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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

1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês

10.1074/jbc.272.43.27015

1083-351X

Michael Höcker, Robert J. Henihan, Stefan Rosewicz, Ernst–Otto Riecken, Zhensheng Zhang, Theodore J. Koh, Timothy C. Wang,

Nuclear Receptors and Signaling

Gastrin stimulates transcription of the human histidine decarboxylase (HDC) gene through binding to the G-protein-coupled cholecystokinin-B/gastrin receptor. We have explored the possibility that mitogen-activated protein kinase cascades play a role in mediating the effects of gastrin on transcription in a gastric cancer (AGS-B) cell line. Gastrin and phorbol 12-myristate 13-acetate (PMA) treatment of AGS-B cells was found to increase the phosphorylation of tyrosine residues of extracellular signal-regulated kinases (ERKs) 1 and 2 and increase ERK activity as determined by thein vitro phosphorylation of myelin basic protein. Reporter gene assays also demonstrated that gastrin and PMA stimulated Elk-1- and c-Myc-dependent transactivation, consistent with gastrin- and PMA-induced activation of ERKs. Overexpression of wild type ERK-1 and ERK-2 or activation of endogenous ERKs using activated MEK-1 (mitogen-activated protein kinase kinase or ERK kinase) overexpression stimulated HDC promoter activity in a dose-dependent fashion. Interruption of the ERK-related pathway using expression vectors for kinase-deficient ERKs or an ERK-specific phosphatase (PAC-1) blocked gastrin- and PMA-stimulated HDC promoter activity. In contrast, inhibition of the Jun kinase pathway using an interfering dominant negative SEK-1 (stress-activated protein kinase/ERK-1) mutant did not inhibit HDC promoter activity. Furthermore, whereas gastrin stimulated phosphorylation of Shc proteins and association with Grb2, activation of the HDC promoter was not influenced by expression of dominant negative Ras (N15 or N17) proteins. However, gastrin stimulated Raf-1 kinase activity, and activation of the HDC promoter was blocked by coexpression of a dominant negative Raf-1 construct. Overall, these data demonstrate that gastrin regulates HDC transcription in a Rafdependent, Ras-independent fashion predominantly through activation of the ERK-related pathway. Gastrin stimulates transcription of the human histidine decarboxylase (HDC) gene through binding to the G-protein-coupled cholecystokinin-B/gastrin receptor. We have explored the possibility that mitogen-activated protein kinase cascades play a role in mediating the effects of gastrin on transcription in a gastric cancer (AGS-B) cell line. Gastrin and phorbol 12-myristate 13-acetate (PMA) treatment of AGS-B cells was found to increase the phosphorylation of tyrosine residues of extracellular signal-regulated kinases (ERKs) 1 and 2 and increase ERK activity as determined by thein vitro phosphorylation of myelin basic protein. Reporter gene assays also demonstrated that gastrin and PMA stimulated Elk-1- and c-Myc-dependent transactivation, consistent with gastrin- and PMA-induced activation of ERKs. Overexpression of wild type ERK-1 and ERK-2 or activation of endogenous ERKs using activated MEK-1 (mitogen-activated protein kinase kinase or ERK kinase) overexpression stimulated HDC promoter activity in a dose-dependent fashion. Interruption of the ERK-related pathway using expression vectors for kinase-deficient ERKs or an ERK-specific phosphatase (PAC-1) blocked gastrin- and PMA-stimulated HDC promoter activity. In contrast, inhibition of the Jun kinase pathway using an interfering dominant negative SEK-1 (stress-activated protein kinase/ERK-1) mutant did not inhibit HDC promoter activity. Furthermore, whereas gastrin stimulated phosphorylation of Shc proteins and association with Grb2, activation of the HDC promoter was not influenced by expression of dominant negative Ras (N15 or N17) proteins. However, gastrin stimulated Raf-1 kinase activity, and activation of the HDC promoter was blocked by coexpression of a dominant negative Raf-1 construct. Overall, these data demonstrate that gastrin regulates HDC transcription in a Rafdependent, Ras-independent fashion predominantly through activation of the ERK-related pathway. The CCK 1The abbreviations used are: CCK, cholecystokinin; HDC, histidine decarboxylase; PMA, phorbol 12-myristate 13-acetate; GAS-RE, gastrin response element; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase or ERK kinase; JNK, Jun kinase; kb, kilobase; hHDC, human HDC; Luc, luciferase; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; WT, wild type; MT, mutant; DN, dominant negative; SEK, stress-activated protein kinase/ERK-1; TK, thymidine kinase; MBP, myelin basic protein. 1The abbreviations used are: CCK, cholecystokinin; HDC, histidine decarboxylase; PMA, phorbol 12-myristate 13-acetate; GAS-RE, gastrin response element; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase or ERK kinase; JNK, Jun kinase; kb, kilobase; hHDC, human HDC; Luc, luciferase; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; WT, wild type; MT, mutant; DN, dominant negative; SEK, stress-activated protein kinase/ERK-1; TK, thymidine kinase; MBP, myelin basic protein.-B/gastrin receptor, a member of the family of G-protein-coupled receptors that contain seven membrane-spanning regions, has a high affinity for both gastrin and cholecystokinin octapeptide (CCK-8) (1Kopin A.S. Lee Y.-M. McBride E.W. Miller L.J. Lu M. Lin H. Kalowski L.F. Beinborn M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3605-3609Crossref PubMed Scopus (471) Google Scholar, 2Wank S.A. Pisenga J.R. deWeerth A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8691-8695Crossref PubMed Scopus (462) Google Scholar, 3Nakata H. Matsui T. Ito M. Taniguchi T. Naribayashi Y. Avima N. Nakamura A. Kinoshita Y. Chiara K. Hosoda S. et al.Biochem. Biophys. Res. Commun. 1992; 187: 1151-1157Crossref PubMed Scopus (116) Google Scholar). Agonist binding to this receptor has been shown to activate a phospholipase C that hydrolyzes phosphatidylinositol bisphosphate, generating inositol 1,4,5-trisphosphate and diacylglycerol, which mobilizes intracellular Ca2+ and activates protein kinase C, respectively (4Seva C. Scemama J.L. Pradayrol L. Sarfati P.D. Vaysse N. Regul. Pept. 1994; 52: 31-38Crossref PubMed Scopus (35) Google Scholar, 5Bertrand V. Bastie M.J. Vaysse N. Pradayrol L. Int. J. Cancer. 1994; 56: 427-432Crossref PubMed Scopus (39) Google Scholar). Whereas CCK-B/gastrin receptors have been detected in both the brain and the pancreas of most mammalian species, the major target of gastrin in the gastrointestinal tract is the enterochromaffin-like cell of the gastric corpus. Gastrin stimulation of enterochromaffin-like cells leads to increased histamine secretion and elevated activity of histidine decarboxylase (HDC). HDC catalyzes the decarboxylation ofl-histidine and thus is the rate-limiting enzyme in the stomach for the generation of histamine, the major gastric acid secretogogue. Studies from a number of groups have now shown that gastrin-stimulated HDC enzymatic activity is paralleled by increased HDC gene expression in the gastric corpus and isolated enterochromaffin-like cells (6Höcker M. Zhang Z. Fenstermacher D.A. Tågerud S. Chulak M.B. Joseph D. Wang T.C. Am. J. Physiol. 1996; 270: G619-G633Crossref PubMed Google Scholar, 7Dimaline R. Sandvik A.K. FEBS Lett. 1991; 281: 20-22Crossref PubMed Scopus (91) Google Scholar, 8Chen E. Monstein H.-J. Nylander Z.-G. Zhao C.-M. Sundler F. Håkanson R. Gastroenterology. 1994; 107: 18-27Abstract Full Text PDF PubMed Google Scholar, 9Sandvik A.K. Dimaline R. Marvik R. Brenna E. Waldum R.H.L. Am. J. Physiol. 1994; 267: G254-G258PubMed Google Scholar). In addition, targeted disruption of the CCK-B/gastrin receptor in mice leads to decreased expression of the HDC gene in the gastric corpus (10Nagata A. Ito M. Iwata N. Kuno J. Takano H. Minowa O. Chihara K. Matsui T. Noda T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11825-11830Crossref PubMed Scopus (219) Google Scholar, 11Langhans N. Rindi G. Chiu M. Rehfeld J.F. Ardman B. Beinborn M. Kopin A.S. Gastroenterology. 1997; 112: 280-286Abstract Full Text PDF PubMed Scopus (185) Google Scholar).To study transcriptional regulation of HDC by gastrin, we developed the AGS-B cell line, a human gastric cancer cell line stably expressing the recombinant human CCK-B/gastrin receptor (6Höcker M. Zhang Z. Fenstermacher D.A. Tågerud S. Chulak M.B. Joseph D. Wang T.C. Am. J. Physiol. 1996; 270: G619-G633Crossref PubMed Google Scholar). Experiments from our laboratory have shown that the HDC promoter is regulated transcriptionally by gastrin preferentially through a protein kinase C-dependent signaling pathway (6Höcker M. Zhang Z. Fenstermacher D.A. Tågerud S. Chulak M.B. Joseph D. Wang T.C. Am. J. Physiol. 1996; 270: G619-G633Crossref PubMed Google Scholar). In addition, recent studies from our laboratory have provided evidence that transcriptional stimulation of the HDC gene by gastrin or phorbol 12-myristate 13-acetate (PMA) is mediated by a 23-nucleotide cis-acting element, the gastrin response element (GAS-RE), which is located downstream of the transcriptional start site (12Zhang Z. Höcker M. Koh T.J. Wang T.C. J. Biol. Chem. 1996; 271: 14188-14197Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Gastrin and PMA treatment of AGS-B cells induces enhanced binding of a nuclear factor to the GAS-RE. In a subsequent study, we found activation of AP-1 to be essential for gastrin-stimulated HDC transcription, although the mechanism is likely indirect (13Höcker M. Zhang Z. Merchant J.M. Wang T.C. Am. J. Physiol. 1997; 35: 6822-6830Google Scholar).The signal transduction pathways that mediate the activation of specific transcriptional effects by the CCK-B/gastrin receptor have not been defined fully. Although the CCK-B/gastrin receptor has been linked to phospholipase C and activation of protein kinase C (4Seva C. Scemama J.L. Pradayrol L. Sarfati P.D. Vaysse N. Regul. Pept. 1994; 52: 31-38Crossref PubMed Scopus (35) Google Scholar, 5Bertrand V. Bastie M.J. Vaysse N. Pradayrol L. Int. J. Cancer. 1994; 56: 427-432Crossref PubMed Scopus (39) Google Scholar), the steps downstream of protein kinase C activation are not entirely clear. Because the mitogen-activated kinase (MAPK) pathway has been characterized as an important signaling route for the transmission of protein kinase C effects on the activity of the AP-1 complex (14Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar), MAPK-regulated signaling could represent an important pathway for mediating the transcriptional effects of gastrin. The best studied members of the MAPK family are the extracellular signal-regulated kinases (ERKs), 42- and 44-kDa serine/threonine kinases encoded by the ERK-2 and ERK-1 genes, respectively (14Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). The ERKs are activated enzymatically through tyrosine and threonine phosphorylation by their upstream activator kinases MEK (MAPK kinase or ERK kinase) in response to growth-promoting factors acting on a variety of cell surface receptors (15Cobb M. Goldsmith E. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar). Following activation, the ERKs up-regulate the biological activity of a number of downstream targets, including transcription factors such as c-Myc, c-Fos, and p62 TCF, through specific phosphorylation of these factors (14Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). The other branch of the MAPK family is represented by the JUN kinases (JNKs), which are activated by tumor necrosis factor α and agents that induce cellular stress but can also be activated through signaling by G-protein-coupled receptors.These observations led us to investigate a possible role for the MAPK cascades in mediating the effects of gastrin on activation of the HDC promoter. We find that the ERK pathway is essential for transmission of the gastrin effects on the transcriptional activity of the human HDC promoter and that the CCK-B/gastrin receptors expressed in AGS-B gastric cancer cells are linked through a Ras-independent, Raf-dependent mechanism to ERK signaling cascades.DISCUSSIONOur studies suggest that the ERK-dependent signaling pathways mediate the stimulatory effects of gastrin on the transcription of the HDC gene. Several different lines of evidence are consistent with this conclusion. Overexpression of wild type ERK-1 and ERK-2 induced HDC promoter activity dose-dependently, suggesting a regulatory function for these kinases on the activity of the HDC promoter. A second approach involved expression of a constitutively active form of MEK-1 which would be expected to activate endogenous ERKs in AGS-B cells. Previous studies have shown that the MEK-1 mutant Δ-N3-S218E-S222D results in constitutive activity as well as loss of responsiveness to upstream activators (20Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-969Crossref PubMed Scopus (1254) Google Scholar). Thus, although overexpression of WT MEK-1 resulted in a minimal increase in HDC promoter activity, the Δ-N3-S218E-S222D mutant form of MEK-1 was able to increase by severalfold the expression of an HDC reporter gene but showed no response to gastrin or PMA. These data support the concept that activation of the endogenous ERKs is sufficient to stimulation transcription of the HDC gene and that gastrin and PMA regulate the HDC promoter through a MEK-1-dependent signaling route.In addition, we found that gastrin and PMA were able to stimulate tyrosine phosphorylation of ERK-1 and ERK-2 and kinase activity in gastric cancer (AGS) cells. Previous studies have shown that ERK activity is elevated after stimulation of CCK-B/gastrin receptors in several different cell types (29Taniguchi T. Matsui T. Ito M. Murayama T. Tsukamoto T. Katakami Y. Chiba T. Chihara K. Oncogene. 1994; 9: 861-867PubMed Google Scholar, 30Seva C. Kowalski-Chauvel A. Blanchet J.-S. Vaysse N. Pradayrol L. FEBS Lett. 1996; 378: 74-78Crossref PubMed Scopus (44) Google Scholar, 31Todisco A. Takeuchi Y. Seva C. Dickinson C.J. Yamada T. J. Biol. Chem. 1995; 270: 28337-28341Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), but they did not address the role in activating important downstream targets. We found that activation of the CCK-B/gastrin receptor in AGS-B cells was sufficient to activate ERK-regulated transcription factors such as c-Myc and Elk-1, consistent with an important role for this pathway in the transmission of gastrin-dependent signals into the nucleus. Importantly, we found that expression of kinase-deficient ERKs or the ERK-specific phosphatase PAC-1 which interfere with the ERK cascade blocked completely gastrin- and PMA-induced activation of the HDC gene. Overall, these findings demonstrate that gastrin activates the ERK cascade and that this pathway represents the major signaling route for gastrin-dependent regulation of the HDC promoter.Because we have shown recently that activation of c-jun and AP-1 leads to increased HDC transcription (13Höcker M. Zhang Z. Merchant J.M. Wang T.C. Am. J. Physiol. 1997; 35: 6822-6830Google Scholar), we also investigated a role for JNK-dependent signaling. The JNKs (JNK-1 and JNK-2) are distant relatives of the MAPKs and were identified initially based on their ability to phosphorylate and activate c-Jun. The murine SEK-1 gene (and the human homologue, MKK4) are upstream kinases that activate JNKs by phosphorylation of the Thr-Pro-Tyr sequence (14Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). JNKs are activated by agents that induce cellular stress, such as UV light and tumor necrosis factor α, although JNKs may also be activated by tyrosine kinase growth factors such as epidermal growth factor in a Ras-dependent manner. Recently, Dabrowskiet al. (32Dabrowski A. Grady T. Logsdon C.D. Williams J.A. J. Biol. Chem. 1996; 271: 5686-5690Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) have shown that JNKs are activated rapidly in isolated rat pancreatic acini after CCK-A receptor activation.Overall, our studies do not suggest an important role for the JNK pathway in the activation of HDC by gastrin. First, although our studies using the GAL4-c-Jun/GAL4-Luc system indicate that c-Jun phosphorylation and transactivation are increased by gastrin stimulation, we have shown that the activation is most likely secondary to ERK activation rather than to activation of JNK pathways. Second, although our data indicate that activation of JNK (via overexpression of SEK-1) can activate HDC promoter activity, this pathway is clearly distinct from the gastrin pathway. Gastrin stimulation of HDC promoter activity was blocked completely by the kinase-deficient ERK-1 mutant, whereas SEK-1 stimulation of HDC was not blocked by the kinase-deficient ERK-1. The lack of effect of the dominant negative SEK-1 mutant, which blocks JNK activation by extracellular stimuli without interfering with the ERK pathway, on gastrin stimulation of HDC promoter activity also suggests that the JNK pathway is probably not required for the gastrin-stimulated response. Finally, neither gastrin nor PMA appeared to be able to stimulate phosphorylation of JNK in AGS-B cells.Recent studies indicate that G-protein-coupled receptors can activate the ERKs through several different mechanisms (33Post G.R. Brown J.H. FASEB J. 1996; 10: 741-749Crossref PubMed Scopus (198) Google Scholar). Gi-coupled receptors, such as the thrombin (34LaMorte V.J. Kennedy E.D. Collins L.R. Goldstein D. Harootunian A.T. Brown J.H. Feramisco J.R. J. Biol. Chem. 1993; 268: 19411-19415Abstract Full Text PDF PubMed Google Scholar), lysophosphatidic acid (35Howe L.R. Marshall C.J. J. Biol. Chem. 1993; 268: 20717-20720Abstract Full Text PDF PubMed Google Scholar), α2-adrenergic (36Alblas J. van Corven E.J. Hordijk P.L. Milligan G. Moolenaar W.H. J. Biol. Chem. 1993; 268: 22235-22238Abstract Full Text PDF PubMed Google Scholar), and m2 muscarinic (37Winitz S. Russell M. Qian N.X. Gardner A. Dwyer L. Johnson G.L. J. Biol. Chem. 1993; 268: 19196-19199Abstract Full Text PDF PubMed Google Scholar), are similar to tyrosine kinase receptors in that they activate the ERKs through a Ras-dependent pathway. Activation of Gi-coupled receptors leads to tyrosine phosphorylation of Shc and its association with Grb2, a protein that links growth factor receptors to the Ras guanine nucleotide exchange factors. Activation of the ERKs by Gi-coupled receptors is mediated by the G-protein ॆγ subunits and thus can be blocked not only by dominant negative RasN17 but also by coexpression of a ॆγ-binding COOH-terminal peptide derived from ॆARK1 (ॆARK1ct) (38Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar). However, the signaling pathways coupling Gq-linked receptors to ERK activation are less clear and may involve Ras-independent pathways. For example, stimulation of ERK activity by Gq-coupled receptors such as m1 muscarinic and the α1-adrenergic is insensitive to the Gॆγ-sequestering ॆARK1cf peptide and RasN17 (38Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar, 39Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Instead, ERK activation by Gq-coupled receptors occurs predominantly through a Ras-insensitive, protein kinase C-dependent pathway (40van Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The GTP-bound α-subunit of the Gq/11 protein activates phosphoinositide hydrolysis and protein kinase C, which then stimulate ERK activation via a poorly understood mechanism involving the activation of Raf-1 kinase (41Kolch W. Heldecker G. Kochs G. Hummel R. Vahldl H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1152) Google Scholar, 42Troppmair J. Brudger J.T. Munoz H. Lloyd P.A. Kyriakis J. Banerjee P. Avruch J. Rapp U.R. J. Biol. Chem. 1994; 269: 7030-7035Abstract Full Text PDF PubMed Google Scholar).Several studies have suggested that the Ras pathway can be activated through agonist stimulation of members of the CCK receptor family (30Seva C. Kowalski-Chauvel A. Blanchet J.-S. Vaysse N. Pradayrol L. FEBS Lett. 1996; 378: 74-78Crossref PubMed Scopus (44) Google Scholar,43Duan R.-D. Williams J.A. Am. J. Physiol. 1994; 267: G401-G408PubMed Google Scholar). However, these studies did not address the issue as to whether activation of Ras is important for receptor-dependent cellular functions such as the transcriptional regulation of cell-specific genes. Our results in AGS-B cells demonstrate that CCK-B/gastrin receptor activation leads to Shc tyrosine phosphorylation association with Grb2, suggesting possible activation of the Ras pathway. However, although these assays support the idea that Ras is most likely activated by gastrin, stimulation of HDC promoter activity was unaffected by the RasN17 or RasN15 dominant negative mutants. In contrast, gastrin-stimulated Raf-1 kinase activity and expression of the dominant negative Raf-1 protein blocked completely gastrin-stimulated activity of the HDC promoter. Thus, taken together, our results suggest that in gastric cells, the CCK-B/gastrin receptor is linked through a Ras-independent, Raf-dependent mechanism to ERK-related signaling pathways, which are essential for the regulation of HDC transcriptional activity by this G-protein-coupled receptor.Finally, overexpression of ERK-1/2 appeared to activate the HDC promoter through the previously identified gastrin response element or GAS-RE, a 23-base pair element located downstream of the start site. The mechanism by which gastrin stimulation activates the GAS-RE remains unclear but presumably involves increased binding by a novel nuclear factor (the GAS-RE-binding protein) (12Zhang Z. Höcker M. Koh T.J. Wang T.C. J. Biol. Chem. 1996; 271: 14188-14197Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Further studies will be required to determine if the GAS-RE-binding protein indeed represents a novel downstream target for the ERK pathway. The CCK 1The abbreviations used are: CCK, cholecystokinin; HDC, histidine decarboxylase; PMA, phorbol 12-myristate 13-acetate; GAS-RE, gastrin response element; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase or ERK kinase; JNK, Jun kinase; kb, kilobase; hHDC, human HDC; Luc, luciferase; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; WT, wild type; MT, mutant; DN, dominant negative; SEK, stress-activated protein kinase/ERK-1; TK, thymidine kinase; MBP, myelin basic protein. 1The abbreviations used are: CCK, cholecystokinin; HDC, histidine decarboxylase; PMA, phorbol 12-myristate 13-acetate; GAS-RE, gastrin response element; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase or ERK kinase; JNK, Jun kinase; kb, kilobase; hHDC, human HDC; Luc, luciferase; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; WT, wild type; MT, mutant; DN, dominant negative; SEK, stress-activated protein kinase/ERK-1; TK, thymidine kinase; MBP, myelin basic protein.-B/gastrin receptor, a member of the family of G-protein-coupled receptors that contain seven membrane-spanning regions, has a high affinity for both gastrin and cholecystokinin octapeptide (CCK-8) (1Kopin A.S. Lee Y.-M. McBride E.W. Miller L.J. Lu M. Lin H. Kalowski L.F. Beinborn M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3605-3609Crossref PubMed Scopus (471) Google Scholar, 2Wank S.A. Pisenga J.R. deWeerth A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8691-8695Crossref PubMed Scopus (462) Google Scholar, 3Nakata H. Matsui T. Ito M. Taniguchi T. Naribayashi Y. Avima N. Nakamura A. Kinoshita Y. Chiara K. Hosoda S. et al.Biochem. Biophys. Res. Commun. 1992; 187: 1151-1157Crossref PubMed Scopus (116) Google Scholar). Agonist binding to this receptor has been shown to activate a phospholipase C that hydrolyzes phosphatidylinositol bisphosphate, generating inositol 1,4,5-trisphosphate and diacylglycerol, which mobilizes intracellular Ca2+ and activates protein kinase C, respectively (4Seva C. Scemama J.L. Pradayrol L. Sarfati P.D. Vaysse N. Regul. Pept. 1994; 52: 31-38Crossref PubMed Scopus (35) Google Scholar, 5Bertrand V. Bastie M.J. Vaysse N. Pradayrol L. Int. J. Cancer. 1994; 56: 427-432Crossref PubMed Scopus (39) Google Scholar). Whereas CCK-B/gastrin receptors have been detected in both the brain and the pancreas of most mammalian species, the major target of gastrin in the gastrointestinal tract is the enterochromaffin-like cell of the gastric corpus. Gastrin stimulation of enterochromaffin-like cells leads to increased histamine secretion and elevated activity of histidine decarboxylase (HDC). HDC catalyzes the decarboxylation ofl-histidine and thus is the rate-limiting enzyme in the stomach for the generation of histamine, the major gastric acid secretogogue. Studies from a number of groups have now shown that gastrin-stimulated HDC enzymatic activity is paralleled by increased HDC gene expression in the gastric corpus and isolated enterochromaffin-like cells (6Höcker M. Zhang Z. Fenstermacher D.A. Tågerud S. Chulak M.B. Joseph D. Wang T.C. Am. J. Physiol. 1996; 270: G619-G633Crossref PubMed Google Scholar, 7Dimaline R. Sandvik A.K. FEBS Lett. 1991; 281: 20-22Crossref PubMed Scopus (91) Google Scholar, 8Chen E. Monstein H.-J. Nylander Z.-G. Zhao C.-M. Sundler F. Håkanson R. Gastroenterology. 1994; 107: 18-27Abstract Full Text PDF PubMed Google Scholar, 9Sandvik A.K. Dimaline R. Marvik R. Brenna E. Waldum R.H.L. Am. J. Physiol. 1994; 267: G254-G258PubMed Google Scholar). In addition, targeted disruption of the CCK-B/gastrin receptor in mice leads to decreased expression of the HDC gene in the gastric corpus (10Nagata A. Ito M. Iwata N. Kuno J. Takano H. Minowa O. Chihara K. Matsui T. Noda T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11825-11830Crossref PubMed Scopus (219) Google Scholar, 11Langhans N. Rindi G. Chiu M. Rehfeld J.F. Ardman B. Beinborn M. Kopin A.S. Gastroenterology. 1997; 112: 280-286Abstract Full Text PDF PubMed Scopus (185) Google Scholar). To study transcriptional regulation of HDC by gastrin, we developed the AGS-B cell line, a human gastric cancer cell line stably expressing the recombinant human CCK-B/gastrin receptor (6Höcker M. Zhang Z. Fenstermacher D.A. Tågerud S. Chulak M.B. Joseph D. Wang T.C. Am. J. Physiol. 1996; 270: G619-G633Crossref PubMed Google Scholar). Experiments from our laboratory have shown that the HDC promoter is regulated transcriptionally by gastrin preferentially through a protein kinase C-dependent signaling pathway (6Höcker M. Zhang Z. Fenstermacher D.A. Tågerud S. Chulak M.B. Joseph D. Wang T.C. Am. J. Physiol. 1996; 270: G619-G633Crossref PubMed Google Scholar). In addition, recent studies from our laboratory have provided evidence that transcriptional stimulation of the HDC gene by gastrin or phorbol 12-myristate 13-acetate (PMA) is mediated by a 23-nucleotide cis-acting element, the gastrin response element (GAS-RE), which is located downstream of the transcriptional start site (12Zhang Z. Höcker M. Koh T.J. Wang T.C. J. Biol. Chem. 1996; 271: 14188-14197Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Gastrin and PMA treatment of AGS-B cells induces enhanced binding of a nuclear factor to the GAS-RE. In a subsequent study, we found activation of AP-1 to be essential for gastrin-stimulated HDC transcription, although the mechanism is likely indirect (13Höcker M. Zhang Z. Merchant J.M. Wang T.C. Am. J. Physiol. 1997; 35: 6822-6830Google Scholar). The signal transduction pathways that mediate the activation of specific transcriptional effects by the CCK-B/gastrin receptor have not been defined fully. Although the CCK-B/gastrin receptor has been linked to phospholipase C and activation of protein kinase C (4Seva C. Scemama J.L. Pradayrol L. Sarfati P.D. Vaysse N. Regul. Pept. 1994; 52: 31-38Crossref PubMed Scopus (35) Google Scholar, 5Bertrand V. Bastie M.J. Vaysse N. Pradayrol L. Int. J. Cancer. 1994; 56: 427-432Crossref PubMed Scopus (39) Google Scholar), the steps downstream of protein kinase C activation are not entirely clear. Because the mitogen-activated kinase (MAPK) pathway has been characterized as an important signaling route for the transmission of protein kinase C effects on the activity of the AP-1 complex (14Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar), MAPK-regulated signaling could represent an important pathway for mediating the transcriptional effects of gastrin. The best studied members of the MAPK family are the extracellular signal-regulated kinases (ERKs), 42- and 44-kDa serine/threonine kinases encoded by the ERK-2 and ERK-1 genes, respectively (14Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). The ERKs are activated enzymatically through tyrosine and threonine phosphorylation by their upstream activator kinases MEK (MAPK kinase or ERK kinase) in response to growth-promoting factors acting on a variety of cell surface receptors (15Cobb M. Goldsmith E. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1657) Google Scholar). Following activation, the ERKs up-regulate the biological activity of a number of downstream targets, including transcription factors such as c-Myc, c-Fos, and p62 TCF, through specific phosphorylation of these factors (14Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). The other branch of the MAPK family is represented by the JUN kinases (JNKs), which are activated by tumor necrosis factor α and agents that induce cellular stress but can also be activated through signaling by G-protein-coupled receptors. These observations led us to investigate a possible role for the MAPK cascades in mediating the effects of gastrin on activation of the HDC promoter. We find that the ERK pathway is essential for transmission of the gastrin effects on the transcriptional activity of the human HDC promoter and that the CCK-B/gastrin receptors expressed in AGS-B gastric cancer cells are linked through a Ras-independent, Raf-dependent mechanism to ERK signaling cascades. DISCUSSIONOur studies suggest that the ERK-dependent signaling pathways mediate the stimulatory effects of gastrin on the transcription of the HDC gene. Several different lines of evidence are consistent with this conclusion. Overexpression of wild type ERK-1 and ERK-2 induced HDC promoter activity dose-dependently, suggesting a regulatory function for these kinases on the activity of the HDC promoter. A second approach involved expression of a constitutively active form of MEK-1 which would be expected to activate endogenous ERKs in AGS-B cells. Previous studies have shown that the MEK-1 mutant Δ-N3-S218E-S222D results in constitutive activity as well as loss of responsiveness to upstream activators (20Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-969Crossref PubMed Scopus (1254) Google Scholar). Thus, although overexpression of WT MEK-1 resulted in a minimal increase in HDC promoter activity, the Δ-N3-S218E-S222D mutant form of MEK-1 was able to increase by severalfold the expression of an HDC reporter gene but showed no response to gastrin or PMA. These data support the concept that activation of the endogenous ERKs is sufficient to stimulation transcription of the HDC gene and that gastrin and PMA regulate the HDC promoter through a MEK-1-dependent signaling route.In addition, we found that gastrin and PMA were able to stimulate tyrosine phosphorylation of ERK-1 and ERK-2 and kinase activity in gastric cancer (AGS) cells. Previous studies have shown that ERK activity is elevated after stimulation of CCK-B/gastrin receptors in several different cell types (29Taniguchi T. Matsui T. Ito M. Murayama T. Tsukamoto T. Katakami Y. Chiba T. Chihara K. Oncogene. 1994; 9: 861-867PubMed Google Scholar, 30Seva C. Kowalski-Chauvel A. Blanchet J.-S. Vaysse N. Pradayrol L. FEBS Lett. 1996; 378: 74-78Crossref PubMed Scopus (44) Google Scholar, 31Todisco A. Takeuchi Y. Seva C. Dickinson C.J. Yamada T. J. Biol. Chem. 1995; 270: 28337-28341Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), but they did not address the role in activating important downstream targets. We found that activation of the CCK-B/gastrin receptor in AGS-B cells was sufficient to activate ERK-regulated transcription factors such as c-Myc and Elk-1, consistent with an important role for this pathway in the transmission of gastrin-dependent signals into the nucleus. Importantly, we found that expression of kinase-deficient ERKs or the ERK-specific phosphatase PAC-1 which interfere with the ERK cascade blocked completely gastrin- and PMA-induced activation of the HDC gene. Overall, these findings demonstrate that gastrin activates the ERK cascade and that this pathway represents the major signaling route for gastrin-dependent regulation of the HDC promoter.Because we have shown recently that activation of c-jun and AP-1 leads to increased HDC transcription (13Höcker M. Zhang Z. Merchant J.M. Wang T.C. Am. J. Physiol. 1997; 35: 6822-6830Google Scholar), we also investigated a role for JNK-dependent signaling. The JNKs (JNK-1 and JNK-2) are distant relatives of the MAPKs and were identified initially based on their ability to phosphorylate and activate c-Jun. The murine SEK-1 gene (and the human homologue, MKK4) are upstream kinases that activate JNKs by phosphorylation of the Thr-Pro-Tyr sequence (14Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). JNKs are activated by agents that induce cellular stress, such as UV light and tumor necrosis factor α, although JNKs may also be activated by tyrosine kinase growth factors such as epidermal growth factor in a Ras-dependent manner. Recently, Dabrowskiet al. (32Dabrowski A. Grady T. Logsdon C.D. Williams J.A. J. Biol. Chem. 1996; 271: 5686-5690Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) have shown that JNKs are activated rapidly in isolated rat pancreatic acini after CCK-A receptor activation.Overall, our studies do not suggest an important role for the JNK pathway in the activation of HDC by gastrin. First, although our studies using the GAL4-c-Jun/GAL4-Luc system indicate that c-Jun phosphorylation and transactivation are increased by gastrin stimulation, we have shown that the activation is most likely secondary to ERK activation rather than to activation of JNK pathways. Second, although our data indicate that activation of JNK (via overexpression of SEK-1) can activate HDC promoter activity, this pathway is clearly distinct from the gastrin pathway. Gastrin stimulation of HDC promoter activity was blocked completely by the kinase-deficient ERK-1 mutant, whereas SEK-1 stimulation of HDC was not blocked by the kinase-deficient ERK-1. The lack of effect of the dominant negative SEK-1 mutant, which blocks JNK activation by extracellular stimuli without interfering with the ERK pathway, on gastrin stimulation of HDC promoter activity also suggests that the JNK pathway is probably not required for the gastrin-stimulated response. Finally, neither gastrin nor PMA appeared to be able to stimulate phosphorylation of JNK in AGS-B cells.Recent studies indicate that G-protein-coupled receptors can activate the ERKs through several different mechanisms (33Post G.R. Brown J.H. FASEB J. 1996; 10: 741-749Crossref PubMed Scopus (198) Google Scholar). Gi-coupled receptors, such as the thrombin (34LaMorte V.J. Kennedy E.D. Collins L.R. Goldstein D. Harootunian A.T. Brown J.H. Feramisco J.R. J. Biol. Chem. 1993; 268: 19411-19415Abstract Full Text PDF PubMed Google Scholar), lysophosphatidic acid (35Howe L.R. Marshall C.J. J. Biol. Chem. 1993; 268: 20717-20720Abstract Full Text PDF PubMed Google Scholar), α2-adrenergic (36Alblas J. van Corven E.J. Hordijk P.L. Milligan G. Moolenaar W.H. J. Biol. Chem. 1993; 268: 22235-22238Abstract Full Text PDF PubMed Google Scholar), and m2 muscarinic (37Winitz S. Russell M. Qian N.X. Gardner A. Dwyer L. Johnson G.L. J. Biol. Chem. 1993; 268: 19196-19199Abstract Full Text PDF PubMed Google Scholar), are similar to tyrosine kinase receptors in that they activate the ERKs through a Ras-dependent pathway. Activation of Gi-coupled receptors leads to tyrosine phosphorylation of Shc and its association with Grb2, a protein that links growth factor receptors to the Ras guanine nucleotide exchange factors. Activation of the ERKs by Gi-coupled receptors is mediated by the G-protein ॆγ subunits and thus can be blocked not only by dominant negative RasN17 but also by coexpression of a ॆγ-binding COOH-terminal peptide derived from ॆARK1 (ॆARK1ct) (38Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar). However, the signaling pathways coupling Gq-linked receptors to ERK activation are less clear and may involve Ras-independent pathways. For example, stimulation of ERK activity by Gq-coupled receptors such as m1 muscarinic and the α1-adrenergic is insensitive to the Gॆγ-sequestering ॆARK1cf peptide and RasN17 (38Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar, 39Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Instead, ERK activation by Gq-coupled receptors occurs predominantly through a Ras-insensitive, protein kinase C-dependent pathway (40van Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The GTP-bound α-subunit of the Gq/11 protein activates phosphoinositide hydrolysis and protein kinase C, which then stimulate ERK activation via a poorly understood mechanism involving the activation of Raf-1 kinase (41Kolch W. Heldecker G. Kochs G. Hummel R. Vahldl H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1152) Google Scholar, 42Troppmair J. Brudger J.T. Munoz H. Lloyd P.A. Kyriakis J. Banerjee P. Avruch J. Rapp U.R. J. Biol. Chem. 1994; 269: 7030-7035Abstract Full Text PDF PubMed Google Scholar).Several studies have suggested that the Ras pathway can be activated through agonist stimulation of members of the CCK receptor family (30Seva C. Kowalski-Chauvel A. Blanchet J.-S. Vaysse N. Pradayrol L. FEBS Lett. 1996; 378: 74-78Crossref PubMed Scopus (44) Google Scholar,43Duan R.-D. Williams J.A. Am. J. Physiol. 1994; 267: G401-G408PubMed Google Scholar). However, these studies did not address the issue as to whether activation of Ras is important for receptor-dependent cellular functions such as the transcriptional regulation of cell-specific genes. Our results in AGS-B cells demonstrate that CCK-B/gastrin receptor activation leads to Shc tyrosine phosphorylation association with Grb2, suggesting possible activation of the Ras pathway. However, although these assays support the idea that Ras is most likely activated by gastrin, stimulation of HDC promoter activity was unaffected by the RasN17 or RasN15 dominant negative mutants. In contrast, gastrin-stimulated Raf-1 kinase activity and expression of the dominant negative Raf-1 protein blocked completely gastrin-stimulated activity of the HDC promoter. Thus, taken together, our results suggest that in gastric cells, the CCK-B/gastrin receptor is linked through a Ras-independent, Raf-dependent mechanism to ERK-related signaling pathways, which are essential for the regulation of HDC transcriptional activity by this G-protein-coupled receptor.Finally, overexpression of ERK-1/2 appeared to activate the HDC promoter through the previously identified gastrin response element or GAS-RE, a 23-base pair element located downstream of the start site. The mechanism by which gastrin stimulation activates the GAS-RE remains unclear but presumably involves increased binding by a novel nuclear factor (the GAS-RE-binding protein) (12Zhang Z. Höcker M. Koh T.J. Wang T.C. J. Biol. Chem. 1996; 271: 14188-14197Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Further studies will be required to determine if the GAS-RE-binding protein indeed represents a novel downstream target for the ERK pathway. Our studies suggest that the ERK-dependent signaling pathways mediate the stimulatory effects of gastrin on the transcription of the HDC gene. Several different lines of evidence are consistent with this conclusion. Overexpression of wild type ERK-1 and ERK-2 induced HDC promoter activity dose-dependently, suggesting a regulatory function for these kinases on the activity of the HDC promoter. A second approach involved expression of a constitutively active form of MEK-1 which would be expected to activate endogenous ERKs in AGS-B cells. Previous studies have shown that the MEK-1 mutant Δ-N3-S218E-S222D results in constitutive activity as well as loss of responsiveness to upstream activators (20Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-969Crossref PubMed Scopus (1254) Google Scholar). Thus, although overexpression of WT MEK-1 resulted in a minimal increase in HDC promoter activity, the Δ-N3-S218E-S222D mutant form of MEK-1 was able to increase by severalfold the expression of an HDC reporter gene but showed no response to gastrin or PMA. These data support the concept that activation of the endogenous ERKs is sufficient to stimulation transcription of the HDC gene and that gastrin and PMA regulate the HDC promoter through a MEK-1-dependent signaling route. In addition, we found that gastrin and PMA were able to stimulate tyrosine phosphorylation of ERK-1 and ERK-2 and kinase activity in gastric cancer (AGS) cells. Previous studies have shown that ERK activity is elevated after stimulation of CCK-B/gastrin receptors in several different cell types (29Taniguchi T. Matsui T. Ito M. Murayama T. Tsukamoto T. Katakami Y. Chiba T. Chihara K. Oncogene. 1994; 9: 861-867PubMed Google Scholar, 30Seva C. Kowalski-Chauvel A. Blanchet J.-S. Vaysse N. Pradayrol L. FEBS Lett. 1996; 378: 74-78Crossref PubMed Scopus (44) Google Scholar, 31Todisco A. Takeuchi Y. Seva C. Dickinson C.J. Yamada T. J. Biol. Chem. 1995; 270: 28337-28341Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), but they did not address the role in activating important downstream targets. We found that activation of the CCK-B/gastrin receptor in AGS-B cells was sufficient to activate ERK-regulated transcription factors such as c-Myc and Elk-1, consistent with an important role for this pathway in the transmission of gastrin-dependent signals into the nucleus. Importantly, we found that expression of kinase-deficient ERKs or the ERK-specific phosphatase PAC-1 which interfere with the ERK cascade blocked completely gastrin- and PMA-induced activation of the HDC gene. Overall, these findings demonstrate that gastrin activates the ERK cascade and that this pathway represents the major signaling route for gastrin-dependent regulation of the HDC promoter. Because we have shown recently that activation of c-jun and AP-1 leads to increased HDC transcription (13Höcker M. Zhang Z. Merchant J.M. Wang T.C. Am. J. Physiol. 1997; 35: 6822-6830Google Scholar), we also investigated a role for JNK-dependent signaling. The JNKs (JNK-1 and JNK-2) are distant relatives of the MAPKs and were identified initially based on their ability to phosphorylate and activate c-Jun. The murine SEK-1 gene (and the human homologue, MKK4) are upstream kinases that activate JNKs by phosphorylation of the Thr-Pro-Tyr sequence (14Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). JNKs are activated by agents that induce cellular stress, such as UV light and tumor necrosis factor α, although JNKs may also be activated by tyrosine kinase growth factors such as epidermal growth factor in a Ras-dependent manner. Recently, Dabrowskiet al. (32Dabrowski A. Grady T. Logsdon C.D. Williams J.A. J. Biol. Chem. 1996; 271: 5686-5690Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) have shown that JNKs are activated rapidly in isolated rat pancreatic acini after CCK-A receptor activation. Overall, our studies do not suggest an important role for the JNK pathway in the activation of HDC by gastrin. First, although our studies using the GAL4-c-Jun/GAL4-Luc system indicate that c-Jun phosphorylation and transactivation are increased by gastrin stimulation, we have shown that the activation is most likely secondary to ERK activation rather than to activation of JNK pathways. Second, although our data indicate that activation of JNK (via overexpression of SEK-1) can activate HDC promoter activity, this pathway is clearly distinct from the gastrin pathway. Gastrin stimulation of HDC promoter activity was blocked completely by the kinase-deficient ERK-1 mutant, whereas SEK-1 stimulation of HDC was not blocked by the kinase-deficient ERK-1. The lack of effect of the dominant negative SEK-1 mutant, which blocks JNK activation by extracellular stimuli without interfering with the ERK pathway, on gastrin stimulation of HDC promoter activity also suggests that the JNK pathway is probably not required for the gastrin-stimulated response. Finally, neither gastrin nor PMA appeared to be able to stimulate phosphorylation of JNK in AGS-B cells. Recent studies indicate that G-protein-coupled receptors can activate the ERKs through several different mechanisms (33Post G.R. Brown J.H. FASEB J. 1996; 10: 741-749Crossref PubMed Scopus (198) Google Scholar). Gi-coupled receptors, such as the thrombin (34LaMorte V.J. Kennedy E.D. Collins L.R. Goldstein D. Harootunian A.T. Brown J.H. Feramisco J.R. J. Biol. Chem. 1993; 268: 19411-19415Abstract Full Text PDF PubMed Google Scholar), lysophosphatidic acid (35Howe L.R. Marshall C.J. J. Biol. Chem. 1993; 268: 20717-20720Abstract Full Text PDF PubMed Google Scholar), α2-adrenergic (36Alblas J. van Corven E.J. Hordijk P.L. Milligan G. Moolenaar W.H. J. Biol. Chem. 1993; 268: 22235-22238Abstract Full Text PDF PubMed Google Scholar), and m2 muscarinic (37Winitz S. Russell M. Qian N.X. Gardner A. Dwyer L. Johnson G.L. J. Biol. Chem. 1993; 268: 19196-19199Abstract Full Text PDF PubMed Google Scholar), are similar to tyrosine kinase receptors in that they activate the ERKs through a Ras-dependent pathway. Activation of Gi-coupled receptors leads to tyrosine phosphorylation of Shc and its association with Grb2, a protein that links growth factor receptors to the Ras guanine nucleotide exchange factors. Activation of the ERKs by Gi-coupled receptors is mediated by the G-protein ॆγ subunits and thus can be blocked not only by dominant negative RasN17 but also by coexpression of a ॆγ-binding COOH-terminal peptide derived from ॆARK1 (ॆARK1ct) (38Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar). However, the signaling pathways coupling Gq-linked receptors to ERK activation are less clear and may involve Ras-independent pathways. For example, stimulation of ERK activity by Gq-coupled receptors such as m1 muscarinic and the α1-adrenergic is insensitive to the Gॆγ-sequestering ॆARK1cf peptide and RasN17 (38Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (404) Google Scholar, 39Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Instead, ERK activation by Gq-coupled receptors occurs predominantly through a Ras-insensitive, protein kinase C-dependent pathway (40van Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The GTP-bound α-subunit of the Gq/11 protein activates phosphoinositide hydrolysis and protein kinase C, which then stimulate ERK activation via a poorly understood mechanism involving the activation of Raf-1 kinase (41Kolch W. Heldecker G. Kochs G. Hummel R. Vahldl H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1152) Google Scholar, 42Troppmair J. Brudger J.T. Munoz H. Lloyd P.A. Kyriakis J. Banerjee P. Avruch J. Rapp U.R. J. Biol. Chem. 1994; 269: 7030-7035Abstract Full Text PDF PubMed Google Scholar). Several studies have suggested that the Ras pathway can be activated through agonist stimulation of members of the CCK receptor family (30Seva C. Kowalski-Chauvel A. Blanchet J.-S. Vaysse N. Pradayrol L. FEBS Lett. 1996; 378: 74-78Crossref PubMed Scopus (44) Google Scholar,43Duan R.-D. Williams J.A. Am. J. Physiol. 1994; 267: G401-G408PubMed Google Scholar). However, these studies did not address the issue as to whether activation of Ras is important for receptor-dependent cellular functions such as the transcriptional regulation of cell-specific genes. Our results in AGS-B cells demonstrate that CCK-B/gastrin receptor activation leads to Shc tyrosine phosphorylation association with Grb2, suggesting possible activation of the Ras pathway. However, although these assays support the idea that Ras is most likely activated by gastrin, stimulation of HDC promoter activity was unaffected by the RasN17 or RasN15 dominant negative mutants. In contrast, gastrin-stimulated Raf-1 kinase activity and expression of the dominant negative Raf-1 protein blocked completely gastrin-stimulated activity of the HDC promoter. Thus, taken together, our results suggest that in gastric cells, the CCK-B/gastrin receptor is linked through a Ras-independent, Raf-dependent mechanism to ERK-related signaling pathways, which are essential for the regulation of HDC transcriptional activity by this G-protein-coupled receptor. Finally, overexpression of ERK-1/2 appeared to activate the HDC promoter through the previously identified gastrin response element or GAS-RE, a 23-base pair element located downstream of the start site. The mechanism by which gastrin stimulation activates the GAS-RE remains unclear but presumably involves increased binding by a novel nuclear factor (the GAS-RE-binding protein) (12Zhang Z. Höcker M. Koh T.J. Wang T.C. J. Biol. Chem. 1996; 271: 14188-14197Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Further studies will be required to determine if the GAS-RE-binding protein indeed represents a novel downstream target for the ERK pathway. We thank John K. Westwick, Melanie H. Cobb, Richard A. Maurer, Kathleen Kelly, Natalie G. Ahn, Leonard I. Zon, John M. Kyriakis, Geoffrey M. Cooper, T. Jake Liang, and Anil K. Rustgi for providing constructs used in these studies. We also thank Ian Rosenberg, Ramnik Xavier, and Katharina Detjen for helpful comments and suggestions. Oligonucleotides were supplied by the Center for the Study of Inflammatory Bowel Disease.