2-A minoethyldiphenyl borate(2-APB)is the most commonly used pharmacological agent in the study of calcium release-activated channels(CRACa);however,its inhibitory mechanism to CRACs remains unclear.To address this is...2-A minoethyldiphenyl borate(2-APB)is the most commonly used pharmacological agent in the study of calcium release-activated channels(CRACa);however,its inhibitory mechanism to CRACs remains unclear.To address this issue,we systematically employed confocal imaging,dual-wavelength excitation photometry and FRET to examine the effects of 2-APB on the dynamic activities and function of STIM1 and Orail,two key components of CRACs.Imaging results support that there are two signaling pathways(Orail-independent and Orail-dependent)for the formation of STM1 puncta.2 APB could dose dependently block Orail-independent but not Oril-dependent STIM1 puncta formation,despite its obvious inhibition effect on store-opented Ca^(2+)entry(SOCE).In addition,we found that although 2-APB could not visibly alter near plasma membrane CAD-eYFP localization,it could completely block CAD-YFP-induced constitutive Ca^(2+)entry and promnote the interaction between Orail and CAD by FRET mea-surements.Therefore,we proposed that inhibitory action of 2-APB on SOCE might attribute to its direct inhbitory effects on Orail channel itself,but not the interference on puncta formation between STIM1 and Orail.展开更多
目的:骨肉瘤是一种常见的恶性骨肿瘤,恶性程度高,往往在早期就会发生远隔器官的转移,从而导致骨肉瘤的预后非常差。Orai1是一类定位于细胞膜,介导钙离子内流的受体依赖性钙通道蛋白。大量研究发现钙通道蛋白Orai1过表达于多种肿瘤细胞,...目的:骨肉瘤是一种常见的恶性骨肿瘤,恶性程度高,往往在早期就会发生远隔器官的转移,从而导致骨肉瘤的预后非常差。Orai1是一类定位于细胞膜,介导钙离子内流的受体依赖性钙通道蛋白。大量研究发现钙通道蛋白Orai1过表达于多种肿瘤细胞,并对维持肿瘤细胞粘附、侵袭、迁移等恶性表型有非常重要的作用。然而,钙通道蛋白Orai1是否参与了骨肉瘤的转移过程,目前未见相关报道。本研究的目的是探究钙通道蛋白Orai1是否在骨肉瘤转移过程中的发挥作用。方法:利用合成的靶向Orai1的小干扰RNA(Orai1 si RNA)片段,转染至人骨肉瘤细胞系Saos-2细胞。在Saos-2细胞中抑制Orai1的表达。采用细胞黏附实验、细胞划痕实验和细胞Transwell实验检测骨肉瘤细胞的黏附、迁移及侵袭等肿瘤细胞转移能力;Western-blot实验检测Saos-2细胞的中黏着斑激酶(FAK)和桩蛋白(Paxillin)的表达水平和磷酸化水平。结果:靶向Orai1 si RNA瞬时转染至骨肉瘤细胞系Saos-2细胞后,Saos-2细胞中Orai1蛋白表达水平和m RNA转录水平均显著下降。并且,在Saos-2细胞中抑制Orai1表达后,Saos-2细胞的黏附能力、迁移能力、及侵袭能力均显著下降。进一步研究发现,在Saos-2细胞中抑制Orai1表达后,Saos-2细胞的FAK和Paxillin磷酸化水平明显下降。结论:Orai1可以促进骨肉瘤细胞的黏附、迁移和侵袭,增加黏着斑的形成,从而促进骨肉瘤的转移。因此,深入研究钙通道蛋白Orai1调控骨肉瘤转移的分子机制,可为骨肉瘤转移的治疗提供新的新方向和新策略。展开更多
The past five years have witnessed the discovery of the endoplasmic reticulum calcium(Ca2+) sensor STIM1 and the plasma membrane Ca2+channel Orai1 as the bona fide molecular components of the store-operated Ca2+ entry...The past five years have witnessed the discovery of the endoplasmic reticulum calcium(Ca2+) sensor STIM1 and the plasma membrane Ca2+channel Orai1 as the bona fide molecular components of the store-operated Ca2+ entry(SOCE) and the Ca2+ release-activated Ca2+current(I CRAC) .It has been known for two decades that SOCE and ICRAC are required for lymphocyte activation as evidenced by severe immunodeficient phenotypes in patients lacking ICRAC.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.展开更多
Calcium is a key regulator of many physiological processes that are perturbed in cancer, such as migration, proliferation and apoptosis. The proteins STIM and Orai mediate store-operated calcium entry(SOCE), the main ...Calcium is a key regulator of many physiological processes that are perturbed in cancer, such as migration, proliferation and apoptosis. The proteins STIM and Orai mediate store-operated calcium entry(SOCE), the main pathway for calcium entry in non-excitable cells. Changes in the expression and function of STIM and Orai have been found in a range of cancer types and thus implicated in disease progression. Here we discuss the role of STIM, Orai and the SOCE pathway in the progression of melanoma and explore how the heterogeneous nature of melanoma may explain the lack of consensus in the field regarding the role of SOCE in the progression of this disease.展开更多
基金supported by the National Natural Sciences Foundation of China(Grant Nos.31371217 and 30871311).
文摘2-A minoethyldiphenyl borate(2-APB)is the most commonly used pharmacological agent in the study of calcium release-activated channels(CRACa);however,its inhibitory mechanism to CRACs remains unclear.To address this issue,we systematically employed confocal imaging,dual-wavelength excitation photometry and FRET to examine the effects of 2-APB on the dynamic activities and function of STIM1 and Orail,two key components of CRACs.Imaging results support that there are two signaling pathways(Orail-independent and Orail-dependent)for the formation of STM1 puncta.2 APB could dose dependently block Orail-independent but not Oril-dependent STIM1 puncta formation,despite its obvious inhibition effect on store-opented Ca^(2+)entry(SOCE).In addition,we found that although 2-APB could not visibly alter near plasma membrane CAD-eYFP localization,it could completely block CAD-YFP-induced constitutive Ca^(2+)entry and promnote the interaction between Orail and CAD by FRET mea-surements.Therefore,we proposed that inhibitory action of 2-APB on SOCE might attribute to its direct inhbitory effects on Orail channel itself,but not the interference on puncta formation between STIM1 and Orail.
文摘目的:骨肉瘤是一种常见的恶性骨肿瘤,恶性程度高,往往在早期就会发生远隔器官的转移,从而导致骨肉瘤的预后非常差。Orai1是一类定位于细胞膜,介导钙离子内流的受体依赖性钙通道蛋白。大量研究发现钙通道蛋白Orai1过表达于多种肿瘤细胞,并对维持肿瘤细胞粘附、侵袭、迁移等恶性表型有非常重要的作用。然而,钙通道蛋白Orai1是否参与了骨肉瘤的转移过程,目前未见相关报道。本研究的目的是探究钙通道蛋白Orai1是否在骨肉瘤转移过程中的发挥作用。方法:利用合成的靶向Orai1的小干扰RNA(Orai1 si RNA)片段,转染至人骨肉瘤细胞系Saos-2细胞。在Saos-2细胞中抑制Orai1的表达。采用细胞黏附实验、细胞划痕实验和细胞Transwell实验检测骨肉瘤细胞的黏附、迁移及侵袭等肿瘤细胞转移能力;Western-blot实验检测Saos-2细胞的中黏着斑激酶(FAK)和桩蛋白(Paxillin)的表达水平和磷酸化水平。结果:靶向Orai1 si RNA瞬时转染至骨肉瘤细胞系Saos-2细胞后,Saos-2细胞中Orai1蛋白表达水平和m RNA转录水平均显著下降。并且,在Saos-2细胞中抑制Orai1表达后,Saos-2细胞的黏附能力、迁移能力、及侵袭能力均显著下降。进一步研究发现,在Saos-2细胞中抑制Orai1表达后,Saos-2细胞的FAK和Paxillin磷酸化水平明显下降。结论:Orai1可以促进骨肉瘤细胞的黏附、迁移和侵袭,增加黏着斑的形成,从而促进骨肉瘤的转移。因此,深入研究钙通道蛋白Orai1调控骨肉瘤转移的分子机制,可为骨肉瘤转移的治疗提供新的新方向和新策略。
基金supported by the National Institutes of Health(Grant No. 5R01HL097111)to Mohamed Trebak
文摘The past five years have witnessed the discovery of the endoplasmic reticulum calcium(Ca2+) sensor STIM1 and the plasma membrane Ca2+channel Orai1 as the bona fide molecular components of the store-operated Ca2+ entry(SOCE) and the Ca2+ release-activated Ca2+current(I CRAC) .It has been known for two decades that SOCE and ICRAC are required for lymphocyte activation as evidenced by severe immunodeficient phenotypes in patients lacking ICRAC.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.
文摘Calcium is a key regulator of many physiological processes that are perturbed in cancer, such as migration, proliferation and apoptosis. The proteins STIM and Orai mediate store-operated calcium entry(SOCE), the main pathway for calcium entry in non-excitable cells. Changes in the expression and function of STIM and Orai have been found in a range of cancer types and thus implicated in disease progression. Here we discuss the role of STIM, Orai and the SOCE pathway in the progression of melanoma and explore how the heterogeneous nature of melanoma may explain the lack of consensus in the field regarding the role of SOCE in the progression of this disease.
