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Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4231
Address all correspondence and requests for reprints to: Michael K. Skinner, Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4231. E-mail: skinner{at}mail.wsu.edu
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Introduction |
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The majority of the Sertoli cell functions are regulated by the gonadotropin FSH (7, 8). FSH acts on Sertoli cells via the protein kinase A pathway (7, 8, 9). The actions of FSH on Sertoli cells may also involve other signal transduction pathways, including protein kinase C and calcium mobilization (10). Together, these signal transduction pathways phosphorylate and activate a number of transcription factors, such as cAMP response element-binding protein (9), C/EBPß (11), c-fos (12), c-myc (13), GATA-1 (14), SF-1 (15), and WIN (16). It is speculated that the activation and combinations of many of these transcription factors are responsible for the transcription of Sertoli cell-specific gene expression.
Recently, Sertoli cells have been shown to express members of the basic helix-loop-helix (bHLH) transcription factor family. The bHLH family of transcription factors are critical cell-type determinants and play important roles in cell growth and differentiation. A basic helix-loop-helix domain that is conserved from yeast to mammals characterizes members of this family (17). The bHLH domain consists of two amphipathic helixes separated by a loop that mediates homo- and heterodimerization adjacent to a DNA-binding region rich in basic amino acids (18). The bHLH dimers bind to an E Box (CANNTG) DNA consensus sequence present in a wide variety of tissue-specific promoters (19, 20). The E box domain has been shown to influence the promoters of a number of Sertoli cell-specific genes, including transferrin (21), c-fos (22), SF-1 (23), and FSH receptor (24).
The bHLH proteins have been classified into two distinct classes. The
ubiquitously expressed class A bHLH proteins consist of E2–2
(25), HEB (26), and E12 and E47 [the
differentially spliced products of the E2A gene (20)],
which dimerize with tissue-restricted and developmentally regulated
class B proteins such as MyoD and neuroD (27, 28).
Previous observations suggest that the Sertoli cells express the class
A proteins E47 (29) and REB
(30) (the rat
isoform of human HEB). Sertoli cell-specific class B bHLH proteins are
yet to be determined. Recent reports suggest that bHLH proteins
regulate FSH-stimulated Sertoli cell gene expression (21, 22, 31).
The members of the Id (inhibitor of differentiation/DNA binding) family modulate the transcriptional activity of class A and B bHLH heterodimers. The four known Id proteins (Id1, Id2, Id3, and Id4) share a homologous HLH domain, but lack the basic DNA binding region (32, 33). Thus, the Id proteins act to sequester bHLH proteins by forming inactive dimers to prevent binding of bHLH proteins to the E-box sites (34, 35, 36). Therefore, Id proteins are largely considered as dominant negative regulators of differentiation pathways (37, 38, 39). Some of the activities of Id2, such as induction of apoptosis, have been shown to reside in the N-terminal domain and are independent of HLH-mediated dimerization (40). Recent studies have also demonstrated that Id2 may act as an inhibitor of proliferation and is required for the determination and maintenance of the differentiated state of alveolar epithelial cells (41). The expression of Id4, unlike other members of the Id family, is tissue-restricted and is expressed primarily in adult brain, kidney, and testis (42, 43, 44).
Interestingly, terminally differentiated Sertoli cells also express Id proteins (45). The induction of Id in various cell types has been studied in response to serum, which is known to induce cell proliferation. Because the Sertoli cell is a terminally differentiated and postmitotic cell type, the expression and function of Id proteins are unclear. The present investigation was designed: to understand whether members of the Id family are differentially expressed in response to FSH, and to determine whether these proteins influence Sertoli cell differentiated functions.
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Materials and Methods |
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Western blotting and immunoprecipitation
Sertoli cells were cultured in 150-mm plates and were treated
with FSH and cAMP as above. After 72 h of treatment, the cells
were washed twice with HBSS and lysed with 1 ml M-PER lysis buffer
(Pierce Chemical Co., Rockford IL) supplemented with
miniprotein protease inhibitor cocktail (Boehringer Ingelheim GmbH, Indianapolis, IN) at 4 C for 30 min. Lysates were
centrifuged at 10,000 x g for 30 min at 4 C, and
supernatants were collected. The protein concentration in the
supernatants was estimated using Bradford’s assay (Bio-Rad Laboratories, Inc., Hercules, CA). Approximately 50–150 µg
protein in SDS sample loading buffer was boiled for 5 min and
electrophoresed on a 4–20% gradient mini-SDS gel (Bio-Rad Laboratories, Inc.). The protein was subsequently transferred
onto nitrocellulose membrane and probed with specific antibodies to
Id1, Id2, Id3, and Id4 (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA). The specific antigen-antibody complex was visualized
using an alkaline phosphatase chemiluminescent detection kit
(Bio-Rad Laboratories, Inc.).
For immunoprecipitation, the Sertoli cells were cultured in 150-mm plates and were treated with FSH and cAMP as above. After 72 h of treatment, the cells were rinsed three times with HBSS and labeled with 50 µl/0.5 ml [35S]methionine for 4 h at 32 C in methionine free DMEM (Life Technologies, Inc.). The cells were then washed with PBS and lysed at 4 C for 30 min with 1 ml M-PER lysis buffer (Pierce Chemical Co.) supplemented with miniprotein protease inhibitor cocktail (Boehringer Ingelheim GmbH). Lysates were centrifuged at 10,000 x g for 30 min at 4 C, and supernatants were collected. Each supernatant was incubated with 5 µl normal rabbit IgG and 50 µl packed protein A-agarose beads (Santa Cruz Biotechnology, Inc.) at 4 C for 1 h. The complex formed was removed by centrifugation at 500 x g for 5 min at 4 C. The supernatant was then incubated with 5 µl of either antirabbit Id1, Id2, Id3, or Id4 antibodies for 4 h at 4 C. Immunoprecipitates were collected on protein A-agarose beads and washed four times with M-PER lysis buffer and two times in TSA buffer (10 mM Tris pH8.0, 140 mM NaCl). The precipitate was dissolved in 30 µl SDS sample buffer subjected to electrophoresis on a 4–20% gradient mini SDS gel. The 14C-methylated protein markers (Amersham Pharmacia Biotech, Piscataway, NJ) was used to determine approximate sizes of the fractionated immune complexes. The gels were fluorographed according the procedure of Skinner and Griswold (50).
RNA preparation
Freshly isolated or cultured Sertoli cells were lysed directly
using TRI Reagent (Sigma, St. Louis, MO). The cell lysate
was then passed several times through a Pasteur pipette to form
homogenous lysate. To avoid any possible contamination of extracellular
material and high-molecular-weight DNA in the final RNA samples, the
homogenate was centrifuged at 12,000 x g for 10 min at
4 C. Total RNA was then isolated from the cell lysate, following the
manufacturer’s protocol for RNA isolation using TRI Reagent. The final
RNA pellet was dissolved in distilled water at a concentration of 1
mg/ml.
PCR
Total RNA (2 µg) was reverse transcribed in a final vol
of 20 µl containing 20 U RNasin (Promega Corp., Madison,
WI); 200 µM each of deoxy-ATP, deoxycycidine
triphosphate, thymidine 5'-triphosphate, and deoxy-GTP; 1 µg oligo dT
(Pharmacia, Peapack, NJ), 10 µM
dithiothreitol, and 200 U MMLV reverse transcriptase (Life Technologies, Inc.) in the MMLV first-strand synthesis buffer
supplied by the manufacturer (Life Technologies, Inc.).
The RNA and oligo dT primer in the buffer were first denatured for 5
min at 65 C, then cooled on ice before addition of nucleotides and
enzyme. The reverse transcriptase reaction was carried out at 37 C for
1 h. PCR was performed using the GeneAmp kit (Perkin-Elmer Cetus, Norwalk, CT) with 30 cycles as follows: 94 C, 1 min
(denaturation); 58C, 2 min (primer annealing); and 72 C, 1 min (primer
extension). Each PCR reaction contained 250 pg reverse-transcribed DNA,
1 µM of each 5' and 3' oligonucleotide primers; 2.5 U
Taq polymerase (AmpliTaq, Perkin-Elmer Cetus), 200 µM of each deoxy-ATP,
deoxycycidine triphosphate, deoxy-GTP, and thymidine
5'-triphosphate.
The primer pair sequences used were obtained from published sequences
of Id1, 2, 3, and 4 (Table 1
) and
synthesized from commercial sources. The possible contamination of RNA
with DNA was distinguished by performing the RT reaction without MMLV
reverse transcriptase. The absence of any product in the amplification
reaction using such a reverse-transcribed preparation indicated the
absence of any contaminating DNA in our RNA samples.
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Subcloning and sequencing
After amplification, the PCR products of each reaction were
subjected to electrophoresis through 1.5% agarose gel in buffer, and
the products were visualized by ethidium bromide staining. The bands
were then dissected out, and the DNA was isolated from the gel using
Glass MAX DNA isolation system (Life Technologies, Inc.).
The purified DNA fragments were subcloned into pGEM-TEZ (Promega Corp.) plasmid. The cloned DNA fragments were sequenced using
standard M13 forward and reverse primers in an automated
fluorescence-based sequencer (PE Applied Biosystems,
Norwalk ,CT). All the sequences reported are consensus of two different
experiments. The sequence alignments [Genetics Computer Group (Madison, WI) DNA analysis software] were carried
out using the available sequences of rat Id1, Id2, and Id3 and mouse
Id4.
Northern blot analysis
Total RNA was extracted from Sertoli cells cultured in 6-well
plates and treated with FSH, cAMP, serum, or vehicle alone (control),
as above, using TRI-Reagent (Sigma). Approximately 10 µg
total RNA was fractionated on a 1% formaldehyde-agarose gel. After
fractionation, the RNA in the gel was transferred onto nylon membrane
(Hybond+ N, Amersham Pharmacia Biotech) in 10 x SSC buffer and UV-cross-linked as
described previously (51). The membranes were then
prehybridized in Quick Hybridization buffer (Stratagene,
La Jolla, CA) for 30 min at 60 C. The hybridization was carried out at
60 C for 1 h with 32P-labeled Id probes. The
pGEM-tEZ plasmids containing the Id complementary DNA (cDNA) fragments
obtained by RT-PCR of Sertoli cell RNA were used as templates to
generate random primed (Stratagene) probes for Northern
blotting. The membrane was subsequently stripped and rehybridized with
the constitutively expressed rat cyclophilin. All the probes were
labeled using prime-it II kit from Stratagene. X-OMAT AR
Film (Eastman Kodak Co., Rochester, NY) was exposed
to the membranes overnight at -80 C, and densitometry values were
obtained by scanning with Imagequant Digital Image analysis system
(Molecular Dynamics, Inc., Sunnyvale, CA). The
densitometric values obtained for Id blots were normalized to the
values of rat cyclophilin so as to control for variation in loading and
determining the magnitude of expression.
Plasmids and antisense oligonucleotides
The CAT reporter plasmid (pUC8-CAT) containing -581 bp
(-581 bp mTf-CAT) was generously provided by Dr. G. Stanley McKnight
(University of Washington, Seattle, WA). The mouse transferrin promoter
used in the present study included the transcriptional initiation site
of the transferrin gene, which is 54 bp upstream of the start site of
translation (52).
The antisense oligonucleotide to rat Id1, 2, and 3 and mouse Id4 was
designed to incorporate 15 bases around and including the translational
initiation site (Table 1
). The scrambled oligonucleotides were
generated by Genetics Computer Group software analysis
package using the respective Id antisense oligonucleotides. The
antisense and the scrambled oligonucleotides (showing no substantial
homology to any known genes, as determined through a BLASTn search)
were synthesized from commercial sources using phosphorothioate
modification. The expression plasmid pCI-neo-Id2 was constructed. The
human Id2 (GenBank Accession No. M97796) PCR primers were designed to
amplify the human Id2 coding sequence (99–468 bp). The 369-bp Id2 PCR
fragment obtained through RT-PCR of human SKOV3 cell line RNA was first
subcloned in pGEM-T-EZ (Promega Corp.) plasmid. The
pGEM-T-EZ plasmid containing the Id2 fragment was then digested with
EcoRI, and the resulting fragment was ligated into
EcoRI-digested pCIneo expression plasmid (Promega Corp.).
Transfections
Sertoli cells, cultured in 24-well plates at the density of
106 cells for 48 h, were transfected with a
reporter gene construct by the calcium phosphate method coupled with
hyper osmotic shock (10% glycerol) as previously described (21, 53). Briefly, 1.5 µg reporter plasmid in 150 µl transfection
buffer [250 mM CaCl2, mixed 1:1
vol/vol with 2 x Hebes (28 mM NaCl, 50 mM
HEPES, and 1.47 mM
Na2HPO4, pH 7.05){rsqb]
was added to each well of a 24-well plate containing 1 x
106 Sertoli cells in 1 ml Ham’s F-12 with 0.01%
BSA, and incubation was performed at 32 C for 4 h. After
incubation, the cells were subjected to a hyper osmotic shock. The
medium was aspirated, and 1 ml 10% glycerol in HBSS (Life Technologies, Inc.) was added. The cells were incubated for 3
min, and the wells were washed twice before fresh Ham’s F-12 was
added. The transfected Sertoli cells were treated with 4
µM of either the antisense or scrambled oligonucleotide
immediately after transfection. Various treatments were added to the
cells 2 h after the addition of antisense oligonucleotides. The
cells were retreated with the oligonucleotide every 12 h, for a
total of 72 h. In each experiment the transfection efficiency was
monitored by transfecting the Sertoli cells with a plasmid containing
the ß-galactosidase gene driven by a CMV promoter. Subsequent
staining and counting the cells expressing ß-galactosidase (blue
color) resulted in approximately 25% transfection efficiency.
CAT assay
Assay of CAT activity was performed as follows: medium was
removed from the wells, and the cells were washed once with PBS. One
hundred microliters of the cell lysis buffer (Promega Corp.) was added to each well, and incubation was carried out
for 15 min at room temperature. The wells were then scraped, and buffer
was collected in 1.5-ml microfuge tubes. Tubes were heated to 65 C for
10 min to inactivate endogenous acetylases and then centrifuged at
12,000 x g for 10 min at 4 C to remove cell debris. An
aliquot of cell extract (54 µl) was mixed with 65 µl
0.25-M Tris (pH 8.0), 25 µg
N-butyryl coenzyme A (5 mg/ml; Sigma), and 0.1
µCi(1 µl) of 14C-chloramphenicol
(ICN, Costa Mesa, CA) and incubated overnight at 37
C. The mixture was extracted once with 300 µl mixed xylenes and
back-extracted with 100 µl 0.25-M Tris (pH
8.0). A 200-µl aliquot of the organic phase was counted in a
scintillation counter to determine the relative amount of CAT activity.
The average conversion of CAT substrate for treated cells ranged
between 20 and 30%. This assay was found to be linear with the protein
concentration used. The CAT reporter plasmid without the mTf promoter
was used as negative control. In response to FSH and dbcAMP, the
relative CAT activity of the negative control plasmid was in the range
of 1.5–2.
Statistical analysis
All transfection data were obtained from a minimum of three
different experiments unless otherwise stated. Each data point (from
treatments) was converted to a relative value, with the mean and
SEM from multiple experiments determined as indicated in
the figure legends. Data were analyzed by an ANOVA with the SAS Institute, Inc. (Durham, NC) statistical package as
indicated in the figure legends. When stated, data were also analyzed
with a Student’s t test, with a comparison between the
control and treatment group.
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Results |
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). As shown in Fig. 1, all four
members of the Id family are expressed in the Sertoli cells. Cloning
and sequencing of the PCR products confirmed the identity of the Id1,
Id2, Id3, and Id4 transcripts. The four Id transcripts were observed
when the cultured Sertoli cells were untreated or treated with FSH or
the cAMP analog dbcAMP for 72 h. This PCR procedure demonstrates
the absence or presence of the Id expression, whereas the Northern blot
procedure below was used to quantitate messenger RNA (mRNA) levels.
Expression of the Id transcripts was also observed in freshly isolated
Sertoli cells, whole 20-day-old testis, and embryonic day-15 testis
(data not shown). To extend this analysis, the expression of the Id
proteins was determined. Western blotting of 50–150 µg total Sertoli
cell protein, extracted after 72 h of culture, was performed with
isoform specific Id antibodies. The immunoreactivity alone was low and
inconsistent, presumably because Id proteins are expressed at low
levels and have a short half-life (54). To circumvent this
problem, immunoprecipitation was performed on total Sertoli cell
proteins metabolically labeled with
35S-methionine. The labeled proteins were
immunoprecipitated with isoform-specific Id antibodies,
electrophoretically separated on SDS polyacrylamide gels and
fluorographed. As shown in Fig. 2, Id1,
Id2, Id3, and Id4 proteins were detectable in Sertoli cells. These
observations demonstrate that all four members of the Id family are
expressed in Sertoli cells. Single bands were observed for Id2 (14
kDa), Id3 (20 kDa), and Id4 (18 kDa), whereas two bands at 16 and 20
kDa were observed for Id1.
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). A single 1.2-kb Id1 (Fig. 3A) and 1.6-kb Id2 (Fig. 4A) transcript was observed after
exposure of the Northern blot. Two Id3 transcripts (Fig. 5A) of approximately 1.8 and 1.4 kb were
observed in Sertoli cells after exposure of the Northern blots. The
presence of two Id3 mRNA transcript sizes in Sertoli cells (Fig. 5A) is
a novel observation (55) and may be attributable to
modifications in the 3' untranslated region. The 3' modification is
presumably a result of differential splicing or alternatively used poly
adenylation sites within the 3' untranslated region. Such a
modification at the 3' end has also been reported previously for
Id4, which results in multiple mRNA transcripts (approximately 1.7,
2.8, and 4 kb) (56, 57). Multiple transcripts of Id4 were
also observed when RNA from Sertoli cells treated with FSH was
hybridized with Id4 cDNA (Fig. 6A). The
Northern blot analysis confirms the RT-PCR (Fig. 1) and
immunoprecipitation (Fig. 2) data, indicating that all four isoforms of
Id are expressed in Sertoli cells. Based on the normalization of the
Northern blots with a constitutively expressed cyclophilin gene, it is
suggested that the Id genes are expressed at different levels. Id2 and
Id3 genes show the highest levels of expression, followed by Id1, and
the expression of Id4 is the lowest in control untreated Sertoli cells
(data not shown). Previous studies have shown cyclophilin to be
constitutively expressed and not affected by hormone treatment of
Sertoli cells (16, 21, 31).
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The actions of FSH on Sertoli cells are primarily through the cAMP
protein kinase A pathway (9). To explore the potential
regulatory role of the cAMP pathway on Id gene expression in Sertoli
cells, a cell permeable cAMP analog known to promote cell
differentiation was added to the cells, and the expression of various
isoforms of Id was evaluated. Similar to the effect of FSH, cAMP
significantly reduced (30%) the expression of Id1 (Fig. 3). Treatment
of Sertoli cells with cAMP had no effect on the levels of Id2 (Fig. 4)
and Id3 (Fig. 5). The effect of cAMP on Id4 expression (all three
transcripts) was more dramatic than FSH, which resulted in more than a
3-fold increase in the levels of Id4 over the controls (Fig. 6). Taken
together, these observations suggest that the expression of Id1 and Id4
in Sertoli cells is influenced by FSH through the cAMP-protein kinase A
pathway.
Previous literature suggests that the expression of Id is inducible by
serum (58). To assess the effect of serum on Sertoli cell
Id expression, the cells were treated with 10% bovine calf serum.
Treatment of Sertoli cells with serum significantly stimulated Id1 and
Id2 expression (Figs. 3 and 4). The expression of the 1.4-kb Id3
transcript was significantly increased in response to serum, whereas
the expression of the larger 1.8-kb transcript remained unchanged. In
contrast, the expression of Id4 (1.7 kb) transcript was significantly
decreased (Fig. 6). Surprisingly, the 4-kb Id4 transcript was virtually
undetectable in the Northern blot analysis after serum treatment (Fig. 6A). However, a novel 1.3-kb Id4 transcript was observed after serum
treatment (Fig. 6A). This novel 1.3-kb Id4 transcript was expressed at
relatively high levels, compared with the 2.8-kb and 1.7-kb Id4
transcripts (Fig. 6A). Observations demonstrate opposing effects of FSH
and serum on expression of the Id genes. The expression of Id1 (Fig. 3)
and Id4 (Fig. 6), in response to these agents, is particularly
interesting and suggests that both these isoforms may have opposing
functions in Sertoli cells.
An antisense approach was used to better understand the role of Id1,
Id2, Id3, and Id4 in regulating Sertoli cell differentiated functions.
Sertoli cells from 20-day-old rats, cultured under serum-free
conditions, were transfected with a proximal transferrin-promoter CAT
construct (Tf-CAT) as a marker of Sertoli cell differentiation
(6, 60). The DNA phosphorothioate modified antisense
oligonucleotides covering the region around the ATG initiation codons
of rat Id1, Id2, and Id3 and mouse Id4 (Table 1) were added to the
Sertoli cells and were subsequently treated with either FSH, cAMP, or
serum. As expected, FSH, cAMP, and serum stimulated the Tf-CAT
activity, reflecting their ability to promote Sertoli cell
differentiation (Fig. 7). In the presence
of Id1 antisense oligonucleotide, the Tf-CAT activity in response to
FSH and cAMP increased over 2-fold and 1.2-fold, respectively, compared
with the corresponding Tf-CAT activity observed in the absence of
antisense oligonucleotide (Fig. 7). The antisense oligonucleotides have
been successfully used to target and reduce individual Id mRNA levels
(37). In contrast to the effects of Id1 antisense
oligonucleotide on transferrin promoter activity, addition of Id2
antisense oligonucleotide to the Sertoli cells transfected with Tf-CAT
reporter plasmid resulted in an inhibition of CAT activity in response
to FSH and cAMP (Fig. 8). The
down-regulation of transferrin promoter activity in the presence of Id2
antisense oligonucleotide suggests that, in Sertoli cells, Id2
expression may be required to maintain a differentiated response. To
address this hypothesis, Sertoli cells were cotransfected with the
transferrin promoter reporter construct and with an Id2 expression
plasmid. The ectopic expression of Id2 resulted in a small, but
significant, increase in transferrin promoter activity (Fig. 9). Taken together, the Id2 antisense and
over-expression data suggest that Id 2 may be involved in the
regulation of transferrin promoter activity in Sertoli cells.
Surprisingly, the addition of Id3 and Id4 antisense oligonucleotides
had no effect on the transferrin promoter activity (data not shown).
However, the lack of an effect of Id4 antisense oligonucleotide may be
because the Id4 antisense oligonucleotide was designed using the 5' end
of the mouse Id4 sequence. The 5' end of the mouse Id4 sequence may not
be homologous to the 5' region of the rat Id4 sequence that is
currently not known. The potential function of Id4 requires further
investigation. The change in the activity of the transferrin promoter
activity in the presence of antisense oligonucleotide is not
attributable to nonspecific effects of the oligonucleotides, because
control-scrambled oligonucleotides (Figs. 7 and 8) and Id3 and Id 4
antisense oligonucleotides (data not shown) had no effect on
transferrin promoter activity.
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Discussion |
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The experiments presented in the current study show that all four Id-family members are expressed in postmitotic and differentiated Sertoli cells. Distinct differences in steady-state expression levels of the Id isoforms under various physiological conditions were observed. Interestingly, Id1 expression was down-regulated by FSH, whereas Id4 expression was up-regulated by FSH. In contrast, serum up-regulated the expression of Id1, 2, and 3, but down-regulated Id4 expression in Sertoli cells. Therefore, the expression of Id isoform genes is differentially regulated in Sertoli cells.
The decrease in the expression of Id1 observed after the treatment of cultured Sertoli cells with FSH was anticipated, because FSH is required to maintain differentiated functions (59). Further evidence that Id1 negatively influences Sertoli cell differentiated function is furnished by the observation that the ectopic expression of Id1 in Sertoli cells blocks FSH-mediated transferrin (21) and c-fos (22) promoter activation. Ectopic expression of Id1 was also shown to strongly inhibit the myelin promoter activity in Schwann cells (63). Conversely, Id1 antisense oligonucleotide, which selectively targets the Id1 mRNA, increases the transferrin promoter activation in response to FSH. The reduction in the levels of Id1 mRNA by an Id1 antisense oligonucleotide can be manifested, in terms of decrease in the Id1 protein levels (37). A decrease in Id1 protein may allow the formation of functional dimers, which can then bind to the E- Box in the proximal transferrin promoter. In contrast, ectopic expression of Id1 in Sertoli cells inhibits Tf-CAT activity, presumably because Id inhibits the binding of bHLH proteins to the E-Box in the transferrin promoter by forming nonfunctional dimers (21). The effect of FSH on Id1 gene expression is mediated primarily through the cAMP-protein kinase A system, because cAMP treatment also reduced Id1 gene expression. A similar decrease in the Id1 mRNA level was also observed in Schwann cells treated with cAMP (63). This experiment supports the role of Id1 as a negative regulator of Sertoli cell differentiation and implicates the HLH family of transcription factors as key regulators of Sertoli cell differentiation.
In general, the Id proteins are expected to have an overlapping function because of their ability to form nonfunctional dimers with differentiation-inducing bHLH proteins. Recent studies suggest that this may not be true, and some Id proteins (i.e. Id2 and Id 4) may, in fact, be required to induce and maintain the differentiated state of a particular cell (41, 64, 65, 66, 67). Id2 is required for the determination and maintenance of the differentiated alveolar epithelial cells (41). The constitutive expressions of Id2 and Id3 mRNA in Sertoli cells suggests that both these proteins may have a significant role in maintaining Sertoli cell function. This hypothesis is supported by the observation that an antisense oligonucleotide to Id2 inhibits transferrin promoter activation, which is in contrast to the effect of Id1 antisense oligonucleotide. The exact mechanism by which Id2 regulates the promoter activity is not known. The speculation is presented that Id2 may inhibit a specific bHLH transcription factor complex that may normally repress Sertoli cell differentiated functions. Id2 was recently shown to homodimerize and inhibit cyclin A promoter activity in alveolar cells (41). The expression of Id3 and the lack of an antisense effect in Sertoli cells is intriguing. It is likely that Id3 may be involved in transcriptional events not directly involved in transferrin promoter activation.
The dynamics of Id4 expression in Sertoli cells is of particular interest. Id4 mRNA expression is up-regulated in response to FSH and cAMP. This observation contrasts sharply with the long-standing view that high levels of Id mRNA in proliferative and undifferentiated cells decrease as they are induced to differentiate (58). The specificity of this Id4 regulation is distinct, because the levels of mRNA encoding Id2 and Id3 remained unchanged, and those of Id1 decreased after treatment of Sertoli cells with FSH or cAMP. The elevated expression of Id4 in response to FSH implicates its role in Sertoli cell differentiation. The mouse Id4 antisense oligonucleotide had no effect on the transferrin promoter activation, possibly because it may not have targeted the rat Id4 mRNA in Sertoli cells. The mechanism by which Id4 influences cellular function remains to be elucidated. Id4 expression is differentially regulated in various cell types. Id4 expression is up-regulated in differentiated adipocytes by hormones such as insulin, dexamethasone, and methyl-isobutyllxanthine (64) but is down-regulated in astrocytes by cAMP (56). These observations suggest that Id4 may selectively dimerize with stage- and cell-specific bHLH proteins in the various cell types. The significance of Id4 in regulating Sertoli cell differentiated functions requires further investigation.
The expression of all four Id isoforms in Sertoli cells in response to serum is entirely opposite to that observed after FSH and cAMP treatment. The effect of serum, a mixture of growth factors, hormones, and mitogens on cells at the concentration used in the present study is generally considered a proliferative signal. The increase in the expression of Id1, Id2, and Id3 in response to serum was anticipated and confirms previous studies that Id proteins are serum-inducible (38, 68, 69). The serum-mediated decrease in the expression of Id4 is intriguing. The observed increase in the expression of Id4 in response to differentiating signals like FSH and a decrease in response to proliferative signals, such as serum, supports the hypothesis that Id4 may be required to maintain or regulate Sertoli cell differentiated functions.
All four Id proteins, when fused to the heterologous GAL4 DNA-binding
domain, can activate GAL4-dependent transcription, which required an
intact HLH activity (70). Cotransfection with exogenous
class A bHLH protein (E-proteins) can greatly potentiate the
trans-activation, which is abolished upon cotransfection
with class B bHLH proteins (70). The Sertoli cells express
high levels of E47 (29) and REB (30) class
A bHLH protein. It is likely that the Id proteins may differentially
bind to Sertoli cell bHLH proteins and influence promoter activation.
Although the HLH domains of all four Id proteins are largely conserved,
the highly divergent C- and N-terminal domains (58) may
have selective binding preferences and trans-activation
potential in Sertoli cells. These transcriptional actions of the Id
isoforms will be of interest to elucidate.
In summary, the postmitotic and differentiated Sertoli cells express high levels of Id. The expression of Id1 and Id4 is regulated by signals that induce differentiation and proliferation, such as FSH and serum, respectively. Contrary to the hypothesis that Id proteins have redundant functions, the observations presented in the current study suggest that Id1 may act to maintain growth potential, whereas Id2 and Id4 may be involved in the differentiation of Sertoli cells. Based on the expression of Id2 and its effect on transferrin promoter, it is speculated that Id2 may have a dual function in Sertoli cells. Id2 may be required to maintain the growth potential of Sertoli cells in response to mitogenic stimuli such as serum, but may also promote differentiation in response to FSH. Therefore, the Id proteins seem to have functions other than simply the inhibition of differentiation. These differential functions of Id may involve molecular mechanisms, such as transcriptional activation, sequestering of bHLH proteins, and other isoform specific functions that remain to be fully understood.
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Acknowledgments |
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Footnotes |
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Received November 3, 2000.
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  P. Viswanathan, M. A. Wood, and W. H. Walker Follicle-Stimulating Hormone (FSH) Transiently Blocks FSH Receptor Transcription by Increasing Inhibitor of Deoxyribonucleic Acid Binding/Differentiation-2 and Decreasing Upstream Stimulatory Factor Expression in Rat Sertoli Cells Endocrinology, August 1, 2009; 150(8): 3783 - 3791. [Abstract] [Full Text] [PDF]
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 E. A. Ahmed, A. D. B.-v. Rijbroek, H. B. Kal, H. Sadri-Ardekani, S. C. Mizrak, A. M.M. v. Pelt, and D. G. d. Rooij Proliferative Activity In Vitro and DNA Repair Indicate that Adult Mouse and Human Sertoli Cells Are Not Terminally Differentiated, Quiescent Cells Biol Reprod, June 1, 2009; 80(6): 1084 - 1091. [Abstract] [Full Text] [PDF]
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 K. J. Gordon, K. C. Kirkbride, T. How, and G. C. Blobe Bone morphogenetic proteins induce pancreatic cancer cell invasiveness through a Smad1-dependent mechanism that involves matrix metalloproteinase-2 Carcinogenesis, February 1, 2009; 30(2): 238 - 248. [Abstract] [Full Text] [PDF]
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 W. Xia, D. D. Mruk, W. M. Lee, and C. Y. Cheng Differential Interactions between Transforming Growth Factor-beta3/TbetaR1, TAB1, and CD2AP Disrupt Blood-Testis Barrier and Sertoli-Germ Cell Adhesion J. Biol. Chem., June 16, 2006; 281(24): 16799 - 16813. [Abstract] [Full Text] [PDF]
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 M. Carroll, M. Hamzeh, and B. Robaire Expression, Localization, and Regulation of Inhibitor of DNA Binding (Id) Proteins in the Rat Epididymis J Androl, March 1, 2006; 27(2): 212 - 224. [Abstract] [Full Text] [PDF]
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 T. Muir, I. Sadler-Riggleman, and M. K. Skinner Role of the Basic Helix-Loop-Helix Transcription Factor, Scleraxis, in the Regulation of Sertoli Cell Function and Differentiation Mol. Endocrinol., August 1, 2005; 19(8): 2164 - 2174. [Abstract] [Full Text] [PDF]
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J. Chaudhary, I. Sadler-Riggleman, J. M. Ague, and M. K. Skinner The Helix-Loop-Helix Inhibitor of Differentiation (ID) Proteins Induce Post-Mitotic Terminally Differentiated Sertoli Cells to Re-Enter the Cell Cycle and Proliferate Biol Reprod, May 1, 2005; 72(5): 1205 - 1217. [Abstract] [Full Text] [PDF]
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 E. Kebebew, M. Peng, P. A. Treseler, O. H. Clark, Q.-Y. Duh, D. Ginzinger, and R. Miner Id1 Gene Expression Is Up-Regulated in Hyperplastic and Neoplastic Thyroid Tissue and Regulates Growth and Differentiation in Thyroid Cancer Cells J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6105 - 6111. [Abstract] [Full Text] [PDF]
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M. J. Scobey, C. A. Fix, and W. H. Walker The Id2 Transcriptional Repressor Is Induced by Follicle-stimulating Hormone and cAMP J. Biol. Chem., April 16, 2004; 279(16): 16064 - 16070. [Abstract] [Full Text] [PDF]
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 L. Shan, M. Yu, C. Qiu, and E. G. Snyderwine Id4 Regulates Mammary Epithelial Cell Growth and Differentiation and Is Overexpressed in Rat Mammary Gland Carcinomas Am. J. Pathol., December 1, 2003; 163(6): 2495 - 2502. [Abstract] [Full Text]
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J. J. Buzzard, N. G. Wreford, and J. R. Morrison Thyroid Hormone, Retinoic Acid, and Testosterone Suppress Proliferation and Induce Markers of Differentiation in Cultured Rat Sertoli Cells Endocrinology, September 1, 2003; 144(9): 3722 - 3731. [Abstract] [Full Text] [PDF]
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M. K. Rao, C. M. Wayne, M. L. Meistrich, and M. F. Wilkinson Pem Homeobox Gene Promoter Sequences that Direct Transcription in a Sertoli Cell-Specific, Stage-Specific, and Androgen-Dependent Manner in the Testis in Vivo Mol. Endocrinol., February 1, 2003; 17(2): 223 - 233. [Abstract] [Full Text] [PDF]
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