©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Neuroendocrine-specific Expression of the Human Prohormone Convertase 1 Gene
HORMONAL REGULATION OF TRANSCRIPTION THROUGH DISTINCT cAMP RESPONSE ELEMENTS (*)

Erik Jansen (§) , Torik A. Y. Ayoubi (¶) , Sandra M. P. Meulemans , Wim J. M. Van de Ven (**)

From the (1)Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven, and Flanders Institute for Biotechnology, Herestraat 49, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5`-terminal region of the human prohormone convertase 1 (PC1) gene and identified and characterized the PC1 promoter region. We found multiple transcription start sites located within a 15-base pair region, 205 base pairs upstream of the translation start codon. The promoter region is not G+C-rich and does not contain a canonical TATA box nor a CAAT box. Transient expression assays with a set of human PC1 gene fragments containing progressive 5` deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, the proximal promoter region confers both basal and hormone-regulated promoter activity. Site-specific mutagenesis experiments demonstrate that two closely spaced cAMP response elements within the proximal promoter region direct cAMP-mediated hormonal regulation of transcription of the PC1 gene.


INTRODUCTION

A large number of secretory proteins is initially synthesized as part of larger precursors, which are post-translationally processed into mature bioactive products. This processing requires the activity of a family of endoproteolytic enzymes that has been discovered recently. The responsible enzymes are subtilisin-like serine endoproteases, and endoproteolysis generally occurs at sites consisting of paired basic amino acid residues. Until now, six prohormone- and proprotein-processing enzymes have been molecularly characterized, most of these by cDNA cloning. With respect to tissue distribution, they can be classified into different subclasses.

The furin enzyme, which is encoded by the FUR gene, can be considered as the mammalian prototype of this enzyme family. The structure of the FUR gene has been analyzed extensively (see Ref. 1, and references therein). The gene is expressed in a wide variety of tissues, and furin appears to be responsible for the cleavage of precursor proteins within the constitutive secretory pathway(2, 3, 4) . Recently, promoter regions, directing FUR gene expression, have been analyzed in detail(5) .

In contrast to furin, the prohormone convertases (PCs)()PC1(6, 7) , also described as PC3 (8), and PC2 (6, 9) are expressed predominantly in cells of (neuro)endocrine origin(10) . Evidence has been provided that PC1 and PC2 are involved in the tissue-specific processing of prohormones and neuropeptide precursors within the regulated secretory pathway, resulting in the release of mature products from their inactive precursor molecules. For example, PC1 and PC2 are capable of correctly cleaving the multifunctional precursor protein POMC, at distinct pairs of basic residues(11, 12) . Moreover, it has been shown that introduction of PC1 antisense RNA in the pituitary corticotroph-derived cell line AtT-20 results in a decrease of POMC processing(13) . This illustrates that PC1 mRNA encodes an enzyme activity essential to the processing of POMC in corticotrophs. According to recent studies, PC1 and PC2 selectively process proinsulin and proglucagon in pancreatic islets(14, 15, 16, 17) .

Understanding the mechanism governing neuroendocrine-specific expression of the human PC1 gene requires knowledge of the promoter functions of the gene. In our effort to elucidate the regulatory mechanism underlying neuroendocrine-specific human PC1 expression, we have cloned and sequenced the 5`-flanking region of the human PC1 gene, identified the transcription start sites, and localized transcriptional control elements. Moreover, the promoter region has been shown to direct neuroendocrine-specific reporter gene expression when analyzed in transfection experiments. The results indicate that tissue-specific expression and hormonal regulation of human PC1 expression is directed through two distinct CREs within the proximal promoter region.


EXPERIMENTAL PROCEDURES

Library Screening and Sequence Analysis

A human genomic library in FixII (Stratagene) was screened with a P-labeled probe spanning 309 bp from the 5`-end of the published human PC1 cDNA sequence(18) . Inserts were subcloned into pGEM vectors (Promega) and characterized by Southern blot analysis according to standard techniques(19) . The 1324-bp nucleotide sequence of the human PC1 gene (Fig. 1B) containing 0.8 kb of 5`-flanking region, the complete first exon, and part of the first intron was sequenced on both strands using the AutoRead Sequencing and Automatic Laser Fluorescence protocols (Pharmacia Biotech Inc.). Evaluation of nucleotide sequence data were performed with computer programs Genepro (Riverside Scientific) and Intelligenetics (IntelliGenetics, Inc.). Scanning of the putative promoter region for consensus binding sites of transcription factors was done using Signal Scan 3.0 (20) and Transcription Factor Data Base release 7.3(21) .


Figure 1: Structure of the human PC1 gene 5`-flanking region. A, restriction map of a DNA fragment containing the first exon and adjacent sequences. Exon 1 is represented as a box with the shaded area corresponding to coding sequences. Relevant restriction endonuclease sites are indicated: B, BamHI; H, HindIII; M, MscI; N, NdeI; Sc, ScaI; S, SmaI; Sp, SphI; X, XbaI. B, DNA sequence of the human PC1 gene promoter. The transcription initiation sites are given in bold and are underlined. The translation initiation codon is indicated in bold. Intron 1 sequences are in lowercase. Putative CRE motifs are doubleunderlined. Putative Sp1 sites are underlined with a singleline.



Primer Extension Analysis

A 30-nucleotide primer (PE-1), complementary to nucleotides -162 to -133 relative to the 3`-end of human PC1 exon 1, was 5`-end-labeled using [-P]ATP (6000 Ci/mmol, DuPont NEN) and T4 polynucleotide kinase (Boehringer Mannheim). The P-labeled PE-1 was annealed to 20 µg of total RNA from a human lung carcinoid tumor (18) or to 20 µg of total RNA from human SCLC cell line NCI-H82 (American Type Culture Collection (ATCC)) and extended by SuperScript II RNase H reverse transcriptase (Life Technologies, Inc.) according to the manufacturer's protocol. The extension products were analyzed on a 6% polyacrylamide sequencing gel containing 8 M urea.

RNase Protection Analysis

RNase protection experiments were performed essentially according to standard protocols(19) . In brief, a genomic fragment, corresponding to nucleotide -288 to -1 relative to the translation initiation codon ATG within exon 1, was subcloned and used as template for in vitro synthesis of antisense RNA probes. The P-labeled RNA probe was purified by denaturing polyacrylamide gel electrophoresis and hybridized to 20 µg of DNase-treated total RNA from a human lung carcinoid tumor. Control for protection specificity was carried out with tRNA. Subsequent RNase A (15 µg/ml) and RNase T1 (150 units/ml) treatment was carried out at 25 or 37 °C. The protected products were analyzed on a 6% sequencing gel.

Northern Blot Analysis

Total RNA was isolated from AtT-20, -TC3, and COS-1 cells using the guanidinium thiocyanate procedure (22). 20 µg of RNA was size-fractionated through a 1% agarose gel, blotted onto Hybond-N membranes (Amersham Corp.), and hybridized to a human PC1 cDNA probe, according to standard procedures(19) .

Reporter Plasmid Constructions

The human PC1-luciferase fusion gene expression plasmids were constructed by subcloning into the polylinker of the promoterless, luciferase encoding plasmid pGL2-Basic (Promega). The 3`-end of all promoter constructs is at position -1 relative to the translation initiation codon in exon 1. Mutational analysis of the proximal promoter elements was done using the Altered Sites in vitro mutagenesis system (Promega). In both CREs, the central AC dinucleotide core was mutated into a TG dinucleotide. Site-specific mutants were verified by sequencing.

Conditions for Cell Culture, Transfections, and Hormonal Incubations

AtT-20 pituitary corticotroph cells (ATCC, CRL 1795), -TC3 insulinoma cells(23) , and COS-1 kidney fibroblasts (ATCC, CRL 1650) were cultured according to the suppliers' protocols. DNAs were purified using anion exchange chromatography (Nucleobond AX, Machery-Nagel, Germany). Unless otherwise indicated, cells were propagated in the prescribed media supplemented with 10% fetal calf serum. Cells were transfected using cationic liposomes (Lipofectamine, Life Technologies) according to the manufacturer's protocol. For each experiment, luciferase activity was determined in duplicate or triplicate wells. The results are expressed as the mean of three to four individual transfection experiments. Cells were harvested at 24 h after start of the transfection, and luciferase reporter enzyme activity driven by the various human PC1 promoter fragments was determined with the Luciferase Assay System (Promega) using a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Hormonal incubations were performed with cells shifted to serum-free medium 24 h prior to transfection. In studying intracellular cAMP signaling, transfected cells were cultured for 6 h in the presence or absence of 10 µM forskolin (Sigma), an adenylate cyclase activator, and 0.1 mM of the phosphodiesterase inhibitor IBMX (Sigma). When indicated, transfected cells were cultured in medium supplemented with 10 µM Rp-cAMP (BioLog, Bremen, Germany), a PKA inhibitor, for time periods indicated. CRF was tested at a final concentration of 10 nM or 100 nM. The dopamine agonist bromocryptine (Sigma), was tested at a 1 µM concentration in transient transfection experiments in which the luciferase constructs were cotransfected with a construct expressing the human dopamine D receptor(24) .


RESULTS

Cloning and Nucleotide Sequence of the 5`-terminal Region of the Human PC1 Gene

Upon screening of a human genomic library with a human PC1 5` cDNA fragment, three independent clones were isolated and shown to be overlapping by restriction site mapping. These clones cover 16 kb of the 5`-flanking region, the complete first exon, and the 5`-part of intron 1. A HindIII fragment, containing 4.5 kb of the upstream region, exon 1, and part of the first intron, was subcloned for restriction sites mapping and sequencing purposes. Fig. 1A shows a schematic representation of this fragment comprising the 5`-part of the human PC1 gene. Fig. 1B shows the 1324-bp sequence established from this clone. A splice donor sequence, CAG/gtaag, that resembles the consensus (C,A)AG/gt(a,g)ag (25) marked the 3`-end of the first exon.

Identification of the Transcription Initiation Sites

Both primer extension and RNase protection studies were performed to determine the transcription initiation sites of the human PC1 gene. Primer extension with primer PE-1 on RNA isolated from a human lung carcinoid tumor, expressing the endogenous PC1 gene (18) resulted in multiple specific products (Fig. 2A), as deduced from comparison with the NCI-H82 cell line, which does not express PC1 at detectable levels(18) . These multiple extension products point toward heterogeneous transcription initiation. The majority of the extension products appeared to be derived from transcripts initiated within a 15-bp region, 205 bp upstream of the translation start site. In order to test whether the extension products reflect legitimate transcription initiation and were not derived from stops of the reverse transcriptase due to secondary structure of the target RNA, RNase protection analysis was performed.


Figure 2: Identification of the transcription initiation sites of the human PC1 gene. A, primer extension analysis. A P-labeled oligonucleotide (PE-1) complementary to nucleotides -162 to -133 relative to the 3`-end of exon 1 was annealed to 20 µg of total RNA isolated from a human lung carcinoid tumor (lane1) or to 20 µg of total RNA from the SCLC cell line NCI-H82 (lane2), and extended by reverse transcriptase. The extension products were analyzed on a 8 M urea, 6% polyacrylamide gel along with molecular size markers (lanesM). The exposure time was 2 days (leftmarkerlane and lane1) or 5 days (lane2 and rightmarkerlane) with an intensifying screen at -80 °C. B, RNase protection analysis. A P-labeled RNA probe complementary to nucleotides -288 to -1 relative to the translation start codon was hybridized to 20 µg of total RNA from a human lung carcinoid tumor (lanes2 and 3) or to 20 µg of tRNA (lane1). Subsequent RNase A and T1 digestion was performed at 25 °C (lane2) or at 37 °C (lanes1 and 3). The protection products were analyzed on a 6% sequencing gel. LaneM, molecular size markers. Lane4, probe alone, no RNases added. Exposure time is 3 h with an intensifying screen at -80 °C.



A single-strand P-labeled RNase protection probe, corresponding to nucleotide -288 to -1 relative to the translation initiation codon, was hybridized to human lung carcinoid RNA. As can be seen in Fig. 2B, RNase protection, like primer extension, resulted in multiple protected fragments, with the major products terminating within a 15-bp region at -205 bp 5` to the translation initiation codon. Minor products with either longer or shorter protected length, were also detected. The results demonstrate that the majority of the human PC1 transcripts originate from transcription starting within a 15-bp region, 205 bp upstream of the ATG translation start codon in exon 1 of the human PC1 gene. In conclusion, the results of the RNase protection studies confirm the positions of the transcription initiation sites as observed in the primer extension experiments (Fig. 2). Thus, the first exon of the human PC1 gene consists of maximally 239 bp of 5`-untranslated sequences and contains the first 60 codons (Fig. 1B).

Structure of the 5`-Flanking Sequence of the Human PC1 Gene

Examination of the proximal 5`-flanking region revealed the absence of canonical TATA and CAAT boxes. In addition, the promoter region is not particular G+C-rich. One of the more upstream, although minor, transcription initiation site matches the terminal deoxynucleotidyltransferase Initiator core sequence, CATTCTGG(26) , which is indicated as initiator element for TATA-less promoters(27) . Scanning of the proximal region for promoter regulatory elements revealed several potential binding sites for transcription factors (Fig. 1B). In more detail, a CRE consensus sequence (TGACGTCA(28) ) and a CRE-like motif containing a consensus CRE 5`-half site (TGACGTgt) are present within a 100-bp region upstream of the transcription initiation sites. A detailed, functional analysis of this region will be presented in the next sections.

Neuroendocrine-specific Expression Directed by the Human PC1 Gene Promoter

To test whether the 5`-flanking sequence of the human PC1 gene is capable of directing neuroendocrine-specific gene expression, a fusion gene construct was made, containing 4.5 kb of 5`-flanking sequence of the human PC1 gene fused to the promoterless luciferase gene as present in pGL2-Basic. The resulting construct was assayed for promoter activity by transient transfections into several cell lines, of neuroendocrine as well as non-neuroendocrine origin. As a negative control, the promoterless luciferase encoding plasmid pGL2-Basic was used in parallel transfections of each cell line. In all cell lines tested, transfection of pGL2-Basic DNA resulted in very low, basal activity. As shown in Fig. 3, the highest levels of human PC1 promoter-driven luciferase activity were observed in the AtT-20 pituitary corticotroph cells and in the -TC3 insulinoma cells. Both cell lines contain high levels of PC1 mRNA (Fig. 4). In contrast, luciferase activity was 10-12-fold lower in the non-neuroendocrine COS-1 cells that do not express PC1. The neuroendocrine-specific expression of the human PC1 promoter constructs suggests that the 4.5-kb 5`-flanking sequence contains signals for directing cell type-specific expression of the human PC1 gene. Sequential deletion of the region between position -4.5 kb and -971 bp had no significant effect on promoter activity (Fig. 3). Additional deletion of the promoter region up to -288 resulted in a 1.8-1.6-fold drop in luciferase activity in AtT-20 and -TC3 cells. Further reduction of the upstream sequence to nucleotide -224 decreased promoter activity by 5-fold in AtT-20 and -TC3 cells and 2-fold in COS-1 cells, indicating that the DNA sequence between -288 and -224 contains positive regulatory elements enhancing PC1 expression. In summary, the region between -288 and -1 represents a minimal core promoter that is capable of directing cell type-specific expression of the human PC1 gene.


Figure 3: Deletion analysis of the human PC1 gene promoter. PC1 gene fragments, containing progressive 5` deletions and a common 3`-end at nucleotide -1 relative to the translation start codon in exon 1, were inserted upstream of the promoterless luciferase gene in the pGL2-Basic vector. Promoter activity of each PC1-luciferase fusion gene in AtT-20 (blackbars), -TC3 (hatchedbars), and COS-1 cells (shadedbars) was determined. Values are the mean ± S.E. (error bar) of at least four independent transfection experiments, each of which was performed in duplicate.




Figure 4: Northern blot analysis of PC1 gene expression. A, Northern blot of 20 µg of total RNA isolated from AtT-20 cells (lane1), -TC3 cells (lane2), and COS-1 cells (lane3). Hybridization with a P-labeled human PC1 cDNA probe. Exposure time was 16 h at room temperature. B, same Northern blot, subsequently hybridized with a mouse actin cDNA probe. Exposure time was 2 h at -80 °C with an intensifying screen.



Hormonal Regulation of Human PC1 Promoter Activity

The proximal promoter region contains a consensus CRE (TGACGTCA, CRE-1) between -283 bp and -276 bp and a CRE-like motif (TGACGTGT, CRE-2) 20 bp downstream. Deletion of the proximal promoter region up to -224 bp resulted in a 5-fold drop in promoter activity when assayed in transient transfection experiments (Fig. 3). In order to investigate whether this region could confer cAMP responsiveness on human PC1 gene expression, we have analyzed CRE-1 and CRE-2 in more detail.

AtT-20 and COS-1 cells, transfected with the -288 and -224 human PC1 promoter-luciferase reporter constructs, were cultured in the presence or absence of forskolin and IBMX for 6 h. In this experiment, the -288 construct showed a specific activation upon incubation with forskolin and IBMX. The human PC1 promoter activity was enhanced 10-12-fold in COS-1 cells and 3-5-fold in AtT-20 cells (data not shown). In addition, complete loss of cAMP-mediated enhancement of PC1 promoter activity occurred when the region between -288 and -224 was deleted, suggesting the involvement of the CRE-1 and CRE-2 in cAMP-mediated transcriptional activation of the proximal human PC1 promoter.

To investigate whether the lower activation in AtT-20 cells is due to constitutive, high intracellular cAMP levels and PKA activity, the experiments were repeated with transfected cells that were preincubated with Rp-cAMP, a specific PKA inhibitor. After an overnight preincubation with Rp-cAMP, the cells were incubated in serum-free medium with or without the addition of forskolin and IBMX for 6 h. As shown in Fig. 5, basal, non-induced human PC1 promoter activity of the -288 construct in AtT-20 cells is reduced 3-fold and cAMP-mediated promoter activation in AtT-20 was now 12-14-fold, as high as in COS-1 cells in the initial experiments, in which cells were not preincubated with Rp-cAMP. No reduction of non-induced activity of the -288 construct was observed in COS-1 cells, probably because low basal activity was already present. The activity of the -224 construct is not influenced by the Rp-cAMP treatment, which stresses the importance of CRE-1 and CRE-2 in the cAMP-mediated response. We also tested the -4.5-kb, the -3.5-kb, and the -971-bp constructs for cAMP-mediated activation of transcription. These constructs displayed a similar level of activation upon incubation with forskolin and IBMX. Since 5`-deletion up to -224 bp completely abolished transcriptional activation, the obtained data indicate that cAMP-mediated transcriptional activation is controlled by regulatory elements within the proximal -288 to -224 bp of the human PC1 promoter region.


Figure 5: Regulation of human PC1 promoter activity by cAMP-mediated mechanisms. Human PC1-promoter-luciferase reporter constructs, containing sequences up to -288 bp or -224 bp and a common 3`-end at nucleotide -1, were tested in transient transfection experiments. A, after transfection, cells were incubated overnight in serum-free medium. The next day, cells were shifted to serum-free medium with (+F/I) or without the addition of 10 µM forskolin and 0.1 mM IBMX. After 6 h, luciferase activity was measured. B, after transfection, cells were preincubated overnight in serum-free medium supplemented with 10 µM of the PKA inhibitor Rp-cAMP. The next day, cells were shifted to serum-free medium with (+F/I) or without the addition of 10 µM forskolin and 0.1 mM IBMX. After 6 h, luciferase activity was measured. The histograms show the mean ± S.E. of the results of at least three independent transfection experiments. , AtT-20; , AtT-20 +F/I; , COS-1; &cjs2110;, COS-1 +F/I.



To further pinpoint the exact sites mediating the observed activation, site-directed mutagenesis was performed to specifically modify one or both CRE motifs by mutating the central core AC dinucleotide to a TG dinucleotide. In double mutants, this completely abolished cAMP-mediated activation of both the -971-bp and -288-bp constructs (Fig. 6). This is in agreement with the previous observations and indicates that no additional elements in the upstream region between -971 and -288 are involved in cAMP-mediated promoter activation. In addition, mutation of CRE-1 almost completely abolished cAMP-mediated promoter activation in AtT-20 cells (Fig. 6A). However, in COS-1 cells this mutant can still be activated 4-fold (Fig. 6B). A differential effect is also observed when the CRE-2 mutant is tested. This construct displayed a 4-fold lower activation in COS-1 cells, whereas in AtT-20 cells, activation was only reduced 2-fold. This suggests differential regulatory mechanisms acting through the distinct elements CRE-1 and CRE-2 in AtT-20 and COS-1 cells.


Figure 6: Mutational analysis of human PC1 promoter activation by cAMP-mediated signal transduction. A, AtT-20 cells were transfected with wild-type (wt) or mutant CRE-1 (mut-1), mutant CRE-2 (mut-2), or double mutant CRE-1 and CRE-2 (mut1-2) human PC1 promoter-luciferase constructs, preincubated with Rp-cAMP, and shifted to medium with (hatchedbars) or without (blackbars) added forskolin and IBMX, and processed as described in the legend of Fig. 5. B, same procedure for COS-1 cells with (cross-hatched bars) or without (shadedbars) the forskolin/IBMX incubation.



In order to study hormonal regulation of the human PC1 gene, we subsequently investigated whether CRF is able to activate human PC1 promoter activity in the pituitary corticotroph cell line AtT-20. This is of particular interest, since ACTH is one of the final products when POMC is processed by PC1 and CRF is a well known ACTH secretogogue. Moreover, it has been reported that in AtT-20 cells, PC1 mRNA is coregulated with its substrate POMC(29) . Binding of CRF to the adenylyl cyclase-coupled CRF receptor induces intracellular cAMP accumulation, which in turn results in activation of the cAMP-dependent PKA(30) . Upon incubation of Rp-cAMP-preincubated AtT-20 cells, cultured in serum-free medium with or without the addition of 10 nM CRF, a specific 7-fold activation of the human PC1 promoter was observed in transient transfection experiments (Fig. 7A). Upon site-specific mutation of the CRE motifs, activation of human PC1 transcription by CRF was completely abolished as observed in the previous forskolin/IBMX induction experiments.


Figure 7: Hormonal regulation of human PC1 promoter activity. A, AtT-20 cells were transfected with the wild-type (wt) or mutant CRE (mut1-2) human PC1 proximal promoter constructs, preincubated with Rp-cAMP, and incubated for 6 h in serum-free medium with or without (blackbar) CRF added to a final concentration of 10 nM (hatchedbars) or 100 nM (shadedbars). B, AtT-20 cells were transfected with the PC1 promoter constructs and a human dopamine D receptor expression construct. The transfected cells were incubated overnight in serum-free medium with (hatchedbars) or without (blackbars) the dopamine agonist bromocryptine (1 µM) added. The next morning, fresh medium with or without bromocryptine was added and cells were assayed for luciferase activity after 6 h.



Dopamine is one of the inhibitory factors of PC1 expression in vivo(29, 31) . It has been shown that this hypothalamic factor, by binding to a specific dopamine D receptor, exerts its effect, at least in part, by decreasing intracellular cAMP levels(32) . In line with the proposed mechanism of action of the D receptor is our observation that in AtT-20 cells, transfected with the dopamine D receptor and incubated with bromocryptine, the luciferase activity driven by the human PC1 promoter is decreased 3-fold to similar basal levels as observed in the previous experiments, in which AtT-20 cells were incubated with the PKA inhibitor Rp-cAMP (Fig. 7B).

In summary, our data provide the first clue on how transcriptional activity of the human PC1 gene is controlled. Hormonal regulation seems to be exerted via CRE motifs within the proximal promoter region, which also directs neuroendocrine-specific expression when analyzed in transfection experiments.


DISCUSSION

In our effort to define DNA regulatory elements controlling neuroendocrine-specific human PC1 gene expression, we have cloned and sequenced the 5`-terminal region of the human PC1 gene, including 732 bp of 5`-flanking sequences, the complete 419-bp first exon, and 173 bp of the first intron.

Using primer extension and RNase protection analysis, we have shown that the human PC1 gene has multiple, dispersed transcription start sites. The majority of the human PC1 encoding mRNAs originates from transcription initiation within a 15-bp region, 205 bp upstream of the translation start codon in exon 1. Primary structure analysis of the upstream sequence reveals the absence of canonical TATA or CAAT boxes. This may explain the observed dispersion of transcription initiation. These characteristics are also found in other neuroendocrine-specific promoters, like the type II sodium channel (33), D dopamine receptor(34) , and brain-specific aldolase C(35) . The sequence surrounding one of the minor transcription start sites of the human PC1 gene is homologous to the Initiator core sequence of the terminal deoxynucleotidyltransferase gene. This element is capable of directing the assembly of a transcription initiation complex at TATA-less promoters, resulting in efficient and discrete transcription initiation within the initiator(26, 27) . The functional significance of this region in the human PC1 gene remains to be established, however, since it appears to constitute only a minor transcription start site. In addition, the 5`-flanking region of the human PC1 gene is not G+C-rich, which is in contrast to the high G+C content of the human PC2 5`-flanking region(36) . This difference could be an important factor in the observed differential regulation of PC1 and PC2 gene expression.

Recently, the mouse PC1 gene was cloned(37) , but no data on promoter activity and its hormonal regulation are available. Sequence comparison of the 5`-upstream region of the human and mouse PC1 genes shows a high degree of sequence conservation. Moreover, the homology of the proximal 5`-flanking region is even higher than the exon 1 sequence conservation, suggesting the presence of promoter regulatory elements within the proximal promoter region.

In this paper, we have demonstrated that the 5`-flanking sequence of the human PC1 gene contains a functional promoter, which is highly active in neuroendocrine cells and displays only low activity in non-neuroendocrine cells. Deletion analysis clearly indicated that the core promoter region between -288 bp and -1 bp relative to the translation start codon exhibited substantial neuroendocrine specificity. Similarly, the core promoters of several other genes, such as POMC(38) , glucagon(39) , synapsin I(40) , and interphotoreceptor retinoid-binding protein (41) have been shown to direct neuroendocrine specificity. Pituitary-specific POMC gene transcription (42) and cell-specific expression of the glucagon gene (43) have been shown to be directed by the interaction of cell-specific transcription factors with distinct DNA elements within the proximal promoter region.

In the present study, we have shown that hormonal regulation of human PC1 gene expression is directed through two distinct CRE motifs, separated by a 20-bp spacer, within the region from bp -288 to -1. In more detail, incubation of transfected AtT-20 cells with forskolin and IBMX, agents known to elevate intracellular cAMP levels, resulted in a 10-12-fold increase of promoter activity. Upon site-specific mutagenesis of both CREs, this activation is completely abrogated. In addition, basal, non-induced promoter activity of these mutants is also substantially reduced to levels that closely correspond to the activity observed when the wild-type human PC1 promoter-luciferase constructs are introduced into COS-1 cells. The importance of the CREs in regulating PC1 gene expression is also stressed by the sequence conservation of the CREs and flanking sequences in mice and man. Various hormones regulate gene expression via a cAMP-dependent signal transduction pathway, which in turn modulates the function of transcription factors that bind to CRE motifs in the respective target genes(44) . In this context, the presence of CREs within the proximal, TATA-less PC1 promoter is interesting, since evidence has been provided for the involvement of CRE binding transcription factors in recruiting components of the transcription initiation complex to cAMP-responsive, TATA-less promoters, similar to Sp1-mediated transcriptional activation(45) .

Of particular physiological importance is our observation that CRF, a potent ACTH secretogogue, enhanced PC1 gene expression through transactivation of the proximal, CRE-containing promoter region. In this context, it is important to note that POMC gene transcription is also enhanced by CRF and that in AtT-20 cells PC1 mRNA has been shown to be coregulated with POMC in response to CRF(29) . We have also established that dopamine D receptor-mediated down-regulation of PC1 promoter activity is mediated through the CREs. Inhibitory control of gene expression by dopamine has also been observed in studies with prolactin promoter constructs(46) . Proper functioning of peptide secreting cells requires coordinate expression of hormone and its respective processing enzyme. The study of regulatory elements in the PC1 promoter will shed light on common regulatory pathways necessary for the functioning of neuroendocrine cells. We are currently characterizing factors involved in the cAMP-mediated regulation of PC1 gene expression.

In conclusion, this paper describes the first features of the neuroendocrine-specific human PC1 promoter. Further analysis of the promoter regulatory elements and their binding factors should help to elucidate the molecular mechanisms underlying tissue specificity and hormone-regulated expression of the human PC1 gene.


FOOTNOTES

*
This work was supported in part by the Geconcentreerde Onderzoekacties 1992-1996, EC Contract ERBCHBGCT 920091, the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-technologisch Onderzoek in de Industrie (IWT), and Stichting Technische Wetenschappen Grant STW-22.2726. This is a publication from the Belgian Programme on Interuniversity Poles of Attraction, initiated by the Belgian State, Prime Minister's Office, Science Policy Programming. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U24128.

§
Holder of a Human Capital and Mobility postdoctoral fellowship from the European Union.

Holder of a postdoctoral fellowship from the IWT.

**
To whom correspondence should be addressed. Tel.: 32-16-345987; Fax: 32-16-346073.

The abbreviations used are: PC, prohormone convertase; bp, base pair(s); CRE, cAMP response element; CRF, corticotropin-releasing factor; IBMX, 3-isobutylmethylxhanthine; kb, kilobase(s); PKA, protein kinase A; POMC, proopiomelanocortin; SCLC, small cell lung cancer; ACTH, adrenocorticotropic hormone.


ACKNOWLEDGEMENTS

We thank Dr. Douglas Hanahan for kindly providing the -TC3 cell line and Marleen Willems for cell culture.


REFERENCES
  1. Van de Ven, W. J. M., Van Duijnhoven, J. L. P., and Roebroek, A. J. M.(1993) Crit. Rev. Oncogen.4, 115-136 [Medline] [Order article via Infotrieve]
  2. Bresnahan, P. A., Leduc, R., Thomas, L., Thorner, J., Gibson, H. L., Brake, A. J., Barr, P. J., and Thomas, G.(1990) J. Cell Biol.111, 2851-2859 [Abstract]
  3. Van de Ven, W. J. M., Voorberg, J. T. L., Fontijn, R., Pannekoek, H., Van den Ouweland, A. M. W., Van Duijnhoven, J. L. P., Roebroek, A. J. M., and Siezen, R. J.(1990) Mol. Biol. Rep.14, 265-275 [Medline] [Order article via Infotrieve]
  4. Day, R., Schäfer, M. K.-H., Cullinan, W. E., Watson, S. J., Chrétien, M., and Seidah, N. G.(1993) Neurosci. Lett.149, 27-30 [CrossRef][Medline] [Order article via Infotrieve]
  5. Ayoubi, T. A. Y., Creemers, J. W. M., Roebroek, A. J. M., and Van de Ven, W. J. M.(1994) J. Biol. Chem.269, 9298-9303 [Abstract/Free Full Text]
  6. Seidah, N. G., Gaspar, L., Mion, P., Marcinkiewicz, M., Mbikay, M., and Chrétien, M.(1990) DNA9, 415-424
  7. Seidah, N. G., Marcinkiewicz, M., Benjannet, S., Gaspar, L., Beaubien, G., Mattei, M. G., Lazure, C., Mbikay, M., and Chrétien, M. (1991) Mol. Endocrinol.5, 111-122 [Abstract]
  8. Smeekens, S. P., Avruch, A. S., Lamendola, J., Chan, S. J., and Steiner, D. F.(1991) Proc. Natl. Acad. Sci. U. S. A.88, 340-344 [Abstract]
  9. Smeekens, S. P. and Steiner, D. F.(1990) J. Biol. Chem.265, 2997-3000 [Abstract/Free Full Text]
  10. Seidah, N. G., Chrétien, M., and Day, R.(1994) Biochimie76, 197-209 [CrossRef][Medline] [Order article via Infotrieve]
  11. Benjannet, S., Rondeau, N., Day, R., Chrétien, M., and Seidah, N. G.(1991) Proc. Natl. Acad. Sci. U. S. A.88, 3564-3568 [Abstract]
  12. Thomas, L., Leduc, R., Thorne, B. A., Smeekens, S. P., Steiner, D. F., and Thomas, G.(1991) Proc. Natl. Acad. Sci. U. S. A.88, 5297-5301 [Abstract]
  13. Bloomquist, B. T., Johnson, R. C., and Mains, R. E.(1992) DNA Cell Biol.11, 791-797 [Medline] [Order article via Infotrieve]
  14. Bennett, D. L., Bailyes, E. M., Nielsen, E., Guest, P. C., Rutherford, N. G., Arden, S. D., and Hutton, J. C.(1992) J. Biol. Chem.267, 15229-15236 [Abstract/Free Full Text]
  15. Smeekens, S. P., Montag, A. G., Thomas, G., Albiges-Rizo, C., Carroll, R., Benig, M., Phillips, L. A., Martin, S., Ohagi, S., Gardner, P., Swift, H. H., and Steiner, D. F.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 8822-8826 [Abstract]
  16. Rouillé, Y., Westermark, G., Martin, S. K., and Steiner, D. F. (1994) Proc. Natl. Acad. Sci. U. S. A.91, 3242-3246 [Abstract]
  17. Mineo, I., Matsumura, T., Shingu, R., Namba, M., Kuwajima, M., and Matsuzawa, Y.(1995) Bioch. Biophys. Res. Commun.207, 646-651 [CrossRef][Medline] [Order article via Infotrieve]
  18. Creemers, J. W. M., Roebroek, A. J. M., and Van de Ven, W. J. M.(1992) FEBS Lett.300, 82-88 [CrossRef][Medline] [Order article via Infotrieve]
  19. Sambrook, J., Fritsch, E. F., and Maniatis, T.(1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  20. Prestridge, D. S.(1993) Comput. Appl. Biosci.9, 113-115 [Abstract]
  21. Ghosh, D.(1993) Nucleic Acids Res.21, 3117-3118 [Abstract]
  22. Chomczynski, P. and Sacchi, N.(1987) Anal. Biochem.162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  23. Efrat, S., Linde, S., Kofod, H., Spector, D., Delannoy, M., Grant, S., Hanahan, D., and Baekkeskov, S.(1988) Proc. Natl. Acad. Sci. U. S. A.85, 9037-9041 [Abstract]
  24. Grandy, D. K., Marchionni, M. A., Makam, H., Stofko, R. E., Alfano, M., Frothingham, L., Fischer, J. B., Burke-Howie, K. J., Bunzow, J. R., Server, A. C., and Civelli, O.(1989) Proc. Natl. Acad. Sci. U. S. A.86, 9762-9766 [Abstract]
  25. Padgett, R. A., Grabowski, P. J., Konarska, M. M., Seiler, S., and Sharp, P. A.(1986) Annu. Rev. Biochem.55, 1119-1150 [CrossRef][Medline] [Order article via Infotrieve]
  26. Smale, S. T., Schmidt, M. C., Berk, A. J., and Baltimore, D.(1990) Proc. Natl. Acad. Sci. U. S. A.87, 4509-4513 [Abstract]
  27. Kaufmann, J., and Smale, S. T.(1994) Genes & Dev.8, 821-829
  28. Montminy, M. R., Sevarino, K. A., Wagner, J. A., Mandel, G., and Goodman, R. H.(1986) Proc. Natl. Acad. Sci. U. S. A.83, 6682-6686 [Abstract]
  29. Bloomquist, B. T., Eipper, B. A., and Mains, R. E.(1991) Mol. Endocrinol.5, 2014-2024 [Abstract]
  30. Chen, R., Lewis, K. A., Perrin, M. H., and Vale, W. W.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 8967-8971 [Abstract]
  31. Day, R., Schäfer, M. K.-H., Watson, S. J., Chrétien, M., and Seidah, N. G.(1992) Mol. Endocrinol.6, 485-497 [Abstract]
  32. Senogles, S. E.(1994) J. Biol. Chem.269, 23120-23127 [Abstract/Free Full Text]
  33. Maue, R. A., Kraner, S. D., Goodman, R. H., and Mandel, G.(1990) Neuron4, 223-231 [Medline] [Order article via Infotrieve]
  34. Minowa, M. T., Minowa, T., Monsma, F. J., Jr., Sibley, D. R., and Mouradian, M. M.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 3045-3049 [Abstract]
  35. Vibert, M., Henry, J., Kahn, A., and Skala, H.(1989) Eur. J. Biochem.181, 33-39 [Abstract]
  36. Ohagi, S., LaMendola, J., LeBeau, M. M., Espinosa, R., III, Takeda, J., Smeekens, S. P., Chan, S. J., and Steiner, D. F.(1992) Proc. Natl. Acad. Sci. U. S. A.89, 4977-4981 [Abstract]
  37. Ftouhi, N., Day, R., Mbikay, M., Chrétien, M., and Seidah, N. G. (1994) DNA Cell Biol.13, 395-407 [Medline] [Order article via Infotrieve]
  38. Jeannotte, L., Trifiro, M. A., Plante, R. K., Chamberland, M., and Drouin, J.(1987) Mol. Cell. Biol.7, 4058-4064 [Medline] [Order article via Infotrieve]
  39. Philippe, J., Drucker, D. J., Knepel, W., Jepeal, L., Misulovin, Z., and Habener, J. F.(1988) Mol. Cell. Biol.8, 4877-4888 [Medline] [Order article via Infotrieve]
  40. Sauerwald, A., Hoesche, C., Oschwald, R., and Killimann, M. W.(1990) J. Biol. Chem.265, 14932-14937 [Abstract/Free Full Text]
  41. Bobola, N., Hirsch, E., Albini, A., Altruda, F., Noonan, D., and Ravazzolo, R.(1995) J. Biol. Chem.270, 1289-1294 [Abstract/Free Full Text]
  42. Therrien, M. and Drouin, J.(1993) Mol. Cell. Biol.13, 2342-2353 [Abstract]
  43. Philippe, J., Morel, C., and Cordier-Bussat, M.(1995) J. Biol. Chem.270, 3039-3045 [Abstract/Free Full Text]
  44. Borelli, E., Montmayeur, J. P., Foulkes, N. S., and Sassone-Corsi, P. (1992) Crit. Rev. Oncogen.3, 321-338 [Medline] [Order article via Infotrieve]
  45. Ferreri, K., Gill, G., and Montminy, M.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 1210-1213 [Abstract]
  46. Elsholz, H. P., Lew, A. M., Albert, P. R., and Sundmark, V. C.(1991) J. Biol. Chem.266, 22919-22925 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.