高血糖促心脏连接蛋白43磷酸化及其对连接通道功能的影响
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摘要
心脏间隙连接通道是心肌细胞之间的特殊连接通道,其功能是完成心肌细胞之间的电化学信息交换,其功能的异常与心律失常的发生有密切关系。心室间隙连接通道主要由连接蛋白43组成。间隙连接通道的功能主要受胞内pH值、胞内Ca2+浓度、连接蛋白磷酸化状态、跨通道电压的调控。新近的一些研究表明高血糖可以促间隙连接通道连接蛋白43磷酸化并使其功能发生改变。但高血糖促连接蛋白43磷酸化的具体位点及其机理尚不清楚。高血糖可以激活蛋白激酶C,而蛋白激酶C又可以使连接蛋白43羧基末端丝氨酸磷酸化,据此推测高血糖可能通过使连接蛋白43羧基末端丝氨酸磷酸化而改变其调控功能。由此,本课题组拟应用基因点突变、抗原再现技术来研究高血糖对间隙连接通道连接蛋白43的促磷酸化作用及其机制,如果能阐明高血糖致心脏间隙连接通道功能异常的机制,那将有助于阐明糖尿病患者心律失常的发生机理并为预防及治疗糖尿病并发心律失常开辟另一崭新的方向。
本研究首先通过构建pcDNA3-野生型连接蛋白43质粒(Wt-Cx43-pcDNA3)、pcDNA3-突变型连接蛋白43质粒(S262F-Cx43-pcDNA3,S368A -Cx43-pcDNA3),分别转染HeLa细胞,经G418筛选后得到表达相应连接蛋白43的细胞株(Wt-Cx43-HeLa细胞,S262F-Cx43-HeLa细胞,S368A -Cx43-HeLa细胞)。应用上述细胞株分两部分进行细胞学实验。第一部分是在筛选后细胞株的培养基中加入不同浓度的葡萄糖(5mmol/L,25mmol/L),应用抗磷酸化Cx43特异性抗体或应用Cx43位点磷酸化抗体通过细胞免疫化学,细胞免疫荧光,及免疫印迹分析Cx43磷酸化情况。对比分析不同葡萄糖浓度对Cx43磷酸化的影响及磷酸化位点。第二部分通过应用Lucifer Yellow划痕试验检测高血糖间隙连接通道功能的改变。
结果表明:1,高血糖能促使Cx43磷酸化;2,应用抗连接蛋白43磷酸化特异性位点的抗体(p-connexin43-Ser368和p-connexin43-Ser262)进行细胞免疫荧光,Western bloting,结果表明高血糖促使连接蛋白43的Ser368位点磷酸化,而对Ser262的无磷酸化作用;3,应用Lucifer Yellow划痕试验检测间隙连接通道功能的改变,结果表明高血糖能降低间隙连接通道传导。综上所述,本实验研究表明(1)高血糖可以促进Cx43的磷酸化,提高Cx43的磷酸化程度;(2)高血糖促使连接蛋白43的Ser368位点磷酸化;(3)高血糖能降低间隙连接通道传导,降低细胞GJ功能。
Gap junctions are collections of plasma membrane channels that enable adjacent cells to exchange cytoplasmic components directly without transit through the extracellular space. These channels allow passage of small molecules (generally less than 1000 Da) such as ions, individual amino acids or short peptides,second messengers (e.g. Ca2+, IP3, cAMP) and other metabolites.Gap junction intercellular communication (GJIC) mediated by connexin43 played an important role in the maintenance of the normal electric activities between cardiac myocytes. The increase in gap junctional communication correlated with the phosphorylation of Cx43. Gap junctional communication was also dependent upon extracellular calcium,entracellular PH value, phosphorylation status of Cx43. Recent work has also illustrated that hyperglycemia can induce phosphorylation of cardiac connexin 43 and change gap junction intercellular communication, but little is known about the mechanism and phosphorylation site of Cx43 hy hyperglycemia. Other investigators previously demonstrated that hyperglycemia induces a persistent actvation of protein kinase C(PKC) in vascular tissues and cells in culture,and actvation of protein kinase C(PKC) lead to serines phosphorylation of connexin 43.So we suggest that hyperglycemia may induces alteration of gap junction intercellular communication throught serines phosphorylation of connexin 43.
In the present research, wild-type connexin43-pcDNA3 (Wt-Cx43-pcDNA3) and site-directed mutant connexin43-pcDNA3 (S262F-Cx43-pcDNA3 , S368A-Cx43-pcDNA3) plasmids were constructed and then transfected into HeLa cells respectively, which undergo further selection with G418 to purify those transfected cell types (Wt-Cx43-HeLa cell, S262F-Cx43-HeLa cell, S368A -Cx43-HeLa cell) respectively. Further experiments were carried out in two parts with these cell types. Firstly, different concentrations of glucose (5mmolL, 25mmolL) were added to the medium of the respective transfected cell types, and phosphorylation status and phosphorylation site were detected to determine the effect of hyperglycemia on Cx43. Secondly,Dye-coupling(evaluated by scrape-loading) was used to detect the gap junction permeability of the respective transfected cell types to evaluate the effect of hyperglycemia on gap junction permeability . Firstly,it was observed that hyperglycemia can induce phosphorylation of cardiac connexin 43, and hyperglycemia can lead to S368 phosphorylation of Cx43 in those of Wt-Cx43-HeLa cells [with anti specific phosphorylated sites connexin43 antibodies (p-connexin43-Ser368)], while no S262 phosphorylation(in those of S262F-Cx43-Hela cells) or S368 phosphorylation of Cx43 (in those of Wt-Cx43-Hela cells or S368A -Cx43-HeLa cells) was detected. Secondly, Dye-coupling(evaluated by scrape-loading) indicated that hyperglycemia decreased gap junction permeability in Wt-Cx43-Hela cells(compared to S368A-Cx43-Hela cells and Wt-Cx43-Hela cells cultured with normal glucose ).Therefore, it was concluded that (1) Hyperglycemia can induce phosphorylation of connexin 43, (2) hyperglycemia can lead to S368 phosphorylation of Cx43. (3)Hyperglycemia decreased gap junction permeability in Wt-Cx43-Hela cells .
本研究首先通过构建pcDNA3-野生型连接蛋白43质粒(Wt-Cx43-pcDNA3)、pcDNA3-突变型连接蛋白43质粒(S262F-Cx43-pcDNA3,S368A -Cx43-pcDNA3),分别转染HeLa细胞,经G418筛选后得到表达相应连接蛋白43的细胞株(Wt-Cx43-HeLa细胞,S262F-Cx43-HeLa细胞,S368A -Cx43-HeLa细胞)。应用上述细胞株分两部分进行细胞学实验。第一部分是在筛选后细胞株的培养基中加入不同浓度的葡萄糖(5mmol/L,25mmol/L),应用抗磷酸化Cx43特异性抗体或应用Cx43位点磷酸化抗体通过细胞免疫化学,细胞免疫荧光,及免疫印迹分析Cx43磷酸化情况。对比分析不同葡萄糖浓度对Cx43磷酸化的影响及磷酸化位点。第二部分通过应用Lucifer Yellow划痕试验检测高血糖间隙连接通道功能的改变。
结果表明:1,高血糖能促使Cx43磷酸化;2,应用抗连接蛋白43磷酸化特异性位点的抗体(p-connexin43-Ser368和p-connexin43-Ser262)进行细胞免疫荧光,Western bloting,结果表明高血糖促使连接蛋白43的Ser368位点磷酸化,而对Ser262的无磷酸化作用;3,应用Lucifer Yellow划痕试验检测间隙连接通道功能的改变,结果表明高血糖能降低间隙连接通道传导。综上所述,本实验研究表明(1)高血糖可以促进Cx43的磷酸化,提高Cx43的磷酸化程度;(2)高血糖促使连接蛋白43的Ser368位点磷酸化;(3)高血糖能降低间隙连接通道传导,降低细胞GJ功能。
Gap junctions are collections of plasma membrane channels that enable adjacent cells to exchange cytoplasmic components directly without transit through the extracellular space. These channels allow passage of small molecules (generally less than 1000 Da) such as ions, individual amino acids or short peptides,second messengers (e.g. Ca2+, IP3, cAMP) and other metabolites.Gap junction intercellular communication (GJIC) mediated by connexin43 played an important role in the maintenance of the normal electric activities between cardiac myocytes. The increase in gap junctional communication correlated with the phosphorylation of Cx43. Gap junctional communication was also dependent upon extracellular calcium,entracellular PH value, phosphorylation status of Cx43. Recent work has also illustrated that hyperglycemia can induce phosphorylation of cardiac connexin 43 and change gap junction intercellular communication, but little is known about the mechanism and phosphorylation site of Cx43 hy hyperglycemia. Other investigators previously demonstrated that hyperglycemia induces a persistent actvation of protein kinase C(PKC) in vascular tissues and cells in culture,and actvation of protein kinase C(PKC) lead to serines phosphorylation of connexin 43.So we suggest that hyperglycemia may induces alteration of gap junction intercellular communication throught serines phosphorylation of connexin 43.
In the present research, wild-type connexin43-pcDNA3 (Wt-Cx43-pcDNA3) and site-directed mutant connexin43-pcDNA3 (S262F-Cx43-pcDNA3 , S368A-Cx43-pcDNA3) plasmids were constructed and then transfected into HeLa cells respectively, which undergo further selection with G418 to purify those transfected cell types (Wt-Cx43-HeLa cell, S262F-Cx43-HeLa cell, S368A -Cx43-HeLa cell) respectively. Further experiments were carried out in two parts with these cell types. Firstly, different concentrations of glucose (5mmolL, 25mmolL) were added to the medium of the respective transfected cell types, and phosphorylation status and phosphorylation site were detected to determine the effect of hyperglycemia on Cx43. Secondly,Dye-coupling(evaluated by scrape-loading) was used to detect the gap junction permeability of the respective transfected cell types to evaluate the effect of hyperglycemia on gap junction permeability . Firstly,it was observed that hyperglycemia can induce phosphorylation of cardiac connexin 43, and hyperglycemia can lead to S368 phosphorylation of Cx43 in those of Wt-Cx43-HeLa cells [with anti specific phosphorylated sites connexin43 antibodies (p-connexin43-Ser368)], while no S262 phosphorylation(in those of S262F-Cx43-Hela cells) or S368 phosphorylation of Cx43 (in those of Wt-Cx43-Hela cells or S368A -Cx43-HeLa cells) was detected. Secondly, Dye-coupling(evaluated by scrape-loading) indicated that hyperglycemia decreased gap junction permeability in Wt-Cx43-Hela cells(compared to S368A-Cx43-Hela cells and Wt-Cx43-Hela cells cultured with normal glucose ).Therefore, it was concluded that (1) Hyperglycemia can induce phosphorylation of connexin 43, (2) hyperglycemia can lead to S368 phosphorylation of Cx43. (3)Hyperglycemia decreased gap junction permeability in Wt-Cx43-Hela cells .
引文
1 Giepmans BN. Gap junctions and connexin-interacting proteins. Cardiovasc Res, 2004, 62(2):233-45.
2 Sohl G, Willecke K. Gap junctions and the connexin protein family. Cardiovasc Res, 2004, 62(2):228-32.
3 Teunissen BE, Bierhuizen MF. Transcriptional control of myocardial connexins. Cardiovasc Res, 2004, 62(2):246-55.
4 Maza J, Das Sarma J, Koval M. Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum. J Biol Chem, 2005, 280(22):21115-21.
5 Lauf U, Giepmans BN, Lopez P, Braconnot S, et al. Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells. Proc Natl Acad Sci U S A, 2002, 99(16):10446-51.
6 Shaw RM, Fay AJ, Puthenveedu MA, et al. Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions. Cell, 2007, 128(3): 547-60.
7 Gaietta G, Deerinck TJ, Adams SR, et al. Multicolor and electron microscopic imaging of connexin trafficking. Science, 2002, 296(5567):503-7.
8 Sosinsky GE, Gaietta GM, Hand G, et al. Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics. Cell Commun Adhes, 2003, 10(4-6):181-6
9 Hunter AW, Jourdan J, Gourdie RG.. Fusion of GFP to the carboxyl terminus of connexin43 increases gap junction size in HeLa cells. Cell Commun Adhes, 2003, 10(4-6):211-4.
10 Hunter AW, Barker RJ, Zhu C, et al. Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion. Mol Biol Cell, 2005, 16(12):5686-98.
11 Berthoud VM, Minogue PJ, Laing JG, et al. Pathways for degradation of connexins and gap junctions. Cardiovasc Res, 2004, 62 (2):256-67.
12细胞分子病理生理学,金惠铭主编,郑州大学出版社,2002年第一版
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14 Cruciani V, Mikalsen SO. Connexins, gap junctional intercellular communication and kinases. Biol-Cell,2002, 94(7-8):433-443.
15 Lampe PD, Lau AF. The effects of connexin phosphorylation on gap junctional communication[J]. Int J Biochem Cell Biol, 2004, 36(7):1171-1186.
16 Ogawa T, Hayashi T, Kyoizumi S, et al. Anisomycin downregulates gap-junctional intercellular communication via the p38 MAP-kinase pathway. J Cell Sci, 2004, 117(Pt 10):2087- 2096.
17 Lau AF. c-Src: Bridging the Gap Between Phosphorylation- and Acidification-Induced Gap Junction Channel Closure. Sci STKE, 2005(291):pe33.
18 Li W, Hertzberg EL, Spray DC. Regulation of Connexin43-Protein Binding in Astrocytes in Response to Chemical Ischemia/Hypoxia. J Biol Chem, 2005, 280(9):7941-7948.
19 Huang YS, Tseng YZ, Wu JC, et al. Mechanism of oleic acid-induced gap junctional disassembly in rat cardiomyocytes. J Mol Cell Cardiol, 2004, 37(3):755-766.
20 Herve JC, Sarrouilhe D. Protein phosphatase modulation of the intercellular junctional communication: Importance in cardiac myocytes. Prog Biophys Mol Biol, 2006, 90 (1-3):225-248
21 Lampe PD, Lau AF. Regulation of gap junctions by phosphorylation of connexins. Arch Biochem Biophys. 2000, 384(2):205-15.
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1. Herve JC, Sarrouilhe D. Protein phosphatase modulation of the intercellular junctional communication: Importance in cardiac myocytes[J]. Prog Biophys Mol Biol,2006,90 (1-3):225-248.
2. Sohl G, Willecke K. Gap junctions and the connexin protein family[J]. Cardiovasc Res,2004,62(2):228-232.
3. Schulz R, Heusch G. Connexin43 and ischemic preconditioning[J]. Cardiovasc Res,2004,62(2):335-344.
4. Saez JC, Berthoud VM, Branes MC, et al. Plasma Membrane Channels Formed by Connexins: Their Regulation and Functions[J]. Physiol Rev,2003 ,83(4):1359-1400.
5. Cooper CD, Lampe PD. Casein Kinase 1 Regulates Connexin-43 Gap Junction Assembly[J]. J Biol Chem,2002,277(47):44962-44968.
6. Lampe PD, Lau AF. The effects of connexin phosphorylation on gap junctional communication[J]. Int J Biochem Cell Biol,2004,36(7):1171-1186.
7. Lampe PD, Kurata WE, Warn-Cramer BJ, et al. Formation of a distinct connexin43phosphoisoform in mitotic cells is dependent upon p34cdc2 kinase[J]. J Cell Sci,1998,111( Pt 6):833-841.
8. Li W, Hertzberg EL, Spray DC. Regulation of Connexin43-Protein Binding in Astrocytes in Response to Chemical Ischemia/Hypoxia[J]. J Biol Chem,2005,280(9):7941-7948.
9. Tenbroek EM, Lampe PD, Solan JL, et al. Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP[J]. J Cell Biol,2001 ,155(7):1307-1318.
10. Yogo K, Ogawa T, Akiyama M, et al. Identification and functional analysis of novel phosphorylation sites in Cx43 in rat primary granulosa cells[J]. FEBS Lett,2002,531(2):132-136.
11. Cruciani V, Mikalsen SO. Connexins, gap junctional intercellular communication and kinases[J]. Biol-Cell,2002,94(7-8):433-443.
12. Lampe PD, TenBroek EM, Burt JM, et al. Phosphorylation of Connexin43 on Serine368 by Protein Kinase C Regulates Gap Junctional Communication[J]. J Cell Biol,2000,149(7):1503-1512.
13. Huang YS, Tseng YZ, Wu JC, et al. Mechanism of oleic acid-induced gap junctional disassembly in rat cardiomyocytes[J]. J Mol Cell Cardiol,2004,37(3):755-766.
14. Doble BW, Dang X, Ping P, et al. Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cellcell contact forming cardiomyocytes[J]. J Cell Sci,2004,117(Pt 3):507-514.
15. Ogawa T, Hayashi T, Kyoizumi S, et al. Anisomycin downregulates gap-junctional intercellular communication via the p38 MAP-kinase pathway[J]. J Cell Sci,2004,117(Pt 10):2087-2096.
16. Giepmans BN. Gap junctions and connexin-interacting proteins[J]. Cardiovasc Res,2004,62(2):233-245.
17. Kanemitsu MY, Jiang W, Eckhart W. Cdc2-mediated Phosphorylation of the Gap Junction Protein,Connexin43, during Mitosis[J]. Cell Growth Differ,1998,9(1):13-21.
18. Lau AF. c-Src: Bridging the Gap Between Phosphorylation- andAcidification-Induced Gap Junction Channel Closure[J]. Sci STKE,2005 ,2005(291):pe33.
19. Mikalsen SO, Kaalhus O, Characterization of pervanadate, an inducer of cellular tyrosine phosphorylation and inhibitor of gap junctional intercellular communication[J]. Biochim Biophys Acta,1996,1290(3):308-318.
2 Sohl G, Willecke K. Gap junctions and the connexin protein family. Cardiovasc Res, 2004, 62(2):228-32.
3 Teunissen BE, Bierhuizen MF. Transcriptional control of myocardial connexins. Cardiovasc Res, 2004, 62(2):246-55.
4 Maza J, Das Sarma J, Koval M. Defining a minimal motif required to prevent connexin oligomerization in the endoplasmic reticulum. J Biol Chem, 2005, 280(22):21115-21.
5 Lauf U, Giepmans BN, Lopez P, Braconnot S, et al. Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells. Proc Natl Acad Sci U S A, 2002, 99(16):10446-51.
6 Shaw RM, Fay AJ, Puthenveedu MA, et al. Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions. Cell, 2007, 128(3): 547-60.
7 Gaietta G, Deerinck TJ, Adams SR, et al. Multicolor and electron microscopic imaging of connexin trafficking. Science, 2002, 296(5567):503-7.
8 Sosinsky GE, Gaietta GM, Hand G, et al. Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics. Cell Commun Adhes, 2003, 10(4-6):181-6
9 Hunter AW, Jourdan J, Gourdie RG.. Fusion of GFP to the carboxyl terminus of connexin43 increases gap junction size in HeLa cells. Cell Commun Adhes, 2003, 10(4-6):211-4.
10 Hunter AW, Barker RJ, Zhu C, et al. Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion. Mol Biol Cell, 2005, 16(12):5686-98.
11 Berthoud VM, Minogue PJ, Laing JG, et al. Pathways for degradation of connexins and gap junctions. Cardiovasc Res, 2004, 62 (2):256-67.
12细胞分子病理生理学,金惠铭主编,郑州大学出版社,2002年第一版
13 Schulz R, Heusch G. Connexin43 and ischemic preconditioning. Cardiovasc Res, 2004,62(2):335-344.
14 Cruciani V, Mikalsen SO. Connexins, gap junctional intercellular communication and kinases. Biol-Cell,2002, 94(7-8):433-443.
15 Lampe PD, Lau AF. The effects of connexin phosphorylation on gap junctional communication[J]. Int J Biochem Cell Biol, 2004, 36(7):1171-1186.
16 Ogawa T, Hayashi T, Kyoizumi S, et al. Anisomycin downregulates gap-junctional intercellular communication via the p38 MAP-kinase pathway. J Cell Sci, 2004, 117(Pt 10):2087- 2096.
17 Lau AF. c-Src: Bridging the Gap Between Phosphorylation- and Acidification-Induced Gap Junction Channel Closure. Sci STKE, 2005(291):pe33.
18 Li W, Hertzberg EL, Spray DC. Regulation of Connexin43-Protein Binding in Astrocytes in Response to Chemical Ischemia/Hypoxia. J Biol Chem, 2005, 280(9):7941-7948.
19 Huang YS, Tseng YZ, Wu JC, et al. Mechanism of oleic acid-induced gap junctional disassembly in rat cardiomyocytes. J Mol Cell Cardiol, 2004, 37(3):755-766.
20 Herve JC, Sarrouilhe D. Protein phosphatase modulation of the intercellular junctional communication: Importance in cardiac myocytes. Prog Biophys Mol Biol, 2006, 90 (1-3):225-248
21 Lampe PD, Lau AF. Regulation of gap junctions by phosphorylation of connexins. Arch Biochem Biophys. 2000, 384(2):205-15.
22 Lampe PD, Lau AF. The effects of connexin phosphorylation on gap junctional communication.Int J Biochem Cell Biol. 2004 Jul;36(7):1171-86.
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