胃泌素(gastrin)係於胃腸道及胰臟所表現之荷爾蒙，其可促進胃酸分泌外，亦能促使胃腸細胞增生及分化，而其轉錄調控是具有發育及組織特異性。已知在AGS（human gastric adenocarcinoma cell）細胞中，EGF可經由Ras-ERK pathway來調控gastrin 基因啟動區之活性表現；並且發現於gastrin基因啟動區上-68至-61 bp位置的Sp1 site為EGF的responsive element。本實驗室先前亦發現以PMA刺激AGS細胞可活化PKC及Raf-MEK-ERK訊息傳遞路徑並促進gastrin 基因啟動區的活化，且此受刺激細胞中之c-Jun蛋白亦能被誘導增加。由於本實驗室研究12(S)-lipoxygenase (12-LOX)基因表現之調控，已確認EGF及PMA均可誘發c-Jun蛋白的生合成，並於更進一步的實驗分析後發現，此c-Jun可結合於12-LOX基因啟動區之Sp1蛋白，而間接對12-LOX基因進行調控。所以本論文除了進一步探討PMA誘導gastrin基因活化方式外，更想瞭解上述之c-Jun/Sp1 交互作用機制是否在AGS cell系統中也參與在PMA引發調控gastrin 基因表現中 。由實驗結果得知PMA刺激AGS細胞後 gastrin mRNA會循不同時間點而有表現增加趨勢。經由gastrin基因啟動區上進行5’-連續性切除的報告基因 (reporter) 分析中發現-82到-40 bp及-40至＋66 bp區間是PMA誘導gastrin基因啟動區活性的重要位置。利用gel shift assay顯示Sp1會結合至-71/-51及-52/-31片段DNA probes。但進一步利用gastrin基因啟動區-142至+66 bp區間上之不同Sp1 sites點突變所得之結果顯示，此Sp1 sites主要影響在此啟動區的basal表現而不參與在PMA誘發的反應中。另外，gastrin基因啟動區上的TATA box及下游區域(-28至+66 bp)對於PMA調控gastrin 蛋白表現亦具有部分重要性。

The peptide hormone gastrin, expressed in the gastrointestinal tract and pancreas, is an important regulator of gastric acid secretion and growth factor of the gastrointestinal mucosa. The expression of gastrin was under tightly developmental and tissue specific control. It was reported that EGF activated the transcription of gastrin in human gastric adenocarcinoma AGS cells through Ras-ERK signaling pathway. Besides, the Sp1 binding site residing at –68 to –61 bp of gastrin promoter plays an important role in the EGF-induced gene expression. Previously, our laboratory had reported that EGF and PMA enhanced the biosynthesis of c-Jun in A431 cells, which subsequently induced the transcription of 12(S)-lipoxygenase by interacting with Sp1. We also found that treatment of the AGS cells with PMA enhanced the expression of c-Jun. Therefore, we want to identify the regulation mechanisms of PMA-induced expression of gastrin and to analyze the role of c-Jun/Sp1 interaction in the regulation of gastrin gene expression in AGS cells. In this report, we found that PMA, an activator of PKC, induced the gene expression of gastrin in a time-dependent manner in AGS cells. Transient transfection with a series of 5’-deletion mutants revealed that the 5’-flanking region spanning from -82 to -40 bp and –40 to +66 bp were important for the PMA-induced gastrin gene activation in AGS cells. From the result of gel shift assay, it indicated that Sp1 will bind with the DNA oligonucleotide probe spanning the promoter region –71/-51 and –52/-31. However, the Sp1 sites residing at -68 to -61 bp and -44 to -40 bp of gastrin promoter were not involved in PMA-induced expression of gastrin gene, but involved in basal expression. Except that, TATA box and the downstream element (-28 to +66 bp) also play an important role in PMA-induced gastrin activation in AGS cells.

Amemiya-Kudo, M., Shimano, H., Yoshikawa, T., Yahagi, N., Hasty, A. H., Okazaki, H., Tamura, Y., Shionoiri, F., Iizuka, Y., Ohashi, K., Osuga, J., Harada, K., Gotoda, T., Sato, R., Kimura, S., Ishibashi, S., and Yamada, N. Promoter analysis of the mouse sterol regulatory element-binding protein-1c gene. J. Biol. Chem. 275, 31078-31085, 2000

Baldwin, G. S., and Shulkes, A. Gastrin, gastrin receptors and colorectal carcinoma. Gut 42, 581-584, 1998

Bundgaard, J. R., Hansen, T. v. O, Friis-Hansen, L., Rourke, I. J., Solinge, W. W. van., Nielsen, F. C., and Refeld J. F. A distal Sp1-element is necessary for maximal activity of the human gastrin gene promoter. FEBS Letters 369, 225-228, 1995

Chang, J. M. (張家銘) Signal transduction of EGF-induced expression of cyclooxygenase 2 in A431 cells. 國立成功大學藥理學研究所碩士論文, 2000

Chen, B. K., Liu, Y. W., Yamamoto, S., and Chang, W. C. Overexpression of Ha-ras enhances the transcription of human arachidonate 12-lipoxygenase promoter in A431 cells. Biochim. Biophys. Acta. 1344, 270-277, 1997

Chen, B. K., Kung, H. C., Tsai, T. Y., and Chang, W. C. Essential role of mitogen-activated protein kinase pathway and c-Jun induction in epidermal growth factor-induced gene expression of human 12-lipoxygenase. Mol. Pharm. 57, 153-161, 2000(A)

Chen, B. K., and Chang, W. C. 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. 97, 10406-10411, 2000 (B)

Chung, D. C., Brand, S. J., and Tillotson, L. G. Mutually exclusive interactions between factors binding to adjacent Sp1 and AT-rich elements regulate gastrin gene transcription in insulinoma cells. J. Biol. Chem. 270, 8829-8836, 1995

Chupreta, S., Du, M., Todisco, A., and Merchant, J. L. EGF stimulates gastrin promoter through activation of Sp1 kinase activity. Am. J. Physiol. 278, C697-708 , 2000

Dembinski, A. B., Johnson, L. R. Growth of the pancreas and gastrointestinal mucosa by secretin, cerulein, and petagastrin. Endocrinology 106, 323-328, 1980
Dockray, G. J. Gastrin and gastric epithelial physiology. J. Phsio. 518(2), 315-324, 1999

Dockray, G. J. Gastrin, growth, and colon neoplasia. Gut 47, 747-748, 2000

Dockray, G. J. The Gastrins: Their Production and Biological activities. Annu. Rev. Physiol. 63, 119-139, 2001

Ford, M. G., Valle, J. D., Soroka, C. J., and Merchant, J. L. EGF receptor activation stimulates endogenous gastrin gene expression in canine G cells and human gastric cell cultures. J. Clin. Invest. 99, 2762-2771, 1997

Hansen, T. v. O., Bundgaard, J. R., Nielsen, F. C., and Rehfeld, J. F. Composite action of three GC/GT boxes in the proximal promoter region is important for gastrin gene transcriiption. Mol. Cell. Endocrino. 155, 1-8, 1999

Hocker, M., Henihan, R. J., Rosewicz, S., Riecken, E., Zhang, Z., Koh, T. J., and Wang, T. C. Gastrin and phorbol 12-myristate 13-acetate regulate the human histidine decarboxylase promoter through raf-dependent activation of extracellular signal-regulated kinase-related signaling pathways in gastric cancer cells. J. Biol. Chem. 272, 27015-27024, 1997

Ishizuka, J., Martines, J., Townsend, C. M. Jr., and Thompson, J. C. The effect of gastrin on growth of human stomach cancer cells. Ann. Surg. 215, 528-535, 1992

Iwase, K., Evers, B. M., Hellmich, M. R., Guo, Y. S., Higashide, S., Kim ,H. J., and Townsend, C. M. Jr. Regulation of growth of human gastric cancer by gastrin and glycine-extended progastrin. Gastroenterology 113, 782-790, 1997
Johnson, L. R. New aspect of the trophic action of gastrointestinal hormones. Gastroenterology 92, 788-792, 1977

Koh, T. J., and Chen, D. Gastrin as a growth factor in the gastrointestinal tract. Regulatory peptides 93, 37-44, 2000

Koh, T. J., Bulitta, C. J., Fleming, J. V., Dockray, G. J., Varro, A., and Wang, T. C. Gastrin is a target of the β-catanin/TCF4 growth-signaling pathway in a model of intestinal polyposis. J. Clin. Invest. 106, 533-539, 2000

Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D., and Rapp, U. R. Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 364, 249-252, 1993

Konturek, S. J., Konturek, P. C., Hartwich, A., and Hahn, E. G. Helicobacter pylori infection and gastrin and cyclooxygenase expression in gastric and colorectal malignancies. Regulatory peptides 93, 13-19, 2000

Majumdar, A., Johnson, L. R. Gastric mucosal cell proliferation during development in rats and effects of gastrin. Am. J. Physiol. 242, G135-G139, 1982

Marks, P., Iyer, G., Cui, Y., and Merchant, J. L. Fos is required for EGF stimulation of the gastrin promoter. Am. J. Physiol. 271, G942-G948, 1996

Merchant, J. L., Demediuk, B., and Brand, S. J. A GC-rich element confers epidermal growth factor responsiveness to transcription from the gastrin promoter. Mol. Cell. Biol. 11, 2686-2696, 1991

Merchant, J. L., Shiotani, A., Mortensen, E. R., Shumaker, D. K., and Abraczinskas, D. R. Epidermal growth factor stimulation of human gastrin promoter reguires Sp1. J Biol. Chem. 270, 6314-6319, 1995

Merchant, J. L., Iyer, G. R., Taylor, B. R., Kitchen, J. R., Mortensen, E. R., Wang, Z., Flintooft, R. J., Michel, J. B., and Bassel-Duby, R. ZBP-89 a Kruppel-like zinc finger protein, inhibits epidermal growth factor induction of the gastrin promoter. Mol. Cell. Biol. 16, 6644-6653, 1996

Merchant, J. L., Du, M., and Todisco, A. Sp1 phosphorylation by Erk2 stimulates DNA binding. Biochem. Biophy. Res. Commun. 254, 454-461, 1999

Miller, L. R., Jacobson, E. D., Johnson, L.R. Effect of petagastrin on gastric mucosal cells grown in tissue culture. Gastroenterology 64, 254-267, 1973

Mortensen, E. R., Marks, P. A., Shiotani, A., and Merchant, J. L. Epidermal growth factor and okadaic acid stimulate Sp1 Proteolysis. J. Biol. Chem. 272, 16540-16547, 1997

Piontek, M., Hengels, K., Porschen, R., and Strohmeyer, G. Protein kinase C and adenylate cyclase as targets for ggrowth inhibition of human gastric cancer cells. J. Cancer Res. Clin. Oncol. 119, 697-699, 1993

Pugh, B. F. Mechanisms of transcription complex assembly. Curr. Opin. Cell. Biol. 8, 303-311, 1996

Raychowdhury, R., Zhang, Z., Hocker, M. and Wang, T. C. Activation of human histidine decarboxylase gene promoter activity by gastrin is mediated by two distinct nuclear factors. J. Biol. Chem. 274, 20961-20969, 1999

Rozengurt, E. and Walsh, J. H. Gastrin, CCK, signaling, and cancer. Annu. Rev. Physiol. 63, 49-76, 2001

Shiotani, A., and Merchant, J. L. cAMP regulates gastrin gene expression. Am. J. Physiol. 269, G458-G464, 1995

Singh, P., Walker, J. P., Townsend, C. M. Jr., and Thompson, J. C. Role of gastrin and gastrin receptors on the growth of a transplanted mouse colon carcinoma (MC26) in BALB/c mice. Cancer Res. 46, 1612-1616, 1986

Singh, P., Rae-Venter, B., Townsend, C. M. Jr., Khalil, T., and Thompson, J. C. Gastrin receptors in normal and malignant gastrointestinal mucosa: age-associated changes. Am. J. Physiol. 249, G761-G769, 1989

Smith, A. M., and Watson, S. A. Gastin and gastrin receptor activation: an early event in the adenoma-carcinoma sequence. Gut 47, 820-824, 2000

Tillotson, L. G., Wang, T. C., amd Brand, S. J. Activation of gastrin transcription in pacreatic insulinoma cells by a CACC promoter element and a 70-kDa sequence-specific DNA-binding protein. J. Biol. Chem. 269, 2234-2240,
1994

Tillotson, L. G. RIN ZF, a novel zinc finger gene, encodes proteins that bind to the CACC element of the gastrin promoter. J. Biol. Chem. 274, 8123-8128, 1999

Townsend, C. M. Jr., Beauchamp, R. D., Singh, P., and Thompson, J. C. Growth factors and intestinal neoplasms. Am. J. Surg. 155, 526-536, 1988

Tsai, S.B. (蔡松柏) Signal transduction of PMA-induced gastrin expression in human adenocarcinoma AGS cells. 國立成功大學藥理學研究所碩士論文, 2001

Wang, T. C., and Brand, S. Islet cell-specific regulatory domain in the gastrin promoter contains adjacent positive and negative DNA elements. J. Biol. Chem. 265, 8908-8914, 1990

Watson, S. A., Durrant, L. G., Crosbie, J. D., and Morris, D. L. The in vitro growth response of primary human colorectal and gastric cancer cells to gastrin. Int. J. Cancer 43, 692-696, 1989 (A)

Watson, S. A., Durrant, L. G., and Morris, D. L. Gastrin: growth enhancing effects on human gastric and colonic tumor cells. Br. J. Cancer 59, 554-558, 1989 (B)

Winsett, O. E., Townsend, C. M. Jr., Glass, E. J., Rae-Venter, B., and Thompson, J. C. Gastrin stimulates growth of colon cancers. Surg. Forum. 33, 384-396, 1982

Zhang, Z., Hocker, M., Koh, T. J., and Wang, T. C. The human histidine decarboxylase promoter is regulated by gastrin and phorbol 12-myristate 13-acetate through a downstream cis-acting element. J. Biol. Chem. 271, 14188-14197, 1996

Zhu, G. H., Wong, B. C. Y., Eggo, M. C., Yuen, S. T., Lai, K. C., and Lam, S. K. Pharmacological inhibition of protein kinase C activity could induce apoptosis in gastric cancer cells by differential regulation of apoptosis-related genes. Digestive disease and science 44, 2020-2026, 1999

Zhu, G. H., Wong, B. C. Y., Eggo, M. C., Ching, C. K., Yuen, S. T., Chan, E. Y. T., Lai, K. C., and Lam, S. K. Non-seroidal anti-inflammatory drug-induced apoptosis in gastric cancer cells id blocked by protein kinase C activation through inhibition of c-myc. Brit.J.Cancer 79, 393-400, 1999

Zhu, G. H., Wong, B. C. Y., Slosberg, E. D., Eggo, M. C., Ching, C. K., Yuen,
S. T., Lai, K. C., Soh, J. W., Weinstein, I. B., and Lam, S. K. Over- expression of protein kinase C-β1 isoenzyme suppresses indomethacin- induced apoptosis in gastric epithelial cells. Gastroenterology 118, 507-514, 2000