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INTRODUCTION |
With its ability to stimulate gonadal steroidogenesis and
gametogenesis, luteinizing hormone
(LH)1 is essential for normal
reproductive function in mammals (1, 2). Luteinizing hormone is a
heterodimeric protein composed of an
glycoprotein hormone subunit
(
GSU) common to all members of the glycoprotein family of hormones
that is non-covalently linked to a unique LH
subunit that confers
its biological specificity (1, 2). Biosynthesis of LH depends on the
coordinated expression of both the
GSU and LH
subunit genes. In
humans, the single copy
GSU gene resides on chromosome 6q12-q21
(Locus ID 1081) while the LH
gene resides amid a cluster of six
chorionic gonadotropin-
genes on chromosome 19q13.32 (Locus ID
1082). In addition to their different locations within the human
genome, the pattern of expression of the genes encoding
GSU and
LH
are temporally and spatially distinct.
During development
GSU is seen throughout Rathke's pouch as early
as embryonic day (e) 9.5 in the mouse (3). By later stages of pituitary
development and in adult mammals,
GSU expression is limited to
thyrotropes and gonadotropes (4). Gonadotrope-specific expression of
the
GSU gene is controlled by an array of regulatory elements,
including the pituitary glycoprotein hormone basal element (5, 6),
basal elements (6), gonadotrope-specific element (7), and tandemly
repeated cAMP response elements (8-10), as well as the intricate
interplay between their cognate binding proteins (6).
In a fashion similar to the
GSU promoter, the LH
gene is
regulated by a combinatorial array of transcription factors and regulatory elements. In this case, however, the elements consist of
those that bind early growth response 1 (Egr1) (11-16), steroidogenic factor 1 (SF-1) (11-20), Pitx1 (21-23), and an as yet unidentified Otx-related homeodomain protein (24), as well as proteins that bind
elements in the distal domain, such as nuclear factor-Y (NF-Y) and Sp1
(16, 25-27). Unlike
GSU, which is a marker of early pituitary
development, expression of the LH
gene is one of the last steps that
defines a mature gonadotrope.
As indicated above, many of the elements and proteins involved in
regulation of LH
gene expression and terminal differentiation of
gonadotropes have been characterized; however, the full scope of
factors required for differentiation to a mature cell that expresses
all phenotypic markers of a gonadotrope, including LH
, have yet to
be identified. To that end, we compared the gene expression of cell
lines that represent two distinct stages of gonadotrope development by
differential display.
T3-1 cells represent early gonadotrope
progenitors that express
GSU and gonadotropin-releasing hormone
(GnRH) receptor but not the unique
-subunits of the
glycoprotein hormones (28). The L
T2 cell line is characterized by
expression of markers of fully differentiated gonadotropes, including
the
-subunits of the gonadotropins (29, 30). Because these two cell
lines represent phenotypic gonadotropes at embryonic days before
(
T3-1) or after (L
T2) the ability to express LH
,
characterization of differentially expressed factors may uncover
components that are essential for expression of the LH
subunit gene
and should refine our understanding of gonadotrope differentiation.
One factor that is more highly expressed in L
T2 cells than
T3-1
cells is the HMG-like nuclear phosphoprotein known as p8 or candidate
of metastasis 1 (com1). The HMG class of proteins to which p8 is
related (HMG-I/Y or HMGA) function as architectural transcription
factors that promote gene activation by relieving histone H1-mediated
repression of transcription (31) and facilitating the formation of
enhanceosomes as a consequence of both protein/DNA and protein/protein
interactions (32). Like p8, these proteins, which have the capacity to
bend, straighten, unwind, and induce loop or supercoil formation in
linear DNA molecules in vitro, are at maximal levels of
expression during embryonic development and in rapidly proliferating
cells (32). While p8 lacks characteristic "A-T hook" DNA binding
domains found in the HMGA class of non-histone chromatin-binding
proteins (32, 33), it does appear to bind DNA, especially upon
phosphorylation (34), and thus, p8 may perform a comparable role within
cells. In the gonadotrope, p8 appears to be a stage-specific component
of the cellular transcriptome that may play a functional role in the
initiation of LH
gene expression. This potential is explored herein.
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EXPERIMENTAL PROCEDURES |
PCR Differential Display--
Total RNA was extracted from
confluent
T3-1 and L
T2 cells using the method of Chomczynski and
Sacchi (35). Removal of chromosomal DNA contamination from samples, PCR
differential display, and reamplification of DNA were performed as
described in the Current Protocols in Molecular Biology (36). Each
assay was performed using one of four degenerate anchored oligo(dT)
primer sets (T12MN; M can be G, A, or C and N is G, A, T,
and C) where each primer set is dictated by the 3' base (N) with
degeneracy in the penultimate (M) position by itself or each in
combination with one of 26 decameric primers that were originally
designed to allow for coverage of the expressed genome (37). Clones of interest were subcloned into the pCR®II-TOPO® vector (Invitrogen, Carlsbad, CA) using the standard protocol described by the
manufacturer. The DNA sequence of each clone was found by
dideoxynucleotide sequencing using Sp6 and/or T7 primers.
Identification of clones was determined by comparing each sequence
against the BLAST database (38).
Plasmid Vectors--
All plasmid DNAs were prepared from
overnight bacterial cultures using Qiagen DNA plasmid columns according
to the protocol of the supplier (Qiagen, Chatsworth, CA). Murine
p8-pcDNA3 was a gift from Juan Iovanna and colleagues (39). To
produce the p8-antisense vector (p8AS-pcDNA3), p8-pcDNA3 was
digested with BamHI and ApaI restriction
endonucleases. The p8 insert was then blunt-ended and ligated into the
EcoRV site of pcDNA3. Clones were dideoxynucleotide
sequenced to verify selection of a reverse orientation clone. The LH
promoter-driven luciferase reporter vector (23) as well as the
GSU
promoter-driven luciferase reporter vector (6) have been described previously.
Cell Lines--
The gonadotrope-derived cell lines,
T3-1 and
L
T2, were maintained in high glucose Dulbecco's modified Eagle's
medium containing 2 mM L-glutamine and
supplemented with 10% fetal bovine serum and antibiotics (complete
medium). To produce the control (C)-L
T2, p8-knockdown
(KD)-L
T2, and p8-overexpressing (OE)-L
T2 cell lines, 24 × 106 L
T2 cells on a 100-mm culture dish were transfected
with 16 µg of pcDNA3, p8AS-pcDNA3, and p8-pcDNA3,
respectively, using 80 µl of LipofectAMINE reagent (Invitrogen) and
medium lacking serum and antibiotics. Stably transfected cells
were selected by introducing 500 µg/ml G418 (Invitrogen) into the
complete medium 3 days following the transfection. The cells
utilized in these studies, which were maintained in the above-described
complete medium supplemented with G418, represent pools of
clones for each cell line derived from the parent L
T2 line.
Transfection Assays--
The day prior to transient
transfection, C-L
T2 and p8-KD-L
T2 cells were plated at a density
of ~2 × 106 cells/35-mm well. Transfections were
carried out using medium lacking serum, antibiotics, and G418
selection medium with 10 µl of LipofectAMINE reagent, 2 µg
each test vector, and 100 ng of pRL-CMV (Promega, Madison, WI), which
was used to normalize data for transfection efficiency. Cell cultures
were incubated with the transfection mixtures for ~18 h at 37 °C
in a humidified atmosphere with 5% CO2. Complete
medium was then added to the cells, which where indicated, were
also supplemented with 100 nM GnRH. Twenty-four hours
following the addition of fresh medium and hormonal treatments,
cells were lysed in passive lysis buffer (Promega) and a
dual-luciferase assay was performed on each cellular lysate as per
standard procedures (Promega). Transient transfections were performed a
minimum of three times with at least two separate plasmid preparations
for each construct that was tested.
Northern Blot Analyses--
For each Northern blot, 10 or 20 µg of total RNA were separated by electrophoresis in a 1% denaturing
agarose gel and transferred to nylon membrane (Hybond-N+;
Amersham Biosciences) by gravity and capillary action. After UV
cross-linking to fix RNA to the nylon, the membrane was prehybridized for approximately 3 h and hybridized with the appropriate
radiolabeled probe overnight at 45 °C in a roller-bottle
hybridization oven (Techne, Inc., Princeton, NJ). The hybridization
solution consisted of 40% deionized formamide, 20 mM
PIPES, 800 mM NaCl, 2 mM EDTA, 4% SDS, 80 µg/ml salmon sperm DNA. All probes were made by radiolabeling of
cDNAs with [32P]deoxy-CTP or ATP (3000 Ci/mmol,
PerkinElmer Life Sciences) using DECAprime II kit as per the
suggestions of the manufacturer (Ambion, Austin, TX). The final washes
following hybridization were in 0.5× SSC, 0.5% SDS at 65 °C. The
membranes were exposed to Biomax MR film (Eastman Kodak Co., Rochester,
NY) for one or more days at
80 °C. In addition, some blots were
exposed to storage phosphor screens (Amersham Biosciences) for
2-18 h followed by densitometric scanning and analysis using a Storm
820 PhosphorImager (Amersham Biosciences). Between
hybridizations, each blot was stripped of radioactivity using the
protocol enclosed with the nylon membrane. Northern blots were
replicated at least three times, and densitometric scanning was
performed on representative blots. Random-prime labeled probes used in
the Northern blot analyses consisted of the full murine p8 cDNA
(39), murine LH
cDNA representing bases 35 through 213 (40),
murine cytoskeletal
-actin cDNA representing bases 1210 through 1657 (41), a murine GnRH receptor cDNA representing bases 247 through
537 (42), and a cDNA encompassing the murine 18 S rRNA contained
within primers included in the QuantumRNA 18 S Internal
Standards (Ambion). In all cases a very small amount of flanking DNA
from the multiple cloning sites of each vector was included when making probes.
In Situ Hybridization--
Timed pregnancies were obtained by
mating CF1 or FVB/N male × CF1 female. Noon on the day of
copulatory plug detection was considered e0.5. Appropriately aged
embryos were frozen in 2-methylbutane at approximately
30 °C and
sectioned at 15 µm on a Hacker-Bright cryostat (Hacker Instruments
and Industries, Fairfield, NJ). In situ hybridization was
performed as described by Cushman et al. (43). Briefly,
sections were warmed to room temperature for 30 min and fixed in 4%
paraformaldehyde/phosphate-buffered saline (PBS) at 37 °C. Following
proteinase K treatment (0.1 µg/ml), sections were fixed again in 4%
paraformaldehyde/PBS and washed in PBS. Sections were acetylated using
a 0.1 M triethanolamine, 0.25% acetic anhydride mixture
and incubated with hybridization solution minus the probe (50%
formamide, 5× SSC, 2% Boehringer blocking powder, 0.1% Triton X-100,
0.5% CHAPS, 1 mg/ml yeast tRNA, 5 mM EDTA, 50 mg/ml
heparin in diethyl pyrocarbonate-treated water) at 55 °C.
Riboprobe was diluted in hybridization solution and allowed to
hybridize overnight at 55 °C in a chamber humidified with 5× SSC.
The next morning, sections were washed in a 50% formamide, 0.5× SSC
mixture at 55 °C followed by a wash in 0.5× SSC at room temperature
and blocked with the following solution in a chamber humidified with
water: 10% heat-inactivated sheep serum; 2% BSA; 0.02% sodium azide
in 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 0.1% Triton X-100. Anti-digoxigenin Fab fragments (Roche Molecular Biochemicals) were diluted 1:1000 in blocking buffer, incubated on
sections for 1 h at room temperature in a humidified chamber and
washed in PBS. After equilibration in several washes of chromagen buffer (100 mM Tris-Cl (pH 9.5), 100 mM NaCl,
50 mM MgCl2), sections were developed overnight
in the chromagen buffer with 4.5 µl/ml 4-nitro blue tetrazolium
chloride and 3.5 µl/ml 5-bromo-4-chloro-3-indolyl-phosphate added as
substrate for alkaline phosphatase activity. Sections were rinsed with
PBS, fixed in 4% paraformaldehyde/PBS, washed, and mounted. In
situ hybridization was repeated at least two times for each
embryonic age.
Probes Used for in Situ Hybridization--
Plasmids were
linearized as follows to generate sense and antisense probes: 435-bp
GSU cDNA (GenBankTM accession number NM0099889, bp
numbers 181 through 616 of
GSU) in pGEM3ZF+ was
linearized with BamHI for antisense and HindIII
for sense probes; 233-bp LH
cDNA (GenBankTM
accession number NM_008497, bp numbers 12 through 245) in
BSK
was linearized with HindIII for antisense
and BamHI for sense probes; and full murine p8 cDNA in
pcDNA3 was linearized with EcoRI for antisense and
ApaI for sense probes. Digoxigenin-labeled riboprobe was
generated with in vitro transcription using 10× DIG
RNA labeling mix (Roche Molecular Biochemicals) and purified with the
RNeasy minikit (Qiagen).
Experimental Animals--
All animal studies were conducted in
accordance with the principles and procedures approved by the
Institutional Animal Care and Use Committee of Case Western Reserve University.
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RESULTS |
p8 mRNA Is Up-regulated in Cells That Represent Mature
Gonadotropes--
To identify factors that may be essential for
maturation of gonadotropes, differential display was employed to
compare the mRNA populations from gonadotrope-derived cells that
represent distinct developmental stages, namely
T3-1 and L
T2.
Because over 300 differentially expressed cDNAs were isolated using
this method, reverse Northern blots were performed to verify the
expression seen by differential display (data not shown). After
reamplification and nucleotide sequencing of several clones, we focused
our attention on clone 100 (Fig.
1A), which was identified as
the 3'-untranslated region of p8 using the BLAST database available
through the National Center for Biotechnology Information (38). p8
shows both sequence and structural similarities with
factors that have proven vital for embryonic development (34, 44). To
confirm the differential expression of p8 in
T3-1 and L
T2 cells,
we examined total RNA from each cell line by Northern blot analysis and
found that mRNA encoding p8 was significantly up-regulated in
L
T2 compared with
T3-1 cells (Fig. 1, B and
C).

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Fig. 1.
p8 mRNA is higher in cells that represent
fully differentiated gonadotropes. A, PCR differential
display comparison between T3-1 cells and L T2 cells illustrates
the band (arrow) found to be up-regulated in cells
representing the mature gonadotrope. This band (clone 100) was found to
be a partial murine p8 cDNA. B, Northern blot analysis
of T3-1 and L T2 cell RNA. Ten micrograms of total RNA each
extracted from cells indicated across the top were hybridized to
radiolabeled murine p8 cDNA. C, ethidium-stained gel
used for Northern blot above. Approximately equal RNA loading was
observed by the intensity of ethidium staining in each 28 S and 18 S
rRNA subunit band.
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Silencing of Endogenous p8 in L
T2 Cells--
To determine the
functional significance of p8 mRNA, a plasmid vector driving
expression of p8-antisense was used to stably transfect L
T2 cells
(p8-KD-L
T2). We anticipated that this aberrant message would form
double-stranded RNA with the endogenous p8 transcript and either
inhibit its translation or target it for degradation, thus allowing for
sequence-specific, post-transcriptional silencing of the p8 gene. Our
data support the latter possibility as no detectable levels of p8
mRNA were observed in p8-KD-L
T2 cells (Fig.
2). In contrast, in L
T2 cells that
were stably transfected with a plasmid encoding the sense strand of p8
(p8-OE-L
T2), a 579-nucleotide p8 mRNA representing the
transcription product from the transfected vector was identified.
Importantly, endogenous p8 transcript (639 nucleotides) was detected in
total RNA from untransfected L
T2 cells as well as cells that have
been stably transfected with the empty, negative control vector
(C-L
T2). Thus, stable transfection of the p8 antisense vector
effectively removed p8 mRNA from L
T2 cells.

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Fig. 2.
Silencing of endogenous p8 in
L T2 cells. Northern blot analysis of RNA
from several gonadotrope-derived cell lines. Ten micrograms of total
RNA extracted from several gonadotrope-derived cell lines indicated
across the top were hybridized to radiolabeled murine p8 and actin
cDNA. Size markers are indicated on the left, and the
positions of transcripts and the 18 S rRNA subunit are indicated on the
right.
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It is known that double-stranded RNA in the cytoplasm of mammalian
cells can trigger profound effects, including a global suppression of
translation and mRNA degradation in a sequence-nonspecific manner
as part of an interferon response (45), as well as apoptosis (46). To
provide evidence that the loss of endogenous p8 was due to
sequence-specific rather than nonspecific gene silencing, Northern blot
hybridization with a radiolabeled
-actin probe was performed (Fig.
2). Levels of detectable
-actin mRNA were comparable across RNA
samples from p8-KD-L
T2 cells, p8-OE-L
T2 cells, as well as
C-L
T2 and normal, untransfected L
T2s, indicating that the product
of this architectural gene, and likely other transcripts, was not
affected by the presence of double-stranded RNA in the p8 knockdown
cell line.
The LH
Promoter Is Non-functional When p8 Is Knocked Down in
L
T2 Cells--
Expression of the p8 gene appears to follow a
pattern that corresponds to LH
expression in gonadotrope-derived
cell lines. To determine the potential impact of p8 on regulation of
the LH
gene, p8-KD-L
T2 and C-L
T2 cells were tested for their
ability to support activity of a LH
promoter-driven luciferase
reporter after transient transfection. As expected, the C-L
T2 cell
line displayed levels of luciferase activity ~4-fold higher than that from cell lines transfected with a promoterless reporter vector (Fig.
3A), levels that are
comparable with normal L
T2 cells (data not shown). In cells lacking
p8 (p8-KD-L
T2), the LH
promoter was non-functional, even in the
presence of 100 nM GnRH (Fig. 3B). To confirm
that the effect of p8 knockdown on the LH
promoter was not due to an
overall hindrance of cellular processes, an
GSU promoter-driven
construct was also tested. This vector was shown to be functional in
p8-KD-L
T2 cells as well as C-L
T2 cells. Thus, p8 appears to be
crucial for expression of LH
in L
T2 cells. However,
overexpression of p8 does not allow for activation of LH
-driven
luciferase expression in heterologous cells (COS7 and
T3-1 cells) or
enhanced activation in homologous cells (L
T2 cells) (data not
shown). While there are many potential explanations for the lack of an
increase in LH
gene expression upon overexpression of p8, the
simplest may be that endogenous p8 in L
T2 cells may already be
exerting a maximal effect on the LH
gene. In the heterologous cells
it is possible that other factors vital to the impact of p8 on LH
promoter activity may be absent. In other words, p8 may be necessary
but not sufficient for expression of the LH
gene, and the mechanism
by which p8 regulates LH
gene expression may be very complex and/or
quite indirect.

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Fig. 3.
The LH promoter is
non-functional when p8 is knocked down in L T2
cells. A, C-L T2 and p8-KD-L T2 stable cell lines
were transiently transfected with promoter-driven luciferase constructs
consisting of the bovine LH promoter (LH -luc), the
human GSU promoter ( GSU-luc), or a promoterless
luciferase plasmid ( -luc). In both the untreated C-L T2
and p8-KD-L T2 cell lines, the -luc construct showed only very low
levels of luciferase activity, as expected. In contrast, the LH -luc
construct displayed normal luciferase activity in C-L T2 cells,
~4-fold higher than the promoterless control, but was non-functional
in the p8-KD-L T2 cells. On the other hand, the GSU-luc construct
displayed its normal, high levels of luciferase in both untreated cell
lines. Values are means ± S.D. of firefly luciferase activity
normalized with renilla luciferase activity. B, C-L T2 and
p8-KD-L T2 stable cell lines were transiently transfected with
LH -luc and treated for 24 h with 100 nM GnRH. The
LH promoter remains non-functional in the p8-KD-L T2 cells, even
in the presence of GnRH. Values are means ± S.D. of firefly
luciferase activity normalized with renilla luciferase activity.
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LH
mRNA Is Down-regulated in p8-KD-L
T2 Cells Even in the
Presence of GnRH--
To determine the importance of p8 on activation
of endogenous genes in gonadotrope-derived cells, we extended our
investigation to include Northern blot analysis of the LH
gene. As
shown in Fig. 4, there is a lack of LH
gene expression in cells that stably express the p8 antisense strand
(p8-KD-L
T2). In contrast, low levels of LH
gene expression were
detected in normal, untreated L
T2 cells, C-L
T2 cells and
p8-OE-L
T2 cells. To further characterize the impact of p8 removal on
LH
activity, the various L
T2 cell lines were treated with 100 nM GnRH for 6 h prior to harvest and collection of RNA
for analysis by Northern blot. While this treatment paradigm increased
LH
gene expression in normal L
T2, C-L
T2, and p8-OE-L
T2
cells, it could not rescue expression of LH
in the p8-KD-L
T2
cells. This provides further functional evidence for the importance of
the p8 transcription factor in LH
gene expression in L
T2
cells.

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Fig. 4.
Even in the presence of GnRH, mRNA
encoding LH is undetectable in
p8-KD-L T2 cells. Northern blot analysis
of RNA from several untreated and GnRH-treated (for 6 h)
gonadotrope-derived cell lines. Twenty and 10 µg of total RNA
extracted from several treated and untreated gonadotrope-derived cell
lines indicated across the top and bottom were
hybridized to radiolabeled murine LH and GnRH receptor
(R), respectively. After stripping each blot, they were
hybridized with radiolabeled murine 18 S rRNA sequence. Message
encoding LH and GnRHR was found to be up-regulated upon GnRH
treatment in all cells containing p8 (p8-OE-L T2, C-L T2, and
L T2). However, LH mRNA was undetectable in both untreated and
GnRH treated p8-KD-L T2 cells, while GnRHR mRNA, which was
expressed in p8-KD-L T2 cells, was not responsive to GnRH. The graph
shows relative signal intensity of LH or GnRHR message normalized to
18S rRNA as measured by exposure to a storage phosphor screen and
scanning on a densitometer.
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Unlike LH
, the GnRH receptor gene is expressed in the L
T2 cells
lacking p8 (Fig. 4). In the absence of GnRH stimulation, this level of
gene expression is higher in p8-KD-L
T2s than p8-OE-L
T2, C-L
T2,
or normal L
T2 cells. However, upon treatment of each cell line with
100 nM GnRH for 6 h, GnRH receptor gene expression is
lowest in the p8-KD-L
T2 cells. While a modest increase in expression
was observed in the other cell lines, the GnRH receptor gene does not
appear to be GnRH responsive in p8-KD-L
T2 cells. In addition, other
genes (
GSU,
-actin, and Egr-1) that are GnRH-responsive in
gonadotropes, normal L
T2 cells, and C-L
T2 cells do not show an
increase in gene expression upon GnRH stimulation in p8-KD-L
T2 cells
(data not shown), opening the intriguing possibility that p8 impacts
the GnRH signaling cascade in L
T2 cells.
Temporal Expression of p8 Gene in Vivo Mirrors That of the
Gonadotrope-derived Cells--
Expression of the p8 gene is very low
in gonadotrope-derived precursor cells (
T3-1), while higher levels
of expression are seen in cells that represent a later stage of
gonadotrope differentiation (L
T2). To determine whether a similar
pattern of temporal expression of p8 mRNA occurs in
vivo, in situ hybridization was performed in murine
tissues at distinct developmental time points (Fig. 5). Whereas p8 was found to be
undetectable in the developing pituitary gland at e13.5 (when
gonadotropes are phenotypically similar to
T3-1 cells) and e15.5,
abundant expression was observed throughout the pituitary gland at
e16.5 (prior to detection of LH
), e17.5 (gonadotropes at this
stage have a phenotype similar to that of L
T2 cells), and e18.5,
indicating that expression of the p8 gene in the developing pituitary
gland is stage-specific and transient, as it was again undetectable in
the pituitary glands of normal adults. Thus, p8 follows a
transient pattern of expression, similar to other factors vital to
pituitary development (47, 48).

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Fig. 5.
Temporal expression of p8 gene in
vivo mirrors that of the gonadotrope-derived cells.
In situ hybridization for detection of mRNA encoding
GSU, LH , and p8 in developing and adult pituitary glands at
e13.5, e15.5, e16.5, e17.5, e18.5, and approximately post-natal day 56. While GSU is detectable in pituitary tissue across all time points
tested, LH is detectable beginning at e17.5 through adulthood. On
the other hand, detection of p8 mRNA is temporal and transient. p8
appears to arise from the ventral aspect of the developing pituitary
gland at e16.5. However, p8 is undetectable in the pituitary glands of
normal adult mice (bar = 300 µm).
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|
As expected,
GSU mRNA was detectable throughout the pituitary
gland at each embryonic day tested as well as in presumptive gonadotropes and thyrotropes of the adult. In addition, while LH
mRNA was observed in the pituitary glands of adult mice and at
later stages of embryonic development (e17.5 and e18.5), we were unable
to detect the transcript at earlier time points, such as e16.5. While
others have been able to detect very low levels of LH
gene
expression in pituitary glands at e16.5 by use of 35S-labeled oligonucleotides (49), we were unable to do so
using digoxygenin-labeled cRNA. However, we were able to detect p8
expression using the same type of probe at this developmental age.
Thus, it appears that expression of the p8 gene precedes LH
gene
expression in the developing pituitary gland.
 |
DISCUSSION |
Like Egr1, SF-1, and Pitx1, which have all been described as
playing a role in terminal differentiation of gonadotropes
(11-14;18-20), the p8 nuclear phosphoprotein may play a functional
role in LH
gene expression as well as a potential role in cellular
differentiation. However, unlike Egr1, SF-1, and Pitx1, p8 expression
in the developing pituitary gland is transient. High levels of
expression can be detected during later embryogenesis, while no p8 is
detectable in the pituitary glands of normal adult mice. Thus, p8 may
represent a true embryonic factor vital to gonadotrope development and
LH
gene expression.
Activation of LH
gene expression likely involves transcription
factors that bind directly to DNA regulatory elements as well as
additional transcriptional regulators that influence the binding or
activity of these proteins, some of which are also likely to affect the
chromatin mechanics of the gene. Clearly, p8, a new member of the HMG
family of chromatin-binding proteins, plays a role in expression of the
LH
gene as its absence in L
T2 cells corresponds to an absence of
LH
gene expression. What is not known, however, is how p8 functions
to direct this expression.
In addition to their ability to relieve histone H1-mediated repression
of transcription, the HMGA class of proteins are also known for their
ability to specifically interact with numerous transcription factors to
form stereospecific multiprotein enhanceosome complexes (50-52). This
allows the architectural transcription factors considerable flexibility
in regulating the expression of a large number of genes (33, 53). p8
has been shown to enhance the activity of other transcription factors
(54) as well as interact directly with both factors and coactivators of transcription (55). Thus, p8 may function in a similar fashion to
activate the LH
promoter, forming an enhanceosome with factors known
to be important for basal expression of LH
. In fact, one of the
numerous factors that HMGA has been shown to interact with is NF-Y
(NF-YA subunit) (56), a factor that is known to be important for
activation of LH
(27) and FSH
(57) gene expression. In this
regard, determining the protein partners of p8 in L
T2 cells may
provide insight toward its function in LH
gene expression.
Alternatively, or perhaps concurrently, p8 may be playing a role in the
remodeling of chromatin during development, moving the gene from
transcriptionally inactive heterochromatic regions to euchromatin,
allowing for the induction of LH
gene expression. This may explain
why p8 is not necessary for expression of the LH
gene in adult mice
and why overexpression of p8 in L
T2 cells does not enhance LH
promoter activity or endogenous LH
gene expression. In addition,
with its mitogenic potential (44, 58) and possible anti-apoptotic role
(59), p8 may be involved in gonadotrope cellular proliferation during
development. Its absence of expression in the adult may be critical to
maintaining/limiting the gonadotrope population within the anterior
pituitary gland as p8 has been shown to be up-regulated in
proliferating and tumorigenic tissues (44, 59-62). A similar temporal
pattern of expression has been observed with other factors vital to
developmental processes (59-62), including other members of the HMGA
family (53).
Determination of pituitary cell types during organogenesis occurs in
response to a series of extrinsic and intrinsic signaling molecules. In
Rathke's pouch, multiple molecular gradients help to establish
distinct patterns of transcription factors that allow for commitment,
positional determination, and differentiation of pituitary cell types.
For example, Rosenfeld and colleagues have shown that a ventral to
dorsal gradient of BMP2 expression in Rathke's pouch induces a ventral
to dorsal gradient of GATA2 expression (63, 64). The high levels of
GATA2 in the most ventral aspect of the developing gland restrict Pit-1
gene expression in presumptive gonadotropes, allowing for induction of
the transcription factors that are critical for differentiation of the
cell type, including SF-1 (64). In fact, in vivo expression
of a dominant-negative GATA2 inhibited terminal differentiation of
gonadotropes while extended expression of GATA2 dorsally expanded the
gonadotrope population. Perhaps like SF-1, expression of the p8 gene is
regulated by GATA2. In this regard, several potential GATA-binding
sites are located in the 5' flanking region of the murine p8 gene (39) that have yet to be characterized.
While p8 is important for LH
gene expression in vitro, a
definitive answer to the significance of p8 for gonadotrope maturation and initiation of LH
gene expression in vivo will lie in
studies involving temporally disregulated expression or targeted
disruption of the p8 gene in mice. In regards to the latter, Iovanna
and colleagues have recently developed knock-out mice to study the role
of p8 on cell growth, apoptosis, and tumor development (65, 66). No
abnormalities were found in organs of these mice by histological
analysis, including liver, lung, intestine, pancreas, testis, brain,
kidney, and heart (65). To this point, the p8 knock-out mice have yet
to be assessed for defects in fertility or gonadotrope development. To
determine whether p8 is a dominant member of a signal transduction
pathway or serves as a component of a complex mechanism (perhaps even
redundant or compensatory) for initiation of LH
gene expression, a
close examination of the pituitary glands, reproductive fitness, and
reproductive system organ development in these mice will be necessary.
In addition, the identification and characterization of p8-responsive
genes may better elucidate the role of p8 in LH
gene expression and the development of gonadotropes from committed precursors to
differentiated cells.
Of the factors that appear to be essential for terminal differentiation
of gonadotropes and expression of LH
, p8 is unique among them
because of the transient component to its expression. p8 gene
expression is detectable at e16.5 in pituitary cells, one developmental
day prior to detection of LH
mRNA in our system. By its temporal
pattern of expression, p8 appears to be a marker of differentiating
gonadotropes that acquire the ability to express LH
. However, p8
appears to be more than simply a marker of the developing gonadotrope
as the knockdown of p8 in L
T2 cells corresponds with an inability of
these cells to express the LH
gene. Thus, our data indicate that p8
is a stage-specific component of the gonadotrope transcriptome that may
play a functional role in LH
gene expression during embryonic
cellular differentiation.