Metformin Attenuates High Glucose‐Induced Coronary Vascular Endothelial Hyper Permeability Via Inhibition of Orai‐1 Mediated Store‐Operated Calcium Entry
Shamsideen AliDo DoanTerrence OjongH M SolomonErsever CorpuzThai HuynhMuhammad HaziqMohd ShahidMohammad R. SiddiquiMohammad NewazSuhail AkhtarZulfiqar AhmadMohammad Tauseef
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Increase in vascular endothelial permeability is an earliest pathological hallmark in diabetes mellitus, which progressively leads to cardiovascular disease. Stromal‐interaction molecule 1 (STIM1), upon sensing the depletion of calcium (Ca 2+ ) from the endoplasmic reticulum (ER) store, organizes as a puncta that triggers store‐operated Ca 2+ entry (SOCE) via plasmalemmal Ca 2+ ‐selective Orai1 channels in endothelial cells. Thus, signals to disrupt endothelial cell‐cell junctions’ integrity and increase in endothelial permeability. Recent studies show that the anti‐diabetic drug metformin confers vascular benefits beyond glycaemia control and reduced the high risk of cardiovascular events in pre‐diabetes patients. However, the precise pharmacological role of metformin and the mechanism to regulate SOCE‐induced endothelial hyper permeability response in coronary vasculature remain enigmatic in the pathogenesis of hyperglycemia. Here, we demonstrate a previously undetermined role of metformin in inhibiting the Orai1 mediated induction of SOCE and thereby preventing the disintegration of vascular endothelial (VE) cadherin in high‐glucose exposed coronary vascular endothelial cells (ECs). Our data showed that exposing the human coronary endothelial cell monolayer to high glucose media (25mM) for 48 hours increased the expression of Orai1, protracted SOCE and the increased in endothelial permeability. We found that SOCE mediated by Orai1 activated the Pyk2 in ECs. Intriguingly, we observed the tyrosine phosphorylation of vascular endothelial‐protein tyrosine phosphatase (VE‐PTP) at tyrosine (Y) 1981 residue downstream of the Pyk2 activation in high glucose milieu. Our results showed that metformin prevented the SOCE by decreasing the expression of Orai1 and thereby abrogated the Pyk2 mediated phosphorylation of VE‐PTP in high glucose condition. Thus, led to the stabilization and maintenance of VE‐cadherin at interendothelial junctions and strengthening the barrier function. Our data identify heretofore unprecedented signaling mechanism by which metformin via inhibiting SOCE, maintains VE‐cadherin integrity and henceforth decreased high glucose induced hyper permeability response in human coronary endothelial cells. Support or Funding Information This work is supported by the Department of Pharmaceutical Sciences, Chicago State University College of Pharmacy (CSU‐COP) research funding to Mohammad Tauseef.Keywords:
Orai1
Vascular permeability
Endothelial Dysfunction
The past five years have witnessed the discovery of the endoplasmic reticulum calcium (Ca(2+)) sensor STIM1 and the plasma membrane Ca(2+) channel Orai1 as the bona fide molecular components of the store-operated Ca(2+) entry (SOCE) and the Ca(2+) release-activated Ca(2+) current (I (CRAC)). It has been known for two decades that SOCE and I (CRAC) are required for lymphocyte activation as evidenced by severe immunodeficient phenotypes in patients lacking I (CRAC). In recent years however, studies have uncovered expression of STIM1 and Orai1 proteins in various tissues and described additional roles for these proteins in physiological functions and pathophysiological conditions. Here, we will summarize novel findings pertaining to the role of STIM1 and Orai1 in the vascular system and discuss their potential use as targets in the therapy of vascular disease.
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AbstractCalcium (Ca2+) entry into non-excitable cells is mainly carried by store-operated channels (SOCs), which serve essential functions ranging from regulation of transcription to cell growth. The best-characterised store-operated current, ICRAC, is the calcium release-activated calcium (CRAC) current initially discovered in T-lymphocytes and mast cells. The search for the molecular components of the CRAC channel lasted over 20 years. Recently STIM1 has been identified as the Ca2+ sensor in the endoplasmic reticulum (ER) that accumulates into punctae close to the plasma-membrane following store-depletion. The identification of STIM1 has been closely followed by the discovery of Orai1 as the CRAC channel pore in human T-cells. Upon punctae formation STIM1 activates Ca2+ influx via Orai1 channels. This review covers functional details concerning the activation cascade of the STIM1 / Orai1 complex from ER Ca2+ sensing to Ca2+ influx through Orai1. Furthermore, functional domains within STIM1 and Orai1 in comparison to their structural homologs STIM2 as well as Orai2 and Orai3, respectively, are displayed together with recent findings on the pore architecture and selectivity filter of Orai channels. A broad tissue expression of STIM and Orai proteins together with substantial effects in STIM1 / Orai1 knock-out mice suggests an essential physiological role in store-operated Ca2+ signaling in human health and disease.
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In non-excitable cells, agonist-induced depletion of intracellular Ca(2+) stores triggers Ca(2+) influx via a process termed store-operated Ca(2+) entry (SOCE). In T-lymphocytes, stromal interaction molecule 1 (STIM1) acts as the intra-store Ca(2+) sensor and Orai1 functions as the Ca(2+)-permeable SOCE channel activated by STIM1 following store depletion. Two functionally distinct Ca(2+) entry pathways exist in skeletal muscle; one activated by store depletion (SOCE) and a second by sustained/repetitive depolarization that does not require store depletion (excitation-coupled Ca(2+) entry, ECCE). However, the role of STIM1 and Orai1 in coordinating SOCE and ECCE activity in skeletal muscle and whether these two Ca(2+) entry pathways represent distinct molecular entities or two different activation mechanisms of the same channel complex is unknown. Here we address these issues using siRNA-mediated STIM1 knockdown, dominant-negative Orai1, and permeation-defective Orai1 to determine the role of STIM1 and Orai1 in store-operated and excitation-coupled Ca(2+) entry in skeletal myotubes. SOCE and ECCE activity were quantified from both intracellular Ca(2+) measurements and Mn(2+) quench assays. We found that STIM1 siRNA reduced STIM1 protein by more than 90% and abolished SOCE activity, while expression of siRNA-resistant hSTIM1 fully restored SOCE. SOCE was also abolished by dominant-negative Orai1 (E106Q) and markedly reduced by expression of a permeation-defective Orai1 (E190Q). In contrast, ECCE was unaffected by STIM1 knockdown, E106Q expression or E190Q expression. These results are the first to demonstrate that SOCE in skeletal muscle requires both STIM1 and Orai1 and that SOCE and ECCE represent two distinct molecular entities.
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Trimeric intracellular cation (TRIC) channels have been proposed to modulate Ca2+ release from the endoplasmic reticulum (ER) and determine oscillatory Ca2+ signals. Here, we report that TRIC-A–mediated amplitude and frequency modulation of ryanodine receptor 2 (RyR2)-mediated Ca2+ oscillations and inositol 1,4,5-triphosphate receptor (IP3R)-induced cytosolic signals is based on attenuating store-operated Ca2+ entry (SOCE). Further, TRIC-A–dependent delay in ER Ca2+ store refilling contributes to shaping the pattern of Ca2+ oscillations. Upon ER Ca2+ depletion, TRIC-A clusters with stromal interaction molecule 1 (STIM1) and Ca2+-release–activated Ca2+ channel 1 (Orai1) within ER–plasma membrane (PM) junctions and impairs assembly of the STIM1/Orai1 complex, causing a decrease in Orai1-mediated Ca2+ current and SOCE. Together, our findings demonstrate that TRIC-A is a negative regulator of STIM1/Orai1 function. Thus, aberrant SOCE could contribute to muscle disorders associated with loss of TRIC-A.
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Endoplasmic reticulum calcium(Ca2+) sensor stromal interaction molecule 1(STIM1) and plasma membrane Ca2+ channel Orai1 are the bona fide molecular components of the store-operated Ca2+ entry(SOCE) and the Ca2+ release-activated Ca2+ current(ICRAC).We have uncovered the protein expression of STIM1 and Orai1 in various tissues and described many kinds of roles for these proteins under physiological and pathophysiological conditions.Here,we summarize the novel findings pertaining to the role of STIM1 and Orai1 in the vascular system and discuss their potential use as targets in the treatment of vascular diseases.
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Stromal interaction molecule 1 (STIM1) and Orai1 have been identified as crucial elements of the store-operated Ca 2+ entry (SOCE) pathway, but the mechanism of their functional interaction remains controversial. It is now well established that, upon depletion of the stores, both molecules can accumulate and colocalize in specific areas (puncta) where the endoplasmic reticulum comes in close proximity to the plasma membrane. Some models propose a direct interaction between STIM1 and Orai1 as the most straightforward mechanism for signal transduction from the stores to the plasma membrane. To test some of the predictions of a conformational coupling model, we assessed how tight the relationships are between STIM1 and Orai1 expression, puncta formation, and SOCE activation. Here we present evidence that STIM1 accumulates in puncta equally well in the presence or absence of Orai1 expression, that STIM1 accumulation is not sufficient for Orai1 accumulation in the same areas, and that normal Ca 2+ release-activated Ca 2+ current ( I CRAC ) can be activated in STIM1-deficient cells. These data challenge the idea of direct conformational coupling between STIM1 and Orai1 as a viable mechanism of puncta formation and SOCE activation and uncover greater complexity in their relationship, which may require additional intermediate elements.
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Store-operated Ca(2+) entry (SOCE) is an ubiquitous mechanism for Ca(2+) entry in eukaryotic cells. This route for Ca(2+) influx is regulated by the filling state of the intracellular Ca(2+) stores communicated to the plasma membrane channels by the proteins of the Stromal Interaction Molecule (STIM) family, STIM1, and STIM2. Store-dependent, STIM1-modulated, channels include the Ca(2+) release-activated Ca(2+) channels, comprised of subunits of Orai proteins, as well as the store-operated Ca(2+) (SOC) channels, involving Orai1, and members of the canonical transient receptor potential family of proteins. Recent studies have revealed the expression of splice variants of STIM1, STIM2, and Orai1 in different cell types. While certain variants are ubiquitously expressed, others, such as STIM1L, show a more restricted expression. The splice variants for STIM and Orai1 proteins exhibit significant functional differences and reveal that alternative splicing enhance the functional diversity of STIM1, STIM2, and Orai1 genes to modulate the dynamics of Ca(2+) signals.
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Store-operated Ca2+ entry (SOCE) is likely the most common mode of regulated influx of Ca2+ into cells. However, only a limited number of pharmacological agents have been shown to modulate this process. 2-Aminoethyldiphenyl borate (2-APB) is a widely used experimental tool that activates and then inhibits SOCE and the underlying calcium release-activated Ca2+ current (I CRAC). The mechanism by which depleted stores activates SOCE involves complex cellular movements of an endoplasmic reticulum Ca2+ sensor, STIM1, which redistributes to puncta near the plasma membrane and, in some manner, activates plasma membrane channels comprising Orai1, -2, and -3 subunits. We show here that 2-APB blocks puncta formation of fluorescently tagged STIM1 in HEK293 cells. Accordingly, 2-APB also inhibited SOCE and I(CRAC)-like currents in cells co-expressing STIM1 with the CRAC channel subunit, Orai1, with similar potency. However, 2-APB inhibited STIM1 puncta formation less well in cells co-expressing Orai1, indicating that the inhibitory effects of 2-APB are not solely dependent upon STIM1 reversal. Further, 2-APB only partially inhibited SOCE and current in cells co-expressing STIM1 and Orai2 and activated sustained currents in HEK293 cells expressing Orai3 and STIM1. Interestingly, the Orai3-dependent currents activated by 2-APB showed large outward currents at potentials greater than +50 mV. Finally, Orai3, and to a lesser extent Orai1, could be directly activated by 2-APB, independently of internal Ca2+ stores and STIM1. These data reveal novel and complex actions of 2-APB effects on SOCE that can be attributed to effects on both STIM1 as well as Orai channel subunits.
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