蛋白磷酸酶4在TNF-α诱导的肝脏胰岛素抵抗中的作用机制研究
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摘要
目的：蛋白磷酸酶4(Protein phosphatase4, PP4)是PP2A家族的重要成员,参与调节许多重要的细胞进程,如中心体的成熟、微管的组织和生长、DNA损伤修复,并与胰岛素受体底物4(Insulin receptor4, IRS4)的降解和肝糖代谢有关。本研究通过建立TNF-a诱导的胰岛素抵抗的细胞和动物模型,分析细胞或肝脏组织中PP4的表达及活性的变化,探讨PP4在TNF-a诱导的肝脏胰岛素抵抗中的作用及其机制。
方法：TNF-α(10ng/ml)刺激人肝癌细胞系HepG2和C57BL/6小鼠原代肝细胞24h,建立胰岛素抵抗的细胞模型；C57BL/6小鼠背部皮下埋植TNF-a(8μg/kg-day)缓释泵处理7天,建立TNF-a诱导的胰岛素抵抗动物模型。用G418筛选得到稳定转染PP4高表达质粒HA-PP4和转染PP4活性抑制突变体质粒FLAG-PP4RL的HepG2细胞克隆；应用PP4-siRNA及PP4-shRNA干扰HepG2细胞与原代肝细胞中PP4的表达,获得PP4低表达的HepG2细胞和原代肝细胞；构建PP4-shRNA腺病毒载体,通过尾静脉注射C57BL/6、鼠,建立肝脏PP4低表达的动物模型。测定小鼠血糖、血脂、胰岛素和TNF-a水平。用蒽酮法检测细胞和肝脏组织中的糖原水平,Real-time PCR测定糖异生相关基因(G6Pase、PEPCK、PGCl-α)的表达,Western blot分析细胞或肝脏组织胰岛素信号通路中JNK、P-JNK、IRS1、P-IRS1、AKT、P-AKT、GSK、P-GSK和PP4的水平,用免疫沉淀结合丝／苏氨酸磷酸酶活性分析法测定PP4的活性。免疫共沉淀结合Western blot检测PP4与IRS1的相互作用。
结果：1.我们首先以2型糖尿病小鼠-db/db小鼠(C57BKS/J背景)为胰岛素抵抗模型,检测到血糖、胰岛素和TNF-a水平显著升高,而胰岛素敏感指数(ISI)降低。蒽酮法检测结果显示肝组织糖原含量显著降低,Western blot结果表明糖原合成和糖异生相关信号通路受损：JNK的磷酸化和IRS1的307-Ser磷酸化水平增强,而GSK、AKT的磷酸化水平下降,同时伴有IRS1表达下降及PEPCK表达增加。这些结果表明db/db小鼠的肝脏发生了胰岛素抵抗。我们还观察到db/db小鼠肝组织中PP4的表达和活性均显著增强,提示PP4可能参与了肝脏胰岛素抵抗的发生。在体外试验中,我们采用10ng/mlTNF-a处理HepG2细胞和C57BL/6小鼠原代肝细胞24h,建立胰岛素抵抗细胞模型。在这两种细胞的胰岛素抵抗模型中,糖原含量显著下降,糖异生相关基因表达增加,细胞的糖原合成信号通路受损： JNK的磷酸化和IRS1的307-Ser磷酸化水平升高,而GSK和AKT的磷酸化水平下降。Western blot和磷酸酶活性分析结果显示PP4的表达和活性均增强。在TNF-a诱导的C57BL/6小鼠胰岛素抵抗动物模型中,血清胰岛素水平升高,ISI降低,肝脏组织中糖原含量显著下降,糖异生相关基因(G6Pase、PEPCK、PGC1-a)的mRNA水平升高。肝脏组织的糖原合成信号通路受损：IRS1和JNK的磷酸化水平显著增加,而GSK和AKT的磷酸化水平及IRS1表达水平显著降低。同时肝脏组织PP4的表达和活性增强。细胞和动物实验结果均提示PP4参与了TNF-a诱导的肝脏胰岛素抵抗的发生。2.为了进一步探讨PP4在TNF-a诱导的肝脏胰岛素抵抗中的作用及机制,我们针对PP4的表达和活性,分别建立了稳定高表达PP4以及PP4活性被抑制的HepG2细胞克隆,应用PP4-siRNA及PP4-shRNA获得PP4低表达的HepG2细胞和原代肝细胞以及肝脏PP4低表达的C57BL/6小鼠。这些细胞和动物的分析结果表明①PP4高表达的HepG2细胞中糖原含量降低,糖原合成信号通路受损；②PP4表达降低可以使HepG2细胞、原代肝细胞以及C57BL/6小鼠中糖原含量增加,糖异生相关基因表达降低,糖原合成信号通路恢复；③抑制PP4活性,HepG2细胞的糖原合成增加,糖原合成信号通路得以恢复。此外,免疫共沉淀及Westernblot的结果显示,在HepG2细胞和原代肝细胞中PP4与IRS1存在着相互调节作用。这种相互调节可能在TNF-a诱导的肝脏胰岛素抵抗中起关键作用。
结论：
1.在肝胰岛素抵抗的细胞和动物模型中,PP4的表达和活性均显著增强；
2.PP4是TNF-α信号通路的正调控因子,促进TNF-α诱导的胰岛素抵；
3.PP4通过两种方式参与TNF-α诱导的胰岛素抵抗。一方面,通过激活JNK调控胰岛素信号通路；另一方面,可能通过与IRS1直接作用而调节TNF-α诱导的胰岛素抵抗。
Objectives:Protein phosphatase4(PP4) is a member of PP2A-family and plays important role in many cellular processes, such as centrosome maturation, microtubule growth and organization, and DNA repair. PP4also involves in the down-regulation of IRS4and hepatic glucose metabolism. In the present study, we established the TNF-a-induced insulin resistance models in vivo and in vitro and analyzed the expression and phosphatase activity of PP4in hepatocytes and liver. Moreover, we explored the role of PP4in TNF-a-induced hepatic insulin resistance.
Methods:We established cellular insulin resistance model by treatment of human HepG2cells and C57BL/6mouse primary hepatocytes with lOng/ml TNF-a for24h. Implanting the TNF-a osmotic pumps to the back subcuticle of mice was used to establish TNF-a-induced insulin resistance animal model. The HepG2cell clones expressing HA-PP4and FLAG-PP4RL were obtained by G418selection. The expression of PP4in HepG2cells and primary hepatocytes was knock-downed by transfection of PP4-siRNA and PP4-shRNA. Moreover, Ad-PP4-shRNA was delivered to the livers in C57BL/6mice via tail veil injection before implanting the TNF-a osmotic pumps to the back subcuticle of mice. Serum insulin and TNF-a levels were detected by ELISA, and glycogen content was measured by Anthrone method. The expression of the G6Pase, PEPCK and PGCl-α was analyzed by Real-time PCR, and the levels of JNK、P-JNK、IRS1、P-IRS1、AKT、P-AKT、 GSK、P-GSK、and PP4were detected by Western blot. Phosphatase activity of PP4was also analyzed with immunoprecipitation and phosphatase activity assay. In addition, the interaction between PP4and IRS1was measured by co-immunoprecipitation and Western blot.
Results:The db/db mice displayed increased levels of serum GLU, insulin and TNF-a, and decreased ISI. We also found that glycogen levels in the liver of db/db mice, suggesting a state of insulin resistance. Importantly, levels of phosphorylated JNK and IRS1, and PEPCK expression were elevated in liver of db/db mice, companied by decreased IRS1expression and phosphorylated levels of GSK and AKT. It is noteworthy that elevated PP4activity and expression were detected in liver of db/db mice, suggesting that PP4may be involved in TNF-a-induced insulin resistance. To extend these observations from db/db mice to the cellular models of insulin resistance, human HepG2cells and C57BL/6mouse primary hepatocytes were treated with TNF-a to induce insulin resistance. These two cellular models displayed a state of insulin resistance, as assessed by declined glycogen content, elevated expression of gluconeogenesis-related genes and impaired insulin signaling. The expression and activity of PP4were also increased. Moreover, C57BL/6mice implanted the TNF-a osmotic pumps to the back subcuticle displayed declined glycogen content, elevated expression of gluconeogenesis-related genes and impaired insulin signaling, companied by reduction of PP4expression and activity. Taken together, these data suggest that PP4may play a key role in TNF-a-induced insulin resistance.
In order to further assess whether PP4is involved in TNF-a-induced insulin resistance and explore the possible mechanism, HA-PP4, PP4-siRNA, PP4-shRNA and PP4RL were used to investigate the effect of PP4expression and activity on TNF-a-induced hepatic insulin resistance. The results suggested that up-regulation of PP4expression resulted in decreased glycogen content in HepG2cells and impaired glycogen synthesis and gluconeogenesis signaling. In contrast, down-regulation of PP4expression led to increase of glycogen content in HepG2cells and C57BL/6mouse primary hepatocytes and liver of C57BL/6mice, companied by restoration of insulin signaling. Similarly, inhibition of PP4activity in HepG2cells also led to elevated glycogen content. Moreover, the interaction between PP4and IRS1was assessed by co-immunoprecipitation and Western blot.
Conclusions:
1. In cellular or animal insulin resistance models, the expression and activity of PP4were increased in hepatocytes and liver.
2. PP4functions as a positive regulator of TNF-a-signaling and a linker between inflammation and hepatic insulin resistance.
3. PP4plays a key role in TNF-a-induced hepatic insulin resistance via regulating IRS1by activation of JNK or interaction with IRS1directly.
方法：TNF-α(10ng/ml)刺激人肝癌细胞系HepG2和C57BL/6小鼠原代肝细胞24h,建立胰岛素抵抗的细胞模型；C57BL/6小鼠背部皮下埋植TNF-a(8μg/kg-day)缓释泵处理7天,建立TNF-a诱导的胰岛素抵抗动物模型。用G418筛选得到稳定转染PP4高表达质粒HA-PP4和转染PP4活性抑制突变体质粒FLAG-PP4RL的HepG2细胞克隆；应用PP4-siRNA及PP4-shRNA干扰HepG2细胞与原代肝细胞中PP4的表达,获得PP4低表达的HepG2细胞和原代肝细胞；构建PP4-shRNA腺病毒载体,通过尾静脉注射C57BL/6、鼠,建立肝脏PP4低表达的动物模型。测定小鼠血糖、血脂、胰岛素和TNF-a水平。用蒽酮法检测细胞和肝脏组织中的糖原水平,Real-time PCR测定糖异生相关基因(G6Pase、PEPCK、PGCl-α)的表达,Western blot分析细胞或肝脏组织胰岛素信号通路中JNK、P-JNK、IRS1、P-IRS1、AKT、P-AKT、GSK、P-GSK和PP4的水平,用免疫沉淀结合丝／苏氨酸磷酸酶活性分析法测定PP4的活性。免疫共沉淀结合Western blot检测PP4与IRS1的相互作用。
结果：1.我们首先以2型糖尿病小鼠-db/db小鼠(C57BKS/J背景)为胰岛素抵抗模型,检测到血糖、胰岛素和TNF-a水平显著升高,而胰岛素敏感指数(ISI)降低。蒽酮法检测结果显示肝组织糖原含量显著降低,Western blot结果表明糖原合成和糖异生相关信号通路受损：JNK的磷酸化和IRS1的307-Ser磷酸化水平增强,而GSK、AKT的磷酸化水平下降,同时伴有IRS1表达下降及PEPCK表达增加。这些结果表明db/db小鼠的肝脏发生了胰岛素抵抗。我们还观察到db/db小鼠肝组织中PP4的表达和活性均显著增强,提示PP4可能参与了肝脏胰岛素抵抗的发生。在体外试验中,我们采用10ng/mlTNF-a处理HepG2细胞和C57BL/6小鼠原代肝细胞24h,建立胰岛素抵抗细胞模型。在这两种细胞的胰岛素抵抗模型中,糖原含量显著下降,糖异生相关基因表达增加,细胞的糖原合成信号通路受损： JNK的磷酸化和IRS1的307-Ser磷酸化水平升高,而GSK和AKT的磷酸化水平下降。Western blot和磷酸酶活性分析结果显示PP4的表达和活性均增强。在TNF-a诱导的C57BL/6小鼠胰岛素抵抗动物模型中,血清胰岛素水平升高,ISI降低,肝脏组织中糖原含量显著下降,糖异生相关基因(G6Pase、PEPCK、PGC1-a)的mRNA水平升高。肝脏组织的糖原合成信号通路受损：IRS1和JNK的磷酸化水平显著增加,而GSK和AKT的磷酸化水平及IRS1表达水平显著降低。同时肝脏组织PP4的表达和活性增强。细胞和动物实验结果均提示PP4参与了TNF-a诱导的肝脏胰岛素抵抗的发生。2.为了进一步探讨PP4在TNF-a诱导的肝脏胰岛素抵抗中的作用及机制,我们针对PP4的表达和活性,分别建立了稳定高表达PP4以及PP4活性被抑制的HepG2细胞克隆,应用PP4-siRNA及PP4-shRNA获得PP4低表达的HepG2细胞和原代肝细胞以及肝脏PP4低表达的C57BL/6小鼠。这些细胞和动物的分析结果表明①PP4高表达的HepG2细胞中糖原含量降低,糖原合成信号通路受损；②PP4表达降低可以使HepG2细胞、原代肝细胞以及C57BL/6小鼠中糖原含量增加,糖异生相关基因表达降低,糖原合成信号通路恢复；③抑制PP4活性,HepG2细胞的糖原合成增加,糖原合成信号通路得以恢复。此外,免疫共沉淀及Westernblot的结果显示,在HepG2细胞和原代肝细胞中PP4与IRS1存在着相互调节作用。这种相互调节可能在TNF-a诱导的肝脏胰岛素抵抗中起关键作用。
结论：
1.在肝胰岛素抵抗的细胞和动物模型中,PP4的表达和活性均显著增强；
2.PP4是TNF-α信号通路的正调控因子,促进TNF-α诱导的胰岛素抵；
3.PP4通过两种方式参与TNF-α诱导的胰岛素抵抗。一方面,通过激活JNK调控胰岛素信号通路；另一方面,可能通过与IRS1直接作用而调节TNF-α诱导的胰岛素抵抗。
Objectives:Protein phosphatase4(PP4) is a member of PP2A-family and plays important role in many cellular processes, such as centrosome maturation, microtubule growth and organization, and DNA repair. PP4also involves in the down-regulation of IRS4and hepatic glucose metabolism. In the present study, we established the TNF-a-induced insulin resistance models in vivo and in vitro and analyzed the expression and phosphatase activity of PP4in hepatocytes and liver. Moreover, we explored the role of PP4in TNF-a-induced hepatic insulin resistance.
Methods:We established cellular insulin resistance model by treatment of human HepG2cells and C57BL/6mouse primary hepatocytes with lOng/ml TNF-a for24h. Implanting the TNF-a osmotic pumps to the back subcuticle of mice was used to establish TNF-a-induced insulin resistance animal model. The HepG2cell clones expressing HA-PP4and FLAG-PP4RL were obtained by G418selection. The expression of PP4in HepG2cells and primary hepatocytes was knock-downed by transfection of PP4-siRNA and PP4-shRNA. Moreover, Ad-PP4-shRNA was delivered to the livers in C57BL/6mice via tail veil injection before implanting the TNF-a osmotic pumps to the back subcuticle of mice. Serum insulin and TNF-a levels were detected by ELISA, and glycogen content was measured by Anthrone method. The expression of the G6Pase, PEPCK and PGCl-α was analyzed by Real-time PCR, and the levels of JNK、P-JNK、IRS1、P-IRS1、AKT、P-AKT、 GSK、P-GSK、and PP4were detected by Western blot. Phosphatase activity of PP4was also analyzed with immunoprecipitation and phosphatase activity assay. In addition, the interaction between PP4and IRS1was measured by co-immunoprecipitation and Western blot.
Results:The db/db mice displayed increased levels of serum GLU, insulin and TNF-a, and decreased ISI. We also found that glycogen levels in the liver of db/db mice, suggesting a state of insulin resistance. Importantly, levels of phosphorylated JNK and IRS1, and PEPCK expression were elevated in liver of db/db mice, companied by decreased IRS1expression and phosphorylated levels of GSK and AKT. It is noteworthy that elevated PP4activity and expression were detected in liver of db/db mice, suggesting that PP4may be involved in TNF-a-induced insulin resistance. To extend these observations from db/db mice to the cellular models of insulin resistance, human HepG2cells and C57BL/6mouse primary hepatocytes were treated with TNF-a to induce insulin resistance. These two cellular models displayed a state of insulin resistance, as assessed by declined glycogen content, elevated expression of gluconeogenesis-related genes and impaired insulin signaling. The expression and activity of PP4were also increased. Moreover, C57BL/6mice implanted the TNF-a osmotic pumps to the back subcuticle displayed declined glycogen content, elevated expression of gluconeogenesis-related genes and impaired insulin signaling, companied by reduction of PP4expression and activity. Taken together, these data suggest that PP4may play a key role in TNF-a-induced insulin resistance.
In order to further assess whether PP4is involved in TNF-a-induced insulin resistance and explore the possible mechanism, HA-PP4, PP4-siRNA, PP4-shRNA and PP4RL were used to investigate the effect of PP4expression and activity on TNF-a-induced hepatic insulin resistance. The results suggested that up-regulation of PP4expression resulted in decreased glycogen content in HepG2cells and impaired glycogen synthesis and gluconeogenesis signaling. In contrast, down-regulation of PP4expression led to increase of glycogen content in HepG2cells and C57BL/6mouse primary hepatocytes and liver of C57BL/6mice, companied by restoration of insulin signaling. Similarly, inhibition of PP4activity in HepG2cells also led to elevated glycogen content. Moreover, the interaction between PP4and IRS1was assessed by co-immunoprecipitation and Western blot.
Conclusions:
1. In cellular or animal insulin resistance models, the expression and activity of PP4were increased in hepatocytes and liver.
2. PP4functions as a positive regulator of TNF-a-signaling and a linker between inflammation and hepatic insulin resistance.
3. PP4plays a key role in TNF-a-induced hepatic insulin resistance via regulating IRS1by activation of JNK or interaction with IRS1directly.
引文
1. Bastard, J. P., et al., Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur Cytokine Netw, 2006.17(1):4-12.
2. Xu, J., et al., Trauma and Hemorrhage-Induced Acute Hepatic Insulin Resistance:Dominant Role of Tumor Necrosis Factor (TNF)-{alpha}. Endocrinology,2008 (online)
3. Plomgaard, P., et al., Associations between insulin resistance and TNF-alpha in plasma, skeletal muscle and adipose tissue in humans with and without type 2 diabetes. Diabetologia,2007.50(12): 2562-71.
4. Erol, A., Insulin resistance is an evolutionarily conserved physiological mechanism at the cellular level for protection against increased oxidative stress. Bioessays,2007.29(8):811-8.
5. Ryden, M. and P. Arner, Tumour necrosis factor-alpha in human adipose tissue -- from signalling mechanisms to clinical implications. J Intern Med,2007.262(4):431-8.
6. Aggarwal, B. B., Tumour necrosis factors receptor associated signalling molecules and their role in activation of apoptosis, JNK and NF-kappaB. Ann Rheum Dis,2000.59 Suppl 1:16-16.
7. Liu, G. and C. M. Rondinone, JNK:bridging the insulin signaling and inflammatory pathway. Curr Opin Investig Drugs,2005.6(10): 979-87.
8. Zick, Y., Ser/Thr phosphorylation of IRS proteins:a molecular basis for insulin resistance. Sci STKE,2005.2005(268):pe4.
9. Zhou, G., et al., Protein phosphatase 4 is involved in tumor necrosis factor-alpha-induced activation of c-Jun N-terminal kinase. J Biol Chem,2002.277(8):6391-8.
10. Hu, M. C., et al., Protein phosphatase X interacts with c-Rel and stimulates c-Rel/nuclear factor kappaB activity. J Biol Chem,1998. 273(50):33561-5.
11. Mihindukulasuriya, K. A., et al., Protein phosphatase 4 interacts with and down-regulates insulin receptor substrate 4 following tumor necrosis factor-alpha stimulation. J Biol Chem,2004.279(45): 46588-94.
12. Sesti, G., et al., Defects of the insulin receptor substrate (IRS) system in human metabolic disorders. Faseb J,2001.15(12): 2099-111.
13. Suzuki, R., et al., Both insulin signaling defects in the liver and obesity contribute to insulin resistance and cause diabetes in Irs2(-/-) mice. J Biol Chem,2004.279(24):25039-49.
14. Valverde, A. M., et al., Molecular mechanisms of insulin resistance in IRS-2-deficient hepatocytes. Diabetes,2003.52(9):2239-48.
15. Zhou, L., et al., Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells. Mol Endocrinol,1999.13(3):505-14.
16. Uchida, T., et al., IRS-4 mediates protein kinase B signaling during insulin stimulation without promoting antiapoptosis. Mol Cell Biol, 2000.20(1):126-38.
17. Qu,B. H., et al., Insulin receptor substrate-4 enhances insulin-like growth factor-I-induced cell proliferation. J Biol Chem,1999. 274(44):31179-84.
18. Denu, J. M., et al. Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide:evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry.1998,37(16):5633-42.
19. Krejsa, C. M., et al.. Impact of oxidative stress on signal transduction control by phosphotyrosine phosphatases. Environ Health Perspect.1998,106 Suppl 5:1179-84
20. Keyse, S. M. Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr Opin Cell Biol. 2000,12(2):186-92.
21. Hitomi.H., et al. Aldosterone suppresses insulin signaling via the downregulation of insulin receptor substrate-1 in vascular smooth muscle cells. Hypertension.2007,50(4):750-755.
22. Birnbaum, M. J. Turning down insulin signaling. J Clin Invest.2001, 108(5):655-659.
23. Dokra, A., et al. Brain IRS2 signaling coordinates life span and nutrient homeostasis. Science.2007,317(5836):369-372.
24. Wilson, C., et al. Extracellular and subcellular regulation of the PI3K/Akt cassette:new mechanisms for controlling insulin and growth factor signalling. Biochem Soc Trans.2007,35(Pt 2):219-221.
25. Dokras, A., et al. Screening women with polycystic ovarian syndrome for metabolic syndrome. Obstet Gynecol,2005,106 (1):131-137.
26. Pedersen, B. K. IL-6 signalling in exercise and disease. Biochem Soc Trans.2007,35(Pt 5):1295-1297.
27. Shah, A., et al. Adipose inflammation, insulin resistance, and cardiovascular disease. J Parenter Enteral Nutr.2008,32(6): 638-644.
28. Hotamisligil, G. S. Inflammatory pathways and insulin action. Int J Obes Relat Metab Disord,2003,27 (Supp13):S53-S55.
29. Stephens, J. M., Lee, J., Pilch,P. F, et al. Tumor necrosis factor-alpha-induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and GLUT4 expression without a loss of insulin receptormediated signal transduction. J Biol Chem,1997,272(2):971-976.
30. Arner, P. The adipocyte in insulin resistance:key molecules and the impact of the thiazolidinediones. Trends Endocrinol Metab.2003, 14(3):137-145.
31. Gasic,S., et al. Tumor necrosis factor alpha stimulates lipolysis in adipocytes by decreasing Gi protein concentrations[J]. J Biol Chem.1999,274 (10):6770-6775.
32. Fasshauer, M., et al. Interleukin (IL)-6 mRNA expression is stimulated by insulin, isoproterenol, tumour necrosis factor alpha, growth hormone, and IL-6 in 3T3-L1 adipocytes. Horm Metab Res,2003, 35(3):147-152.
33. Ruan, H., et al. Insulin resistance in adipose tissue:direct and indirect effects of tumor necrosis factor-alpha. Cytokine Growth Factor Rev.2003,14(5):447-455.
34. Yudkin, J. S. Inflammation, obesity, and the metabolic syndrome. Horm Metab Res.2007,39(10):707-709.
35. Lorenzo,M., et al. Insulin resistance induced by tumor necrosis factor-alpha in myocytes and brown adipocytes.J Anim Sci,2008, 86(14 Suppl)-.E94-E104.
36. Luca, C., et al. Inflammation and insulin resistance. FEBS Lett, 2008,582 (1):97-105.
37. Zhou, G., et al. Protein Phosphatase 4 Is a Positive Regulator of Hematopoietic Progenitor Kinase 1.J Biol Chem.2004,279(47): 49551-49561.
38. Yeh, P. Y., et al. Suppression of MEK/ERK signalling pathway enhances cisplatin-induced NF-κB activation by protein phosphatase 4-mediated NF-jB p65 Thr dephosphorylation. J Biol Chem.2004, 279(25):26143-26148.
39. Yoon, Y. S., et al. Suppressor of MEK null (SMEK)/protein phosphatase 4 catalytic subunit (PP4C) is a key regulator of hepatic gluconeogenesis. Proc Nat1 Acad Sci USA.2010,107(41):17704-17709.
40. Khalil,M., et al. Human hepatocytes cell lines proliferating as cohesive spheroid colonies in alginate markedly upregulate beth synthetic and detoxificatory liver function. J Hepatol. 2001,297:68-77.
41. Giraud, J. et al. Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling. J Biol Chem. 2004,279(5):3447-54.
1. Hu, M. C., Shui, J. W., Mihindukulasuriya, K. A., et al. Genomic structure of the mouse PP4 gene:a developmentally regulated protein phosphatase. Gene.2001,278(1-2):89-99.
2. Wade, T., Miyata, T., Inagi, R., et al. Cloning and characterization of a novel subunit of protein serine/threonine phosphatase 4 from mesengial cells. J Am Soc Nephrol.2001,12(12):2601-2608.
3. Zhang, X., Ozawa,Y., Lee, H., et al. Histone deacetylase 3(HDAC3) activity is regulated by interaction with protein serine/threonine phosphatase 4. Genes Dev.2005,19(7):827-839.
4. Gingras, A. C., Caballero, M., Zarske, M., et al. A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity. Mol Cell Proteomics. 2005,4 (11):1725-1740.
5. Venkitaramani, D. V., Fulton, D. B., Andreotti, A. H., et al. Mapping the Ca2+-dependent binding of an invertebrate homolog of protein phosphatase 4 regulatory subunit 2 to the small EF-hand protein, calsensin. Biochimica et Biophysica Acta.2006,1763(3):322-329.
6. Lee, D. H., Pan, Y., Kanner, S., et al. A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination. Nat Struct Mol Biol.2010,17(3):365-372.
7. Wu, H. I., Brown, J. A., Dorie, M. J., et al. Genome-wide identification of genes conferring resistance to the anticancer agents cisplatin, oxaliplatin, and mitomycin C. Cancer Res.2004,64(11):3940-3948.
8. Mendoza, M. C., Booth, E.0., Shaulsky, G., et al. MEK1 and Protein Phosphatase 4 Coordinate Dictyosteliura Development and Chemotaxis. Mol Cell Biol.2007,27(10):3817-3827.
9. Sousa-Nunes, R., Chia, W., Somers, W. G.. Protein Phosphatase 4 mediates localization of the Miranda complex during Drosophila neuroblast asymmetric divisions. Genes Dev.2009,23(3):359-372.
10. Chen,G.I., Tisayakorn,S., Jorgensen,C., et al. PP4R4/KIAA1622 Forms a Novel Stable Cytosolic Complex with Phosphoprotein Phosphatase 4. J Biol Chem.2008,283(43):29273-29284.
11. Kloeker, S., Wadzinski, B. E. Purification and identification of a novel subunit of protein serine/threonine phosphatase 4. J Biol Chem.1999, 274(9):5339-5347.
12. Kong, M., Fox, C. J., Mu, J., et al. The PP2A-associated protein a 4 is an essential inhibitor of apoptosis. Science.2004,306(5696):695-698.
13. Hastie, C. J., Carnegie, G. K., Morrice, N., et al. A novel 50 kDa protein forms complexes with protein phosphatase 4 and is located at centrosomal microtubule organizing centres. Biochem J.2000,347(3):845-855.
14. Zhou, G., Mihindukulasuriya, K. A., Hu, M. C., et al. Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced Activation of c-Jun N-terminal Kinase. J Biol Chem.2002,277(8):6391-6398.
15. Helps, N. R., Brewis, N. D., Lineruth, K., et al. Protein phosphatase 4 is an essential enzyme required for organization of microtubules at centrosomes in Drosophila embryos. J Cell Sci.1998,111(Pt 10):1331-1340.
16. Martin-Granados.C., Philp,A., Oxenham, S. K., et al. Depletion of protein phosphatase 4 in human cells reveals essential roles in centrosome maturation, cell migration and the regulation of Rho GTPases. Int J Biochem Cell Biol.2008,40(10):2315-2332.
17. Toyo-oka, K., Mori, D., Yano, Y., et al. Protein phosphatase 4 catalytic subunit regulates Cdkl activity and microtubule organization via NDEL1 dephosphorylat ion. J Cell Biol.2008,180(6):1133-1147.
18. Han, X., Gomes, J. E., Birmingham, C. L., et al. The Role of Protein Phosphatase 4 in Regulating Microtubule Severing in the Caenorhabditis elegans Embryo. Genetics.2009,181(3):933-943.
19. Carnegie, G. K., Sleeman, J. E., Morrice, N., et al. Protein phosphatase 4 interacts with the survival of motor neurons complex and enhances the temporal localisation of snRNPs. J Cell Sci.2003,116(Pt 10):1905-1913.
20. Yang, J., Fan, G. H., Wadzinski, B. E., et al. Protein phosphatase 2A interacts with and directly dephosphorylates RelA. J Biol Chem.2001, 276(51):47828-47833.
21. Yeh, P. Y., Yeh, K. H., Chuang, S. E., et al. Suppression of MEK/ERK signalling pathway enhances cisplatin-induced NF-κB activation by protein phosphatase 4-mediated NF-jB p65 Thr dephosphorylation. J Biol Chem.2004,279(25):26143-26148.
22. You, D. J., Kim, Y. L., Park, C. R., et al. Regulation of I κ B Kinase by G β L through Recruitment of the Protein Phosphatases. Mol Cells.2010, 30(6):527-532.
23. Chen,L., Dong, W., Zou, T., et al. Protein phosphatase 4 negatively regulates LPS cascade by inhibiting ubiquitination of TRAF6. FEBS Lett. 2008,582(19):2843-2849.
24. Zhou, G., Boomer, J. S., Tan, T. H. Protein Phosphatase 4 Is a Positive Regulator of Hematopoietic Progenitor Kinase 1. J Biol Chem.2004,279(47): 49551-49561.
25. Mihindukulasuriya, K. A., Zhou, G., Qin, J., et al. Protein Phosphatase 4 Interacts with and Down-regulates Insulin Receptor Substrate 4 following Tumor NecrosisFactor- α Stimulation. J Biol Chem.2004, 279(45):46588-46594.
26. Shui.J. W., Hu, M. C., Tan, T. H. Conditional Knockout Mice Reveal an Essential Role of Protein Phosphatase 4 in Thymocyte Development and Pre-T-Cell Receptor Signaling. Mol Cell Biol.2007,27(1):79-91.
27. Huang, S., Shu, L., Easton,J., et al. Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity. J Biol Chem.2004, 279(35):36490-36496
28. Mourtada-Maarabouni, M., Williams, G. T. Protein phosphatase 4 regulates apoptosis, proliferation and mutation rate of human cells Biochim Biophys Acta.2008,1783(8):1490-1502.
29. Chowdhury, D., Xu, X., Zhong, X., et al. A PP4-phosphatase complex dephosphorylates gamma-H2AX generated during DNA replication. Mol Cell.2008,31(1):33-46.
30. Zhang, W., Durocher, D. De novo telomere formation is suppressed by the Mecl-dependent inhibition of Cdc13 accumulation at DNA breaks. Genes Dev. 2010,24(5):502-515.
31. Yoon, Y. S., Lee, M. W., Ryu, D., et al. Suppressor of MEK null (SMEK)/protein phosphatase 4 catalytic subunit (PP4C) is a key regulator of hepatic gluconeogenesis Proc Natl Acad Sci USA.2010,107(41):17704-17709.
32. Wang, B., Zhao,A., Sun,L, et al. Protein phosphatase PP4 is overexpressed in human breast and lung tumors. Cell Res.2008,18(9):974-977.
33. Gingras, A. C., Caballero, M., Zarske, M., et al. A Novel, Evolutionarily Conserved Protein Phosphatase Complex Involved in Cisplatin Sensitivity. Mol Cell Proteomics. 2005,4(11):1725-1740.
34. Sumiyoshi, E., Sugimoto, A., Yamamoto, M. Protein phosphatase 4 is required for centrosome maturation in mitosis and sperm meiosis in C-elegans. J Cell Sci.2002,115(Pt7):1403-1410.
35. Cohen, P. T., Philp, A., Vazquez-Martin, C. Protein phosphatase 4 -from obscurity to vital functions. FEBS Lett.2005,579(15):3278-3286.
2. Xu, J., et al., Trauma and Hemorrhage-Induced Acute Hepatic Insulin Resistance:Dominant Role of Tumor Necrosis Factor (TNF)-{alpha}. Endocrinology,2008 (online)
3. Plomgaard, P., et al., Associations between insulin resistance and TNF-alpha in plasma, skeletal muscle and adipose tissue in humans with and without type 2 diabetes. Diabetologia,2007.50(12): 2562-71.
4. Erol, A., Insulin resistance is an evolutionarily conserved physiological mechanism at the cellular level for protection against increased oxidative stress. Bioessays,2007.29(8):811-8.
5. Ryden, M. and P. Arner, Tumour necrosis factor-alpha in human adipose tissue -- from signalling mechanisms to clinical implications. J Intern Med,2007.262(4):431-8.
6. Aggarwal, B. B., Tumour necrosis factors receptor associated signalling molecules and their role in activation of apoptosis, JNK and NF-kappaB. Ann Rheum Dis,2000.59 Suppl 1:16-16.
7. Liu, G. and C. M. Rondinone, JNK:bridging the insulin signaling and inflammatory pathway. Curr Opin Investig Drugs,2005.6(10): 979-87.
8. Zick, Y., Ser/Thr phosphorylation of IRS proteins:a molecular basis for insulin resistance. Sci STKE,2005.2005(268):pe4.
9. Zhou, G., et al., Protein phosphatase 4 is involved in tumor necrosis factor-alpha-induced activation of c-Jun N-terminal kinase. J Biol Chem,2002.277(8):6391-8.
10. Hu, M. C., et al., Protein phosphatase X interacts with c-Rel and stimulates c-Rel/nuclear factor kappaB activity. J Biol Chem,1998. 273(50):33561-5.
11. Mihindukulasuriya, K. A., et al., Protein phosphatase 4 interacts with and down-regulates insulin receptor substrate 4 following tumor necrosis factor-alpha stimulation. J Biol Chem,2004.279(45): 46588-94.
12. Sesti, G., et al., Defects of the insulin receptor substrate (IRS) system in human metabolic disorders. Faseb J,2001.15(12): 2099-111.
13. Suzuki, R., et al., Both insulin signaling defects in the liver and obesity contribute to insulin resistance and cause diabetes in Irs2(-/-) mice. J Biol Chem,2004.279(24):25039-49.
14. Valverde, A. M., et al., Molecular mechanisms of insulin resistance in IRS-2-deficient hepatocytes. Diabetes,2003.52(9):2239-48.
15. Zhou, L., et al., Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells. Mol Endocrinol,1999.13(3):505-14.
16. Uchida, T., et al., IRS-4 mediates protein kinase B signaling during insulin stimulation without promoting antiapoptosis. Mol Cell Biol, 2000.20(1):126-38.
17. Qu,B. H., et al., Insulin receptor substrate-4 enhances insulin-like growth factor-I-induced cell proliferation. J Biol Chem,1999. 274(44):31179-84.
18. Denu, J. M., et al. Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide:evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry.1998,37(16):5633-42.
19. Krejsa, C. M., et al.. Impact of oxidative stress on signal transduction control by phosphotyrosine phosphatases. Environ Health Perspect.1998,106 Suppl 5:1179-84
20. Keyse, S. M. Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr Opin Cell Biol. 2000,12(2):186-92.
21. Hitomi.H., et al. Aldosterone suppresses insulin signaling via the downregulation of insulin receptor substrate-1 in vascular smooth muscle cells. Hypertension.2007,50(4):750-755.
22. Birnbaum, M. J. Turning down insulin signaling. J Clin Invest.2001, 108(5):655-659.
23. Dokra, A., et al. Brain IRS2 signaling coordinates life span and nutrient homeostasis. Science.2007,317(5836):369-372.
24. Wilson, C., et al. Extracellular and subcellular regulation of the PI3K/Akt cassette:new mechanisms for controlling insulin and growth factor signalling. Biochem Soc Trans.2007,35(Pt 2):219-221.
25. Dokras, A., et al. Screening women with polycystic ovarian syndrome for metabolic syndrome. Obstet Gynecol,2005,106 (1):131-137.
26. Pedersen, B. K. IL-6 signalling in exercise and disease. Biochem Soc Trans.2007,35(Pt 5):1295-1297.
27. Shah, A., et al. Adipose inflammation, insulin resistance, and cardiovascular disease. J Parenter Enteral Nutr.2008,32(6): 638-644.
28. Hotamisligil, G. S. Inflammatory pathways and insulin action. Int J Obes Relat Metab Disord,2003,27 (Supp13):S53-S55.
29. Stephens, J. M., Lee, J., Pilch,P. F, et al. Tumor necrosis factor-alpha-induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and GLUT4 expression without a loss of insulin receptormediated signal transduction. J Biol Chem,1997,272(2):971-976.
30. Arner, P. The adipocyte in insulin resistance:key molecules and the impact of the thiazolidinediones. Trends Endocrinol Metab.2003, 14(3):137-145.
31. Gasic,S., et al. Tumor necrosis factor alpha stimulates lipolysis in adipocytes by decreasing Gi protein concentrations[J]. J Biol Chem.1999,274 (10):6770-6775.
32. Fasshauer, M., et al. Interleukin (IL)-6 mRNA expression is stimulated by insulin, isoproterenol, tumour necrosis factor alpha, growth hormone, and IL-6 in 3T3-L1 adipocytes. Horm Metab Res,2003, 35(3):147-152.
33. Ruan, H., et al. Insulin resistance in adipose tissue:direct and indirect effects of tumor necrosis factor-alpha. Cytokine Growth Factor Rev.2003,14(5):447-455.
34. Yudkin, J. S. Inflammation, obesity, and the metabolic syndrome. Horm Metab Res.2007,39(10):707-709.
35. Lorenzo,M., et al. Insulin resistance induced by tumor necrosis factor-alpha in myocytes and brown adipocytes.J Anim Sci,2008, 86(14 Suppl)-.E94-E104.
36. Luca, C., et al. Inflammation and insulin resistance. FEBS Lett, 2008,582 (1):97-105.
37. Zhou, G., et al. Protein Phosphatase 4 Is a Positive Regulator of Hematopoietic Progenitor Kinase 1.J Biol Chem.2004,279(47): 49551-49561.
38. Yeh, P. Y., et al. Suppression of MEK/ERK signalling pathway enhances cisplatin-induced NF-κB activation by protein phosphatase 4-mediated NF-jB p65 Thr dephosphorylation. J Biol Chem.2004, 279(25):26143-26148.
39. Yoon, Y. S., et al. Suppressor of MEK null (SMEK)/protein phosphatase 4 catalytic subunit (PP4C) is a key regulator of hepatic gluconeogenesis. Proc Nat1 Acad Sci USA.2010,107(41):17704-17709.
40. Khalil,M., et al. Human hepatocytes cell lines proliferating as cohesive spheroid colonies in alginate markedly upregulate beth synthetic and detoxificatory liver function. J Hepatol. 2001,297:68-77.
41. Giraud, J. et al. Nutrient-dependent and insulin-stimulated phosphorylation of insulin receptor substrate-1 on serine 302 correlates with increased insulin signaling. J Biol Chem. 2004,279(5):3447-54.
1. Hu, M. C., Shui, J. W., Mihindukulasuriya, K. A., et al. Genomic structure of the mouse PP4 gene:a developmentally regulated protein phosphatase. Gene.2001,278(1-2):89-99.
2. Wade, T., Miyata, T., Inagi, R., et al. Cloning and characterization of a novel subunit of protein serine/threonine phosphatase 4 from mesengial cells. J Am Soc Nephrol.2001,12(12):2601-2608.
3. Zhang, X., Ozawa,Y., Lee, H., et al. Histone deacetylase 3(HDAC3) activity is regulated by interaction with protein serine/threonine phosphatase 4. Genes Dev.2005,19(7):827-839.
4. Gingras, A. C., Caballero, M., Zarske, M., et al. A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity. Mol Cell Proteomics. 2005,4 (11):1725-1740.
5. Venkitaramani, D. V., Fulton, D. B., Andreotti, A. H., et al. Mapping the Ca2+-dependent binding of an invertebrate homolog of protein phosphatase 4 regulatory subunit 2 to the small EF-hand protein, calsensin. Biochimica et Biophysica Acta.2006,1763(3):322-329.
6. Lee, D. H., Pan, Y., Kanner, S., et al. A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination. Nat Struct Mol Biol.2010,17(3):365-372.
7. Wu, H. I., Brown, J. A., Dorie, M. J., et al. Genome-wide identification of genes conferring resistance to the anticancer agents cisplatin, oxaliplatin, and mitomycin C. Cancer Res.2004,64(11):3940-3948.
8. Mendoza, M. C., Booth, E.0., Shaulsky, G., et al. MEK1 and Protein Phosphatase 4 Coordinate Dictyosteliura Development and Chemotaxis. Mol Cell Biol.2007,27(10):3817-3827.
9. Sousa-Nunes, R., Chia, W., Somers, W. G.. Protein Phosphatase 4 mediates localization of the Miranda complex during Drosophila neuroblast asymmetric divisions. Genes Dev.2009,23(3):359-372.
10. Chen,G.I., Tisayakorn,S., Jorgensen,C., et al. PP4R4/KIAA1622 Forms a Novel Stable Cytosolic Complex with Phosphoprotein Phosphatase 4. J Biol Chem.2008,283(43):29273-29284.
11. Kloeker, S., Wadzinski, B. E. Purification and identification of a novel subunit of protein serine/threonine phosphatase 4. J Biol Chem.1999, 274(9):5339-5347.
12. Kong, M., Fox, C. J., Mu, J., et al. The PP2A-associated protein a 4 is an essential inhibitor of apoptosis. Science.2004,306(5696):695-698.
13. Hastie, C. J., Carnegie, G. K., Morrice, N., et al. A novel 50 kDa protein forms complexes with protein phosphatase 4 and is located at centrosomal microtubule organizing centres. Biochem J.2000,347(3):845-855.
14. Zhou, G., Mihindukulasuriya, K. A., Hu, M. C., et al. Protein Phosphatase 4 Is Involved in Tumor Necrosis Factor-α-induced Activation of c-Jun N-terminal Kinase. J Biol Chem.2002,277(8):6391-6398.
15. Helps, N. R., Brewis, N. D., Lineruth, K., et al. Protein phosphatase 4 is an essential enzyme required for organization of microtubules at centrosomes in Drosophila embryos. J Cell Sci.1998,111(Pt 10):1331-1340.
16. Martin-Granados.C., Philp,A., Oxenham, S. K., et al. Depletion of protein phosphatase 4 in human cells reveals essential roles in centrosome maturation, cell migration and the regulation of Rho GTPases. Int J Biochem Cell Biol.2008,40(10):2315-2332.
17. Toyo-oka, K., Mori, D., Yano, Y., et al. Protein phosphatase 4 catalytic subunit regulates Cdkl activity and microtubule organization via NDEL1 dephosphorylat ion. J Cell Biol.2008,180(6):1133-1147.
18. Han, X., Gomes, J. E., Birmingham, C. L., et al. The Role of Protein Phosphatase 4 in Regulating Microtubule Severing in the Caenorhabditis elegans Embryo. Genetics.2009,181(3):933-943.
19. Carnegie, G. K., Sleeman, J. E., Morrice, N., et al. Protein phosphatase 4 interacts with the survival of motor neurons complex and enhances the temporal localisation of snRNPs. J Cell Sci.2003,116(Pt 10):1905-1913.
20. Yang, J., Fan, G. H., Wadzinski, B. E., et al. Protein phosphatase 2A interacts with and directly dephosphorylates RelA. J Biol Chem.2001, 276(51):47828-47833.
21. Yeh, P. Y., Yeh, K. H., Chuang, S. E., et al. Suppression of MEK/ERK signalling pathway enhances cisplatin-induced NF-κB activation by protein phosphatase 4-mediated NF-jB p65 Thr dephosphorylation. J Biol Chem.2004,279(25):26143-26148.
22. You, D. J., Kim, Y. L., Park, C. R., et al. Regulation of I κ B Kinase by G β L through Recruitment of the Protein Phosphatases. Mol Cells.2010, 30(6):527-532.
23. Chen,L., Dong, W., Zou, T., et al. Protein phosphatase 4 negatively regulates LPS cascade by inhibiting ubiquitination of TRAF6. FEBS Lett. 2008,582(19):2843-2849.
24. Zhou, G., Boomer, J. S., Tan, T. H. Protein Phosphatase 4 Is a Positive Regulator of Hematopoietic Progenitor Kinase 1. J Biol Chem.2004,279(47): 49551-49561.
25. Mihindukulasuriya, K. A., Zhou, G., Qin, J., et al. Protein Phosphatase 4 Interacts with and Down-regulates Insulin Receptor Substrate 4 following Tumor NecrosisFactor- α Stimulation. J Biol Chem.2004, 279(45):46588-46594.
26. Shui.J. W., Hu, M. C., Tan, T. H. Conditional Knockout Mice Reveal an Essential Role of Protein Phosphatase 4 in Thymocyte Development and Pre-T-Cell Receptor Signaling. Mol Cell Biol.2007,27(1):79-91.
27. Huang, S., Shu, L., Easton,J., et al. Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity. J Biol Chem.2004, 279(35):36490-36496
28. Mourtada-Maarabouni, M., Williams, G. T. Protein phosphatase 4 regulates apoptosis, proliferation and mutation rate of human cells Biochim Biophys Acta.2008,1783(8):1490-1502.
29. Chowdhury, D., Xu, X., Zhong, X., et al. A PP4-phosphatase complex dephosphorylates gamma-H2AX generated during DNA replication. Mol Cell.2008,31(1):33-46.
30. Zhang, W., Durocher, D. De novo telomere formation is suppressed by the Mecl-dependent inhibition of Cdc13 accumulation at DNA breaks. Genes Dev. 2010,24(5):502-515.
31. Yoon, Y. S., Lee, M. W., Ryu, D., et al. Suppressor of MEK null (SMEK)/protein phosphatase 4 catalytic subunit (PP4C) is a key regulator of hepatic gluconeogenesis Proc Natl Acad Sci USA.2010,107(41):17704-17709.
32. Wang, B., Zhao,A., Sun,L, et al. Protein phosphatase PP4 is overexpressed in human breast and lung tumors. Cell Res.2008,18(9):974-977.
33. Gingras, A. C., Caballero, M., Zarske, M., et al. A Novel, Evolutionarily Conserved Protein Phosphatase Complex Involved in Cisplatin Sensitivity. Mol Cell Proteomics. 2005,4(11):1725-1740.
34. Sumiyoshi, E., Sugimoto, A., Yamamoto, M. Protein phosphatase 4 is required for centrosome maturation in mitosis and sperm meiosis in C-elegans. J Cell Sci.2002,115(Pt7):1403-1410.
35. Cohen, P. T., Philp, A., Vazquez-Martin, C. Protein phosphatase 4 -from obscurity to vital functions. FEBS Lett.2005,579(15):3278-3286.