From the
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.
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)
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.
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.
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.
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
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
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
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.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Dr. Douglas Hanahan for kindly providing the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)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) .
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) .
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.
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).
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.
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.
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.
/EMBL Data Bank with accession number(s) U24128.
-TC3 cell line and Marleen Willems for cell culture.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.