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Endocrinology Vol. 139, No. 3 1184-1196
Copyright © 1998 by The Endocrine Society

ARTICLES

Analysis of Molecular Mechanisms Controlling Neuroendocrine Cell Specific Transcription of the Chromogranin A Gene1

Lucie Canaff2, Sarah Bevan2,3, Damian G. Wheeler, Andrew J. Mouland, Robert P. Rehfuss, John H. White4 and Geoffrey N. Hendy5

Departments of Medicine (L.C., S.B., A.J.M., R.P.R., G.N.H.), Physiology (D.G.W., J.H.W., G.N.H.), and Human Genetics (G.NH.), McGill University and Royal Victoria Hospital, Montréal, Québec H3A 1A1, Canada

Address all correspondence and requests for reprints to: Dr. Geoffrey N. Hendy, Calcium Research Laboratory, Room H4–67, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Québec H3A 1A1, Canada. E-mail: gnhendy{at}medcor.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chromogranin A (CgA), a member of the granin/secretogranin family of acidic glycoproteins that play multiple roles in the process of regulated secretion of peptide hormones and neurotransmitters, is specifically expressed in endocrine and neuroendocrine cells. We previously cloned and characterized the human (h) CgA gene and showed that nucleotides -55 to +32 relative to the transcriptional start site that contain a consensus cAMP element (CRE) and TATA-box motif were sufficient for neuroendocrine cell-specific expression. Here, we examined the role of the well conserved CRE in basal and cAMP-stimulated transcription in neuroendocrine cells. Transient transfection studies with hCgA gene promoter/chloroamphenical acetyl transferase (CAT) reporter constructs were conducted in a panel of neuroendocrine cell lines as well as in nonendocrine cell lines. Deletion or mutation of the CRE resulted in loss of neuroendocrine cell specific transcriptional activity. Mutation of a well conserved region (the TG-box) located between the CRE and the TATA box had no effect or resulted in only a modest decrease in activity. Mutation of the CRE in 5'-extended (-2300 to +32 and -700 to +32) constructs resulted in a 50–75% decrease in basal activity in neuroendocrine cells. This emphasized the importance of the CRE in basal transcription and also suggested that other elements between -700 and -55 may act independently of the CRE to contribute to full basal activity in some neuroendocrine cells. Dibutyryl cAMP stimulated transcriptional activity in neuroendocrine cells, and this was abolished by mutation of the CRE. In the presence of a PKA inhibitor, dibutyryl cAMP-induced activity was completely abolished and basal activity was decreased by up to 85%. Similar protein-DNA complexes were formed in gel retardation assays with a CgA-CRE oligonucleotide and nuclear extracts from both neuroendocrine and nonendocrine cells. A predominant complex that was supershifted by addition of a CREB antibody was identical in all cell types. By immunoblot analysis, levels of total CREB protein and phosphorylated (Ser 133) CREB did not differ between neuroendocrine and nonendocrine cells. Phosphorylated CREB was increased by forskolin treatment, an effect that was blocked by a PKA-inhibitor. Expression of the transcriptional cointegrator, CREB-binding protein (CBP), assessed by both RT-PCR and Western blot analysis, did not differ between neuroendocrine and nonendocrine cells. In summary, the CRE in the hCgA gene proximal promoter is critical for both basal and cAMP-induced expression in neuroendocrine cells via a PKA-mediated pathway. However, the neuroendocrine specificity of hCgA gene transcription mediated by the CRE is not a function of levels of total CREB or phosphorylated CREB or its cointegrator CBP. Specificity may be achieved by a PKA-responsive CRE-binding protein other than CREB expressed specifically in neuroendocrine cells, expression of a repressor molecule that binds CREB in nonendocrine cells, or may lie downstream of a CRE-binding protein, e.g. in the activity or amount of cointegrators other than CBP, which are required to couple transactivators to the basal transcriptional machinery.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHROMOGRANIN A (CgA) is a member of the chromogranin/secretogranin (granin) family of acidic secretory glycoproteins that are expressed in all endocrine and neuronal cells. Granins are efficiently directed to granules of the regulated secretory pathway where they are co-stored with the resident hormone or neurotransmitter until release from the cell by exocytosis. Granins have been proposed to play multiple roles in the secretory process. They act intracellularly by modulating prohormone processing and targeting peptide hormones or neurotransmitters to secretory granules of the regulated pathway, and they function as proproteins that are processed to biologically active peptides that act extracellularly to modulate hormone secretion in an autocrine or paracrine manner (see Refs. 1–4 for review).

We previously isolated and characterized the human (h) CgA gene (5). Its organization is very similar to that of the mouse (6) and bovine (7) genes, and the proximal promoter region is strongly conserved across species (5, 6, 7, 8). We (5) and others (6) showed that 2-kb of the 5'-flanking region of the human and mouse CgA genes conferred neuroendocrine cell-specific transcriptional activity upon reporter genes in transient transfection studies. We defined a minimal promoter construct containing 87 bp of the human CgA gene (nucleotides -55 to +32 relative to the transcriptional start site) directed neuroendocrine cell specific activity of a chloramphenicol acetyl transferase (CAT) gene. This portion of the hCgA gene promoter contains a consensus cAMP response element (CRE) and a TATA-box motif. These two elements are conserved in the CgA gene across species and are also found in the gene promoters of other members of the granin family such as chromogranin B (CgB) (9) and secretogranin II (SgII) (10, 11). This indicated that the CRE in addition to the TATA box may play a critical role in the expression of the granins. Evidence in support of this has been presented by Wu et al. (12) for the mouse (m) CgA gene and by us (13, 14) for the hCgA gene.

In the present study, we carried out gene transfer experiments with hCgA gene promoter/CAT reporter gene constructs in a variety of human and rodent neuroendocrine cell lines to further understand the role of the CRE and another conserved sequence (the TG-box) just downstream of the CRE in the minimal promoter region in the regulation of CgA gene expression. Our results demonstrate that the CRE plays a key role in basal as well as in cAMP-stimulated expression of the hCgA gene in neuroendocrine cells, whereas the TG-box plays a much lesser role and only contributes to basal transcriptional activity in selected neuroendocrine cell types. However, we found by gel retardation assays and immunoblot analyses equivalent amounts of PKA-sensitive CREB in neuroendocrine and nonendocrine cells. Expression of the CREB-binding protein (CBP) was similar in neuroendocrine and nonendocrine cells. In addition, we have determined that the distal promoter contains additional positively acting, cell-specific regulatory elements that function independently of the CRE. Finally, we have identified a sequence downstream of the transcriptional start site, which is not conserved across species, and contributes positively to basal expression of the CgA/CAT reporter gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All cell lines, with the exception of the BEN cell line, which was kindly given by Dr. L. J. Deftos (San Diego, CA), were from the American Type Culture Collection (Rockville, MD). DMEM, antibiotics, horse serum, and FBS were from Life Technologies, Inc. Kaign’s modified Ham’s F12 medium (F12K) was from Irvine Scientific (Irvine, CA). [14C]chloramphenicol (45 Ci/mmol) was from ICN Biomedicals (Baie d’Urfé, Québec, Canada). The lipofectin reagent (a 50:50 suspension of (1,2-dioleoyloxy-3-(trimethylamminio)-propane and dioleoylphosphatidylethanolamine in sterile, pyrogen-free water) for transfection experiments was kindly provided by Dr. J. R. Silvius (McGill University) or was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Restriction enzymes and modifying enzymes were from Pharmacia LKB Biotechnology, Inc. (Baie d’Urfé, Québec, Canada) or Life Technologies, Inc. Acetyl coenzyme A and the ß-galactosidase expression vector, pCH110, were from Pharmacia. Cyclic dibutyryl-A-3:5-MP was from Boehringer Mannheim and H-89 was from Biomol (Plymouth Meeting, PA).

Plasmid constructs
Human CgA promoter deletion constructs, p2300CAT, p700CAT, p450CAT, p260CAT, and p55CAT contained hCgA gene fragments, from 2316 bp to 55 bp upstream to 32 bp downstream of the start of transcription, cloned into the CAT expression vector, pBLCAT3. These constructs were prepared as described previously (5). The hCgA promoter constructs, p55(-CRE)CAT, p55(-TG)CAT, p55(-CRE/-TG)CAT, and p31CAT were created by ligating BamHI-XhoI fragments generated by PCR into pBLCAT3. The forward primer for the p55(-CRE)CAT fragment 5'-AAGTCAGGATCCGCCAGTGTCATTTCCGG-3', is composed of six nucleotides flanking a BamHI site followed by nucleotides -53 to -37, but with nucleotides -51 to -48 (TGAC) replaced by CAGT. The forward primer for the p55(-TG)CAT fragment 5'-AACTAGGGATCCGCTGACGTCATTGATCTGGTC-3', is composed of six nucleotides flanking a BamHI site followed by nucleotides -53 to -33, but with nucleotides -41 to -37 (TCCGG) replaced by GATCT. The forward primer for the p55(-CRE/-TG)CAT fragment 5'-AACGCGGGATCCGCCAGTGTCATTGATCTGGTCG-3' is composed of six nucleotides flanking a BamHI site followed by nucleotides -53 to -32, but with nucleotides -51 to -48 (TGAC) and nucleotides -41 to -37 (TCCGG) replaced by CAGT and GATCT, respectively. The forward primer for the p31CAT fragment 5'-TTTCCGGGATCCGGGTATATA-3' is composed of six nucleotides flanking a BamHI site followed by nucleotides -31 to -23. For all fragments the reverse primer was 5'-CACGGGGCTCGAGCAC-3', which is the complement of +25 to +41 and contains the XhoI site. Fragments were amplified by PCR, cleaved with BamHI and XhoI, gel-purified and ligated into BamHI-XhoI cut pBLCAT3.

Human CgA promoter constructs p2300(-CRE)CAT and p700(-CRE)CAT were the same as constructs p2300CAT and p700CAT, respectively, except that in the CRE motif, the sequence TGAC was mutated to CAGT. This mutation in the CRE was the same as that made in the shorter hCgA-CRE mutant constructs. Construct p700(-CRE)CAT was created by ligating a HindIII-XhoI fragment of 753 bp, generated by PCR, into HindIII and XhoI cleaved pBLCAT3. Recombinant p2300(-CRE)CAT was created by ligating a HindIII-HindIII fragment of 1481-bp (cleaved from p2300CAT, spanning from the HindIII site of pBLCAT3, just upstream from the hCgA insert, to the HindIII site at nucleotide -721 of the hCgA promoter) into P700(-CRE)CAT digested with HindIII. The HindIII-XhoI fragment of 753 nucleotides was generated by site-directed mutagenesis using PCR and spanned nucleotides -721 to +32 of the hCgA gene.

Human CgA gene constructs 3' extended to nucleotide +198 were made as follows. Recombinants p2300/198CAT, p450/198CAT, p55/198CAT, and p31/198CAT were made by inserting a hCgA gene fragment (+33 to +198) into the XhoI site at hCgA gene position +32 of the plasmids p2300CAT, p450CAT, p55CAT, and p31CAT, respectively. Construct p55/198–33CAT was made by inserting the +33 to +198 fragment in the antisense direction, into the XhoI site at hCgA gene position +32 of p55CAT. Recombinant p33–198/55CAT was made by inserting the hCgA +33 to +198 fragment in the sense direction, into the SalI site 28 bp upstream to the -55 to +32 hCgA sequence of p55CAT. The hCgA gene +33 to +198 fragment was created by PCR. The forward primer was 5'-CTGCAGTGCTCGAGCCCCGT-3'. This sequence is hCgA gene +20 to +39 with an additional XhoI site. The reverse primer was 5'-AGTTGCCTCGAGCAGCTGGCGGTGT-3'. This comprises six nucleotides flanking an XhoI site, followed by complementary hCgA gene sequence +194 to +182. The PCR amplified fragment was digested with XhoI, gel-purified, and ligated into hCgA-CAT plasmids as described above.

Plasmid DNAs were prepared by the alkaline lysis/cesium chloride gradient method or by alkaline lysis followed by affinity chromatography (Qiagen Inc., Chatsworth, CA). All hCgA-CAT constructs were verified by dideoxynucleotide sequencing.

Cell culture and transient transfection
Five neuroendocrine cell types were used: AtT20, a mouse pituitary corticotroph cell line; BEN, a human lung carcinoma cell line; GH4C1, a rat pituitary cell line; PC-12, a rat pheochromocytoma cell line and TT, a human medullary thyroid carcinoma cell line. Two nonendocrine cell lines were used: NIH 3T3, a mouse fibroblast cell line and Rat-2, a rat fibroblast cell line. BEN cells were maintained in RPMI with the addition of 10% FBS and L-glutamine. PC-12 cells were maintained in DMEM with the addition of 10% FBS and 5% horse serum. TT cells were maintained in Ham’s F12 medium with 10% FBS. All other cell lines were maintained in DMEM with 10% FBS. All maintenance media contained 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B.

For transfections, cells were trypsinized, seeded at 5 x 105 cells/35 mm tissue culture dish and incubated overnight. Medium was aspirated and cells were washed twice with serum-free DMEM. Transfections with 6 µg of the phCgACAT constructs, with the positive control SV40 early region promoter-driven plasmid pSV2CAT or the positive control Rous Sarcoma Virus LTR promoter/enhancer-driven plasmid pRSVCAT and the negative control promoterless plasmid, pBLCAT3, were done in parallel experiments in all cell lines. Each transfection involved cotransfection with 2 µg of control plasmid, pCH110, in which the gene for ß-galactosidase is under the control of the SV40 early promoter. DNA was transfected by lipofection (15) in the majority of cells. However, TT cells (10 x 106 cells) were transfected by electroporation with a single pulse of 250 V/4 mm, 960 microfarads using the Bio-Rad (Mississauga, Ontario, Canada) Cell Porator with an extended capacitance unit. Forty-eight hours after transfection, cells were washed once with ice-cold PBS and harvested in 1 ml of PBS by scraping into 1.5 ml Eppendorf tubes. Cells were spun at 500 x g at 4 C for 7 min and resuspended in 250 µl of 0.5 M Tris-HCl, pH 7.5. Cells were lysed by three freeze-thaw cycles, and the DNA and cell debris were removed by centrifugation at 16,000 x g for 20 min at 4 C. Cell lysates were assayed for protein using the BioRad protein assay kit and assayed for ß-galactosidase and CAT activity as described below. CAT activities were normalized to ß-galactosidase activity, and the data are presented as the fold-activity relative to that of the promoterless pBLCAT3 vector.

CAT and ß-galactosidase assays
CAT assays were performed with 5 to 20 µg of protein essentially as described (16). Reactions were incubated at 37 C for 0.5 to 4 h. For the longer incubations, 1 µg of 4 mM acetyl-CoA was added after 2 h of incubation. After thin layer chromatography of the samples, acetylation of chloramphenicol was first visualized with autoradiograph film and then quantitated by liquid scintillation counting. The CAT measurements were within the linear part of the response range. ß-galactosidase assays were done by measuring an increase in optical density at 420 nm using O-nitrophenyl-ß-D-galactopyranoside as substrate (16). For a single experiment duplicate or triplicate estimations were made, and the data were expressed as mean ± SEM of three to four independent experiments.

DNA mobility shift assays
Cells were harvested for gel retardation assays by washing with 5 ml ice-cold PBS followed by scraping the cells in 1 ml PBS. Cells were centrifuged at 2,000 rpm for 10 min at 4 C, and the pellets were resuspended in 100 µl high salt extraction buffer [25 mM Tris (pH 7.9), 0.3 mM DTT, 0.1 mM EDTA, 420 mM NaCl, 10% glycerol]. Cells were lysed by three cycles of freeze/thaw and cell debris removed by centrifugation at 10,000 rpm for 10 min at 4 C. Cell nuclear extracts were made by the protocol of Dignam et al. (17).

For gel retardation assays, cell nuclear extracts were added to incubations as indicated in the figures. Samples were incubated for 15 min on ice in 10 µl 25 mM Tris-HCl (pH 8.0), 1 mM DTT, 50 mM KCl, 20% glycerol containing 1 µg poly (dI-dC) and then for a further 20 min at 23 C after the addition of 50,000–100,000 cpm (5–10 fmol) of 32P-end-labeled double-stranded oligonucleotide. The nucleotide sequences of the CgA-CRE, CgA-CREmut 1, CgA-CREmut 2, CgA-CREmut 3, SS-CRE, AP-1, and SP-1 oligonucleotides are shown in Table 1Go. Specific antibodies to the CRE-binding protein (CREB) (New England Biolabs, Beverly, MA) were also added to some reactions. Samples were loaded on 5% polyacrylamide gels equilibrated in 25 mM Tris-HCl/31.1 mM boric acid/1 mM EDTA (pH 8.0) and electrophoresed at 8 V/cm. Gels were dried before autoradiography.


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  		Table 1. Oligonucleotides used in gel retardation assays

 
RT-PCR
Preparation of total RNA. After 48 h in culture, cells were scraped off with Trizol (GIBCO-BRL, Burlington, Ontario, Canada) and total RNA was isolated as described by the manufacturer. The total RNA isolated was then treated with DNAse I (Pharmacia) for 30 min at 37 C. The RNA was purified by a phenol/chloroform extraction and ethanol precipitation.

RT. Five-microgram RNA samples were reverse transcribed with recombinant superscript II RNAse H (GIBCO-BRL) using oligo (dT) 15–18 (Pharmacia) in a total volume of 20 µl. Five microliters of the RT mixture was subjected to standard PCR procedures.

Samples were assessed for CBP using the primers 5'-AGTGGAATTCAAAACACAATTGGTTCTGTTGGTGCAGGGCA-3' (forward) and 5'-TAAAGCTGGCTGGTTACCCAGGATGCCTTGCTTATGTAAACG-3' (reverse). These sequences are highly conserved between human, mouse, and rat CBP.

The ß-actin primers generated a 500-bp fragment whereas the CBP primers amplified a 704-bp fragment encoding a.a. 450 to 684 of the CREB binding region. The PCR mixture contained 2 mM MgCl2, 0.2 mM of each dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 25 pmol of forward and reverse primers and 2.5U/100 µl of Taq polymerase (GIBCO-BRL). Thirty cycles (94 C, 45 sec; 58 C, 45 sec; 72 C, 1 min) were performed with a programmable thermocycler (GeneAmp PCR system 9600, Perkin-Elmer Cetus, Mississauga, Ontario, Canada). Aliquots were taken after 15, 20, and 25 cycles.

Western immunoblots
Cells were lysed in triple detergent buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% NaN3, 0.1% SDS, 100 µg/ml PMSF, 1 µg/ml aprotonin, 0.1% NP-40, 0.5% sodium deoxycholate for 5 min at 0 C. The cell lysates were spun at 12,000 x g, 2 min, 4 C and the supernatants were stored at -80 C. Aliquots were electrophoresed through 12% SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Bio-Rad). Membranes were rinsed in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20), blocked with 5% dried milk powder in TBST for 1–2 h and incubated with antisera for either phospho-specific CREB (Ser 133) or total CREB (Ser 133 phosphorylation state-independent) according to manufacturer’s instructions (NEB Inc., Beverly, MA) or antibodies directed against epitopes in either the NH2-terminus or COOH-terminal portion of CBP (Upstate Biotechnology, Lake Placid, NY) or antihuman ß-tubulin monoclonal antibody (Cedarlane Laboratories Limited, Hornby, Ontario, Canada). Antibody-antigen complexes were detected by chemiluminescence using the Phototope-Star Western blot detection kit (NEB Inc., Beverly, MA), or the LumiGlo chemiluminescent substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The hCga gene exon I sequence augments expression in an orientation and position-dependent manner
Whereas the exon I untranslated regions of the human and bovine CgA genes are well conserved (Fig. 1AGo), those of the human and rodent CgA genes are not (Fig. 1BGo). In a previous study of the mouse (m) CgA gene promoter (8), it was found that inclusion of exon I sequence extending to close to the translation initiation codon in promoter constructs led to enhanced reporter gene activity in the single cell line (of neuroendocrine origin) tested. In our present study, we examined this issue further by extending hCgA gene promoter constructs to position +198, just upstream of the translation initiation codon. Transient transfection studies were carried out in human neuroendocrine BEN cells, rodent neuroendocrine GH4C1 cells, and the nonendocrine Rat2 cell line using the series of constructs shown in Fig. 2AGo.



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  		Figure 1. Comparison of exon I untranslated regions of the human, bovine, and mouse CgA genes. A, Alignment of exon I untranslated regions of the human and bovine CgA genes (hCgA and bCgA respectively). B, Alignment of exon I untranslated regions of the human and mouse CgA genes (hCgA and mCgA, respectively). Nucleotides are numbered from the transcription initiation site (+1). Untranslated nucleotides are shown in upper case. Translated nucleotides are shown in lower case (atg = translation initiation codon).

 


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  		Figure 2. Human CgA gene exon I sequence augments expression in an orientation and position-dependent manner. A, Constructs containing portions (from -2300 to -31) to +198 of the hCgA gene promoter inserted upstream of the CAT gene in pBLCAT3 were prepared as described in Materials and Methods. In addition, p55/198–33CAT in which nucleotides -55 to +32, followed by nucleotides +198 to +33 in the antisense orientation, and p33–198/55CAT, in which nucleotides +33 to +198 were inserted upstream of -55 to +32, in the sense orientation, were also prepared. The gray boxes represent the +33 to +198 sequence and the arrows inside the boxes indicate its orientation. Partial restriction map of the hCgA gene is shown at the top. Restriction sites shown are: B, BamHI; Xb, XbaI; Xh, XhoI; Pv, PvuII. B, Recombinants shown in A in which the +33 to +198 sequence is in its normal position and orientation were transiently transfected into neuroendocrine cell lines BEN and GH4C1 and into nonendocrine cell line Rat2. Cells were harvested 48 h after transfection, and cell lysates were assayed for CAT activity. C, Recombinants shown in A in which +33 to +198 is in the reverse orientation or is inserted upstream of position -55, were transiently transfected into neuroendocrine BEN and GH4C1 cells, or into nonendocrine cell line Rat2. Cells were harvested 48 h after transfection and cell lysates were assayed for CAT activity. In both B and C, results are mean ± SEM expressed relative to the activity of pBLCAT3.

 
The 3'-extended constructs (p2300/198CAT, p450/198CAT, p55/198CAT) were all more active than the corresponding recombinants terminating at +32 (p2300CAT, p450CAT, p55CAT) in BEN and GH4C1 neuroendocrine cells, although the relative fold-increase was most marked with the p55/198 construct and less so with the 5'-extended constructs (Fig. 2BGo). Importantly, the p31/198CAT construct demonstrated only baseline activity. Therefore the +33 to +198 sequence does not function independently of the proximal promoter elements but rather, cooperates with them to augment their activity. Enhanced activity of the shortest 3'-extended construct (p55/198CAT), but none of the others, was seen in the Rat2 nonendocrine cell line (Fig. 2BGo).

In BEN cells, the stimulatory effect of +33 to +198 was abolished or greatly reduced in constructs in which the sequence was either inverted (p55/198–33CAT) or moved upstream of position -55 (p33–198/55CAT). This suggests that this element does not function as a classical transcriptional enhancer in these cells. It is likely therefore that the major effect of the +33 to +198 sequence in BEN cells is either to enhance the stability of the messenger RNA (mRNA) or to increase its ability to be translated. However, in GH4C1 cells, the +33 to +198 sequence retained {approx}50% of its activity after inversion or placement upstream of the CRE. This suggests that this element may act as an enhancer to regulate transcription in certain cell types.

Neuroendocrine cell-specific transcriptional activity of the human CgA gene promoter
Putative binding sites for transcription factors within the 5'-flanking region of the hCgA gene are shown in Table 2Go. Constructs containing portions (from -2300 to -31) to +32 of the hCgA gene promoter inserted upstream of the CAT gene (Fig. 3Go) were transiently transfected into a variety of neuroendocrine and nonendocrine cell lines to determine the minimal portion of the 5'-flanking region of the hCgA gene necessary for neuroendocrine cell-specific transcriptional activity. Constructs with 2300 bp to 55 bp of 5'-flanking sequence were several-fold more active than a promoterless CAT construct in all neuroendocrine cell lines (Fig. 3BGo), but showed activity no greater than pBLCAT3 in nonendocrine cells (Fig. 3CGo). The p31CAT construct, which retains the TATA-box but lacks the CRE-containing region was inactive in neuroendocrine as well as nonendocrine cells (Fig. 3Go, B and C). The observation that the hCgA-CAT constructs were inactive in nonendocrine cells indicated that the neuroendocrine cell-specific activity is not due to a nonendocrine cell repressor mechanism.


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  		Table 2. Putative binding sites for transcription factors in the hCgA -2300 to +198 sequence1

 


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  		Figure 3. Neuroendocrine cell-specific transcriptional activity of the human CgA gene promoter. A, Constructs containing portions (from -2300 to -31) to +32 of the hCgA gene promoter inserted upstream of the CAT gene in pBLCAT3. Partial restriction map of the hCgA gene is shown at the top. Restriction sites shown are: B, BamHI; H, HindIII; Xb, XbaI; Xh, XhoI. B, Recombinants shown in A were transiently transfected into neuroendocrine cell lines TT, BEN, PC12, GH4C1, and AtT20. Cells were harvested 48 h after transfection and cell lysates were assayed for CAT activity. Results are mean ± SEM for all neuroendocrine cell lines tested expressed relative to the activity of pBLCAT3. C, Recombinants shown in A were transiently transfected into nonendocrine cell lines Rat2 and NIH 3T3. Experiments were performed and results expressed as described in B above.

 
Within each particular neuroendocrine cell type the relative activities of the series of deletion constructs varied by no more than 2- to 3-fold (Fig. 3BGo), indicating that portions of the distal promoter may have modest enhancer or repressor activity. However, no consistent pattern was observed among cell types, suggesting that these effects are cell specific. Most importantly, these observations emphasized the fact that for all neuroendocrine cell-types the proximal promoter, -55 to -32 sequence containing the CRE, was critical for basal transcriptional activity.

The CRE is critical for basal transcriptional activity in neuroendocrine cells
The hCgA gene promoter from -55 to +1 is shown in Fig. 4AGo. In addition to the TATA-box, this region contains the CRE (-51 to -44) and another well conserved sequence designated the TG-box (-43 to -34) (see Fig. 7Go in Ref.5). To determine the importance of these motifs in basal transcriptional activity, hCgA (-55 to +32)CAT constructs mutated in either the CRE, or the TG-box, or both (Fig. 4AGo) were transiently transfected into neuroendocrine and nonendocrine cell-lines. In all neuroendocrine cell lines, mutation of the TG-box [p55(-TG)CAT] either had no effect (in BEN, GH4C1, and AtT20 cells), or resulted in an approximately 2-fold reduction in activity (PC12 and TT cells) (Fig. 4BGo). However, in all cell lines mutation of the CRE [p55(-CRE)CAT] or combined mutation of the CRE and TG-box [p55(-CRE/-TG)CAT] dramatically reduced transcriptional activity (Fig. 4BGo). In nonendocrine Rat2 (Fig. 4BGo) and NIH3T3 (data not shown) cells the wild-type and mutated hCgA constructs were no more active than pBLCAT3. These results emphasized the critical role played by the CRE, but not the TG-box, in basal transcription in all neuroendocrine cells tested.



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  		Figure 4. The CRE is critical for basal transcriptional activity in neuroendocrine cell lines. A, The hCgA gene promoter sequence from -55 to +1. The CRE (-51 to -44) and the TG-box (-43 to -34) are overlined, and the TATA motif is boxed. Human CgA(-55 to +32)CAT recombinants mutated either in the CRE [p55(-CRE)CAT], or in the TG-box [p55(-TG)CAT], or both [p55(-CRE/-TG)CAT] were constructed as described in Materials and Methods. B, Recombinants shown in A were transiently transfected into neuroendocrine cell lines BEN, TT, PC-12, GH4C1, and AtT20, and nonendocrine cell line Rat2. Cells were harvested 48 h after transfection and cell lysates were assayed for CAT activity. Results are mean ± SEM for all cell lines tested relative to the activity of pBLCAT3.

 


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  		Figure 7. Comparison of protein-DNA complexes formed in gel retardation assays with a CgA-CRE oligonucleotide and nuclear extracts from both neuroendocrine and nonendocrine cells. A, The gel retardation assays show that similar protein-DNA complexes formed with nuclear extracts from neuroendocrine GH4C1, TT, and nonendocrine N1H3T3, Rat-2 cell lines, and [32P]-labeled CgA-CRE-WT oligonucleotide spanning -56 to -39 of the human CgA gene promoter (see Table 1Go). Addition of anti-CREB antibody (+) shifted the complex of faster mobility (->) to one of slower mobility (S->), which was identical in neuroendocrine and nonendocrine extracts. B, Oligonucleotides mutated in the CRE (CgA-CRE-MUT1, CgA-CRE-MUT2) were unable to form the same protein-DNA complexes as CgA-CRE-WT in both neuroendocrine AtT-20 and nonendocrine Rat-2 cell extracts whereas oligonucleotide CgA-CRE-MUT3, mutated outside the CRE, was able to do so. C, Competition analysis of oligonucleotides containing or lacking CREs in displacing binding of the CgA-CRE to neuroendocrine or nonendocrine extracts. Extracts of BEN cells or PC-12 cells were incubated with radiolabeled CgA-CRE-WT probe either alone or with 100x of each of the following competitor oligonucleotides: SS-CRE, SP-1 or AP-1 (see Table 1Go).

 
It is also noteworthy that mutation of the CRE or the TG-box did not lead to enhanced expression in nonendocrine cell lines that might have been anticipated if a repressor functioned through this region in nonendocrine cells. The demonstration that the minimal hCgA promoter constructs containing a mutated CRE are as active in nonendocrine cells as in neuroendocrine cells provides strong evidence that a significant proportion of the tissue-specific activity of the minimal hCgA promoter is mediated by the positive action of the CRE in neuroendocrine cells rather than the negative action of a putative repressor in nonendocrine cells.

Role of the CRE in basal transcriptional activity in the 5'-extended hCgA promoter
Having shown that the CRE is important for neuroendocrine cell-specific transcriptional activity in the context of the minimal promoter construct, p55CAT, we next assessed the importance of the CRE in 5'-extended constructs. Human CgA (-2300 to +32) and (-700 to +32) gene promoter constructs mutated in the CREs (see Fig. 5AGo) were transiently transfected into neuroendocrine and nonendocrine cell lines. In all neuroendocrine cell lines tested the mutated CRE constructs [p2300(-CRE)CAT and p700(-CRE)CAT] were less active than the wild-type p2300CAT and p700CAT constructs (Fig. 5BGo). The relative reductions in activity were 50% in BEN cells, 65% in AtT20 cells, and 75% in GH4C1 cells (Fig. 5BGo). In nonendocrine Rat2 cells, the activities of wild-type p2300CAT, p700CAT, mutants p2300(-CRE)CAT, and p700(-CRE)CAT were similar to that of the promoterless construct, pBLCAT3 (Fig. 5BGo). We therefore concluded that the CRE is important for basal activity in neuroendocrine cells even in the context of the 5'-extended hCgA promoter. The results also indicate that other elements between -700 and -55 act independently of the CRE, and contribute to full basal activity to varying extents depending upon the particular neuroendocrine cell type.



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  		Figure 5. Role of the CRE in basal transcription in the 5'-extended hCgA promoter. A, Recombinants p2300(-CRE)CAT and p700(-CRE)CAT. Mutations (indicated by asterisks) were made in the CRE (double underlined). The TATA-box is underlined. Partial restriction map of the hCgA gene is shown above the hCgA-CAT constructs. Arrow indicates start of transcription. Restriction sites shown are: B, BamHI; H, HindIII; Xh, XhoI. B, Recombinants shown in A, and p2300CAT, p700CAT, and pBLCAT3 were transiently transfected into neuroendocrine cell lines BEN, GH4C1, and AtT20, and into the nonendocrine cell line, Rat2. Cells were harvested 48 h after transfection, and cell lysates were assayed for CAT activity. Results are mean ± SEM for all cell lines tested expressed relative to the activity of pBLCAT3.

 
The human CgA promoter is responsive to cAMP: role of the CRE and protein kinase A
Given the critical role of the CRE in basal transcription of the hCgA gene we assessed whether this motif mediates cAMP responsiveness of the hCgA gene. Neuroendocrine BEN cells were transiently transfected with both wild type and CRE-mutated hCgA (-2300 to +32) and (-55 to +32) constructs in the absence and presence of 1 mM dibutyryl cAMP. The wild-type p2300CAT (data not shown) and p55CAT constructs were stimulated 2-fold over basal by dibutyryl cAMP, an effect that was abolished by mutation of the CRE (Fig. 6AGo). The control pSS-70 construct containing a CRE, showed high basal activity which was only slightly stimulated by 1 mM dibutyryl cAMP (Fig. 6AGo).



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  		Figure 6. The human CgA promoter is responsive to cAMP: role of the CRE and protein kinase A. A, BEN neuroendocrine cells were transiently transfected with p55CAT, p55(-CRE)CAT, pBLCAT3, and pSS70CAT. Twenty-four hours after transfection, the medium was exchanged with fresh medium only or medium containing 1 mM dibutyryl cAMP as indicated. Cells were harvested 48 h later, and cell lysates were assayed for CAT activity. Results are mean ± SEM expressed relative to the activity of pBLCAT3. B, Neuroendocrine BEN cells were transiently transfected with p55CAT, p31CAT, and pBLCAT3. Twenty-four hours after transfection, the culture medium was exchanged for fresh medium only or medium containing 10 µM H-89, a selective protein kinase A inhibitor, as indicated. Cells were harvested 48 h after transfection, and CAT activity of cell lysates measured. Results are mean ± SEM expressed relative to the activity of pBLCAT3. C, Neuroendocrine BEN cells were transiently transfected with p55CAT, p31CAT, and pBLCAT3. Twenty-four hours after transfection, the medium was exchanged for fresh medium only, medium with addition of 1 mM dibutyryl cAMP only, or medium with addition of both 1 mM dibutyryl cAMP and 10 µM H-89. Cells were harvested 48 h after transfection and CAT activity of cell lysates measured. Results are mean ± SEM expressed relative to the activity of pBLCAT3.

 
Given the modest effect of dibutyryl cAMP under these conditions, the role of protein kinase A in controlling both basal and cAMP-inducible CgA gene expression was investigated. The effect of the highly selective PKA inhibitor H-89 (18) on CgA expression was tested in the absence and presence of 1 mM dibutyryl cAMP. Basal transcriptional activities of p2300CAT (data not shown) and p55CAT were reduced by 63% and 85%, respectively, in the presence of 10 µM H-89 (Fig. 6BGo). In an experiment in which 1 mM dibutyryl cAMP stimulated transcriptional activity of p55CAT over 2-fold, addition of the PKA inhibitor reduced activity to 46% of unstimulated control (Fig. 6CGo). Neither 1 mM dibutyryl cAMP nor 10 µM H-89 had any effect on the activities of p31CAT and pBLCAT3. Taken together, our results show that the protein kinase A pathway is important for both basal and stimulated expression of the hCgA gene under these conditions.

Similar protein-DNA complexes form on the hCgA CRE in neuroendocrine and nonendocrine cells
To determine whether the same or different protein-DNA complexes form on the hCgA gene CRE in neuroendocrine and nonendocrine cells, we performed a series of gel retardation assays. As shown in Fig. 7AGo similar protein-DNA complexes formed with nuclear extracts from neuroendocrine GH4C1, TT and nonendocrine NIH3T3, Rat-2 cell lines and a [32P]-labeled CgA-CRE-WT oligonucleotide. Similar results were obtained with extracts from neuroendocrine AtT-20, BEN, PC-12, and nonendocrine COS-7 cells (data not shown). Addition of CREB antibody supershifted the same predominant complex in both neuroendocrine and nonendocrine cells. The specificity of complex formation was demonstrated by the inability of [32P]-labeled CRE mutant oligonucleotides to form the same protein-DNA complexes as [32P]-labeled CRE-CgA-WT oligonucleotide with neuroendocrine AtT-20 and nonendocrine Rat-2 extracts (Fig. 7BGo). Similar results were obtained with neuroendocrine GH4C1 nuclear extracts (data not shown). When several oligonucleotides containing either CRE, CRE-like, or unrelated sequences were tested for their ability to compete with the [32P]-labeled CgA-CRE-WT oligonucleotide only the somatostatin gene CRE was able to do so as shown with neuroendocrine BEN and PC-12 extracts in Fig. 7CGo. Similar results were obtained with neuroendocrine AtT-20 and GH4C1 cells and nonendocrine Rat-2 cells.

Basal and forskolin-stimulated phosphorylated CREB is expressed equivalently in neuroendocrine and nonendocrine cells
Gel retardation experiments suggested that similar amounts of CREB were expressed in neuroendocrine and nonendocrine cells. These observations were supported by Western blot analysis (Fig. 8AGo). Similar amounts of total CREB and phospho-CREB (Ser 133) were found in neuroendocrine AtT-20, BEN, GH4C1, PC-12, and nonendocrine N1H3T3 and Rat-2 cells. The basal levels of phospho-CREB (Ser 133) were markedly stimulated in all cells by combined forskolin and IBMX treatment and, as shown in Fig. 8BGo, this stimulation was blocked by H89, a PKA-selective inhibitor. Thus, we find no correlation between cell-specific CgA promoter activity and levels of functional CREB.



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  		Figure 8. Western immunoblot of cell extracts from neuroendocrine and nonendocrine cells treated with forskolin and IBMX using phospho-CREB or CREB antibodies. A, Upper panel, Phospho-CREB; lower panel, total CREB expression in neuroendocrine AtT-20, BEN, GH4C1, PC-12 and nonendocrine N1H3T3, Rat 2 cells either untreated or treated with 10 µM forskolin and 100 µM IBMX for 2 h. B, Upper panel, Phospho-CREB; lower panel, total CREB expression in neuroendocrine BEN and nonendocrine Rat 2 cells either untreated or treated with 10 µM forskolin and 100 µM IBMX with and without 10 µM H89.

 
CREB binding protein expression in neuroendocrine and nonendocrine cells
The mechanisms governing cell-specific expression of the CgA promoter were further probed by analysis of expression of CBP in neuroendocrine and nonendocrine cells. Expression of CBP mRNA was probed by semiquantitative RT-PCR analysis of mRNA levels by amplification of a 704-bp band encoding the CREB binding region of CBP. Strikingly, no difference was detected in levels of CBP mRNA in neuroendocrine and nonendocrine cells (Fig. 9Go). Protein levels were assessed by Western blotting using antibodies specific to the N-terminal or C-terminal regions of CBP. Abundant levels of full length CBP (265 kDa) were detected with both antibodies (Figs. 10Go, A and B) in neuroendocrine (AtT-20, BEN, GH4CI, PC-12, TT) and nonendocrine cells (Cos-7, N1H3T3, Rat-2, Ros).



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  		Figure 9. Neuroendocrine and nonendocrine cells express equivalent amounts of CBP mRNA. RT-PCR analysis was performed on RNA extracted from neuroendocrine AtT-20, BEN, GH4C1, PC-12, and TT cells, and nonendocrine COS-7, NIH3T3, Rat-2, and Ros cells. The CBP PCR product is 704-bp and that of ß-actin is 500-bp. The sizes of DNA markers (lane M) are shown in bp.

 


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  		Figure 10. Western immunoblot of cell extracts from neuroendocrine and nonendocrine cells using CBP antibodies. Protein extracts from neuroendocrine AtT-20, BEN, GH4C1, PC-12, and TT cells, and nonendocrine COS-7, NIH3T3, Rat-2, and Ros cells were subjected to 8% SDS-PAGE and immunoblotted. A, NH2-terminal CBP antibody; B, COOH-terminal CBP antibody; and C, ß-tubulin antibody.

 
Taken together, our results show that neuroendocrine specific expression of CgA cannot be accounted for by enhanced levels of CREB or its cointegrator CBP in neuroendocrine cells. This suggests that as-yet-unidentified neuroendocrine specific factors acting through the CRE are responsible.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Chromogranin A is widely expressed in endocrine and neuronal cells, and elucidating the basis of its neuroendocrine cell-specific expression is likely to reveal general mechanisms important for the expression of many other neuroendocrine gene products. In pursuing this goal, we previously cloned and characterized the human CgA gene and showed that 87-bp of the proximal promoter region (-55 to +32 relative to the transcription start site) were sufficient for neuroendocrine cell-specific expression. Comparison of the proximal promoter sequences of the human (5, 19), bovine (7), and rodent (6, 8) CgA genes reveals that the most well conserved elements are a purine-rich GAGA sequence, a CRE, a sequence we have designated the TG-box, and the TATA-box element. The GAGA sequence lies upstream of position -55, which defines the 5' boundary of the minimal neuroendocrine-specific promoter sequence. In the present study, we demonstrated that deletion of -55 to -32, which contains the CRE and the TG-box leads to complete loss of neuroendocrine cell-specific expression. The activity of a reporter construct driven by the hCgA -31 to +32 sequence was the same as promoterless constructs in both neuroendocrine and nonendocrine cells indicating that activation of the CgA gene involves enhancer action via specific positively acting neuroendocrine cell-transacting factors rather than release of negative repressor activity in nonendocrine cells.

Mutation of the CRE in the minimal promoter construct p55CAT resulted in complete loss of the neuroendocrine cell-specific activity. In 5'-extended constructs, p2300CAT and p700CAT, mutation of the CRE resulted in a 50–75% decrease in basal activity in neuroendocrine cells. This emphasizes the important role played by the CRE in basal transcription but also suggests that other elements between -700 and -55 of the human CgA gene may act independently of the CRE to contribute to full basal activity in some neuroendocrine cell types. The importance of the CRE was also shown by the demonstration that the -55 to -32 sequence confers strong basal neuroendocrine-cell specific transcriptional activity on a heterologous adenovirus-2 major late promoter driving a CAT reporter gene, and this activity is stimulated further by cAMP (our unpublished results).

Human CgA gene transcription was stimulated by dibutyryl cAMP in the neuroendocrine BEN cell line and this effect was abolished by mutation of the CRE. In addition, the stimulation was completely blocked by addition of a PKA inhibitor, which also had a marked inhibitory effect on basal activity. The relatively modest induction of hCgA gene expression in BEN cells by cAMP is likely to be a reflection of a system already at high basal activity, and therefore unable to be stimulated further to any great extent. In fact, the majority of studies that have examined the responsiveness of CgA biosynthesis to cAMP or forskolin, which directly stimulates adenylate cyclase, have either shown a modest increase (7, 20) or no effect (20, 21, 22, 23). An exception to this is a recent study of the mouse CgA gene in which up to several-fold increases in its expression were noted in neuroendocrine PC12 cells treated with dibutyryl cAMP (12). This may be due to differences in responsiveness of one cell type relative to another, or to the specific tissue culture conditions employed. Therefore, as is the case for other genes like those for tyrosine hydroxylase (24), prohormone convertase 1 (25), prodynorphin (26) SgII (27), and renin (28, 29) basal, and tissue-specific transcriptional activity of the CgA gene is dependent upon the CRE.

A large number of proteins, which all belong to the basic-leucine zipper class of transcription factors, bind to the consensus CRE sequence, and modulate transcriptional activity (30, 31, 32). These include the cAMP response element binding protein (CREB), activating transcription factors (ATFs), and cAMP response element modulator (CREM). Activation of the PKA-pathway leads to phosphorylation of CREB/ATF-1 family members (e.g. phosphorylation of CREB at Ser 133), which is required for them to activate transcription. In our study, the results obtained with the PKA selective inhibitor H89, support those obtained with the mutated CRE constructs, suggesting that the activity comes from phosphorylated CRE-binding proteins. It is noteworthy that mutation of the CRE or addition of H89 lead to similar reductions in hCgA gene expression. This suggests that ubiquitously expressed PKA-regulated CRE-binding proteins such as CREB or ATF-1, are important for CgA gene expression. It is likely, however, that no single CRE-binding protein is solely responsible for mediating CgA gene transcription.

CREB binds the CREs of mouse CgA (12), mouse CgB (33) and human CgA (the present studies, and Ref.34) genes and ATF-1 binds the human CgA gene (unpublished data). Our gel retardation assays showed that the protein-DNA complexes formed with an oligonucleotide spanning the hCgA gene CRE and nuclear extracts from neuroendocrine and nonendocrine cells were similar with no clear-cut differences between cell types. The predominant complex was supershifted with CREB antibody in all cells, and the specificity of binding to the CRE was shown by the inability of CRE mutants to form the same complex and competition experiments with other CRE, AP-1 and SP-1 containing oligonucleotides. Nolan et al. (34) found similar CREB-containing protein-DNA complexes with an oligonucleotide spanning the human CgA gene promoter CRE and nuclear extract from neuroendocrine BEN cells but did not examine nonendocrine cell extracts. Wu et al. (12) compared results in gel retardation assays with an oligonucleotide spanning the mouse CgA gene promoter CRE and nuclear extracts from neuroendocrine AtT20 and nonendocrine N1H3T3 cells. They found that the entire complex formed with AtT20 nuclear extract supershifted to a new single complex of slower mobility with CREB antibody, whereas only a portion of the complex formed with N1H3T3 nuclear extract was supershifted by CREB antibody, and two supershifted complexes were observed. Therefore, while there is general agreement that the predominant protein-DNA complexes formed with either neuroendocrine or nonendocrine nuclear extracts and oligonucleotides spanning either the human or mouse CgA gene promoter CRE contains CREB, the importance of selective binding to the CRE by other factors in a nonendocrine cell as suggested by Wu et al. (12) was not supported in our study using several different neuroendocrine and nonendocrine cells. Recent studies on the neuroendocrine cell-specific expression of the proprotein convertase 1 (PC1) gene suggest that the CRE-elements in its proximal promoter bind novel neuroendocrine factors in addition to known CRE binding proteins like CREB (35).

The mechanism of tissue specificity of CgA gene transcription potentially could be due to either the amount or phosphorylation status of CREB, or the relative amount or activity of so-called coactivators or cointegrators such as CBP, which are recruited by phosphorylated CREB, and couple this transactivation to the basal transcriptional machinery. In the present study, we have excluded the former possibility by demonstrating that there was no difference in total CREB or in phosphorylated CREB levels in neuroendocrine and nonendocrine cells. Moreover, phosphorylated CREB was increased by forskolin, and the effect of this treatment was blocked by a PKA-inhibitor in both types of cells. With respect to the latter possibility, we show that expression of CBP at both the mRNA and protein levels is similar in neuroendocrine and nonendocrine cells, thus making it unlikely that the amount of CBP is the critical factor in determining neuroendocrine cell expression of CgA. However, it will be important in future studies to assess whether in nonendocrine cells there is a block in the correct association of CBP with the transcriptional machinery.

The human and bovine CgA genes show almost perfect homology in the 5'-flanking sequence between the CRE and TATA-box, which we have designated the TG-box, suggesting that this region may be important in binding trans-acting factors which act either independently or in association with proteins bound to the CRE. The rodent genes also exhibit very high homology with this TG-box region just downstream of the CRE; however, they also have an insertion of more than twenty nucleotides just upstream of the TATA-box relative to the human and bovine genes. We found that mutation of the TG-box in the human gene had a modest or no effect on transcriptional activity in either neuroendocrine or nonendocrine cells. In the mouse CgA gene, mutation of this region caused a 50% reduction in expression in PC12 and AtT20 cells (21). This can be compared with our present study in which mutation of the TG-box had either no effect (BEN, GH4C1, AtT20, and Rat2 cells) or resulted in a 50% decrease in transcriptional activity (TT and PC12 cells) (see Fig. 2BGo). In all neuroendocrine cells, with the exception of TT cells, mutation of the CRE alone was sufficient to reduce transcriptional activity to basal, indicating that the promoter effects of the CRE predominated over those of the TG-box. However, in TT cells mutation of both the CRE and TG-box was required to obtain complete reduction to basal transcriptional activity. Thus, while the CRE is the preeminent modulator of basal CgA gene expression, a contribution is also made by the TG-box in some neuroendocrine cell types.

Transcriptional regulation of the human CgA gene has been studied by others (19, 34). In an initial report that focused on distal sequences from -726 to -455, Nolan et al. (19) noted that in transient transfection studies in neuroendocrine BEN cells, whereas hCgA/GH reporter constructs containing sequence from -726 to -590 demonstrated high basal activity, deleted constructs from -566 to -455 showed 5-fold lower activity. Constructs deleted to positions closer to the transcriptional start site were not studied. These authors concluded at this time (19), that elements within the -590 to -566 sequence were important for basal transcriptional activity. In a later study (34), these authors suggested that the sequence from -570 to -555, rather than sequences upstream of -566 as emphasized in their initial report (19), acted as a specific neuroendocrine cell enhancer. It can be concluded from our studies that other elements between -700 and -55 may contribute either enhancer or repressor activities but, with the exception of BEN cells, the magnitude of these effects relative to overall promoter activity is slight, and the sequences involved vary from one neuroendocrine cell type to the other (see Fig. 5BGo). Similar conclusions can be drawn from studies of the mouse CgA gene promoter (12).

In summary, we have shown that the CRE of the human CgA gene plays a pivotal role in mediating both the basal and cAMP-induced expression of this gene in neuroendocrine cells. Other elements that play a lesser role include the TG-box lying just downstream of the CRE and more distal promoter sequences which may contribute to full transcriptional activity in particular neuroendocrine cell types. The mechanism of tissue-specific control mediated by the CRE is presently unknown. It could be due to a PKA-responsive CRE-binding protein other than CREB in neuroendocrine cells, expression of a repressor molecule that binds CREB in nonendocrine cells, or to the relative amount or activity of coactivators or cointegrators other than CBP, which are required to couple transactivators to the basal transcriptional machinery (36, 37, 38).


   Acknowledgments
 
We thank Dr. Leonard J. Deftos for the BEN cell line, and Pamela Kirk, Carmen Ferrara-Wilson and Rosa Foschi for preparation of the manuscript.


   Footnotes
 
1 This work was supported by Medical Research Council of Canada Grants MT-9315 and MT-11704 and a grant from the Kidney Foundation of Canada. Back

2 These authors contributed equally to this study. Back

3 Supported in part by the Royal Victoria Hospital Research Institute. Back

4 Chercheur-boursier of the Fonds de la Recherche en Santé du Québec (FRSQ). Back

5 Scientist of the Medical Research Council of Canada. Back

Received August 18, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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