This study demonstrates that HBx binds to PPARγ in the cytoplasm and interferes nuclear translocation of PPARγ, to cause suppression of the transcriptional activity and pro-apoptotic property of PPARγ.
1. ΗBx directly interacts with PPARγ in vitro and in vivo.
2. The subcellular localization of PPARγ was redistributed to the cytoplasm by HBx.
3. Transgenic expression of HBx (HBx-TG) in C57BL/6 mice did not alter expression of PPARγ in the liver tissues, while the amount of nuclear PPARγ was significantly reduced in the liver of HBx-TG mice compared to the normal control.
4. HBx inhibits the DNA binding activity of PPARγ.
5. The transcriptional activity of PPARγ was dose-dependently inhibited by HBx.
6. Cell viability was decreased with PPARγ ligand in HepG2, but this effect was not observed in HepG2.2.15.
39
7. Survival in HepG2 cells infected with Ad-GFP-HBx was significantly increased as compared with that in HepG2 infected with Ad-GFP after treatment with troglitazone.
HBx prevents the translocation to the nucleus and binding of PPARγ to DNA due to the complex formation in the cytoplasm, thus inhibits the transcriptional activity of PPARγ. It suggests that HBx modulates the function of PPARγ via protein-protein interaction and this modulation may play a role in the pathogenesis of HBV-associated liver diseases and contribute to the HBV-associated hepatocarcinogenesis.
40
References
1. Feitelson MA. Hepatitis B virus in hepatocarcinogenesis. J Cell Physiol 1999;181:188-202.
2. Nakatake H, Chisaka O, Yamamoto S, Matsubara K, Koshy R. Effect of X protein on transactivation of hepatitis B virus promoters and on viral replication. Virology 1993;195:305-14.
3. Kim CM, Koike K, Saito I, Miyamura T, Jay G. HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature 1991;351:317-20.
4. Seifer M, Gerlich WH. Increased growth of permanent mouse fibroblasts in soft agar after transfection with hepatitis B virus DNA. Arch Virol 1992;126:119-28.
5. Koike K, Moriya K, Yotsuyanagi H, Shintani Y, Fujie H, Tsutsumi T, et al.
Compensatory apoptosis in preneoplastic liver of a transgenic mouse model for viral hepatocarcinogenesis. Cancer Lett 1998;134:181-6.
6. Chisari FV, Klopchin K, Moriyama T, Pasquinelli C, Dunsford HA, Sell S, et al.
Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 1989;59:1145-56.
7. Takada S, Shirakata Y, Kaneniwa N, Koike K. Association of hepatitis B virus X protein with mitochondria causes mitochondrial aggregation at the nuclear periphery, leading to cell death. Oncogene 1999;18:6965-73.
8. Su F, Schneider RJ. Hepatitis B virus HBx protein sensitizes cells to apoptotic
41
killing by tumor necrosis factor alpha. Proc Natl Acad Sci U S A 1997;94:8744-9.
9. Su F, Theodosis CN, Schneider RJ. Role of NF-kappaB and myc proteins in apoptosis induced by hepatitis B virus HBx protein. J Virol 2001;75:215-25.
10. Yun C, Lee JH, Park H, Jin YM, Park S, Park K, et al. Chemotherapeutic drug, adriamycin, restores the function of p53 protein in hepatitis B virus X (HBx) protein-expressing liver cells. Oncogene 2000;19:5163-72.
11. Truant R, Antunovic J, Greenblatt J, Prives C, Cromlish JA. Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation. J Virol 1995;69:1851-9.
12. Elmore LW, Hancock AR, Chang SF, Wang XW, Chang S, Callahan CP, et al.
Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis. Proc Natl Acad Sci U S A 1997;94:14707-12.
13. Koike K, Moriya K, Iino S, Yotsuyanagi H, Endo Y, Miyamura T, et al. High-level expression of hepatitis B virus HBx gene and hepatocarcinogenesis in transgenic mice.
Hepatology 1994;19:810-9.
14. Yu DY, Moon HB, Son JK, Jeong S, Yu SL, Yoon H, et al. Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus X-protein. J Hepatol 1999;31:123-32.
15. Diao J, Garces R, Richardson CD. X protein of hepatitis B virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis. Cytokine Growth Factor Rev 2001;12:189-205.
42
16. Cheong JH, Yi M, Lin Y, Murakami S. Human RPB5, a subunit shared by eukaryotic nuclear RNA polymerases, binds human hepatitis B virus X protein and may play a role in X transactivation. EMBO J 1995;14:143-50.
17. Qadri I, Maguire HF, Siddiqui A. Hepatitis B virus transactivator protein X interacts with the TATA- binding protein. Proc Natl Acad Sci U S A 1995;92:1003-7.
18. Haviv I, Shamay M, Doitsh G, Shaul Y. Hepatitis B virus pX targets TFIIB in transcription coactivation. Mol Cell Biol 1998;18:1562-9.
19. Perini G, Oetjen E, Green MR. The hepatitis B pX protein promotes dimerization and DNA binding of cellular basic region/leucine zipper proteins by targeting the conserved basic region. J Biol Chem 1999;274:13970-7.
20. Kong HJ, Hong SH, Lee MY, Kim HD, Lee JW, Cheong J. Direct binding of hepatitis B virus X protein and retinoid X receptor contributes to phosphoenolpyruvate carboxykinase gene transactivation. FEBS Lett 2000;483:114-8.
21. Tugwood JD, Issemann I, Anderson RG, Bundell KR, McPheat WL, Green S. The mouse peroxisome proliferator activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl CoA oxidase gene. EMBO J 1992;11:433-9.
22. Zhang B, Marcus SL, Sajjadi FG, Alvares K, Reddy JK, Subramani S, et al.
Identification of a peroxisome proliferator-responsive element upstream of the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl- CoA dehydrogenase. Proc Natl Acad Sci U S A 1992;89:7541-5.
23. Aldridge TC, Tugwood JD, Green S. Identification and characterization of DNA
43
elements implicated in the regulation of CYP4A1 transcription. Biochem J 1995;306:473-9.
24. Vanden Heuvel JP. Peroxisome proliferator-activated receptors: a critical link among fatty acids, gene expression and carcinogenesis. J Nutr 1999;129:575S-80S.
25. Fajas L, Auboeuf D, Raspe E, Schoonjans K, Lefebvre AM, Saladin R, et al. The organization, promoter analysis, and expression of the human PPARgamma gene. J Biol Chem 1997;272:18779-89.
26. Fajas L, Fruchart JC, Auwerx J. PPARgamma3 mRNA: a distinct PPARgamma mRNA subtype transcribed from an independent promoter. FEBS Lett 1998;438:55-60.
27. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, et al. Ligand binding and co-activator assembly of the peroxisome proliferator- activated receptor-gamma.
Nature 1998;395:137-43.
28. Juge-Aubry C, Pernin A, Favez T, Burger AG, Wahli W, Meier CA, et al. DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements. Importance of the 5'-flanking region. J Biol Chem 1997;272:25252-9.
29. DiRenzo J, Soderstrom M, Kurokawa R, Ogliastro MH, Ricote M, Ingrey S, et al.
Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors.
Mol Cell Biol 1997;17:2166-76.
30. Rosen ED, Spiegelman BM. PPARgamma : a nuclear regulator of metabolism, differentiation, and cell growth. J Biol Chem 2001;276:37731-4.
44
31. Zhu Y, Alvares K, Huang Q, Rao MS, Reddy JK. Cloning of a new member of the peroxisome proliferator-activated receptor gene family from mouse liver. J Biol Chem 1993;268:26817-20.
32. Braissant O, Foufelle F, Scotto C, Dauca M, Wahli W. Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat. Endocrinology 1996;137:354-66.
33. Desvergne B, Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 1999;20:649-88.
34. Wu Z, Xie Y, Morrison RF, Bucher NL, Farmer SR. PPARgamma induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPalpha during the conversion of 3T3 fibroblasts into adipocytes. J Clin Invest 1998;101:22-32.
35. Brown KK, Henke BR, Blanchard SG, Cobb JE, Mook R, Kaldor I, et al. A novel N-aryl tyrosine activator of peroxisome proliferator-activated receptor-gamma reverses the diabetic phenotype of the Zucker diabetic fatty rat. Diabetes 1999;48:1415-24.
36. Reddy JK, Rao MS, Azarnoff DL, Sell S. Mitogenic and carcinogenic effects of a hypolipidemic peroxisome proliferator, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14, 643), in rat and mouse liver. Cancer Res 1979;39:152-61.
37. Peters JM, Cattley RC, Gonzalez FJ. Role of PPAR alpha in the mechanism of action of the nongenotoxic carcinogen and peroxisome proliferator Wy-14,643. Carcinogenesis 1997;18:2029-33.
38. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell
45 1996;87:159-70.
39. He TC, Chan TA, Vogelstein B, Kinzler KW. PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 1999;99:335-45.
40. Okano H, Shiraki K, Inoue H, Yamanaka T, Deguchi M, Sugimoto K, et al.
Peroxisome proliferator-activated receptor gamma augments tumor necrosis factor family-induced apoptosis in hepatocellular carcinoma. Anticancer Drugs 2002;13:59-65.
41. DuBois RN, Gupta R, Brockman J, Reddy BS, Krakow SL, Lazar MA. The nuclear eicosanoid receptor, PPARgamma, is aberrantly expressed in colonic cancers. Carcinogenesis 1998;19:49-53.
42. Kilgore MW, Tate PL, Rai S, Sengoku E, Price TM. MCF-7 and T47D human breast cancer cells contain a functional peroxisomal response. Mol Cell Endocrinol 1997;129:229-35.
43. Mueller E, Sarraf P, Tontonoz P, Evans RM, Martin KJ, Zhang M, et al. Terminal differentiation of human breast cancer through PPAR gamma. Mol Cell 1998;1:465-70.
44. Kubota T, Koshizuka K, Williamson EA, Asou H, Said JW, Holden S, et al. Ligand for peroxisome proliferator-activated receptor gamma (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo. Cancer Res 1998;58:3344-52.
45. Sarraf P, Mueller E, Jones D, King FJ, DeAngelo DJ, Partridge JB, et al.
Differentiation and reversal of malignant changes in colon cancer through PPARgamma. Nat Med 1998;4:1046-52.
46. Altiok S, Xu M, Spiegelman BM. PPARgamma induces cell cycle withdrawal:
inhibition of E2F/DP DNA- binding activity via down-regulation of PP2A. Genes Dev
46 1997;11:1987-98.
47. Elstner E, Muller C, Koshizuka K, Williamson EA, Park D, Asou H, et al. Ligands for peroxisome proliferator-activated receptorgamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc Natl Acad Sci U S A 1998;95:8806-11.
48. Sells MA, Chen ML, Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A 1987;84:1005-9.
49. Kim HI, Cha JY, Kim SY, Kim JW, Roh KJ, Seong JK, et al. Peroxisomal proliferator-activated receptor-gamma upregulates glucokinase gene expression in beta-cells.
Diabetes 2002;51:676-85.
50. Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, et al. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci U S A 1994;91:7355-9.
51. Spandau DF, Lee CH. Trans-activation of viral enhancers by the hepatitis B virus X protein. J Virol 1988;62:427-34.
52. Kim HI, Kim JW, Kim SH, Cha JY, Kim KS, Ahn YH. Identification and functional characterization of the peroxisomal proliferator response element in rat GLUT2 promoter.
Diabetes 2000;49:1517-24.
53. Kim J, Chwae YJ, Kim MY, Choi IH, Park JH, Kim SJ. Molecular basis of HLA-C recognition by p58 natural killer cell inhibitory receptors. J Immunol 1997;159:3875-82.
54. Wu Q, Li Y, Liu R, Agadir A, Lee MO, Liu Y, et al. Modulation of retinoic acid
47
sensitivity in lung cancer cells through dynamic balance of orphan receptors nur77 and COUP-TF and their heterodimerization. EMBO J 1997;16:1656-69.
55. Jiang S, Song MJ, Shin EC, Lee MO, Kim SJ, Park JH. Apoptosis in human hepatoma cell lines by chemotherapeutic drugs via Fas-dependent and Fas-independent pathways. Hepatology 1999;29:101-10.
56. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 1998;95:2509-14.
57. Fajas L, Debril MB, Auwerx J. Peroxisome proliferator-activated receptor-gamma:
from adipogenesis to carcinogenesis. J Mol Endocrinol 2001;27:1-9.
58. Altucci L, Gronemeyer H. Nuclear receptors in cell life and death. Trends Endocrinol Metab 2001;12:460-8.
59. Makino Y, Yoshikawa N, Okamoto K, Hirota K, Yodoi J, Makino I, et al. Direct association with thioredoxin allows redox regulation of glucocorticoid receptor function. J Biol Chem 1999;274:3182-8.
60. Moilanen AM, Poukka H, Karvonen U, Hakli M, Janne OA, Palvimo JJ.
Identification of a novel RING finger protein as a coregulator in steroid receptor-mediated gene transcription. Mol Cell Biol 1998;18:5128-39.
61. Tsutsumi T, Suzuki T, Shimoike T, Suzuki R, Moriya K, Shintani Y, et al. Interaction of hepatitis C virus core protein with retinoid X receptor alpha modulates its transcriptional activity. Hepatology 2002;35:937-46.
62. Nagy L, Kao HY, Chakravarti D, Lin RJ, Hassig CA, Ayer DE, et al. Nuclear
48
receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell 1997;89:373-80.
63. Glass CK, Rose DW, Rosenfeld MG. Nuclear receptor coactivators. Curr Opin Cell Biol 1997;9:222-32.
64. Folkers GE, van der Saag PT. Adenovirus E1A functions as a cofactor for retinoic acid receptor beta (RAR beta) through direct interaction with RAR beta. Mol Cell Biol 1995;15:5868-78.
65. Sista ND, Barry C, Sampson K, Pagano J. Physical and functional interaction of the Epstein-Barr virus BZLF1 transactivator with the retinoic acid receptors RAR alpha and RXR alpha. Nucleic Acids Res 1995;23:1729-36.
66. Henkler F, Hoare J, Waseem N, Goldin RD, McGarvey MJ, Koshy R, et al.
Intracellular localization of the hepatitis B virus HBx protein. J Gen Virol 2001;82:871-82.
67. Wang XW, Forrester K, Yeh H, Feitelson MA, Gu JR, Harris CC. Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc Natl Acad Sci U S A 1994;91:2230-4.
68. Mueller E, Smith M, Sarraf P, Kroll T, Aiyer A, Kaufman DS, et al. Effects of ligand activation of peroxisome proliferator-activated receptor gamma in human prostate cancer. Proc Natl Acad Sci U S A 2000;97:10990-5.
69. Tsubouchi Y, Sano H, Kawahito Y, Mukai S, Yamada R, Kohno M, et al. Inhibition of human lung cancer cell growth by the peroxisome proliferator-activated receptor-gamma agonists through induction of apoptosis. Biochem Biophys Res Commun 2000;270:400-5.
49
70. Butler R, Mitchell SH, Tindall DJ, Young CY. Nonapoptotic cell death associated with S-phase arrest of prostate cancer cells via the peroxisome proliferator-activated receptor gamma ligand, 15-deoxy-delta12,14-prostaglandin J2. Cell Growth Differ 2000;11:49-61.
50 국문요약
HBx 에 의한 PPARγ 의 전사활성 조절
최 윤 희
연세대학교 대학원 의과학사업단
<지도 김 세 종 교수>
B 형 간염바이러스 X 단백질(HBx)은 여러 세포 내 단백질과 상호 결합함 으로써 그 생물학적 기능을 수행하며, 간암발생과정에 중요한 역할을 하는 것 으로 알려져 왔다. PPARγ는 핵 수용체의 한 종류로, 세포내의 대사과정, 세포의
증식과 사멸 및 종양화에 관여하는 전사인자이다. 본 연구에서는 HBx 가
PPARγ와 세포질 내에서 결합하여 핵으로의 translocation 을 차단함으로써, PPARγ의 전사활성과 세포고사 유도 기능을 억제함을 밝혔다. HBx 와 PPARγ가 상호결합함을 관찰하였고, 사람 간세포주인 Chang 세포에 HBx 와 PPARγ를 동 시에 발현시킨 결과, PPARγ가 HBx 와 co-localization 하며 세포질 내에 더 많이 존재하는 것을 확인함으로써, 정상적으로 주로 핵 내에 존재하는 PPARγ가 핵 내로 translocation 되지 못하는 것을 관찰할 수 있었다. 대조 마우스와 HBx 형 질전환 마우스에서 HBx 과발현에 따른 PPARγ의 발현량에는 차이가 없음에도 불구하고, 핵 내 존재하는 PPARγ는 HBx 형질전환 쥐에서 상당히 감소되어 있
51
었다. 또한 HBx 는 PPARγ의 DNA 결합을 억제하였으며, 재조합 HBx 단백질을 핵단백질과 반응시켰을 때, HBx 농도 의존적으로 PPARγ의 DNA 결합능이 감소
되는 것을 확인하였다. PPARγ의 response element 를 포함하는 reporter 의 활성도 HBx 의 농도에 따라 현저히 억제됨을 관찰함으로써 HBx 에 의한 PPARγ 전사활 성억제를확인하였다. 세포증식을 억제하는 기능을 갖고 있는 것으로 알려진
PPARγ의 리간드인 troglitazone 을 사용하여 HepG2 와 HepG2.2.15 세포의 세포증 식을 비교해 본 결과, HepG2 세포에서는 troglitazone 에 의해 세포증식이 억제되 었음에 반해, HBV 가 형질 도입된 HepG2.2.15 세포에서는 이러한 증식억제 효 과를 볼 수 없었다. HepG2 세포주에 아데노바이러스를 이용한 HBx 이입시킨 결 과 또한 troglitazone 에 의한 세포증식억제가 유도되지 않았다. 이상의 결과로 HBx 는 PPARγ과 세포질 내에서 복합체를 이루어, PPARγ의 고유한 기능인 전사 활성 기능과 세포고사 유도 기능을 억제함을 확인하였고, 이러한 결과는 B 형 간염바이러스 감염으로 야기되는 간질환의 병인과정에 중요한 역할을 담당할 것으로 생각된다.
핵심되는 말 : HBx, PPARγ, 전사활성, 단백질−단백질간상호작용