Cx43及其磷酸化蛋白在帕金森病细胞表达
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摘要
目的探讨缝隙连接蛋白43(Cx43)及其磷酸化蛋白在过表达α-突触核蛋白(α-Syn)的帕金森病细胞模型表达及其意义。方法分别在SH-SY5Y细胞中过表达空载(空载组)、野生型α-Syn(WTα-Syn组)以及人突变型A53Tα-Syn(A53Tα-Syn组),应用Western Blot技术检测各组Cx43蛋白及磷酸化Cx43蛋白(p-Cx43)的表达。结果与空载组相比较,WTα-Syn组以及A53Tα-Syn组的细胞内Cx43蛋白表达增加(F=6.37,q=4.04、5.76,P<0.05),p-Cx43/Cx43升高,差异有显著性(F=23.43,q=5.73、9.65,P<0.01);与WTα-Syn组相比,A53Tα-Syn组的细胞内p-Cx43/Cx43升高(q=3.61,P<0.05)。结论 Cx43及p-Cx43蛋白表达水平的改变可能参与了帕金森病的发病过程。
Objective To investigate the expression and significance of connexin 43(Cx43) and its phosphorylated form(p-Cx43) in Parkinson disease(PD) cell model with overexpression of α-Synuclein(α-Syn). Methods Overexpression of empty vector, wild-type α-Syn, and human mutant A53 T α-Syn was performed in SH-SY5 Y cells(named empty vector group, WT α-Syn group, and A53 T α-Syn group, respectively). Western Blot was used to determine the expression of Cx43 and p-Cx43 proteins. Results Compared with the empty vector group, the WT α-Syn group and A53 T α-Syn group had significantly higher Cx43 expression and p-Cx43/Cx43 ratio(F=6.37,q=4.04-5.76,P<0.05;F=23.43,q=5.73-9.65,P<0.01); the A53 T α-Syn group had a significantly higher p-Cx43/Cx43 ratio than the WT α-Syn group(q=3.61,P<0.05). Conclusion The changes in Cx43 and p-Cx43 levels may be involved in the development of PD.
Objective To investigate the expression and significance of connexin 43(Cx43) and its phosphorylated form(p-Cx43) in Parkinson disease(PD) cell model with overexpression of α-Synuclein(α-Syn). Methods Overexpression of empty vector, wild-type α-Syn, and human mutant A53 T α-Syn was performed in SH-SY5 Y cells(named empty vector group, WT α-Syn group, and A53 T α-Syn group, respectively). Western Blot was used to determine the expression of Cx43 and p-Cx43 proteins. Results Compared with the empty vector group, the WT α-Syn group and A53 T α-Syn group had significantly higher Cx43 expression and p-Cx43/Cx43 ratio(F=6.37,q=4.04-5.76,P<0.05;F=23.43,q=5.73-9.65,P<0.01); the A53 T α-Syn group had a significantly higher p-Cx43/Cx43 ratio than the WT α-Syn group(q=3.61,P<0.05). Conclusion The changes in Cx43 and p-Cx43 levels may be involved in the development of PD.
引文
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[27]MUSIL L S,CUNNINGHAM B A,EDELMAN G M,et al.Differential phosphorylation of the gap junction protein connexin43in junctional communication-competent and-deficient cell lines[J].Journal of Cell Biology,1990,111(5Pt 1):2077-2088.
[28]YOGO K,OGAWA T,AKIYAMA M A,et al.PKA implicated in the phosphorylation of Cx43induced by stimulation with FSH in rat granulosa cells[J].Journal of Reproduction and Development,2006,52(3):321-328.
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[31]CAMERON S J,MALIK S,AKAIKE M,et al.Regulation of epidermal growth factor-induced connexin 43 gap junction communication by big mitogen-activated protein kinase1/ERK5but not ERK1/2kinase activation[J].The Journal of Biological Chemistry,2003,278(20):18682-18688.
[32]THEVENIN A F,KOWAL T J,FONG J T,et al.Proteins and mechanisms regulating gap-junction assembly,internalization,and degradation[J].Physiology(Bethesda,Md.),2013,28(2):93-116.
[33]SOLAN J L,LAMPE P D.Kinase programs spatiotemporally regulate gap junction assembly and disassembly:effects on wound repair[J].Seminars in Cell&Developmental Biology,2016,50:40-48.
[34]FALK M M,BELL C L,KELLS A M,et al.Molecular mechanisms regulating formation,trafficking and processing of annular gap junctions[J].BMC Cell Biology,2016,Suppl1:22-32.
[2]BEITZ J M.Parkinson’s disease:a review[J].Frontiers in Bioscience-Landmark,2014,6:65-74.
[3]LEX K M,KUNDT F S,LORENZL S.Using tube feeding and levodopa-carbidopa intestinal gel application in advanced Parkinson’s disease[J].British Journal of Nursing(Mark Allen Publishing),2018,27(5):259-262.
[4]ALERTE T N,AKINFOLARIN A A,FRIEDRICH E E,et al.Alpha-synuclein aggregation alters tyrosine hydroxylase phosphorylation and immunoreactivity:lessons from viral transduction of knockout mice[J].Neuroscience Letters,2008,435(1):24-29.
[5]CAO Zhentang,WU Yufeng,LIU Genliang,et al.alpha-Synuclein in salivary extracellular vesicles as a potential biomarker of Parkinson’s disease[J].Neuroscience Letters,2019,696:114-120.
[6]ZHANG Guoxin,XIA Yun,WAN Fang,et al.New perspectives on roles of alpha-Synuclein in Parkinson’s disease[J].Frontiers in Aging Neuroscience,2018,10:370-378.
[7]AVSHALUMOVA L J,KORIAKOS A.Overview of skin diseases linked to connexin gene mutations[J].International Journal of Dermatology,2014,53(2):192-205.
[8]EPIFANTSEVA I,SHAW R M.Intracellular trafficking pathways of Cx43gap junction channels[J].Biochimica et Biophysica Acta-Biomembranes,2018,1860(1):51-52.
[9]VINKEN M.Introduction:connexins,pannexins and their channels as gatekeepers of organ physiology[J].Cellular and Molecular Life Sciences,2015,72(15):2775-2778.
[10]NAGY J I,RASH J E.Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS[J].Brain Research Reviews,2000,32(1):51-52.
[11]BENNETT M V,BARRIO L C,BARGIELLO T A,et al.Gap-junctions-new tools,new answers,new questions[J].Neuron,1991,6(3):305-320.
[12]LAMPE P D,LAU A F.The effects of connexin phosphorylation on gap junctional communication[J].The International Journal of Biochemistry&Cell Biology,2004,36(7):1171-1186.
[13]SHL G,WILLECKE K.Gap junctions and the connexin protein family[J].Cardiovascular Research,2004,62(2):228-232.
[14]JOHNSON R G,REYNHOUT J K,TENBROEK E M,et al.Gap junction assembly:roles for the formation plaque and regulation by the C-Terminus of connexin43[J].Molecular Biology of the Cell,2011,22(1):71-86.
[15]SOLAN J L,LAMPE P D.Connexin43 phosphorylation:structural changes and biological effects[J].The Biochemical Journal,2009,419(2):261-272.
[16]LIN J H,YANG J,LIU S J,et al.Connexin mediates gap junction-independent resistance to cellular injury[J].Journal of Neuroscience,2003,23(2):430-441.
[17]FONSECA C G,GREEN C R,NICHOLSON L F.Upregulation in astrocytic connexin 43gap junction levels may exacerbate generalized seizures in mesial temporal lobe epilepsy[J].Brain Research,2002,929(1):105-116.
[18]SURMEIER D J,OBESO J A,HALLIDAY G M.Selective neuronal vulnerability in Parkinson disease[J].Nature Reviews Neuroscience,2017,18(2):101-113.
[19]BEYER E C,BERTHOUD V M.Gap junction gene and protein families:connexins,innexins,and pannexins[J].Biochimica et Biophysica Acta-Biomembranes,2018,1860(1):5-8.
[20]FARBER N M,PEREZ-LLORET S,GAMZU E R.Design and development of a novel supportive care product for the treatment of sialorrhea in Parkinson’s disease[J].Current Topics in Medicinal Chemistry,2015,15(10):939-954.
[21]SAEZ J C,BERTHOUD V M,BRANES M C,et al.Plasma membrane channels formed by connexins:their regulation and functions[J].Physiological Reviews,2003,83(4):1359-1400.
[22]VANSLYKE J K,MUSIL L S.Dislocation and degradation from the ER are regulated by cytosolic stress[J].Journal of Cell Biology,2002,157(3):381-394.
[23]ZUNDORF G,KAHLERT S,REISER G.Gap-junction blocker carbenoxolone differentially enhances NMDA-induced cell death in hippocampal neurons and astrocytes in co-culture[J].Journal of Neurochemistry,2007,102(2):508-521.
[24]VINKEN M,DECROCK E,LEYBAERT L,et al.Non-channel functions of connexins in cell growth and cell death[J].Biochimica et Biophysica Acta-Biomembranes,2012,1818(8,SI):2002-2008.
[25]GREER K,CHEN Jiang,BRICKLER T,et al.Modulation of gap junction-associated Cx43in neural stem/progenitor cells following traumatic brain injury[J].Brain Research Bulletin,2017,134:38-46.
[26]SOLAN J L,LAMPE P D.Specific Cx43phosphorylation events regulate gap junction turnover in vivo[J].FEBS Letters,2014,588(8):1423-1429.
[27]MUSIL L S,CUNNINGHAM B A,EDELMAN G M,et al.Differential phosphorylation of the gap junction protein connexin43in junctional communication-competent and-deficient cell lines[J].Journal of Cell Biology,1990,111(5Pt 1):2077-2088.
[28]YOGO K,OGAWA T,AKIYAMA M A,et al.PKA implicated in the phosphorylation of Cx43induced by stimulation with FSH in rat granulosa cells[J].Journal of Reproduction and Development,2006,52(3):321-328.
[29]BERTHOUD V M,LEDBETTER M L,HERTZBERG E L,et al.Connexin43in MDCK cells:regulation by a tumor-promoting phorbol ester and Ca2+[J].European Journal of Cell Biology,1992,57(1):40-50.
[30]COOPER C D,LAMPE P D.Casein kinase 1regulates connexin-43gap junction assembly[J].The Journal of Biological Chemistry,2002,277(47):44962-44968.
[31]CAMERON S J,MALIK S,AKAIKE M,et al.Regulation of epidermal growth factor-induced connexin 43 gap junction communication by big mitogen-activated protein kinase1/ERK5but not ERK1/2kinase activation[J].The Journal of Biological Chemistry,2003,278(20):18682-18688.
[32]THEVENIN A F,KOWAL T J,FONG J T,et al.Proteins and mechanisms regulating gap-junction assembly,internalization,and degradation[J].Physiology(Bethesda,Md.),2013,28(2):93-116.
[33]SOLAN J L,LAMPE P D.Kinase programs spatiotemporally regulate gap junction assembly and disassembly:effects on wound repair[J].Seminars in Cell&Developmental Biology,2016,50:40-48.
[34]FALK M M,BELL C L,KELLS A M,et al.Molecular mechanisms regulating formation,trafficking and processing of annular gap junctions[J].BMC Cell Biology,2016,Suppl1:22-32.
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