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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
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-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are accompanied by a rapid increase in the circulating levels of LH (14) (Khan, S. A., L. Jaeger, and I. Huhtaniemi, unpublished observations).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,
C/EBPß, C/EBP
, and C/EBP
, and two less related proteins, Ig/EBP
(C/EBP
) and CHOP (C/EBP
) (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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C/EBP, or C/EBP in Leydig cells at any stage of development (data
not shown). However, as shown in Fig. 1A
, 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. 1B, 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. 1B, 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. 2, 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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), 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. 3B). 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. 4). 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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), 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. 5B), 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. 1
and 5), 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. 6).
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, 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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, 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. 8B). 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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.
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Acknowledgments |
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Footnotes |
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Received August 25, 1997.
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Y. Du, S. Tsai, J. R. Keller, and S. C. Williams Identification of an Interleukin-3-regulated Aldoketo Reductase Gene in Myeloid Cells Which May Function in Autocrine Regulation of Myelopoiesis J. Biol. Chem., March 15, 2000; 275(10): 6724 - 6732. [Abstract] [Full Text] [PDF]
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Y. Pohnke, R. Kempf, and B. Gellersen CCAAT/Enhancer-binding Proteins Are Mediators in the Protein Kinase A-dependent Activation of the Decidual Prolactin Promoter J. Biol. Chem., August 27, 1999; 274(35): 24808 - 24818. [Abstract] [Full Text] [PDF]
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E. Silverman, S. Eimerl, and J. Orly CCAAT Enhancer-binding Protein beta and GATA-4 Binding Regions within the Promoter of the Steroidogenic Acute Regulatory Protein (StAR) Gene Are Required for Transcription in Rat Ovarian Cells J. Biol. Chem., June 18, 1999; 274(25): 17987 - 17996. [Abstract] [Full Text] [PDF]
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A. J. Reinhart, S. C. Williams, B. J. Clark, and D. M. Stocco SF-1 (Steroidogenic Factor-1) and C/EBP{beta} (CCAAT/Enhancer Binding Protein-{beta}) Cooperate to Regulate the Murine StAR (Steroidogenic Acute Regulatory) Promoter Mol. Endocrinol., May 1, 1999; 13(5): 729 - 741. [Abstract] [Full Text]
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M. Kanzaki and P. L. Morris Growth Hormone Regulates Steroidogenic Acute Regulatory Protein Expression and Steroidogenesis in Leydig Cell Progenitors Endocrinology, April 1, 1999; 140(4): 1681 - 1686. [Abstract] [Full Text]
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N. D. Angerer, Y. Du, D. Nalbant, and S. C. Williams A Short Conserved Motif Is Required for Repressor Domain Function in the Myeloid-specific Transcription Factor CCAAT/Enhancer-binding Protein epsilon J. Biol. Chem., February 12, 1999; 274(7): 4147 - 4154. [Abstract] [Full Text] [PDF]
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H. A. LaVoie, J. C. Garmey, and J. D. Veldhuis Mechanisms of Insulin-Like Growth Factor I Augmentation of Follicle-Stimulating Hormone-Induced Porcine Steroidogenic Acute Regulatory Protein Gene Promoter Activity in Granulosa Cells Endocrinology, January 1, 1999; 140(1): 146 - 153. [Abstract] [Full Text]
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S. C. Williams, Y. Du, R. C. Schwartz, S. R. Weiler, M. Ortiz, J. R. Keller, and P. F. Johnson C/EBPepsilon Is a Myeloid-specific Activator of Cytokine, Chemokine, and Macrophage-Colony-stimulating Factor Receptor Genes J. Biol. Chem., May 29, 1998; 273(22): 13493 - 13501. [Abstract] [Full Text] [PDF]
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A. J. Lincoln, Y. Monczak, S. C. Williams, and P. F. Johnson Inhibition of CCAAT/Enhancer-binding Protein alpha and beta Translation by Upstream Open Reading Frames J. Biol. Chem., April 17, 1998; 273(16): 9552 - 9560. [Abstract] [Full Text] [PDF]
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S. L. Gyles, C. J. Burns, B. J. Whitehouse, D. Sugden, P. J. Marsh, S. J. Persaud, and P. M. Jones ERKs Regulate Cyclic AMP-induced Steroid Synthesis through Transcription of the Steroidogenic Acute Regulatory (StAR) Gene J. Biol. Chem., September 7, 2001; 276(37): 34888 - 34895. [Abstract] [Full Text] [PDF]
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