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Cloning and Functional Analysis of calcineurin Subunits A and B in Development and Fecundity of Nilaparvata lugens(Stål)

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Rice science 2022年第2期29卷 143-154页

作者： WANG Weixia ZHU Tingheng WAN Pinjun WEI Qi HE Jiachun LAI Fengxiang FU Qiang State Key Laboratory of Rice Biology China National Rice Research InstituteHangzhou 310006China College of Biotechnology and Bioengineering Zhejiang University of TechnologyHangzhou 310014China

Serine/threonine phosphatase calcineurin(CN)is a unique but confounding calcium/calmodulin-mediated enzyme,which is composed of a catalytic subunit A(CNA)and a regulatory subunit B(CNB).We cloned six transcripts for CNA named from NlCNA-X1 to NlCNA-X6,one CNB named NlCNB1 and one CNB homologous gene NlCNBH1 from Nilaparvata lugens.All of them are constitutively transcripted in various tissues and developmental stages.The primary structure of the six isoforms showed obvious differences in the length and composition of the amino acid sequence between the two binding domains of Ca^(2+)/calmodulin(CaM)and CNB.Ca^(2+)-binding EF-hand motifs were found in NlCNB1 and NlCNBH1.The specific gene silencing of NlCNA,NlCNB1 and NlCNBH1 respectively by RNAi resulted in drastical reduction in survival rate,female weight,eclosion rate and fecundity of N.lugens.These results showed that NlCNA,NlCNB1 and NlCNBH1 were required for N.lugens growth and reproduction.The negative effects of NlCNB1 silence on nymph mortality(97%),molting malformation(90%)and female sterile(50%)were more serious than those of NlCNA or NlCNBH1.qRT-PCR and enzyme-linked immunosorbent assay(ELISA)analyses indicated that the nymphs with silenced NlCNA,NlCNB1 or NlCNBH1 showed impaired hormone and energy metabolism.In nymphs,the contents of 20-hydroxyecdysone(20E)after NlCNB1 RNAi and phenoloxidase after NlCNA RNAi were particularly decreased.These results suggested that NlCNA is involved in immunity of N.lugens by regulation of phenoloxidase,while NlCNB1 may control the growth and development of N.lugens by 20E signaling pathway in addition to interact with CNA.Injection of 70 ng/μL dsNlCNB1 resulted in 77.0%down regulation of NlCNB1,and the nymph mortality was up to 57.9%at 10 d after injection.Therefore,NlCNB1 could be a potential candidate target used for strategy design in control of N.lugens.Our results revealed the importance of CN in the regulation of the growth and development of N.lugens,which provided a basis for further study of the molecular mech

关键词： Nilaparvata lugens calcineurin calcineurin catalytic subunit calcineurin regulatory subunit 20-hydroxyecdysone phenoloxidase

calcineurin is involved in cardioprotection induced by ischemic postconditioning through attenuating endoplasmic reticulum stress

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Chinese Medical Journal 2011年第20期124卷 3334-3340页

作者： CHEN Yi-hong WU Xu-dong YAO Shu-tong SUN Seng LIU Xiu-hua Department of Pathophysiology Chinese People＇s LiberationArmy General Hospital Beijing 100853 China

Background Ischemic postconditioning （I-postC） is a newly discovered and more amenable cardioprotective strategy capable of protecting the myocardium from ischemia/reperfusion （I/R） injury. Endoplasmic reticulum （ER） is a principal site for secretary protein synthesis and calcium storage. Myocardial I/R causes ER stress and emerging studies suggest that the cardioprotection has been linked to the modulation of ER stress. The aim of the present study was to determine whether cardioprotection of I-postC involves reduction in ER stress through calcineurin pathway. Methods In the in vivo model of rat myocardial I/R, myocardial infarct size was measured by triphenyltetrazolium chloride （TTC） staining and apoptosis was detected using terminal eoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling （TUNEL） assay. Expression of calreticulin, C/EBP homologous protein （CHOP）, caspase-12, and activation of caspase-12 in myocardium were detected by Western blotting. The activity and expression of calcineurin in myocardium were also detected. Results I-postC protected the I/R heart against apoptosis, myocardial infarction, and leakage of lactate dehydrogenase （LDH） and creatine kinase-MB （CK-MB）. I-postC suppressed I/R-induced ER stress, as shown by a decrease in the expression of calreticulin and CHOP, and caspase-12 activation. I-postC downregulated calcineurin activation in myocardium subjected to I/R. Conclusion I-postC protects myocardium from I/R injury by suppressing ER stress and calcineurin pathways are not associated with the I-postC-induced suppression of ER stress-related apoptosis.

关键词： ischemic postconditioning endoplasmic reticulum stress calcineurin rats myocardial infarction

calcineurin/NFAT信号通路上调5型磷酸二酯酶的表达及介导内皮素-1诱导的肺动脉平滑肌细胞增殖

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南方医科大学学报 2013年第1期33卷 26-29页

作者：卢家美王小闯谢新明韩冬李少军李满祥西安交通大学医学院第二附属医院呼吸病研究室陕西西安710004 西安交通大学医学院第二附属医院呼吸内科陕西西安710004 西安交通大学医学院第二附属医院急诊医学科陕西西安710004

目的探讨calcineurin/NFAT信号通路是否介导内皮素-1(ET-1)诱导的5型磷酸二酯酶(PDE5)的表达及肺动脉平滑肌细胞(PASMCs)的增殖;同时验证calcineurin抑制剂环孢素A及PDE5抑制剂西地那非能否抑制ET-1刺激的PASMCs增殖。方法以ET-1刺激PAS... 详细信息

目的探讨calcineurin/NFAT信号通路是否介导内皮素-1(ET-1)诱导的5型磷酸二酯酶(PDE5)的表达及肺动脉平滑肌细胞(PASMCs)的增殖;同时验证calcineurin抑制剂环孢素A及PDE5抑制剂西地那非能否抑制ET-1刺激的PASMCs增殖。方法以ET-1刺激PASMCs增殖,分别于ET-1刺激前给予环孢素A或西地那非抑制calcineurin或PDE5的活性。采用calcineurin活性检测试剂盒检测其活性,免疫印迹法检测PDE5的表达,酶联免疫吸附法检测cGMP的含量,3H-TdR渗入法检测PASMCs的增殖。结果 ET-1可激活原代培养的PASMCs中calcineurin的活性,上调PDE5表达,降低cGMP的水平。calcineurin特异性抑制剂环孢素A可阻断ET-1的上述作用;抑制PDE5的活性可逆转ET-1导致的cGMP含量减少。而环孢素A及西地那非可分别抑制ET-1刺激的PASMCs增殖。结论 ET-1可通过激活calcineurin/NFAT信号通路介导ET-1诱发的PDE5表达,进而降低cGMP含量,引起PASMCs增殖;而抑制calcineurin或PDE5可提高cGMP水平,抑制ET-1诱导的PASMCs增殖。

关键词： calcineurin NFAT信号通路 5型磷酸二酯酶内皮素-1 细胞增殖肺动脉平滑肌细胞

Elevation of gene expression of calcineurin,calmodulin and calsyntenin in oxidative stress induced PC12 cells

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Genes & Diseases 2021年第1期8卷 87-93页

作者： P.Aravind Sarojini R.Bulbule N.Hemalatha R.L.Babu K.S.Devaraju Department of Biochemistry Karnatak UniversityPavate NagarDharwad580003India Department of Biochemistry and Nutrition CFTRIV V MohollaMysore570020India Department of Bioinformatics and Biotechnology Akkamahadevi Women’s UniversityVijayapuraKarnataka586108India

In normal physiological conditions,reactive oxygen and nitrogen species are used as important signaling molecules in the cell.However,in excess it causes the disruption of cell resulting in their death.Oxidative stress causes influx in intracellular calcium levels leading to higher concentrations of calcium in the cell.This accelerated calcium affects both the mitochondria and nuclei leading to excitotoxicity in neurons.Intracellular calcium levels are controlled by voltage dependent calcium channels located in the plasma membrane,calcium stores like endoplasmic/sarcoplasmic reticulum and majorly by calcium binding proteins.Our study was aimed at analyzing the gene expression of major calcium binding proteins namely calcineurin,calmodulin,calreticulin,synaptotagamin and calsyntenin in stress induced PC 12 cells.Rotenone(1 mM),Peroxynitrite(10 mM),H_(2)O_(2)(100 mM)and High glucose(33 mM)were used to induce oxidative stress in PC12 cells.Results obtained from the study suggest that calcineurin,calmodulin and calsyntenin gene expression were enhanced compared to the control due to oxidative stress.However,synaptotagmin and calreticulin gene expression were down regulated.Further,Akt protein expression(stress marker)was enhanced in PC12 cells with all other stress inducers except in hyperglycemic condition.

关键词： Akt calcineurin Calmodulin Oxidative stress Peroxynitrite

Role of the Ca^2＋-calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

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Chinese Medical Journal 2018年第3期131卷 330-338页

作者： Xiu-Ping Xu Yong-Ming Yao Guang-Ju Zhao Zong-Sheng Wu Jun-Cong Li Yun-Long Jiang Zhong-Qiu Lu Guang-Liang Hong Emergency Center The First Affiliated Hospital of Wenzhou Medical University Wenzhou Zhejiang 325000 China Department of Microbiology and Immunology Burns Institute First Hospital Affiliated to the Chinese PLA General Hospital Beijing 100048 China

Background：Mitofusin-2 （MFN2）,a well-known mitochondrial fusion protein,has been shown to participate in innate immunity,but its role in mediating adaptive immunity remains poorly characterized.In this study,we explored the potential role of MFN2 in mediating the immune function of T lymphocytes.Methods：We manipulated MFN2 gone expression in Jurkat cells via lentiviral transduction of MFN2 small interfering RNA （siRNA） or full-length MFN2.After transduction,the immune response and its underlying mechanism were determined in Jurkat cells.One-way analysis of variance and Student＇s t-test were performed to determine the statistical significance between the groups.Results：Overexpression of MFN2 enhanced the immune response of T lymphocytes by upregulating Ca2＋（359.280 ± 10.130 vs.266.940 ± 10.170,P =0.000）,calcineurin （0.513 ± 0.014 vs.0.403 ± 0.020 nmol/L,P =0.024）,and nuclear factor of activated T cells （NFATs） activation （1.040 ± 0.086 vs.0.700 ± 0.115,P =0.005）,whereas depletion of MFN2 impaired the immune function ofT lymphocytes by downregulating Ca2＋（141.140 ± 14.670 vs.267.060 ± 9.230,P =0.000）,calcineurin （0.054 ± 0.030 nmol/L vs.0.404 ± 0.063 nmol/L,P =0.000）,and NFAT activation （0.500 ± 0.025 vs.0.720 ± 0.061,P =0.012）.Furthermore,upregulated calcineurin partially reversed the negative effects ofMFN2 siRNA on T cell-mediated immunity evidenced by elevations in T cell proliferation （1.120 ± 0.048 vs.0.580 ± 0.078,P =0.040）,interleukin-2 （IL-2） production （473.300 ± 24.100 vs.175.330 ± 12.900 pg/ml,P =0.000）,and the interferon-γ/IL-4 ratio （3.080 ± 0.156 vs.0.953 ± 0.093,P =0.000）.Meanwhile,calcineurin activity inhibitor depleted the positive effects of overexpressed MFN2 on T cells function.Conclusions：Our findings suggest that MFN2 may regulate T cell immune functions primarily through the Ca2＋-calcineurin-NFAT pathway.MFN2 may represent a potential therapeutic target for T cell immune dysfunction-related diseases.

关键词： calcineurin Calcium Jurkat Cells Mitofusin-2 Protein Nuclear Factor of Activated T cells

Taking out the garbage:cathepsin D and calcineurin in neurodegeneration

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Neural Regeneration Research 2017年第11期12卷 1776-1779页

作者： Andreas Aufschnaiter Verena Kohler Sabrina Büttner Institute of Molecular Biosciences University of GrazHumboldtstraBe 508010 GrazAustria Department of Molecular Biosciences The Wenner-Gren InstituteStockholm UniversitySvante Arrheniusvag 20C106 91 StockholmSweden

Cellular homeostasis requires a tightly controlled balance between protein synthesis, folding and degradation. Especially long-lived, post-mitotic cells such as neurons depend on an efficient proteostasis system to maintain cellular health over decades. Thus, a functional decline of processes contributing to protein degradation such as autophagy and general lysosomal proteolytic capacity is connected to several age-associated neurodegenerative disorders, including Parkinson＇s, Alzheimer＇s and Huntington＇s diseases. These so called proteinopathies are characterized by the accumulation and misfolding of distinct proteins, subsequently driving cellular demise. We recently linked efficient lysosomal protein breakdown via the protease cathep- sin D to the Ca2＋/calmodulin-dependent phosphatase calcineurin. In a yeast model for Parkinson＇s disease, functional calcineurin was required for proper trafficking of cathepsin D to the lysosome and for recycling of its endosomal sorting receptor to allow further rounds of shuttling. Here, we discuss these findings in relation to present knowledge about the involvement of cathepsin D in proteinopathies in general and a possible connection between this protease, calcineurin signalling and endosomal sorting in particular. As dysregulation of Ca2＋ homeostasis as well as lysosomal impairment is connected to a plethora of neurode- generative disorders, this novel interplay might very well impact pathologies beyond Parkinson＇s disease.

关键词： neurodegeneration Parkinson’s disease α-synuclein cathepsin D calcineurin retromer yeast lysosome endosomal sorting

calcineurin和TRPC6参与ET-1诱导的肺动脉平滑肌细胞增殖

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南京医科大学学报（自然科学版） 2013年第9期33卷 1191-1195页

作者：王小闯李满祥党晓燕李萍彭卓高彦霞古长维西安交通大学医学院第二附属医院急诊医学科陕西西安710004 西安交通大学医学院第二附属医院呼吸科陕西西安710004 西安交通大学医学院第二附属医院呼吸病研究室陕西西安710004

目的:探讨calcineurin和瞬时感受器电位6(TRPC6)的相互作用以及对内皮素-1(ET-1)诱导的肺动脉平滑肌细胞增殖的影响。方法:以ET-1刺激肺动脉平滑肌细胞(PASMCs)检测calcineurin活性以及TRPC6的表达;分别于ET-1刺激前给予calcineurin特... 详细信息

目的:探讨calcineurin和瞬时感受器电位6(TRPC6)的相互作用以及对内皮素-1(ET-1)诱导的肺动脉平滑肌细胞增殖的影响。方法:以ET-1刺激肺动脉平滑肌细胞(PASMCs)检测calcineurin活性以及TRPC6的表达;分别于ET-1刺激前给予calcineurin特异性抑制剂环孢素A(CsA)和TRPC6 siRNA处理,测定calcineurin活性、TRPC6的表达以及细胞的增殖情况;采用calcineurin活性检测试剂盒检测其活性,Western blot和RT-PCR检测TRPC6的表达,MTT法检测PASMCs的增殖。结果:ET-1可激活原代培养的PASMCs中calcineurin的活性,上调TRPC6的表达;CsA和TRPC6 siRNA可以分别降低TRPC6表达和calcineurin活性;也可以抑制ET-1刺激的PASMCs增殖。结论:calcineurin与TRPC6参与ET-1诱导的肺动脉平滑肌细胞增殖并相互影响。

关键词： calcineurin NFAT信号通路 TRPC6 ET-1 肺动脉平滑肌细胞

Differentiation of expression profi les of two calcineurin subunit genes in chicken skeletal muscles during early postnatal growth depending on anatomical location of muscles and breed

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Journal of Integrative Agriculture 2016年第5期15卷 1085-1094页

作者： SHAN Yan-ju XU Wen-juan SHU Jing-ting ZHANG Ming SONG Wei-tao TAO Zhi-yun ZHU Chun-hong LI Hui-fang Key Laboratory for Poultry Genetics and Breeding of Jiangsu Province Institute of Poultry Science Chinese Academy of Agricultural Science

calcineurin（Cn or CaN） is implicated in the control of skeletal muscle fiber phenotype and hypertrophy. However, little information is available concerning the expression of Cn in chickens. In the present study, the expression of two Cn subunit genes（Cn Aα and Cn B1） was quantified by q PCR in the lateral gastrocnemius（LG, mainly composing of red fast-twitch myofibers）, the soleus（mainly composing of red slow-twitch myofibers） and the extensor digitorum longus（EDL, mainly composing of white fast-twitch myofibers） from Qingyuan partridge chickens（QY, slow-growing chicken breed） and Recessive White chickens（RW, fast-growing chicken breed） on different days（1, 8, 22, 36, 50 and 64 days post-hatching）. Although Cn Aα and Cn B1 gene expressions were variable with different trends in different skeletal muscles in the two chicken breeds during postnatal growth, it is highly muscle phenotype and breed specific. In general, the levels of Cn Aα and Cn B1 gene expressions of the soleus were lower than those of EDL and LG in both chicken breeds at the same stages. Compared between the two chicken breeds, the levels of Cn Aα gene expression of the three skeletal muscles in QY chickens were higher than those in RW chickens on days 1 and 22. However, on day 64, the levels of both Cn Aα and Cn B1 gene expressions of the three skeletal muscles were lower in QY chickens than those in RW chickens. Correlation analysis of the levels of Cn Aα and Cn B1 gene expressions of the same skeletal muscle showed that there were positive correlations for all three skeletal muscle tissues in two chicken breeds. These results provide some valuable clues to understand the role of Cn in the development of chicken skeletal muscles, with a function that may be related to meat quality.

关键词： calcineurin chicken skeletal muscle expression

calcineurin,NFAT2,COX-2和VEGF-A在体外培养正常黑素细胞和不同黑素瘤细胞系中的表达

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中国皮肤性病学杂志 2012年第8期26卷 667-669页

作者：徐玉荣廖文俊蔡越简强任婧刘启方第四军医大学西京皮肤医院陕西西安710032 第四军医大学航空航天生理学教研室陕西西安710032

目的检测calcineurin/NFAT信号途径中calcineurin,NFAT2,COX-2和VEGF-A分子在正常人黑素细胞和不同黑素瘤细胞系中的表达,初步探讨它们与黑素瘤发生、发展的关系。方法选取正常人原代黑素细胞(MC)及来源于原位(WM793B)、侵袭性(LIBR)及... 详细信息

目的检测calcineurin/NFAT信号途径中calcineurin,NFAT2,COX-2和VEGF-A分子在正常人黑素细胞和不同黑素瘤细胞系中的表达,初步探讨它们与黑素瘤发生、发展的关系。方法选取正常人原代黑素细胞(MC)及来源于原位(WM793B)、侵袭性(LIBR)及转移性(SK-MEL-5)的黑素瘤细胞系,采用RT-PCR,Westernblot检测四种细胞中calcineurin,NFAT2,COX-2和VEGF-A的表达情况。结果与正常人黑素细胞相比,在不同黑素瘤细胞系中calcineurin,NFAT2,COX-2和VEGF-A4个分子明显高表达。结论 calcineurin/NFAT信号途径可能与黑素瘤的发生有关。

关键词：黑素细胞黑素瘤 calcineurin NFAT2 COX-2 VEGF-A