Regulation of neuronal cholecystokinin gene

Scand J Clin Lab Invest 2001; 61, Suppl 234: 61–67

Regulation of neuronal cholecystokinin gene transcription T H O M A S v. O . H A N S E N & F I N N C . N I E L S E N Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark.

Hansen TvO, FC Nielsen. Regulation of neuronal cholecystokinin gene transcription. Scand J Clin Lab Invest 2001; 61, Suppl 234: 61–67. Cholecystokinin (CCK) is a neuroendocrine peptide expressed in I-cells of the small intestine and in central and peripheral neurons. Whereas intestinal CCK is involved in the release of pancreatic enzymes and the contraction of the gallbladder, cerebral CCK is implicated in a variety of functions, such as feeding behaviour, anxiety and memory. The expression of CCK is developmentally regulated. Brain CCK mRNA levels are low before birth, but increase markedly shortly after birth and reach adult like patterns of expression three weeks after birth during the final maturation of the central nervous system. In the adult, several substances induce neuronal CCK mRNA expression via activation of transcription factors binding to regulatory elements in the CCK promoter. Recent studies have examined the signaling pathways, transcription factors and regulatory elements involve d in cAMP, fibroblast growth factor-2, and calciuminduced CCK gene transcription in neuronal cells. The review describes the signaling pathways and transcription factors involved in neuronal CCK gene transcription. Key words: CCK, promoter region, regulatory elements, CREB, cAMP, FGF-2, calcium, signaling pathways, PKA, MAPK, ERK, p38 Thomas v. O. Hansen, Department of Clinical Biochemistry, KB3014, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail: [email protected]

Cholecystokinin (CCK) is a neuroendocrine peptide expressed in the I-cells of the small intestinal mucosa and in neurons of the central nervous system (CNS) [1]. Intestinal CCK regulates the release of pancreatic enzymes and contraction of the gallbladder, while neuronal CCK has been implicated in a variety of CNS functions such as feeding behaviour, anxiety and memory [2, 3]. The expression of CCK is developmentally regulated. In mouse and rat brain, CCK mRNA and protein levels are low before birth, but

increase steadily postnatally during neuronal maturation until adulthood [4–6]. In the adult brain CCK is among the most abundant neuropeptides [7, 8] and particularly high mRNA and protein levels are expressed in the cerebral cortex, thalamus, and hippocampus, although significant quantities occur in almost any region of the brain [5, 9]. Neuronal CCK mRNA levels are increased by cAMP, growth factors, dopamine, estrogens, and seizures [10–14], mainly via activation of transcription factors binding to regulatory ele61

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T. v. O. Hansen and F. C. Nielsen

- 1 00 G AG C AC GT GT T AC TG C CA GTC TG CG T CAGC G TT GGG TA AA T AC AT G AC TG C TC G TG CA CA A TG AC G GT CAG AC GC A GTCG C AA CCC AT TT A TG TA C TG AC E -box

C R E /TR E

-50 G CC G AC GC GC C GG GC G GG GC TAT TT A AGAG A CA GCC GC CC G CT GG T CC TC C GG C TG CG CG G CC CG C CC CG ATA AA T TCTC T GT CGG CG GG C GA CC A GG AG TATA -box

FIG. 1. Sequence of the proximal part of the human CCK promoter. The putative TATA-box and the Ebox element are indicated in grey, while the CRE/ TRE is indicated in black. The transcriptiona l start site is indicated with an arrow.

ments in the CCK promoter. The proximal human CCK promoter contains at least two conserved regulatory sites, namely an E-box element and a combined cAMP response element (CRE)/12-O-tetradecanoylphorbol-13acetate (TPA) response element (TRE) (Fig. 1) [15, 16]. The CRE/TRE binding site in the CCK promoter consists of the consensus sequence 5’CTGCGTCAGC-3’, which is identical to the TRE-296 of the c-fos gene, and the CRE-2 element of the proenkephalin gene [17, 18]. The element has a strong preference for the transcription factor CREB and multiple extracellular signals converge on the CRE/TRE in neuronal cells [15, 19, and Hansen et al., unpublished]. This review summarizes the signaling pathways and transcription factors involve d in regulated CCK gene transcription in neuronal cells. CCK gene transcription is activated by fibroblast growth factor-2 (FGF-2), cAMP, and calcium influx. In the following section a brief introduction to the signaling pathways activated by these substances and their role in CREB-dependent CCK gene transcription will be presented. The FGF-2 signaling pathway FGF-2 (also known as basic FGF) is produced in vast areas of the CNS, where it promotes cell migration, differentiation, neuronal survival, and developmental processes [20]. In the rat, adult FGF-2 levels and patterns of distribution are reached at postnatal day 26 [21] and this coincides with the appearance of transmitter-

active CCK peptides [6], suggesting that FGF2 could play a role in CCK production in late development. FGF-2 binding induces FGF receptor dimerization and autophosphorylation of tyrosine residues and recruitment of adaptor proteins, such as the SH2-domain-containing phospholipase C- (PLC- ) and FRS2 [22, 23]. Whereas PLC- activates protein kinase C (PKC), FRS2 recruits the adaptor protein Grb2, which in turn binds the guanine nucleotide exchange factor (GEF) SOS linking the FGF receptor to the Ras signaling pathway. One target of Ras is the highly conserved extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascade, which is involve d in a variety of cell functions such as cell growth, proliferation, and differentiation [24]. The ERK MAPK cascade consists of a three-kinase module, involving the MAPK-kinase-kinase (MAP3K) Raf, the MAPK/ERK-kinases (MEKs) and finally the ERK MAPKs, which is activated via dual phosphorylation of the threonine- and tyrosine motif in the activation loop. ERK activates several cytoplasmic and nuclear proteins, including RSK2, a member of the pp90 ribosomal S6 kinase family [25]. Upon activation both ERK and RSK2 are rapidly translocated to the nucleus where ERK activates another relative of RSK2, termed MSK1 [26]. RSK2 and MSK1 in turn phosphorylate and activate the transcription factor CREB [26, 27]. FGF-2 has previously been shown to activate the p38 MAPK signaling pathway as well, and the p38 MAPKs were subsequently shown to be important for differentiation and developmental processes [19, 28–31]. This observation was surprising since the p38 MAPKs were regarded as a group of stress-activated protein kinases, due to their activation by proinflammatory cytokines and environmental stress [32]. The upstream cascade involved in growth factor mediated p38 MAPK activation is still not entirely clear, but it may involve several MAP3Ks, including MLK3 (which is activated by the Rho subfamily of GTPases) and the MAPK-kinases MKK3, MKK4, MKK6, and MKK7 [33]. The downstream targets include MAPKAP-kinase 2 and MSK1, which both are CREB kinases [26, 28]. The role of FGF-2 in neuronal CCK gene transcription has been examined in SK- N-MC

Regulation of CCK gene transcription cells transfected with CCK promoter constructs and treated with recombinant FGF-2 [19]. FGF2 increased CCK transcription via the CRE/ TRE in the proximal CCK promoter region. Cotransfection of a dominant negative Ras mutant inhibited FGF-2 induced CCK transcription, suggesting that FGF-2 mediates its effects via activation of Ras. Further downstream activation is mediated by the ERK MAPK and the p38 MAPK signaling pathways, which eventually lead to CREB phosphoryla tion and CREB-dependent CCK gene transcription. The results suggest that the ERK and p38 MAPK pathways proceed in parallel, which is in agreement with recent data showing that nerve growth factor (NGF) mediated phosphorylation of CREB also proceeds via the ERK and p38 MAPK pathways [34]. As mentioned above the signaling pathways that activate p38 are not completely understood, but since a dominant negative Ras mutant can inhibit signaling, it is possible that Ras controls p38 via phosphatidylinosito l 3-kinase (PI3-K) mediated activation of the Rho subfamily of small GTPases [35]. Although the above study has focused on the effects by FGF-2, the mechanisms may be extended to nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which have been demonstrated to activate the MAPK pathways in a similar way as FGF-2 [27, 34, 36] and to induce CCK expression in different brain areas [12]. Th e cA MP sign aling p athwa y A large number of signals in the CNS are transduced via binding of hormones, neurotransmitters, or neuropeptides to the seven-transmembrane domain class of receptors, which activate receptor-associated GTP-binding proteins (Gproteins). The Gs protein stimulates the activation of the adenylate cyclase enzyme family, which converts ATP to cAMP. The major target of cAMP in most cells is the cAMP-dependent protein kinase A (PKA), which upon binding of cAMP releases its two catalytic subunits that become translocated to the nucleus and phosphorylates CREB [37]. cAMP also activates the ERK MAPK signalling pathway. Both PKA-independent and PKA-dependent signaling pathways have been

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proposed to be involved in this process. PKAindependent activation involves a family of cAMP-binding guanine nucleotide exchange factors (cAMP-GEFs), which are activated by cAMP binding. The cAMP-GEFs in turn directly activate the Ras family member Rap1, which is an upstream target of the Raf family member B-Raf and subsequently the ERK MAPK signaling pathway. PKA-dependent Rap1 activation may involve a PKA induced increase in intracellular calcium levels, PKA induced phosphorylation of Rap1, which induces the association of B-Raf with Rap1, or PKA induced phosphorylatio n of B-Raf [38]. Finally, cAMP has also been shown to activate Ras in a PKAindependent manner [39], probably via activation of cAMP-Ras-GEFs. The role of cAMP in neuronal CCK gene transcription has been examined in both SK-NMC and PC12 cells transfected with CCK promoter constructs and treated with forskolin, an activator of adenylate cyclase [15, 19, and Hansen et al., unpublished]. In both cell lines, forskolin potently activated the CCK promoter via the CRE/TRE in the proximal promoter region. PKA mimicked the effects of forskolin in SK-N-MC cells [19], suggesting that PKA is mediating the effects by forskolin. Treatment with PKA and ERK MAPK inhibitors revealed that forskolin-induced CCK promoter activation and CREB-dependent transcription were mediated via PKA, but also via cAMP activation of the ERK MAPK pathway in PC12 cells (Hansen et al., unpublished). Forskolin activated ERK in a PKA-independent manner (Hansen, unpublished), suggesting that cAMP activates ERK via cAMP-GEFs in PC12 cells. Taken together, the studies suggest that cAMP targets the CCK promoter and CREB-dependent transcription via activation of PKA and via activation of the ERK MAPK pathway in a PKA-independent manner. The calcium signaling pathway Calcium is an important signaling molecule in the central nervous system, where a transient increase in the intracellular calcium concentrations induces changes in gene expression via activation of transcription factors. Calcium flows into neurons from the extracellular space

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through neurotransmitter receptors or voltagegated calcium channels or it can be released from intracellular stores [40]. The major calcium binding protein is calmodulin, which is found in both cytoplasmic and nuclear compartments. The calcium/calmodulin complex activates several protein kinases, including the calcium/calmodulin-dependent protein kinases (CaMKs). One member of this family, CaMKIV is predominantly located in the nucleus and has been shown to be an important regulator of gene expression via its ability to phosphorylate and activate transcription factors such as CREB [41]. Calcium influx has moreover been shown to activate the ERK MAPK pathway [42]. The exact mechanism is still unclear, but may involve the calcium-regulated guanine-nucleotide releasing factor Ras-GRF, calcium-Rap1-GEFs, or EGF-independent tyrosine phosphorylatio n of EGF receptors that culminate in Ras activation [43–45]. Finally, calmodulin has been shown to modulate the activity of adenylate cyclases, increasing the intracellular levels of cAMP and thereby activating PKA [46]. To examine the role of calcium in regulated CCK gene expression, PC12 cells were transfected with CCK promoter constructs and treated with KCl (Hansen et al., unpublished), that stimulates the opening of L-type voltagesensitive calcium channels. KCl activated CCK transcription via the CRE/TRE in the proximal promoter region. Treatment with inhibitors of PKA, ERK MAPK, and CaMKIV revealed that calcium-induced promoter activation was mediated by the sequential activation of PKA and ERK MAPK signaling pathways, whereas activation did not involve CaMKIV. Calciuminduced ERK activation was inhibited by a PKA inhibitor (Hansen, unpublished) indicating that calcium-sensitive adelylate cyclases were involved in ERK MAPK activation. In conclusion, the data suggest that calcium activates the CCK promoter and CREB-dependent transcription via a sequential activation of calcium sensitive adelylate cyclases, PKA, and the ERK signaling pathway.

S y n e r g i s t i c a c t i va t i o n o f C C K g e n e tran scrip tio n The combined treatment with either forskolin/ FGF-2 or forskolin/KCl synergistically stimulated CCK promoter activation [19, and Hansen et al., unpublished]. Forskolin and FGF-2 treatment induced a combined activation of PKA, ERK MAPK, and p38 MAPK and these kinases converge on CREB resulting in increased phosphorylation of CREB Ser-133, increased CREB-dependent transcription and ultimately synergistic activation of CCK gene transcription. Combined treatment with forskolin and KCl results in the activation of PKA and ERK MAPK and induces synergistic activation of the ERK MAPK pathway (Hansen, unpublished), of CREB-dependent transcription, and of CCK promoter activation. However, forskolin and KCl did not induce a synergistic increase in the level of CREB phosphorylation, suggesting that one or more targets downstream of CREB could be important for synergisticly induced CCK promoter activation and CREB-dependent transcription. The signaling pathways leading to cumulative activation of CCK gene transcription are outlined in Fig. 2. Fu nc tio n of CREB an d CCK in neurons The cAMP, FGF-2, and calcium signaling pathways all mediate their effects via the CRE/TRE in the proximal part of the human CCK promoter. Although the element is a potential binding site for both the CREB/ATF and the AP-1 families of transcription factors, all available data indicate that promoter activation is almost exclusively mediated by CREB [19, and Hansen et al., unpublished]. CREB belongs to the CREB/ATF family, which also includes the transcription factors CREM, ATF1, ATF-2 (also called CRE-BP1), ATF-3, and ATF-4 (also known as CREB2) [47]. A common feature of this family is the basic leucine zipper (bZIP) domain. The leucine zipper repeat is involved in the formation of homo- and heterodimers, while the basic region just N-terminal to the leucine zipper is involved in the sequence-specific interaction with DNA. CREB is ubiquitousl y expressed

Regulation of CCK gene transcription Ca 2+ channel

Hormone/Neurotransmitter receptor

Plasma Membrane

Growth factor receptor

Calcium sensitive cyclase

Adenylate cyclase

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SOS

Ca 2+ RasGRF

cAMP

Ca 2+-Rap1-GEFs cAMP-Rap1-GEFs

Rap1

Ras ?

Raf PKA

MEK ERK

Nucleus

RSK2 PKAc

CCK gene

P

P

MKK3/6 p38

MSK1 MAPKAP-K2/3

CREB CRE

FIG. 2. Model of the signaling pathways leading to cumulative activation of the CCK gene promoter. Forskolin, neurotransmitter s or neuropeptide s stimulate adenylat e cyclas e activity and cAMP production , which leads to the translocatio n of the catalyti c subunit of PKA to the nucleus, where it phosphorylate s CREB on Ser-133. Moreover, the PKA pathway activates the ERK MAPK signalin g pathway either directly by PKA or via cAMPRap1-GEFs. KCl induces depolarization , which results in calcium influx via L-type voltage-sensitiv e calcium channels . Calcium can subsequentl y activate the ERK MAPK pathway via activation of Rap1 or Ras. Rap1 can be activated either by calcium-Rap1-GEFs or by cAMP-Rap1-GEFs via calcium/calmoduli n sensitive adenylate cyclases, whereas Ras can be activated by RasGRFs. Receptor binding of FGF-2 is followed by stimulation of Ras, which activates the ERK MAPK and the p38 MAPK pathways. The downstrea m activators of ERK and p38 include RSK-2, MAPKAP kinase-2, and MSK1, respectively, which all have been demonstrate d to be CREB kinases [26–28, 34]. Cross-talk between the PKA, ERK and p38 MAPK pathways results in increased CREB and CBP activatio n and ultimately to increased CCK gene transcription .

and binds to CREs as a dimer. CREB is activated by phosphorylation of Ser-133 [48]. Phosphorylate d CREB recruits the transcriptional co-activator CBP, which is a 265-kDa nuclear protein that promotes transcription via direct interaction with components of the basal transcriptional machinery and via its intrinsic histone acetyltranferase activity [49]. CBP has recently been proposed to be a target for several kinases, including PKA, CaMKIV, ERK MAPK, and RSK2 [50], suggesting that both CREB and CBP phosphorylation could be necessary for maximal gene expression. The CREB family of transcription factors is critical for a variety of neuronal responses,

including memory, learning, and neuronal plasticity during development. Similar to CCK-B receptor-deficient mice, mice with a targeted mutation in CREB had deficient long-term memory [3, 51], suggesting that CREB could play an important role in the induction of CCK during memory processes. Moreover, CREdependent regulation is essential for the control of neuronal plasticity during postnatal neocortical development [52]. Since CCK mRNA levels reach adult-like patterns three weeks after birth [4–6], brain CCK may be an important CRE-regulated gene during the final maturation of the CNS. Finally, CCK, CREB and CBP are co-expressed in large parts of the

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mature CNS. CCK is expressed in major parts of the neocortex, hippocampus and the thalamus [5, 9] and CBP has been localized to CCK neurons in the hippocampus and the thalamus [53]. Taken together, the spatio-temporal and functional relationship between the factors may indicate that CRE-dependent transcriptional activation of CCK is of major significance in long-term memory and neuronal maturation. References 1 Rehfeld JF. Cholecystokinin . In: Schultz SG, Makhlouf GM, Rauner D, eds. Handboo k of Physiology: The Gastrointestina l System. Bethesda: American Physiologica l Society, 1989: 337–58. 2 Fink H, Rex A, Voits M, Voigt JP. Major biological actions of CCK – a critical evaluatio n of research findings . Exp Brain Res 1998; 123: 77– 83. 3 Sebret A, Lena I, Crete D, Matsui T, Roques BP, Dauge V. Rat hippocampa l neurons are criticall y involved in physiologica l improvement of memory processe s induced by cholecystokinin- B receptor stimulation . J Neurosci 1999; 19: 7230– 7. 4 Hasegawa M, Usui H, Araki K, Kuwano R, Takahashi Y. Developmenta l and regiona l change s of cholecystokini n mRNA in rat brains. FEBS Lett 1986; 194: 224–6. 5 De Belleroch e J, Bandopadhya y R, King A, Malcolm AD, O’Brien K, Premi BP, Rashid A. Regional distributio n of cholecystokini n messenger RNA in rat brain during development : quantitatio n and correlatio n with cholecystoki nin immunoreactivit y. Neuropeptide s 1990; 15: 201–12. 6 Mogense n NW, Hilsted L, Bardram L, Rehfeld JF. Procholecystokini n processin g in rat cerebral cortex during development . Brain Res Dev Brain Res 1990; 54: 81–6. 7 Rehfeld JF. Immunochemical studies on cholecystokinin . II. Distribution and molecula r heterogeneit y in the central nervous system and small intestin e of man and hog. J Biol Chem 1978; 253: 4022–30. 8 Crawley JN. Comparative distributio n of cholecystokinin and other neuropeptides . Why is this peptide different from all other peptides ? Ann N Y Acad Sci 1985; 448: 1–8. 9 Larsson LI, Rehfeld JF. Localization and molecular heterogeneit y of cholecystokini n in the central and periphera l nervous system. Brain Res 1979; 165: 201–18. 10 Monstein HJ, Folkesson R, Geijer T. Procholecystokinin and proenkephali n A mRNA expression is modulated by cyclic AMP and noradrena line. J Mol Endocrinol 1990; 4: 37–41.

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