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γ-Secretase 활성억제단백질인 TMP21의 과발현이 신경세포주에서 NGF 수용체 신호전달과정에 미치는 영향

Overexpression of TMP21 Could Induce not only Downregulation of TrkA/ERK Phosphorylation but also Upregulation of p75NTR/RhoA Expression on NGF Receptor Signaling Pathway

  • 최선일 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 지승완 (식품의약품안전평가원 실험동물자원과) ;
  • 허윤경 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 김지은 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 남소희 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 황인식 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 이혜련 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 구준서 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 이영주 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 이언필 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 최해욱 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 김홍성 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 이재호 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 정영진 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 이수해 (식품의약품안전평가원 실험동물자원과) ;
  • 심선보 (식품의약품안전평가원 실험동물자원과) ;
  • 황대연 (부산대학교 생명자원과학대학 바이오소재과학과)
  • Choi, Sun-Il (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Jee, Seung-Wan (Department of Laboratory Animal Resources, National Institute of Food and Drug Safety Evaluation) ;
  • Her, Youn-Kyung (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Kim, Ji-Eun (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Nam, So-Hee (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Hwang, In-Sik (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Lee, Hye-Ryun (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Goo, Jun-Seo (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Lee, Young-Ju (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Lee, Eon-Pil (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Choi, Hae-Wook (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Kim, Hong-Sung (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Lee, Jae-Ho (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Jung, Young-Jin (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Lee, Su-Hae (Department of Laboratory Animal Resources, National Institute of Food and Drug Safety Evaluation) ;
  • Shim, Sun-Bo (Department of Laboratory Animal Resources, National Institute of Food and Drug Safety Evaluation) ;
  • Hwang, Dae-Youn (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University)
  • 투고 : 2011.04.20
  • 심사 : 2011.08.06
  • 발행 : 2011.08.30

초록

TMP21은 AD의 원인으로 작용하는 A${\beta}$-42 펩타이드 생성에 중요한 ${\gamma}$-secretase 활성을 억제하는 p24 family에 속하는 type I 막 단백질이다. 본 연구에서는 TMP21이 세포의 성장과 분화에 중요한 NGF 수용체 신호전달과정에 미치는 영향을 분석하고자 인간의 TMP21 cDNA를 합성하고, CMV promoter 조절 하에 hTMP21를 클로닝하여, CMV/hTMP21 벡터를 제조하였다. 그리고 이들 벡터를 B35 neuroblastoma에서 과발현시킨 후 ${\gamma}$-secretase 구성단백질과 NGF 수용체 연관 단백질의 변화를 관찰하였다. 그 결과, 4종류의 ${\gamma}$-secretase 구성단백질의 발현은 vehicle transfectants보다 CMV/hTMP21 transfectants에서 유의적으로 감소하였다. 또한 NGF low affinity 수용체인 $p75^{NTR}$과 downstream 단백질인 RhoA의 양은 NGF를 처리하지 않은 TMP21 transfectants에서 유의적으로 증가하였으나 NGF 처리에 의해 감소되었다. High affinity NGF 수용체인 TrkA의 인산화도 NGF 처리가 없는 경우 유의적으로 감소하였으나 NGF 처리에 의해 증가되었다. 또한 downstream 신호전달 과정 중에서 ERK의 인산화는 TrkA와 유사한 발현변화를 나타내었으나 Akt 인산화는 NGF의 처리에 의해 더욱 증가하였다. 이러한 결과는 TMP21이 neuroblastoma에서 NGF 수용체 신호전달과정를 조절하는 중요한 단백질로서 작용함을 제시하며, AD의 작용기전 연구에 중요한 기초자료를 제공할 것으로 사료된다.

Transmembrane protein 21 (TMP21) is a member of the p24 cargo protein family and has been shown to modulate ${\alpha}$-secretase-mediated A${\beta}$ production which was specifically observed in the brains of subjects with Alzheimer's disease (AD). In order to investigate whether TMP21 could affect nerve growth factor (NGF) receptor signaling pathway, the alteration of NGF receptors and their downstream proteins were detected in TMP21 over-expressed cells. CMV/hTMP21 vector used in this study was successfully expressed into TMP21 proteins in B35 cells after lipofectamin transfection. Expressed TMP21 proteins induced the down-regulation of ${\gamma}$-secretase complex components including Presenlin-1 (PS-1), PS-2, Nicastrin (NST), Pen-2 and APH-1. Also, the expression level of NGF receptor $p75^{NTR}$ and RhoA were significantly higher in CMV/hTMP21 transfectants than vehicle transfectants, while their levels returned to vehicle levels after NGF treatment. However, the phosphorylation of NGF receptor TrkA was dramtically decreased in NGF No-treated CMV/hTMP21 transfectants compared with vehicle transfectants, and increased in NGF treated CMV/hTMP21 transfectants. In TrkA downstream signaling pathway, the phosphorylation level of ERK was also decreased in CMV/hTMP21 transfectants, while the phosphorylation of Akt was increased in the same transfectants. Furthermore, NGF treatment induced the increase of phosphorylation level of Akt and ERK in CMV/hTMP21 transfectants. Therefore, these results suggested that over-expression of TMP21may simultaneously induce the up-regulation of $p75^{NTR}$/RhoA expression and the down-regulation of TrkA/ERK phosphorylation through the inhibition of ${\gamma}$-secretase activity.

키워드

참고문헌

  1. Barlowe, C. 2000. Traffic COPs of the early secretory pathway. Traffic 1, 371-377. https://doi.org/10.1034/j.1600-0854.2000.010501.x
  2. Blum, R., P. Feick, M. Puype, J. Vandekerckhove, R. Klengel, W. Nastainczyk, and I. Schulz. 1996. Tmp21 and p24A, two type I proteins enriched in pancreatic microsomal membranes, are members of a protein family involved in vesicular trafficking. J. Biol. Chem. 271, 17183-17189. https://doi.org/10.1074/jbc.271.29.17183
  3. Blum, R., F. Pfeiffer, P. Feick, W. Nastainczyk, B. Kohler, K. H. Schäfer, and I. Schulz. 1999. Intracellular localization and in vivo trafficking of p24A and p23. J. Cell Sci. 112, 537-548.
  4. Chen, F., H. Hasegawa, G. Schmitt-Ulms, T. Kawarai, C. Bohm, T. Katayama, Y. Gu, N. Sanjo, M. Glista, E. Rogaeva, Y. Wakutani, R. Pardossi-Piquard, X. Ruan, A. Tandon, F. Checler, P. Marambaud, K. Hansen, D. Westaway, P. St George-Hyslop, and P. Fraser. 2006. TMP21 is a presenilin complex component that modulates $\gamma$-secretase but not ε-secretase activity. Nature 440, 1208-1212. https://doi.org/10.1038/nature04667
  5. Choi, S. I., J. H. Park, Y. K. Her, Y. K. Lee, J. E. Kim, S. H. Nam, J. S. Goo, M. J. Jang, H. S. Lee, H. J. Son, C. Y. Lee, and D. Y. Hwang. 2010. Effects of water extract of Liriope platyphylla on the m RNA expression and protein secretion of nerve growth factors. Korean J. Medicinal Crop Sci. 18, 291-297.
  6. De Strooper, B. 2003. Aph-1, Pen-2, and Nicastrin with Presenilin Generate an Active $\gamma$-Secretase Complex. Neuron 38, 9-12. https://doi.org/10.1016/S0896-6273(03)00205-8
  7. De Strooper, B., P. Saftig, K. Craessaerts, H. Vanderstichele, G. Guhde, W. Annaert, K. Von Figura, and F. Van Leuven. 1998. Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391, 387-390. https://doi.org/10.1038/34910
  8. di Mola, F. F., H. Friess, Z. W. Zhu, A. Koliopanos, T. Bley, P. Di Sebastiano, P. Innocenti, A. Zimmermann, and M. W. Büchler. 2000. Nerve growth factor and Trk high affinity receptor (TrkA) gene expression in inflammatory bowel disease. Int. J. Gastroenterol. Hepatol. 46, 670-679.
  9. Dolcini, V., J. Dunys, J. Sevalle, F. Chen, M. V. Guillot-Sestier, P. St George-Hyslop, P. E. Fraser, and F. Checler. 2008. TMP21 regulates A$\beta$ production but does not affect caspase-3, p53, and neprilysin. Biochem. Biophys. Res. Commun. 371, 69-74. https://doi.org/10.1016/j.bbrc.2008.03.151
  10. Einarsdottir, E., A. Carlsson, J. Minde, G. Toolanen, O. Svensson, G. Solders, G. Holmgren, D. Holmberg, and M. Holmberg. 2004. A mutation in the nerve growth factor beta gene (NGFB) causes loss of pain perception. Hum. Mol. Genet. 13, 799-805. https://doi.org/10.1093/hmg/ddh096
  11. Freund-Michel, V. and N. Frossard. 2008. The nerve growth factor and its receptors in airway inflammatory diseases. Pharmacol. Ther. 117, 52-76. https://doi.org/10.1016/j.pharmthera.2007.07.003
  12. Gu, L., W. Yao, Z. Yan, L. Xie, D. Sun, D. Li, Z. Zeng, and Z. Wen. 2003. Effects of TFAR19 gene on the growth and biorheological properties of mouse erythroleukemia cell line MEL. Sci. China C. Life. Sci. 46, 293-301.
  13. Hatchett, C. S., S. Tyler, D. Armstrong, D. Dawbarn, and S. J. Allen. 2007. Familial Alzheimer's disease presenilin 1 mutation M146V increases gamma secretase cutting of p75NTR in vitro. Brain Res. 1147, 248-255. https://doi.org/10.1016/j.brainres.2007.02.002
  14. Heese, K., J. W. Low, and N. Inoue. 2006. Nerve growth factor, Neural stem cells and Alzheimer's disease. Neurosignals 15, 1-12. https://doi.org/10.1159/000094383
  15. Hwang, D. Y., K. R. Chae, T. S. Kang, J. H. Hwang, C. H. Lim, H. K. Kang, J. S. Goo, M. R. Lee, H. J. Lim, S. H. Min, J. Y. Cho, J. T. Hong, C. W. Song, S. G. Paik, J. S. Cho, and Y. K. Kim. 2002. Alterations in behavior, amyloid $\beta$-42, caspase-3, and Cox-2 in mutant PS2 transgenic mouse model of Alzheimer's disease. FASEB J. 16, 805-813. https://doi.org/10.1096/fj.01-0732com
  16. Hwang, D. Y., J. S. Cho, C. K. Kim, S. B. Shim, S. W. Jee, S. H. Lee, S. J. Seo, S. Y. Choi, and Y. K. Kim. 2006. Changes in presenilin 2-binding Wnt proteins, behavior, amyloid-$\beta$ 42, $\gamma$-secretase activity, and testosterone sensitivity in transgenic mice coexpressing tetracycline-controlled transactivator and human mutant presenilin 2. Neuromolecular Med. 8, 415-432. https://doi.org/10.1385/NMM:8:3:415
  17. Iwatsubo, T. 2004. The $\gamma$-secretase complex: machinery for intramembrane proteolysis. Curr. Opin. Neurobiol. 14, 379-383. https://doi.org/10.1016/j.conb.2004.05.010
  18. Perini, G., V. Della-Bianca, V. Politi, G. Della Valle, I. Dal-Pra, F. Rossi, and U. Armato. 2002. Role of p75 neurotrophin receptor in the neurotoxicity by $\beta$-amyloid peptides and synergistic effect of inflammatory cytokines. J. Exp. Med. 195, 907-918. https://doi.org/10.1084/jem.20011797
  19. Russo, C., V. Dolcini, S. Salis, V. Venezia, N. Zambrano, T. Russo, and G. Schettini. 2002. Signal transduction through tyrosine-phosphorylated C-terminal Fragments of amyloid precursor protein via an enhanced interaction with Shc/Grb2 adaptor proteins in reactive astrocytes of Alzheimer's disease brain. J. Biol. Chem. 277, 35282-35288. https://doi.org/10.1074/jbc.M110785200
  20. Seo, S. J., D. Y. Hwang, J. S. Cho, K. R. Chae, C. K. Kim, S. B. Shim, S. W. Jee, S. H. Lee, J. S. Sin, S. Y. Choi, J. Kim, and Y. K. Kim. 2007. PEN-2 overexpression induces $\gamma$-secretase protein and its activity with amyloid $\beta$-42 production. Neurochem Res. 32, 1016-1023. https://doi.org/10.1007/s11064-006-9262-0
  21. Shirotani, K., D. Edbauer, M. Kostka, H. Steiner, and C. Haass. 2004. Immature nicastrin stabilizes APH-1 independent of PEN-2 and presenilin: identification of nicastrin mutants that selectively interact with APH-1. J. Neurochem. 89, 1520-1527. https://doi.org/10.1111/j.1471-4159.2004.02447.x
  22. Shirotani, K., D. Edbauer, S. Prokop, C. Haass, and H. Steiner. 2004. Identification of distinct $\gamma$-secretase complexes with different APH-1 variants. J. Biol. Chem. 279, 41340-41345. https://doi.org/10.1074/jbc.M405768200
  23. Susen, K. and A. Blöchl. 2005. Low concentrations of aggregated $\beta$-amyloid induce neurite formation via the neurotrophin receptor p75. J. Mol. Med. 83, 720-735. https://doi.org/10.1007/s00109-005-0671-3
  24. Tarr, P. E., R. Roncarati, G. Pelicci, P. G. Pelicci, and L. D'Adamio. 2002. Tyrosine phosphorylation of the $\beta$-amyloid precursor protein cytoplasmic tail promotes interaction with Shc. J. Biol. Chem. 277, 16798-16804. https://doi.org/10.1074/jbc.M110286200
  25. Tsui-Pierchala, B. A. and D. D. Ginty. 1999. Characterization of an NGF-P-TrkA retrograde-signaling complex and age-dependent regulation of TrkA phosphorylation in sympathetic neurons. J. Neurosci. 19, 8207-8218.
  26. Vassar, R. 2004. BACE1: the $\beta$-secretase Enzyme in Alzheimer's Disease. J. Mol. Neurosci. 23, 105-114. https://doi.org/10.1385/JMN:23:1-2:105
  27. Verdile, G., S. E. Gandy, and R. N. Martins. 2007. The role of presenilin and its interacting proteins in the biogenesis of Alzheimer's beta amyloid. Neurochem. Res. 32, 609-623. https://doi.org/10.1007/s11064-006-9131-x
  28. Vetrivel, K. S., P. Gong, J. W. Bowen, H. Cheng, Y. Chen, M. Carter, P. D. Nguyen, L. Placanica, F. T. Wieland, Y. M. Li, M. Z. Kounnas, and G. Thinakaran. 2007. Dual roles of the transmembrane protein p23/TMP21 in the modulation of amyloid precursor protein metabolism. Mol. Neurodegener. 2, 4. https://doi.org/10.1186/1750-1326-2-4
  29. Wang, Y. J., X. Wang, J. J. Lu, Q. X. Li, C. Y. Gao, X. H. Liu, Y. Sun, M. Yang, Y. Lim, G. Evin, J. H. Zhong, C. Masters, and X. F. Zhou. 2011. p75NTR regulates $A{\beta}$ deposition by increasing $A{\beta}$ production but inhibiting $A{\beta}$ aggregation with Its extracellular domain. J. Neurosci. 31, 2292-2304. https://doi.org/10.1523/JNEUROSCI.2733-10.2011
  30. Yu, G., M. Nishimura, S. Arawaka, D. Levitan, L. Zhang, A. Tandon, Y. Q. Song, E. Rogaeva, F. Chen, T. Kawarai, A. Supala, L. Levesque, H. Yu, D. S. Yang, E. Holmes, P. Milman, Y. Liang, D. M. Zhang, D. H. Xu, C. Sato, E. Rogaev, M. Smith, C. Janus, Y. Zhang, R. Aebersold, L. S. Farrer, S. Sorbi, A. Bruni, P. Fraser, and S. t. George-Hyslop. 2000. Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and ${\beta}APP$ processing. Nature 407, 48-54. https://doi.org/10.1038/35024009