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Properties and hormonal regulation of two structurally related cAMP phosphodiesterases from the rat Sertoli cell.

Properties and hormonal regulation of two structurally related cAMP phosphodiesterases from the... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 27, Issue of September 18370-18377.1991 25, PP. 0 1991 by The American Society for Biochemistry and Molecular Biologv, Inc. Printed in U. 5’. A. Properties and Hormonal Regulation of Two Structurally Related cAMP Phosphodiesterases from the Rat Sertoli Cell* (Received for publication, January 2, 1991) Johannes V. Swinnen, Kallen E. Tsikalas, and Marco Conti$ From the Laboratories for Reproductive Biology, Departments of Pediatrics and Physiology, University of North Carolina, .. . Chapel Hill, North Carolina 27599 Upon exposure to follicle-stimulating hormone The responsiveness of the target cell to hormones is contin- (FSH), the gonadotropin-responsive Sertoli cell ex- uously adjusted in synchrony with changes in the extracellular presses increased rolipram-sensitive CAMP-specific environment. The mechanisms involved in this adaptation phosphodiesterase (CAMP-PDE) activity. To under- are complex and involve changes in the components of the stand the mechanisms leading to this activation, the signal transduction machinery at the plasma membrane level CAMP-PDEs present in the Sertoli cell were character- as well as changes in the enzymes involved in intracellular ized and their regulation studied. Comparison of the signalling. Phosphodiesterases (PDEs),’ the enzymes that conceptual translates of two groups of PDE cDNA degrade cyclic nucleotides might play an important role in clones isolated from a Sertoli cell cDNA library these processes. In most cells, including the Sertoli cell (l), (ratPDE3 and ratPDE4) showed that the encoded pro- degradation of cyclic nucleotides is carried out by multiple teins were structurally similar, containing a core re- PDEs with different properties in terms of affinity for cAMP gion of 455 amino acids with a sequence identity of and cGMP, regulation of hydrolytic activity, and sensitivity 87%. The amino and carboxyl termini were divergent. to inhibitors (2). Although several types of PDEs are able to Expression of these cDNAs in Escherichia coli and hydrolyze CAMP, it is now recognized that two families of monkey COS-7 cells demonstrated that the encoded PDEs have a high affinity for this nucleotide. One of these CAMP-PDEs had similar affinities for the cAMP sub- forms, referred to as cGMP-inhibited PDE is sensitive to the strate and were equally sensitive to a number of PDE selective inhibitor cilostamide and the cardiotonic milrinone inhibitors (rolipram > Ro 20-1724 > cilostamide). FSH (2). In several cell types, this enzyme is membrane bound and stimulation of the Sertoli cell produced an increased rapidly activated by insulin and P-adrenergic agonists (3). rate of transcription of the ratPDE3 gene and elevated The other type, termed CAMP-specific PDE (CAMP-PDE) is mRNA levels for ratPDE3 and to a lesser extent of insensitive to cGMP and is selectively inhibited by the anti- ratPDE4. The increase in mRNA levels was detected depressant rolipram (2). Also this latter form of PDE has after 1 h of stimulation. Forskolin, cholera toxin, and been shown to be regulated by hormones. In the Sertoli cell, M,02’-dibutyryl cAMP produced a similar increase in FSH via an increase in cAMP causes the activation of a PDE rate of transcription and elevated mRNA levels, indi- with the properties of a CAMP-PDE (1,4). Similar hormone- cating that this activation is mediated by an increase dependent regulation of cAMP hydrolyzing activity has also in intracellular CAMP. RatPDE4 mRNA levels were been demonstrated in glioma and astrocytoma cell lines (5- 10 ng of FSH/ml, whereas maximal upon exposure to 8), in fibroblasts (9, lo), granulosa (11), and lymphoma cells ratPDE3 mRNA levels could be further elevated with (12). The CAMP-PDE which in the rat Sertoli cell is stimu- higher FSH concentrations. The intensity of an im- lated by FSH, is thought to be involved in the refractoriness munoreactive band with characteristics identical to a that follows hormone stimulation (13-15). It is now clear that purified CAMP-PDE, correlated with the increased the family of the CAMP-PDEs is composed of multiple related cAMP hydrolytic activity after FSH or dibutyryl forms. Molecular cloning data (16-18) have demonstrated cAMP treatment, demonstrating that changes in that in the rat there are at least four genes encoding cAMP CAMP-PDE protein levels are involved in this regula- phosphodiesterases which have been highly conserved tion. These data provide evidence that multiple CAMP- PDE forms are expressed in the rat Sertoli cell. Al- through evolution (16, 17). It has been shown that the steady state mRNA level for one of these PDEs (ratPDE3) is regu- though differences in the pattern of activation of these lated by hormones and cAMP (19). Here we have studied in forms were observed, these data show, that in the rat Sertoli cell, the CAMP-PDE activity is regulated by detail the mechanisms involved in this regulation. In addition, hormones via a novel mechanism that involves a the finding that another CAMP-PDE gene (ratPDE4) is ex- CAMP-dependent activation of transcription of a PDE pressed in the Sertoli cell prompted us to study the properties gene. and the regulation of this latter PDE in response to FSH. Here we report that the expression of both these structurally * This work was supported by National Institutes of Health Grant related CAMP-PDEs is under the control of hormones. We HD20788. The costs of publication of this article were defrayed in show that differences in expression occur at least in part at part by the payment of page charges. This article must therefore be the level of gene transcription and that an increase in PDE hereby marked “advertisement” in accordance with 18 U.S.C. Section activity follows changes in PDE protein concentration. 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted The abbreviations used are: PDE, cyclic nucleotide phosphodies- to the GenBankTM/EMBL Data Bank with accession number($ terase; CAMP-PDE, CAMP-specific phosphodiesterase; SDS, sodium MZ35349-M25350. dodecyl sulfate; Bt2cAMP, N‘,OO”-dibutyryl CAMP; FSH, follicle- $ To whom correspondence should be addressed Laboratories for stimulating hormone; EGTA, [ethylenebis(oxyethylenenitrilo)]tetra- Reproductive Biology, CB 7500 MacNider 202H, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-6795. acetic acid; PBS, phosphate-buffered saline. This is an Open Access article under the CC BY license. Hormonal Regulation of CAMP Phosphodiesterases 18371 cells were rinsed with PBS and lysed for RNA extraction, or collected EXPERIMENTAL PROCEDURES with a rubber policeman and homogenized in an all-glass Dounce homogenizer in a buffer containing 20 mM Tris.Cl (pH 8.01, 10 mM Materials EDTA, 0.2 mM EGTA, 50 mM benzamidine, 0.5 pg/ml leupeptin, 0.7 Crotalus atrox snake venon and lysozyme were purchased from pg/ml pepstatin, and 2 mM phenylmethylsulfonyl fluoride (homoge- Sigma; Pansorbin cells from Behring Diagnostics; Ro 20-1724 (4-(3- nization buffer). After centrifugation for 15 min at 14,000 X g, the butoxy-4-methoxybenzyl)-2-imidazolidinone) was from Hoffmann- supernatant and the pellet fractions were subjected to Western blot LaRoche; rolipram (4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyr- analysis, immunoprecipitation, or PDE assay. rolidone) was from Berlex, NJ; cilostamide (N-cyclohexy-4-[(1,2- dihydro-2-oxo-6-quinolyl)oxy]-N-methylbutyramide) was provided Sertoli Cell Cultures by Dr. H. Hidaka, Nagoya University School of Medicine, Showa- Ku, Nagoya 466 Japan; AG 1-X8 resin was obtained from Bio-Rad Sertoli cell cultures from 15- to 17-day-old Sprague-Dawley rats [2,8-,”H]cAMP (20-50 Ci/mmol), ‘*‘I-labeled protein A (2-10 pCi/pg), were prepared as described (26) and maintained in medium without [&”P]CTP (3000 Ci/mmol), and [a-”PIUTP (800 Ci/mmol) were serum for 4 days. On the fourth day of culture the cells were rinsed purchased from Du Pont-New England Nuclear; guanidium thiocya- with Hank’s balanced salt solution, the medium was replaced and nate from Fluka; CsCl from Gallard and Schlesinger; immobilon from FSH (NIDDK ov-FSH S16), N6,O””dibutyryl CAMP, forskolin, chol- Millipore; Biotrans from ICN; a random primed DNA labeling kit era toxin, or vehicle were added to the medium. At the indicated time was purchased from Boehringer Mannheim; p2523 columns from PBS and lysed for after treatment, the cells were rinsed twice with 5prime-3prime; and dextran sulfate M, 500,000 was from Pharmacia. RNA extraction. For immunoprecipitation, Western blot analysis, or PDE assay, the cells were scraped in homogenization buffer with a Sequence Analysis rubber policeman and homogenized in an all-glass Dounce homoge- nizer. Cellular fractions were prepared by centrifugation at 14,000 X The cloning of PDEs from a rat Sertoli cell cDNA library has been g for 15 min. described (16). The complete ratPDE3 DNA sequence (19) and also a partial cDNA sequence of two ratPDE4 clones have been published PDE Assay (16). These clones have been completely sequenced and also two additional ratPDE4 clones (ratPDE4.3 and ratPDE4.4) have been PDE assays were performed basically as described (27) and as partially characterized. The sequences were aligned using the detailed previously (19). Cell extracts in homogenization buffer were MicroGenie software package (Beckmann). adjusted to 40 mM Tris.Cl (pH 8.0), 10 mM MgC12, 1.25 mM 2- mercaptoethanol, and 1 p~ [“HICAMP (0.1 pCi/reaction). After in- Prokaryotic Expression cubation at 34 “C for 3-10 min the reactions were terminated by 1 volume of a 40 mM Tris. C1 (pH 7.5) solution containing addition of To express ratPDE3 and ratPDE4 cDNAs in bacteria, the prokar- 10 mM EDTA and heat denaturation. To each reaction tube 50 pg of R. Crowl) yotic expression vector pRC23 (20) (kindly provided by Dr. C. atrox snake venom was added. After incubation at 34 “C for 20 was used. This vector contains the h phage PL promoter followed by min the reaction products were separated by anion exchange chro- a ribosome-binding site and an EcoRI cloning site. The distance matography on AG 1-X8 resin and the amount of released radiola- between the ribosome-binding site and the AUG codon is critical for beled adenosine was quantitated by scintillation counting. PDE ac- efficient translation (21). Because the EcoRI site in cDNA clone tivity determinations were performed in triplicate at three different ratPDE3.1 precedes the putative initiation AUG codon by 150 bases time intervals. For the inhibitor studies, cell extracts were incubated (19), a new EcoRI site was created 3 bases 5’ of the AUG by the in the presence of lo-” to lo-’ M Ro 20-1724, rolipram, or cilostamide polymerase chain reaction. cDNA clone ratPDE4.2 starts with an which were dissolved in dimethyl sulfoxide. Control tubes contained GcoRI site 5 bases upstream of the putative AUG and was ligated equal amounts (2.5%) of dimethyl sulfoxide. For the determination into the EcoRI site of pRC23 without further manipulation. Esche- of the K, of the enzyme (28) cell extracts were incubated with 0.1 richia coli DH5a bacteria were transformed (22) with the pRC23 pCi of [“HICAMP and 0.1-50 p~ unlabeled cAMP for 2, 4, 6, and 8 compatible plasmid pRK248cIts (provided by Dr. R. Crowl) which min. The PDE activity is expressed as the rate of cAMP hydrolysis/ encodes a temperature-sensitive hcIAt2 repressor (23) and confers min. tetracyclin resistance to the cells. Tetracyclin resistant pRK248cIts containing DH5a bacteria were transformed with the pRC23-rat- RNA Extraction and Northern Blot Analysis PDE3 and pRC23-ratPDE4 constructs and grown in the presence of 30 “C. Expression of ampicillin (the selectable marker of pRC23) at Total RNA was prepared as described (29). Sertoli or COS-7 cells the ratPDEs by shifting the temperature from 30 to 42 “C for 3 h was were lysed in a buffer containing 50% (w/v) guanidine thiocyanate, monitored by polyacrylamide gel electrophoresis followed by Coo- 16.7 mM Na citrate, 0.5% (w/v) Na lauryl sarcosine, 80 mM 2- massie Brilliant Blue staining or Western blot analysis (with Klll mercaptoethanol, and 0.1% (v/v) Sigma antifoam A. This cell lysate antiserum, see below) and PDE assays. For PDE assay, the bacteria a solution containing 5.7 M cesium chloride and was layered on top of were pelleted, resuspended in 20 mM Tris and 1 mM EDTA, and lysed 167 mM Na acetate (pH 5.0) (30). The RNA was pelleted by centrif- with 5 mg/ml of lysozyme. The solution was adjusted to 0.2 mM ugation for 16 h at 100,000 X g. After centrifugation the RNA pellet EGTA, 50 mM benzamidine, 0.5 pg/ml leupeptin, 0.7 pg/ml pepstatin, was redissolved in ddH,O and ethanol precipitated. Poly(A)’ RNA and 2 mM phenylmethylsulfonyl fluoride. was prepared by oligo(dT)-cellulose chromatography as described (31). Five micrograms of poly(A)+ RNA or 10-20 pg of total RNA Eukaryotic Expression was denatured by incubation at 50 “C for 1 h in a buffer containing The pCMV5 expression in vector (24) drives the expression of 50% (v/v) dimethyl sulfoxide, 20% (v/v) glyoxal, and 20 mM sodium cDNAs cloned into the multiple cloning site in between the human phosphate (pH 6.8) (31). After separation on a 1% agarose gel in 10 cytomegalovirus major immediate early gene promoter and enhancers mM sodium phosphate (pH 6.8), the gel was blotted onto a Biotrans and the transcription termination and polyadenylation signals of the nylon membrane (32) and baked at 80 “C for 1 h. Blots were prehy- human growth hormone. The ratPDE3.1 and ratPDE4.1 cDNAs were bridized in 50% v/v) formamide, 50 mM sodium phosphate (pH 6.5), inserted into the EcoRI site of the plasmid. After transformation, 5 X SSC (20 X SSC, 3 M NaCl and 0.3 M Na:, citrate), 5 X Denhardt’s ampicillin-resistant DH5a bacteria were tested for the presence and (100 X Denhardt’s, 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrroli- the orientation of the ratPDE cDNAs. Plasmids were prepared from done, 2% (w/v) bovine serum albumin, fraction V), 250 pg/ml of 1 liter of bacterial cultures using p2523 columns according to the boiled sonicated salmon sperm DNA, 0.5% (w/v) SDS, and 1% (w/v) protocol of the manufacturer. Monkey kidney cells (COS-7) were glycine at 42 “C for 4 h. RatPDE3, ratPDE4, and actin cDNA clones grown in high glucose Dulbecco’s modified Eagle’s medium containing were radiolabeled (33) using a random primed labeling kit and follow- 5% (v/v) fetal calf serum. When the cells reached a density of ing the manufacturers’ instructions. The blots were consecutively approximately 40% of confluency, the medium was aspirated and incubated at 42 “C for 16-20 h with radiolabeled probe at 1-2 X lo6 medium without serum was added. The cells were transfected using cpm/ml of hybridization buffer (50% (v/v) formamide, 20 mM sodium the DEAE-dextran method (25) with 10 pg of plasmid DNA. Four phosphate (pH 6.5), 5 X SSC, 1 X Denhardt’s, 100 pg/ml of boiled hours after the addition of the DNA the cells were treated with 10% sonicated salmon sperm DNA, 0.5% (w/v) SDS, and 10% (w/v) dimethyl sulfoxide in phosphate-buffered saline (PBS), rinsed with dextran sulfate. The membranes were washed twice for 20-30 min at PBS, and incubated in complete medium for 48 h. “Mock” transfected 65-68 in 0.1 X SSC, 0.1% (w/v) SDS and autoradiographed using cells were treated exactly the same way but did not receive DNA. The intensifying screens. 18372 Hormonal Regulation of CAMP Phosphodiesterases Nuclear Run-off sequence of ratPDE4 (data not shown, submitted to Gen- Bank) is compiled of two independent cDNA clones from the Nuclear run-off assays were performed basically as described (34). Isolation of Nuclei-Sertoli cells from 17-day-old male Sprague- unamplified library. No in-frame termination codon 5' of the Dawley rats were cultured in medium without serum. On the fourth first ATG was found in the available ratPDE4 sequence. day of culture the medium was changed and the cells were treated Therefore, it is not clear whether this first ATG is the correct with 500 ng/ml of FSH for different lengths of time (0 min, 30 min, start site. The sequence surrounding this ATG in ratPDE4.1 3 h, 24 h) or with 500 ng/ml of FSH, 1 mM B2cAMP, 100 pM forskolin, fits the Kozak consensus sequence since it has a purine in or 100 ng/ml of cholera toxin for 3 h. After the appropriate incubation time the medium was removed and the cells were rinsed twice with position -3 (35). The putative ratPDE4 major open reading ice-cold PBS. The cells were collected by scraping with a rubber frame therefore encodes a protein with a calculated molecular policeman and centrifugation for 5 min at 500 X g. For each nuclear mass of 64.273 kDa or larger. The first three codons following run-off reaction nuclei from approximately 5 X lo7 cells were prepared the most 5' ATG encode the same amino acids around the by Dounce homogenization as described (34). putative initiation codon in the ratPDE3.1 cDNA (Fig. 1). Binding Plasmids to Nitrocellulose-Plasmids containing cDNA inserts of c-fos, ubiquitin, ratPDE3, or ratPDE4 were linearized with After a short stretch of unrelated sequences at the amino the appropriate restriction enzymes, phenol and chloroform ex- terminus, the similarity between ratPDE3 and ratPDE4 re- tracted, and ethanol precipitated. The linearized plasmids were de- sumes and continues for about 454 amino acids (453 for natured in 0.1 M NaOH for 30 min at room temperature and neutral- ratPDE3 and 455 for ratPDE4). In this region the sequence ized by addition of 10 volumes of 6 X SSC (20 X SSC, 3 M NaCl, 0.3 M Na3 citrate). Five micrograms of each plasmid was spotted onto identity is 87%. The carboxyl termini of both putative pro- nitrocellulose filters using a slot blot apparatus. The filters were teins are less homologous although both are highly negatively baked for 2 h at 80 "C under vacuum. charged (Fig. 1) and contain sequences that fit the consensus Nuclear Run-off Reactions-These reactions were performed as sequence for phosphorylation by casein kinase I1 (36). Both described (34) and the total 3zP incorporation into newly transcribed putative proteins also contain a sequence of 4 consecutive RNA was estimated by scintillation counting. After hybridization to the nitrocellulose strips containing the linearized immobilized plas- basic amino acids, similar to the nuclear localization signal of mids, the filters were washed twice in 2 X SSC for 1 h at 65 "C and single stranded unhybridized RNA was digested by RNase A. After one more wash in 2 X SSC at 37 "C for 1 h the filters were air-dried and exposed to x-ray film. The radioactive spots were excised from the membranes and counted. Immunoprecipitation Fixed Staphylococcus aureus cells (Pansorbin) were prewashed twice in PBS. For one immunoprecipitation 50 J of Pansorbin was incubated for 1 h on ice with an equal volume of polyclonal antiserum Klll (1/100 dilution in PBS plus 0.05% bovine serum albumin). This rabbit antiserum was raised against a synthetic peptide (peptide 2224) based on the deduced amino acid sequence of ratPDE3, but recognizes both recombinant ratPDE3 and ratPDE4 proteins expressed in bac- teria or COS-7 cells. The Pansorbin-antibody complexes were washed once with 20 mM Tris. C1 (pH 7.8), 0.5 M NaCl and twice with 20 mM Tris.Cl (pH 7.8). Soluble extracts from Sertoli cells from three 35- mm dishes were added to the Pansorbin-antibody complexes and incubated for 1.5 h on ice. After three washes with PBS the Pansor- bin-antibody-antigen complexes were pelleted at 14,000 X g. The pellets were resuspended in 45 p1 of PBS containing 1% SDS and incubated at room temperature for 10 min to release the antibody- antigen complexes from the Pansorbin. After centrifugation at 14,000 X g the supernatant was collected, 2 X sample buffer was added (1 X sample buffer, 62.5 mM Tris.Cl, pH 6.8, 10% (v/v) glycerol, 2% SDS (w/v), 715 mM 2-mercaptoethanol, 0.0025% (w/v) bromphenol blue), and the samples were subjected to SDS-polyacrylamide gel electro- phoresis. Western Blot Analysis To cellular fractions or immunoprecipitates from Sertoli cells, volume of 2 X sample buffer was added. The samples were heated to 100 "C for 5 min and subjected to electrophoresis in a 10% SDS- polyacrylamide gel. After electrophoresis the proteins were blotted onto an immobilon membrane which was blocked in a PBS solution containing 0.05% (v/v) Tween 20 and 5% (w/v) dry nonfat milk. Klll antiserum at a dilution of 1/100 was incubated with the mem- brane in blocking buffer and afterwards rinsed three times with a FIG. 1. Comparison of the deduced amino acid sequences of PBS washing solution containing 1% (v/v) Nonidet P-40 and 0.2% ratPDE3 and ratPDE4 from the rat Sertoli cell. Amino acid (v/v) Tween 20. Finally, the membrane was incubated with '''1- sequences of the major open reading frames deduced from ratPDE3 labeled protein A (0.5 pCi/ml) in blocking buffer, rinsed 5 times with (19) and ratPDE4 cDNAs were aligned. Amino acid residues are PBS washing solution, and autoradiographed. represented by the single letter code. Gaps have been introduced in the sequences to optimize the alignment. Regions of sequence identity RESULTS have been boxed. Vertical arrowheads delineate the highly homologous region of 455 amino acids. The underlined amino acid residues rep- Sequence Comparison of RatPDE3 and RatPDE4"As pre- resent the sequence of a synthetic peptide (K111) that was used to viously reported (16), two groups of cDNA clones (ratPDE3 raise polyclonal antibodies. A putative nuclear translocation signal and ratPDE4) were isolated from a cDNA library derived sequence is indicated by small arrows. Horizontal arrowheads deline- from cultured 15-day-old Sprague-Dawley rat Sertoli cells ate the region of ratPDE4 shown in Fig. 2. Putative sites for phos- phorylation by casein kinase I1 are indicated by asterisks. that had been treated with 500 PM Bt,cAMP (19). The DNA Hormonal Regulation of CAMP Phosphodiesterases 18373 the SV40 large T antigen (37) and other nuclear proteins (38- both PDEs (1.9 and 2.5 p~ cAMP for ratPDE3 and ratPDE4, 42). respectively). Velocity determined in crude extracts ranged A cDNA clone (DPD) similar to the ratPDE4 clones has 1 nmol/min/mg of protein (Table I). In between 0.5 and also been isolated from rat brain (18). 525 codons of the open addition, both recombinant enzymes displayed similar sensi- reading frame and the available 3"untranslated region of the tivities to a variety of PDE inhibitors, rolipram and Ro 20- ratPDE4 and DPD clones are identical (Fig. 2). The 5' por- 1724 being more effective inhibitors than cilostamide or mil- tions of the putative open reading frames are unrelated (Fig. rinone (Table I). 2). The 5' portion of our ratPDE4 sequence was derived from Since expression in prokaryotic cells does not allow the two independent clones (ratPDE4.1 and ratPDE4.2) from a same post-translational modifications as in eukaryotic cells, Sertoli cell unamplified library. In addition, the same se- the two cDNAs were also expressed in monkey kidney (COS- quence was present in a genomic clone derived from a rat 7) cells. Affinities for cAMP of the two PDEs recovered from cosmid library (data not shown), ruling out the possibility of COS-7 cell transfection were very similar to those determined cloning artifacts. from the bacterial expression (ratPDE3 K,, = 1.81; ratPDE4 Characterization of the Recombinant PDE Activity Encoded K,, = 2.85). However, although Northern and Western blot by RatPDE4 and Comparison with RatPDE3-The high de- analysis indicated that transfection efficiency, transcription, gree of homology between the ratPDE3 and the ratPDE4 and translation of the two constructs in COS-7 cells was sequences suggests that both enzymes have similar kinetics comparable, differences were observed in the amount and in and physicochemical characteristics. To verify this possibility, intracellular distribution of the CAMP-PDE activity re- both PDEs were expressed in prokaryotic as well as in eukar- covered (PDE activity in picomoles/min . mg of protein ? S.E., yotic cells. n = 10: mock supernatant: 179 & 26; mock pellet: 49 & 15; To express the two cDNAs in E. coli, cDNAs were modified ratPDE3 supernatant: 716 ? 90; ratPDE3 pellet: 171 k 25; to arrange the putative initiation methionine immediately ratPDE4 supernatant: 308 & 56; ratPDE4 pellet: 143 & 11). after the ribosomal-binding site of the pRC23 vector. Trans- Further experiments are required to clarify these findings. formation of E. coli DH5a with pRC23-ratPDE3 and pRC23- Regulation of Steady State mRNA Levels for RatPDI33 and ratPDE4 led to the appearance of major immunoreactive RatPDE4 in Sertoli Cells-Previous biochemical characteriza- bands (with the Klll antibody, see "Experimental Proce- tion of the Sertoli cell PDEs indicated that rolipram-sensitive dures") of 71 and 68 kDa, respectively (data not shown). Less PDEs were present in this cell, mostly after hormonal stim- intense immunoreactive bands of lower molecular weight were ulation (13). The cloning data suggested that mainly ratPDE3 often recovered, suggesting degradation of the recombinant and ratPDE4 were expressed in the Sertoli cell (16). To protein. The immunoreactivity was associated with the ap- further assess this possibility, Sertoli cell mRNA was hybrid- to the PDE cDNA clones available. These Northern blot pearance of high affinity PDE activity in the bacterial lysates. ized cAMP hydrolysis by both recombinant enzymes followed Mi- analyses indicated that ratPDE3 and ratPDE4 are the two chaelis-Menten kinetics and Lineweaver-Burke plots were dominant ratPDEs expressed in these cells, and that the linear with substrate concentrations ranging between 0.1 and cAMP analog treatment has major effects on mRNA levels. 50 PM. The K, values for cAMP (Table I) were similar for Sertoli cells from 15-day-old rats cultured for 4 days in defined medium expressed ratPDE3 mRNA levels that were at the limit of detection by Northern blot analysis (Figs. 3 and 4). PDE4: MKEQGGTVSGAGSSRGGG 0 D~A H~L Q~L Q P N After 24 h treatment with the cAMP analog Bt, CAMP, DPD : SLRIVRNNPTLLTNLHGAPNKRSPAASQAP however, the ratPDE3 steady state transcript level is in- PDf4: YLSVCLFAEESYQKLAHETLEELDWCLD ..... creased at least 100-fold (19). To test if this is also the case DPD : VTRVSL 00 QEESYQKLAMETLEELDWCLD I ..... dbcAMP dbcAMP dbcAMP FIG. 2. Comparison of the deduced amino acid sequences of I- + ' I" + ' I- +t ratPDE4 and DPD. Amino acid sequences of the major open kb reading frames deduced from ratPDE4 and DPD (18) cDNAs were - compared using the MicroGenie align routine. Amino acid residues of the amino-terminal region of ratPDE4 (as delineated in Fig. 1) and of the corresponding region of DPD are represented by the single letter code. Gaps have been introduced in the sequences to optimize -9 alignments. Regions of sequence identity have been boxed. ao TABLE I Comparison of the properties of recombinant ratPDE3 and ratPDE4 expressed in E. coli Recombinant Recombinant Properties ratPDE3 ratPDE4 Calculated molecular mass 66,249 Da 64,273 Da SDS-PAGE 71 K,,, CAMP (phi) 1.9 2.5 ND" K,,, cGMP (pM) ND actin ratPM3 ratPDM Specific activity extract 593 k 65 851 f 143 FIG. 3. Regulation of ratPDE3 and ratPDE4 steady state (pmol/min. mg of pro- mRNA levels by dibutyryl cAMP in the rat Sertoli cell. Sertoli tein) cells from 15-day-old Sprague-Dawley rats were cultured for 4 days Inhibitors (ED,) in pM) in medium without serum (26). The cells were incuhat.ed in the cGMP >50 >50 absence or presence of 0.5 mM Bt,cAMP. After 24 h, poly(A)' RNA Methylisobutylxanthine 18.5 f 2.0 21.0 f 3.0 was prepared and (5 pg) separated on a 1% agarose gel, blotted, and Rolipram 0.6 f 0.1 0.4 k 0.1 hybridized with '"P-labeled ratPDE3 cDNA and autoradiographed. RO 20-1724 1.6 f 0.9 1.2 & 0.2 After removal of the probe, the membrane was rehyhridized with Cilostamide 38.6 f 5.1 29.0 f 2.7 ratPDE4 cDNA and consecutively with actin cDNA. Size markers " ND, not detectable. were radiolabeled HindIII-digested X DNA fragments. kDa 68 kDa 18374 Hormonal Regulation of CAMP Phosphodiesterases were present under basal conditions, while ratPDE3 tran- scripts were not detectable under these experimental condi- tions. Marked differences in hormone dependence of the two PDEs were also present when rats were treated with FSH in vivo. Whereas ratPDE3 mRNA levels undergo a large increase after FSH injection, no significant changes in ratPDE4 mRNA could be detected under these conditions (data not shown). Regulation of Transcription of RatPDE3 and RatPDE4- To test whether the differences in regulation of steady state mRNA levels for ratPDE3 and ratPDE4 are caused by differ- ences in mRNA stability or by differential regulation of gene transcription, nuclear run-off assays were performed. The FIG. 4. Dose dependence of the FSH stimulation of ratPDE3 rate of transcription of c-fos and ubiquitin was measured as and ratPDE4 steady state mRNA levels in the rat Sertoli cell. positive and negative control for the FSH effects. In the three On the fourth day of culture in defined medium, primary Sertoli cells were rinsed and incubated for 24 h with increasing concentrations of different experiments performed, Sertoli cell nuclei isolated FSH. Total RNA was prepared, separated on a 1% agarose gel, blotted prior to FSH treatment showed low levels of ratPDE3 and onto a nylon membrane, and hybridized with :"P-labeled ratPDE3 ratPDE4 transcription (Table 11). The transcription rate of cDNA and autoradiographed. After removal of the probe, the mem- ratPDE3 increased earlier than 30 min after FSH treatment, brane was rehybridized with ratPDE4 cDNA and consecutively with reached a maximum at 3 h, and returned towards basal within actin cDNA. The mRNA abundance as determined by densitometric 24 h. The rate of transcription of ratPDE4 did not signifi- scanning of the autoradiograms are expressed in arbitrary units. cantly change during the time period studied. Furthermore, dibutyryl CAMP, forskolin, and cholera toxin produced an increase in the ratPDE3 rate of transcription similar to FSH (Table 111). This indicates that hormone effects on PDE gene TABLE I1 Time course of FSH regulation of ratPDE3 and ratPDE4 rate of transcription 4.Okb". ----~--~~y-~qw--~ rotPDE4 Nuclear run-off assays were performed as described under "Exper- J. imental Procedures." Five micrograms of plasmids containing rat- FIG. 5. Time course of the regulation of the ratPDE3 and PDE3, ratPDE4, c-fos, or ubiquitin cDNAs were immobilized on ratPDE4 steady state mRNA levels by FSH in the rat Sertoli nylon filters. Sertoli cells from 17-day-old Sprague-Dawley rats were cell. Sertoli cells from 15-day-old Sprague-Dawley rats were cultured treated with 500 ng/ml of FSH for the indicated lengths of time, in defined medium. On the fourth day of culture the cells were treated nuclei were prepared, and incubated with [n-'"P]UTP. The total :'T with 100 ng of FSH/ml of medium. At the indicated times total RNA incorporation into newly transcribed RNA was estimated by scintil- was prepared. Ten micrograms of RNA of each time point was lation counting. After hybridization to the nylon strips containing glyoxylated and separated on a 1% agarose gel. After blotting onto a the plasmids, the filters were washed and exposed to x-ray film. The nylon membrane the blot was hybridized with '"P-labeled ratPDE3 radioactive spots were excised from the membranes and counted. cDNA and autoradiographed. After removal of the ratPDE3 probe, These counts were divided by the total number of counts in the the membrane was rehybridized with radiolabeled ratPDE4 cDNA hybridization solution to express the rate of transcription in parts and autoradiographed. per million. The rate of transcription of c-fos and ubiquitin was measured as positive and negative control for the FSH effects. for ratPDE4, the same Northern blots were hybridized with Specific RNA concentration after FSH Immobilized radiolabeled ratPDE4 cDNAs of similar specific activities as addition DNA the ratPDE3 cDNAs and autoradiographed. The ratPDE4 0 90 min 3h 24 h steady state transcript level was readily detectable under basal PPm conditions and was increased only about 5-fold as determined RatPDE3 ND" 3.97 12.93 1.07 by spectrophotometric scanning (Fig. 3). These differences RatPDE4 1.19 0.63 1.15 0.83 were confirmed in several other Northern blot experiments 10.27 2.17 19.31 2.65 c-fos from independent Sertoli cell cultures and were also evident 3.03 4.03 4.03 6.07 Ubiquitin after FSH or forskolin treatment (Figs. 4 and 5, and data not " ND, not detectable. shown). The increase in steady state levels of ratPDE3 and ratPDE4 mRNA by FSH is dose-dependent and occurs at TABLE I11 physiological concentrations of FSH ranging between 1 and Regulation of ratPDE3 and ratPDE4 rate of transcription by FSH, 100 ng/ml. Whereas ratPDE4 mRNA levels were maximal &CAMP, forskolin, and cholera toxin upon exposure of the cells to 10 ng of FSH/ml of medium, Nuclear run-off assays were performed as described in Table 11. Sertoli cells from 17-day-old Sprague-Dawley rats were incuhated in ratPDE3 mRNA levels could be further elevated with higher the presence or absence of 500 ng/ml FSH, 1 mM Bt,cAMP, 100 pM FSH concentrations (Fig. 4). forskolin, or 100 ng/ml cholera toxin for 3 h. When Sertoli cells were exposed to FSH, the AMP-PDE Specific RNA concentration activity increases after a time lag of 1 h and reaches a Immobilized maximum at 18-24 h (15). The time course of changes in the DNA None FSH Rt,cAMP Forskolin ratPDE steady state mRNA levels in primary Sertoli cell cultures treated with 100 ng/ml FSH is reported in Fig. 5. ppm After a time lag of about 1 h the level of transcripts increased, 0.88 8.31 10.54 8.69 8.80 RatPDE3 RatPDE4 1.57 2.08 2.68 3.01 2.51 a maximum between 3 and 12 h, and then gradually reached c-fos 2.48 7.48 8.73 decreased to near basal levels in a time span of 3 days. 4.15 2.00 2.43 2.93 2.53 Uhiquitin Confirming the above reported data, ratPDE4 transcripts 8.74 9.04 Hormonal Regulation of CAMP Phosphodiesterases 18375 analyzed by immunoblotting. Even after enriching the PDE protein by immunoprecipitation, no detectable band could be observed under basal conditions (Fig. 7A). Conversely, an - immunoreactive 67-kDa band appeared after FSH and dibu- 97 - tyryl cAMP treatment (Fig. 7A). The intensity of these bands correlated with the levels of CAMP-PDE activity measured in 68- - the supernatants before immunoprecipitation (Fig. 7B). Fi- nally, the size of this band was identical to that of a purified -I " " CAMP-PDE from the Sertoli cell (Fig. 7A).' 45 - " " DISCUSSION -peptide + peptide B peptide Biochemical characterization and molecular cloning of the kDa - + CAMP-PDEs has uncovered an unexpected complexity of this 200 - family of enzymes. In the rat there are at least four genes 97 - encoding cGMP-insensitive, rolipram- and Ro 20-1724-inhib- 68" ited CAMP-PDEs (16), and data suggesting alternate splicing open the possibility to expression of an even larger number 45 - of PDE proteins (16, 17, 19). Transcripts derived from two of these genes (ratPDE3 and ratPDE4) are present in the rat Sertoli cell in culture. Although differences in basal levels and 29 - in the degree of hormone stimulation are present, the expres- 18 - sion of both ratPDE3 and ratPDE4 is under the control of follicle-stimulating hormone and CAMP. The data presented FIG. 6. Immunoblot analysis of soluble fractions (A) and here demonstrate that the increase in cAMP hydrolyzing partially purified CAMP-PDE preparations (B) from the rat activity observed in this cell is the result of an increase in Sertoli cell with Klll antiserum. On the fourt,h day of culture rate of CAMP-PDE gene transcription, accumulation of in defined medium, primary Sertoli cells were incubated in the pres- mRNA, and neosynthesis of the CAMP-PDE protein. These ence or absence of 1 mM Bt2cAMP for 24 h. The cells were scraped with a rubber policeman and homogenized in an all-glass Dounce ia w homogenizer. After fractionation of the cell extract by centrifugation at 14,000 X g for 15 min, the supernatant proteins (A) and a CAMP- PDE protein preparation, partially purified from the rat Sertoli celp (R), were subjected to SDS-polyacrylamide gel electrophoresis. After blotting onto nylon membranes, duplicate blots were incubated with the Klll antiserum (raised against a synthetic ratPDE peptide) or with the Klll antiserum preincubated with excess peptide used for 68 - PDE immunizations. Immunoreactive proteins were visualized by incubat- I IgG ing the blots with "'1-labeled protein A and autoradiography. transcription are mediated by the activation of the CAMP- dependent pathway. Regulation of the RatPDE Protein Levels-To determine whether the increase in ratPDE mRNA results in an increase B 'W in PDE protein content, Sertoli cell extracts were subjected to immunoblot analysis. A polyclonal antiserum (K111) was ~ I mi raised against a synthetic peptide based on the sequence of ratPDE3. This antiserum recognizes both recombinant rat- PDE3 and ratPDE4 proteins (data not shown). Although :j immunoblot analysis of crude Sertoli cell extracts showed several bands, only the signals of a 67 and a 54-kDa polypep- ! -1 m o7 tide were abolished by competing the antibody with the pep- :I tide used for immunization (Fig. 6A). The 67-kDa protein i ;/ " . . " i' " _._ level was markedly affected by Bt'cAMP treatment, being 0 ..-.> ry. mw undetectable in extracts from unstimulated Sertoli cells (Fig. 6A). To confirm that the 67-kDa protein corresponds to the FIG. 7. Regulation of CAMP-PDE protein levels (A) and cAMP hydrolyzing activity (B) by FSH and dibutyryl cAMP CAMP-PDE, extracts from stimulated Sertoli cells were par- in the soluble fraction of the rat Sertoli cell. Sertoli cells from tially purified by high performance liquid chromatography' 15-day-old Sprague-Dawley rats were cultured in defined medium. and the peak fraction of CAMP-PDE activity was subjected On the fourth day of culture the cells were incubated in the presence to immunoblot analysis. Under these conditions only the or absence of 500 ng/ml FSH or 1 mM Bt2cAMP. Twenty-four hours immunoreactivity of a 67-kDa polypeptide was present and after the additions the cells were scraped with a rubber policeman the signal was competed by the peptide (Fig. 6B). Using an and homogenized in an all-glass Dounce homogenizer. This cell extract was fractionated by centrifugation at 14,000 X g for 15 min. alternative approach to increase the immunoblot signal, sol- The supernatant fractions were subjected to PDE assays and immu- uble extracts from control, FSH-, or dibutyryl CAMP-stimu- noprecipitation and the anti-CAMP-PDE antibody K111. The im- lated Sertoli cells were first immunoprecipitated with the munoprecipitated proteins were solubilized and separated on a 10% Klll antiserum, and the immunoprecipitated proteins were polyacrylamide gel. CAMP-PDE protein purified from the rat Sertoli cell2 was loaded on the same gel. After blotting onto a nylon mem- ' M. Conti, J. Odeh, J. V. Swinnen, and M. E. Svoboda, submitted brane, the blot was incubated with the Klll antibody and ""I-labeled for publication. protein A and autoradiographed. 18376 Hormonal Regulation of CAMP Phosphodiesterases data, then, provide evidence for a novel mechanism of regu- ratPDE3 gene transcription is slower than that observed for lation of PDEs that involves hormone-dependent CAMP- c-fos. The transcription of c-fos reaches a maximum at the mediated changes in the rate of a PDE gene transcription. 30-min time point, while ratPDE3 transcription peaks only A molecular cloning approach has demonstrated that the after 3 h. It is then to be expected that the FSH-dependent Sertoli cell expresses ratPDE3 and ratPDE4. As previously CAMP-mediated transcription of the two genes involves dif- shown (19), ratPDE3 encodes a protein with a calculated ferent steps. Although the basic mechanism might be the molecular mass of 66.2 kDa, while conceptual translation of same, e.g. phosphorylation of a cAMP response element- the ratPDE4 cDNAs is consistent with a PDE protein of 64.2 binding protein, additional regulatory steps either delay the kDa or higher molecular mass. Although no in-frame up- activation of ratPDE3 gene transcription, or cause a more stream termination codon was found in the cDNA clones that rapid termination of c-fos transcription. The possibility we have available, preliminary sequence available from a should also be considered that the c-fos gene product might genomic clone demonstrates the presence of a stop codon 108 be involved in the modulation of transcription of the ratPDE3 bases upstream from the putative initiation methionine. gene. In fact, the c-fosljun complex is a transcription activator Nevertheless, further studies are needed to unequivocally that binds to an AP-1 regulatory element (45), and hormonal prove that the ATG that we have selected codes for the activation of the Sertoli cell leads to increased levels of both initiation methionine also in uiuo. On the other hand, these fos andjun mRNAs (47). CAMP-PDEs might have multiple initiation methionines (17). The finding that cAMP regulates the expression of CAMP- The ratPDE4 cDNAs isolated from the rat Sertoli cell cDNA PDEs explains earlier reports on cell lines with diminished library have sequences identical to the DPD clone isolated by protein kinase A and phosphodiesterase activity. The kin- complementation from a rat brain library except for the first and PPD mutant strains of S49 lymphoma cells, for instance, 40 amino acids of the putative open reading frames (18). have a decreased protein kinase A activity in comparison with Although cloning artifacts cannot be completely excluded, it the wild type cells. The kinase defect is associated with a is possible that alternate splicing of a single ratPDE4 gene decrease in cAMP hydrolytic activity, indicating that protein occurs in brain and testis. Evidence suggesting alternate splic- kinase A mediates, probably by regulating transcription fac- ing or multiple start sites have also been reported for other tors, the expression of certain PDE forms (48, 49). CAMP-PDE encoding cDNAs (17, 19). In addition, most cell The hypothesis that CAMP-PDE activation is dependent types studied express multiple transcripts of the ratPDE on new synthesis of the PDE protein is here proven by the genes, some of which are tissue specific (16). There is, then, use of polyclonal antibodies. It is, however, still possible that additional modifications such as phosphorylation are neces- potential for the expression of a large number of rolipram- sensitive CAMP-PDEs. That alternate splicing might be a sary to express the full activity of the PDE. One puzzling finding is that although ratPDE4 mRNA is expressed in the property of PDEs is also suggested by data on other PDE families. It has been reported that two distinct amino termini Sertoli cell under basal conditions, no immunoreactive PDE have been found for a Ca2+/calmodulin-dependent PDE (43). protein could be detected in the Sertoli cell soluble fraction. Based on the sequence similarity in the central portion of It is possible that ratPDE4 protein levels are below the the proteins, it was expected that the kinetic properties of sensitivity of the immunodetection employed in our study. both enzymes are very similar. This was confirmed here by An alternative possibility is that the ratPDE4 protein is not present in the soluble cell fraction, but compartmentalizes in prokaryotic expression of the two PDEs. The two enzymes have a similar K,,, for cAMP and similar sensitivity to different other subcellular districts. This latter possibility would also reconcile differences found in the stimulated Sertoli cell. inhibitors. Therefore, the catalytic centers are structurally and functionally very similar. It is possible that the amino While high levels of both ratPDE3 and ratPDE4 mRNA are and carboxyl termini, being different in the two proteins, present under these culture conditions, only one immuno- serve different functions in the two CAMP-PDEs. reactive band could be detected even after immunoprecipita- Both ratPDE3 and ratPDE4 transcript steady state levels tion. Other reports have demonstrated the presence of a low K,,, CAMP-PDE in the particulate fraction of the brain (50). were increased by FSH after a lag of about 1 h and remained 2 days. This is in agreement with the Consistent with this view also is the finding that rolipram elevated for at least (51). appearance of the cAMP hydrolyzing activity in the Sertoli binding to membrane-bound sites was detected in brain Also in the rat liver, a membrane-bound PDE is sensitive to cell supernatant (15). The increase in ratPDE3 mRNA levels or FSH was consistently larger than rolipram (52, 53). in response to Bt,cAMP The time course of this CAMP-mediated activation of these that of ratPDE4. Nuclear run-off experiments showed that a CAMP-PDEs in the Sertoli cell is very different from that 10-fold increase in the rate of ratPDE3 gene transcription followed FSH treatment in uitro. Conversely, the basal tran- described for the CAMP-dependent activation of a cGMP- inhibited PDE from human platelets (54-56) and rat adipo- scription rate of ratPDE4 was at the limit of detection and cytes (57, 58). This latter enzyme is membrane bound and FSH activation of transcription was small and not significant. very rapidly activated by phosphorylation. In a cell where the It is, then, possible that also mRNA stabilization plays a role two modes of regulation coexist, there will be a complex in the regulation of the steady state mRNA levels of these genes. The FSH effect on the ratPDE gene transcription is pattern of PDE activation, with early and delayed increases in PDE activity. The exact significance of this complexity is mimicked by cAMP analogs and other cAMP increasing unclear, but there is evidence that the long term PDE acti- agents, indicating that this hormonal activation of transcrip- vation is involved in the regulation of the hormone respon- tion is mediated by CAMP. Transcriptional activation of cAMP responsive genes such as c-fos is commonly thought to siveness of the Sertoli cell (15). The homologous and heter- ologous desensitization of the rat Sertoli cell is in part or occur via CAMP-dependent phosphorylation of cAMP re- completely reverted after inhibition of the PDEs by Ro 20- sponse element-binding proteins (44) or by regulating the 1724 (13). This indicates that these CAMP-PDEs serve to levels of trans-acting factors that bind to the AP1 and/or AP2 elements (45, 46). Similar regulatory sequences can therefore control the responsiveness of the Sertoli cell to hormones. On the other hand, experiments with in uitro model systems also be expected in the 5’-flanking region of the ratPDE3 gene. It is worth noting, however, that the time course of containing hormone-sensitive adenylate cyclase, CAMP-de- Hormonal Regulation of cAMP Phosphodiesterases 18377 Nucleic Acids Res. 12, 5707-5717 pendent protein kinase 11, and cyclic nucleotide phosphodi- 26. Monaco, L., and Conti, M. (1986) Biol. Reprod. 35, 258-266 esterase support the hypothesis that rapid cAMP turnover 27. Thompson, W. J., and Appleman, M. M. (1971) Biochemistry 10, may function as a mechanism for signal amplification by the 311-316 CAMP-dependent protein kinase (59). These hypotheses can 28. Lineweaver, H., and Burk, D. (1934) J. Am. Chem. SOC. 56,658- now be tested by studying other hormone-responsive cell systems that express a different subset of CAMP-PDEs and 29. Chirgwin, J. 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L., Dahlback, H., Jornvall, H., and A. 87,533-537 Russel, D. W. (1989) J. Biol. Chem. 264, 8222-8229 59. Leiser, M., Fleischer, N., and Erlichman, J. (1986) J. Biol. Chem. 25. Lopata, M. A., Cleveland, D. W., and Sollner-Webb, B. (1984) 261, 15486-15490 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Properties and hormonal regulation of two structurally related cAMP phosphodiesterases from the rat Sertoli cell.

Swinnen, J.V.; Tsikalas, K.E.; Conti, M.
Journal of Biological Chemistry – Sep 1, 1991
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 27, Issue of September 18370-18377.1991 25, PP. 0 1991 by The American Society for Biochemistry and Molecular Biologv, Inc. Printed in U. 5’. A. Properties and Hormonal Regulation of Two Structurally Related cAMP Phosphodiesterases from the Rat Sertoli Cell* (Received for publication, January 2, 1991) Johannes V. Swinnen, Kallen E. Tsikalas, and Marco Conti$ From the Laboratories for Reproductive Biology, Departments of Pediatrics and Physiology, University of North Carolina, .. . Chapel Hill, North Carolina 27599 Upon exposure to follicle-stimulating hormone The responsiveness of the target cell to hormones is contin- (FSH), the gonadotropin-responsive Sertoli cell ex- uously adjusted in synchrony with changes in the extracellular presses increased rolipram-sensitive CAMP-specific environment. The mechanisms involved in this adaptation phosphodiesterase (CAMP-PDE) activity. To under- are complex and involve changes in the components of the stand the mechanisms leading to this activation, the signal transduction machinery at the plasma membrane level CAMP-PDEs present in the Sertoli cell were character- as well as changes in the enzymes involved in intracellular ized and their regulation studied. Comparison of the signalling. Phosphodiesterases (PDEs),’ the enzymes that conceptual translates of two groups of PDE cDNA degrade cyclic nucleotides might play an important role in clones isolated from a Sertoli cell cDNA library these processes. In most cells, including the Sertoli cell (l), (ratPDE3 and ratPDE4) showed that the encoded pro- degradation of cyclic nucleotides is carried out by multiple teins were structurally similar, containing a core re- PDEs with different properties in terms of affinity for cAMP gion of 455 amino acids with a sequence identity of and cGMP, regulation of hydrolytic activity, and sensitivity 87%. The amino and carboxyl termini were divergent. to inhibitors (2). Although several types of PDEs are able to Expression of these cDNAs in Escherichia coli and hydrolyze CAMP, it is now recognized that two families of monkey COS-7 cells demonstrated that the encoded PDEs have a high affinity for this nucleotide. One of these CAMP-PDEs had similar affinities for the cAMP sub- forms, referred to as cGMP-inhibited PDE is sensitive to the strate and were equally sensitive to a number of PDE selective inhibitor cilostamide and the cardiotonic milrinone inhibitors (rolipram > Ro 20-1724 > cilostamide). FSH (2). In several cell types, this enzyme is membrane bound and stimulation of the Sertoli cell produced an increased rapidly activated by insulin and P-adrenergic agonists (3). rate of transcription of the ratPDE3 gene and elevated The other type, termed CAMP-specific PDE (CAMP-PDE) is mRNA levels for ratPDE3 and to a lesser extent of insensitive to cGMP and is selectively inhibited by the anti- ratPDE4. The increase in mRNA levels was detected depressant rolipram (2). Also this latter form of PDE has after 1 h of stimulation. Forskolin, cholera toxin, and been shown to be regulated by hormones. In the Sertoli cell, M,02’-dibutyryl cAMP produced a similar increase in FSH via an increase in cAMP causes the activation of a PDE rate of transcription and elevated mRNA levels, indi- with the properties of a CAMP-PDE (1,4). Similar hormone- cating that this activation is mediated by an increase dependent regulation of cAMP hydrolyzing activity has also in intracellular CAMP. RatPDE4 mRNA levels were been demonstrated in glioma and astrocytoma cell lines (5- 10 ng of FSH/ml, whereas maximal upon exposure to 8), in fibroblasts (9, lo), granulosa (11), and lymphoma cells ratPDE3 mRNA levels could be further elevated with (12). The CAMP-PDE which in the rat Sertoli cell is stimu- higher FSH concentrations. The intensity of an im- lated by FSH, is thought to be involved in the refractoriness munoreactive band with characteristics identical to a that follows hormone stimulation (13-15). It is now clear that purified CAMP-PDE, correlated with the increased the family of the CAMP-PDEs is composed of multiple related cAMP hydrolytic activity after FSH or dibutyryl forms. Molecular cloning data (16-18) have demonstrated cAMP treatment, demonstrating that changes in that in the rat there are at least four genes encoding cAMP CAMP-PDE protein levels are involved in this regula- phosphodiesterases which have been highly conserved tion. These data provide evidence that multiple CAMP- PDE forms are expressed in the rat Sertoli cell. Al- through evolution (16, 17). It has been shown that the steady state mRNA level for one of these PDEs (ratPDE3) is regu- though differences in the pattern of activation of these lated by hormones and cAMP (19). Here we have studied in forms were observed, these data show, that in the rat Sertoli cell, the CAMP-PDE activity is regulated by detail the mechanisms involved in this regulation. In addition, hormones via a novel mechanism that involves a the finding that another CAMP-PDE gene (ratPDE4) is ex- CAMP-dependent activation of transcription of a PDE pressed in the Sertoli cell prompted us to study the properties gene. and the regulation of this latter PDE in response to FSH. Here we report that the expression of both these structurally * This work was supported by National Institutes of Health Grant related CAMP-PDEs is under the control of hormones. We HD20788. The costs of publication of this article were defrayed in show that differences in expression occur at least in part at part by the payment of page charges. This article must therefore be the level of gene transcription and that an increase in PDE hereby marked “advertisement” in accordance with 18 U.S.C. Section activity follows changes in PDE protein concentration. 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted The abbreviations used are: PDE, cyclic nucleotide phosphodies- to the GenBankTM/EMBL Data Bank with accession number($ terase; CAMP-PDE, CAMP-specific phosphodiesterase; SDS, sodium MZ35349-M25350. dodecyl sulfate; Bt2cAMP, N‘,OO”-dibutyryl CAMP; FSH, follicle- $ To whom correspondence should be addressed Laboratories for stimulating hormone; EGTA, [ethylenebis(oxyethylenenitrilo)]tetra- Reproductive Biology, CB 7500 MacNider 202H, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-6795. acetic acid; PBS, phosphate-buffered saline. This is an Open Access article under the CC BY license. Hormonal Regulation of CAMP Phosphodiesterases 18371 cells were rinsed with PBS and lysed for RNA extraction, or collected EXPERIMENTAL PROCEDURES with a rubber policeman and homogenized in an all-glass Dounce homogenizer in a buffer containing 20 mM Tris.Cl (pH 8.01, 10 mM Materials EDTA, 0.2 mM EGTA, 50 mM benzamidine, 0.5 pg/ml leupeptin, 0.7 Crotalus atrox snake venon and lysozyme were purchased from pg/ml pepstatin, and 2 mM phenylmethylsulfonyl fluoride (homoge- Sigma; Pansorbin cells from Behring Diagnostics; Ro 20-1724 (4-(3- nization buffer). After centrifugation for 15 min at 14,000 X g, the butoxy-4-methoxybenzyl)-2-imidazolidinone) was from Hoffmann- supernatant and the pellet fractions were subjected to Western blot LaRoche; rolipram (4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyr- analysis, immunoprecipitation, or PDE assay. rolidone) was from Berlex, NJ; cilostamide (N-cyclohexy-4-[(1,2- dihydro-2-oxo-6-quinolyl)oxy]-N-methylbutyramide) was provided Sertoli Cell Cultures by Dr. H. Hidaka, Nagoya University School of Medicine, Showa- Ku, Nagoya 466 Japan; AG 1-X8 resin was obtained from Bio-Rad Sertoli cell cultures from 15- to 17-day-old Sprague-Dawley rats [2,8-,”H]cAMP (20-50 Ci/mmol), ‘*‘I-labeled protein A (2-10 pCi/pg), were prepared as described (26) and maintained in medium without [&”P]CTP (3000 Ci/mmol), and [a-”PIUTP (800 Ci/mmol) were serum for 4 days. On the fourth day of culture the cells were rinsed purchased from Du Pont-New England Nuclear; guanidium thiocya- with Hank’s balanced salt solution, the medium was replaced and nate from Fluka; CsCl from Gallard and Schlesinger; immobilon from FSH (NIDDK ov-FSH S16), N6,O””dibutyryl CAMP, forskolin, chol- Millipore; Biotrans from ICN; a random primed DNA labeling kit era toxin, or vehicle were added to the medium. At the indicated time was purchased from Boehringer Mannheim; p2523 columns from PBS and lysed for after treatment, the cells were rinsed twice with 5prime-3prime; and dextran sulfate M, 500,000 was from Pharmacia. RNA extraction. For immunoprecipitation, Western blot analysis, or PDE assay, the cells were scraped in homogenization buffer with a Sequence Analysis rubber policeman and homogenized in an all-glass Dounce homoge- nizer. Cellular fractions were prepared by centrifugation at 14,000 X The cloning of PDEs from a rat Sertoli cell cDNA library has been g for 15 min. described (16). The complete ratPDE3 DNA sequence (19) and also a partial cDNA sequence of two ratPDE4 clones have been published PDE Assay (16). These clones have been completely sequenced and also two additional ratPDE4 clones (ratPDE4.3 and ratPDE4.4) have been PDE assays were performed basically as described (27) and as partially characterized. The sequences were aligned using the detailed previously (19). Cell extracts in homogenization buffer were MicroGenie software package (Beckmann). adjusted to 40 mM Tris.Cl (pH 8.0), 10 mM MgC12, 1.25 mM 2- mercaptoethanol, and 1 p~ [“HICAMP (0.1 pCi/reaction). After in- Prokaryotic Expression cubation at 34 “C for 3-10 min the reactions were terminated by 1 volume of a 40 mM Tris. C1 (pH 7.5) solution containing addition of To express ratPDE3 and ratPDE4 cDNAs in bacteria, the prokar- 10 mM EDTA and heat denaturation. To each reaction tube 50 pg of R. Crowl) yotic expression vector pRC23 (20) (kindly provided by Dr. C. atrox snake venom was added. After incubation at 34 “C for 20 was used. This vector contains the h phage PL promoter followed by min the reaction products were separated by anion exchange chro- a ribosome-binding site and an EcoRI cloning site. The distance matography on AG 1-X8 resin and the amount of released radiola- between the ribosome-binding site and the AUG codon is critical for beled adenosine was quantitated by scintillation counting. PDE ac- efficient translation (21). Because the EcoRI site in cDNA clone tivity determinations were performed in triplicate at three different ratPDE3.1 precedes the putative initiation AUG codon by 150 bases time intervals. For the inhibitor studies, cell extracts were incubated (19), a new EcoRI site was created 3 bases 5’ of the AUG by the in the presence of lo-” to lo-’ M Ro 20-1724, rolipram, or cilostamide polymerase chain reaction. cDNA clone ratPDE4.2 starts with an which were dissolved in dimethyl sulfoxide. Control tubes contained GcoRI site 5 bases upstream of the putative AUG and was ligated equal amounts (2.5%) of dimethyl sulfoxide. For the determination into the EcoRI site of pRC23 without further manipulation. Esche- of the K, of the enzyme (28) cell extracts were incubated with 0.1 richia coli DH5a bacteria were transformed (22) with the pRC23 pCi of [“HICAMP and 0.1-50 p~ unlabeled cAMP for 2, 4, 6, and 8 compatible plasmid pRK248cIts (provided by Dr. R. Crowl) which min. The PDE activity is expressed as the rate of cAMP hydrolysis/ encodes a temperature-sensitive hcIAt2 repressor (23) and confers min. tetracyclin resistance to the cells. Tetracyclin resistant pRK248cIts containing DH5a bacteria were transformed with the pRC23-rat- RNA Extraction and Northern Blot Analysis PDE3 and pRC23-ratPDE4 constructs and grown in the presence of 30 “C. Expression of ampicillin (the selectable marker of pRC23) at Total RNA was prepared as described (29). Sertoli or COS-7 cells the ratPDEs by shifting the temperature from 30 to 42 “C for 3 h was were lysed in a buffer containing 50% (w/v) guanidine thiocyanate, monitored by polyacrylamide gel electrophoresis followed by Coo- 16.7 mM Na citrate, 0.5% (w/v) Na lauryl sarcosine, 80 mM 2- massie Brilliant Blue staining or Western blot analysis (with Klll mercaptoethanol, and 0.1% (v/v) Sigma antifoam A. This cell lysate antiserum, see below) and PDE assays. For PDE assay, the bacteria a solution containing 5.7 M cesium chloride and was layered on top of were pelleted, resuspended in 20 mM Tris and 1 mM EDTA, and lysed 167 mM Na acetate (pH 5.0) (30). The RNA was pelleted by centrif- with 5 mg/ml of lysozyme. The solution was adjusted to 0.2 mM ugation for 16 h at 100,000 X g. After centrifugation the RNA pellet EGTA, 50 mM benzamidine, 0.5 pg/ml leupeptin, 0.7 pg/ml pepstatin, was redissolved in ddH,O and ethanol precipitated. Poly(A)’ RNA and 2 mM phenylmethylsulfonyl fluoride. was prepared by oligo(dT)-cellulose chromatography as described (31). Five micrograms of poly(A)+ RNA or 10-20 pg of total RNA Eukaryotic Expression was denatured by incubation at 50 “C for 1 h in a buffer containing The pCMV5 expression in vector (24) drives the expression of 50% (v/v) dimethyl sulfoxide, 20% (v/v) glyoxal, and 20 mM sodium cDNAs cloned into the multiple cloning site in between the human phosphate (pH 6.8) (31). After separation on a 1% agarose gel in 10 cytomegalovirus major immediate early gene promoter and enhancers mM sodium phosphate (pH 6.8), the gel was blotted onto a Biotrans and the transcription termination and polyadenylation signals of the nylon membrane (32) and baked at 80 “C for 1 h. Blots were prehy- human growth hormone. The ratPDE3.1 and ratPDE4.1 cDNAs were bridized in 50% v/v) formamide, 50 mM sodium phosphate (pH 6.5), inserted into the EcoRI site of the plasmid. After transformation, 5 X SSC (20 X SSC, 3 M NaCl and 0.3 M Na:, citrate), 5 X Denhardt’s ampicillin-resistant DH5a bacteria were tested for the presence and (100 X Denhardt’s, 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrroli- the orientation of the ratPDE cDNAs. Plasmids were prepared from done, 2% (w/v) bovine serum albumin, fraction V), 250 pg/ml of 1 liter of bacterial cultures using p2523 columns according to the boiled sonicated salmon sperm DNA, 0.5% (w/v) SDS, and 1% (w/v) protocol of the manufacturer. Monkey kidney cells (COS-7) were glycine at 42 “C for 4 h. RatPDE3, ratPDE4, and actin cDNA clones grown in high glucose Dulbecco’s modified Eagle’s medium containing were radiolabeled (33) using a random primed labeling kit and follow- 5% (v/v) fetal calf serum. When the cells reached a density of ing the manufacturers’ instructions. The blots were consecutively approximately 40% of confluency, the medium was aspirated and incubated at 42 “C for 16-20 h with radiolabeled probe at 1-2 X lo6 medium without serum was added. The cells were transfected using cpm/ml of hybridization buffer (50% (v/v) formamide, 20 mM sodium the DEAE-dextran method (25) with 10 pg of plasmid DNA. Four phosphate (pH 6.5), 5 X SSC, 1 X Denhardt’s, 100 pg/ml of boiled hours after the addition of the DNA the cells were treated with 10% sonicated salmon sperm DNA, 0.5% (w/v) SDS, and 10% (w/v) dimethyl sulfoxide in phosphate-buffered saline (PBS), rinsed with dextran sulfate. The membranes were washed twice for 20-30 min at PBS, and incubated in complete medium for 48 h. “Mock” transfected 65-68 in 0.1 X SSC, 0.1% (w/v) SDS and autoradiographed using cells were treated exactly the same way but did not receive DNA. The intensifying screens. 18372 Hormonal Regulation of CAMP Phosphodiesterases Nuclear Run-off sequence of ratPDE4 (data not shown, submitted to Gen- Bank) is compiled of two independent cDNA clones from the Nuclear run-off assays were performed basically as described (34). Isolation of Nuclei-Sertoli cells from 17-day-old male Sprague- unamplified library. No in-frame termination codon 5' of the Dawley rats were cultured in medium without serum. On the fourth first ATG was found in the available ratPDE4 sequence. day of culture the medium was changed and the cells were treated Therefore, it is not clear whether this first ATG is the correct with 500 ng/ml of FSH for different lengths of time (0 min, 30 min, start site. The sequence surrounding this ATG in ratPDE4.1 3 h, 24 h) or with 500 ng/ml of FSH, 1 mM B2cAMP, 100 pM forskolin, fits the Kozak consensus sequence since it has a purine in or 100 ng/ml of cholera toxin for 3 h. After the appropriate incubation time the medium was removed and the cells were rinsed twice with position -3 (35). The putative ratPDE4 major open reading ice-cold PBS. The cells were collected by scraping with a rubber frame therefore encodes a protein with a calculated molecular policeman and centrifugation for 5 min at 500 X g. For each nuclear mass of 64.273 kDa or larger. The first three codons following run-off reaction nuclei from approximately 5 X lo7 cells were prepared the most 5' ATG encode the same amino acids around the by Dounce homogenization as described (34). putative initiation codon in the ratPDE3.1 cDNA (Fig. 1). Binding Plasmids to Nitrocellulose-Plasmids containing cDNA inserts of c-fos, ubiquitin, ratPDE3, or ratPDE4 were linearized with After a short stretch of unrelated sequences at the amino the appropriate restriction enzymes, phenol and chloroform ex- terminus, the similarity between ratPDE3 and ratPDE4 re- tracted, and ethanol precipitated. The linearized plasmids were de- sumes and continues for about 454 amino acids (453 for natured in 0.1 M NaOH for 30 min at room temperature and neutral- ratPDE3 and 455 for ratPDE4). In this region the sequence ized by addition of 10 volumes of 6 X SSC (20 X SSC, 3 M NaCl, 0.3 M Na3 citrate). Five micrograms of each plasmid was spotted onto identity is 87%. The carboxyl termini of both putative pro- nitrocellulose filters using a slot blot apparatus. The filters were teins are less homologous although both are highly negatively baked for 2 h at 80 "C under vacuum. charged (Fig. 1) and contain sequences that fit the consensus Nuclear Run-off Reactions-These reactions were performed as sequence for phosphorylation by casein kinase I1 (36). Both described (34) and the total 3zP incorporation into newly transcribed putative proteins also contain a sequence of 4 consecutive RNA was estimated by scintillation counting. After hybridization to the nitrocellulose strips containing the linearized immobilized plas- basic amino acids, similar to the nuclear localization signal of mids, the filters were washed twice in 2 X SSC for 1 h at 65 "C and single stranded unhybridized RNA was digested by RNase A. After one more wash in 2 X SSC at 37 "C for 1 h the filters were air-dried and exposed to x-ray film. The radioactive spots were excised from the membranes and counted. Immunoprecipitation Fixed Staphylococcus aureus cells (Pansorbin) were prewashed twice in PBS. For one immunoprecipitation 50 J of Pansorbin was incubated for 1 h on ice with an equal volume of polyclonal antiserum Klll (1/100 dilution in PBS plus 0.05% bovine serum albumin). This rabbit antiserum was raised against a synthetic peptide (peptide 2224) based on the deduced amino acid sequence of ratPDE3, but recognizes both recombinant ratPDE3 and ratPDE4 proteins expressed in bac- teria or COS-7 cells. The Pansorbin-antibody complexes were washed once with 20 mM Tris. C1 (pH 7.8), 0.5 M NaCl and twice with 20 mM Tris.Cl (pH 7.8). Soluble extracts from Sertoli cells from three 35- mm dishes were added to the Pansorbin-antibody complexes and incubated for 1.5 h on ice. After three washes with PBS the Pansor- bin-antibody-antigen complexes were pelleted at 14,000 X g. The pellets were resuspended in 45 p1 of PBS containing 1% SDS and incubated at room temperature for 10 min to release the antibody- antigen complexes from the Pansorbin. After centrifugation at 14,000 X g the supernatant was collected, 2 X sample buffer was added (1 X sample buffer, 62.5 mM Tris.Cl, pH 6.8, 10% (v/v) glycerol, 2% SDS (w/v), 715 mM 2-mercaptoethanol, 0.0025% (w/v) bromphenol blue), and the samples were subjected to SDS-polyacrylamide gel electro- phoresis. Western Blot Analysis To cellular fractions or immunoprecipitates from Sertoli cells, volume of 2 X sample buffer was added. The samples were heated to 100 "C for 5 min and subjected to electrophoresis in a 10% SDS- polyacrylamide gel. After electrophoresis the proteins were blotted onto an immobilon membrane which was blocked in a PBS solution containing 0.05% (v/v) Tween 20 and 5% (w/v) dry nonfat milk. Klll antiserum at a dilution of 1/100 was incubated with the mem- brane in blocking buffer and afterwards rinsed three times with a FIG. 1. Comparison of the deduced amino acid sequences of PBS washing solution containing 1% (v/v) Nonidet P-40 and 0.2% ratPDE3 and ratPDE4 from the rat Sertoli cell. Amino acid (v/v) Tween 20. Finally, the membrane was incubated with '''1- sequences of the major open reading frames deduced from ratPDE3 labeled protein A (0.5 pCi/ml) in blocking buffer, rinsed 5 times with (19) and ratPDE4 cDNAs were aligned. Amino acid residues are PBS washing solution, and autoradiographed. represented by the single letter code. Gaps have been introduced in the sequences to optimize the alignment. Regions of sequence identity RESULTS have been boxed. Vertical arrowheads delineate the highly homologous region of 455 amino acids. The underlined amino acid residues rep- Sequence Comparison of RatPDE3 and RatPDE4"As pre- resent the sequence of a synthetic peptide (K111) that was used to viously reported (16), two groups of cDNA clones (ratPDE3 raise polyclonal antibodies. A putative nuclear translocation signal and ratPDE4) were isolated from a cDNA library derived sequence is indicated by small arrows. Horizontal arrowheads deline- from cultured 15-day-old Sprague-Dawley rat Sertoli cells ate the region of ratPDE4 shown in Fig. 2. Putative sites for phos- phorylation by casein kinase I1 are indicated by asterisks. that had been treated with 500 PM Bt,cAMP (19). The DNA Hormonal Regulation of CAMP Phosphodiesterases 18373 the SV40 large T antigen (37) and other nuclear proteins (38- both PDEs (1.9 and 2.5 p~ cAMP for ratPDE3 and ratPDE4, 42). respectively). Velocity determined in crude extracts ranged A cDNA clone (DPD) similar to the ratPDE4 clones has 1 nmol/min/mg of protein (Table I). In between 0.5 and also been isolated from rat brain (18). 525 codons of the open addition, both recombinant enzymes displayed similar sensi- reading frame and the available 3"untranslated region of the tivities to a variety of PDE inhibitors, rolipram and Ro 20- ratPDE4 and DPD clones are identical (Fig. 2). The 5' por- 1724 being more effective inhibitors than cilostamide or mil- tions of the putative open reading frames are unrelated (Fig. rinone (Table I). 2). The 5' portion of our ratPDE4 sequence was derived from Since expression in prokaryotic cells does not allow the two independent clones (ratPDE4.1 and ratPDE4.2) from a same post-translational modifications as in eukaryotic cells, Sertoli cell unamplified library. In addition, the same se- the two cDNAs were also expressed in monkey kidney (COS- quence was present in a genomic clone derived from a rat 7) cells. Affinities for cAMP of the two PDEs recovered from cosmid library (data not shown), ruling out the possibility of COS-7 cell transfection were very similar to those determined cloning artifacts. from the bacterial expression (ratPDE3 K,, = 1.81; ratPDE4 Characterization of the Recombinant PDE Activity Encoded K,, = 2.85). However, although Northern and Western blot by RatPDE4 and Comparison with RatPDE3-The high de- analysis indicated that transfection efficiency, transcription, gree of homology between the ratPDE3 and the ratPDE4 and translation of the two constructs in COS-7 cells was sequences suggests that both enzymes have similar kinetics comparable, differences were observed in the amount and in and physicochemical characteristics. To verify this possibility, intracellular distribution of the CAMP-PDE activity re- both PDEs were expressed in prokaryotic as well as in eukar- covered (PDE activity in picomoles/min . mg of protein ? S.E., yotic cells. n = 10: mock supernatant: 179 & 26; mock pellet: 49 & 15; To express the two cDNAs in E. coli, cDNAs were modified ratPDE3 supernatant: 716 ? 90; ratPDE3 pellet: 171 k 25; to arrange the putative initiation methionine immediately ratPDE4 supernatant: 308 & 56; ratPDE4 pellet: 143 & 11). after the ribosomal-binding site of the pRC23 vector. Trans- Further experiments are required to clarify these findings. formation of E. coli DH5a with pRC23-ratPDE3 and pRC23- Regulation of Steady State mRNA Levels for RatPDI33 and ratPDE4 led to the appearance of major immunoreactive RatPDE4 in Sertoli Cells-Previous biochemical characteriza- bands (with the Klll antibody, see "Experimental Proce- tion of the Sertoli cell PDEs indicated that rolipram-sensitive dures") of 71 and 68 kDa, respectively (data not shown). Less PDEs were present in this cell, mostly after hormonal stim- intense immunoreactive bands of lower molecular weight were ulation (13). The cloning data suggested that mainly ratPDE3 often recovered, suggesting degradation of the recombinant and ratPDE4 were expressed in the Sertoli cell (16). To protein. The immunoreactivity was associated with the ap- further assess this possibility, Sertoli cell mRNA was hybrid- to the PDE cDNA clones available. These Northern blot pearance of high affinity PDE activity in the bacterial lysates. ized cAMP hydrolysis by both recombinant enzymes followed Mi- analyses indicated that ratPDE3 and ratPDE4 are the two chaelis-Menten kinetics and Lineweaver-Burke plots were dominant ratPDEs expressed in these cells, and that the linear with substrate concentrations ranging between 0.1 and cAMP analog treatment has major effects on mRNA levels. 50 PM. The K, values for cAMP (Table I) were similar for Sertoli cells from 15-day-old rats cultured for 4 days in defined medium expressed ratPDE3 mRNA levels that were at the limit of detection by Northern blot analysis (Figs. 3 and 4). PDE4: MKEQGGTVSGAGSSRGGG 0 D~A H~L Q~L Q P N After 24 h treatment with the cAMP analog Bt, CAMP, DPD : SLRIVRNNPTLLTNLHGAPNKRSPAASQAP however, the ratPDE3 steady state transcript level is in- PDf4: YLSVCLFAEESYQKLAHETLEELDWCLD ..... creased at least 100-fold (19). To test if this is also the case DPD : VTRVSL 00 QEESYQKLAMETLEELDWCLD I ..... dbcAMP dbcAMP dbcAMP FIG. 2. Comparison of the deduced amino acid sequences of I- + ' I" + ' I- +t ratPDE4 and DPD. Amino acid sequences of the major open kb reading frames deduced from ratPDE4 and DPD (18) cDNAs were - compared using the MicroGenie align routine. Amino acid residues of the amino-terminal region of ratPDE4 (as delineated in Fig. 1) and of the corresponding region of DPD are represented by the single letter code. Gaps have been introduced in the sequences to optimize -9 alignments. Regions of sequence identity have been boxed. ao TABLE I Comparison of the properties of recombinant ratPDE3 and ratPDE4 expressed in E. coli Recombinant Recombinant Properties ratPDE3 ratPDE4 Calculated molecular mass 66,249 Da 64,273 Da SDS-PAGE 71 K,,, CAMP (phi) 1.9 2.5 ND" K,,, cGMP (pM) ND actin ratPM3 ratPDM Specific activity extract 593 k 65 851 f 143 FIG. 3. Regulation of ratPDE3 and ratPDE4 steady state (pmol/min. mg of pro- mRNA levels by dibutyryl cAMP in the rat Sertoli cell. Sertoli tein) cells from 15-day-old Sprague-Dawley rats were cultured for 4 days Inhibitors (ED,) in pM) in medium without serum (26). The cells were incuhat.ed in the cGMP >50 >50 absence or presence of 0.5 mM Bt,cAMP. After 24 h, poly(A)' RNA Methylisobutylxanthine 18.5 f 2.0 21.0 f 3.0 was prepared and (5 pg) separated on a 1% agarose gel, blotted, and Rolipram 0.6 f 0.1 0.4 k 0.1 hybridized with '"P-labeled ratPDE3 cDNA and autoradiographed. RO 20-1724 1.6 f 0.9 1.2 & 0.2 After removal of the probe, the membrane was rehyhridized with Cilostamide 38.6 f 5.1 29.0 f 2.7 ratPDE4 cDNA and consecutively with actin cDNA. Size markers " ND, not detectable. were radiolabeled HindIII-digested X DNA fragments. kDa 68 kDa 18374 Hormonal Regulation of CAMP Phosphodiesterases were present under basal conditions, while ratPDE3 tran- scripts were not detectable under these experimental condi- tions. Marked differences in hormone dependence of the two PDEs were also present when rats were treated with FSH in vivo. Whereas ratPDE3 mRNA levels undergo a large increase after FSH injection, no significant changes in ratPDE4 mRNA could be detected under these conditions (data not shown). Regulation of Transcription of RatPDE3 and RatPDE4- To test whether the differences in regulation of steady state mRNA levels for ratPDE3 and ratPDE4 are caused by differ- ences in mRNA stability or by differential regulation of gene transcription, nuclear run-off assays were performed. The FIG. 4. Dose dependence of the FSH stimulation of ratPDE3 rate of transcription of c-fos and ubiquitin was measured as and ratPDE4 steady state mRNA levels in the rat Sertoli cell. positive and negative control for the FSH effects. In the three On the fourth day of culture in defined medium, primary Sertoli cells were rinsed and incubated for 24 h with increasing concentrations of different experiments performed, Sertoli cell nuclei isolated FSH. Total RNA was prepared, separated on a 1% agarose gel, blotted prior to FSH treatment showed low levels of ratPDE3 and onto a nylon membrane, and hybridized with :"P-labeled ratPDE3 ratPDE4 transcription (Table 11). The transcription rate of cDNA and autoradiographed. After removal of the probe, the mem- ratPDE3 increased earlier than 30 min after FSH treatment, brane was rehybridized with ratPDE4 cDNA and consecutively with reached a maximum at 3 h, and returned towards basal within actin cDNA. The mRNA abundance as determined by densitometric 24 h. The rate of transcription of ratPDE4 did not signifi- scanning of the autoradiograms are expressed in arbitrary units. cantly change during the time period studied. Furthermore, dibutyryl CAMP, forskolin, and cholera toxin produced an increase in the ratPDE3 rate of transcription similar to FSH (Table 111). This indicates that hormone effects on PDE gene TABLE I1 Time course of FSH regulation of ratPDE3 and ratPDE4 rate of transcription 4.Okb". ----~--~~y-~qw--~ rotPDE4 Nuclear run-off assays were performed as described under "Exper- J. imental Procedures." Five micrograms of plasmids containing rat- FIG. 5. Time course of the regulation of the ratPDE3 and PDE3, ratPDE4, c-fos, or ubiquitin cDNAs were immobilized on ratPDE4 steady state mRNA levels by FSH in the rat Sertoli nylon filters. Sertoli cells from 17-day-old Sprague-Dawley rats were cell. Sertoli cells from 15-day-old Sprague-Dawley rats were cultured treated with 500 ng/ml of FSH for the indicated lengths of time, in defined medium. On the fourth day of culture the cells were treated nuclei were prepared, and incubated with [n-'"P]UTP. The total :'T with 100 ng of FSH/ml of medium. At the indicated times total RNA incorporation into newly transcribed RNA was estimated by scintil- was prepared. Ten micrograms of RNA of each time point was lation counting. After hybridization to the nylon strips containing glyoxylated and separated on a 1% agarose gel. After blotting onto a the plasmids, the filters were washed and exposed to x-ray film. The nylon membrane the blot was hybridized with '"P-labeled ratPDE3 radioactive spots were excised from the membranes and counted. cDNA and autoradiographed. After removal of the ratPDE3 probe, These counts were divided by the total number of counts in the the membrane was rehybridized with radiolabeled ratPDE4 cDNA hybridization solution to express the rate of transcription in parts and autoradiographed. per million. The rate of transcription of c-fos and ubiquitin was measured as positive and negative control for the FSH effects. for ratPDE4, the same Northern blots were hybridized with Specific RNA concentration after FSH Immobilized radiolabeled ratPDE4 cDNAs of similar specific activities as addition DNA the ratPDE3 cDNAs and autoradiographed. The ratPDE4 0 90 min 3h 24 h steady state transcript level was readily detectable under basal PPm conditions and was increased only about 5-fold as determined RatPDE3 ND" 3.97 12.93 1.07 by spectrophotometric scanning (Fig. 3). These differences RatPDE4 1.19 0.63 1.15 0.83 were confirmed in several other Northern blot experiments 10.27 2.17 19.31 2.65 c-fos from independent Sertoli cell cultures and were also evident 3.03 4.03 4.03 6.07 Ubiquitin after FSH or forskolin treatment (Figs. 4 and 5, and data not " ND, not detectable. shown). The increase in steady state levels of ratPDE3 and ratPDE4 mRNA by FSH is dose-dependent and occurs at TABLE I11 physiological concentrations of FSH ranging between 1 and Regulation of ratPDE3 and ratPDE4 rate of transcription by FSH, 100 ng/ml. Whereas ratPDE4 mRNA levels were maximal &CAMP, forskolin, and cholera toxin upon exposure of the cells to 10 ng of FSH/ml of medium, Nuclear run-off assays were performed as described in Table 11. Sertoli cells from 17-day-old Sprague-Dawley rats were incuhated in ratPDE3 mRNA levels could be further elevated with higher the presence or absence of 500 ng/ml FSH, 1 mM Bt,cAMP, 100 pM FSH concentrations (Fig. 4). forskolin, or 100 ng/ml cholera toxin for 3 h. When Sertoli cells were exposed to FSH, the AMP-PDE Specific RNA concentration activity increases after a time lag of 1 h and reaches a Immobilized maximum at 18-24 h (15). The time course of changes in the DNA None FSH Rt,cAMP Forskolin ratPDE steady state mRNA levels in primary Sertoli cell cultures treated with 100 ng/ml FSH is reported in Fig. 5. ppm After a time lag of about 1 h the level of transcripts increased, 0.88 8.31 10.54 8.69 8.80 RatPDE3 RatPDE4 1.57 2.08 2.68 3.01 2.51 a maximum between 3 and 12 h, and then gradually reached c-fos 2.48 7.48 8.73 decreased to near basal levels in a time span of 3 days. 4.15 2.00 2.43 2.93 2.53 Uhiquitin Confirming the above reported data, ratPDE4 transcripts 8.74 9.04 Hormonal Regulation of CAMP Phosphodiesterases 18375 analyzed by immunoblotting. Even after enriching the PDE protein by immunoprecipitation, no detectable band could be observed under basal conditions (Fig. 7A). Conversely, an - immunoreactive 67-kDa band appeared after FSH and dibu- 97 - tyryl cAMP treatment (Fig. 7A). The intensity of these bands correlated with the levels of CAMP-PDE activity measured in 68- - the supernatants before immunoprecipitation (Fig. 7B). Fi- nally, the size of this band was identical to that of a purified -I " " CAMP-PDE from the Sertoli cell (Fig. 7A).' 45 - " " DISCUSSION -peptide + peptide B peptide Biochemical characterization and molecular cloning of the kDa - + CAMP-PDEs has uncovered an unexpected complexity of this 200 - family of enzymes. In the rat there are at least four genes 97 - encoding cGMP-insensitive, rolipram- and Ro 20-1724-inhib- 68" ited CAMP-PDEs (16), and data suggesting alternate splicing open the possibility to expression of an even larger number 45 - of PDE proteins (16, 17, 19). Transcripts derived from two of these genes (ratPDE3 and ratPDE4) are present in the rat Sertoli cell in culture. Although differences in basal levels and 29 - in the degree of hormone stimulation are present, the expres- 18 - sion of both ratPDE3 and ratPDE4 is under the control of follicle-stimulating hormone and CAMP. The data presented FIG. 6. Immunoblot analysis of soluble fractions (A) and here demonstrate that the increase in cAMP hydrolyzing partially purified CAMP-PDE preparations (B) from the rat activity observed in this cell is the result of an increase in Sertoli cell with Klll antiserum. On the fourt,h day of culture rate of CAMP-PDE gene transcription, accumulation of in defined medium, primary Sertoli cells were incubated in the pres- mRNA, and neosynthesis of the CAMP-PDE protein. These ence or absence of 1 mM Bt2cAMP for 24 h. The cells were scraped with a rubber policeman and homogenized in an all-glass Dounce ia w homogenizer. After fractionation of the cell extract by centrifugation at 14,000 X g for 15 min, the supernatant proteins (A) and a CAMP- PDE protein preparation, partially purified from the rat Sertoli celp (R), were subjected to SDS-polyacrylamide gel electrophoresis. After blotting onto nylon membranes, duplicate blots were incubated with the Klll antiserum (raised against a synthetic ratPDE peptide) or with the Klll antiserum preincubated with excess peptide used for 68 - PDE immunizations. Immunoreactive proteins were visualized by incubat- I IgG ing the blots with "'1-labeled protein A and autoradiography. transcription are mediated by the activation of the CAMP- dependent pathway. Regulation of the RatPDE Protein Levels-To determine whether the increase in ratPDE mRNA results in an increase B 'W in PDE protein content, Sertoli cell extracts were subjected to immunoblot analysis. A polyclonal antiserum (K111) was ~ I mi raised against a synthetic peptide based on the sequence of ratPDE3. This antiserum recognizes both recombinant rat- PDE3 and ratPDE4 proteins (data not shown). Although :j immunoblot analysis of crude Sertoli cell extracts showed several bands, only the signals of a 67 and a 54-kDa polypep- ! -1 m o7 tide were abolished by competing the antibody with the pep- :I tide used for immunization (Fig. 6A). The 67-kDa protein i ;/ " . . " i' " _._ level was markedly affected by Bt'cAMP treatment, being 0 ..-.> ry. mw undetectable in extracts from unstimulated Sertoli cells (Fig. 6A). To confirm that the 67-kDa protein corresponds to the FIG. 7. Regulation of CAMP-PDE protein levels (A) and cAMP hydrolyzing activity (B) by FSH and dibutyryl cAMP CAMP-PDE, extracts from stimulated Sertoli cells were par- in the soluble fraction of the rat Sertoli cell. Sertoli cells from tially purified by high performance liquid chromatography' 15-day-old Sprague-Dawley rats were cultured in defined medium. and the peak fraction of CAMP-PDE activity was subjected On the fourth day of culture the cells were incubated in the presence to immunoblot analysis. Under these conditions only the or absence of 500 ng/ml FSH or 1 mM Bt2cAMP. Twenty-four hours immunoreactivity of a 67-kDa polypeptide was present and after the additions the cells were scraped with a rubber policeman the signal was competed by the peptide (Fig. 6B). Using an and homogenized in an all-glass Dounce homogenizer. This cell extract was fractionated by centrifugation at 14,000 X g for 15 min. alternative approach to increase the immunoblot signal, sol- The supernatant fractions were subjected to PDE assays and immu- uble extracts from control, FSH-, or dibutyryl CAMP-stimu- noprecipitation and the anti-CAMP-PDE antibody K111. The im- lated Sertoli cells were first immunoprecipitated with the munoprecipitated proteins were solubilized and separated on a 10% Klll antiserum, and the immunoprecipitated proteins were polyacrylamide gel. CAMP-PDE protein purified from the rat Sertoli cell2 was loaded on the same gel. After blotting onto a nylon mem- ' M. Conti, J. Odeh, J. V. Swinnen, and M. E. Svoboda, submitted brane, the blot was incubated with the Klll antibody and ""I-labeled for publication. protein A and autoradiographed. 18376 Hormonal Regulation of CAMP Phosphodiesterases data, then, provide evidence for a novel mechanism of regu- ratPDE3 gene transcription is slower than that observed for lation of PDEs that involves hormone-dependent CAMP- c-fos. The transcription of c-fos reaches a maximum at the mediated changes in the rate of a PDE gene transcription. 30-min time point, while ratPDE3 transcription peaks only A molecular cloning approach has demonstrated that the after 3 h. It is then to be expected that the FSH-dependent Sertoli cell expresses ratPDE3 and ratPDE4. As previously CAMP-mediated transcription of the two genes involves dif- shown (19), ratPDE3 encodes a protein with a calculated ferent steps. Although the basic mechanism might be the molecular mass of 66.2 kDa, while conceptual translation of same, e.g. phosphorylation of a cAMP response element- the ratPDE4 cDNAs is consistent with a PDE protein of 64.2 binding protein, additional regulatory steps either delay the kDa or higher molecular mass. Although no in-frame up- activation of ratPDE3 gene transcription, or cause a more stream termination codon was found in the cDNA clones that rapid termination of c-fos transcription. The possibility we have available, preliminary sequence available from a should also be considered that the c-fos gene product might genomic clone demonstrates the presence of a stop codon 108 be involved in the modulation of transcription of the ratPDE3 bases upstream from the putative initiation methionine. gene. In fact, the c-fosljun complex is a transcription activator Nevertheless, further studies are needed to unequivocally that binds to an AP-1 regulatory element (45), and hormonal prove that the ATG that we have selected codes for the activation of the Sertoli cell leads to increased levels of both initiation methionine also in uiuo. On the other hand, these fos andjun mRNAs (47). CAMP-PDEs might have multiple initiation methionines (17). The finding that cAMP regulates the expression of CAMP- The ratPDE4 cDNAs isolated from the rat Sertoli cell cDNA PDEs explains earlier reports on cell lines with diminished library have sequences identical to the DPD clone isolated by protein kinase A and phosphodiesterase activity. The kin- complementation from a rat brain library except for the first and PPD mutant strains of S49 lymphoma cells, for instance, 40 amino acids of the putative open reading frames (18). have a decreased protein kinase A activity in comparison with Although cloning artifacts cannot be completely excluded, it the wild type cells. The kinase defect is associated with a is possible that alternate splicing of a single ratPDE4 gene decrease in cAMP hydrolytic activity, indicating that protein occurs in brain and testis. Evidence suggesting alternate splic- kinase A mediates, probably by regulating transcription fac- ing or multiple start sites have also been reported for other tors, the expression of certain PDE forms (48, 49). CAMP-PDE encoding cDNAs (17, 19). In addition, most cell The hypothesis that CAMP-PDE activation is dependent types studied express multiple transcripts of the ratPDE on new synthesis of the PDE protein is here proven by the genes, some of which are tissue specific (16). There is, then, use of polyclonal antibodies. It is, however, still possible that additional modifications such as phosphorylation are neces- potential for the expression of a large number of rolipram- sensitive CAMP-PDEs. That alternate splicing might be a sary to express the full activity of the PDE. One puzzling finding is that although ratPDE4 mRNA is expressed in the property of PDEs is also suggested by data on other PDE families. It has been reported that two distinct amino termini Sertoli cell under basal conditions, no immunoreactive PDE have been found for a Ca2+/calmodulin-dependent PDE (43). protein could be detected in the Sertoli cell soluble fraction. Based on the sequence similarity in the central portion of It is possible that ratPDE4 protein levels are below the the proteins, it was expected that the kinetic properties of sensitivity of the immunodetection employed in our study. both enzymes are very similar. This was confirmed here by An alternative possibility is that the ratPDE4 protein is not present in the soluble cell fraction, but compartmentalizes in prokaryotic expression of the two PDEs. The two enzymes have a similar K,,, for cAMP and similar sensitivity to different other subcellular districts. This latter possibility would also reconcile differences found in the stimulated Sertoli cell. inhibitors. Therefore, the catalytic centers are structurally and functionally very similar. It is possible that the amino While high levels of both ratPDE3 and ratPDE4 mRNA are and carboxyl termini, being different in the two proteins, present under these culture conditions, only one immuno- serve different functions in the two CAMP-PDEs. reactive band could be detected even after immunoprecipita- Both ratPDE3 and ratPDE4 transcript steady state levels tion. Other reports have demonstrated the presence of a low K,,, CAMP-PDE in the particulate fraction of the brain (50). were increased by FSH after a lag of about 1 h and remained 2 days. This is in agreement with the Consistent with this view also is the finding that rolipram elevated for at least (51). appearance of the cAMP hydrolyzing activity in the Sertoli binding to membrane-bound sites was detected in brain Also in the rat liver, a membrane-bound PDE is sensitive to cell supernatant (15). The increase in ratPDE3 mRNA levels or FSH was consistently larger than rolipram (52, 53). in response to Bt,cAMP The time course of this CAMP-mediated activation of these that of ratPDE4. Nuclear run-off experiments showed that a CAMP-PDEs in the Sertoli cell is very different from that 10-fold increase in the rate of ratPDE3 gene transcription followed FSH treatment in uitro. Conversely, the basal tran- described for the CAMP-dependent activation of a cGMP- inhibited PDE from human platelets (54-56) and rat adipo- scription rate of ratPDE4 was at the limit of detection and cytes (57, 58). This latter enzyme is membrane bound and FSH activation of transcription was small and not significant. very rapidly activated by phosphorylation. In a cell where the It is, then, possible that also mRNA stabilization plays a role two modes of regulation coexist, there will be a complex in the regulation of the steady state mRNA levels of these genes. The FSH effect on the ratPDE gene transcription is pattern of PDE activation, with early and delayed increases in PDE activity. The exact significance of this complexity is mimicked by cAMP analogs and other cAMP increasing unclear, but there is evidence that the long term PDE acti- agents, indicating that this hormonal activation of transcrip- vation is involved in the regulation of the hormone respon- tion is mediated by CAMP. Transcriptional activation of cAMP responsive genes such as c-fos is commonly thought to siveness of the Sertoli cell (15). The homologous and heter- ologous desensitization of the rat Sertoli cell is in part or occur via CAMP-dependent phosphorylation of cAMP re- completely reverted after inhibition of the PDEs by Ro 20- sponse element-binding proteins (44) or by regulating the 1724 (13). This indicates that these CAMP-PDEs serve to levels of trans-acting factors that bind to the AP1 and/or AP2 elements (45, 46). Similar regulatory sequences can therefore control the responsiveness of the Sertoli cell to hormones. On the other hand, experiments with in uitro model systems also be expected in the 5’-flanking region of the ratPDE3 gene. It is worth noting, however, that the time course of containing hormone-sensitive adenylate cyclase, CAMP-de- Hormonal Regulation of cAMP Phosphodiesterases 18377 Nucleic Acids Res. 12, 5707-5717 pendent protein kinase 11, and cyclic nucleotide phosphodi- 26. Monaco, L., and Conti, M. (1986) Biol. Reprod. 35, 258-266 esterase support the hypothesis that rapid cAMP turnover 27. Thompson, W. J., and Appleman, M. M. (1971) Biochemistry 10, may function as a mechanism for signal amplification by the 311-316 CAMP-dependent protein kinase (59). These hypotheses can 28. Lineweaver, H., and Burk, D. (1934) J. Am. Chem. SOC. 56,658- now be tested by studying other hormone-responsive cell systems that express a different subset of CAMP-PDEs and 29. Chirgwin, J. 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