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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 1Go). Adult testis (Fig. 1Go) 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. 1Go). However, only PDE4D1 and PDE4D2 variants were clearly induced by FSH (Fig. 1Go). 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.

 
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. 2AGo). 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. 2BGo. 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.

 
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. 2BGo). Sequences similar to inducible activator protein 2 (AP2) and activator protein 1 (AP1) sites were also identified (Fig. 2BGo). 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. 3Go). 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.

 
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. 2BGo 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 1Go). 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 1Go). 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 1Go). 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

 
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. 4Go). 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. 4Go). 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. 4Go). 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. 4Go). 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).

 
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. 5Go). 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. 5Go). 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).

 
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. 6Go). 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.

 
To confirm the cAMP-dependent regulation of the promoter, different pharmacological treatments were used that increase cAMP levels in the Sertoli cell (Fig. 7Go). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{alpha} (33, 34), prodynorphin (35), and urokinase (36). Only in the inhibin-{alpha} 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Earl’s MEM, Hank’s buffer, trypsin, gentamycin, penicillin, and streptomycin were from GIBCO (Grand Island, NY); collagenase was from Worthington (Freehold, NJ). [{alpha}-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 ({lambda} 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 {lambda} 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 1.5–2 105 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 Eagle’s 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" (9306–28).


    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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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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