肝癌衍生生長因子(hepatoma derived growth factor;HDGF)是一種新的生長因子，於肝癌細胞株中被發現，具有刺激纖維母細胞和肝癌細胞生長的能力。HDGF在結構、氨基酸序列和功能上類似於HMG蛋白。HDGF被認為在細胞週期和腫瘤增殖上扮演關鍵的角色。最近更發現HDGF在腎臟胚胎發育中扮演重要角色，並認為此因子為血管內皮增生因子。此外HDGF也和細胞間的相互作用(cell-cell interaction)及細胞移動(cell migration)有關；除此之外HDGF在血管的發育及疾病發生時具有刺激平滑肌細胞增殖的能力，且HDGF帶有兩個細胞核定位訊號(nuclear localization sequence；NLS)序列具nuclear targeting之功能，可刺激細胞生長。本研究利用HDGF的HATH domain來探討其進入NIH 3T3細胞的作用機制:由dominant negative dynamin細胞實驗顯示dynamin並非HATH domain進入細胞所必須;利用螢光顯微鏡的技術發現HATH domain會與巨胞飲作用(macropinocytosis)之標的物Dextran有位置重疊(co-localization)的情形。所以我們更進一步以巨胞飲作用的抑制劑證明HATH domain進入細胞的路徑會受抑制劑作用而遭到阻斷，所以推論HATH domain會經由類似巨胞飲作用的機制進入NIH 3T3細胞。

HDGF (hepatoma-derived growth factor) is a novel growth factor, identified from conditioned medium of hepatoma cell line. HDGF has growth stimulating activity for fibroblast and some hepatoma cells. HDGF, a novel defined growth factor with mitogenic effect, has homology protein sequence as HMG (high mobility group) protein and their three dimension structures appeared to be similar to each other. Recently, elevated HDGF expression was found in developing kidneys but less was found in adult kidney. In addition, HDGF expression was found to be correlated with angiogenic status of tissues. Thus, it is speculated that HDGF plays a role during embryonic development and angiogenesis. HDGF also plays a role in cell-cell interaction and cell migration. HDGF is a growth factor that is involved in stimulating vascular smooth muscle cells (SMCs) proliferation during development and in disease. HDGF contains a true bipartite nuclear localization sequence necessary for nuclear targeting. HDGF is sciential factor in stimulating of DNA replication and cell proliferation of vascular smooth muscle cell. Here, we investigate the NIH 3T3 cells internalization mechanism of the HATH domain of HDGF.
We found HATH domain internalization is dynamin-independent. By using confocal microscopy, we show that HATH domain was colocalized with Dextran. The different mechanisms of uptake and cellular processing were studied by treating with macropinocytosis pathway inhibitor to investigate HATH domain internalization. Since these inhibitors can block HATH domain uptake, we suggest that the internalization mechanism of HATH domain may be similar to macropinocytosis.

第五章 參考文獻

1. Nakamura, H., Izumoto, Y., Kambe, H., Kuroda, T., Mori, T., Kawamura, K. et al. (1994) Molecular cloning of complementary DNA for a novel human hepatoma-derived growth factor. Its homology with high mobility group-1 protein. J. Biol. Chem. 269：25143–25149.

2. Enomoto, H., Yoshida, K., Kishima, Y., Kinoshita, T., Yamamoto, M.,
Everett, A. D., Miyajima, A., and Nakamura, H. (2002). Hepatoma-derived growth factor is highly expressed in developing liver and promotes fetal hepatocyte proliferation. Hepatology. 36：1519-1527.

3. Oliver, J. A. & Al-Awqati, Q. (1998) An endothelial growth factor involved in rat renal development. J. Clin. Invest. 102：1208–1219.

4. Nakamura, H., Kambe, H., Egawa, T., Kimura, Y., Ito, H., Hayashi, E.,
Yamamoto, H., Sato, J., and Kishimoto, S. (1989) Partial purification and characterization of human hepatoma-derived growth factor. Clin. Chim. Acta. 183：273-284.

5. Shih-Che Sue1, Jeou-Yuan Chen1, Shao-Chen Lee2, Wen-guey Wu2 and Tai-huang Huang1,3* (2004) Solution Structure and Heparin Interaction of Human Hepatoma-derived Growth Factor. J. Mol. Biol. 343：1365-1377

6. Izumoto, Y., Kuroda, T., Harada, H., Kishimoto, T., and Nakamura, H. (1997). Hepatoma-derived growth factor belongs to a gene family in mice showing significant homology in the amino terminus. Biochem Biophys Res Commun. 238：26-32.

7. Ge, H., Si, Y. & Roeder, R. G. (1998) Isolation of cDNAs encoding novel transcription coactivators p52 and p75 reveals an alternate regulatory mechanism of transcriptional activation. EMBO J. 17：6723–6729.

8. Dietz, F., Franken, S., Yoshida, K., Nakamura, H., Kappler, J. & Gieselmann, V. (2002) The family of hepatoma-derived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies. Biochem. J. 366：491–500.
9. Kuroda, T., Tanaka, H., Nakamura, H., Nishimune, Y. & Kishimoto, T. (1999) Hepatoma-derived growth factor-related protein (HRP)-1 gene in spermatogenesis in mice. Biochem. Biophys. Res. Commun. 262：433–437.

10. Bustin, M., Lehn, D. A., and Landsman, D. (1990) Structural features of the HMG chromosomal proteins and their genes. Biochim. Biophys. Acta. 1049：231-243.

11. Shinohara, T., Singh, D. P. & Fatma, N. (2002) LEDGF, a survival factor, activates stress-related genes. Prog. Retin. Eye Res. 21：341–358.

12. Ikegame, K., Yamamoto, M., Kishima, Y., Ecomoto, H., Yoshida, K., Suemura, M. et al. (1999). A new member of a hepatoma derived growth factor gene family can translocate to the nucleus. Biochem. Biophys. Res. Commun. 266：81–87.

13. Javaherian, K., Sadeghi, M., and Liu, L. F. (1979) Nonhistone proteins HMG1 and HMG2 unwind DNA double helix. Nucleic Acids Res. 6：3569-3580.

14. Kuehl, L., Rechsteiner, M., and Wu, L. (1985) Relationship between the structure of chromosomal protein HMG1 and its accumulation in the cell nucleus. J. Biol. Chem. 260：10361-10368.

15. Shafritz, D. A. (1990) Regulation of liver gene expression during various physiologic and pathophysiologic states. Prog. Liver. Dis. 9：39-55

16. Thanos, D., and Maniatis, T. (1992) The high mobility group protein HMG I(Y) is required for NF-kappa B-dependent virus induction of the human IFN-beta gene. Cell. 71：777-789.

17. Everett, A. D. (2001) Identification, cloning, and developmental expression of hepatoma-derived growth factor in the developing rat heart. Dev. Dyn. 222：450-458.

18. Everett, A. D., Stoops, T., and McNamara, C. A. (2001) Nuclear targeting is required for hepatoma-derived growth factor-stimulated mitogenesis in vascular smooth muscle cells. J. Biol. Chem. 276： 37564-37568.

19. Kishima, Y., Yamamoto, H., Izumoto, Y., Yoshida, K., Enomoto, H., Yamamoto, M., Kuroda, T., Ito, H., Yoshizaki, K., and Nakamura, H. (2002a) Hepatoma-derived growth factor stimulates cell growth after translocation to the nucleus by nuclear localization signals. J. Biol. Chem. 277：10315-10322.

20. Everett, A. D., Lobe, D. R., Matsumura, M. E., Nakamura, H., and McNamara, C. A. (2000) Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development. J. Clin Invest. 105：567-575.

21. Matsuyama, A., Inoue, H., Shibuta, K., Tanaka, Y., Barnard, G. F., Sugimachi, K., and Mori, M. (2001) Hepatoma-derived growth factor is associated with reduced sensitivity to irradiation in esophageal cancer. Cancer Res 61：5714-5717.

22. Kishima, Y., Yoshida, K., Enomoto, H., Yamamoto, M., Kuroda, T., Okuda, Y., Uyama, H., and Nakamura, H. (2002b). Antisense oligonucleotides of hepatoma- derived growth factor (HDGF) suppress the proliferation of hepatoma cells. Hepatogastroenterology. 49：1639-1644.

23. Qiu, C., Sawada, K., Zhang, X., and Cheng, X. (2002) The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds. Nat. Struct. Biol. 9(3)：217-224

24. Nameki, N., Tochio, N., Koshiba, S., Inoue, M., Yabuki, T., Aoki, M., Seki, E., Matsuda, T., Fujikura, Y., Saito, M., Ikari, M., Watanabe, M., Terada, T., Shirouzu, M., Yoshida, M., Hirota, H., Tanaka, A., Hayashizaki, Y., Guntert, P., Kigawa, T., and Yokoyama, S. (2005) Solution structure of the PWWP domain of the hepatoma-derived growth factor family. Protein Sci. 14(3)：756-764

25. Slater, L. M., Allen, M. D., and Bycroft, M. (2003) Structural variation in PWWP domains. J. Mol. Biol. 330(3)：571-576

26. Ge, Y. Z., Pu, M. T., Gowher, H., Wu, H. P., Ding, J. P., Jeltsch, A., and Xu, G. L. (2004) Chromatin Targeting of de Novo DNA Methyltransferases by the PWWP Domain J. Biol. Chem. 279(24)：25447-25454

27. Kambe, H., Kishima, Y., Kuroda, T., Enomoto, H., Ogawa, H., and Nakmaura, H. (2000) Protein kinase C inhibitor, H-7 suppresses the growth activity of hepatoma-derived growth factor. Hepatogastroenterology. 47(36)：1645-164

28. Mukherjee, S.,Ghosh, R.N. Maxfield, F.R. (1997) Endocytosis. Physiol. Rev. 77(3)：759-803

29. Conner, Sean D. and Sandra L. Schmid. (2003) Regulated portals of entry into the cell. Nature. 422:37-44

30. W. H. Lewis. (1931) Pinocytosis. Bull. Johns Hopkins Hosp. 49：17-36

31. Sean D. Conner and Sandra L. Schmild. (2003) Regulated portals of entry into the cell. Nature. 6：422; 37-44

32. H. J.-P. Ryser and W.-C. Shen, (1986) Drug-poly(lysine) conjugates: their potential for chemotherapy and for the study of endocytosis, In G. Gregoriadis, J. Senior nad G. Poste (eds.), Targeting of drugs with synthetic systems, Plenum Publ. Co., New York, pp. 103-121

33. Pearse, B.M. (1988) Receptors compete for adaptors found in plasma membrane coated pits. EMBO Journal. 7(11)：3331-6

34. van der Bliek, A.M. and Meyerowitz, E.M. (1991) Dynamin-like protein encode by the Drosophila shibire gene associated with vesicular traffic. Nature. 351: 411-414

35. Hinshaw, J.E., Schmid, S.L. (1995) Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature. 374(6518)： 190-2

36. Schmid, Sandra L. (1997) Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu. Rev. Biochem. 66:511-548

37. Mousavi, Seyed Ali, Lene Malerod, Trond Berg, and Rune Kjeken. (2004) Clathrin-dependent endocytosis. Biochem. J. 377:1-16

38. Goldstein, J.L., Brown, M.S., Anderson, R.G., Russell, D.W., Schneider, W.J. (1985) Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1：1-39

39. Mellman, Ira. (1996) Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12:575-625

40. Anderson, R.G., Brown, M.S., Beisiegel, U., Goldstein, J.L. (1982) Surface distribution and recycling of the low density lipoprotein receptor as visualized with antireceptor antibodies. J. Cell Biol. 93(3)： 523-31

41. Anderson, R.G., Brown, M.S., Goldstein, J.L. (1977) Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell. 10(3)：351-64

42. Dunn, W.A., Hubbard, A.L. (1984) Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics. J. Cell Biol. 98(6), 2148-59

43. Sharma, D.K., Brown, J.C., Choudhury, A., Peterson, T.E., Holicky, E., Marks, D.L., Simari, R., Parton, R.G., and Pagano, R.E. (2004) Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol. Mol. Biol. Cell. 15：3114-3122

44. Anderson, R. G. (1998) The caveolae membrane system. Annu. Rev. Biochem. 67：199–225

45. Razani, B., Woodman, S. E. & Lisanti, M. P. (2002) Caveolae: From cell biology to animal physiology. Pharmacol. Rev. 54：431–467

46. Pelkmans, L., Puntener, D. & Helenius, A. (2002) Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science. 296：535–539

47. Thomsen, P., Roepstorff, K., Stahlhut, M. & van Deurs, B. (2002) Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol. Biol. Cell. 13：238–250

48. Ridley, A. J. (2001) Rho proteins: Linking signaling with membrane trafficking. Traffic. 2：303–310

49. Mellman, I. &Steinman, R. M. (2001) Dendritic cells: Specialized and regulated antigen processing machines. Cell 106：255–258

50. Steele-Mortimer, O., Knodler, L. A. & Finlay, B. B. (2000) Poisons, ruffles and rockets: Bacterial pathogens and the host cell cytoskeleton. Traffic. 1：107–118

51. Swanson, J.A., Colin, C. (1995) Macropinocytosis. Trends Cell Biol. 5(11):424-8.

52. Shpetner, H.S. and Vallee, R.B. (1989) Identification of dynamin, a novel mechanochemical enzyme that mediates interations between microtubules. Cell. 59:421-432

53. Chen, M.S., Obar, R.A., Schroder, C.C., Austin, T.W., Poodry, C.A., Wadsworth, S.C., and Vallee, R.B. (1991) Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature. 351:583-586

54. Sven Thoms and Ralf Erdmann. (2005) Dynamin-related proteins and Pexll proteins in peroxisome divison and proliferation. FEBS Journal. 272： 5169-5181

55. Kelly, R.B. (1999) New twists for dynamin. Nat Cell Biol. 1：E8-E9

56. 王佳蕙.(2005) Membrane Pore Formation and Internalization of Cobra Cardiotoxin in Myocytes: Mechanism and Cytotoxicity. 博士論文

57. Sallusto, F., Cella, M., Danieli, C., Lanzavecchia, A. (1995) Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182:389-400

58. West, M.A., Bretscher, M.S. & Watts, C. (1989) Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells. J. Cell Biol. 109：2731–2739

59. Rubtsova, S.N., et al. (1998) Disruption of actin microfilaments by cytochalasin D leads to activation of p53. FEBS Lett. 430：353-357

60. Urbanik, E., and Ware, B.R. (19889) Actin filament capping and cleaving activity of cytochalasins B, D, E, and H. Arch. Biochem. Biophys. 269： 181

61. Kirkham, M., and Parton, R.G. (2005) Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers. Biochim Biophys Acta. 1746：349-63 Review

62. Huth, U.S., Schubert, R., and Peschka-Suss, R. (2006) Investigating the uptake and intracellular fate of pH-sensitive liposomes by flow cytometry and spectral bio-imaging. J Control Release. 110：490-540