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Endocrinology Vol. 140, No. 2 835-843
Copyright © 1999 by The Endocrine Society

ARTICLES

Isoform-Specific Regulation of the CCAAT/Enhancer-Binding Protein Family of Transcription Factors by 3',5'-Cyclic Adenosine Monophosphate in Sertoli Cells1

Line M. Grønning, Maria K. Dahle, Kristin A. Taskén, Sven Enerbäck, Lars Hedin, Kjetil Taskén and Helle K. Knutsen

Institute of Medical Biochemistry (L.M.G., M.K.D., K.A.T., K.T., H.K.K.), University of Oslo, N-0317 Oslo, Norway; Institutes of Molecular Biology (S.E.) and Physiology (L.H.), University of Gøteborg, S-41390 Gøteborg, Sweden

Address all correspondence and requests for reprints to: Line M. Grønning, M.Sc., Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112 Blindern, N-0317 Oslo, Norway. E-mail: l.m.gronning{at}basalmed.uio.no


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The C/EBP (CCAAT/enhancer-binding protein) family of transcription factors is important for differentiation, lipid biosynthesis, and metabolism. Here, we demonstrate for the first time the presence of C/EBP {alpha}, ß, {delta}, and {zeta} messenger RNA (mRNA) and protein in Sertoli cell primary cultures. Treatment with FSH or 8-CPTcAMP strongly induced C/EBP ß mRNA above basal levels with rapid and transient kinetics in Sertoli cell primary cultures as well as in whole testes from hypophysectomized rats. Whereas C/EBP ß mRNA was induced approximately 50-fold, C/EBP {delta} mRNA was induced 5- to 8-fold by cAMP in Sertoli cells. Messenger RNA for C/EBP ß and {delta} were induced by inhibition of protein synthesis with cycloheximide and cycloheximide acted synergistically with cAMP. Immunoblots with C/EBP antibodies demonstrated a strong induction of C/EBP ß, {delta}, and {zeta} by cAMP. Electrophoretic mobility shift analysis of nuclear proteins from cAMP-treated Sertoli cells using a C/EBP consensus oligonucleotide and antibodies revealed specific binding of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP ß antibody. Transfections of Sertoli cells with a C/EBP reporter construct showed approximately 3-fold induction of reporter gene activity by cAMP. In contrast, the reporter gene vector with a mutated form of the C/EBP binding site, was almost unresponsive to cAMP in transfections of Sertoli cells. Furthermore, C/EBP ß expression increased the activities of two promoters known to be cAMP-responsive in Sertoli cells. Thus, the early induction of C/EBP isoforms by cAMP may play a role in FSH-dependent regulation of late response genes in Sertoli cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN SERTOLI CELLS, the somatic cells of the seminiferous tubules of the testis, a number of processes such as structural maturation, metabolic activities and gene transcription are stimulated by FSH. Furthermore, FSH is implicated in the terminal differentiation of Sertoli cells and normally initiates the first wave of spermatogenesis. Although spermatogenesis can be maintained by high levels of testosterone alone, FSH leads to an increased secretion of a variety of proteins, e.g. androgen binding protein, transferrin, and plasminogen activator, which are necessary for spermatogenesis (1). Binding of FSH to its G protein-coupled receptor activates adenylyl cyclase leading to production of cAMP, which subsequently activates cAMP-dependent protein kinase (PKA) (2). Transcriptional activation by cAMP is partly mediated through the cAMP-response element, CRE (TGACGTCA), which is bound by various CREB, CREM and ATF-1 isoforms, some of which activate transcription, whereas others inhibit transcription. The transcriptional activation potency of this leucine zipper family of transcription factors is enhanced by PKA-dependent phosphorylation of a highly conserved phosphorylation-site in the kinase-inducible domain (KID) (e.g. Ser 133 in CREB) (3). CRE-regulated genes have been shown to respond to cAMP both with rapid and sustained kinetics (4, 5). However, several genes with delayed kinetics appear to be regulated by cAMP through an intermediate transcription factor induced by cAMP with rapid kinetics and acting on a "second generation" of genes.

The CCAAT/enhancer binding proteins (C/EBP) is a family of transcription factors associated with differentiation, which belongs to the leucine zipper group of transcription factors. They are encoded by six different genes and denoted C/EBP {alpha}, ß, {delta}, {gamma}, {zeta}, and crp1 (6). The messenger RNA (mRNA) of C/EBP ß (also called LAP: liver enriched activator protein) can be translated from a different down-stream initiation codon within the same reading frame, giving rise to a transcriptional repressor (LIP: liver enriched inhibitor protein) containing the 145 C-terminal amino acids including the dimerization and DNA binding domains, but lacking the N-terminal activation domain (7). The role of C/EBP isoforms during proliferation and differentiation have been well described for liver and fat cells (8, 9). C/EBP ß [also called NF-IL6, IL-6DBP, GPE-BP, CRP2, AGP/EBP or NF-M; for review see (6)] has been implicated in female reproduction, and LH-dependent regulation of C/EBP ß has recently been shown to be necessary for ovulation (10, 11). Suire et al. (12) showed, in cotransfection assays, that overexpression of C/EBP {alpha} (C/EBP) and C/EBP {delta} (NF-IL6ß, CRP3) in Sertoli cells stimulated transcription from the cAMP-regulated transferrin promoter containing a C/EBP binding site. This may implicate C/EBP isoforms in the cAMP-regulation of transferrin. However, no C/EBP isoform mRNAs or proteins have so far been reported in Sertoli cells. In this study, we demonstrate for the first time presence of C/EBP isoforms in Sertoli cells of the testis. We further show an immediate and strong cAMP-mediated induction of C/EBP ß both at mRNA and protein level in rat Sertoli cell primary cultures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation and stimulation of cell cultures
Primary cultures of rat Sertoli cells and peritubular cells were made from testes of 19 days old Sprague Dawley rats (B&K Universal AS, Nittedal, Norway) according to the method of Dorrington et al. with some modifications (13). The cells were plated in 6-well plates (35 mm/well) for transfections or in 10-cm culture dishes (Nunc, Copenhagen, Denmark) for RNA and protein analysis and cultured in modified MEM containing Eagle’s MEM (Gibco BRL, Grand Island, NY, 212090–022) with addition of streptomycin (100 mg/liter), penicillin (105 IU/liter), fungizone (0.25 mg/liter), L-glutamine (2 mM) and FCS (10% Gibco BRL 11099–117) at 32 C in a humidified atmosphere with 5% CO2. After three days, the cells were incubated further in serum-free modified MEM. After 2 days of culture in serum-free medium, the medium was changed, and incubation was continued in presence or absence of FSH (1 µg/ml ovine FSH-S17, NIH, Bethesda, MD), 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) (Sigma Chemical Co., C-3912, St. Louis, MO) and/or cycloheximide (5 µg/ml; Sigma Chemical Co. C-6255). Preovulatory granulosa cells, obtained as previously reported (14), extracts of liver (rat/human) and adipose (human) tissues were used for comparison in the immunoblot assays. The cells and tissues were homogenized as previously described (15).

Animals
Immature Sprague Dawley rats were selected to the same weight at day 20 and hypophysectomized by Møllegaard Breeding Center Ltd (Copenhagen, Denmark). More than 80% of the animals appeared completely hypophysectomized as judged by testes size and weight at day 29, which was 65 ± 5 g. Animals were injected sc with 250 µg FSH-S17 in 0.9% saline with 0.1% BSA.

RNA extraction and Northern analysis
Whole testes of untreated and FSH-injected hypophysectomized rats, and control rats were homogenized in guanidine isiothiocyanate and centrifuged at 500 x g for 5 min. Total RNA from Sertoli cells or whole testes was extracted by the guanidine isothiocyanate/CsCl method as previously described (13, 16). Northern blot analysis was performed using 20 µg total RNA that was denatured in 50% (vol/vol) formamide and 6% (vol/vol) formaldehyde and subjected to electrophoresis in a 1.5% (wt/vol) agarose gel containing 6.7% formaldehyde. Ethidium bromide staining of the gel verified equal loading in each lane. cDNA probes for C/EBP {alpha} (2.6 kb; mouse) (17), C/EBP ß (1.5 kb; mouse) (7), C/EBP {delta} (1.0 kb; mouse) (18) and C/EBP {zeta} (CHOP) (0.64 kb; human) (19) were labeled with [{alpha}-32P]dCTP using megaprime DNA labeling system (Amersham RPN 1607, Arlington Heights, IL) to a specific activity of 0.5–1.0 x 109 cpm/µg. Hybridization was performed with 50% formamide at 42 C according to the ICN procedure. After hybridization, the filters were washed four times in a solution containing 2 x standard saline citrate (SSC; 300 mM NaCl and 30 mM sodium citrate, pH 7.0) with 0.1% SDS at 25 C for 5 min and twice in 0.1 to 0.5 x SSC with 0.1% SDS at 50 C for 30 min. Northern blots were subjected to phosphoimaging or autoradiography using Hyperfilm MP from Amersham, Buckinghamshire, UK. The signal intensities of suitably exposed films were estimated by the use of a densitometer (OmniMedia Scanner; XRS, 6cx, software: Bioimage; Ann Arbor, MI).

Immunoblotting
Sertoli cells (12 x 106 cells) were washed in 5 ml cold PBS and then scraped in 500 µl of a buffer containing 10 mM potassium phosphate, pH 6.8, 1 mM EDTA, 10 mM 3-[(3-cholamidopropyl)dimethyl-ammonio]1-propane sulfonate (CHAPS; Sigma Chemical Co. C-3023), Pefablock (1 mg/ml, Boehringer Mannheim, 1429868, Mannheim, Germany), leupeptin (1 mg/ml, Sigma Chemical Co. L-2884) and pepstatin A (1 mg/ml, Sigma Chemical Co. P-4265). Cell suspensions were sonicated three times for 10 sec (Heat Systems Ultrasonics, NY) and centrifuged for 5 min at 12,000 x g. Supernatants were stored at -70 C until analysis. Samples were diluted in SDS sample buffer before loading on a one-dimensional SDS-polyacrylamide gel (4.5% stacking gel, 12% separating gel; Novex system, San Diego, CA). Thirty-five micrograms of total protein were loaded in each lane, subjected to electrophoresis, and subsequently transferred to polyvinyldifluoride membranes (Millipore Corp., Bedford, MA) by electroblotting (Novex system). The membranes were then blocked in a solution containing PBS, 0.1% MgCl2, 0.3% Tween-20, and 0.2% I-Block (Tropix, Bedford, MA) and incubated with rabbit polyclonal antibodies against C/EBP {alpha}, ß, {delta}, and {zeta} (CHOP, GADD153) (1:1000) (Santa Cruz Biothechnology, Inc., Santa Cruz, CA). Immunoreactive proteins were visualized by chemiluminescense using an alkaline phosphatase-conjugated secondary antibody (1:40.000) (Tropix) and CDP-Star (Tropix) as substrate.

Preparation of nuclear extracts
Sertoli cells (12 x 106 cells) were scraped in HBSS containing 0.1% fatty acid free BSA, harvested by centrifugation at 320 x g at 4 C for 5 min, and washed in cold PBS. Cell pellets were resuspended in 450 µl hypotonic buffer (10 mM Tris, pH 7.6, 10 mM NaCl, 3 mM MgCl2) followed by addition of 50 µl 5% NP-40 lysis buffer (Sigma Chemical Co. N-3516) and the nuclei pelleted by centrifugation at 130 x g at 4 C for 5 min. Nuclei were resuspended in 1 ml hypotonic buffer followed by centrifugation at 130 x g at 4 C for 5 min. Nuclei pellets were resuspended in 100 µl of a buffer containing 5 mM HEPES, pH 7.9, 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF and extracted with NaCl (400 mM) while mixing for 30 min at 4 C followed by centrifugation at 30 000 x g for 20 min at 4 C. The supernatant was stored at -70 C until analysis.

DNA protein complex analysis
Electrophoretic mobility shift assays (EMSAs) were performed using double-stranded 32P end-labeled C/EBP consensus oligonucleotide (5' GATCGATTGCGCAATC 3'). For each reaction, 2 x 104 cpm of labeled probe was incubated with 2.5 µg of crude nuclear proteins from Sertoli cells, and 0.5 µg of poly dI-dC in a buffer containing 5 mM HEPES, pH 7.9, 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF with 120 mM KCl and 5 mM MgCl2 at room temperature for 15 min. Competition experiments were performed in the presence of 300-fold molar excess of unlabeled probe or with mutated C/EBP sequence (5' GATCGAGACTAGTCTC 3'). Supershift experiments were performed by incubation of nuclear extract/DNA with C/EBP ß antibody (Santa Cruz Biotechnology, Inc.) for 30 min at 4 C. Samples were run in 6% nondenaturing polyacrylamide gels at 150 V in Tris-glycine buffer (50 mM Tris, pH 8.5, 380 mM glycine, 2 mM EDTA) at 4 C. Subsequently, gels were dried and subjected to autoradiography.

Plasmid constructions
Constructs containing a single copy of the C/EBP binding site or a mutated form of the consensus C/EBP site (5' GATCGAGACTAGTCTC 3') inserted in front of the herpes simplex thymidine kinase promoter (-81 to +52) fused to firefly luciferase reporter gene in the vector pT81, were used for transfections. The plasmid pCATControl (Promega Corp., Madison, WI, E1011) was cotransfected as internal control. The vector pT81 was a kind gift from Dr. Johan Lund (University of Bergen, Norway). A construct containing the basal promoter and cAMP-responsive region of the rat RIIß 5'-flanking region (-723 to -123) in front of a CAT-reporter gene (pCATbasic; Promega Corp.) (20) and a construct containing the cAMP-responsive region of the rat phosphodiesterase (PDE) 4D1/2 5'-flanking region (-1540 to +2) in front of a luciferase reporter gene (pGL2Basic; Promega Corp.) (21) were cotransfected with either the CMV-C/EBP ß (NF-IL6) expression vector (22) or the CMV-containing pCRTM3 vector (Invitrogen Corp., San Diego, CA). The CMV-C/EBP ß expression vector was a kind gift from Dr. Shizuo Akira (Hyogo College of Medicine, Hyogo, Japan). The PDE4D1/2 reporter construct was kindly provided by Drs. Elena Vicini (University of Rome La Sapiensa, Rome, Italy) and Marco Conti (Stanford University Medical Center, University La Sapienza, Rome, Italy, Stanford, CA).

Transfections, luciferase assays, and CAT assays
Transient tranfections of Sertoli cell- and peritubular cell primary cultures were carried out after 2 days of culture in serum-free medium. Lipofectamine-mediated transfections were performed essentially as recommended by the manufacturer (Gibco BRL) using 2 µg DNA (1.5 µg reporter and 0.5 µg internal control) with 5 µl lipofectamine in 1 ml serum-free modified MEM without antibiotics per 35-mm well. After 3 h, media were changed to modified MEM containing antibiotics. After 18 h, cells were stimulated with 8-CPTcAMP (100 µM). Cotransfections in Sertoli cells were performed with either the CMV-C/EBP ß expression vector (250 ng) or the vector pCRTM3 (250 ng) with 1.5 µg reporter plasmid. Total amount of DNA was adjusted to 2 µg with the pUC18 plasmid (CLONTECH Laboratories, Inc., Palo Alto, CA, catalog no. 6110–1). Cells were harvested in reporter lysis buffer (Promega Corp., E397A) after 28 h of stimulation. Luciferase activities were measured using luciferase assay reagent (Promega Corp., E3971) and an LKB Wallac 1251 luminometer (LKB, Helsinki, Finland). CAT-activities were measured according to the organic phase extraction method with some modifications (23).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FSH induces mRNA for C/EBP ß in Sertoli cell primary cultures
Hybridization of Northern blots with RNA from Sertoli cell primary cultures with 32P-labeled probes for C/EBP {alpha}, ß, {delta}, and {zeta} demonstrated very low or undetectable levels of C/EBP isoform mRNAs under basal conditions. Figure 1Go shows a representative Northern blot of C/EBP ß. Treatment with FSH-S17 (1 µg/ml) strongly induced C/EBP ß mRNA with rapid and transient kinetics with maximum induction approximately 100-fold above basal levels at 1.5 h of stimulation. The C/EBP ß mRNA level was decreased to control levels at 12 h of stimulation with FSH.



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  		Figure 1. Time-dependent regulation of C/EBP ß mRNA by FSH in Sertoli cell primary cultures. Sertoli cells were incubated in the absence (B; same level at 1.5–12 h) or presence of ovine FSH-S17 (1 µg/ml) for upto 12 h. Total cellular RNA was prepared and examined by Northern analysis. The blot was hybridized with a 32P-labeled cDNA probe for C/EBP ß. One representative of two independent experiments is shown. C/EBP {delta} mRNA was induced 3- to 5-fold with similar kinetics on the same blots (not shown).

 
cAMP induces mRNA for C/EBP ß and {delta} in Sertoli cell primary cultures
Figure 2AGo shows representative Northern blots of C/EBP ß and {delta}. Treatment with 8-CPTcAMP (100 µM) induced C/EBP ß mRNA to high levels with rapid and transient kinetics (Fig. 2AGo, upper panel). Densitometric analysis from several experiments demonstrated that C/EBP ß mRNA levels were induced by cAMP to approximately 50-fold above basal levels with maximum at 1 to 2 h, which were decreased to control levels at 24 h (Fig. 2BGo). C/EBP {delta} mRNA was also induced with a transient and rapid time course (Fig. 2AGo, lower panel). However, the magnitude of stimulation was only 5- to 8-fold, and the induction was sustained for up to 4 h, whereafter an inhibitory effect of prolonged incubation with cAMP was consistently observed at 12 to 48 h. C/EBP {alpha} and {zeta} mRNA was detected only at very low levels and appeared not to be regulated by cAMP.



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  		Figure 2. Time-dependent regulation of mRNA for C/EBP ß and {delta} by cAMP in Sertoli cell primary cultures. A, Sertoli cells were incubated in the absence (B) or presence of 8-CPTcAMP (100 µM) (S) for up to 48 h. Total cellular RNA was prepared and examined by Northern analysis. The blots were hybridized with 32P-labeled cDNA probes for C/EBP ß and C/EBP {delta}. One representative of three independent experiments is shown. Levels of C/EBP ß mRNA based on densitometric scanning results from three individual experiments (Basal; open symbols, 8-CPTcAMP-stimulated; filled symbols; data from all three experiments were plotted) are shown in (B) as arbitrary units relative to the level of C/EBP ß mRNA in cells stimulated with 8-CPTcAMP for 2 h. Densitometric analysis of C/EBP {delta} (not shown) revealed a 5- to 8-fold up-regulation by cAMP in one blot. However, lower levels of expression in untreated controls precluded quantitative densitometric analysis of other experiments with similar apparent regulation.

 
Concentration-dependent regulation of C/EBP ß mRNA by cAMP in Sertoli cell primary cultures was examined at 2 h. Figure 3Go demonstrates that maximal induction of C/EBP ß mRNA (approximately 50-fold) was obtained at 100 µM of 8-CPTcAMP. Half-maximal induction was at approximately 10 µM of 8-CPTcAMP, which is consistent with effects of cAMP on other parameters in Sertoli cells as well as in other cell systems (2, 24, 25).



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  		Figure 3. Concentration-dependent effects of 8-CPTcAMP on C/EBP ß mRNA in Sertoli cells. Sertoli cells were incubated with increasing concentrations of 8-CPTcAMP (3–300 µM) for 2 h. Total cellular RNA was prepared and examined by Northern analysis. A representative Northern blot with its corresponding densitometric scanning data are shown. The experiment was repeated twice with similar results.

 
Effect of inhibition of protein synthesis on cAMP-mediated induction of C/EBP ß mRNA
The cAMP-mediated induction of C/EBP ß was examined in the absence and presence of an inhibitor of protein synthesis (cycloheximide). Figure 4Go depicts densitometric scanning results of a Northern blot hybridized with a C/EBP ß complementary DNA (cDNA) probe. C/EBP ß mRNA was induced approximately 4-fold by 8-CPTcAMP (suboptimal dose; 30 µM) at 6 h. In contrast, combined treatment of 8-CPTcAMP and cycloheximide in cells that had been pretreated with cycloheximide for 2 h before addition of 8-CPTcAMP for 6 h, resulted in a synergistic induction of C/EBP ß mRNA to levels 2- to 3-fold above levels of C/EBP ß mRNA in cells treated with 8-CPTcAMP alone. Furthermore, treatment with protein synthesis inhibitor alone resulted in a strong induction of C/EBP ß mRNA levels to 10-fold above basal levels. Similarly, C/EBP {delta} mRNA was induced by cycloheximide and strongly induced with the combined treatment of cycloheximide and cAMP. In addition, C/EBP {zeta} mRNA was detected at low levels and weakly induced with rapid (1 h) and transient kinetics by cAMP in the presence of cycloheximide (data not shown). C/EBP {alpha} mRNA was detected at low levels and weakly induced with late kinetics (6–12 h) by cAMP in the presence of cycloheximide (data not shown). Cycloheximide alone did not induce C/EBP {zeta} and {alpha} mRNAs.



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  		Figure 4. Effect of cycloheximide on 8-CPTcAMP-mediated induction of C/EBP ß mRNA. Sertoli cells were incubated in the absence (B) or presence of 8-CPTcAMP (cAMP; 30 µM), cycloheximide (cyc; 5 µg/ml) or a combination of cycloheximide and 8-CPTcAMP where cycloheximide (8 h) was added 2 h before cAMP (6 h). Total cellular RNA was prepared and examined by Northern analysis. Relative levels of C/EBP ß mRNA are depicted from densitometric scanning of suitably exposed autoradiograms. One representative of three individual experiments is shown.

 
C/EBP ß (LAP/LIP), {delta} and {zeta} protein are induced by cAMP in Sertoli cell primary cultures
We further investigated the expression of immunoreactive isoforms for C/EBP in Sertoli cells. Immunoblots with C/EBP ß antibody (Fig. 5AGo), demonstrated detectable levels of C/EBP ß (LAP/LIP) protein under basal conditions (2 h after change of medium). However, the basal level of C/EBP ß protein was increased after 8 h in culture. Treatment with 8-CPTcAMP (100 µM) for 2 h strongly induced C/EBP ß with maximal induction after 4 to 8 h of stimulation with 8-CPTcAMP. C/EBP ß protein was also detected in rat preovulatory granulosa cells obtained 6 h after stimulation by hCG in vivo, in rat and human liver extracts and human adipose tissue extracts. Levels of C/EBP ß in Sertoli cells under basal conditions were equivalent to the observed levels in rat liver and hCG-stimulated granulosa cells. Three isoforms of C/EBP ß/LAP were observed in the molecular weight range of 34–39 kDa (Fig. 5AGo; upper and lower panel). The upper panel of Fig. 5AGo is a longer exposure, showing the induction of the truncated, nontransactivating form of C/EBP ß (LIP, 20 kDa), which was induced with the same kinetics as LAP. Sertoli cell C/EBP {alpha} immunoreactive protein comigrated with C/EBP {alpha} in rat liver and was not induced by 8-CPTcAMP (Fig. 5BGo, upper panel). Granulosa cells showed low levels of an immunoreactive band with slightly higher mobility than in Sertoli cells and rat liver. Additional 30-, 20-, and 19-kDa immunoreactive bands were observed in granulosa cells and rat liver. C/EBP {delta} (Fig. 5BGo, middle panel) was induced from low levels under basal conditions with the same kinetics as C/EBP ß. C/EBP {zeta} was induced with slower kinetics, from low basal levels, to a weak induction after 8 h of stimulation with 8-CPTcAMP (Fig. 5BGo, lower panel). Levels of C/EBP {delta} and {zeta} in Sertoli cells were considerably lower in Sertoli cells compared with the expression in rat granulosa cells and liver, respectively. The identity of the slower migrating band detected by the C/EBP {delta} antibody in granulosa cells is unknown. This antibody was specific for rodent C/EBP {delta}, explaining the lack of detection of C/EBP {delta} in human liver and adipose tissue. All antibodies to C/EBP isoforms also detected a band at approximately 50 kDa (not shown), known as C/EBP reactive material (crm) the identity of which is unknown (18).



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  		Figure 5. Time-dependent cAMP-mediated regulation of C/EBP ß (LAP/LIP), {alpha}, {delta}, and {zeta} in Sertoli cell primary cultures. Sertoli cells were incubated in the absence (control) or presence of 8-CPTcAMP (100 µM) for 2, 4, or 8 h. Total protein extracts were prepared and examined by immunoblotting, using antibodies to C/EBP ß (LAP/LIP) (A), C/EBP {alpha}, C/EBP {delta} and C/EBP {zeta} (B). Two different exposures are shown to resolve both the LAP and LIP forms of C/EBP ß. Rat preovulatory granulosa cells, rat and human liver, and human adipose tissue were used as controls. Brackets indicate migration of specific C/EBP isoforms. Arrows indicate mobility of See-Blue molecular weight markers (Novex). Whereas the levels of the unregulated C/EBP {alpha} and {zeta} were examined once, regulation of levels of C/EBP ß and {delta} were examined in several independent experiments (n = 5 (ß); n = 3 ({delta}), of which one representative experiment is shown).

 
FSH induces C/EBP ß mRNA in whole testes of hypophysectomized rats
To study whether C/EBP ß is induced by FSH in vivo, groups of hypophysectomized rats (n = 3) were either injected with FSH-S17 (250 µg) or left untreated and the animals were killed 6 h later. Figure 6Go shows Northern blot analysis of C/EBP ß levels in total RNA from testes of a representative hypophysectomized (Hypox) rat (lane 1), a hypophysectomized rat injected with 250 µg FSH-S17 (lane 2), and a control rat (lane 3). Treatment with FSH induced C/EBP ß mRNA approximately 10-fold and 7-fold above levels in hypophysectomized- and control rats, respectively. C/EBP ß levels in control rats were approximately 1.5-fold higher than in hypophysectomized rats.



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  		Figure 6. FSH induces C/EBP ß mRNA in whole testes of hypophysectomized (Hypox) rats. Rats were hypophysectomized at day 20, completely hypophysectomized animals were randomized into two groups at day 29, injected with FSH (250 µg ovine FSH-S17) or left untreated and killed 6 h later. Total RNA from whole testes of untreated (-FSH, lane 1), rats injected with FSH (+FSH, lane 2) and control rats was prepared and examined by Northern analysis. The blot was hybridized with 32P-labeled probe for C/EBP ß. Data from one representative of three individual animals in each group is shown.

 
EMSA reveals specific C/EBP DNA binding in nuclear extracts from cAMP-treated Sertoli cells
Specific binding of nuclear proteins from unstimulated and cAMP-stimulated Sertoli cells to a 32P-labeled consensus C/EBP oligonucleotide was examined (Fig. 7Go). Binding to the labeled C/EBP consensus site was strongly induced by cAMP (lane 2 vs. lane 6). Proteins binding to the labeled DNA-fragment could only be displaced by the homologous unlabeled probe (lanes 3 and 7) and not by the mutated oligo (lanes 4 and 8). Addition of an antibody to C/EBP ß supershifted the majority of specific complexes (lanes 5 and 9). Incubation with an antibody to C/EBP {delta} resulted in a supershift where only a minor fraction of the specific complexes shifted mobility (data not shown).



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  		Figure 7. Specific C/EBP DNA binding induced in nuclear extracts from 8-CPTcAMP-treated Sertoli cells. EMSA and supershift experiment with a C/EBP oligonucleotide (5' GAT CGATTG CGC AAT C 3') as the 32P-labeled probe and the C/EBP oligonucleotide (+; lanes 3 and 7) or mutant (5' GATCGAGACTAGTCTC 3') (mut; lanes 4 and 8) as the competitor (300 x molar excess). A C/EBP ß antibody directed against the carboxy terminus of rat C/EBP ß was used for the supershift experiment (lane 5 and 9). Complex formation was analyzed using 2.5 µg protein from nuclear extracts of unstimulated (lanes 2 to 5) or cAMP-stimulated (8-CPTcAMP, 100 µM, 6 h; lanes 6 to 9) Sertoli cells. Lane 1 is probe in the absence of nuclear extracts. One representative of five individual experiments is shown.

 
Increased C/EBP reporter activity by cAMP is specific to Sertoli cells and is not observed in peritubular cells
Sertoli- and peritubular cell primary cultures were transfected with luciferase reporter vector-constructs containing one single copy of the C/EBP binding site, or a mutated form of the same C/EBP site inserted upstream of the thymidine kinase promoter (Fig. 8Go). Transfections using the construct containing the C/EBP binding site showed an approximately 3-fold induction of luciferase activity by 8-CPTcAMP. In contrast, transfections with the reporter gene vector with a mutated form of the C/EBP binding site, demonstrated lower basal level of expression and was almost unresponsive to cAMP in Sertoli cells (Fig. 8AGo). Furthermore, both constructs containing the C/EBP binding site and the mutated binding site were unresponsive to 8-CPTcAMP when transfected in peritubular cells (Fig. 8BGo). Both CAT and luciferase activities were consistently higher in peritubular cells, due to a higher transfection efficiency in these cells.



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  		Figure 8. Increased C/EBP reporter activity in cAMP-stimulated Sertoli cells. Constructs (1.5 µg) containing a single copy of the C/EBP binding site or a mutated form of the C/EBP site inserted in front of the thymidine kinase/luciferase reporter gene in the vector pT81 were transfected into Sertoli cells (A) and myoid peritubular cells (B). Twenty hours after transfection, the cells were either left untreated (open bars) or treated with 8-CPTcAMP (100 µM) for 28 h (hatched bars). Data represent luciferase activity normalized for CAT activity directed by a cotransfected control plasmid. (Data represents mean ± SEM of three separate transfections performed in triplicate).

 
C/EBP ß cotransfections increases reporter activities driven by the PKA/RIIß- and PDE4D1/2 promoters in Sertoli cells
To examine the effect of C/EBP ß on known cAMP-responsive genes, we cotransfected C/EBP ß expression vector with the previously characterized cAMP-responsive 5'-flanking region of the gene encoding the RIIß subunit of protein kinase A (20) and the promoter directing expression of the phosphodiesterase (PDE) 4D1/2 isoforms (21). Table 1Go shows that expression of C/EBP ß increased the reporter activities of RIIß and PDE4D1/2 4.1- and 3.9-fold, respectively. Expression of C/EBP ß was verified by Western blotting analysis (not shown). Furthermore, C/EBP ß expression increased the activity of the C/EBP reporter 3.7-fold (Table 1Go).


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  		Table 1. C/EBP ß cotransfection increases reporter activities driven by the PKA/RIIß- and PDE4D1/2 promoters in Sertoli cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study reports for the first time the presence of several members of the C/EBP transcription factor family at the mRNA- and protein level in Sertoli cell primary cultures. Treatment with 8-CPTcAMP induced all isoforms of C/EBP with the exception of C/EBP {alpha}. The responses to cAMP were different both in kinetics and in magnitude. Furthermore, we show that C/EBP ß is induced by FSH in vivo as evidenced by the induction of C/EBP ß mRNA in whole testes of hypophysectomized rats injected with FSH. Finally, expression of C/EBP ß increased the activity of two FSH/cAMP responsive promoters, indicating that up-regulation of C/EBP may be implicated in FSH-dependent activation of late response genes in Sertoli cells.

Transcriptional activation of certain genes containing functional CRE, AP-2 or SRE elements occurs rapidly and does not require protein synthesis (26, 27). The various isoforms in the C/EBP family represent different proteins translated from distinct genes on separate chromosomes (6). The rapid kinetics of the cAMP-mediated induction of C/EBP ß and {delta} mRNA, together with the synergistic induction by the combination of cycloheximide and cAMP, indicates that C/EBP ß and {delta} are immediate early genes in Sertoli cells, and that their regulation by cAMP is not dependent on de novo protein synthesis. In fact, the induction of C/EBP ß mRNA is faster than the induction of c-fos in Sertoli cells (28). Furthermore, C/EBP ß has been shown to be involved in regulation of c-fos in NIH 3T3 fibroblasts (29). Thus, cAMP-mediated transcriptional regulation of C/EBP is probably directly activated by preexisiting factors modified via phosphorylation by PKA. In addition to transcriptional activation of C/EBP by cAMP, we cannot rule out the possibility that cAMP might have a stabilizing effect on C/EBP mRNA transcripts. C/EBP ß and {delta} has been reported to be regulated by lipopolysaccarides or inflammatory cytokines such as IL-1, IL-6, and TNF with rapid kinetics in mouse liver, kidney, lung, and spleen (22, 30, 31). cAMP has been shown to increase C/EBP {delta} mRNA during fetal lung development and with sustained kinetics in osteoblasts (32, 33). The induction of all C/EBP isoform mRNAs by treatment with cycloheximide in the present study, indicate that the basal levels of C/EBP mRNAs are under the control of proteins with rapid turnover controlling mRNA stability and/or transcription.

The 5'-flanking region of the rat C/EBP ß gene contains at least two putative CREB binding sites (34). Niehof et al. (34) showed in NIH 3T3 fibroblasts and Neuro 217 cells that CREB was able to induce transcription of C/EBP ß through binding to two CREB binding sites in its promoter. The 5'-flanking region of the rat C/EBP {delta} gene was recently characterized (35, 36), revealing one putative CREB binding site (37). The C/EBP {alpha} promoter contains no CREB binding site but has a Myc/USF site that is implicated in the tissue specific expression of this isoform (38). The presence or absence of CRE binding sites in the C/EBP promoter regions is consistent with the differential regulation of C/EBP isoforms by cAMP. Furthermore, cis-acting elements other than CRE and trans-acting factors other than CREB isoforms may be involved in cAMP-mediated regulation of C/EBP genes. However, a molecular explanation for the differential regulation of C/EBP isoforms by cAMP will have to await a more detailed examination of the respective promoters.

The C/EBP ß-gene encodes three in-frame methionines that can potentially give rise to three translation products of 39 (LAP), 36 (LAP), and 20 kDa (LIP) (7). It has not been established whether 39-kDa LAP and 36-kDa LAP are different translation products initiating at the first (+1) and second (+24) methionine or whether 39-kDa LAP is a more highly phosphorylated form of 36-kDa LAP. In Sertoli cells, one isoform of LAP was observed under basal conditions with a molecular weight of about 36 kDa that comigrated with immunoreactive C/EBP ß in granulosa cells. After stimulation with cAMP, two new isoforms were observed with molecular weights of approximately 39 kDa and 34 kDa. The occurrence of three LAP forms with different mobilities in cAMP-stimulated Sertoli cells could be due to phosphorylation/dephosphorylation induced by PKA, and/or cAMP could possibly direct alternative initiation of translation, indicating that the 39-kDa form is an inducible translation product in Sertoli cells. Immunoreactive C/EBP ß also migrated as three bands with similar mobilities in rat but not human liver. The mobility of the C/EBP ß protein has varied between the cell types studied (39, 40, 41), reflecting cell- and species-specific differences of the protein. Trautwein et al. (42) demonstrated that phosphorylation of LAP at Ser105 by PKA has no influence on DNA binding, whereas PKA-mediated phosphorylation of other serine residues located in the C-terminal DNA-binding domain, inhibited DNA binding, suggesting that site-specific phosphorylations of LAP modulate transactivation of its target genes. Furthermore, PKA-mediated phosphorylation of Ser 299 has been shown to facilitate nuclear translocation of C/EBP ß (40, 43). Preliminary studies indicate a distinct nuclear translocation of C/EBP ß upon stimulation with cAMP in Sertoli cells (data not shown). C/EBP ß also contains phosphorylation sites for PKC (37) and calmodulin-dependent protein kinase II (44).

The C/EBP ß/LIP is a transcriptional repressor that is translated from the same mRNA species as LAP by using a different down-stream AUG within the same reading frame (7). In this study, we show that the levels of C/EBP ß/LAP under basal conditions were similar to the levels in preovulatory granulosa cells stimulated with hCG and the levels in rat liver extracts. However, in the latter tissue, apparently all three isoforms of LAP were present also in the absence of cAMP. C/EBP ß/LIP was not detected in preovulatory granulosa cells, consistent with earlier publications stating that the LAP isoform is the most abundant and the dominating isoform during gonadotropin-induced C/EBP ß expression (45). In our study, the basal levels of C/EBP ß/LAP and C/EBP {zeta} appeared to increase with the time in culture (2 h vs. 8 h), suggesting that autocrine factors secreted by Sertoli cells possibly mediated the slight induction observed. LIP (20 kDa) was induced with the same kinetics as LAP in Sertoli cells, but not as strongly as LAP. The molecular weight of LIP in Sertoli cells correlates with the molecular weight of LIP in Hep G2 cells (7). The ratio of LAP to LIP increased after treatment with cAMP with maximum stimulation of both LAP/LIP observed at 8 h. In vitro studies by Descombes et al. (7) showed that this ratio increased about 5-fold during terminal liver differentiation suggesting that regulation of LIP levels may serve to modulate transcriptional activation by LAP. In Sertoli cells, cAMP/FSH may activate transcription of a number of C/EBP responsive genes by increasing the LAP/LIP ratio. This hypothesis is confirmed by the observation that cAMP induces activity of a C/EBP reporter when transfected into Sertoli cells. Furthermore, the members of the C/EBP family may exist both as heterodimers, and homodimers (30, 46). The various homo- and heterodimers may possess different transactivating and binding activities as well as selectivity toward specific C/EBP sites. The strong cAMP-mediated induction of C/EBP ß could alter the proportion of homo- and heterodimerization and affect the expression of genes controlled by specific C/EBP factors. In addition, the ability of various C/EBP isoforms to activate or repress a promoter is dependent on the composition and context of the C/EBP cis-element and association with cofactors or factors binding to neighboring sites (29, 47, 48, 49, 50, 51)

C/EBP {zeta} (CHOP, GADD153) is a stress-inducible protein that, upon heterodimerization with other family members of C/EBP, is proposed to play a dual role by inhibiting the ability of C/EBP proteins to bind several known C/EBP sites and to activate binding to a specific DNA-binding site for heterodimers of C/EBP {zeta} complexed with C/EBP {alpha}/ß/{delta} (52, 53). In Sertoli cells, the kinetics of cAMP-mediated induction of C/EBP {zeta} were slower than that of C/EBP ß and {delta}. One can speculate that C/EBP {zeta} may serve as an inhibitor of transcriptional activation mediated by C/EBP and by C/EBP ß and {delta} in Sertoli cells.

In conclusion, we report that C/EBP isoforms are present in Sertoli cells and that they are strongly increased by FSH and cAMP with rapid kinetics. Furthermore, the observation that testicular C/EBP are decreased by hypophysectomy and increased in FSH-injected animals indicate that C/EBP levels are regulated in vivo by FSH. In addition, this regulation results in a secondary, cAMP-mediated cell-specific increase in transcription from a C/EBP reporter. Finally, C/EBP ß cotransfection with reporter constructs containing the cAMP-responsive regions of two genes (RIIß, PDE4D1/2) known to respond to cAMP with slow kinetics in Sertoli cells, resulted in significantly increased promoter activities. Although cAMP induced both promoters more strongly than C/EBP ß, cAMP-induced C/EBP ß may significantly contribute to the regulation of these promoters. The difference in response may be due to the action of additional cAMP-regulated factors. Thus, the cAMP-mediated induction of C/EBP isoforms may be a physiological mechanism for FSH-dependent regulation of late response genes.


   Acknowledgments
 
We greatly appreciate the skillful technical assistance of Gladys Josefsen and Guri Opsahl.


   Footnotes
 
1 This work was supported by the Norwegian Cancer Society, The Norwegian Research Council, Anders Jahres Foundation for the Promotion of Science, The Swedish Medical Research Council, and Novo Nordisk Foundation Committee. Back

Received June 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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