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Endocrinology Vol. 139, No. 1 272-279
Copyright © 1998 by The Endocrine Society

ARTICLES

Luteinizing Hormone-Dependent Gene Regulation in Leydig Cells May Be Mediated by CCAAT/Enhancer-Binding Protein-ß1

Demet Nalbant, Simon C. Williams, Douglas M. Stocco and Shafiq A. Khan

Department of Cell Biology and Biochemistry (D.N., S.C.W., D.M.S., S.A.K.) and Southwest Cancer Center (S.C.W., S.A.K.), University Medical Center, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Address all correspondence and requests for reprints to: Shafiq A. Khan, Ph.D., Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, Texas 79430. E-mail: cbbsak{at}wpoffice.net.ttuhsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leydig cells are located in the interstitium of the testis and function as the primary site for testosterone biosynthesis. Leydig cell development and steroidogenic function are dependent upon pituitary-derived LH. Circulating LH levels in prepubertal mammals are low but rise sharply during puberty, inducing terminal differentiation of immature Leydig cells into adult Leydig cells. The molecular mechanisms involved in LH action on differentiation specific gene expression and initiation of steroidogenic function in immature Leydig cells are poorly understood. Members of the CCAAT/enhancer-binding protein (C/EBP) family of basic region/leucine zipper transcription factors have previously been implicated as regulators of terminal differentiation in several cell types. In the present study we have investigated the possible involvement of C/EBP proteins in regulating LH-dependent gene expression in Leydig cells. We have detected the expression of one family member, C/EBPß, in Leydig cells. C/EBPß messenger RNA and protein levels were significantly higher in mature adult Leydig cells than in immature cells, displaying an expression pattern similar to those of other developmentally regulated genes in Leydig cells such as steroidogenesis acute regulatory (StAR) protein and 3ß-hydroxysteroid dehydrogenase. C/EBPß messenger RNA and protein levels also increased when immature Leydig cells were treated with either hCG, a functional analog of LH (hCG/LH), or (Bu)2cAMP. To confirm that hCG/LH and (Bu)2cAMP were acting specifically on Leydig cells, we studied their effects on C/EBPß expression in an established Leydig cell line (MA-10). hCG and (Bu)2cAMP treatment also induced the expression of C/EBPß and StAR in MA-10 cells, coincident with stimulation of steroid production in these cells. (Bu)2cAMP treatment did not alter the subcellular localization of C/EBPß protein in MA-10 cells, suggesting that the increase is due to stimulation of C/EBPß expression. We conclude that expression of C/EBPß is regulated by hCG/LH in Leydig cells and that C/EBPß may play a significant role in LH-regulated Leydig cell differentiation and function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEYDIG cells undergo two distinct periods of proliferation and differentiation in most mammals; the first occurs during fetal life, and the second precedes puberty (1, 2). During the prepubertal period, three well defined developmental stages of Leydig cells have been observed in the rat testis that exhibit distinct morphological, biochemical, and functional characteristics (3, 4, 5, 6). Initially, an ill defined population of mesenchymal stem cells proliferates and differentiates to give rise to precursor Leydig cells (PLC), which retain mesenchymal morphology but acquire LH receptors and the ability to synthesize steroidogenic enzymes, albeit at a low level. PLCs subsequently differentiate into immature Leydig cells, which attain a more rounded structure, acquire lipid droplets, and are capable of producing testosterone that is rapidly metabolized into 5{alpha}-reduced products. The immature Leydig cells then undergo an additional round of proliferation and terminally differentiate into nondividing adult-type Leydig cells (7, 8). Adult Leydig cells are distinguished from their immature precursors by a higher abundance of smooth ER and the absence of lipid droplets (7). They also express a higher number of LH receptors on their surface and acquire full steroidogenic potential.

Several in vivo studies in rodents and primates have shown that functional differentiation of Leydig cells is primarily dependent on LH (7, 9, 10, 11); however, the mechanisms of action of LH at the distinct stages of Leydig cell development remain unclear. Proliferation and differentiation of stem cells into PLCs do not require LH, whereas the differentiation of PLCs into immature Leydig cells and their subsequent proliferation and differentiation into adult Leydig cells are dependent upon LH (7, 8, 12). Circulating levels of LH are low during the period when immature Leydig cells proliferate, and it is likely that any effects of LH at this stage are indirect and are probably mediated through locally produced growth factors and cytokines (13). However, terminal differentiation and acquisition of the full steroidogenic potential of adult-type Leydig cells are accompanied by a rapid increase in the circulating levels of LH (14) (Khan, S. A., L. Jaeger, and I. Huhtaniemi, unpublished observations). The hypothesis that increased levels of LH are required for terminal differentiation, but not proliferation, of Leydig cells is supported by analysis of rats after neonatally induced hypothyroidism. LH levels remain extremely low in this animal model, and adult testes are characterized by the presence of large numbers of Leydig cells that display reduced steroidogenic potential (15).

The LH receptor is a member of the G protein-coupled pituitary glycoprotein subfamily of transmembrane receptors (16). Binding of LH to its receptor results in stimulation of adenylate cyclase and leads to increased intracellular levels of cAMP, activation of protein kinase A, and phosphorylation of cellular proteins involved in cAMP-dependent gene expression and function. Classically, cAMP-dependent effects on gene expression are mediated at the transcriptional level through members of the cAMP response element (CRE)-binding protein (CREB) family of basic region/leucine zipper (bZIP) family of transcription factors (17). However, many cAMP-responsive genes in Leydig cells and other steroidogenic cells do not contain consensus cAMP response elements in their control regions, indicating that the effects of LH on both Leydig cell differentiation and function are likely to involve other factors (18, 19). Consequently, we have been interested in identifying nuclear transcription factors that are targets of LH signaling pathways in Leydig cells and have concentrated on members of the CCAAT/enhancer binding protein (C/EBP) family of bZIP transcription factors.

The C/EBP family consists of four closely related members, C/EBP{alpha}, C/EBPß, C/EBP{delta}, and C/EBP{epsilon}, and two less related proteins, Ig/EBP (C/EBP{gamma}) and CHOP (C/EBP{zeta}) (20). C/EBP proteins bind to the sequence ATTGCGCAAT and variants thereof (21) and are expressed in a limited, partially overlapping, pattern in mammalian tissues, predominantly in terminally differentiated cells such as adipocytes, hepatocytes, macrophages, and eosinophils. C/EBPß is widely expressed in mammalian tissues and has been implicated in the regulation of both tissue-specific genes during differentiation of hepatocytes, adipocytes, and myeloid cells and of inducible genes activated during inflammatory responses (20). Both the level of expression and the subcellular localization of C/EBPß have been shown to be regulated by changes in intracellular cAMP levels in several cell types, and C/EBPß has been implicated as a critical regulator of subsets of cAMP-inducible genes (22, 23). In this study, we investigated whether C/EBPß might be a component of LH-dependent signaling pathways in Leydig cell differentiation and function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of primary Leydig cells
Male Wistar Crl:(W1) BR rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and maintained under controlled light and temperature. Food and water were provided ad libitum. Rats were killed by CO2 asphyxiation, and testes were removed. In cases where RNA and protein was prepared from freshly isolated Leydig cells, a Percoll gradient purification method was used (15, 24). Briefly, decapsulated testes from 20- and 70-day-old rats were incubated for 15 min in culture medium (MEM with Earle’s salts, supplemented with nonessential amino acids and antibiotics, Life Technologies, Grand Island, NY) containing 0.25 mg/ml collagenase (Sigma Chemical Co., St. Louis, MO). The interstitial cells were separated from seminiferous tubules by sedimentation at unit gravity for 5 min. The supernatant containing the interstitial cells was removed and centrifuged at 600 x g for 10 min. After two subsequent washes in culture medium, interstitial cells were then purified over a 55% Percoll gradient (15, 24). Typically, the number of Leydig cells obtained from this method varied between 0.4–1 x 106 cells/testis depending on the age of the animals, and the purity of the Leydig cell preparations was greater than 90% as judged by 3ß-hydroxysteroid dehydrogenase (3ßHSD) staining.

In experiments to investigate the hormonal regulation of C/EBPß expression in vitro, immature Leydig cells were isolated without Percoll gradient purification from 20-day-old rats as described previously (13, 25, 26). After collagenase dispersal of decapsulated testes, the cells were plated into six-well culture dishes (Corning, Corning, NY) as 1-ml aliquots in culture medium (2 x 106 cells/well). After 1 h, the Leydig cells had attached to the surface of the plate, whereas contaminating cells (Sertoli and germ cells) remained unattached. At this time, the medium was discarded, and the cells were washed three times to remove contaminating cells. Fresh medium was added, and the cells were cultured for 48 h at 37 C. The medium was collected for analysis of steroid levels, and fresh medium was added to the cells. At this stage the cell preparation contained more than 90% Leydig cells.

Hormonal stimulation of primary and MA-10 Leydig cells
Immature Leydig cells were incubated in culture medium and incubated with or without 1 IU/ml hCG (Pregnyl, Organon, West Orange, NJ), 0.5 mM (Bu)2cAMP (Sigma), 1 IU/ml FSH (Metrodin, Serono Laboratories, Randolph, MA), or 1 µM testosterone (Sigma) for different time periods. MA-10 cells (27) were a gift from Dr. M. Ascoli (University of Iowa, Iowa City, IA) and were cultured in Waymouth’s MB 752/1 medium containing 15% heat-inactivated horse serum (Life Technologies). MA-10 cells were treated with hCG and/or (Bu)2cAMP as described above for immature Leydig cells.

Total RNA isolation and Northern analysis
Total RNA was prepared from primary Leydig cells or MA-10 cells, using a modified guanidine isothiocyanate/phenol extraction pro-cedure. Cells were lysed in 0.5 ml lysis buffer [4 M guanidine isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% Sarcosyl, and 0.1 M ß-mercaptoethanol], and 50 µl 2 M sodium acetate (pH 4.0), 0.5 ml water-saturated phenol, and 0.3 ml chloroform-isoamyl alcohol (24:1, vol/vol) were added sequentially. After mixing and incubation on ice for 20 min, the mixture was centrifuged at 10,000 x g for 20 min at 4 C. The aqueous phase was removed to a fresh microcentrifuge tube, and 0.5 ml isopropanol was added and incubated at -20 C for 1 h. RNA was sedimented by centrifugation at 10,000 x g for 20 min, resuspended in 0.4 ml lysis buffer, and reprecipitated with 1 vol isopropanol as before. The RNA pellet was washed with 70% ethanol and resuspended in water. For Northern analyses, 10 µg total RNA was electrophoresed through a 1% agarose-formaldehyde gel and transferred to nylon membranes (Micron Separations, Westborough, MA). Membranes were hybridized for 16–24 h at 42 C in hybridization buffer [0.6 mg/ml herring sperm DNA (Amresco, Solon, OH), 0.05% BSA, 0.1% SDS, 5 x SSC (0.75 M NaCl and 75 mM Na3 citrate, pH 7.0), and 12.5 mM NaPO4, pH 6.6] and washed once with 2 x SSC at 42 C, once with 2 x SSC-1% SDS at 42 C, and once with 2 x SSC-1% SDS at 65 C. Membranes were first exposed to a phosphor screen, then analyzed on a Molecular Dynamics model 445SI PhosphorImager (Sunnyvale, CA) and subsequently exposed to autoradiographic film (X-Omat AR, Eastman Kodak, Rochester, NY) at -70 C using an intensifying screen. The C/EBPß-specific probe was a 450-bp NcoI/HindIII fragment derived from pMEXCRP2 (28) containing the 3'-portion of the C/EBPß-coding region. DNA fragments were labeled with [32P]deoxy-CTP using the Prime-It II random primer labeling kit (Stratagene, La Jolla, CA). Ethidium bromide-stained gels were photographed to demonstrate equal loading of RNA samples in different lanes.

Protein isolation and Western analysis
Total cellular proteins were isolated by isopropanol precipitation of the phenol phase from the first step of the RNA isolation procedure. Protein pellets were washed three times in 0.3 M guanidine-HCl in 95% ethanol and subsequently dissolved in 1% SDS. Nuclear and cytosolic proteins were isolated from MA-10 cells as described previously (29). Protein concentrations were determined using either the protein assay kit (Bio-Rad Laboratories, Hercules, CA) or the BCA protein assay kit (Pierce Chemical Co., Rockford, IL). Total cellular, cytoplasmic, or nuclear proteins were separated by SDS-PAGE in 10 or 12% gels and then transferred to nitrocellulose membranes (Micron Separations, Westborough, MA). All gels were run in duplicate, and one gel from each pair was stained with Coomassie blue to ensure that equal amounts of protein were loaded in each lane. Membranes were blocked in TBS (100 mM Tris-HCl, pH 7.5, containing 0.9% NaCl) containing 4% Carnation non fat dry milk (Carnation, Los Angeles, CA) and probed with polyclonal antisera specific for C/EBPß, steroidogenic acute regulatory protein (StAR), and 3ßHSD for 1 h. The C/EBPß and StAR antisera were rabbit antipeptide antisera directed against the first 14 amino acids of rat C/EBPß (30) or amino acids 88–98 of mouse StAR (31). The rabbit antibody against human 3ßHSD (32) was a gift from Dr. Ian Mason (Glasgow, Scotland). The blots were washed three times in TBS containing 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated goat antirabbit IgG (1:20,000) for 30 min. After three more washes, immune complexes were detected using the SuperSignal chemiluminescence detection kit (Pierce). Band intensities were determined by densitometric scanning of gel images using a BioImage Visage 2000 (BioImage, Ann Arbor, MI) computer-assisted image analysis system.

RIA
Quantitation of progesterone was performed by RIA, as previously described (33), directly on aliquots of the medium from control and treated cells. Antibodies to progesterone were obtained from Holly Hills Biological (Hillsboro, OR). Analysis of the RIA data was performed using a computer program specifically designed for this purpose. The data were expressed as nanograms of progesterone per mg protein.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C/EBPß is expressed in rat Leydig cells
To determine whether any members of the C/EBP family might be candidate regulators of Leydig cell differentiation and/or function, we first examined their expression patterns in immature and adult Leydig cells purified by cell fractionation from rat testis. Whole cell protein extracts and total RNA were prepared and analyzed by Western and Northern blotting. To date, we have been unable to detect C/EBP{alpha}, C/EBP{delta}, or C/EBP{epsilon} in Leydig cells at any stage of development (data not shown). However, as shown in Fig. 1AGo, C/EBPß messenger RNA (mRNA) was detected in RNA prepared from both immature and adult cells, with the level in adult cells being consistently higher. The difference in C/EBPß levels at these developmental stages was more evident in Western blots. C/EBPß protein was barely detectable in extracts from immature cells (Fig. 1BGo, lane 1), but was easily detected in extracts from adult cells (lane 2). Confirmation that the protein detected in these experiments was truly C/EBPß came from side by side comparison with a recombinant form of C/EBPß expressed in HeLa cells (Fig. 1BGo, lane 3) and the use of two independent anti-C/EBPß antisera (data not shown). Leydig cell C/EBPß migrates with a mol wt of 34,000, corresponding to the size of a C/EBPß polypeptide initiating at the second in-frame initiation codon in the C/EBPß-coding sequence. We have not detected either the 38,000 (full length liver activator protein) or 20,000 (liver-inhibitory protein) forms of C/EBPß that may potentially be synthesized from C/EBPß mRNA by use of alternative translation initiation codons (20). To confirm the identity and developmental status of our purified Leydig cell preparation, protein levels of two markers of differentiated Leydig cells, StAR and 3ßHSD, were also assayed in the immature and adult cell extracts by Western blotting. As shown in Fig. 2Go, A and B, StAR and 3ßHSD proteins were both low to undetectable in immature cells, but were easily detectable in adult cells. These results confirm the purity of our cell preparations and indicate that C/EBPß is expressed in a differentiation-specific pattern in rat Leydig cells.



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  		Figure 1. Developmental changes in C/EBPß expression in Leydig cells. A, Analysis of C/EBPß mRNA levels. Total RNA was isolated from purified Leydig cells from either 20-day-old (immature Leydig cells) or 70-day-old (adult type Leydig cells) rats and subjected to Northern blot analysis using a C/EBPß-specific probe. The positions of the 1.8-kilobase C/EBPß mRNA species and 18S and 28S ribosomal RNAs are indicated. A photograph of the ethidium bromide-stained gel is shown as a loading control. Some minor cross-reactivity with 28S RNA was observed. B, Analysis of C/EBPß protein levels. Whole cell lysates were prepared from immature (lane 1) and adult (lane 2) Leydig cells and analyzed by Western blotting using a rabbit antipeptide antiserum that specifically recognizes C/EBPß. A control extract (lane 3) prepared from HeLa cells transfected with a C/EBPß expression vector was run alongside to indicate the position of the 34,000 mol wt isoform of C/EBPß. The migration of mol wt markers (x103) is indicated.

 


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  		Figure 2. Coordinate increases in StAR and 3ßHSD protein levels in primary Leydig cell extracts. Whole cell extracts from immature and adult-type Leydig cells were analyzed by Western blotting using antiserum specific for either StAR (A) or 3ßHSD (B). The positions of the StAR and 3ßHSD proteins are indicated. Elevated levels of both proteins correlated with the acquisition of the differentiated phenotype of the adult-type cells.

 
As LH is the primary regulator of Leydig cell development, we next tested whether expression of C/EBPß might be regulated in response to LH stimulation. Immature Leydig cells were purified from testes of 20-day-old rats and cultured in the absence or presence of the LH homolog, hCG, or (Bu)2cAMP for 4 h. Whole cell protein extracts and total RNA were prepared and analyzed as described above. C/EBPß mRNA levels were consistently higher in cells treated with (Bu)2cAMP (Fig. 3AGo), and C/EBPß protein levels again displayed a more dramatic difference, being essentially undetectable in untreated cells and significantly elevated in cells treated with either (Bu)2cAMP or hCG (Fig. 3BGo). To demonstrate the specificity of the LH/hCG effect on C/EBPß expression in these cells, parallel cultures of cells were treated with FSH or testosterone. C/EBPß protein and mRNA were unchanged in cells treated with either agent (data not shown). StAR protein levels were also examined in extracts from control and (Bu)2cAMP-treated cells and displayed expression patterns similar to that of C/EBPß, being expressed at low to undetectable levels in control cells but at high levels in treated cells (Fig. 4Go). In addition, elevated levels of testosterone were detected in the medium of treated cells compared with those in control cells (data not shown). These data suggest that expression of C/EBPß is regulated by LH in Leydig cells and identify it as a candidate regulator of Leydig cell-specific gene expression.



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  		Figure 3. C/EBPß mRNA and protein levels are stimulated by hCG and (Bu)2cAMP treatment in immature Leydig cells. A, Total RNA was prepared from immature Leydig cells cultured for 4 h in either the absence (Cont) or presence of (Bu)2cAMP. C/EBPß mRNA levels were determined by Northern blot analysis as described in Fig. 1Go. B, Whole cell lysates were prepared from immature Leydig cells cultured in the absence (lane 1) or presence of hCG (lane 2) or (Bu)2cAMP (lane 3) for 4 h. C/EBPß protein levels were analyzed as described in Fig. 1Go.

 


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  		Figure 4. StAR protein levels increase in response to (Bu)2cAMP treatment in immature Leydig cells. StAR protein levels were determined by Western analysis of lysates prepared from immature Leydig cells cultured for 4 h in the absence or presence of (Bu)2cAMP. The position of the 30,000 mol wt StAR protein is indicated with an arrow.

 
Expression of C/EBPß is also regulated by LH/hCG and (Bu)2cAMP in MA-10 Leydig tumor cells
The methods used here for Leydig cell purification consistently yield cultures in which the contribution of Leydig cells is greater than 90%. However, as C/EBPß has previously been shown to be expressed in macrophages, which are the primary contaminating cell type seen in our cultures, we wished to confirm that the effects we observed on C/EBPß expression represented direct effects of LH/hCG and (Bu)2cAMP on Leydig cells. For these studies, we investigated the effects of LH/hCG and cAMP on C/EBPß expression in MA-10 cells, a murine Leydig tumor cell line. Duplicate cultures of MA-10 cells were incubated in the absence or presence of hCG or (Bu)2cAMP for 4 h, and C/EBPß expression was examined by Western and Northern blot analyses. C/EBPß protein levels were elevated 4.5- and 2.7-fold in cells incubated in the presence of (Bu)2cAMP and hCG, respectively (Fig. 5AGo), mirroring the relative effectiveness of these two agents in stimulating C/EBPß protein levels in immature Leydig cells. Quantitation of C/EBPß mRNA levels in MA-10 cells treated with (Bu)2cAMP for 4 h revealed a similar 4-fold increase (Fig. 5BGo), suggesting that stimulation probably occurs at the level of transcription. Basal levels of C/EBPß were relatively higher in MA-10 cells than in immature rat Leydig cells (compare Figs. 1Go and 5Go), presumably reflecting the origin of MA-10 cells from an adult mouse tumor. To demonstrate that the MA-10 cells were responding appropriately to (Bu)2cAMP stimulation, progesterone levels were analyzed by RIA of culture medium from control cells and cells stimulated with (Bu)2cAMP for 1–3 h. As expected, progesterone production increased in a time-dependent fashion in response to (Bu)2cAMP (Fig. 6Go).



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  		Figure 5. C/EBPß protein and mRNA levels are also stimulated by hCG and (Bu)2cAMP in MA-10 cells. A, Duplicate cultures of MA-10 cells were incubated for 4 h in the absence (Control) or presence of (Bu)2cAMP or hCG. Whole cell lysates were prepared and analyzed by Western blotting using the C/EBPß-specific antiserum. C/EBPß protein levels were quantitated by densitometric scanning and are expressed as an average of both samples relative to the level in control cells. The aberrant migration of the C/EBPß band in lane 6 was due to a small defect in the gel. B, C/EBPß mRNA levels were analyzed by Northern blotting of total RNA prepared from control (lane 1) and (Bu)2cAMP-treated (lane 2) MA-10 cells as described in Fig. 1Go. PhosphorImager quantitation revealed 4-fold higher mRNA levels in the treated cells, in agreement with the increase in protein levels. Rat liver RNA (lane 3) was included as a size marker for C/EBPß mRNA.

 


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  		Figure 6. (Bu)2cAMP stimulates steroid synthesis in MA-10 cells. Culture medium was removed from MA-10 cells at hourly intervals after (Bu)2cAMP addition, and progesterone production was quantitated by RIA. Progesterone levels are expressed as nanograms of progesterone per mg protein and represent the mean ±SD of three independent experiments.

 
Increased intracellular cAMP levels have been shown to regulate the expression of C/EBPß in two ways, either by stimulating the expression of the c/ebpß gene at the transcriptional level or by altering the subcellular distribution of preexisting C/EBPß molecules from the cytoplasm to the nucleus. As a first step toward understanding the mechanisms controlling C/EBPß expression in Leydig cells, nuclear and cytoplasmic extracts were prepared from control and (Bu)2cAMP-treated MA-10 cells and analyzed by Western blotting. As shown in Fig. 7Go, C/EBPß protein was entirely localized to the nucleus under both conditions and displayed a 4-fold increase in response to (Bu)2cAMP similar to that seen in whole cell extracts. This experiment indicates that cAMP-dependent increases in C/EBPß protein levels are not due to changes in cellular localization, but are more likely to result from stimulation of c/ebpß gene transcription.



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  		Figure 7. (Bu)2cAMP stimulation does not affect the subcellular distribution of C/EBPß in MA-10 cells. Cytoplasmic (C; lanes 1 and 3) and nuclear (N; lanes 2 and 4) fractions were prepared from control and (Bu)2cAMP-treated MA-10 cells, and C/EBPß protein levels and location were determined by Western blot analysis. C/EBPß protein was only detected in the nuclear fractions, and the cAMP-dependent increase was similar to that seen in whole cell extracts (4-fold).

 
To begin to elucidate the potential function(s) of C/EBPß in Leydig cells, we investigated the kinetics of the increase in C/EBPß protein levels in response to (Bu)2cAMP in MA-10 cells. Whole cell extracts were prepared from MA-10 cells at various times after (Bu)2cAMP addition, and C/EBPß protein levels were analyzed by Western blotting. As shown in Fig. 8AGo, C/EBPß protein levels increased rapidly in response to (Bu)2cAMP, reaching a maximal level after approximately 4 h and remaining elevated over unstimulated levels after 24 h. Similar patterns of C/EBPß expression were seen in three independent experiments (data not shown). A second, slower migrating, C/EBPß-specific band was also detected, and its intensity increased coordinately with the main band (indicated with an asterisk). As this band disappears with phosphatase treatment (data not shown), we hypothesize that this represents a hyperphosphorylated form of the protein and not a larger isoform of C/EBPß. As (Bu)2cAMP is known to stimulate steroid production in MA-10 cells, we compared the expression pattern of C/EBPß with that of the StAR protein, which is critically required for steroidogenesis in these cells. StAR protein levels were first detectable after 4 h of stimulation and continued to increase at subsequent time points (Fig. 8BGo). These data suggest that C/EBPß may be involved in regulating the expression of subsets of genes whose functions are required for steroid synthesis in Leydig cells.



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  		Figure 8. Coordinate induction of C/EBPß and StAR protein levels in MA-10 cells. Whole cell extracts were prepared from MA-10 cells cultured in the absence or presence of (Bu)2cAMP for the indicated times. The upper panel shows a representative Western blot of C/EBPß protein levels over this time course; these were quantitated by densitometric scanning and are expressed relative to the level in unstimulated cells. C/EBPß protein levels consistently increased to maximal levels after 4 h of cAMP treatment and remained elevated even after 24 h. The asterisk in the upper panel indicates the position of a putative hyperphosphorylated form of C/EBPß. The lower panel shows Western analysis of the same samples using a StAR-specific antiserum. StAR protein was first detected at 4 h and continued to increase thereafter. A cross-reacting species was occasionally detected just below the StAR band.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented here provide the first evidence that C/EBPß is expressed in Leydig cells and demonstrate that its gene expression is a target for LH-dependent signaling pathways. Although C/EBPß mRNA had previously been detected by Northern analysis of total RNA prepared from whole testis (30, 34, 35), localization of C/EBPß to Leydig cells allows the assignment of tentative functions of this protein in this organ. Several in vivo and in vitro studies have established LH as the primary hormonal stimulus for proliferation and differentiation of Leydig cells in the immature testis (7, 8, 12). Although other pituitary hormones, such as FSH, PRL, and GH have also been implicated in Leydig cell development, the effects of these hormones appear to be indirect (3, 36, 37). Testosterone, which is produced by Leydig cells in response to LH, has also been shown to participate in the functional maturation of Leydig cells; however, testosterone alone does not appear to be sufficient for full differentiation of the Leydig cells (7, 15). Consequently, identification of the intracellular targets of LH-activated signaling pathways should reveal important information concerning the mechanisms by which Leydig cell differentiation and function are controlled. We have observed that adult-type Leydig cells express significantly higher levels of C/EBPß than immature cells, and LH/hCG induces the expression of C/EBPß in immature Leydig cells. This observation raises the possibility that LH-induced expression of C/EBPß may be required for the functional differentiation of immature cells into adult-type Leydig cells. Furthermore, as LH maintains the functional integrity of adult-type cells, it is logical to assume that C/EBPß may play a role in the regulation of LH-dependent processes in these cells. C/EBPß is an attractive candidate for these roles because it has been identified as a regulator of cell-specific genes in multiple tissues, including hepatocytes, adipocytes, macrophages, and eosinophils, and of inducible genes during inflammatory responses, including acute phase response genes in the liver and cytokine genes in macrophages (20, 38). The target genes for C/EBPß in Leydig cells are not known at present, and functional mapping of regulatory elements in the promoters of Leydig cell-specific genes should determine whether C/EBP-binding sites are contained therein.

Previously, C/EBPß has been shown to be targeted by a number of different signaling pathways and to be regulated at the level of both gene expression and transcriptional activity. Of particular relevance to these studies, increases in intracellular cAMP levels have been shown to regulate C/EBPß via two mechanisms. First, treatment of the PC-12 pheochromocytoma cell line with forskolin caused a redistribution of preformed C/EBPß molecules from the cytoplasm to the nucleus (23). Second, cAMP has been reported to stimulate transcription of the C/EBPß gene in a number of cell types, including hepatocytes and astrocytes (22, 39). In the present study, treatment with (Bu)2cAMP did not affect the intracellular location of C/EBPß in MA-10 cells, suggesting that (Bu)2cAMP stimulates the rate of synthesis of C/EBPß. Recently, studies on the sequences controlling cAMP-dependent activation of C/EBPß expression identified two variant CREs in the C/EBPß promoter. Although these sequences displayed only a five of eight match with the consensus CRE sequence, both were efficiently bound by CREB (40). Analysis of the promoter sequences responsible for LH-dependent increases in C/EBPß mRNA levels should reveal whether these variant CREs are functional in Leydig cells.

Apart from being regulated at the transcriptional level, c/ebpß gene expression may also be regulated at a posttranscriptional stage, in particular at the level of translational initiation. It has been shown that small open reading frames in the 5'-untranslated region of both the c/ebpß and c/ebp{alpha} genes regulate the choice of translation start site (41) or, alternatively, may inhibit translation altogether (Lincoln, A. J., Y. Monczak, S. C. Williams, and P. F. Johnson, unpublished observations). Comparison of the mRNA and protein levels for C/EBPß in Leydig cells reveals that although both show coordinate increases in response to cAMP, the magnitude of the increase may differ. This is particularly evident in immature Leydig cells, in which C/EBPß mRNA is easily detectable by Northern blotting, but C/EBPß protein levels are extremely low. Consequently, although C/EBPß mRNA levels increase about 4-fold in adult cells or in response to (Bu)2cAMP, the increase in the level of C/EBPß protein is significantly greater. While this disparity could be explained by other mechanisms, such as differences in the stability of C/EBPß protein, it may indicate that expression of C/EBPß during differentiation of Leydig cells is controlled at both the transcriptional and translational levels.

The activity of C/EBPß may also be controlled at the posttranslational level via phosphorylation by a large number of protein kinases, including protein kinase A, leading to stimulation of DNA-binding and/or transcriptional activities of C/EBPß (23, 42, 43, 44). Although the specific site(s) of phosphorylation by protein kinase A is unknown, it is possible that C/EBPß activity, as well as expression, may be increased in cAMP-stimulated Leydig cells. We observed a significant increase in the intensity of a slower migrating, C/EBPß-specific band in Western blots, which appears to represent a hyperphosphorylated form of the protein. It will be important to identify residues within C/EBPß that are phosphorylated in response to cAMP-stimulated signaling pathways in Leydig cells and to test the effects of these modifications on its DNA-binding and transcriptional activities. Finally, C/EBPß has also been shown to synergistically activate transcription of certain genes in combination with a number of additional promoter-bound transcription factors, including two members of the nuclear hormone receptor superfamily, the estrogen and glucocorticoid receptors (45, 46), and it is possible that it may participate in similar interactions with other nuclear hormone receptors in Leydig cells. Various studies have implicated other members of this family, including steroidogenic factor-1 and the androgen receptor, as important regulators of Leydig cell differentiation and function (7, 47).

Our data suggest that C/EBPß may be an important regulator of both Leydig cell differentiation and function. Clearly, elucidation of the exact role of C/EBPß will require the identification of its target genes in these cells. The coordinate induction of genes for C/EBPß, StAR, and 3ßHSD in primary and immortalized Leydig cells raises the possibility that C/EBPß might be directly involved in regulating the expression of these and other genes involved in steroid hormone synthesis. As mentioned above, many of these genes, which include a number of members of the cytochrome P450 superfamily, do not contain consensus CREs in their promoter regions (18, 19). Promoter mapping studies have identified numerous cAMP response sequences in steroid hydroxylase genes, suggesting that multiple factors, perhaps including C/EBPß, may participate in this process. For example, cAMP-dependent stimulation of transcription of the StAR gene is controlled by elements located within 253 bp of the transcription start site (48). Although this region contains a binding site for steroidogenic factor-1, disruption of this site in the mouse promoter only affected basal, not cAMP-dependent, expression. Perusal of the sequence of the murine and human StAR promoter regions revealed the presence of an evolutionarily conserved sequence (G/ATGAGGCAAT) that displays significant homology to the consensus C/EBP binding site (S. C. Williams and S. R. King, unpublished observations), raising the possibility that C/EBPß may be involved in regulating StAR gene expression in response to tropic hormone stimulation. In addition, C/EBPß has been shown to regulate the expression of a number of cytochrome P450 genes in other tissues (49, 50, 51, 52, 53, 54, 55), supporting the hypothesis that it may also regulate genes from this family, including steroid hydroxylases, in Leydig cells.

Finally, firm evidence that C/EBPß is an important component of LH-dependent signaling pathways in other steroidogenic tissues has come from the analysis of mice lacking C/EBPß due to targeted mutation of the c/ebpß gene. Apart from their previously reported defects in immune functions (56, 57), female C/EBPß nullizygous mice are infertile due to defects in ovarian function (58). This phenotype has been attributed to a defect in LH signaling in ovarian granulosa cells that is manifested by aberrant regulation of both the PGS-2 and P450 aromatase genes, two genes previously identified as C/EBP targets (51, 54, 59). These mice should provide a powerful model system for analysis of the role of C/EBPß in Leydig cell differentiation and function and male reproductive development.


   Acknowledgments
 
We thank Drs. Peter Johnson and Esta Sterneck for sharing unpublished results on C/EBPß knockout mice, and Jeannine Lincoln and Steven King for critical reading of the manuscript. We acknowledge the expert technical assistance of Rebecca Ball, Nicholas Angerer, and Deborah Alberts.


   Footnotes
 
1 This work was supported by a Grant-In-Aid from the American Heart Association, Texas Affiliate; a Texas Tech University Health Science Center Seed Grant (to S.C.W.); a Texas Tech University Health Science Center Seed Grant (to S.A.K.); and NIH Grant HD-17481 (to D.M.S.). Back

Received August 25, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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