calcium entry in T cells. In this issue of Immunity, Omilusik et al. (2011) record Ca(V)1.4-mediated voltage-gated calcium currents in T cells and address their role for T cell development and function.

Profiling calcium signals of in vitro polarized human effector CD4+ T cells.

Differentiation of naïve CD4+ T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca2+ signals, mainly mediated by the store operated Ca2+ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4+ T cell subsets show differential Ca2+ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4+ effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca2+ release activated Ca2+ currents (ICRAC) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca2+ profiles of helper CD4+ Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4+ T cells.

Reaction-diffusion model for STIM-ORAI interaction: The role of ROS and mutations.

Release of Ca2+ from endoplasmatic retriculum (ER) Ca2+ stores causes stromal interaction molecules (STIM) in the ER membrane and ORAI proteins in the plasma membrane (PM) to interact and form the Ca2+ release activated Ca2+ (CRAC) channels, which represent a major Ca2+ entry route in non-excitable cells and thus control various cell functions. It is experimentally possible to mutate ORAI1 proteins and therefore modify, especially block, the Ca2+ influx into the cell. On the basis of the model of Hoover and Lewis (2011), we formulate a reaction-diffusion model to quantify the STIM1-ORAI1 interaction during CRAC channel formation and analyze different ORAI1 channel stoichiometries and different ratios of STIM1 and ORAI1 in comparison with experimental data. We incorporate the inhibition of ORAI1 channels by ROS into our model and calculate its contribution to the CRAC channel amplitude. We observe a large decrease of the CRAC channel amplitude evoked by mutations of ORAI1 proteins.

A calcium optimum for cytotoxic T lymphocyte and natural killer cell cytotoxicity.

KEY POINTS: Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca2+ ]i (between 122 and 334 nm) and [Ca2+ ]o (between 23 and 625 µm) for efficient cancer cell elimination, well below blood plasma Ca2+ levels. As predicted from these results, partial down-regulation of the Ca2+ channel Orai1 in CTLs paradoxically increases perforin-dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca2+ optimum compatible with this low Ca2+ optimum for efficient cancer cell killing, whereas the Ca2+ optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca2+ ]o . We propose that a partial inhibition of Ca2+ signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination. ABSTRACT: Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to protect the human body against cancer. Ca2+ is a key metabolic factor for lymphocyte function and cancer homeostasis. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity against cancer cells and found that CTLs have a bell-shaped Ca2+ dependence with an optimum for cancer cell elimination at rather low [Ca2+ ]o (23-625 µm) and [Ca2+ ]i (122-334 nm). This finding predicts that a partial inhibition of Orai1 should increase (rather than decrease) cytotoxicity of CTLs at [Ca2+ ]o higher than 625 µm. We tested this hypothesis in CTLs and indeed found that partial down-regulation of Orai1 by siRNA increases the efficiency of cancer cell killing. We found two mechanisms that may account for the Ca2+ optimum of cancer cell killing: (1) migration velocity and persistence have a moderate optimum between 500 and 1000 µm [Ca2+ ]o in CTLs, and (2) lytic granule release at the immune synapse between CTLs and cancer cells is increased at 146 µm compared to 3 or 800 µm, compatible with the Ca2+ optimum for cancer cell killing. It has been demonstrated in many cancer cell types that Orai1-dependent Ca2+ signals enhance proliferation. We propose that a decrease of [Ca2+ ]o or partial inhibition of Orai1 activity by selective blockers in the tumour microenvironment could efficiently reduce cancer growth by simultaneously increasing CTL and NK cell cytotoxicity and decreasing cancer cell proliferation.

The STIM-Orai Pathway: Regulation of STIM and Orai by Thiol Modifications.

Cysteines are among the least abundant amino acids found in proteins. Due to their unique nucleophilic thiol group, they are able to undergo a broad range of chemical modifications besides their known role in disulfide formation, such as S-sulfenylation (-SOH), S-sulfinylation (-SO(2)H), S-sufonylation (-SO(3)H), S-glutathionylation (-SSG), and S-sulfhydration (-SSH), among others. These posttranslational modifications can be irreversible and act as transitional modifiers or as reversible on-off switches for the function of proteins. Disturbances of the redox homeostasis, for example, in situations of increased oxidative stress, can contribute to a range of diseases. Because Ca2+ signaling mediated by store-operated calcium entry (SOCE) is involved in a plethora of cellular responses, the cross-talk between reactive oxygen species (ROS) and Ca2+ is critical for homeostatic control. Identification of calcium regulatory protein targets of thiol redox modifications is needed to understand their role in biology and disease.

Changing calcium: CRAC channel (STIM and Orai) expression, splicing, and posttranslational modifiers.

A wide variety of cellular function depends on the dynamics of intracellular Ca(2+) signals. Especially for relatively slow and lasting processes such as gene expression, cell proliferation, and often migration, cells rely on the store-operated Ca(2+) entry (SOCE) pathway, which is particularly prominent in immune cells. SOCE is initiated by the sensor proteins (STIM1, STIM2) located within the endoplasmic reticulum (ER) registering the Ca(2+) concentration within the ER, and upon its depletion, cluster and trap Orai (Orai1-3) proteins located in the plasma membrane (PM) into ER-PM junctions. These regions become sites of highly selective Ca(2+) entry predominantly through Orai1-assembled channels, which, among other effector functions, is necessary for triggering NFAT translocation into the nucleus. What is less clear is how the spatial and temporal spread of intracellular Ca(2+) is shaped and regulated by differential expression of the individual SOCE genes and their splice variants, their heteromeric combinations and pre- and posttranslational modifications. This review focuses on principle mechanisms regulating expression, splicing, and targeting of Ca(2+) release-activated Ca(2+) (CRAC) channels.

Facilitation of Orai3 targeting and store-operated function by Orai1.

Orai1 subunits interacting with STIM1 molecules comprise the major components responsible for calcium release-activated calcium (CRAC) channels. The homologs Orai2 and Orai3 yield smaller store-operated currents when overexpressed and are mostly unable to substitute Orai1. Orai3 subunits are also essential components of store independent channel complexes and also tune inhibition of ICRAC by reactive oxygen species. Here we use patch-clamp, microscopy, Ca(2+)-imaging and biochemical experiments to investigate the interdependence of Orai2, Orai3 and Orai1. We demonstrate that store-operation and localization of Orai3 but not of Orai2 to STIM1 clusters in HEK cells or to the immunological synapse in T cells is facilitated by Orai1 while Orai3's store-independent activity remains unaffected. On the other hand, one Orai3 subunit confers redox-resistance to heteromeric channels. The inefficient store operation of Orai3 is partly due to the lack of three critical C-terminal residues, the insertion of which improves interaction with STIM1 and abrogates Orai3's dependence on Orai1. Our results suggest that Orai3 down-tunes efficient STIM1 gating when in a heteromeric complex with Orai1.

Visualizing Quantum Dot Labeled ORAI1 Proteins in Intact Cells Via Correlative Light and Electron Microscopy.

ORAI1 proteins are ion channel subunits and the essential pore-forming units of the calcium release-activated calcium channel complex essential for T-cell activation and many other cellular processes. In this study, we used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to image plasma membrane expressed ORAI1 proteins in whole Jurkat T cells in the liquid state. Utilizing a stably transfected Jurkat T cell clone expressing human ORAI1 with an extracellular human influenza hemagglutinin (HA) tag we investigated if liquid-phase STEM can be applied to detect recombinant surface expressed protein. Streptavidin coated quantum dots were coupled in a one-to-one stoichiometry to ORAI1 proteins detected by biotinylated anti-HA fragmented antibody fragments. High-resolution electron microscopic images revealed the individual label locations from which protein pair distances were determined. These data were analyzed using the pair correlation function and, in addition, an analysis of cluster size and frequency was performed. ORAI1 was found to be present in hexamers in a small fraction only, and ORAI1 resided mostly in monomers and dimers.