Sp1是屬於基本的轉錄因子，經由結合至目標基因啟動子富含GC區域以調控基因的轉錄活性，Sp1會將其他轉錄因子帶到目標基因啟動子且參與許多基因的轉錄調控。我們之前的研究指出，Sp1與熱休克蛋白九十α的交互作用對於A431細胞中12(S)-lipoxygenase的表現是重要的。而本研究中，我們發現利用熱休克蛋白九十抑制劑geldanamycin (GA)處理A431細胞時，可以降低ERK1/2及JNK活性，緊接著造成Sp1去磷酸化且導致12(S)-lipoxygenase的表現向下調節。在ERK1/2磷酸化Sp1的位子進行突變(T453A/T739A)，會降低Sp1被帶到12(S)-lipoxygenase啟動子。除此之外，Sp1在細胞有絲分裂期間可以和熱休克蛋白九十分佈在一起。在處理GA之後，Sp1在telophase期間沒有與子代的染色質再度分佈在一起，此現象會造成子代Sp1蛋白質的降解，且是透過ubiquitination的機制而導致Sp1蛋白質的不穩定性。還有，在處理GA之後的有絲分裂時期(M phase)之細胞，Sp1蛋白質也會呈現明顯的降解現象；但是在處理GA之後的間期(interphase)之細胞，Sp1蛋白質沒有降解現象。因此，細胞在進行有絲分裂時，Sp1必須透過熱休克蛋白九十將某些激脢活化而幫助Sp1磷酸化，使得Sp1順利通過有絲分裂而分給子代，防止Sp1被ubiquitin降解的命運。我們的結論是熱休克蛋白九十可以調控Sp1的DNA 鍵結能力和Sp1蛋白質的穩定性，因此對於Sp1調控其目標基因(12(S)-lipoxygenase)的轉錄活性是很重要的。

Simian virus 40 promoter factor 1 (Sp1) is a basic transcriptional factor that binds to the GC-rich region in the promoter of its target gene. It is involved in the transcription of numerous genes by recruiting other transcriptional factors to the promoters of target genes. Our previous study revealed that the interaction of heat shock protein (Hsp) 90 and Sp1 is important for the expression of 12(S)-lipoxygenase in A431 cells. In the present study, we found that treating A431 cells with geldanamycin (GA), an Hsp90 inhibitor, attenuated Erk1/2 and JNK activities which then resulted in the dephosphorylation of Sp1 and the downregulation of 12(S)-lipoxygenase. Mutation at the ERK1/2-phosphorylated residues of Sp1 (T453A/T739A) attenuated the recruitment of Sp1 to 12(S)-lipoxygenase promoter. In addition, Sp1 and Hsp90 co-localized during the mitotic period. After GA treatment, Sp1 did not be re-co-localized with the daughter chromosome during the telophase, which resulted in Sp1 degradation via an ubiquitination-dependent pathway. Furthermore, GA treatment also significantly reduced the level of Sp1 in the mitotic period, but GA treatment did not affect the level of Sp1 in the interphase. Therefore, Hsp90 was necessary to activate kinases that modulate Sp1 phosphorylation during mitosis. Then Hsp90 protected Sp1 from ubiquitination, which completed mitosis. We conclude that Hsp90 was important for the 12(S)-lipoxygenase transcriptional activity because it modulated the DNA binding affinity and stability of Sp1.

1. Abdelrahim, M., and Safe, S. (2005). Cyclooxygenase-2 inhibitors decrease vascular endothelial growth factor expression in colon cancer cells by enhanced degradation of Sp1 and Sp4 proteins. Mol Pharmacol 68, 317-329.
2. Benasciutti, E., Pages, G., Kenzior, O., Folk, W., Blasi, F., and Crippa, M. P. (2004). MAPK and JNK transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription. Blood 104, 256-262.
3. Bieker, J. J. (2001). Kruppel-like factors: three fingers in many pies. J Biol Chem 276, 34355-34358.
4. Bonello, M. R., and Khachigian, L. M. (2004). Fibroblast growth factor-2 represses platelet-derived growth factor receptor-alpha (PDGFR-alpha) transcription via ERK1/2-dependent Sp1 phosphorylation and an atypical cis-acting element in the proximal PDGFR-alpha promoter. J Biol Chem 279, 2377-2382.
5. Capdevila, J., Yadagiri, P., Manna, S., and Falck, J. R. (1986). Absolute configuration of the hydroxyeicosatetraenoic acids (HETEs) formed during catalytic oxygenation of arachidonic acid by microsomal cytochrome P-450. Biochem Biophys Res Commun 141, 1007-1011.
6. Chang, W. C. (2003). Cell signaling and gene regulation of human 12(S)-lipoxygenase expression. Prostaglandins Other Lipid Mediat 71, 277-285.
7. Chang, W. C., Liu, Y. W., Ning, C. C., Suzuki, H., Yoshimoto, T., and Yamamoto, S. (1993). Induction of arachidonate 12-lipoxygenase mRNA by epidermal growth factor in A431 cells. J Biol Chem 268, 18734-18739.
8. Chen, B. K., and Chang, W. C. (2000). Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase. Proc Natl Acad Sci U S A 97, 10406-10411.
9. Chun, R. F., Semmes, O. J., Neuveut, C., and Jeang, K. T. (1998). Modulation of Sp1 phosphorylation by human immunodeficiency virus type 1 Tat. J Virol 72, 2615-2629.
10. Citri, A., Kochupurakkal, B. S., and Yarden, Y. (2004). The achilles heel of ErbB-2/HER2: regulation by the Hsp90 chaperone machine and potential for pharmacological intervention. Cell Cycle 3, 51-60.
11. Clark, J. D., Schievella, A. R., Nalefski, E. A., and Lin, L. L. (1995). Cytosolic phospholipase A2. J Lipid Mediat Cell Signal 12, 83-117.
12. Courey, A. J., Holtzman, D. A., Jackson, S. P., and Tjian, R. (1989). Synergistic activation by the glutamine-rich domains of human transcription factor Sp1. Cell 59, 827-836.
13. DeBoer, C., Meulman, P. A., Wnuk, R. J., and Peterson, D. H. (1970). Geldanamycin, a new antibiotic. J Antibiot (Tokyo) 23, 442-447.
14. Dunah, A. W., Jeong, H., Griffin, A., Kim, Y. M., Standaert, D. G., Hersch, S. M., Mouradian, M. M., Young, A. B., Tanese, N., and Krainc, D. (2002). Sp1 and TAFII130 transcriptional activity disrupted in early Huntington's disease. Science 296, 2238-2243.
15. Dynan, W. S., and Tjian, R. (1983). The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell 35, 79-87.
16. Eustace, B. K., Sakurai, T., Stewart, J. K., Yimlamai, D., Unger, C., Zehetmeier, C., Lain, B., Torella, C., Henning, S. W., Beste, G., et al. (2004). Functional proteomic screens reveal an essential extracellular role for hsp90 alpha in cancer cell invasiveness. Nat Cell Biol 6, 507-514.
17. Garfinkel, M. D., Sollars, V. E., Lu, X., and Ruden, D. M. (2004). Multigenerational selection and detection of altered histone acetylation and methylation patterns: toward a quantitative epigenetics in Drosophila. Methods Mol Biol 287, 151-168.
18. Gasc, J. M., Renoir, J. M., Faber, L. E., Delahaye, F., and Baulieu, E. E. (1990). Nuclear localization of two steroid receptor-associated proteins, hsp90 and p59. Exp Cell Res 186, 362-367.
19. Gething, M. J., and Sambrook, J. (1992). Protein folding in the cell. Nature 355, 33-45.
20. Hamberg, M., and Samuelsson, B. (1974). Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Natl Acad Sci U S A 71, 3400-3404.
21. Hammarstrom, S., Hamberg, M., Samuelsson, B., Duell, E. A., Stawiski, M., and Voorhees, J. J. (1975). Increased concentrations of nonesterified arachidonic acid, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2alpha in epidermis of psoriasis. Proc Natl Acad Sci U S A 72, 5130-5134.
22. Harrison, S. M., Houzelstein, D., Dunwoodie, S. L., and Beddington, R. S. (2000). Sp5, a new member of the Sp1 family, is dynamically expressed during development and genetically interacts with Brachyury. Dev Biol 227, 358-372.
23. Huang, Y. C., Chen, J. Y., and Hung, W. C. (2004). Vitamin D3 receptor/Sp1 complex is required for the induction of p27Kip1 expression by vitamin D3. Oncogene 23, 4856-4861.
24. Hubert, D. A., Tornero, P., Belkhadir, Y., Krishna, P., Takahashi, A., Shirasu, K., and Dangl, J. L. (2003). Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein. Embo J 22, 5679-5689.
25. Hung, J. J., Wang, Y. T., and Chang, W. C. (2006). Sp1 deacetylation induced by phorbol ester recruits p300 to activate 12(S)-lipoxygenase gene transcription. Mol Cell Biol 26, 1770-1785.
26. Hung, J. J., Wu, C. Y., Liao, P. C., and Chang, W. C. (2005). Hsp90alpha recruited by Sp1 is important for transcription of 12(S)-lipoxygenase in A431 cells. J Biol Chem 280, 36283-36292.
27. Iwamoto, I., Yamazaki, H., Nakagawa, N., Kimura, A., Tomioka, H., and Yoshida, S. (1990). Differential effects of two C-terminal peptides of substance P on human neutrophils. Neuropeptides 16, 103-107.
28. Jez, J. M., Chen, J. C., Rastelli, G., Stroud, R. M., and Santi, D. V. (2003). Crystal structure and molecular modeling of 17-DMAG in complex with human Hsp90. Chem Biol 10, 361-368.
29. Kadonaga, J. T., Carner, K. R., Masiarz, F. R., and Tjian, R. (1987). Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51, 1079-1090.
30. Kang, H. T., Ju, J. W., Cho, J. W., and Hwang, E. S. (2003). Down-regulation of Sp1 activity through modulation of O-glycosylation by treatment with a low glucose mimetic, 2-deoxyglucose. J Biol Chem 278, 51223-51231.
31. Kimmins, S., and MacRae, T. H. (2000). Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins. Cell Stress Chaperones 5, 76-86.
32. Leggett, R. W., Armstrong, S. A., Barry, D., and Mueller, C. R. (1995). Sp1 is phosphorylated and its DNA binding activity down-regulated upon terminal differentiation of the liver. J Biol Chem 270, 25879-25884.
33. Liang, P., and MacRae, T. H. (1997). Molecular chaperones and the cytoskeleton. J Cell Sci 110 ( Pt 13), 1431-1440.
34. Liao, W. C., Geng, Y., and Johnson, L. F. (1994). In vitro transcription of the TATAA-less mouse thymidylate synthase promoter: multiple transcription start points and evidence for bidirectionality. Gene 146, 183-189.
35. Lin, L. L., Lin, A. Y., and Knopf, J. L. (1992). Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc Natl Acad Sci U S A 89, 6147-6151.
36. Lindquist, S., and Craig, E. A. (1988). The heat-shock proteins. Annu Rev Genet 22, 631-677.
37. Liu, Y. W., Arakawa, T., Yamamoto, S., and Chang, W. C. (1997). Transcriptional activation of human 12-lipoxygenase gene promoter is mediated through Sp1 consensus sites in A431 cells. Biochem J 324 ( Pt 1), 133-140.
38. Louvion, J. F., Warth, R., and Picard, D. (1996). Two eukaryote-specific regions of Hsp82 are dispensable for its viability and signal transduction functions in yeast. Proc Natl Acad Sci U S A 93, 13937-13942.
39. Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M., and Neckers, L. M. (2000). The heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized ATP-binding domain in the carboxyl terminus of the chaperone. J Biol Chem 275, 37181-37186.
40. Merchant, J. L., Du, M., and Todisco, A. (1999). Sp1 phosphorylation by Erk 2 stimulates DNA binding. Biochem Biophys Res Commun 254, 454-461.
41. Milanini-Mongiat, J., Pouyssegur, J., and Pages, G. (2002). Identification of two Sp1 phosphorylation sites for p42/p44 mitogen-activated protein kinases: their implication in vascular endothelial growth factor gene transcription. J Biol Chem 277, 20631-20639.
42. Miyamoto, T., Ogino, N., Yamamoto, S., and Hayaishi, O. (1976). Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J Biol Chem 251, 2629-2636.
43. Mortensen, E. R., Marks, P. A., Shiotani, A., and Merchant, J. L. (1997). Epidermal growth factor and okadaic acid stimulate Sp1 proteolysis. J Biol Chem 272, 16540-16547.
44. Muramatsu, T., Komori, K., Sakurai, N., Yamada, K., Awasaki, Y., Fukuda, K., and Oda, T. (1996). Primary structure of mannuronate lyases SP1 and SP2 from Turbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability. J Protein Chem 15, 709-719.
45. Pearl, L. H., and Prodromou, C. (2000). Structure and in vivo function of Hsp90. Curr Opin Struct Biol 10, 46-51.
46. Prodromou, C., Roe, S. M., O'Brien, R., Ladbury, J. E., Piper, P. W., and Pearl, L. H. (1997). Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90, 65-75.
47. Rohlff, C., Ahmad, S., Borellini, F., Lei, J., and Glazer, R. I. (1997). Modulation of transcription factor Sp1 by cAMP-dependent protein kinase. J Biol Chem 272, 21137-21141.
48. Russell, L. C., Whitt, S. R., Chen, M. S., and Chinkers, M. (1999). Identification of conserved residues required for the binding of a tetratricopeptide repeat domain to heat shock protein 90. J Biol Chem 274, 20060-20063.
49. Sanchez, E. R., Hirst, M., Scherrer, L. C., Tang, H. Y., Welsh, M. J., Harmon, J. M., Simons, S. S., Jr., Ringold, G. M., and Pratt, W. B. (1990). Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90. J Biol Chem 265, 20123-20130.
50. Scheibel, T., Weikl, T., and Buchner, J. (1998). Two chaperone sites in Hsp90 differing in substrate specificity and ATP dependence. Proc Natl Acad Sci U S A 95, 1495-1499.
51. Schneider, C., Sepp-Lorenzino, L., Nimmesgern, E., Ouerfelli, O., Danishefsky, S., Rosen, N., and Hartl, F. U. (1996). Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90. Proc Natl Acad Sci U S A 93, 14536-14541.
52. Shi, Q., Le, X., Abbruzzese, J. L., Peng, Z., Qian, C. N., Tang, H., Xiong, Q., Wang, B., Li, X. C., and Xie, K. (2001). Constitutive Sp1 activity is essential for differential constitutive expression of vascular endothelial growth factor in human pancreatic adenocarcinoma. Cancer Res 61, 4143-4154.
53. Sreedhar, A. S., Soti, C., and Csermely, P. (2004). Inhibition of Hsp90: a new strategy for inhibiting protein kinases. Biochim Biophys Acta 1697, 233-242.
54. Sugawara, T., Nomura, E., Nakajima, A., and Sakuragi, N. (2004). Characterization of binding between SF-1 and Sp1: predominant interaction of SF-1 with the N-terminal region of Sp1. J Endocrinol Invest 27, 133-141.
55. Suske, G. (1999). The Sp-family of transcription factors. Gene 238, 291-300.
56. Szpirer, J., Szpirer, C., Riviere, M., Levan, G., Marynen, P., Cassiman, J. J., Wiese, R., and DeLuca, H. F. (1991). The Sp1 transcription factor gene (SP1) and the 1,25-dihydroxyvitamin D3 receptor gene (VDR) are colocalized on human chromosome arm 12q and rat chromosome 7. Genomics 11, 168-173.
57. Taatjes, D. J., Naar, A. M., Andel, F., 3rd, Nogales, E., and Tjian, R. (2002). Structure, function, and activator-induced conformations of the CRSP coactivator. Science 295, 1058-1062.
58. Vicart, A., Lefebvre, T., Imbert, J., Fernandez, A., and Kahn-Perles, B. (2006). Increased chromatin association of Sp1 in interphase cells by PP2A-mediated dephosphorylations. J Mol Biol 364, 897-908.
59. Wang, Y. N., and Chang, W. C. (2003). Induction of disease-associated keratin 16 gene expression by epidermal growth factor is regulated through cooperation of transcription factors Sp1 and c-Jun. J Biol Chem 278, 45848-45857.
60. Wu, Y., Zhang, X., and Zehner, Z. E. (2003). c-Jun and the dominant-negative mutant, TAM67, induce vimentin gene expression by interacting with the activator Sp1. Oncogene 22, 8891-8901.
61. Xiao, H., Hasegawa, T., and Isobe, K. (2000). p300 collaborates with Sp1 and Sp3 in p21(waf1/cip1) promoter activation induced by histone deacetylase inhibitor. J Biol Chem 275, 1371-1376.
62. Yahara, I., Minami, Y., and Miyata, Y. (1998). The 90-kDa stress protein, Hsp90, is a novel molecular chaperone. Ann N Y Acad Sci 851, 54-60.
63. Yamamoto, S. (1992). Mammalian lipoxygenases: molecular structures and functions. Biochim Biophys Acta 1128, 117-131.
64. Young, J. C., Agashe, V. R., Siegers, K., and Hartl, F. U. (2004). Pathways of chaperone-mediated protein folding in the cytosol. Nat Rev Mol Cell Biol 5, 781-791.