β-arrestin1/2抑制SphK1蛋白质表达研究
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摘要
神经鞘氨醇激酶1(SphK1)在鞘脂类代谢中起关键作用.研究表明,SphK1在肝癌等多种肿瘤细胞中过度表达,促进肿瘤细胞生长与转移,而阻断SphK1促进肿瘤细胞凋亡.因此,SphK1可以作为肝癌治疗的潜在靶点,但到目前为止,SphK1蛋白质降解调控机制并不清楚.采用Western blot、RT-PCR和siRNA干扰等方法研究SphK1蛋白质降解机制,发现β-arrestin1/2可能通过泛素化途径促进SphK1蛋白质降解.蛋白酶体抑制剂MG132能明显上调外源性和内源性SphK1蛋白水平,过表达β-arrestin1/2显著下调人源胚胎肾细胞HEK293T细胞和人肝癌细胞HepG2细胞中SphK1蛋白水平,且呈剂量依赖性,MG132可以缓解β-arrestin1/2对SphK1的降解作用;β-arrestin1/2干扰片段处理后,HEK293与HepG2细胞内内源性SphK1蛋白表达显著升高.证明β-arrestin1/2可以通过蛋白质泛素化方式促进SphK1降解,这为设计SphK1特异性抑制剂提供理论依据.
Sphingosine kinase type 1(SphK1)is a critical regulator of sphingolipid metabolites.Increasing evidence indicates that SphK1 is highly expressed in human liver cancer,and up regulation of SphK1 expression promotes cancer cell growth and metastasis.In contrast,down regulation of SphK1 expression triggers cancer cell apoptosis suggests the SphK1 could be a potential target for liver cancer therapy.However,the underlying mechanism forSphK1 degradation remains unclear.Here,western blotting,quantitative RT-PCR,and small interfering RNA(siRNA)technologies were used to investigate the mechanism of SphK1 degradation.As a result,the expression of both exogenous and endogenous SphK1 were significantly up-regulated when HEK293 Tcells and HepG2 cells were treated with the proteasome inhibitor MG132.Moreover,the forced over-expression ofβ-arrestin1/2 dose-dependently suppressed SphK1 protein level,and this effect could be attenuated by MG132.Knockdown ofβ-arrestin1/2 expressions in HEK293 Tcells and HepG2 cells led to a significant increase in cellular SphK1 protein level.In conclusion,β-arrestin1/2 could promote SphK1 degradation probably through ubiquitin-mediated pathway,the result of which offers theoretical basis for the development of specific SphK1 inhibitor.
Sphingosine kinase type 1(SphK1)is a critical regulator of sphingolipid metabolites.Increasing evidence indicates that SphK1 is highly expressed in human liver cancer,and up regulation of SphK1 expression promotes cancer cell growth and metastasis.In contrast,down regulation of SphK1 expression triggers cancer cell apoptosis suggests the SphK1 could be a potential target for liver cancer therapy.However,the underlying mechanism forSphK1 degradation remains unclear.Here,western blotting,quantitative RT-PCR,and small interfering RNA(siRNA)technologies were used to investigate the mechanism of SphK1 degradation.As a result,the expression of both exogenous and endogenous SphK1 were significantly up-regulated when HEK293 Tcells and HepG2 cells were treated with the proteasome inhibitor MG132.Moreover,the forced over-expression ofβ-arrestin1/2 dose-dependently suppressed SphK1 protein level,and this effect could be attenuated by MG132.Knockdown ofβ-arrestin1/2 expressions in HEK293 Tcells and HepG2 cells led to a significant increase in cellular SphK1 protein level.In conclusion,β-arrestin1/2 could promote SphK1 degradation probably through ubiquitin-mediated pathway,the result of which offers theoretical basis for the development of specific SphK1 inhibitor.
引文
[1]Bosch F X,Ribes J,Díaz M,et al.Primary liver cancer:Worldwide incidence and trends.Gastroenterology,2004,127(5,Suppl 1):S5-S16.
[2]Sun W Y,Hu S S,Wu J J,et al.Down-regulation ofβ-arrestin2promotes tumour invasion and indicates poor prognosis of hepatocellular carcinoma.Scientific Reports,2016,6:35609.
[3]Ogretmen B,Hannun YA.Biologically active sphingolipids in cancer pathogenesis and treatment.Nature Reviews.Cancer,2004,4(8):604-616.
[4]Pitson S M,Moretti P A B,Zebol J R,et al.Activation of sphingosine kinase 1by ERK1/2-mediated phosphorylation.The EMBO Journal,2003,22(20):5491-5500.
[5]French K J,Schrecengost R S,Lee B D,et al.Discovery and evaluation of inhibitors of human sphingosine kinase.Cancer Research,2003,63(18):5962-5969.
[6]Benovic J L,Kühn H,Weyand I,et al.Functional desensitization of the isolated beta-adrenergicreceptorby the beta-adrenergicreceptor kinase:Potential roleof an analog of the retinal protein arrestin(48-kDa protein).Proceedings of theNational Academy Sciences of the United States of America,1987,84(24):8879-8882.
[7]Gurevich V V,Gurevich E V.The structural basis of arrestin-mediated regulation of G-protein-coupled receptors.Pharmacology&Therapeutics,2006,110(3):465-502.
[8]Moore C A C,Milano S K,Benovic J L.Regulation of receptor trafficking by GRKs and arrestins.Annual Review of Physiology,2007,69:451-482.
[9]Sambrook J,Russell D W.分子克隆实验指南.黄培堂译.北京:科学出版社,2002,1713-1718.(Sambrook J,Russell D W.Molecular cloning.Huang P T.Beijing:Science Press,2002,1713-1718.)
[10]Heinemeyer W,Ramos P C,Dohmen R J.The ultimate nanoscale mincer:Assembly,structure and active sites of the 20Sproteasome core.Cellular and Molecular Life Sciences(CMLS),2004,61(13):1562-1578.
[11]Nixon R A.The role of autophagy in neurodegenerative disease.Nature Medicine,2013,19(8):983-997.
[12]Shida D,Takabe K,Kapitonov D,et al.Targeting SphK1as a new strategy against cancer.Current Drug Targets,2008,9(8):662-673.
[13]Xia P,Gamble J R,Wang L J,et al.An oncogenic role of sphingosine kinase.Current Biology,2000,10(23):1527-1530.
[14]Le Scolan E,Pchejetski D,Banno Y,et al.Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression.Blood,2005,106(5):1808-1816.
[15]Johnson K R,Johnson K Y,Crellin H G,et al.Immunohistochemical distribution of sphingosine kinase 1in normal and tumor lung tissue.The Journal of Histochemistry and Cytochemistry:Official Journal of the Histochemistry Society,2005,53(9):1159-1166.
[16]Liu F,Verin A D,Wang P Y,et al.Differential regulation of sphingosine-1-phosphate-and VEGF-induced endothelial cell chemotaxis.Involvement of Giα2-linked Rho kinase activity.American Journal of Respiratory Cell and Molecular Biology,2001,24(6):711-719.
[17]Visentin B,Vekich J A,Sibbald B J,et al.Validation of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in reducing growth,invasion,and angiogenesis in multiple tumor lineages.Cancer Cell,2006,9(3):225-238.
[18]Schwartz G K,Ward D,Saltz L,et al.A pilot clinical/pharmacological study of the protein kinase C-specific inhibitor safingol alone and in combination with doxorubicin.Clinical Cancer Research,1997,3(4):537-543.
[19]Gamble J R,Xia P,Hahn C N,et al.Phenoxodiol,an experimental anticancer drug,shows potent antiangiogenic properties in addition to its antitumour effects.International Journal of Cancer,2006,118(10):2412-2420.
[20]Chen W,Ma L,Bian J J,et al.Involvement of general control nonderepressible kinase 2 in cancer cell apoptosis by posttranslational mechanisms.Molecular Biology of the Cell,2015,26(6):1044-1057.
[21]Girnita L,Girnita A,Larsson O.Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1receptor.Proceedings of the National Academy Sciences of the United States of America,2003,100(14):8247-8252.
[22]Mukherjee A,Veraksa A,Bauer A,et al.Regulation of Notch signalling by non-visualβ-arrestin.Nature Cell Biology,2005,7(12):1191-1201.
[2]Sun W Y,Hu S S,Wu J J,et al.Down-regulation ofβ-arrestin2promotes tumour invasion and indicates poor prognosis of hepatocellular carcinoma.Scientific Reports,2016,6:35609.
[3]Ogretmen B,Hannun YA.Biologically active sphingolipids in cancer pathogenesis and treatment.Nature Reviews.Cancer,2004,4(8):604-616.
[4]Pitson S M,Moretti P A B,Zebol J R,et al.Activation of sphingosine kinase 1by ERK1/2-mediated phosphorylation.The EMBO Journal,2003,22(20):5491-5500.
[5]French K J,Schrecengost R S,Lee B D,et al.Discovery and evaluation of inhibitors of human sphingosine kinase.Cancer Research,2003,63(18):5962-5969.
[6]Benovic J L,Kühn H,Weyand I,et al.Functional desensitization of the isolated beta-adrenergicreceptorby the beta-adrenergicreceptor kinase:Potential roleof an analog of the retinal protein arrestin(48-kDa protein).Proceedings of theNational Academy Sciences of the United States of America,1987,84(24):8879-8882.
[7]Gurevich V V,Gurevich E V.The structural basis of arrestin-mediated regulation of G-protein-coupled receptors.Pharmacology&Therapeutics,2006,110(3):465-502.
[8]Moore C A C,Milano S K,Benovic J L.Regulation of receptor trafficking by GRKs and arrestins.Annual Review of Physiology,2007,69:451-482.
[9]Sambrook J,Russell D W.分子克隆实验指南.黄培堂译.北京:科学出版社,2002,1713-1718.(Sambrook J,Russell D W.Molecular cloning.Huang P T.Beijing:Science Press,2002,1713-1718.)
[10]Heinemeyer W,Ramos P C,Dohmen R J.The ultimate nanoscale mincer:Assembly,structure and active sites of the 20Sproteasome core.Cellular and Molecular Life Sciences(CMLS),2004,61(13):1562-1578.
[11]Nixon R A.The role of autophagy in neurodegenerative disease.Nature Medicine,2013,19(8):983-997.
[12]Shida D,Takabe K,Kapitonov D,et al.Targeting SphK1as a new strategy against cancer.Current Drug Targets,2008,9(8):662-673.
[13]Xia P,Gamble J R,Wang L J,et al.An oncogenic role of sphingosine kinase.Current Biology,2000,10(23):1527-1530.
[14]Le Scolan E,Pchejetski D,Banno Y,et al.Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression.Blood,2005,106(5):1808-1816.
[15]Johnson K R,Johnson K Y,Crellin H G,et al.Immunohistochemical distribution of sphingosine kinase 1in normal and tumor lung tissue.The Journal of Histochemistry and Cytochemistry:Official Journal of the Histochemistry Society,2005,53(9):1159-1166.
[16]Liu F,Verin A D,Wang P Y,et al.Differential regulation of sphingosine-1-phosphate-and VEGF-induced endothelial cell chemotaxis.Involvement of Giα2-linked Rho kinase activity.American Journal of Respiratory Cell and Molecular Biology,2001,24(6):711-719.
[17]Visentin B,Vekich J A,Sibbald B J,et al.Validation of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in reducing growth,invasion,and angiogenesis in multiple tumor lineages.Cancer Cell,2006,9(3):225-238.
[18]Schwartz G K,Ward D,Saltz L,et al.A pilot clinical/pharmacological study of the protein kinase C-specific inhibitor safingol alone and in combination with doxorubicin.Clinical Cancer Research,1997,3(4):537-543.
[19]Gamble J R,Xia P,Hahn C N,et al.Phenoxodiol,an experimental anticancer drug,shows potent antiangiogenic properties in addition to its antitumour effects.International Journal of Cancer,2006,118(10):2412-2420.
[20]Chen W,Ma L,Bian J J,et al.Involvement of general control nonderepressible kinase 2 in cancer cell apoptosis by posttranslational mechanisms.Molecular Biology of the Cell,2015,26(6):1044-1057.
[21]Girnita L,Girnita A,Larsson O.Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1receptor.Proceedings of the National Academy Sciences of the United States of America,2003,100(14):8247-8252.
[22]Mukherjee A,Veraksa A,Bauer A,et al.Regulation of Notch signalling by non-visualβ-arrestin.Nature Cell Biology,2005,7(12):1191-1201.