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. 1997 May 15;324(Pt 1):133–140. doi: 10.1042/bj3240133

Transcriptional activation of human 12-lipoxygenase gene promoter is mediated through Sp1 consensus sites in A431 cells.

Y W Liu 1, T Arakawa 1, S Yamamoto 1, W C Chang 1
PMCID: PMC1218409  PMID: 9164849

Abstract

The functional 5' flanking region of the human 12-lipoxygenase in epidermoid carcinoma A431 cells was characterized. By a primer extension method, the transcription initiation sites were mapped at -47 adenosine, -48 guanosine and -55 guanosine upstream of the ATG translation start codon. Transient transfection with a series of 5' and 3' deletion constructs showed that the 5' flanking region spanning from -224 to -100 bp was important for the basal expression of 12-lipoxygenase gene. Gel mobility shift assays with antibodies of transcription factors showed that both Sp1 and Sp3 required highly GC-rich Sp1 sites within this region for binding. Disruption of two Sp1 recognition motifs residing at -158 to -150 bp and -123 to -114 bp by site-directed mutagenesis markedly reduced the basal 12-lipoxygenase promoter activity and abolished the retarded bands in a gel-shift assay, indicating that these two Sp1-binding sites were essential for gene expression. The same two Sp1-binding sites in this promoter region were also responsible for epidermal growth factor (EGF)-induced expression of 12-lipoxygenase gene. Moreover, EGF also induced the transcriptional activation of luciferase driven by SV40 early promoter, which contained rich Sp1-binding sites. Taken together, the results suggest that two specific Sp1 consensus sites are involved in the mediation of the basal promoter activity as well as EGF induction of the 12-lipoxygenase gene and that Sp1 and Sp3 transcription factors might have a role in their regulation.

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Selected References

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  1. Arakawa T., Nakamura M., Yoshimoto T., Yamamoto S. The transcriptional regulation of human arachidonate 12-lipoxygenase gene by NF kappa B/Rel. FEBS Lett. 1995 Apr 17;363(1-2):105–110. doi: 10.1016/0014-5793(95)00293-i. [DOI] [PubMed] [Google Scholar]
  2. Arakawa T., Oshima T., Kishimoto K., Yoshimoto T., Yamamoto S. Molecular structure and function of the porcine arachidonate 12-lipoxygenase gene. J Biol Chem. 1992 Jun 15;267(17):12188–12191. [PubMed] [Google Scholar]
  3. Benoist C., Chambon P. In vivo sequence requirements of the SV40 early promotor region. Nature. 1981 Mar 26;290(5804):304–310. doi: 10.1038/290304a0. [DOI] [PubMed] [Google Scholar]
  4. Chang W. C., Liu Y. W., Ning C. C., Suzuki H., Yoshimoto T., Yamamoto S. Induction of arachidonate 12-lipoxygenase mRNA by epidermal growth factor in A431 cells. J Biol Chem. 1993 Sep 5;268(25):18734–18739. [PubMed] [Google Scholar]
  5. Chang W. C., Nakao J., Orimo H., Murota S. Effects of reduced glutathione on the 12-lipoxygenase pathways in rat platelets. Biochem J. 1982 Mar 15;202(3):771–776. doi: 10.1042/bj2020771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang W. C., Ning C. C., Lin M. T., Huang J. D. Epidermal growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells. J Biol Chem. 1992 Feb 25;267(6):3657–3666. [PubMed] [Google Scholar]
  7. Chen B. K., Liu Y. W., Yamamoto S., Chang W. C. Overexpression of Ha-ras enhances the transcription of human arachidonate 12-lipoxygenase promoter in A431 cells. Biochim Biophys Acta. 1997 Feb 18;1344(3):270–277. doi: 10.1016/s0005-2760(96)00151-8. [DOI] [PubMed] [Google Scholar]
  8. Chen X. S., Kurre U., Jenkins N. A., Copeland N. G., Funk C. D. cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localizations of murine 12-lipoxygenases. J Biol Chem. 1994 May 13;269(19):13979–13987. [PubMed] [Google Scholar]
  9. Daniel S., Zhang S., DePaoli-Roach A. A., Kim K. H. Dephosphorylation of Sp1 by protein phosphatase 1 is involved in the glucose-mediated activation of the acetyl-CoA carboxylase gene. J Biol Chem. 1996 Jun 21;271(25):14692–14697. doi: 10.1074/jbc.271.25.14692. [DOI] [PubMed] [Google Scholar]
  10. Dynan W. S., Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983 Nov;35(1):79–87. doi: 10.1016/0092-8674(83)90210-6. [DOI] [PubMed] [Google Scholar]
  11. Funk C. D., Funk L. B., FitzGerald G. A., Samuelsson B. Characterization of human 12-lipoxygenase genes. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3962–3966. doi: 10.1073/pnas.89.9.3962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gill G., Pascal E., Tseng Z. H., Tjian R. A glutamine-rich hydrophobic patch in transcription factor Sp1 contacts the dTAFII110 component of the Drosophila TFIID complex and mediates transcriptional activation. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):192–196. doi: 10.1073/pnas.91.1.192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hamberg M., Samuelsson B. Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3400–3404. doi: 10.1073/pnas.71.9.3400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hammarström S., Hamberg M., Samuelsson B., Duell E. A., Stawiski M., Voorhees J. J. 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. 1975 Dec;72(12):5130–5134. doi: 10.1073/pnas.72.12.5130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hussain H., Shornick L. P., Shannon V. R., Wilson J. D., Funk C. D., Pentland A. P., Holtzman M. J. Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis. Am J Physiol. 1994 Jan;266(1 Pt 1):C243–C253. doi: 10.1152/ajpcell.1994.266.1.C243. [DOI] [PubMed] [Google Scholar]
  17. Izumi T., Hoshiko S., Rådmark O., Samuelsson B. Cloning of the cDNA for human 12-lipoxygenase. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7477–7481. doi: 10.1073/pnas.87.19.7477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jackson S. P., Tjian R. O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell. 1988 Oct 7;55(1):125–133. doi: 10.1016/0092-8674(88)90015-3. [DOI] [PubMed] [Google Scholar]
  19. Kollmar R., Farnham P. J. Site-specific initiation of transcription by RNA polymerase II. Proc Soc Exp Biol Med. 1993 Jun;203(2):127–139. doi: 10.3181/00379727-203-43583. [DOI] [PubMed] [Google Scholar]
  20. Leggett R. W., Armstrong S. A., Barry D., Mueller C. R. Sp1 is phosphorylated and its DNA binding activity down-regulated upon terminal differentiation of the liver. J Biol Chem. 1995 Oct 27;270(43):25879–25884. doi: 10.1074/jbc.270.43.25879. [DOI] [PubMed] [Google Scholar]
  21. Li J. M., Nichols M. A., Chandrasekharan S., Xiong Y., Wang X. F. Transforming growth factor beta activates the promoter of cyclin-dependent kinase inhibitor p15INK4B through an Sp1 consensus site. J Biol Chem. 1995 Nov 10;270(45):26750–26753. doi: 10.1074/jbc.270.45.26750. [DOI] [PubMed] [Google Scholar]
  22. Liu Y. W., Chen B. K., Chen C. J., Arakawa T., Yoshimoto T., Yamamoto S., Chang W. C. Epidermal growth factor enhances transcription of human arachidonate 12-lipoxygenase in A431 cells. Biochim Biophys Acta. 1997 Jan 7;1344(1):38–46. doi: 10.1016/s0005-2760(96)00128-2. [DOI] [PubMed] [Google Scholar]
  23. Lu J., Lee W., Jiang C., Keller E. B. Start site selection by Sp1 in the TATA-less human Ha-ras promoter. J Biol Chem. 1994 Feb 18;269(7):5391–5402. [PubMed] [Google Scholar]
  24. Merchant J. L., Shiotani A., Mortensen E. R., Shumaker D. K., Abraczinskas D. R. Epidermal growth factor stimulation of the human gastrin promoter requires Sp1. J Biol Chem. 1995 Mar 17;270(11):6314–6319. doi: 10.1074/jbc.270.11.6314. [DOI] [PubMed] [Google Scholar]
  25. Nakamura M., Ueda N., Kishimoto K., Yoshimoto T., Yamamoto S., Ishimura K. Immunocytochemical localization of platelet-type arachidonate 12-lipoxygenase in mouse blood cells. J Histochem Cytochem. 1995 Mar;43(3):237–244. doi: 10.1177/43.3.7868854. [DOI] [PubMed] [Google Scholar]
  26. Nugteren D. H. Arachidonate lipoxygenase in blood platelets. Biochim Biophys Acta. 1975 Feb 20;380(2):299–307. doi: 10.1016/0005-2760(75)90016-8. [DOI] [PubMed] [Google Scholar]
  27. Pushpa-Rekha T. R., Burdsall A. L., Oleksa L. M., Chisolm G. M., Driscoll D. M. Rat phospholipid-hydroperoxide glutathione peroxidase. cDNA cloning and identification of multiple transcription and translation start sites. J Biol Chem. 1995 Nov 10;270(45):26993–26999. doi: 10.1074/jbc.270.45.26993. [DOI] [PubMed] [Google Scholar]
  28. Takahashi Y., Reddy G. R., Ueda N., Yamamoto S., Arase S. Arachidonate 12-lipoxygenase of platelet-type in human epidermal cells. J Biol Chem. 1993 Aug 5;268(22):16443–16448. [PubMed] [Google Scholar]
  29. Toussaint C., Bousquet-Lemercier B., Garlatti M., Hanoune J., Barouki R. Testis-specific transcription start site in the aspartate aminotransferase housekeeping gene promoter. J Biol Chem. 1994 May 6;269(18):13318–13324. [PubMed] [Google Scholar]
  30. Yamamoto S. Mammalian lipoxygenases: molecular structures and functions. Biochim Biophys Acta. 1992 Oct 30;1128(2-3):117–131. doi: 10.1016/0005-2760(92)90297-9. [DOI] [PubMed] [Google Scholar]
  31. Yoshimoto T., Arakawa T., Hada T., Yamamoto S., Takahashi E. Structure and chromosomal localization of human arachidonate 12-lipoxygenase gene. J Biol Chem. 1992 Dec 5;267(34):24805–24809. [PubMed] [Google Scholar]
  32. Yoshimoto T., Miyamoto Y., Ochi K., Yamamoto S. Arachidonate 12-lipoxygenase of porcine leukocyte with activity for 5-hydroxyeicosatetraenoic acid. Biochim Biophys Acta. 1982 Dec 13;713(3):638–646. [PubMed] [Google Scholar]

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