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Center for Reproductive Biology, Washington State University School of Molecular Biosciences, Pullman, Washington 99164-4231
Address all correspondence and requests for reprints to: Dr. Michael K. Skinner, Center for Reproductive Biology, Washington State University School of Molecular Biosciences, Pullman, Washington 99164-4231. E-mail: skinner{at}mail.wsu.edu
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Introduction |
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Recently, a novel member of the WH family, rWIN, was cloned from a rat pancreatic endocrine cell line (11). During embryonic development, rWIN and its human homolog, HFH-11 (12), are expressed in proliferating mesenchymal and epithelial cells of the lung, liver, intestine, renal cortex, and pancreas. In the adult tissues, rWIN expression is high in the testis and lung, whereas the expression of HFH-11 is restricted to adult thymus and testis. During embryogenesis, the expression of Trident, the mouse homolog of rWIN/HFH-11, is observed in all proliferative cells, but not in resting cells (13). The role of Trident in cellular proliferation is based on the observation that Trident is expressed in the cells entering the S phase and is then phosphorylated in the M phase of the cell cycle (13). The targeted disruption of the Trident gene in mice results in postnatal death due to polyploidy in the developing myocardium, leading to circulatory defects (14). Therefore, Trident appears to have a role in DNA replication during the G2 and M phases of the cell cycle. Whether Trident is expressed in the adult mouse testis remains to be determined, but, similar to HFH-11, it is highly expressed in the thymus (13). The particular cell type(s) in the testis that expresses rWIN is not known, but its human homolog, HFH-11, is highly expressed in spermatocytes and spermatids (12). In contrast, spermatogonia undergoing active proliferation reportedly do not express HFH-11. Another cell type in the seminiferous tubule is the Sertoli cell. The Sertoli cells have epithelial characteristics and in postpubertal testis provide cytoarchitectural support to the developing germ cells (15). Most, if not all, Sertoli cell functions are regulated by the gonadotropin FSH.
The Sertoli cells continue to proliferate prepubertally until the onset
of puberty and formation of the blood-testis barrier (16). After
formation of the blood-testis barrier, the Sertoli cells become
postmitotic and terminally differentiated. Sertoli cell proliferation
and pre- and postpubertal gene expression are in part regulated by the
gonadotropin FSH (17). The molecular mechanisms regulating the
embryonic mesenchymal to epithelial transition of Sertoli cells and
maintenance of the epithelial phenotype of Sertoli cells are largely
unknown. Therefore, transcription factors that may be potentially
involved in regulating Sertoli cell differentiation are being
investigated. One approach has been to use the promoters of a number of
Sertoli cell genes and analyze for conserved cis elements. A
response element found to be highly conserved in a number of Sertoli
cell genes was an E box response element. The E box element is a
hexanucleotide consensus that binds transcription factors of the basic
helix loop helix family (18). The two ubiquitously expressed bHLH
proteins, REB
and the E2A gene product E47, were shown to be
expressed in Sertoli cells (19, 20). To identify Sertoli cell-specific
bHLH transcription factors, a Sertoli cell complementary DNA (cDNA)
library was screened with the conserved bHLH domain of REB under
conditions of low stringency. One of the clones isolated with a 1023-bp
insert when searched for homology with known sequences in the GenBank
emerged as the rWIN sequence (11). The isolation of rWIN clone from the
Sertoli cell cDNA library suggested that rWIN may be expressed in
Sertoli cells. The current study was designed to investigate the
expression and action of rWIN in Sertoli cells and its potential role
in regulating Sertoli cell differentiated functions. Interestingly,
rWIN was found to have a role in mediating the actions of FSH on
Sertoli cells.
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Materials and Methods |
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Sertoli cell cDNA library
The rat Sertoli cell cDNA library was constructed in HybriZapII
by Stratagene from polyadenylated RNA isolated from
purified Sertoli cells of 20-day-old rats. The HybriZap library is
primarily designed for use in the yeast two-hybrid screening, but also
allows for nucleic acid screening. The standard procedures for nucleic
acid screening were followed as mentioned previously (25). Individual
plaques (1.5 x 106) were screened with the
170-bp REB PCR fragment corresponding to the bHLH domain and end
labeled with [
-32P]ATP and T4 polynucleotide
kinase. The positive clones were excised from the Hybrizap II library
to obtain pAD plasmid-containing clones. The pAD plasmids were analyzed
by restriction mapping and sequencing. The sequences were subjected to
a FASTA and BLASTn searches against known sequences in GenBank.
RNA preparation
Freshly isolated or cultured Sertoli cells were lysed directly
using Tri-Reagent (T9424, Sigma, St. Louis, MO). The cell
lysate was then passed several times through a Pasteur pipette to form
homogenous lysate. The whole tissues (skeletal muscle, brain, and
detunicated testis) were placed in Tri-Reagent (5%, wt/vol) and
homogenized in a tissue homogenizer (Tissue Tearor, BioSpec Products, Inc., Bartlesville, OK). To avoid any possible
contamination of extracellular material and high mol wt 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 and whole tissue homogenate 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 volume of
20 µl containing 20 U RNasin (Promega Corp., Madison,
WI); 200 µM each of deoxy (d)-ATP, dCTP, dTTP, and dGTP;
1 µg oligo(deoxythymidine) (Pharmacia, Piscataway, NJ), 10
µM dithiothreitol, and 200 U Moloney murine leukemia
virus (MMLV) reverse transcriptase (BRL, Gaithersburg, MD) in the MMLV
first strand synthesis buffer supplied by the manufacturer (BRL). The
RNA and oligo(deoxythymidine) 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 Corp./Cetus, Emeryville, CA) with 30 cycles
as follows: 94 C for 1 min (denaturation), 58 C for 2 min (primer
annealing), and 72 C for 1 min (primer extension). Each PCR reaction
contained 250 pg reverse transcribed DNA, 1 µM of each
5'- and 3'-oligonucleotide primer, 2.5 U Taq polymerase
(AmpliTaq, Perkin-Elmer Corp.), and 200
µM of each dATP, dCTP, dGTP, and dTTP. After
amplification, the product of each reaction was subjected to
electrophoresis through 1.5% agarose gel in buffer, and the products
were visualized by ethidium bromide staining.
The primer pair sequences used were obtained from published sequences
of rWIN (GenBank accession no. U83112) and synthesized from commercial
sources. Primer pairs were designed to amplify the N-terminal region of
the rWIN protein containing the winged helix domain (Fig. 1
). 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. The sequence and
position of the primers were as follows: WIN 5'-primer (507), 5'-TTG
GGA CCA AAG CCA GGG GCT AAG; and WIN 3'-primer (1218), 5'-CTT TCG CCG
TGC GCC TAG TGG GAG TTC.
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, the rWIN-specific primers were designed to include
all of the exons, as reported previously (11). Each RT reaction was
performed using three different samples. The PCR-based amplification
reactions were carried out in duplicate on each reverse transcribed RNA
sample. Simultaneous PCR reactions were also carried out using primers
designed to rat cyclophilin to monitor the efficiency of the PCR (26).
Cyclophilin was faithfully amplified in all of the PCR reactions,
indicating consistency in the quality of RT and PCR reactions. The data
presented, therefore, are representative of three different RT-PCR
reactions carried out in duplicate.
Subcloning and sequencing
The PCR fragments were run on 1.5% agarose gel, stained with
ethidium bromide, and visualized. The bands were then dissected out,
and the DNA was isolated from the gel using Glass MAX DNA isolation
system (BRL). The purified DNA fragments were subcloned into pCR 2.1
(Invitrogen, San Diego, CA) plasmid. The cloned DNA
fragments were sequenced using standard M13 forward and reverse primers
in an automated fluorescence-based sequencer (PE Applied Biosystems, Foster City, CA). Internal sequences were obtained
using nested primers that were designed based on previous sequences.
All of the sequences reported are the consensus of two different
experiments. The sequence alignments (GCG DNA analysis software,
Genetics Computer Group, Madison, WI) were carried out
using the available sequence of rWIN.
Northern blot analysis
Total RNA was extracted from cultured Sertoli cells [treated
with FSH, cAMP, or vehicle alone (control)], freshly isolated Sertoli
cells, germ cells, and various tissues as described above using
Tri-Reagent (Sigma). For time-course experiments the RNA
was extracted from cultured Sertoli cells treated with FSH or cAMP for
various time intervals (see figure legends). Approximately 10 µg
total RNA were fractionated on a 1% formaldehyde-agarose gel. After
fractionation, the RNA in the gel was transferred onto a nylon membrane
(Hybond+ N, Amersham Pharmacia Biotech, Arlington Heights, IL) in 10 x SSC (standard
saline citrate) buffer and UV-cross linked as described previously
(25). The membranes were then prehybridized in Quick Hybridization
buffer (Stratagene) for 30 min at 60 C. The hybridization
was carried out at 60 C for 1 h with
32P-labeled rWIN probe obtained by RT-PCR of
Sertoli cell RNA. The membrane was subsequently stripped and
rehybridized with rat cyclophilin. All of the probes were labeled using
the Prime-It II kit from Stratagene.
Plasmids and antisense oligonucleotides
The chloramphenicol acetyltransferase (CAT) reporter plasmid
(pUC8-CAT) containing -581 bp (-581 bp mTf-CAT) was provided by Dr.
G. Stanley McKnight (University of Washington, Seattle, WA) (27). The
3-kb mouse transferrin promoter was ligated into PGL2 basic plasmid
(Promega Corp.) as previously described (28). 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 (29).
The antisense oligonucleotide to rWIN was designed to incorporate 15 bases around and including the translational initiation site. The scrambled oligonucleotide was generated using the GCG software analysis package with the rWIN antisense oligonucleotide. Both oligonucleotides were synthesized from commercial sources using phosphothioate modification. The expression plasmid WH-pREP7 was made by cloning the 711-bp PCR product into the NotI site of the pREP7 plasmid (Invitrogen).
Transfection
Sertoli cells cultured in 24-well plates at a density of
106 cells for 48 h were transfected with a
reporter gene construct by the calcium phosphate method coupled with
hyperosmotic shock (10% glycerol) as previously described (27).
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)] were
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 hyperosmotic 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. Various treatments were subsequently added, and cells were
incubated for 48 h before harvesting for CAT assays. For antisense
experiments the transfected Sertoli cells were treated with the
antisense, sense, or scrambled oligonucleotide immediately after
transfection and 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 cytomegalovirus
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.) were 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)
[14C]chloramphenicol (ICN Biomedicals, Inc., 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 from
20–30%. This assay was linear with the protein concentration
used.
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 CAT activity (control CAT
activity = 1), with the mean and SEM from multiple
experiments determined as indicated in the figure legends. Data were
analyzed by an ANOVA and/or Student’s t test with the SAS
statistical package (SAS Institute, Inc., Cary, NC) as
indicated in the figure legends. The CAT reporter plasmids without mTf
promoter were used as negative controls. In response to FSH and
(Bu)2cAMP, the relative CAT activity of the
negative control plasmid ranged from 1.5–2.
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Results |
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(30) under conditions of low
stringency. The rWIN was identified due to nonspecific interactions in
the screen. The rWIN clone, initially termed 6N2–2-1 in the plasmid
pAD-GAL4, had an insert of 1023 bp. Sequence analysis and translation
in all reading frames indicated that a consensus bHLH domain was absent
from the clone 6N2–2-1. Comparison of the 6N2–2-1 sequence against
known sequences in GenBank and EST databases revealed that 6N2–2-1 was
identical to the recently reported gene rWIN (GenBank accession no.
U83112) isolated from the rat insulinoma (pancreatic endocrine) cell
line. The reported full length of rWIN is approximately 3439 bp and
codes for a 771-amino acid protein with potential start and stop
nucleotide sites positioned at 87 and 2400 bp, respectively (11). The
clone 6N2–2-1 therefore consisted entirely of the 3'-untranslated
region of rWIN (Fig. 1).
Two different approaches, RT-PCR and Northern blot analysis, were used
to confirm that rWIN is expressed in Sertoli cells. Oligonucleotide
primers were designed toward the 5'-end of the rWIN sequence to amplify
a 711-bp region by RT-PCR. The 711-bp PCR product corresponds to the
nucleotides between 507 and 1218 and spans the DNA-binding domain of
rWIN, including the potential exons 4, 5, and 6 as reported previously
(11) (Fig. 1).
As shown in Fig. 2, two PCR products,
designated A and B, of approximately 700 bp were detected in the
reverse transcribed RNA obtained from the whole testis, cultured
Sertoli cells, and germ cells. To determine their identities, PCR
products A and B were isolated, subcloned, and sequenced. Sequence
analysis revealed that both PCR products were nearly identical, except
that the smaller PCR product B lacked the 45-bp exon 6 (Fig. 3). Interestingly, exon 4 was absent in
both PCR products A and B (Fig. 3). rWIN expression was not observed in
other tissues examined, such as brain, liver, kidney, lungs, and muscle
(Fig. 2). The PCR data shown in Fig. 2 suggest that rWIN is also
expressed in Sertoli cells. The expression of rWIN, as determined by
PCR, was also observed in the testis of prenatal day 18 embryos and
postnatal day 0 and 10 rats (Fig. 2). The rWIN transcript was
previously shown to be expressed in the adult rat testis (11), and its
human homolog HFH-11 was localized to the spermatocytes and spermatids
within the seminiferous epithelium (12). The embryonic testis primarily
consists of mitotically active and differentiating Sertoli cells,
interstitial cells, and spermatogonia in a stage of mitotic arrest.
Previous studies have shown that HFH-11 in the testis is expressed
primarily in differentiating germ cells, but not spermatogonia, which
suggests that the cell type(s) expressing rWIN in the embryonic testis
is potentially the Sertoli cells and/or interstitial cells (12).
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, rWIN
expression was observed in the cultured Sertoli cells (control) or in
cultured Sertoli cells treated with the gonadotropin FSH.
Interestingly, the rWIN transcript was also observed in the RNA
obtained from germ cells. The fact that only a single transcript was
seen in Sertoli cells with the Northern blot as opposed to two
transcripts seen in the PCR reaction suggests that the size
fractionation of RNA was not sufficient to resolve the two transcripts
with a 45-bp size difference due to the presence of exon 6 (Fig. 4A).
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).
A time-course experiment was performed to determine the dynamics of the
down-regulation of rWIN expression by FSH. Cultured Sertoli cells were
treated with FSH for different periods of time, and the total RNA was
used to measure the expression of rWIN and transferrin genes (Fig. 5A). Transferrin was used as a marker for
Sertoli cell differentiation and as an example of a gene that is
up-regulated by FSH and cAMP. The quantitation of Northern blot data by
densitometric analysis indicated that FSH maximally stimulated rWIN
expression within 30 min to 1 h (P < 0.001
compared with WIN expression at time zero), which returned to basal
levels by 4 h. In contrast, an increase in transferrin expression
was observed only after 8 h of FSH treatment (Fig. 5B). Similar
results were observed with (Bu)2cAMP stimulation
of Sertoli cells (Fig. 5B). Therefore, the expression of rWIN precedes
the expression of transferrin and suggests a role for rWIN as a
potential immediate early gene involved in the cellular/nuclear events
leading to activation of the transferrin gene. The role of rWIN as an
immediate early gene was supported by the observation that treatment of
Sertoli cells with cycloheximide (5 µg/ml) for 2 h superinduced
rWIN expression (data not shown).
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). The
cells were treated with the antisense oligonucleotide (4
µM) every 12 h for a total of 72 h before
harvesting cells for the CAT assay. As a control the cells were either
not treated with an antisense oligonucleotide or were treated with
scrambled rWIN or a sense oligonucleotide. FSH and cAMP significantly
stimulated CAT activity above basal levels in the absence of any
oligonucleotide (Fig. 6). The presence of
scrambled or sense oligonucleotide did not significantly change FSH- or
cAMP-stimulated transferrin promoter activity compared with the control
value (Fig. 6). However, in the presence of an antisense
oligonucleotide a significant repression of FSH-stimulated transferrin
promoter activity was observed (Fig. 6). The observations suggest that
rWIN expression is sensitive to antisense inhibition and appears to
be involved in FSH-stimulated transferrin promoter activation.
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, overexpression of the rWIN DNA-binding
domain significantly down-regulated the -581 kb proximal transferrin
promoter activity in response to FSH or cAMP. Similar results were
obtained when the 3-kb proximal transferrin promoter was
cotransfected with WH-pREP7 overexpression plasmid (data not shown).
Transfection of various amounts (200 ng to 2 µg) of WH-pREP7 plasmid
showed a concentration-dependent decrease in transferrin promoter
activity (data not shown). The optimum concentration of 1 µg rWIN
expression plasmid was then used for all of the overexpression
experiments. The activity of the mouse transferrin promoter in response
to FSH did not change significantly in the presence or absence of pREP7
plasmid alone. To determine whether the down-regulation was due to the
direct binding of rWIN to a corresponding cis element(s) in
the transferrin promoter, the promoter was analyzed for the rWIN
DNA-binding domain AGATTGAGTA (11). The absence of a consensus rWIN
DNA-binding domain in the transferrin promoter suggests that the
down-regulation may not be due to the direct binding of rWIN to its
corresponding cis element on the transferrin promoter.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Long term stimulation (72 h) of cultured Sertoli cells with FSH decreased rWIN expression. The dynamics of the regulation of rWIN expression suggest that rWIN is transiently up-regulated within 30 min of FSH stimulation. Such a transient increase in rWIN expression is analogous to the induction of the immediate early gene c-fos (31). The expression of the transferrin gene correlates with Sertoli cell differentiation and is up-regulated only after rWIN expression approaches basal levels, which is approximately 4 h after FSH stimulation. The transient increase in rWIN expression precedes the increase in transferrin gene expression. Observations suggest a role for rWIN as a potential immediate early gene that may be required to switch on the network of regulatory genes involved in Sertoli cell differentiation.
The treatment of Sertoli cells with an antisense oligonucleotide down-regulates FSH-induced transferrin promoter activity and provides direct evidence for the role of rWIN in regulating Sertoli cell gene expression. Constitutive expression of the rWIN DNA-binding domain also down-regulated FSH-induced transferrin promoter activity. The data obtained from antisense and overexpression experiments support the time course of rWIN and transferrin gene expression in response to FSH. The transient up-regulation of rWIN within 30 min of FSH stimulation can be blocked by antisense oligonucleotides to rWIN, which may be required to switch on the subsequent regulatory network of genes involved in or leading to transferrin gene expression. The repression of transferrin promoter activity by the constitutively expressed rWIN DNA-binding domain represents a condition in which rWIN expression is neither transient nor under any hormonal control. The down-regulation of rWIN that precedes the transferrin expression may be a critical step, as has been shown with c-fos expression. Such a down-regulation of rWIN may be required for the initiation of Sertoli cell differentiated functions. The ability of rWIN to act as a transcriptional repressor is consistent with the proposed function of HFH-11. Overexpression of HFH-11B, a spliced variant that lacks exon B, is a transcriptional repressor, whereas the HFH-11A isoform, incorporating both exons A and B, has no transcriptional activity (12). The rWIN sequence used in the present study lacked both exon 4 and exon 6, which may have resulted in the transcriptional repression observed in the present study.
The regulatory network of genes controlling transferrin gene expression appear to involve intermediate transcription factors regulated by rWIN, because a consensus rWIN-binding domain (AGATTGAGTA, SAAB5–2) (11) was absent from the mouse transferrin promoter used in the present study. It is speculated that rWIN activity may be dependent on FSH-mediated phosphorylation events. Observations suggest that rWIN may function as an immediate early gene whose transient expression is important for FSH actions on Sertoli cell function. Both the time-course, antisense inhibition, and cycloheximide experiments support this conclusion.
The expression of HFH-11 in the cortical epithelium and mesenchyme of the embryonic kidney is transient and is absent in the adult (12). Such a transient expression of HFH-11 is also observed in the proliferating cells of the embryonic intestine, lung, and liver. The persistent expression of rWIN in both the embryonic and adult testes is a novel observation and suggests a role for rWIN in regulating the differentiation of a particular cell type(s) in the testis. The role of HFH-11 in cellular differentiation is suggested by the studies involving human colon carcinoma cell line Coco-2. HFH-11 is expressed in Coco-2 cells that are beginning to differentiate, implying that HFH-11 may regulate genes mediating the transition between proliferating intestinal epithelial cells and enterocyte differentiation that occurs after stimulation of the cell cycle (12). A similar role for rWIN in Sertoli cells may be predicted. In Sertoli cells, rWIN may be involved in initiating and subsequently maintaining Sertoli cell differentiation. Support for this hypothesis is provided by the observations that rWIN is involved in regulating the transferrin gene, which in the current study is used as a marker of Sertoli cell differentiation. Interestingly, the expression of rWIN is also regulated by FSH.
Apart from maintaining Sertoli cell differentiation in the postpubertal testis, FSH also regulates Sertoli cell division during embryonic and prepubertal stages of development (16). The molecular mechanisms involved in such a biphasic action of FSH from proliferation followed by differentiation are largely unknown. The expression of rWIN in postpubertal and possibly embryonic and postnatal Sertoli cells provides important insights into the molecular mechanisms involved in the control of Sertoli cell function. The low levels of rWIN and a transient increase in its expression may be critical events for the Sertoli cells to maintain their differentiated stage.
In conclusion, the observations presented demonstrate that the expression of rWIN in Sertoli cells is regulated by FSH. rWIN may regulate Sertoli cell gene expression and, in particular, the expression of transferrin. Future studies will be focused on understanding the possible role of rWIN during mesenchymal to epithelial transition of Sertoli cells, identification of intermediate gene targets of rWIN, the possible hormonal regulation of differential splicing, and its significance during various stages of Sertoli cell development. Analysis of testicular morphology in Trident-/- knockout mice will also be performed (14).
Received February 4, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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results
in reduced transcription of hepatocyte-specific genes. Mol Cell Biol 18:4245–4351 in rat testicular Sertoli
cells. Biol Reprod 60:1244–1250This article has been cited by other articles:
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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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 M. K. Dahle, L. M. Gronning, A. Cederberg, H. K. Blomhoff, N. Miura, S. Enerback, K. A. Tasken, and K. Tasken Mechanisms of FOXC2- and FOXD1-mediated Regulation of the RIalpha Subunit of cAMP-dependent Protein Kinase Include Release of Transcriptional Repression and Activation by Protein Kinase Balpha and cAMP J. Biol. Chem., June 14, 2002; 277(25): 22902 - 22908. [Abstract] [Full Text] [PDF]
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 J. Chaudhary, J. Johnson, G. Kim, and M. K. Skinner Hormonal Regulation and Differential Actions of the Helix-Loop-Helix Transcriptional Inhibitors of Differentiation (Id1, Id2, Id3, and Id4) in Sertoli Cells Endocrinology, May 1, 2001; 142(5): 1727 - 1736. [Abstract] [Full Text]
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