Regulation of store-operated channel molecules ORAI and STIM by oxidative stress in blood vessels

ORAI and STIM genes are recently identified store-operated calcium channel molecules that play important roles in human physiology. In this thesis, the effects of oxidative stress conditions including high glucose, homocysteine and H₂O₂ on the expression of ORAI and STIM, Ca²⁺ influx, ORAI channel activity and potential underlying mechanisms were investigated using cell models and in vivo tissue samples from diabetic patients and mice.ORAI1-3 and STIM1-2 were detected in vascular endothelial cells and smooth muscle cells using RT-PCR, western blotting and immunostaining. Their expression was upregulated by chronic treatment with high glucose in cell models. The upregulation was also observed in human aorta from Type 2 diabetic patients and kidney tissues from streptozotocin-induced and Akita Type 1 diabetic mouse models. The high glucose-induced gene upregulation was prevented by the calcineurin inhibitor cyclosporin A and store-operated channel blocker diethylstilbestrol. H₂O₂ also upregulated ORAI1-3 and STIM1-2, however, homocysteine increased STIM1-2 expression, but downregulated ORAI1-3.Ca²⁺ influx and ORAI channel activity were investigated using Ca²⁺ imaging and whole-cell patch clamp. Chronic treatment with high glucose enhanced storeoperated Ca²⁺ influx in endothelial cells, but there was no effect if treated acutely. In HEK-293 cells overexpressing STIM1/ORAI1-3, high glucose had no acute effect on ORAI1-3 currents, but homocysteine decreased the currents. The cytosolic STIM1 movement was monitored by live-cell fluorescence imaging. Oxidative stress did not change STIM1-EYFP translocation and clustering after Ca²⁺ store-depletion. The effect of hyperosmolarity on STIM and ORAI expression and channel activity was also investigated. Hyperosmolarity inhibited ORAI1-3 currents and downregulated ORAI1-3 and STIM1-2 gene expression, but did not alter cytosolic STIM1-EYFP translocation.It is concluded that store-operated channel molecules, STIMs and ORAIs, are new proteins regulated by oxidative stress, especially in diabetes, which may provide a novel concept for the abnormality of Ca²⁺ homeostasis in blood vessels from patients with diabetes

Ca2+ Influx through TRPC Channels Is Regulated by Homocysteine–Copper Complexes

An elevated level of circulating homocysteine (Hcy) has been regarded as an independent risk factor for cardiovascular disease; however, the clinical benefit of Hcy lowering-therapy is not satisfying. To explore potential unrevealed mechanisms, we investigated the roles of Ca2+ influx through TRPC channels and regulation by Hcy–copper complexes. Using primary cultured human aortic endothelial cells and HEK-293 T-REx cells with inducible TRPC gene expression, we found that Hcy increased the Ca2+ influx in vascular endothelial cells through the activation of TRPC4 and TRPC5. The activity of TRPC4 and TRPC5 was regulated by extracellular divalent copper (Cu2+) and Hcy. Hcy prevented channel activation by divalent copper, but monovalent copper (Cu+) had no effect on the TRPC channels. The glutamic acids (E542/E543) and the cysteine residue (C554) in the extracellular pore region of the TRPC4 channel mediated the effect of Hcy–copper complexes. The interaction of Hcy–copper significantly regulated endothelial proliferation, migration, and angiogenesis. Our results suggest that Hcy–copper complexes function as a new pair of endogenous regulators for TRPC channel activity. This finding gives a new understanding of the pathogenesis of hyperhomocysteinemia and may explain the unsatisfying clinical outcome of Hcy-lowering therapy and the potential benefit of copper-chelating therapy

Involvement of TRPC Channels in Lung Cancer Cell Differentiation and the Correlation Analysis in Human Non-Small Cell Lung Cancer

The canonical transient receptor potential (TRPC) channels are Ca2+-permeable cationic channels controlling the Ca2+ influx evoked by G protein-coupled receptor activation and/or by Ca2+ store depletion. Here we investigate the involvement of TRPCs in the cell differentiation of lung cancer. The expression of TRPCs and the correlation to cancer differentiation grade in non-small cell lung cancer (NSCLC) were analyzed by real-time PCR and immunostaining using tissue microarrays from 28 patient lung cancer samples. The association of TRPCs with cell differentiation was also investigated in the lung cancer cell line A549 by PCR and Western blotting. The channel activity was monitored by Ca2+ imaging and patch recording after treatment with all-trans-retinoic acid (ATRA). The expression of TRPC1, 3, 4 and 6 was correlated to the differentiation grade of NSCLC in patients, but there was no correlation to age, sex, smoking history and lung cancer cell type. ATRA upregulated TRPC3, TRPC4 and TRPC6 expression and enhanced Ca2+ influx in A549 cells, however, ATRA showed no direct effect on TRPC channels. Inhibition of TRPC channels by pore-blocking antibodies decreased the cell mitosis, which was counteracted by chronic treatment with ATRA. Blockade of TRPC channels inhibited A549 cell proliferation, while overexpression of TRPCs increased the proliferation. We conclude that TRPC expression correlates to lung cancer differentiation. TRPCs mediate the pharmacological effect of ATRA and play important roles in regulating lung cancer cell differentiation and proliferation, which gives a new understanding of lung cancer biology and potential anti-cancer therapy. © 2013 Jiang et al

High glucose enhances store-operated calcium entry by upregulating ORAI/STIM via calcineurin-NFAT signalling

© 2014, Springer-Verlag Berlin Heidelberg. Abstract: ORAI and stromal interaction molecule (STIM) are store-operated channel molecules that play essential roles in human physiology through a coupling mechanism of internal Ca 2+ store to Ca 2+ influx. However, the roles of ORAI and STIM in vascular endothelial cells under diabetic conditions remain unknown. Here, we investigated expression and signalling pathways of ORAI and STIM regulated by high glucose or hyperglycaemia using in vitro cell models, in vivo diabetic mice and tissues from patients. We found that ORAI1-3 and STIM1-2 were ubiquitously expressed in human vasculatures. Their expression was upregulated by chronic treatment with high glucose (HG, 25 mM d-glucose), which was accompanied by enhanced store-operated Ca 2+ influx in vascular endothelial cells. The increased expression was also observed in the aortae from genetically modified Akita diabetic mice (C57BL/6-Ins2 Akita /J) and streptozocin-induced diabetic mice, and aortae from diabetic patients. HG-induced upregulation of ORAI and STIM genes was prevented by the calcineurin inhibitor cyclosporin A and NFATc3 siRNA. Additionally, in vivo treatment with the nuclear factor of activated T cells (NFAT) inhibitor A-285222 prevented the gene upregulation in Akita mice. However, HG had no direct effects on ORAI1-3 currents and the channel activation process through cytosolic STIM1 movement in the cells co-expressing STIM1-EYFP/ORAIs. We concluded that upregulation of STIM/ORAI through Ca 2+ -calcineurin-NFAT pathway is a novel mechanism causing abnormal Ca 2+ homeostasis and endothelial dysfunction under hyperglycaemia. Key message: ORAI1-3 and STIM1-2 are ubiquitously expressed in vasculatures and upregulated by high glucose.Increased expression is confirmed in Akita (Ins2 Akita /J) and STZ diabetic mice and patients.Upregulation mechanism is mediated by Ca 2+ /calcineurin/NFATc3 signalling.High glucose has no direct effects on ORAI1-3 channel activity and channel activation process

Genetic insights into the impact of complement in Alzheimer's disease

The presence of complement activation products at sites of pathology in post-mortem Alzheimer’s disease (AD) brains is well known. Recent evidence from genome-wide association studies (GWAS), combined with the demonstration that complement activation is pivotal in synapse loss in AD, strongly implicates complement in disease aetiology. Genetic variations in complement genes are widespread. While most variants individually have only minor effects on complement homeostasis, the combined effects of variants in multiple complement genes, referred to as the “complotype”, can have major effects. In some diseases, the complotype highlights specific parts of the complement pathway involved in disease, thereby pointing towards a mechanism; however, this is not the case with AD. Here we review the complement GWAS hits; CR1 encoding complement receptor 1 (CR1), CLU encoding clusterin, and a suggestive association of C1S encoding the enzyme C1s, and discuss difficulties in attributing the AD association in these genes to complement function. A better understanding of complement genetics in AD might facilitate predictive genetic screening tests and enable the development of simple diagnostic tools and guide the future use of anti-complement drugs, of which several are currently in development for central nervous system disorders

The complement system in neurodegenerative diseases

Complement is an important component of innate immune defence against pathogens and crucial for efficient immune complex disposal. These core protective activities are dependent in large part on properly regulated complement-mediated inflammation. Dysregulated complement activation, often driven by persistence of activating triggers, is a cause of pathological inflammation in numerous diseases, including neurological diseases. Increasingly, this has become apparent not only in well-recognized neuroinflammatory diseases like multiple sclerosis but also in neurodegenerative and neuropsychiatric diseases where inflammation was previously either ignored or dismissed as a secondary event. There is now a large and rapidly growing body of evidence implicating complement in neurological diseases that cannot be comprehensively addressed in a brief review. Here, we will focus on neurodegenerative diseases, including not only the ‘classical’ neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, but also two other neurological diseases where neurodegeneration is a neglected feature and complement is implicated, namely, schizophrenia, a neurodevelopmental disorder with many mechanistic features of neurodegeneration, and multiple sclerosis, a demyelinating disorder where neurodegeneration is a major cause of progressive decline. We will discuss the evidence implicating complement as a driver of pathology in these diverse diseases and address briefly the potential and pitfalls of anti-complement drug therapy for neurodegenerative diseases

Novel monoclonal antibodies against mouse C1q: characterisation and development of a quantitative ELISA for mouse C1q

Recent studies have identified roles for complement in synaptic pruning, both physiological during development and pathological in Alzheimer’s disease (AD). These reports suggest that C1q initiates complement activation on synapses and C3 fragments then tag them for removal by microglia. There is an urgent need to characterise these processes in rodent AD models; this requires the development of reagents and methods for detection and quantification of rodent C1q in fluids and pathological tissues. These will enable better evaluation of the role of C1q in disease and its value as disease biomarker. We describe the generation in C1q-deficient mice of novel monoclonal antibodies against mouse and rat C1q that enabled development of a sensitive, specific, and quantitative ELISA for mouse and rat C1q capable of measuring C1q in biological fluids and tissue extracts. Serum C1q levels were measured in wild-type (WT), C1q knockout (KO), C3 KO, C7 KO, Crry KO, and 3xTg and APPNL-G-F AD model mice through ageing. C1q levels significantly decreased in WT, APPNL-G-F, and C7 KO mice with ageing. C1q levels were reduced in APPNL-G-F compared to WT at all ages and in 3xTg at 12 months; C3 KO and C7 KO, but not Crry KO mice, also demonstrated significantly lower C1q levels compared to matched WT. In brain homogenates, C1q levels increased with age in both WT and APPNL-G-F mice. This robust and adaptable assay for quantification of mouse and rat C1q provides a vital tool for investigating the expression of C1q in rodent models of AD and other complement-driven pathologies

Complement receptor 1 is expressed on brain cells and in the human brain

Genome wide association studies (GWAS) have highlighted the importance of the complement cascade in pathogenesis of Alzheimer's disease (AD). Complement receptor 1 (CR1; CD35) is among the top GWAS hits. The long variant of CR1 is associated with increased risk for AD; however, roles of CR1 in brain health and disease are poorly understood. A critical confounder is that brain expression of CR1 is controversial; failure to demonstrate brain expression has provoked the suggestion that peripherally expressed CR1 influences AD risk. We took a multi‐pronged approach to establish whether CR1 is expressed in brain. Expression of CR1 at the protein and mRNA level was assessed in human microglial lines, induced pluripotent stem cell (iPSC)‐derived microglia from two sources and brain tissue from AD and control donors. CR1 protein was detected in microglial lines and iPSC‐derived microglia expressing different CR1 variants when immunostained with a validated panel of CR1‐specific antibodies; cell extracts were positive for CR1 protein and mRNA. CR1 protein was detected in control and AD brains, co‐localizing with astrocytes and microglia, and expression was significantly increased in AD compared to controls. CR1 mRNA expression was detected in all AD and control brain samples tested; expression was significantly increased in AD. The data unequivocally demonstrate that the CR1 transcript and protein are expressed in human microglia ex vivo and on microglia and astrocytes in situ in the human brain; the findings support the hypothesis that CR1 variants affect AD risk by directly impacting glial functions

Terminal complement pathway activation drives synaptic Loss in Alzheimer’s disease models

Complement is involved in developmental synaptic pruning and pathological synapse loss in Alzheimer’s disease. It is posited that C1 binding initiates complement activation on synapses; C3 fragments then tag them for microglial phagocytosis. However, the precise mechanisms of complement-mediated synaptic loss remain unclear, and the role of the lytic membrane attack complex (MAC) is unexplored. We here address several knowledge gaps: (i) is complement activated through to MAC at the synapse? (ii) does MAC contribute to synaptic loss? (iii) can MAC inhibition prevent synaptic loss? Novel methods were developed and optimised to quantify C1q, C3 fragments and MAC in total and regional brain homogenates and synaptoneurosomes from WT and AppNL−G−F Alzheimer’s disease model mouse brains at 3, 6, 9 and 12 months of age. The impact on synapse loss of systemic treatment with a MAC blocking antibody and gene knockout of a MAC component was assessed in Alzheimer’s disease model mice. A significant increase in C1q, C3 fragments and MAC was observed in AppNL−G−F mice compared to controls, increasing with age and severity. Administration of anti-C7 antibody to AppNL−G−F mice modulated synapse loss, reflected by the density of dendritic spines in the vicinity of plaques. Constitutive knockout of C6 significantly reduced synapse loss in 3xTg-AD mice. We demonstrate that complement dysregulation occurs in Alzheimer’s disease mice involving the activation (C1q; C3b/iC3b) and terminal (MAC) pathways in brain areas associated with pathology. Inhibition or ablation of MAC formation reduced synapse loss in two Alzheimer’s disease mouse models, demonstrating that MAC formation is a driver of synapse loss. We suggest that MAC directly damages synapses, analogous to neuromuscular junction destruction in myasthenia gravis

The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels: 4-Chloro-3-ethylphenol inhibits ORAI channels

BackgroundDepletion of the Ca2+ store by ryanodine receptor (RyR) agonists induces store‐operated Ca2+ entry (SOCE). 4‐Chloro‐3‐ethylphenol (4‐CEP) and 4‐chloro‐m‐cresol (4‐CmC) are RyR agonists commonly used as research tools and diagnostic reagents for malignant hyperthermia. Here, we investigated the effects of 4‐CEP and its analogues on SOCE.Experimental ApproachSOCE and ORAI1‐3 currents were recorded by Ca2+ imaging and whole‐cell patch recordings in rat L6 myoblasts and in HEK293 cells overexpressing STIM1/ORAI1‐3.Key Results4‐CEP induced a significant release of Ca2+ in rat L6 myoblasts, but inhibited SOCE. The inhibitory effect was concentration‐dependent and more potent than its analogues 4‐CmC and 4‐chlorophenol (4‐ClP). In the HEK293 T‐REx cells overexpressing STIM1/ORAI1‐3, 4‐CEP inhibited the ORAI1, ORAI2 and ORAI3 currents evoked by thapsigargin. The 2‐APB‐induced ORAI3 current was also blocked by 4‐CEP. This inhibitory effect was reversible and independent of the Ca2+ release. The two analogues, 4‐CmC and 4‐ClP, also inhibited the ORAI1‐3 channels. Excised patch and intracellular application of 4‐CEP demonstrated that the action site was located extracellularly. Moreover, 4‐CEP evoked STIM1 translocation and subplasmalemmal clustering through its Ca2+ store‐depleting effect via the activation of RyR, but no effect on STIM1 redistribution was observed in cells co‐expressing STIM1/ORAI1‐3.Conclusion and Implications4‐CEP not only acts as a RyR agonist to deplete the Ca2+ store and trigger STIM1 subplasmalemmal translocation and clustering, but also directly inhibits ORAI1‐3 channels. These findings demonstrate a novel pharmacological property for the chlorophenol derivatives that act as RyR agonists