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Endocrinology Vol. 141, No. 10 3668-3678
Copyright © 2000 by The Endocrine Society

ARTICLES

Neuroendocrine Cell Type-Specific and Inducible Expression of the Chromogranin B Gene: Crucial Role of the Proximal Promoter1

Nitish R. Mahapatra, Manjula Mahata, Arun K. Datta, Hans-Hermann Gerdes, Wieland B. Huttner, Daniel T. O’Connor and Sushil K. Mahata

Department of Medicine (N.R.M., M.M., D.T.O’C., S.K.M.), University of California, and San Diego Veterans Affairs Healthcare System, San Diego, California 92161; Department of Molecular Biology (A.K.D.), The Scripps Research Institute, La Jolla, California 92037; and Department of Neurobiology (H.-H.G., W.B.H.), Heidelberg University, 69120 Heidelberg, Germany

Address all correspondence and requests for reprints to: Sushil K. Mahata, Ph.D., Department of Medicine (9111H), University of California, 3350 La Jolla Village Drive, San Diego, California 92161-9111H. E-mail: smahata{at}ucsd.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chromogranin B, a soluble acidic secretory protein, is widely distributed in neuroendocrine and neuronal cells, although not in other cell types. To identify the elements governing such widespread, yet selective, expression of the gene, we characterized the isolated mouse chromogranin B promoter. 5'-Promoter deletions localized neuroendocrine cell type-specific expression to the proximal chromogranin B promoter (from -216 to -91 bp); this region contains an E box (at [-206 bp]CACCTG[-201 bp]), four G/C-rich regions (at [-196 bp]CCCCGC[-191 bp], [-134 bp]CCGCCCGC[-127 bp], [-125 bp]GGCGCCGCC[-117 bp], and [-115 bp]CGGGGC[-110 bp]), and a cAMP response element (CRE; at [-102 bp]TGACGTCA[-95 bp]). A 60-bp core promoter region, defined by an internal deletion from -134 to -74 bp upstream of the cap site and spanning the CRE and three G/C-rich regions, directed tissue-specific expression of the gene. The CRE motif directed cell type-specific expression of the chromogranin B gene in neurons, whereas three of the G/C-rich regions played a crucial role in neuroendocrine cells. Both the endogenous chromogranin B gene and the transfected chromogranin B promoter were induced by preganglionic secretory stimuli (pituitary adenylyl cyclase-activating polypeptide, vasoactive intestinal peptide, or a nicotinic cholinergic agonist), establishing stimulus-transcription coupling for this promoter. The adenylyl cyclase activator forskolin, nerve growth factor, and retinoic acid also activated the chromogranin B gene. Secretagogue-inducible expression of chromogranin B also mapped onto the proximal promoter; inducible expression was entirely lost upon internal deletion of the 60-bp core (from -134 to -74 bp). We conclude that CRE and G/C-rich domains are crucial determinants of both cell type-specific and secretagogue-inducible expression of the chromogranin B gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHROMOGRANIN B is a major member of the chromogranin/secretogranin secretory protein family, which also includes chromogranin A and secretogranin II. Chromogranin B was first characterized as a 105- to 113-kDa tyrosine-sulfated secretory protein (p105/p113) in PC12 pheochromocytoma cells (1) and was subsequently detected in the bovine adrenal medulla (2, 3). The chromogranin/secretogranin proteins each have widespread neuroendocrine expression (4, 5).

Five exons in the mouse genome encode the 657-amino acid protein chromogranin B (6). The mature chromogranin B proteins in human, cow, and rat are 657, 626, and 655 amino acids in length, respectively (4, 5). Exon 1 encodes the 5'-untranslated region of the messenger RNA (mRNA) and most of the signal peptide (6); exon 2 encodes the last 4 amino acids of the signal peptide and first 12 amino acids of the mature protein; exon 3 encodes 2 highly conserved cysteines, which form an intramolecular disulfide loop; exon 4 is the longest exon, encoding amino acids 44–631 and generating 4 peptides, known as GAWK, PE-11, BAM-1745, and CCB (4); and exon 5 contains the carboxyl-terminus of the protein, including the last dibasic amino acid pair, the carboxyl-terminal antibacterial peptide secretolytin (7), and the 3'-untranslated region of the mRNA.

Chromogranin B together with secretogranin II play a role in the sorting process to the regulated secretory pathway (5, 8, 9). The disulfide-bonded loop of chromogranin B has been implicated to direct sorting of this protein from the trans-Golgi network to dense core secretory granules (8, 9), although other trafficking molecule receptors, such as carboxypeptidase E (10), or membrane lipid rafts (11) may also be important.

The mouse chromogranin B gene has been isolated (6), and the locus (Chgb) has been positioned to human chromosome 20 pter-p12, mouse chromosome 2, and rat chromosome 3 (12). Studies of steady state mRNA levels indicate that endogenous (chromosomal) chromogranin B gene expression is activated by cAMP or forskolin (13, 14), reserpine (15), membrane depolarization (16), kainic acid (17), and nerve growth factor (NGF) (18).

Here we explore factors governing the activity of the chromogranin B gene to yield its widespread, yet neuroendocrine-selective, pattern of expression. We therefore characterized the mouse chromogranin B gene promoter (to -2788 bp upstream of the cap site [+1]) to identify the molecular basis for its neuroendocrine cell type-specific expression. We found that four G/C-rich domains in the proximal chromogranin B promoter, at [-196 bp]CCCCGC[-191 bp], [-115 bp]CGGGGC[-110 bp], [-125 bp]GGCGCCGCC[-117 bp], and [-134]CCGCCCGC[-127 bp], play important roles in neuroendocrine cell type-specific expression of the gene. A cAMP response element (CRE) at [-102 bp]TGACGTCA[-95 bp] appears to be crucial for chromogranin B expression in neurons. Activation of chromogranin B gene expression by preganglionic neuronal secretory stimuli is also dependent upon these same proximal promoter domains.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sequencing of the mouse chromogranin B promoter
A 2908-bp genomic DNA clone fragment containing the 5'-upstream region of the chromogranin B gene (6) was sequenced. Double stranded DNA sequencing was performed by the automated fluorescent sequencing method, using oligonucleotide primers either to vector sequences or to mouse genomic chromogranin B sequences. Eight microliters of Prism Dye-Terminator Ready Reaction Mix from PE Applied Biosystems/Perkin-Elmer Corp. (Foster City, CA), which contains buffer, nucleotides, dye terminators, and a specially modified FS AmpliTaq polymerase, were added to 500 ng DNA template plus 3.2 pmol of primers. Amplification was performed on an MJ Research, Inc., DNA Engine PTC200 (Waltham, MA), with a hot start at 96 C. Then 25 cycles were run at 96 C for 10 sec, 52 C for 5 sec, and 60 C for 4 min. The samples were purified on G50 microspin Sepharose columns (Pharmacia Biotech, Piscataway, NJ) to remove excess dye-nucleotides and lyophilized in a Speed-Vac (Savant Instruments Inc., Holbrook, NY) for 30 min. Three microliters of a formamide and a blue dextran dye (5:1 ratio) solution was added to the pellet, vortexed, heated to 96–100 C to get rid of secondary structures, and then quick-chilled. The samples were loaded on a 6% polyacrylamide gel (Bio-Rad Laboratories, Inc., reagents) for 12 h at 30 watts on an ABI 373A autosequencer (PE Applied Biosystems). The data were collected and analyzed using ABI 2.1.1 software.

Construction of a series of chromogranin B 5'-promoter deletion/luciferase reporter plasmids for transfection
All PCR-derived fragments were verified by resequencing. An approximately 6-kbp SstI-HindIII fragment of a mouse chromogranin B genomic clone, spanning the promoter region, was subcloned in pXP2 (19). This plasmid was named pXP2-CgB and was used to make a series of 5'-promoter deletion/luciferase reporter plasmids in the firefly luciferase reporter vector pGL3-Basic (Promega Corp., Madison, WI).

pGL3-CgB2788 (-2788 to +34 bp). The pXP2-CgB promoter plasmid was digested with EcoRI, and 5'-overhangs were made blunt with mung bean nuclease, followed by digestion with XhoI. The EcoRI (blunt)-XhoI fragment was then ligated into the blunt-ended MluI and XhoI sites of the pGL3-Basic vector.

pGL3-CgB1302 (-1302 to +34 bp). The pXP2-CgB construct was digested with NdeI, and 5'-overhangs were made blunt, followed by digestion with XhoI. The 1336-bp fragment was ligated into the blunt-ended MluI and XhoI sites of the pGL3-Basic vector.

pGL3-CgB697 (-697 to +34 bp). The pXP2-CgB construct was used as a template to PCR-amplify the -697 to +23 bp region of the promoter using a 5' (upstream)-oligonucleotide primer and a 3' (downstream)-primer. The 5'-primer terminated in a KpnI site, whereas the 3'-primer terminated in an XhoI site at +23 bp downstream from the cap site. The PCR-amplified fragment was then subcloned between the KpnI and XhoI sites of the pGL3-Basic vector. Sequence analysis of this construct revealed deletion of a highly G/C-rich promoter region from -134 to -74 bp upstream of the cap site; in addition, the 3'-end extended only up to +17 bp instead of +23 bp. As this fragment contained the 60-bp deletion from -134 to -74 bp, as described below, we designated this plasmid pGL3-CgB{Delta}697. There is a PstI site at the -291 bp position of the mouse chromogranin B promoter. This site was used to excise a 325-bp PstI-XhoI fragment from the pXP2-CgB construct; this fragment was then substituted for the PstI-XhoI fragment of pGL3-CgB{Delta}697. The resulting plasmid was thus named pGL3-CgB697.

pGL3-CgB388 (-388 to +34 bp). This promoter region was amplified by PCR using pXP2-CgB as a template, a 5'-primer that terminated in a KpnI site, and the 3'-primer, as described below for pGL3-CgB{Delta}697. The PCR product was subcloned between the KpnI and XhoI sites of the pGL3-Basic vector. Like the pGL3-CgB{Delta}697 construct, sequence analysis of this construct revealed the absence of the strong GC-rich promoter region starting from -134 to -74 bp upstream of the cap site. As this fragment contained an internal deletion of 60 bp (deletion from -134 to -74 bp) as described above, we designated this plasmid pGL3-CgB{Delta}388.The pGL3-CgB388 construct was made from pGL3-CgB{Delta}388 using an approach (insertion of PstI/XhoI fragment) similar to that used for making the pGL3-CgB697 construct.

pGL3-CgB291 (-291 to +34 bp). The pXP2-CgB construct was digested with PstI, and 3'-overhangs were blunt-ended with mung bean nuclease, followed by digestion with XhoI. This 325-bp fragment was then subcloned between the blunt-ended MluI and XhoI sites of the pGL3-Basic vector.

pGL3-CgB216 (-216 to +34 bp). The pXP2-CgB construct was digested with BglII, and the 250-bp fragment was subcloned between the BglII site of the pGL3-Basic vector.

pGL3-CgB146 (-146 to +23 bp). This promoter region was amplified by PCR using pXP2-CgB as template, a 5'-primer that terminated in a KpnI site, and the same 3'-primer as that described above for pGL3-CgB{Delta}697. The PCR product of 169 bp was subcloned between the KpnI and XhoI sites of the pGL3-Basic vector.

pGL3-CgB107 (-107 to +23 bp). We amplified this promoter region by PCR using pXP2-CgB as template, a 5'-primer that terminated in a KpnI site, and the same 3'-primer as that described above for pGL3-CgB{Delta}697. The KpnI site was blunt-ended with mung bean nuclease, and the 130-bp PCR product was subcloned between the MluI (made blunt) and XhoI sites of the pGL3-Basic vector.

pGL3-CgB91 (-91 to +23 bp). This promoter fragment was amplified by PCR using pXP2-CgB as template, a 5'-primer that terminated in a KpnI site, and the same 3'-primer, as described above for pGL3-CgB{Delta}697. The PCR product of 114 bp was subcloned between the KpnI and XhoI sites of the pGL3-Basic vector.

pGL3-CgB91.I (-91 to +34 bp). The pGL3-CgB697 construct was digested with KpnI and BlpI, and the resulting overhangs were made blunt with mung bean nuclease and reclosed with T4 DNA ligase. This construct contains 125 bp of chromogranin B promoter.

pGL3-CgB56 (-56 to +17 bp). The pGL3-CgB{Delta}697 construct was digested with KpnI and SmaI and reclosed with T4 DNA ligase. This construct contains 73 bp of chromogranin B promoter.

pGL3-CgB 56.1 (-56 to +34 bp). The pGL3-CgB697 construct was digested with KpnI and SmaI, followed by recircularization with T4 DNA ligase. This construct contains 90 bp of chromogranin B promoter.

Construction of a series of chromogranin B internal deletions (-134 to -74 bp) in 5'-promoter deletion/luciferase reporter plasmids for transfection
pGL3-CgB{Delta}697 (-697 to +17 bp). The pXP2-CgB construct was used as a template to PCR-amplify the -697 to +23 bp region of the promoter using a 5' (upstream)-oligonucleotide primer and a 3' (downstream)-primer. The 5'-primer terminated in a KpnI site, whereas the 3'-primer terminated in a XhoI site at +23 bp downstream from the cap site. The PCR-amplified fragment was then subcloned between the KpnI and XhoI sites of the pGL3-Basic vector. Sequence analysis of this construct revealed deletion of a highly G/C-rich promoter region from -134 to -74 bp upstream of the cap site; in addition, the 3'-end extended only up to +17 bp instead of +23 bp. As this fragment contained the 60-bp deletion from -134 to -74 bp, as described above, we designated this plasmid pGL3-CgB{Delta}697.

pGL3-CgB{Delta}388 (-388 to +23 bp). This promoter region was amplified by PCR using pXP2-CgB as template, a 5'-primer that terminated in a KpnI site, and the same 3'-primer as that described above for pGL3-CgB{Delta}697. The PCR product was subcloned between the KpnI and XhoI sites of the pGL3-Basic vector. Like the pGL3-CgB{Delta}697 construct, sequence analysis of this construct revealed the absence of the strong GC-rich promoter region starting from -134 to -74 bp upstream of the cap site. As there was an internal deletion of 60 bp from -134 to -74 bp in this PCR-generated fragment, as described above, we designated this plasmid pGL3-CgB{Delta}388.

pGL3-CgB{Delta}220 (-220 to +23 bp). This promoter region was amplified by PCR using pXP2-CgB as template, a 5'-primer that terminated in a KpnI site, and the same 3'-primer as that described above for pGL3-CgB{Delta}697. The PCR product was subcloned between the KpnI and XhoI sites of the pGL3-Basic vector. Like pGL3-CgB{Delta}697 and pGL3-CgB{Delta}388 constructs, sequence analysis of this construct revealed the absence of the strong GC-rich promoter region starting from -134 to -74 bp upstream of the cap site. As this fragment had an internal deletion of 60 bp (from -134 to -74 bp), as described above, we designated this plasmid pGL3-CgB{Delta}220.

Cell culture and transfections
Neuroendocrine and neuronal cells. Rat pheochromocytoma (PC12 cells), mouse adrenal tyrosine hydroxylase-expressing (PATH-2 cells), mouse gonadotrope ({alpha}T3–1 cells), mouse anterior pituitary corticotrope (AtT20 cells), rat somatotrope (GC cells) and lactotrope (235 cells), mouse GnRH-producing hypothalamic (GT1–7 cells), and central tyrosine hydroxylase-producing (Cath-a cells) cells were described in detail in an earlier communication (20). Human neuroblastoma (SK-N-SH cells) and Syrian hamster insulin-producing pancreatic islet (HIT-T15 cells) cells were obtained from American Type Culture Collection (Manassas, VA) and grown in high glucose DMEM with 10% heat-inactivated FCS and 1% penicillin/streptomycin. The dorsal root ganglion hybrid cell line F-11 (21) was obtained from G. Dawson, University of Chicago (Chicago, IL), and grown in high glucose DMEM with 10% heat-inactivated FCS and 1% penicillin/streptomycin (100% stocks were 10,000 U/ml penicillin G and 10,000 µg/ml streptomycin sulfate; Life Technologies, Inc., Gaithersburg, MD).

Control cells. The NIH-3T3 (nonneuroendocrine, control) fibroblast cell line was obtained from American Type Culture Collection and grown in high glucose DMEM with 10% heat-inactivated FCS and 1% penicillin/streptomycin. COS-1 cells (simian virus 40 large T antigen-transformed kidney fibroblast cell line), 293T cells (human adenovirus 5-transformed kidney epithelial cell line), and HB cells (human fibroblast cell line) were obtained from American Type Culture Collection.

Supercoiled plasmid DNA for transfection was grown in Escherichia coli strain DH-5{alpha} and purified on columns (QIAGEN, Chatsworth, CA). One day before transfection, cells were split onto poly-D-lysine (Sigma, St. Louis, MO)-coated 6-cm plastic plates at 40–50% cell confluence. Cells were transfected with 2.5 µg supercoiled luciferase reporter plasmid DNA/plate, using the polycationic method (Superfect reagent, QIAGEN). Cells were harvested 16–20 h after transfection for constitutive as well as inducible expressions. Cell extracts were prepared and assayed for protein and luciferase (20). To control for differences in transfection efficiency between plasmids and between cell lines, we used the Promega Corp. Dual-Luciferase reporter assay system, in which chromogranin B promoter/firefly luciferase reporter transfections were accompanied by cotransfection of another luciferase reporter plasmid, pRL-CMV (Promega Corp.), expressing the Renilla luciferase (Rluc) reporter driven by the cytomegalovirus immediate-early enhancer/promoter region. Firefly and Renilla luciferase activities were distinguished sequentially, with the Stop and Glo reagent, by their cofactor dependence: beetle luciferin for firefly luciferase, and coelenterazine for Renilla luciferase.

Northern blot analysis of mRNA
Total RNA was isolated from cells by guanidinium thiocyanate extraction (RNAzol B, Tel-Test, Friendswood, TX). RNA samples (10–20 µg/lane) were size-fractionated on denaturing 1% agarose-formaldehyde gels, transferred to nitrocellulose membranes, and fixed with UV irradiation (StrataLinker, Stratagene, La Jolla, CA). The integrity of the RNA was judged by the appearance of 28S and 18S ribosomal RNA bands on the ethidium bromide-stained gel. The blots were prehybridized, hybridized, and washed as previously described (20).

Random primer-labeled complementary DNA (cDNA) probes were a 2028-bp rat chromogranin B cDNA (22) and a 381-bp mouse cyclophilin cDNA (23), used as a normalizing probe for a housekeeping (constitutively expressed) mRNA. Expression of mRNAs was quantified using a StrataScan 7000 densitometer (Stratagene) and normalized to cyclophilin gene expression (20).

Chemicals
Nicotine and forskolin were obtained from Sigma. Synthetic pituitary adenylyl cyclase-activating polypeptide (PACAP-38) and vasoactive intestinal peptide (VIP) were obtained from Peninsula Laboratories, Inc. (Belmont, CA). NGF (2.5S, murine, natural) was obtained from Life Technologies, Inc. Retinoic acid was purchased from Calbiochem (San Diego, CA).

Data presentation and statistical analysis
Secretagogue potency was estimated as the EC50 (concentration required to produce a half-maximal effect), using the program Kaleidagraph (Synergy/Abelbeck Software, Reading, PA). We chose secretagogue doses based on 10-fold (log10) dose-response curves for each agent, and then conducted subsequent studies at submaximal doses that were at or above the EC50 values for each drug. Transfection experiments were repeated at least three times, with three plates per condition in each experiment. Results are expressed as the mean ± 1 SEM. Descriptive and inferential statistics were performed with the program InStat (GraphPad Software, Inc., San Diego, CA). Student’s t tests or ANOVAs, followed by Tukey-Kramer multiple comparison tests, were used, as appropriate. Significance was determined at the P <= 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sequence of the mouse chromogranin B promoter
Sequence analysis of 2908 bp of the 5'-flanking region of the mouse chromogranin B gene revealed several consensus matches for cis-acting transcriptional control elements (Fig. 1Go). The results are reported as base pairs upstream of the cap site (+1): 1) the TATA box at [-33 bp]TTCATAA[-27 bp]; 2) a cAMP response element (CRE) (24) at [-102 bp]TGACGTCA[-95 bp]; 3) eight G/C-rich elements of 6 consecutive bp or more (potential binding sites for such factors as Sp1, Ap2, or Egr1) at -8/-2 bp, -45/-39 bp, -62/-53 bp, -83/-64 bp, -115/-110 bp, -125/-117 bp, -134/-127 bp, and -196/-191 bp [one of these G/C-rich sites contains a perfect (9/9 bp) consensus match for an Egr1 recognition site: -79/-71 bp (25); another G/C-rich site overlaps a perfect (10/10 bp) consensus match (GGGRNNYYCC; IUPAC nomenclature) for a nuclear factor-{kappa}B site: [-83 bp]GGGGCGCCCC[-74 bp] (25); several of these G/C-rich sites overlap partial matches for Ap2 motifs (e.g. GSSWGSCC; IUPAC nomenclature) (25)]; and 4) an E-box (CANNTG; IUPAC nomenclature) at [-206 bp]CACCTG[-201 bp] (25).



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  		Figure 1. Mouse chromogranin B promoter sequence. Nucleotides are numbered from the transcription initiation site (+1) of the gene. Note the positions of the TATA box at [-33 bp]TTCATAA[-27 bp], the CRE at [-102 bp]TGACGTCA[-95 bp], and eight G/C-rich elements of 6 or more consecutive bp (potential binding sites for such factors as Sp1, Ap2, or Egr1) at [--8 bp]CCGCGCC[-2 bp], [-45 bp]CCCCGCC[-39 bp], [-62 bp]CGCCCCCGGG[-53 bp], [-83 bp]GGGGCGCCCCCGCCCGCCGC[-64 bp], [-115bp]CGGGGC[-110 bp], [-125 bp]GGCGCCGCC[-117 bp], [-134]CCGCCCGC[-127 bp], and [-196 bp]CCCCGC[-191 bp]. One of these G/C-rich sites is a perfect (9/9 bp) consensus match for an Egr1 recognition site: [-79 bp]CGCCCCCGC[-71 bp]. One of these G/C-rich sites overlaps a perfect (10/10 bp) consensus match (GGGRNNYYCC; IUPAC nomenclature) for a nuclear factor-{kappa}B site: [-83 bp]GGGGCGCCCC[-74 bp]. Several of these G/C-rich sites overlap partial matches for Ap2 motifs (e.g. GSSWGSCC; IUPAC nomenclature). There is an E box (CANNTG; IUPAC nomenclature) at [-206 bp]CACCTG[-201 bp]. Transcription factor binding site consensus sequences are shown in bold.

 
Expression of the endogenous chromogranin B gene in neuroendocrine cells
Basal neuroendocrine expression. We investigated the expression of the endogenous chromogranin B gene by Northern blots in clonal cell lines. A high level of steady state chromogranin B mRNA was observed in adrenomedullary PC12 pheochromocytoma cells (Figs. 2Go and 3Go), pituitary (AtT20 corticotrope, and GC somatotrope) cells (Fig. 2Go), although not in control (nonneuroendocrine, COS-1) cells (Fig. 2Go).



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  		Figure 2. Northern blot analyses of chromogranin B mRNA expression in neuroendocrine [PC12 (rat adrenal pheochromocytoma), GC (rat somatotrope), and AtT20 (mouse corticotrope)] vs. control (nonneuroendocrine COS-1, kidney fibroblasts) cells. The position of 18S ribosomal RNA migration is shown on the right. Cyclophilin (housekeeping) mRNA is also shown, for normalization between cell types.

 


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  		Figure 3. Northern blot analyses of chromogranin B mRNA in PC12 cells treated with vehicle (mock), forskolin (10 µM; 16 h), retinoic acid (100 µM; 16 h), nicotine (1 mM; 16 h), PACAP (0.1 µM; 16 h), NGF (100 ng/ml; 16 h), or VIP (1 µM; 16 h). The position of 18S ribosomal RNA migration is shown on the right. The densitometric values of the steady state mRNA for chromogranin B are normalized to values obtained for cyclophilin mRNA.

 
Secretagogue-inducible expression. As acetylcholine is a physiological stimulus for chromaffin cell secretion (26) of catecholamines and costored proteins, including chromogranin B, we investigated whether nicotine can stimulate expression of the chromogranin B gene. Nicotine (1000 µM; 16 h) augmented expression of the chromogranin B gene by 2.0-fold in PC12 cells (Fig. 3Go).

Besides cholinergic stimuli, peptidergic agents, such as PACAP and VIP, also play significant roles in secretion of catecholamines and on transcription of several genes in chromaffin cells, including chromogranin A (27). Therefore, we tested the effects of PACAP and VIP on the expression of the chromogranin B gene and found that PACAP and VIP stimulated expression of the chromogranin B gene by 4.9- and 4.4-fold, respectively (Fig. 3Go).

As the chromogranin B promoter contains a CRE box at -102/-95 bp (Fig. 1Go) that was reported to be functional (14), we tested its regulation by activation of the protein kinase A pathway. The adenylyl cyclase activator forskolin (10 µM; 16 h) increased expression of the chromogranin B gene by 5.8-fold in PC12 cells (Fig. 3Go).

As the neurotropin NGF induces expression of the chromogranin A (28), secretogranin II (18), and vgf (29) genes, we tested whether NGF stimulation is general among the chromogranins. NGF treatment for 16 h caused a 2.7-fold increase in the steady state chromogranin B mRNA level (Fig. 3Go).

Retinoic acid stimulates expression of the chromogranin A gene (30). Although the mouse chromogranin B promoter does not contain a perfect consensus motif for a retinoic acid response element (AGGTCA/TGACCT) (25, 31), a G/C-rich Sp1 motif may be important for retinoic acid-induced expression of at least some genes (32). As the chromogranin B promoter has multiple G/C-rich sites, we tested its retinoic acid response and found 2.3-fold up-regulation of the endogenous chromogranin B gene (Fig. 3Go). Northern blot results were confirmed in a second set of independent experiments.

Expression of the transfected chromogranin B promoter: 5'-promoter deletion mutants
A 2788-bp chromogranin B promoter (fused to a luciferase reporter) conferred expression in neuroendocrine cells, including cell lines of the adrenal medulla (rat PC12 and mouse Path-2 cells; Fig. 4Go), pituitary (AtT20, GC, {alpha}T3–1, and 235; Fig. 4Go), endocrine pancreatic islet (HIT-T15), and neurons including neuroblastoma (Cath-a, GT1–7, SK-N-SH, and F-11; Fig. 4Go), but not in control (nonneuroendocrine) cells, such as COS-1 fibroblasts (Fig. 4Go), NIH-3T3 fibroblasts (Fig. 4Go), 293T epithelial cells (Fig. 4Go), and HB fibroblasts (Fig. 4Go).



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  		Figure 4. Deletion analysis of mouse chromogranin B promoter domains in neuroendocrine (PC12, Path-2, GC, {alpha}T3–1, AtT20, 235, and HIT-T15) and neuronal (Cath-a, GT1–7, SK-N-SH, and F-11) vs. control fibroblast (COS-1, 3T3, 293T, and HB) cell lines. Promoter fragments were subcloned into the polylinker region of the promoterless luciferase reporter vector pGL3-Basic (Promega Corp.), with reference to the transcription initiation (cap) site, as +1. For example, CgB1302 (i.e. -1302 in the figure) spans a region from 1302 bp upstream (5') of the cap site to +34 bp downstream (3') of the cap site. The promoter deletion/luciferase reporter constructs were cotransfected with another luciferase reporter plasmid pRL-CMV (Promega Corp.) expressing the Renilla luciferase. The results are expressed as ratios of firefly/Renilla luciferase activities and are the mean ± 1 SEM (n = 3 transfections for each deletion). Compare with Fig. 1Go for CgB promoter motifs.

 
Expression in neuroendocrine cells was preserved, or even enhanced, upon progressive 5'-deletions down to -216 bp upstream of the cap site, after which specific expression in neuronal and neuroendocrine cells began to decrease. A 38.3–59.1% fall in promoter activity (P < 0.001) was seen after deletion from -216 to -146 bp (which contains the G/C-rich region [-196 to -191 bp]) in neuronal and neuroendocrine cells. An even more dramatic decrease (53–85%; P < 0.001) in promoter activity was noticed upon deletion from -146 to -107 bp; this domain contains three G/C-rich regions at -115/-110, -125/-117, and -134/-127 bp (Figs. 1Go and 4Go). By contrast, in neurons and neuroblastoma (Cath-a, GT1–7, SK-N-SH, and F-11), promoter activity remained unaltered after this G/C-rich deletion (Fig. 4Go). For neurons, a dramatic decrease (59–72%; P < 0.001) in promoter activity was seen after deletion from -107 to -91 bp (Fig. 4Go); of note, this region contains a CRE site at -102/-95 bp, although no G/C-rich domain.

Internal deletion of 60 bp (-134 to -74 bp; a region spanning not only the CRE motif at -102/-95 bp, but also four G/C-rich domains at -83/-74, -115/-110, -125/-117, and -134/-127 bp) caused dramatic decreases in promoter activity in neuroendocrine PC12 and AtT20 cells (P < 0.001), although not in nonneuroendocrine COS-1 cells (not significant; see Fig. 5Go). Other neuroendocrine cell lines (Path-2, GC, 235, {alpha}T3–1, Cath-a, and GT1–7) displayed similar results (data not shown).



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  		Figure 5. Effect of internal deletion of a mouse chromogranin B promoter domain on its expression in neuroendocrine vs. control (fibroblast) cell lines. The plasmids (CgB{Delta}697, CgB{Delta}388, and CgB{Delta}220) bearing an internal deletion of 60 bp (from -134 to -74 bp) and their respective controls (CgB697, CgB388, and CgB220) were transfected along with pRL-CMV expressing Renilla luciferase. The results are expressed as ratios of firefly/Renilla luciferase activities and are the mean ± 1 SEM (n = 3 transfections for each deletion). Compare with Fig. 1Go for CgB promoter motifs.

 
Promoter fragments differing by up to 17 bp in length downstream from the cap site (+17, +23, or +34 bp) did not differ in ability to drive luciferase expression when the promoter length upstream of the cap site was controlled (e.g. -91 bp; data not shown).

Change of neuroendocrine to neuronal phenotypes by NGF treatment results in a partial shift in tissue-specifier element
As G/C-rich regions (-134 to -74 bp) seemed to be the principal tissue-specific elements in determining chromogranin B expression in neuroendocrine cells (Fig. 4Go), whereas the CRE motif -102/-95 bp was more crucial in neurons (Fig. 4Go), we used PC12 cells to test whether conversion of a neuroendocrine to a neuronal phenotype by NGF would also change the tissue specifier elements toward CRE. We treated PC12 cells with NGF (100 ng/ml) for 2 days before splitting for transfection; transfected with CgB147, CgB107, or empty vector on day 3; and harvested on day 4 for luciferase assay. NGF was maintained throughout the experimental period. We have shown that such a NGF dose and time exposure result in neurite sprouting from PC12 cells (28). The -107 bp (CRE-containing) chromogranin B promoter displayed equivalent activity in the presence or absence of NGF (Fig. 6Go); by contrast, the -146 bp promoter (containing four additional G/C-rich regions) was 158% more active in the absence of NGF (P < 0.004). Thus, conversion from a neuroendocrine (PC12) to a neurite-bearing phenotype seemed to reduce dependence on the G/C-rich region of the chromogranin B promoter.



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  		Figure 6. Effect of NGF treatment on partial shift of tissue-specific elements from neuroendocrine to neuronal ones. PC12 cells were treated for 3 days with NGF (100 ng/ml) and transfected with CgB146, CgB107, and pGL3-Basic vector along with a Renilla luciferase reporter driven by the cytomegalovirus immediate-early enhancer/promoter region. The cells were treated with NGF during the entire period and were harvested on day 4 for dual luciferase assay. The results are expressed as ratios of firefly/Renilla luciferase activities and are the mean ± 1 SEM (n = 3 transfections for each deletion). Compare with Fig. 1Go for CgB promoter motifs.

 
Inducible expression of the chromogranin B promoter: search for cis elements mediating secretagogue inducibility
(a) Adenylyl cyclase activation by forskolin. The transfected chromogranin B promoter displayed 9.6-fold forskolin inducibility (P < 0.001) in neuroendocrine PC12 cells (EC50, 0.84 µM; Table 1Go), whereas no inducibility was seen in nonneuroendocrine COS-1 cells (data not shown). The CRE, a likely forskolin target, is at [-102 to -95 bp]. All promoter deletion mutants downstream (3') of the CRE box failed to respond to forskolin (not significant; Fig. 7AGo).


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  		Table 1. Relative potencies and efficacies of secretagogues for activation of the transfected chromogranin B promoter

 


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  		Figure 7. Effect of secretagogue on the activity of chromogranin B promoter. A, chromogranin B promoter expression in response to nicotine (1 mM), retinoic acid (100 µM), and forskolin (10 µM). B, Chromogranin B promoter expression in response to NGF (0.1 µg/ml), VIP (1 µM), and PACAP (0.1 µM). PC12 cells were transfected with chromogranin B promoter progressive 5'-deletion mutant/luciferase constructs and treated with vehicle (mock) or secretagogue for 18 h. Results are the mean ± 1 SEM (n = 3 transfections for each plasmid) and are expressed as relative light units (RLU) in 1 x 106 cells (n = 3 transfections for each plasmid). Compare with Fig. 1Go for CgB promoter motifs.

 
(b) Nicotinic cholinergic stimulation. In PC12 cells, we found a 2.7-fold increase in chromogranin B-transfected promoter activity after nicotine treatment (P < 0.001; EC50, 118 µM; Table 1Go), compared with a 2.0-fold induction of the endogenous chromogranin B gene (Fig. 3Go). No inducibility was seen in nonneuroendocrine COS-1 cells (not significant; data not shown). During serial 5'-deletions, the nicotine response was maximal at -216 bp (P < 0.001), but was entirely lost beyond -107 bp (not significant; Fig. 7AGo). Thus, both the CRE and the G/C-rich promoter regions seemed to play a role in the response to nicotine.

(c) Preganglionic peptidergic stimulation
(i) PACAP. In addition to the classical preganglionic neurotransmitter acetylcholine, noncholinergic transmitters such as PACAP may stimulate catecholamine secretion and biosynthetic enzyme transcription in adrenal medullary chromaffin and PC12 cells (27, 33). In PC12 cells, PACAP stimulated (EC50, 4.08 nM) the transfected chromogranin B promoter by 8.8-fold (P < 0.001; Table 1Go). No inducibility was seen in nonneuroendocrine COS-1 cells (not significant; data not shown). During 5'-promoter deletions, the PACAP response was retained up to -107 bp (P < 0.05), but was entirely lost by -91 bp (not significant; Fig. 7BGo); the -107 bp/-91 bp region contains the CRE box at -102/-95 bp.

(ii) VIP. Like PACAP, VIP stimulates the secretion of catecholamine and induces the biosynthesis of neuropeptides. In PC12 cells (Fig. 7BGo), VIP stimulated (EC50, 0.53 µM) the transfected chromogranin B promoter by 7.34-fold (P < 0.001). During 5'-promoter deletions, the VIP response was retained up to -107 bp (P < 0.001), but was entirely lost by -91 bp (not significant; Fig. 7BGo).
(d) NGF. We found a 1.8-fold increase (P < 0.001) in transfected chromogranin B promoter activity upon exposure to NGF (100 ng/ml; EC50, 17 ng/ml; Table 1Go) compared with a 2.7-fold increment in endogenous gene expression (Fig. 3Go). Promoter 5'-deletion mutants indicated that these growth factor responses were retained up to -107 bp (P < 0.001), but were entirely lost by -91 bp (not significant; Fig. 7BGo).

(e) Retinoic acid. We found that retinoic acid dose dependently stimulated chromogranin B promoter activity (by 4-fold, P < 0.001; EC50, 10.6 µM; Table 1Go). Promoter 5'-deletion mutants indicated that the retinoic acid responses were retained up to -107 bp (P < 0.001; Fig. 7AGo). Although the promoter region upstream of -107 bp does not contain a classical retinoic acid response motif (AGGTCA/TGACCT) (25, 31), it does contain several G/C-rich motifs, which might convey retinoic acid responses (32).

Internal promoter deletions
Inducible expression of the chromogranin B promoter by NGF (0.1 µg/ml), PACAP (0.1 µM), and VIP (1 µM) was entirely lost (P = NS) when an internal deletion of 60 bases (-134 to -74 bp) was made in -697, -388, or -216 bp of the chromogranin B promoter (Fig. 8Go). As noted above, this region spans not only the CRE motif at -102/-95 bp, but also four different G/C-rich domains at -83/-74, -115/-110, -125/-117, and -134/-127 bp (Fig. 1Go); indeed, the region from -134 to -74 bp contains 49/60 G or C residues, for 82% G/C content. Similar dependence on this deleted region was noted for the responses to nicotine (1 mM), retinoic acid (100 µM), and forskolin (10 µM; data not shown).



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  		Figure 8. Effect of internal deletion of chromogranin B promoter domain on secretagogue-induced expression in PC12 cells. PC12 cells were transfected with plasmids (CgB{Delta}697, CgB{Delta}388, and CgB{Delta}220) bearing an internal deletion of 60 bp (from -134 to -74 bp) and their respective controls (CgB697, CgB388, and CgB220) and were treated with vehicle (mock) or secretagogue for 18 h. Results are the mean ± SEM (n = 3 transfections for each plasmid) and are expressed as relative light units (RLU) in 1 x 106 cells (n = 3 transfections for each plasmid). Compare with Fig. 1Go for CgB promoter motifs.

 
Secretagogue potency and efficacy
In comparing the relative potency (EC50 values) vs. the efficacy (maximal or ceiling effect) of several secretagogues (nicotine, PACAP, VIP, forskolin, NGF, or retinoic acid) to activate the transfected chromogranin B promoter (Table 1Go), PACAP displayed the greatest potency (EC50, 4.08 nM), whereas the adenylyl cyclase activator forskolin had the greatest efficacy (9.6-fold increase in luciferase light units over control; Table 1Go). Thus, cAMP-dependent stimuli are prominent in their ability to activate chromogranin B expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The chromogranin/secretogranin secretory proteins are widely distributed in neuroendocrine and neuronal cells, although not in other cell types (4, 34). What factors govern such widespread, yet selective, neuroendocrine expression of this protein family? The promoters of chromogranins A and B and secretogranin II share only the CRE and TATA regions in common (35), and the CRE region is particularly crucial for neuroendocrine cell type-specific expression of mouse (35) and human (36) chromogranin A. CRE domains also play critical roles in tissue-specific expression of other neuroendocrine genes, such as tyrosine hydroxylase (37), neurotropin-inducible vgf (29), and the {alpha}1B-adrenergic receptor (38). For the well studied chromogranin A promoter, a number of secretagogue responses also map onto the CRE box, including responses to cAMP (35), nicotinic cholinergic stimulation (39), membrane depolarization (40), protein kinase C activation (40), NGF (28), and the neuropeptide PACAP (27). We have recently shown that a CRE domain is indispensable for tissue-specific and inducible expression of the mouse secretogranin II gene (20). Do specific domains play similar roles in the chromogranin B promoter?

Northern blot analysis of steady-state chromogranin B mRNA revealed neuroendocrine expression in adrenomedullary (rat PC12), corticotrope (mouse AtT20), and somatotrope (rat GC) cells, but no expression was detected in control (COS-1 kidney fibroblast) cells. These results are consistent with earlier observations that chromogranin B is expressed principally in endocrine, neuroendocrine, and neuronal cell types (4).

Regions underlying cell type specificity of chromogranin B
Our studies with the transfected chromogranin B promoter also verified cell type-specific expression. Chromogranin B promoter expression in neuroendocrine cells was preserved, upon progressive 5'-deletions down to -216 bp upstream of the cap site, after which specific expression began to decline. A 38.3–59.1% fall in promoter activity was seen in both neurons and neuroendocrine cells after deletion from -216 to -146 bp, a region containing one G/C-rich motif at -196/-191 bp. A more dramatic decrease (53–85%) in promoter activity was noticed after deletion from -146 to -107 bp, although only in neuroendocrine cells, not in neurons; this region contains three G/C-rich motifs at -115/-110, -125/-117, and -134/-127 bp. The implication is that G/C-rich elements are crucial in directing tissue-specific expression of the chromogranin B gene in neuroendocrine cells, although perhaps not in neurons. By contrast, neuronal (Cath-a, GT1–7, SK-N-SH, and F-11) chromogranin B promoter activity remained unaltered after deletion from -146 to -107 bp, but a dramatic decrease (59–72%) in promoter activity was seen after deletion from -107 to -91 bp. Of note, the CRE in this promoter is at -102/-95 bp, indicating that the CRE region, even in the absence of the upstream G/C-rich motifs, seems to be sufficient for neuron-specific expression of the chromogranin B gene.

Chromogranin B expression in neuroendocrine cells was entirely abolished by an internal promoter deletion of 60 bp (from -134 to -74 bp) spanning both the CRE and the G/C-rich sites, thereby confirming the importance of these sites in directing tissue-specific expression of the chromogranin B gene.

Regions underlying secretagogue inducibility
As chromogranin B gene expression responds to cAMP (14), we tested its regulation by the protein kinase A pathway, using the adenylyl cyclase stimulator forskolin. Forskolin augmented both the endogenous gene (5.8-fold) as well as the transfected chromogranin B promoter (9.6-fold) in PC12 cells. These findings are in agreement with previous findings on the cAMP pathway in PC12 or bovine chromaffin cells and neurons (13, 14). The forskolin inducibility of chromogranin B in PC12 cells was preserved until 5'-deletions passed -107 bp, removing the CRE at -102/-95 bp. All promoter deletion mutants downstream (3') of the CRE failed to respond to forskolin, confirming that the CRE mediates the effects of forskolin.

Catecholamine secretion from chromaffin cells is regulated by both cholinergic (acetylcholine released from splanchnic nerve) (26) and peptidergic (substance P, PACAP, and VIP, also contained in the splanchnic nerve) (27, 41, 42) preganglionic neuronal stimuli. As chromogranin B is costored and cosecreted with catecholamines in response to such stimuli (4), we investigated whether cholinergic or peptidergic perturbations of secretion modify chromogranin B gene expression. Chromaffin cell secretagogues [nicotine (2.0-fold), PACAP (4.9-fold), and VIP (4.4-fold)] augmented expression of both the endogenous chromogranin B gene and the transfected chromogranin B promoter. A significant fall in secretagogue (nicotine, PACAP, or VIP) inducibility was seen upon deletion from -146 to -107 bp. This region contains three G/C-rich domains. During each treatment (nicotine, PACAP, or VIP), secretagogue responses were retained up to -107 bp, but were entirely lost after -91 bp; the -107 bp/-91 bp region contains the CRE domain at -102/-95 bp. Previously, we found that CRE domains mediated PACAP-induced trans-activation of the chromogranin A (27) and secretogranin II (20) genes. PACAP and VIP seem to use CRE domains in trans-activating other genes, such as interleukin-10 (43), and proenkephalin A (44).

Peptidergic stimulations were both more potent (lower EC50 values) and more effective (greater maximal or ceiling effect) than nicotinic cholinergic stimulation in activating chromogranin B gene expression. These findings are consistent with the characteristically much higher affinity for agonists at G protein-coupled receptors (such as the PACAP/VIP receptor family) compared with extracellular ligand-gated ion channels (such as the nicotinic cholinergic receptor) (45, 46).

NGF had time-dependent effects on chromogranin B expression. In acute (16- to 18-h) NGF exposure experiments (endogenous gene in Fig. 3Go; transfected promoter in Fig. 7BGo), NGF activated chromogranin B expression. By contrast, in the more chronic NGF experiment reported in Fig. 6Go, PC12 cells were predifferentiated into a neurite-bearing state by 72 h of NGF pretreatment before transfection with the chromogranin B promoter/luciferase reporter plasmids. In this setting of chronic NGF differentiation, the dependence of chromogranin B expression on the G/C-rich region (between -134 and -107 bp) was diminished.

Secretagogue-inducible expression of the chromogranin B gene was also severely disrupted after an internal deletion of 60 bp (from -134 to -74 bp) within the CgB697, CgB388, and CgB220 promoter/reporter constructs. These results further refine the dependence of secretagogue inducibility on this region, which harbors not only the CRE box, but also three G/C-rich domains.

In conclusion, we identified crucial regions in the proximal chromogranin B promoter that direct both basal (constitutive neuroendocrine-specific) as well as secretagogue-inducible expression of the gene. Chromogranin B seems to be unique within the chromogranin/secretogranin protein family (20, 35, 47) in having different tissue-specific promoter elements for neuroendocrine vs. neuronal cells. In this promoter, preganglionic stimulus-transcription coupling (39) seems to occur, although peptidergic secretory stimuli (such as PACAP and VIP) are far more potent than a nicotinic cholinergic stimulus in triggering expression of the gene.


   Footnotes
 
1 This work was supported by the Department of Veterans Affairs and the NIH (Grant DA11311 to S.K.M.; Grants HL-55583 and HL-58120 to D.T.O’C.). Back

Received March 23, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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