Characterization of an Intronic Promoter of a Cyclic Adenosine 3',5'-Monophosphate (cAMP)-Specific Phosphodiesterase Gene that Confers Hormone and cAMP Inducibility
Elena Vicini and
Marco Conti
Division of Reproductive Biology (M.C.), Department of
Gynecology and Obstetrics, Stanford University Medical Center,
Stanford, California 94305,
Institute of Histology and General
Embriology (E.V.), School of Medicine, University La Sapienza,
00161 Rome, Italy
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ABSTRACT
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In the Sertoli cell, FSH stimulates
transcription of a cAMP-phosphodiesterase (PDE) gene (PDE4D) and
accumulation of corresponding mRNA and PDE protein. The regulation of
this PDE gene is an important component of the desensitization state
induced by this hormone. Given the ubiquitous nature of this regulation
controlling cAMP levels, the molecular basis for the PDE4D induction
was further investigated. FSH stimulation of the Sertoli cell causes
the accumulation of only two of the four known PDE4D mRNAs (PDE4D1
and PDE4D2). The promoter controlling the expression of these two
messages was identified and characterized. An EcoRI
fragment containing a coding exon as well as 5'-upstream sequence of
the PDE4D1/2 mRNA was isolated from rat genomic libraries and
sequenced. No TATA box was identified, but GC-rich regions were present
upstream of the putative translation start site. RNAse protection and
PCR analysis indicated the presence of at least two distinct cap sites.
This genomic region had promoter activity when transfected both in
Sertoli and MA-10 cells. Deletion mutation indicated that basal
promoter activity was contributed by regions upstream of both cap
sites. Transcription from this promoter was activated by FSH and
(Bu)2cAMP, and elements responsible for cAMP
regulation were present upstream from the second cap site. These data
demonstrate that an intronic promoter that is cAMP- and
hormone-inducible directs the expression of these truncated PDE
proteins.
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INTRODUCTION
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Maintaining intracellular cAMP levels within a narrow range of
concentrations is crucial for growth and differentiation of the hormone
target cell. Extracellular cues and intracellular mechanisms of
homeostasis control these cAMP levels by regulating both synthesis and
degradation of this second messenger. One of these homeostatic
mechanisms involves a feedback loop whereby changes in intracellular
cAMP concentrations modulate the activity or the expression of cyclic
nucleotide phosphodiesterases (1). As progress has been made in
understanding the complexity of the phosphodiesterase (PDE) family of
enzymes, it is becoming clear that multiple biochemical mechanisms are
involved in this feedback, underscoring the importance of a tight
control of cAMP degradation.
Although several isoenzymes from different PDE families may be involved
in the homeostatic regulation of cAMP levels, it has long been
recognized that the cAMP-specific rolipram-sensitive (family 4) PDEs
are an essential component of this feedback. In a large variety of
cells, an increase in intracellular cAMP is followed by an increase in
the activity of one or more cAMP-PDE forms (1). These isoenzymes are
encoded by four distinct genes. FSH stimulation of the Sertoli cell
causes an increase in PDE activity that is associated with a large
increase in PDE4D and PDE4B mRNAs (2, 3). Although message
stabilization may play a role, run-on studies have indicated that an
increased transcription is the primary cause for the increase in the
PDE4D mRNA levels (3). Translation of the accumulated message causes
the appearance of a 67 kDa PDE protein in extracts of the Sertoli cell
(4). The activity of this PDE protein is responsible for the transient
accumulation of cAMP and for induction of a desensitized state (5, 6).
Similar regulation of this PDE4D or of the other PDE4 PDEs has been
observed in several other hormone-responsive cells (2, 7, 8) as well as
inflammatory cells (9, 10, 11). In the latter, this induction is thought to
play a crucial role in the control of the inflammatory process (12, 13). In addition to this long-term induction of PDE isoforms,
posttranslational modifications may be involved in short-term changes
in PDE4 activity (1).
The characterization of the mRNAs derived from the PDE4D gene has led
to the discovery that considerable heterogeneity is present at the 5'
end of the different transcripts. On the basis of cDNA sequencing (2, 14), RNAse protection (15), or PCR analysis (16, 17), it has been
hypothesized that either alternate splicing or the presence of
different promoters controlling different transcriptional units are at
the origin of this heterogeneity. The PDE4D mRNAs that accumulate in
the immature Sertoli cell under basal or after FSH stimulation were
analyzed by PCR. Results indicate that two of the four PDE4D mRNA
species thus far described are expressed in these cells (16). These
have been termed PDE4D1 and PDE4D2. The PDE4D1 and PDE4D2 messages
differ only in the presence or absence of a short intron and therefore
must originate from the same start site and same promoter (16). The
other two known mRNAs derived from the PDE4D gene (PDE4D3 and PDE4D4)
are present at very low levels or could not be detected in these
immature Sertoli cells (17). In the thyroid cell line FRTL-5, PCR
analysis indicated that TSH stimulates the accumulation of PDE4D1 and
PDE4D2 but has minor effects on the levels of the PDE4D3 mRNA expressed
under basal conditions (17). These findings have prompted the
hypothesis that FSH or TSH is regulating the activity of only one of
the several promoters that control different transcriptional units in
the PDE4D gene. In the present study we have identified this promoter
and studied its properties including hormone and cAMP inducibility.
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RESULTS
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FSH Induces Accumulation of PDE4D1 and PDE4D2 but not PDE4D3 or
PDE4D4 mRNAs
Several different PDE4D mRNAs with diverging 5'-end have been
described (18). Previous PCR analysis suggested that PDE4D1 and PDE4D2
mRNAs are predominant in the Sertoli cell cultured in the presence of
FSH (17). To further define the identity of the FSH-inducible mRNAs
expressed in the Sertoli cells, RNA protection was performed with
probes corresponding to the known PDE4D mRNA variants (Fig. 1
). PolyA+-RNAs isolated from treated and
untreated immature Sertoli cells in primary cultures were hybridized
with 5'-terminus probes that are specific for the different variants
and with a probe that recognized all the variants in their 3'-end
common region (Fig. 1
). Adult testis (Fig. 1
) mRNA or FRTL-S cell mRNA
(data not shown) was used as positive control for the expression of
PDE4D3 and PDE4D4 variants. FSH treatment caused a more than 100-fold
increase in PDE4D mRNA as shown by the protected fragments using the
common probe (Fig. 1
). However, only PDE4D1 and PDE4D2 variants were
clearly induced by FSH (Fig. 1
). Even after overexposure of the gel, a
protected fragment corresponding to PDE4D3 and PDE4D4 could not be
detected by this assay. This finding is consistent with the observation
that hormonal treatment causes the appearance of a 67-kDa PDE protein
in extracts of the Sertoli cell. When expressed in a heterologous
system, the PDE4D2 mRNA is translated into a polypeptide with
immunochemical and biochemical properties identical to the native
FSH-induced 67-kDa protein (17). The other putative promoters present
in the PDE4D gene are not active under basal conditions and are not
activated by FSH stimulation. Ribonuclease protection assay (RPA)
analysis of the FRTL-5 cell mRNA after TSH stimulation confirmed the
induction of PDE4D1 and PDE4D2 in these cells (data not shown).

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Figure 1. RNAse Protection Analysis of the PDE4D Variants
Expressed in Control and FSH-Stimulated Sertoli Cell Culture
Primary Sertoli cell cultures from 15-day-old animals were stimulated
for 8 h either with 1 µg/ml ovine (o) FSH or vehicle. At the end
of stimulation PolyA+-RNA was extracted as detailed in
Materials and Methods. A, Schematic representation of
the different PDE4D variants. Arrows indicated the
probes used in the RPA experiment. B, Autoradiography of the
polyacrylamide gel electrophoresis of protected fragments. A
representative experiment of the three performed is reported. The
migration of the protected fragments for each variant expressed in
Sertoli cell is reported on the sides of the autoradiography.
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Structure of the Rat Genomic Clones including the Putative
Translation Start Site of the PDE4D1 mRNA
Several different genomic fragments corresponding to
the 5'-untranslated region of the PDE4D1 message have been isolated by
screening rat genomic libraries (16) (Fig. 2A
). Several
positive clones were characterized by restriction digestion and by
subcloning and sequencing. A rat genomic EcoRI fragment
containing the two first exons of the message was sequenced in its
entirety. The sequence containing 1540 bp upstream from the putative
initiation of translation is reported in Fig. 2B
. A search for TATA box
consensus in the 5'-untranslated region (UT) yielded negative results.
In contrast, three regions of high CG content (CG>95%) were
identified at -390, -450, and -690 upstream from the putative
initiation of translation. These regions resembled GC islands often
observed in promoters that lack a TATA box (19). In these promoters, an
element identified as Inr plays a crucial function in positioning
several components of the RNA polymerase II (20). A potential element
similar to the TdT-Inr (20, 21) was identified surrounding base -341
(TCACTCG).


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Figure 2. Structure of the PDE4D Gene
A, Schematic representation of the PDE4D gene exon intron structure and
of the promoter region controlling the expression of the PDE4D1 and
PDE4D2. The restriction map was determined by restriction digestion and
confirmed by sequence analysis. Coding regions of exons are represented
as filled bars and are numbered with arabic
numbers. The presence of additional upstream exons with
relative promoters is inferred by the RNA analysis, by RPA or PCR.
Introns are denominated with capital letters. The
downward arrow indicates the 5'-end of the longest cDNA
retrieved from a Sertoli cell library. B, Sequence of the rat genomic
region flanking the putative translation start site of the PDE4D1 PDE
variant. Bases are numbered from the putative translation start site.
ATG is marked by a downward arrow. Putative cap sites are
marked as arrows. Potential regulatory elements are
boxed. GC-rich regions are marked by arrows with
broken lines. The position of intron A spliced out in PDE4D2 is
marked by the arrow with continuous line.
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A search comparing the rat promoter with the sequence of known
cis-acting elements found in eukaryotic promoters indicated
the presence of several potential regulatory elements. Sequences
identical to SP1core were present in two regions of the sequence (Fig. 2B
). Sequences similar to inducible activator protein 2 (AP2) and
activator protein 1 (AP1) sites were also identified (Fig. 2B
). A cAMP
response element (CRE) consensus site (22) was observed around position
-83. Potentially interesting is the presence of sequences homologous
to NF-KB sites present in the promoter at position -180. It has been
reported that FSH regulates the expression of this transcription factor
(23). Furthermore, TNF treatment causes an increase in PDE4D mRNA in
thyroid cells (24).
Comparison of the sequence upstream from the putative initiation of
translation was highly conserved between mouse and rat genomic clones
(data not shown). One conspicuous difference between the mouse and rat
sequence is the insertion of a CA repeat in the mouse (data not shown).
Several attempts to determine whether this insert was present in some
of the rat alleles not represented in the library failed, suggesting
that this repeat may be present exclusively in the mouse gene.
Identification of the Transcription Start Sites
Previous attempts to identify the transcription start sites using
primer extension suggested the presence of several start sites (16).
This is a common finding in promoters that lack a TATA box and contain
GC-rich islands (19). The presence of the GC-rich region rendered
difficult the identification of the number and the exact location of
the initiation of transcription. In an attempt to circumvent this
problem, RNAse protection was used to map the transcription start
site/s using mRNA derived from FSH-stimulated Sertoli cells (Fig. 3
). All the probes used generated either one or two
protected fragments. The 5'-boundary of the shorter fragment
corresponds to nucleotide -340 ± 10 from the initiation of
translation. The sequence TGATTCAT in this region conforms with the
signature for a cap site and to an "Inr" sequence (19, 20).
However, the consistent finding of an additional protected fragment
indicates the presence of one or more additional cap sites upstream
from the one identified at -340. This is in agreement with previously
published primer extension data in which two or more extended products
were observed (16). The exact location of the upstream cap site could
not be identified because probes corresponding to region -304 to -700
did not produce a consistent pattern of protection. This may be
attributable to the high GC content of the region.

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Figure 3. RNAse Protection Analysis of the 5'-Region of the
PDE4D1/PDE4D2 mRNA Expressed in Sertoli Cells
Primary Sertoli cell cultures from 15-day-old animals were stimulated
with 1 µg/ml oFSH for 24 h. The stimulation was terminated by
aspiration of the medium and by adding cell lysis buffer. PolyA+-RNA
was extracted as detailed in the Materials and Methods.
A, Schematic representation of the genomic fragment upstream from the
putative translation start site of PDE4D1 and corresponding transcripts
were identified. Probes are depicted as wavy lines;
filled boxes at the end correspond to the sequence from
the vector included in the probe. In panels B and C, autoradiographies
of the polyacrylamide gel electrophoresis of protected fragments are
reported. Two representative experiments of the four performed are
reported. Average size of the protected fragments are reported on the
left of the autoradiography.
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As an alternative approach to identify the 5'-regions of the gene
included in the mRNA, PCR was used to amplify mRNA from stimulated
Sertoli cells. This analysis confirmed that at least a 300 bp sequence
upstream to the first cap site is present in the message expressed in
the Sertoli cell (data not shown). Again, a consistent pattern of
amplification of the region encompassing the GC region could not be
obtained.
Functional Characterization of the PDE4D1 Promoter
To determine whether the genomic region identified by RNAse
protection can indeed function as a promoter, the EcoRI
fragment identified in Fig. 2B
was subcloned upstream of the coding
region of the luciferase cDNA. The promoter activity was studied by
transfection and by measuring the luciferase activity in primary
Sertoli cell cultures in view of the observed large stimulation of
transcription for this gene. Owing to the fact that the transfection
efficiency of the primary Sertoli cell culture is low, the MA-10 Leydig
tumor cell line was also used to confirm the activity obtained in a
more efficient transfection system. A 1.6-kb fragment upstream from the
putative translation start site of PDE4D1 induced luciferase expression
in both transfection systems. A 30-fold increase in luciferase
production was obtained there as compared with a promoterless construct
(Table 1
). The activity was approximately one third of
the activity of a promoter of a different PDE4 gene, PDE4B2, which is
also expressed in the Sertoli cell (Table 1
). This latter promoter
contains a TATA box and, therefore, is expected to direct transcription
more efficiently. The activity of the PDE4D1 promoter was about one
sixth of that obtained when a strong SV40 promoter was used (Table 1
).
Similar results were obtained in MA-10 cells even if the transfection
efficiency was higher in this latter system (data not shown). Treatment
of the transfected cells with (Bu)2cAMP enhanced the
promoter activity. This stimulation is time-dependent, and it is
maximal after 12 h of treatment (data not shown). After this
stimulation, the promoter activity in the presence of
(Bu)2cAMP was approximately one third of that of the strong
SV40 promoter. In all the following experiments, stimulation was
determined after 12 h of incubation.
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Table 1. Comparison of the Basal Promoter Activity of
the 5'-Flanking Region of PDE4D1 with Other Promoters Active in Primary
Sertoli Cell Culture
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Analysis of the Basal and Stimulated Promoter Activity
In view of the presence of more than one cap site, different
constructs containing the proximal and distal cap sites were used to
determine basal and stimulated promoter activity (Fig. 4
). An increase in luciferase expression was observed
when the proximal cap site was present in the construct. Inclusion of
the region where the upstream cap sites are present produced a further
increase in basal transcription activity (Fig. 4
). Conversely,
constructs in which the proximal cap site was deleted (1.2 and 0.9
PDE4D1/2) had consistently less activity than constructs including this
region (1.4 and 1.1 PDE4D1/2) (Fig. 4
). This observation is consistent
with the RPA data. Inclusion of most of the 5'-UT produces a further
increase in basal transcription activity, a statistically significant
increase that suggests the presence of basal transcription enhancer in
the 5'-UT of exon 1 of this region. Similar results were obtained after
transfection in MA-10 cells (Fig. 4
). From these experiments it was
concluded that this region of genomic DNA has basal promoter activity.
The pattern of promoter activity was also consistent with the
hypothesis that multiple cap sites contribute to the overall
transcription.

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Figure 4. Basal Transcription Activity of the 5'-Flanking
Region of PDE4D1 in Sertoli Cells and MA-10 Cells: A Deletion Analysis
A, Schematic representation of the 5'-flanking region of PDE4D1
variant. Bases are numbered from the putative translation start site.
ATG is marked in bold. Putative cap sites are marked as
arrows. Potential regulatory elements are
boxed. In panel B a representation of the PDE-luc
plasmids containing different 5'- and 3'-deletions of the -1540/+ 2 bp
genomic region is reported. C, Transient luciferase expression in
Sertoli and MA-10 cells transfected with the PDE-luc plasmids described
in panel B. Luciferase activity is expressed as RLU corrected by
ß-gal activity. Data are the mean ± SE from the
number of experiments indicated in parentheses
(individual points determined in duplicate).
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The Sertoli cell has demonstrated that the PDE4D1 mRNA accumulates
after stimulation of the cells with FSH. Thus, if the promoter is
correctly identified, its activity should be regulated by cAMP and FSH.
To test this hypothesis, the cAMP inducibility of this promoter was
studied by incubating Sertoli cells transfected with the different
constructs in the presence of 1 mM (Bu)2cAMP
(Fig. 5
). Constructs containing the 5'-UT and the
proximal cap site up to base -381 were not inducible by
(Bu)2cAMP. Conversely, the transcription activity of all
the constructs that included a region from -380 to -1218 was
stimulated by (Bu)2cAMP. Maximal stimulation varied between
3- and 6-fold over basal activity (Fig. 5
). Inclusion of the 5'-UT
region that contains a putative CRE consensus site produced on average
the highest stimulation; however, this was not statistically different
from the stimulation observed with a construct lacking this region.
Similar results were obtained when FSH was used to measure cAMP levels
in the cell (data not shown).

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Figure 5. Cyclic AMP Inducibility of the PDE4D1 Promoter: A
Deletion Analysis
A, Schematic representation of the 5'-flanking region of PDE4D1
variant. Bases are numbered from the putative translation start site.
ATG are marked in bold. Putative cap sites are marked as
arrows. Potential regulatory elements are
boxed. In panel B a representation of the PDE-luc
plasmids containing different 5'- and 3'- deletions of the -1540/+ 2
bp genomic region is presented. C, Cells were transfected with the
plasmids indicated in panel B. After the glycerol shock, cells were
incubated either with 1 mM (Bu)2cAMP or vehicle
for 12 h. The luciferase activity fold stimulation is calculated
as ratio of the RLU measured in treated cells vs.
untreated cells. Data are the mean ± SE of the number
of experiments indicated in parentheses (individual
points determined in duplicate).
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Previous studies on the mRNA indicated that FSH stimulated PDE4D mRNA
accumulation in a concentration-dependent fashion. To test whether the
proximal promoter identified was equally sensitive to FSH, cells
transfected with the 1.4 PDE4D1/2 construct were incubated with
increasing FSH concentrations, and the luciferase activity was measured
(Fig. 6
). FSH stimulated the transcription approximately
4-fold with an EC50 of 1 ng/ml. This concentration was in
the same range observed for the induction of the message (3).

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Figure 6. FSH Stimulation of the PDE4D1/2 Promoter
Sertoli cell cultures were transfected by the calcium phosphate method
with 1.5 µg/ml 1.5PDE4D1/2. Cells were incubated with the indicated
concentration of oFSH or vehicle. Luciferase activity is expressed as
RLU. Data are the mean ± SE of three experiments.
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To confirm the cAMP-dependent regulation of the promoter, different
pharmacological treatments were used that increase cAMP levels in the
Sertoli cell (Fig. 7
). The activity of the 1.5-kb
promoter was stimulated by forskolin, cholera toxin, and 8-bromo-cAMP,
but not by 8-bromo-cGMP. On the contrary,
12-O-tetradodecanoyl-phorbol-13-acetate (TPA), an activator
of protein kinase C, marginally stimulated the activity of this
promoter but potentiated the effect of forskolin and
(Bu)2cAMP.

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Figure 7. Effects of Different Stimuli on the PDE4D1/2
Promoter
Sertoli cell cultures were transfected by the calcium phosphate method
with 1.5 µg/ml 1.5PDE4D1/2. After the glycerol shock, cells were
incubated with the indicated regulators at the following final
concentrations: 1 mM (Bu)2cAMP; 100
µM forskolin; 30 ng/ml cholera toxin; 100 nM
TPA; 1 mM 8-bromo-cAMP; 1 mM 8-bromo-cGMP. The
luciferase activity fold stimulation is calculated as ratio of the RLU
measured in treated cells vs. untreated cells. Data are
the mean ± SE of three experiments.
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DISCUSSION
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The studies described have identified a cAMP- and FSH-inducible
promoter in the PDE4D gene. This intronic promoter controls the
expression of one of the several transcriptional units present in this
gene. Transcription of this unit generates mRNAs that encode a PDE
protein variant of 68- to 72-kDa mass. The identification of this
cAMP-inducible promoter conclusively demonstrates that a feedback loop
controlling cAMP levels is operating in the cell. Thus, cAMP regulates
the activity of this PDE4D1/D2 promoter controlling the expression of a
PDE which, in turn, degrades and inactivates cAMP. This homeostatic
mechanism is important during cell adaptation and desensitization (18).
Similar regulations of PDE4 mRNAs by cAMP or agonists that
regulate cAMP have been demonstrated in glioma cells (2), U937 monocyte
cell line (25), MonoMac 10 cell line (11), myoblasts (8), and FRTL-5
cells (7).
In spite of the several different approaches used, for technical
reasons it was impossible to conclusively identify the cap sites used
in the PDE4D1/D2 promoter. However, the data thus far collected
indicate that multiple cap sites may be used. This is consistent with
the absence of a TATA box and the presence of GC-rich islands in this
region of the sequence. One cap site was identified at -340 from the
putative initiation of translation, while one or more may be present
upstream of base -593. That sequences up to -340 bp are included in
some mRNAs is also supported by the PCR analysis. An alternative
explanation of the RPA analysis would be that the -340 bp is an
exon/intron boundary and that an additional noncoding exon may be
splicing at this point. The possibility that this region corresponds to
an intron/exon boundary cannot be formally excluded. However, the
boundary mapped by RPA does not contain any splicing signature but has
sequence corresponding to a cap site. In addition, the sequence
upstream of -340 is highly conserved between rat and mouse. This is a
further indication that this is not an intronic sequence but a
functionally relevant sequence. Furthermore, some of the mRNAs
expressed in the Sertoli cell contain some sequence upstream from this
site. Cumulatively, these findings are more in line with the presence
of multiple cap sites, the predominant one located at -340. Finally,
the region upstream from -340 has clear basal promoter activity when
assayed with the luciferase reporter (30-fold increase in luciferase
production over a promoterless construct).
The demonstration that an inducible promoter activity can be detected
with the region upstream from the sequence of PDE4D1 conclusively
defines one of the possible transcriptional units present in this gene.
The fact that additional 5'-sequences are present in other mRNAs
implies that exons upstream of the promoter identified here are present
in the gene. Preliminary characterization of additional genomic clones
that we have isolated confirm this hypothesis (S. L. Jin and M. Conti,
manuscript in preparation). According to these findings, the promoter
directing the PDE4D1/PDE4D2 mRNA transcription is located in an intron.
This genomic organization is reminiscent of the structure of the
Drosophila dunce gene (26), which is the ancestor of the PD4
genes. Deletion mutations of the 5'-region of the dunce gene have shown
that different promoters contribute to the expression in the central
nervous system and the reproductive tract of the fly (27). It should be
noted that the intronic promoter identified here has not been described
in the fly.
Previous runoff experiments indicated that FSH stimulates the
transcription of the PDE4D gene approximately 10-fold (3). The
transfection experiments with the luciferase reported here show a clear
stimulation of transcription of these constructs by both FSH and cAMP
analogs (5- to 6-fold stimulation). This stimulation is not as dramatic
as what is observed with the accumulation of the PDE4D mRNA. These
differences may depend on the fact that 1) elements inhibitory of basal
transcription are present in this promoter upstream of -1500 and; 2)
that message stabilization plays an important role in the control of
the steady state levels of PDE4D1 mRNA. Regardless of the quantitative
aspects of the stimulation, these data demonstrate that FSH stimulates
the transcription of this gene from this promoter with a consequent
increase in PDE4D1/PDE4D2 mRNA. We have shown that only the PDE4D2
protein accumulates in Sertoli cells after FSH stimulation. At present
it is not clear why PDE4D1 mRNA is not translated. It is possible that
the intron retained in PDE4D1 inhibits the translocation of the message
from the nucleus to the cytoplasm, thus preventing or delaying
translation. According to this view, only messages with the intron
removed are translated into a protein. That unspliced intron sequences
serve to control the rate of translation has been shown for several
mRNAs (28, 29).
At present it is not clear which promoter elements in this promoter are
responsible for the FSH stimulation of transcription. The promoters of
several genes induced by FSH have been previously characterized. Among
these are the promoter for aromatase (30, 31), RII (32), inhibin-
(33, 34), prodynorphin (35), and urokinase (36). Only in the
inhibin-
and the aromatase promoters have CRE elements been clearly
implicated in the FSH activation. The promoter that we have described
here contains the sequence CGACTCA complementary to TGAGTCG where only
the last base is different from a consensus CRE. This sequence is,
however, located in the 5'-UT region that we have shown by PCR and RPA
to be included in the mRNA. Deletion experiments indicate that this
element is not necessary for the FSH induction, but that constructs
containing this 5'-UT region produce the highest stimulation. The exact
role of this 5'-UT region needs to be further investigated. It is
likely that several different enhancer elements contribute to this
activation of transcription by FSH. Several putative AP-2 consensus
sites (37) are present at -378, -770, -788, and -1208, and these
may be involved in the FSH activation. The synergistic effect of TPA
and (Bu)2cAMP on this promoter is consistent with this AP-2
involvement in the activation. Interestingly, the region
-530CGGGAGGGGCGGT-518 upstream from the first
cap site has considerable homologies with an element involved in cAMP
stimulation in the human urokinase gene (36). This promoter is
activated by FSH when transfected in mouse Sertoli cells. This region
is also similar to other GC-rich elements that are cAMP-inducible (38, 39). Further experiments are necessary to determine the role of this
region of the PDE4D1 promoter in hormone activation.
In summary, our findings demonstrate the presence of an intronic
promoter in the PDE4D gene that directs the expression of a truncated
PDE form (17). This truncated PDE is a component of a feedback loop
present in the cell and is involved in the termination of the hormone
stimulus or desensitization (18). The organization of this gene is
strikingly similar to that of other genes involved in the
cAMP-dependent pathway. For instance, the CRE modulator (CREM), a
transcription factor that mediates the cAMP regulation of gene
expression, contains several promoters. A cAMP-regulated intronic
promoter in this CREM gene directs the expression of a truncated
protein, inducible cAMP early repressor (ICER), that functions as
transcriptional suppressor (40). Therefore, both in the PDE4D and in
the CREM gene, a cAMP-regulated intronic promoter is involved in the
termination/modulation of the cAMP signal. It remains to be determined
whether the PDE promoter characterized is the only inducible promoter
present in this gene and whether additional signal transduction
pathways regulate these promoters.
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MATERIALS AND METHODS
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Materials
Restriction enzymes were purchased from Boehringer Mannheim
(Indianapolis, IN). Plasmid preparation kits were from Qiagen
(Chatsworth, CA). The GeneAmp PCR reagent kit and Taq
polymerase were from Perkin-Elmer Cetus Instruments (Norwalk, CT).
pGL2-Control vector was from Promega (Madison, WI); pCMV-ßgal vector
was a gift from Dr. T. Hsu (Stanford University, Stanford, CA). Tissue
culture supplies including Earls MEM, Hanks buffer, trypsin,
gentamycin, penicillin, and streptomycin were from GIBCO (Grand Island,
NY); collagenase was from Worthington (Freehold, NJ).
[
-32P]UTP (3000Ci/mmol) was manufactured by NEN
Research Products (Boston, MA). All other chemicals used were
analytical grade and purchased from Sigma (St. Louis, MO) or Bio-Rad
(Richmond, CA).
Plasmid Constructions
pGL2-Basic, a promoterless luciferase vector (Promega) was used
to test the various sequences of the ratPDE3/IVD gene for promoter
activity. The different length fragments of the putative gene promoter
were isolated from a genomic clone (
DASH II-ratPDE 3.2) containing
the 5'-flanking region previously characterized (16). To subclone the
genomic fragments into the pGL2-Basic vector, two different strategies
were used. In some cases the fragment was first subcloned into pB KS II
vector (Stratagene, La Jolla, CA), and excised using restriction sites
compatible with pGl2-Basic polylinker, and the insert was transferred
to this latter vector. This strategy was used to obtain 1.2PDE3-luc
(-1540/-354) and 0.9PDE3-luc (-1218/-354) constructs, where the
coordinates are specified relative to the first AUG (+1). In other
cases, the DNA fragments were obtained by PCR using the
DASH
II-ratPDE 3.2 genomic clone as a template and synthetic
oligonucleotides containing the appropriate restriction sites.
1.5PDE3-luc (-1540/+ 2), 1.4PDE3-luc (-1540/-121), 1.1PDE3-luc
(-1218/-121), 0.3PDE3-luc (-381/-121), and 0.2PDE3-luc
(-299/-121) constructs were achieved with this cloning strategy.
To perform the RPA, four different genomic fragments were subcloned in
pB KS II vector. The sequences were amplified by PCR from the l DASH
II-ratPDE 3.2 genomic clone and subcloned in the SmaI site
of the vector. The sequences of the primers used and the corresponding
constructs are listed below: oligonucleotide A
(5'-GAGCCGGGGTCTGCGGGACG-3') and oligonucleotide B
(5'-CGCACATGAGGGCTGCTCCTTCATATTGCAGAGC-3') for pBS KS II-A (-299/+24)
construct; oligonucleotide C (5'-GACTTGAGCGACAAAACAGGAAA-3') and
oligonucleotide A for the pBS KS II-B (-381/+24) construct;
oligonucleotide D (5'-TCCCGGCTGCGCTTCAAAGCAGTGG-3') and oligonucleotide
E (5'-TTTCCTGTTTTGTCGCTCAAGTC-3') for pBS KS II-C (-593/-359)
construct; oligonucleotide F (5'-GACTTGAGCGACAAAACAGGAAA-3') and
oligonucleotide G (5'-GCAAGGCCAACTTTGGCACG-3') for pBS KS II-D
(-381/-121) construct.
Ribonuclase Protection Assay
Run-off transcripts were synthesized from each linearized
template using a Transcription in vitro System Kit (Promega)
and either T3 or T7 polymerase. The full-length single-stranded RNA
probes were purified by acrylamide gel electrophoresis. Poly (A+) RNA
was purified from Sertoli cell culture treated for 24 h with 1
mM (Bu)2cAMP, using a Quick Prep mRNA
Purification Kit (Pharmacia) according to the supplied protocol. The
RPA (41) was performed with RPA II Kit (Ambion, Austin, TX) using 5
µg extracted mRNA and 1.52 105 cpm of labeled probe for
each reaction. Nuclease-resistant probes were visualized by gel
electrophoresis \[5% acrylamide, 8 M urea and 90
mM Tris Borate, 2 mM EDTA (TBE)\] and
autoradiography.
Sertoli Cell Culture and Transfection
Sertoli cell cultures were prepared from 15 day-old
Sprague-Dawley rats following a procedure previously reported (42), and
cells were seeded on 90-mm plastic tissue culture dishes in a
serum-free Eagles MEM supplemented with glutamine, nonessential amino
acids, pyruvic acid, gentamycin, streptomycin, and penicillin.
Incubation was carried out at 32 C in a controlled atmosphere of 95%
air-5% CO2, and cultures were used for transfection
experiments 3 days later. Cells were cotransfected with the
CaPO4-DNA coprecipitate technique using 15 µg reporter
construct per plate (1.5 µg/ml) and 3 µg pCMV-ßgal (0.3 µg/ml)
to allow normalization to ß-galactosidase expression. After 4 h
the medium was aspirated and the cultures were subjected for 2 min to
10% glycerol hyperosmotic shock and fed with fresh medium containing
or lacking 1 mM (Bu)2cAMP. After 12 h,
cells were harvested and lysates assayed for luciferase and
ß-galactosidase activity as described below.
Assay of Luciferase and ß-Galactosidase Activity
Individual dishes were washed twice with PBS then scraped in 1x
Reporter Lysis Buffer (Promega). The cell lysates were centrifuged
(16,000 x g, 2 min) at 4 C, and the supernatants were
assayed. Luciferase activity (43) was performed in duplicate, mixing 20
µl cell extract with 100 µl Luciferase Assay Reagent (Promega). The
produced light was measured in an Auto Climat Lumat LB 952 T/16
luminometer (Berthold, Nashua, NH) and expressed as relative light
units (RLU). ß-Galactosidase assay was performed in duplicate, by
adding to the cell extracts an equal volume of Assay 2x Buffer
(Promega). The samples were incubated at 37 C until a yellow color
developed. In each assay a standard curve with different amounts of
purified-galactosidase enzyme (Promega) was performed. After the
incubation the absorbance of the samples was read at 420 nm in a
spectrophotometer (Beckman, Fullerton, CA). ß-Galactosidase
milliunits in each sample were calculated using the standard curve.
Luciferase activity (RLU) was normalized relative to ß-galactosidase
activity (milliunits) to correct for differences in transfection
efficiency.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Drs. Catherine Jin and Cristiana Caniglia for
the help in cloning and characterization of the mouse PDE4D promoter
and Caren Spencer for the editorial review of the manuscript.
These studies were supported by NIH Grant HD-20788 from the National
Institute of Child Health and Human Development. E.V. was supported in
part by grants from the Italian National Research Council (CNR)
targeted projects "Clinical Applications and Oncological Research"
(ACRO Contract 051601087) and from the "Istituto Superiore della
Sanita" (930628).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Marco Conti, M.D., Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, 300 Pasteur Drive, Room A344, Stanford, California 94305-5317.
Received for publication July 15, 1996.
Accepted for publication March 13, 1997.
 |
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