Mitogen-Activated Protein Kinase and Protein Kinase A Signaling ...

Mitogen-Activated Protein Kinase and Protein Kinase A Signaling Pathways Stimulate Cholecystokinin Transcription via Activation of Cyclic Adenosine 3*,5*-Monophosphate Response Element-Binding Protein

Thomas v. O. Hansen, Jens F. Rehfeld, and Finn C. Nielsen Department of Clinical Biochemistry Rigshospitalet DK-2100 Copenhagen Ø, Denmark

Cholecystokinin (CCK) is a potent neuropeptide expressed in the small intestine and in the central nervous system. We have examined the effect of basic fibroblast factor (bFGF) and forskolin on CCK gene transcription and depicted the signaling pathways that lead to promoter activation. bFGF and forskolin stimulated promoter activity via a cAMP response element (CRE)/12-O-tetradecanoylphorbol-13-acetate response element (TRE) located 80 bp upstream from the transcription initiation site. In nuclear extracts from unstimulated as well as stimulated cells, only CRE-binding protein (CREB) and activating transcription factor-1 (ATF-1) bound to the CRE/TRE, and activation was associated with phosphorylation of CREB serine-133 and ATF-1 serine-63. In murine F9 cells, CREB stimulated promoter activity 10-fold in the presence of protein kinase A (PKA), and in SK-N-MC cells activation was inhibited 60–70% by a dominant negative CREB mutant. In contrast, ATF-1 had no effect in F9 cells and exhibited a dominant negative effect in SK-N-MC cells. bFGF stimulation led to phosphorylation of the p38 mitogen-activated protein kinase (MAPK), and the extracellular signalregulated kinase (ERK) MAPK and promoter activation, phosphorylation of CREB, and GAL4CREB-dependent transcription were selectively prevented by a dominant negative Ras-mutant, the p38 MAPK-specific inhibitor SB203580, and the MAP/ERK kinase 1 (MEK1) inhibitor PD098059. Forskolin stimulation proceeded via the PKA pathway, and to a minor extent via the p38 and ERK MAPK pathways. We conclude that bFGF and forskolin stimulate the CCK gene promoter via the

CRE/TRE(280) in the proximal promoter region. Signaling proceeds through the p38 MAPK, the ERK MAPK, and the PKA-signaling pathways, which leads to cumulative phosphorylation and activation of CREB. We propose that bFGF in combination with neurotransmitters/neuropeptides coupling to the PKA-signaling pathway play an important role in the control of CCK gene expression. (Molecular Endocrinology 13: 466–475, 1999)

INTRODUCTION Cholecystokinin (CCK) is an important neuroendocrine peptide expressed in the endocrine I cells of the small intestine and in central and peripheral neurons (for a review see Ref. 1). CCK is the most abundant neuropeptides in the mammalian brain (2, 3), and in humans particularly high levels are expressed in the neocortical and hippocampal neurons, although significant quantities occur in almost any region of the brain (for a review see Ref. 4). Whereas the role of intestinal CCK in the release of pancreatic enzymes and contraction of the gall bladder is well established, the role of cerebral CCK is not entirely understood. Consistent with its widespread expression, however, CCK has been proposed to regulate a variety of central nervous system functions, including feeding behavior, anxiety, analgesia, and memory functions. Despite the physiological significance of CCK, the factors involved in the control of CCK transcription are essentially unknown. The proximal upstream regulatory sequences of the human CCK gene exhibit at least three conserved and functional elements (5, 6). A Sp1 site is located close to the putative TATA box, and further upstream the promoter exhibits a combined cAMP response element (CRE)/12-O-tetradecanoylphorbol-13-acetate response

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element (TRE) and an E box (Fig. 1). The CRE/TRE motif exhibits a core sequence of 59-CTGCGTCA-39, which is identical to the TRE(2296) of the c-fos gene (7) and the CRE-2 element of the proenkephalin gene (8, 9). CRE/ TRE elements are recognized by numerous transcription factors, including members of the CRE-binding protein (CREB)/activating transcription factor (ATF) and the AP-1 family of transcription factors (for review see Refs. 10 and 11). The factors readily form heterodimers that posses distinct activating potentials and are stimulated by different signaling pathways. Therefore, the CRE/TRE element is likely to play a key role in the control of promoter activity and consequently CCK gene expression. In this study we have examined the effects of basic fibroblast growth factor (bFGF) and forskolin on CCK gene expression. bFGF has previously been demonstrated to stimulate the proenkephalin gene promoter in combination with cAMP (12), indicating that bFGF may play a role in the control of neuropeptide synthesis. We show that bFGF and forskolin stimulate the CCK gene promoter via phosphorylation of CREB bound to a conserved CRE/TRE in the proximal promoter region. Activation proceeds through the p38 mitogen-activated protein kinase (MAPK), the extracellular signal-regulated kinase (ERK) MAPK, and the

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protein kinase A (PKA)-signaling pathways, leading to a cumulative phosphorylation and activation of CREB.

RESULTS Activation of the CCK Gene Promoter by bFGF and Forskolin To examine the effect of bFGF and forskolin on CCK gene promoter activity, human SK-N-MC cells were transfected with the CCK gene promoter constructs CCK-200, CCK-100, or CCK-67 and treated with 25 ng/ml bFGF, 10 mM forskolin, or both (Fig. 2A). bFGF stimulated chloramphenicol acetyl transferase (CAT) activity of the constructs CCK-200 and CCK-100 about 2-fold, whereas, forskolin increased CAT activity 5-fold. No effect of bFGF or forskolin was observed on the CCK-67 construct. The combined treatment with bFGF and forskolin was followed by a 12-fold increase in CAT activity. The effect of bFGF was dose dependent, and half-maximal stimulation was obtained at a concentration of 0.8 nM bFGF (Fig. 2B). Since the response element appeared to be located in the region between 2100 and 267 bp, which contains the previously identified CRE/TRE element (5, 6), this element was mutated (CTGCGTCA 3 CTGCTGAA). Mutation reduced activation 75%, indicating that the CRE/TRE element is the major element mediating the effects of bFGF and forskolin. CREB and ATF-1 Bind to the CRE/TRE in the CCK Gene Promoter

Fig. 1. Structure of the Proximal Upstream Regulatory Domain of the Human CCK Gene The TATA box, the Sp1(239), the CRE/TRE(280), and the E box(297) elements are indicated.

To identify the transcription factors involved in promoter activation, we examined the binding of members of the CREB/ATF and AP-1 family of transcription

Fig. 2. Activation of the CCK Gene Promoter by bFGF and Forskolin A, SK-N-MC cells were transfected with the CCK 2200, 2100, 267, or 2100DCRE/TRE constructs and incubated with 25 ng/ml bFGF, 10 mM forskolin, or both as indicated. After 6 h, CAT and luciferase activity were measured. The results are presented as fold activation over basal (unstimulated cells) and represents the mean 6 SEM of three experiments. B, Dose-response curve of the additive bFGF activation. SK-N-MC cells were transfected with the CCK-100 construct and treated with 10 mM forskolin and increasing concentrations of bFGF as indicated. CAT and luciferase activity were measured after 6 h. The results were normalized as above and are mean 6 SEM of three experiments.

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Fig. 4. CREB serine-133 Is Necessary for Activation by bFGF and Forskolin SK-N-MC cells were transfected with 5 3 GAL4-TATAluciferase reporter plasmid and GAL4-CREB or GAL4-CREB(Ala-133) expression vectors as indicated. Six hours before harvesting the cells were stimulated with either 25 ng/ml bFGF, 10 mM forskolin, or both as indicated. Luciferase activity was measured after 6 h. The results are stated as fold activation over basal (GAL4-TATA-luciferase) and represents the mean 6 SEM of three experiments.

Fig. 3. CREB and ATF-1 Bind to the CRE/TRE Element and Are Phosphorylated upon Stimulation with bFGF and Forskolin A, SK-N-MC cells were treated with 25 ng/ml bFGF, 10 mM forskolin, or both for 6 h, after which crude nuclear extracts were prepared. [g-32P]ATP-labeled CCK(285)-(266) probe was incubated without nuclear extract (lane 1), with nuclear extract from unstimulated cells (lane 2), with nuclear extract from bFGF-stimulated cells (lane 3), with nuclear extract from forskolin-stimulated cells (lane 4), or with nuclear extract from bFGF- and forskolin-stimulated cells (lane 5). B, Binding of CREB and ATF-1 to the CRE/TRE element. Crude nuclear extracts from unstimulated cells, cells treated with 25 ng/ml bFGF, 10 mM forskolin, or both, were incubated with [g-32P]ATP-labeled CCK(285)-(266) probe and antibodies recognizing CREB (lane 2), ATF-1 (lane 3), ATF-3 (lane 4), the Jun family (c-Jun, JunB, JunD) (lane 5), and the Fos family (c-Fos, FosB, Fra-1, Fra-2) (lane 6), respectively. Supershifts are indicated with an asterisk. C, Increased phosphorylation of CREB and ATF-1 upon stimulation by bFGF and forskolin. SK-N-MC cells were unstimulated (lane 1), stimulated with either 25 ng/ml bFGF (lane 2), 10 mM forskolin (lane 3), or both (lane 4). Western blot analysis was performed on cell extracts using either anti-phospho-CREB/ATF-1, anti-CREB, or antiATF-1 antibodies.

factors to the CRE/TRE element by electrophoretic mobility shift assay and supershift analysis. Incubation of crude nuclear extracts from unstimulated SK-N-MC cells with the CCK(285)-(2-66) oligonucleotide, comprising the CRE/TRE, identified a single retarded DNA-

protein complex (Fig. 3A). The binding pattern remained unchanged, when nuclear extracts from bFGF-stimulated, forskolin-stimulated, or bFGF/forskolin stimulated-SK-N-MC cells were used. The subsequent supershift analysis with antibodies recognizing CREB, ATF-1, ATF-3, the Jun family (c-Jun, JunB, JunD), and the Fos family (c-Fos, FosB, Fra-1, Fra-2) showed that only CREB and ATF-1 were associated with the CRE/TRE in both unstimulated and stimulated SK-N-MC cells (Fig. 3B). Since the binding of CREB and ATF-1 remained unchanged, we examined whether activation was associated with phosphorylation of CREB and ATF-1 using an antibody specific for the serine-133-phosphorylated form of CREB and the serine-63-phosphorylated form of ATF-1 (Fig. 3C). Addition of bFGF or forskolin increased phosphorylation of CREB and ATF-1, and stimulation with both agents further increased in the level of phosphorylation. The relative immunoreactivity of ATF-1 was approximately 5-fold lower than that of CREB (data not shown). In agreement with the supershift analysis, the Western analysis with antibodies recognizing both phosphorylated and dephosphorylated forms of CREB or ATF-1 showed that the total amount of CREB or ATF-1 was unchanged upon stimulation (Fig. 3C). The functional significance of the serine-133 phosphorylation was further examined by coexpression of GAL4-CREB fusion proteins and the GAL4-TATA-luciferase reporter plasmid (Fig. 4). The GAL4 reporter alone was unaffected by stimulation but coexpression of GAL4-CREB resulted in a 4-fold stimulation by bFGF, a 10-fold stimulation by forskolin, and a 22-fold stimulation by bFGF and forskolin. In contrast, coexpression of GAL4-CREB(Ala-133), in which serine-133 is mutated to alanine, was insensitive to stimulation by either bFGF, forskolin, or both. We infer that transcriptional


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Fig. 5. Stimulation of the CCK Gene Promoter by CREB A, F9 cells were transfected with CCK-100 reporter plasmid and CREB or ATF-1 expression vectors in the absence or presence of PKA expression vector as indicated. After 48 h, the cells were harvested and analyzed for CAT and luciferase activity. The results are stated as fold activation over basal (CCK-100) and represents the mean 6 SEM of three experiments. B, SK-N-MC cells were transfected with CCK-100 reporter plasmid and the indicated amount of ATF-1 expression vector in the absence or presence of PKA expression vector as indicated. The results are stated as fold activation over basal (CCK-100) and represent the mean 6 SEM of two experiments. C, Effect of KCREB on CCK promoter stimulation. SK-N-MC cells were cotransfected with CCK-100 reporter plasmid and KCREB or CREB expression vectors as indicated. Six hours before harvesting, the cells were stimulated with 10 mM forskolin or 10 mM forskolin and 25 ng/ml bFGF, after which CAT and luciferase activity were measured. The results represent the mean 6 SEM of three experiments.

activation by bFGF and forskolin involves phosphorylation of CREB serine-133 and ATF-1 serine-63. CREB Activates the CCK Gene Promoter To directly establish the functional significance of CREB and ATF-1 in promoter activation, CCK-100 reporter plasmid, CREB, and ATF-1 were coexpressed in murine F9 cells, which in the undifferentiated state have been shown to contain low levels of functional CREB and therefore are suitable for CREB studies (13). Coexpression of CREB with or without PKA was followed by a 4- and 10-fold increase in promoter activity, respectively (Fig. 5A). The significance of endogenous CREB, moreover, was examined by expression of a dominant negative CREB mutant, KCREB, in SK-N-MC cells. The mutant exhibits a point mutation within the DNA-binding domain that leads to the formation of inactive heterodimers with endogenous CREB and ATF-1 (14). As shown in Fig. 5C, stimulation by bFGF and forskolin, as well as forskolin alone, was inhibited with 60–70% by the expression of KCREB.

ATF-1 had no significant effect on promoter activity alone or in combination with PKA (Fig. 5A) or on CREB/ PKA activation (data not shown) in F9 cells. In SKN-MC cells, ATF-1 caused a dose-dependent repression of basal and PKA-induced transcription (Fig. 5B). The results indicate that CREB is the major transactivating factor involved in bFGF and forskolin-stimulated CCK transcription. bFGF Induction of the CCK Gene Promoter Requires the Ras-Signaling Pathway To depict the signaling pathways that mediates CCK gene promoter activation, CCK-100 reporter plasmid and a dominant negative Ras mutant, Ha-Ras (Asn17), which competes with endogenous cellular p21Ras for upstream activators (15, 16), were cotransfected into SK-N-MC cells (Fig. 6A). Coexpression of Ha-Ras (Asn-17) inhibited bFGF stimulation 90%, and the combined bFGF and forskolin stimulation by 40%, but had no effect on forskolin stimulation, indicating that Ha-Ras (Asn-17) interferes with bFGF signaling. Fur-

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Fig. 6. bFGF Stimulates the CCK Gene Promoter via a Ras-Dependent Signaling Pathway A, SK-N-MC cells were cotransfected with CCK-100 reporter plasmid and the dominant negative Ras(Asn-17) expression vector as indicated. Six hours before harvesting, the cells were stimulated with 25 ng/ml bFGF, 10 mM forskolin, or both and subsequently analyzed for CAT and luciferase activity. The results are stated as fold activation over basal (CCK-100) and represent the mean 6 SEM of three experiments. B, Synergistic activation of the CCK gene promoter by oncogenic Ras and PKA. SK-N-MC cells were cotransfected with CCK-100 reporter plasmid, oncogenic Ras(Val-12), and PKA expression vectors as indicated. CAT and luciferase activity were measured after 6 h. The results are stated as fold activation over basal (CCK-100) and represent the mean 6 SEM of three experiments.

thermore, we examined the effect of oncogenic Ras on CCK gene promoter activity. Cotransfection of oncogenic Ras and the catalytic subunit of PKA caused a synergistic activation of CCK gene expression (Fig. 6B), similar to the activation observed with bFGF and forskolin. We infer that Ras is required for bFGF activation of the CCK gene promoter. bFGF Stimulates CCK Transcription via the p38and the ERK-MAPK Pathways To identify putative downstream targets of bFGF-Ras signaling, SK-N-MC cells transfected with CCK-100 reporter construct were stimulated with bFGF in the presence of kinase inhibitors. Neither inhibitors of protein kinase C (chelerythrine chloride), protein kinase A (H-89), phosphoinositide 3-kinase (wortmannin), p70 S6 kinase (rapamycin), or calmodulin kinase (KN-62) inhibited stimulation (data not shown). Therefore, the role of the MAPK pathways, including the p38 and the ERK MAPKs, was examined (Fig. 7A). The p38 MAPK inhibitor SB203580 (17, 18) and the MAP/ERK kinase 1 (MEK1) inhibitor PD098059 (19) selectively prevented the cumulative effect of bFGF. Since these results indicated that bFGF would activate both the p38 MAPK- and the ERK MAPK-signaling pathways, the effect of bFGF on ERK and p38 phosphorylation was examined. Western blot analysis using antibodies recognizing p38 phosphorylated on threonine-180/tyrosine-182, and ERK1/2 phosphorylated on threonine202/tyrosine-204 (Fig. 7B), showed that both p38 and ERK1/2 phosphorylation was increased after stimulation with bFGF, whereas the total amount of p38 or ERK1/2 was unchanged. bFGF-induced CREB phos-

phorylation was also examined (Fig. 7D). Pretreatment of the cells with either SB203580 or PD098059 reduced phosphorylation, whereas combined treatment with SB203580 and PD098059 completely blocked phosphorylation. Finally, the bFGF-stimulated GAL4CREB transcription was also inhibited by both SB203580 and PD098059 (Fig. 7E). Whereas separate addition of SB203580 and PD098059 caused a partial inhibition, both inhibitors almost completely blocked bFGF-induced CREB activity. Since we observed a minor (;20%) decrease in forskolin stimulation after incubation with SB203580 and PD098059 (Fig. 7A), we examined whether forskolin leads to activation of the p38 and ERK. Figure 7C shows the Western analysis of p38 and ERK in SKN-MC cells stimulated with forskolin. Whereas the total level of the proteins remained unchanged, an increase in the phosphorylation of both p38 and ERK was observed. We infer that bFGF stimulates CCK gene expression via the p38- and the ERK MAPK-signaling pathways, whereas, forskolin activation involves PKA and p38 and ERK MAPK.

DISCUSSION Synergy between growth factors and neurotransmitters in control of neuropeptide expression may be important for both differentiation and survival of endocrine cells and neurons and their neurophysiological actions. Here we report that bFGF and forskolin activate the CCK gene promoter via phosphorylation of CREB bound to a conserved CRE/TRE in the proximal


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Fig. 7. Activation of the CCK Gene Promoter by bFGF and Forskolin Involves the p38 and the MEK/ERK MAPK Signaling Pathways A, SK-N-MC cells were transfected with CCK-100 reporter plasmid and 6 h before harvesting, the cells were stimulated with 25 ng/ml bFGF or 10 mM forskolin or 10 mM forskolin and 25 ng/ml bFGF in the presence of 10 mM SB203580, 20 mM PD098059, or both as indicated. The cells were analyzed for CAT and luciferase activity, and results are stated as fold activation over basal and represent the mean 6 SEM of three experiments. B, bFGF increase phosphorylation of p38 and ERK1/2. SK-N-MC cells were incubated in culture medium (lane 1) or stimulated with 25 ng/ml bFGF (lane 2) or incubated with either 10 mM SB203580 (p38) or 20 mM PD098059 (ERK1/2) before addition of 25 ng/ml bFGF (lane 3). Cell extracts were examined by Western analysis with anti-phospho-p38, anti-p38, anti-phospho-ERK1/2, or anti-ERK1/2 antibodies as indicated. C, Forskolin increases phosphorylation of p38 and ERK1/2. SK-N-MC cells were incubated in culture medium (lane 1) or 10 mM forskolin (lane 2) before cell extracts were examined by Western analysis with anti-phospho-p38, anti-p38, anti-phospho-ERK1/2, or anti-ERK1/2 antibodies as indicated. D, SB203580 and PD098059 inhibit bFGF-induced CREB phosphorylation. Western blot analysis with anti-phosphoCREB, anti-CREB, or anti-ATF-1 antibodies was performed on unstimulated SK-N-MC cells (lane 1), cells stimulated with 25 ng/ml bFGF (lane 2), and cells pretreated with 10 mM of SB203580 (lane 3), 20 mM of PD098059 (lane 4), or both (lane 5) before stimulation with 25 ng/ml bFGF. E, GAL4-CREB-dependent transcription is inhibited by SB203580 and PD098059. SK-N-MC cells were cotransfected with 5 3 GAL4-TATA-luciferase reporter plasmid and GAL4-CREB expression vector. Seven hours before harvesting the cells were preincubated with 25 mM SB203580 and/or 50 mM PD098059 as indicated for 60 min. The cells were then stimulated with 25 ng/ml bFGF and subsequently analyzed for luciferase activity. The results are stated as fold activation over basal (CCK-100) and represent the mean 6 SEM of three experiments.

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promoter region. Activation proceeds through the p38 MAPK-, the Ras/ERK MAPK-, and the PKA-signaling pathways, which converge and phosphorylate CREB on serine-133 (Fig. 8). The CCK CRE/TRE motif consists of a core consensus sequence of 59-CTGCGTCA-39, which, like the CRE-2 element of the proenkephalin gene, may bind CREB/ATF and factors belonging to the c-Jun and c-Fos family of transcription factors (6). bFGF and cAMP have previously been demonstrated to activate the proenkephalin promoter via binding of ATF-3/cJun heterodimers (12). The mechanism of CCK gene promoter activation is different, since stimulation is mainly mediated by CREB. Promoter activation correlated to CREB serine-133 phosphorylation (13), which is essential for the association of the CREB-binding protein (CBP) required for trans-activation (20–22). CREB and the catalytic subunit of PKA, moreover, were shown to directly activate CCK gene expression in F9 cells, and coexpression of a dominant negative CREB mutant, which forms inactive heterodimers with wild-type CREB and ATF-1 (14), inhibited activation by bFGF and forskolin. ATF-1 also bound to the CCK CRE/TRE and, like CREB, it was phosphorylated upon stimulation with bFGF and forskolin. We did not, however, observe any promoter activation by ATF-1 even after coexpression with PKA. The reason for the distinct behavior of CREB and ATF-1 in this system is unclear but it has recently been suggested that ATF-1 can reduce promoter activation by heterodimerizing

Fig. 8. Model of the Signaling Pathways Leading to Cumulative Activation of the CCK Gene Promoter by bFGF and Forskolin Receptor binding of bFGF is followed by stimulation of Ras that activates the MEK/ERK pathway and the p38 MAPK pathway. The downstream activators of ERK and p38 MAPK are likely to be the family of pp90 ribosomal S6 kinases (RSK) and the MAPK-activated protein (MAPKAP) kinase 2, respectively, which have been demonstrated to phosphorylate CREB at serine-133 (30, 33, 36, 39). Forskolin or putative neurotransmitter substances or neuropeptides stimulate adenylate cyclase activity and cAMP production, after which PKA is activated and phosphorylates CREB at serine-133. Moreover, the PKA pathway cross-talk with the p38 and ERK MAPK. Ultimately, trans-activation is accomplished by association of CREB with CBP and activation of transcription.

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with CREB-1 (23). Factors belonging to the Jun or Fos family of transcription factors were not detected in the band-shift analysis, supporting the conception that CCK CRE/TRE and the identical proenkephalin CRE-2 are likely to have a preference for CREB, because they exhibit a C residue (CTGCGTCA) in the core of the element (9, 24). It should be noted, however, that CREB has been recently demonstrated to regulate Fos transcription (25), and some of the dominant negative effect of KCREB may be related to Fos. Finally, CCK promoter activation by forskolin/cAMP may involve the direct recruitment of CBP (26). This may explain why bFGF, which fails to induce cAMP, is a weaker activator of CREB- dependent transcription than forskolin, even though both factors promote phosphorylation of CREB on serine 133. Although the PKA-signaling pathway, leading to phosphorylation of CREB, is well established (for a review see Ref. 27), the events after growth factor stimulation are less well characterized. Receptor binding of bFGF is followed by receptor dimerization, autophosphorylation, and activation of phospholipase C (28), as well as the MAPK-signaling pathways in a Ras-dependent manner (29–31). Since coexpression of Ha-Ras (Asn-17) inhibited activation by bFGF and oncogenic Ras caused a synergistic activation of the promoter together with the catalytic subunit of PKA, we inferred that CCK gene promoter activation was associated with activation of the MAPK pathways. Mammalian cells contain at least three well characterized MAPK cascades, which regulate the activity of the ERK MAPKs, the Ras/MEK/ERK pathway, the stress-activated protein kinase/c-Jun amino-terminal kinase (SAPK/JNK) MAPKs, and the p38 MAPKs (for a review see Ref. 32). While the ERK MAPK cascade has been implicated in the activation by various growth factors, the SAPK/JNK and the p38 MAPK pathways were originally shown to mediate stress responses. However, recent studies have shown that the p38 MAPK pathway can also be activated by growth factors (30, 33). Stimulation of CCK transcription and CREB phosphorylation by bFGF was inhibited by the p38 MAPK inhibitor SB203580 and the MEK1 inhibitor PD098059, indicating that both the p38 MAPK pathway and the Ras/MEK/ERK pathway are involved in promoter activation. Likewise, we could demonstrate that bFGF stimulated phosphorylation of both ERK and p38, which are associated with activation of the kinases (34, 35). Inhibition of GAL4-CREB transcription and CREB phosphorylation, moreover, was additive, indicating that the p38 MAPK pathway and the Ras/MEK/ ERK pathway proceed in parallel. This is in agreement with recent data showing that nerve growth factormediated phosphorylation of CREB also proceeds via the p38 MAPK pathway and the Ras/MEK/ERK pathways (36). The signaling pathways that activate p38 are not completely understood, but since signaling can be inhibited by a dominant Ras mutant, it is possible that Ras controls p38 via activation of Rho (37, 38). The downstream activators of ERK and p38 MAPK are likely to be the family of pp90 ribosomal S6 kinases


(RSK) and the MAPK-activated protein kinase 2 (MAPKAP kinase-2), respectively, which have all been demonstrated to phosphorylate CREB at serine-133 (30, 33, 36, 39). Forskolin stimulation involved not only the classical PKA pathway since about 20% of the stimulation can be attributed to activation of p38 and ERK. Although only a minor stimulation proceeded via these pathways, they may be physiologically relevant under certain conditions. In fact, cAMP and PKA have recently been demonstrated to stimulate differentiation of PC12 cells via activation of ERK (40). We find that forskolin phosphorylates p38 more potently than ERK, and this pathway is therefore likely to mediate additional effects of cAMP and PKA. Moreover, the results predict that additive effects of growth factors and cAMP may occur at the level of p38 and ERK. The CCK gene promoter CRE/TRE is conserved from shark and bullfrog to man and is likely to play a central role in the control of CCK gene expression. Our data indicate that it serves to integrate signals from a variety of extracellular factors, including growth and neurotrophic factors as well as transmitters, and generate a finely tuned output of CCK RNA. Signaling from the MAPK- and PKA-signaling pathways are integrated by CREB, localized in situ on the CCK CRE/ TRE. CREB has been reported to play a key role in both neurotrophin responses and memory functions (41–44) and, based on a comparison with the physiological roles of CCK, it is also likely that CREB could be involved in regulation of feeding behavior and anxiety control. Although this study has focused on the effects of bFGF, the mechanism may be extended to nerve growth factor and brain-derived neurotrophic factor and other growth factors in the gastrointestinal channel, which have been demonstrated to activate the MAPK pathways in a similar way as bFGF (33, 41). Stimulation of CCK gene expression by bFGF and neurotransmitters, coupling to the PKA-signaling pathway, may be of importance in several situations. bFGF is produced in vast areas of the central nervous system (45) and could play a role in CCK production in late development. In the rat, adult bFGF levels and patterns of distribution are reached at postnatal day 28 (46), and this coincides with the production of transmitter-active CCK peptides (47). Moreover, unlike most neurotransmitters, bFGF and other growth factors are not released from stored granules in short bursts, but are secreted in a constitutive manner over longer time periods. During adult life it may be envisioned that the function of bFGF is mainly to upregulate the response of the CCK gene promoter to neuropeptides and smaller neurotransmitters that couple to the PKA-signaling pathway. Finally, it is possible that cumulative actions may be relevant during regeneration in which bFGF and CCK have been demonstrated to be expressed at high levels (48, 49). In conclusion, we show that bFGF and forskolin stimulate CCK transcription via the p38 MAPK-, ERK MAPK-, and PKA-signaling pathways and enhanced

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phosphorylation and activation of CREB. We propose that bFGF or other growth and neurotrophic factors, in combination with neurotransmitter/neuropeptide coupling to the PKA signal pathway, plays an important role in the control of endocrine and neuronal CCK gene expression.

MATERIALS AND METHODS Plasmid Constructions The human CCK gene promoter constructs, CCK-200, CCK100, CCK-67, and 100DCRE/TRE, consist of 59-promoter fragments inserted into the pCAT vector (Promega, Madison, WI) as recently described (6). Thymidine kinase (TK)-luciferase was constructed by inserting a HindIII-BglII fragment of pRL-TK (Promega) containing the TK promoter into pGL3 basic (Promega). The mammalian expression vectors encoding CREB and KCREB (14), Ha-Ras (Asn-17) (15), oncogenic Ras (Val-12) (50), and the catalytic subunit of PKA (51) were kind gifts from Richard H. Goodman, Larry A. Feigh, Robert A. Weinberg, and G. Stanley McKnight, respectively. Fulllength ATF-1 cDNA was amplified from SK-N-MC cell RNA using the oligonucleotide primers 59-GTCGTAGCGGCCGCTTATGGAAGATTCCCACAAGAGTACCACG-39 and 59CAGCTGAAGCTTAATCAAACACTTTTATTGGAATAAAGATC-39 and subcloned into pcDNA3.11 (Invitrogen, Carlsbad, CA). The sequence was verified by sequencing. The 5 3 GAL4-TATA-luciferase reporter plasmid, the GAL4-CREB4–283, and the GAL4CREB4–283(Ala-133) expression vectors (52, 53) were kind gifts from Richard A. Maurer. Cell Culture and Transient DNA Transfections Human SK-N-MC neuroblastoma cells were maintained as described (6). One day before transfection 2.5 3 106 SKN-MC cells were seeded in 100-mm culture dishes. Five micrograms of CCK reporter plasmid, 2 mg TK-luciferase, 10 mg CREB, 10 mg KCREB, or 1 mg PKA expression vector when indicated, and pBluescript (Stratagene, La Jolla, CA) to a total of 20 mg were cotransfected using the calcium phosphate coprecipitation method (54). For the GAL4 assay, 5 mg 5 3 GAL4-TATA-luciferase reporter plasmid, 2 mg GAL4CREB or GAL4-CREB(Ala-133) expression vector, 0.5 mg pRL-TK (Promega), and pBluescript (Stratagene) to a total of 20 mg were cotransfected. Six hours before harvesting, the cells were stimulated with either 25 ng/ml bFGF (Amersham, Arlington Heights, IL), 10 mM forskolin (Sigma Chemical Co., St. Louis, MO), or both. SB203580 (10–25 mM) (Calbiochem, San Diego, CA), 20–50 mM PD098059 (Calbiochem), or dimethylsulfoxide were added together with the stimulant when indicated. Murine F9 cells were cultured in DMEM (Life Technologies, Gaithersburg, MD) containing 15% FBS and 1.0 mM sodium pyruvate at 10% CO2 and 37 C. Cells were seeded at 1 3 106/100-mm culture dish coated with 0.1% gelatin 1 day before transfection. Each plate was transfected with 30 mg of DNA, including 5 mg CCK-100 reporter plasmid, 2 mg TKluciferase, 10 mg CREB or ATF-1 expression vector, 5 mg PKA expression vector, and pBluescript (Stratagene) as described above. At the end of incubation the cells were harvested and analyzed for CAT and luciferase activity. All values were normalized to luciferase activity. Preparation of Nuclear Extract and Electrophoretic Mobility Shift Assays Nuclear extracts from SK-N-MC cells were prepared as described previously (55). The protein concentration was deter-

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mined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). [g-32P]ATP labeled double-stranded oligonucleotides (4 3 104 cpm), corresponding to the CCK gene promoter 59-flanking sequence 285 to 266 relative to the transcription start site (59-CCAGTCTGCGTCAGCGTTGG-39), were incubated with 5 mg of nuclear extract in 10 mM HEPES (pH 7.9), 100 mM KCl, 0.05 mM EDTA, 1 mM dithiothreitol, 2.5 mM MgCl2, 6% glycerol, 2 mg of (dI-dC), in a total volume of 20 ml for 30 min at room temperature. For supershift assays, 5 ml of anti-CREB (sc-271), 1 ml of antiATF-1 (sc-243x), 5 ml of anti-ATF-3 (sc-188), 1 ml of anti-Jun (sc-44x), or 1 ml of anti-Fos (sc-253x) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the binding reaction and incubated for an additional 60 min at 4 C before the loading of the gel. DNA-protein complexes were analyzed on a 4% nondenaturing polyacrylamide gel in 0.5 3 TBE buffer (1 3 TBE is 130 mM Tris, 89 mM boric acid, and 2 mM EDTA) and exposed to PhosphoImager (Fuji, Tokyo, Japan) for quantitative analysis. Western Blot Analysis For detection of proteins, approximately 2.5 3 106 SK-N-MC cells were grown in media containing 0.5% serum for 2 days before stimulating with 25 ng/ml bFGF (Amersham), 10 mM forskolin (Sigma), or both for 15 min. SB203580 (10 mM) (Calbiochem), 20 mM PD098059 (Calbiochem), or dimethylsulfoxide were added 1 h before stimulation when indicated. The cells were washed with cold PBS and lysed by addition of 500 ml SDS loading buffer [100 mM Tris-HCl (pH 6.8), 4% SDS, 0.2 M dithiothreitol, 0.2% bromophenol blue, and 20% glycerol] followed by boiling for 5 min. Proteins were separated on 10% SDS polyacrylamide gels and transferred to polyvinyl difluoride Immobilon-P membranes (Milipore, Bedford, MA). After blocking with 5% nonfat dry milk in 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature, filters were incubated with anti-CREB (sc271) (1:500 dilution) (Santa Cruz Biotechnology), anti-ATF-1 (sc-243) (1:1000) (Santa Cruz Biotechnology), anti-p38 (1: 1000 dilution) (New England Biolabs, Beverly, MA), antiERK1/2 (1:1000 dilution) (New England Biolabs), anti-phospho-CREB/ATF-1 (1:1000 dilution) (New England Biolabs), anti-phospho-p38 (1:1000 dilution) (New England Biolabs), or anti-phospho-ERK1/2 antibody (1:1000 dilution) (New England Biolabs) in blocking solution overnight at 4 C. After three washes in wash buffer [10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20], the membranes were incubated with conjugated antimouse or antirabbit IgG-horseradish peroxidase (1:2000 dilution) (New England Biolabs) in blocking solution for 1 h at room temperature. The membranes were then washed three times with wash buffer, and detection of immunoreactive proteins was performed with lumiGLO chemiluminescent reagent according to the manufacturer’s instruction (New England Biolabs).

Acknowledgments Richard H. Goodman, Larry A. Feigh, Robert A. Weinberg, G. Stanley McKnight, and Richard A. Maurer are gratefully acknowledged for the gift of plasmids.

Received September 10, 1998. Revision received November 13, 1998. Accepted December 8, 1998. Address requests for reprints to: Finn Cilius Nielsen, Department of Clinical Biochemistry, Rigshospitalet, DK-2100 Copenhagen Ø. E-mail: [email protected]. This study was supported by grants from the Danish Medical Research Council, the Danish Biotechnology Program for Signal Peptide Research, the John and Birthe Meyer Foundation, and the NOVO Nordisk Foundation.

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REFERENCES 1. Rehfeld JF 1989 Cholecystokinin. In: Schultz SG, Makhlouf GM, Rauner D, (eds) Handbook of Physiology: The Gastrointestinal System. American Physiological Society, Bethesda, MD, pp 337–358 2. Rehfeld JF 1978 Immunochemical studies on cholecystokinin. II. Distribution and molecular heterogeneity in the central nervous system and small intestine of man and hog. J Biol Chem 253:4022–4030 3. Crawley JN 1985 Comparative distribution of cholecystokinin and other neuropeptides. Ann NY Acad Sci 448:1–8 4. Rehfeld JF, Nielsen FC 1995 Molecular forms and regional distribution of cholecystokinin in the central nervous system. In: Bradwejn J, Vasar E (eds) Cholecyctokinin and Anxiety: From Neuron to Behavior. SpringerVerlag, Heidelberg, Germany, pp 33–56 5. Haun RS, Dixon JE 1990 A transcriptional enhancer essential for the expression of the rat cholecystokinin gene contains a sequence identical to the 2296 element of the human c-fos gene. J Biol Chem 265:15455–15463 6. Nielsen FC, Pedersen K, Hansen TV, Rourke IJ, Rehfeld JF 1996 Transcriptional regulation of the human cholecystokinin gene: composite action of upstream stimulatory factor, Sp1, and members of the CREB/ATF- AP-1 family of transcription factors. DNA Cell Biol 15:53–63 7. Scho¨nthal A, Buscher M, Angel P, Rahmsdorf HJ, Ponta H, Hattori K, Chiu R, Karin M, Herrlich P 1989 The Fos and Jun/AP-1 proteins are involved in the downregulation of Fos transcription. Oncogene 4:629–636 8. Comb M, Birnberg NC, Seasholtz A, Herbert E, Goodman HM 1986 A cyclic AMP- and phorbol ester-inducible DNA element. Nature 323:353–356 9. Comb M, Mermod N, Hyman SE, Pearlberg J, Ross ME, Goodman HM 1988 Proteins bound at adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcription. EMBO J 7:3793–3805 10. Angel P, Karin M 1991 The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1072:129–157 11. Delmas V, Molina CA, Lalli E, de Groot R, Foulkes NS, Masquilier D, Sassone-Corsi P 1994 Complexity and versatility of the transcriptional response to cAMP. Rev Physiol Biochem Pharmacol 124:1–28 12. Tan Y, Low KG, Boccia C, Grossman J, Comb MJ 1994 Fibroblast growth factor and cyclic AMP (cAMP) synergistically activate gene expression at a cAMP response element. Mol Cell Biol 14:7546–7556 13. Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675–680 14. Walton KM, Rehfuss RP, Chrivia JC, Lochner JE, Goodman RH 1992 A dominant repressor of cyclic adenosine 39,59monophosphate (cAMP)- regulated enhancer-binding protein activity inhibits the cAMP-mediated induction of the somatostatin promoter in vivo. Mol Endocrinol 6:647–655 15. Feig LA, Cooper GM 1988 Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol 8:3235–3243 16. Farnsworth CL, Feig LA 1991 Dominant inhibitory mutations in the Mg(21)-binding site of RasH prevent its activation by GTP. Mol Cell Biol 11:4822–4829 17. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, Strickler JE, McLaughlin MM, Siemens IR, Fisher SM, Livi GP, White JR, Adams JL, Young PR 1994 A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739–746 18. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, Lee JC 1995 SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS


Lett 364:229–233 19. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR 1995 PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J Biol Chem 270:27489–27494 20. Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, Goodman RH 1993 Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855–859 21. Kwok RP, Lundblad JR, Chrivia JC, Richards JP, Bachinger HP, Brennan RG, Roberts SG, Green MR, Goodman RH 1994 Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370:223–226 22. Arias J, Alberts AS, Brindle P, Claret FX, Smeal T, Karin M, Feramisco J, Montminy M 1994 Activation of cAMP and mitogen responsive genes relies on a common nuclear factor. Nature 370:226–229 23. Ellis MJC, Catherine Lindon A, Flint KJ, Jones NC, Goodbourn S 1995 Activating transcription factor-1 is a specific antagonist of the cyclic adenosine 39,59-monophosphate (cAMP) response element-binding protein-1mediated response to cAMP. Mol Endocrinol 9:255–265 24. Konradi C, Kobierski LA, Nguyen TV, Heckers S, Hyman SE 1993 The cAMP-response-element-binding protein interacts, but Fos protein does not interact, with the proenkephalin enhancer in rat striatum. Proc Natl Acad Sci USA 90:7005–7009 25. Ahn S, Olive M, Aggarwal S, Krylov D, Ginty DD, Vinson C 1998 A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos. Mol Cell Biol 18:967–977 26. Chawla S, Hardingham GE, Quinn DR, Bading H 1998 CBP: a signal-regulated transcriptional coactivator controlled by nuclear calcium and CaM kinase IV. Science 281:1505–1509 27. Montminy MR 1997 Transcriptional regulation by cyclic AMP. Annu Rev Biochem 66:807–822 28. Burgess WH, Dionne CA, Kaplow J, Mudd R, Friesel R, Zilberstein A, Schlessinger J, Jaye M 1990 Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase. Mol Cell Biol 10:4770–4777 29. Shaoul E, Reich-Slotky R, Berman B, Ron D 1995 Fibroblast growth factor receptors display both common and distinct signaling pathways. Oncogene 10:1553–1561 30. Tan Y, Rouse J, Zhang A, Cariati S, Cohen P, Comb MJ 1996 FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2. EMBO J 15:4629–4642 31. Kouhara H, Hadari YR, Spivak-Kroizman T, Schilling J, Bar-Sagi D, Lax I, Schlessinger J 1997 A lipid-anchored Grb2-binding protein that links FGF-receptor activation to the Ras/MAPK signaling pathway. Cell 89:693–702 32. Robinson MJ, Cobb MH 1997 Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 9:180–186 33. Xing J, Ginty DD, Greenberg ME 1996 Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science 273:959–963 34. Payne DM, Rossomando AJ, Martino P, Erickson AK, Her JH, Shabanowitz J, Hunt DF, Weber MJ, Sturgill TW 1991 Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J 10:885–892 35. Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, Davis RJ 1995 Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270:7420–7426 36. Xing J, Kornhauser JM, Xia Z, Thiele EA, Greenberg ME 1998 Nerve growth factor activates extracellular signalregulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphor-

475

ylation. Mol Cell Biol 18:1946–1955 37. Ginty DD, Bonni A, Greenberg ME 1994 Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB. Cell 77:713–725 38. Vojtek AB, Cooper JA 1995 Rho family members: activators of MAP kinase cascades. Cell 82:527–529 39. Iordanov M, Bender K, Ade T, Schmid W, Sachsenmaier C, Engel K, Gaestel M, Rahmsdorf HJ, Herrlich P 1997 CREB is activated by UVC through a p38/HOG-1-dependent protein kinase. EMBO J 16:1009–1022 40. Vossler MR, Yao H, York RD, Pan M-G, Rim CS, Stork PJS 1997 cAMP activates MAP kinase and ELK-1 through a B-Raf and Rap1-dependent pathway. Cell 89:73–82 41. Finkbeiner S, Tavazoie SF, Maloratsky A, Jacobs KM, Harris KM, Greenberg ME 1997 CREB: a major mediator of neuronal neurotrophin responses. Neuron 19:1031–1047 42. Yin JC, Wallach JS, Del Vecchio M, Wilder EL, Zhou H, Quinn WG, Tully T 1994 Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila. Cell 79:49–58 43. Bourtchuladze R, Frenguelli B, Blendy J, Cioffi D, Schutz G, Silva AJ 1994 Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element-binding protein. Cell 79:59–68 44. Yin JC, Del Vecchio M, Zhou H, Tully T 1995 CREB as a memory modulator: induced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila. Cell 81:107–115 45. Baird A 1994 Potential mechanisms regulating the extracellular activities of basic fibroblast growth factor (FGF2). Mol Reprod Dev 39:43–48 46. Kuzis K, Reed S, Cherry NJ, Woodward WR, Eckenstein FP 1995 Developmental time course of acidic and basic fibroblast growth factors’ expression in distinct cellular populations of the rat central nervous system. J Comp Neurol 358:142–53 47. Mogensen NW, Hilsted L, Bardram L, Rehfeld JF 1990 Procholecystokinin processing in the rat cerebral cortex during development. Dev Brain Res 54:81–86 48. Leonard S, Luthman D, Logel J, Luthman J, Antle C, Freedman R, Hoffer B 1993 Acidic and basic fibroblast growth factor mRNAs are increased in striatum following MPTP-induced dopamine neurofiber lesion: assay by quantitative PCR. Brain Res Mol Brain Res 18:275–284 49. Palkovits M 1995 Neuropeptide messenger plasticity in the CNS neurons following axotomy. Mol Neurobiol 10:91–103 50. Shih C, Weinberg RA 1982 Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell 29:161–169 51. Uhler MD, McKnight GS 1987 Expression of cDNAs for two isoforms of the catalytic subunit of cAMP-dependent protein kinase. J Biol Chem 262:15202–15207 52. Sun P, Enslen H, Myung PS, Maurer RA 1994 Differential activation of CREB by Ca21/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity. Genes Dev 8:2527–2539 53. Sun P, Maurer RA 1995 An inactivating point mutation demonstrates that interaction of cAMP response element binding protein (CREB) with the CREB binding protein is not sufficient for transcriptional activation. J Biol Chem 270:7041–7044 54. Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752 55. Andrews NC, Faller DV 1991 A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 19:2499