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-Subunit Transcription in T3–1 Gonadotropes1
Department of Clinical Biochemistry, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, London E1 2AD, United Kingdom
Address all correspondence and requests for reprints to: J. M. Burrin, Department of Clinical Biochemistry, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Turner Street, London E1 2AD, United Kingdom. E-mail: j.m.burrin{at}mds.qmw.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit synthesis and
release from pituitary cells. We have used T3–1 clonal gonadotropes
to investigate the intracellular mechanisms involved in PACAP
regulation of -subunit gene transcription; and using deletion,
mutation, and heterologous constructs of the -promoter linked to a
luciferase reporter gene, we have defined DNA sequences responsive to
PACAP. Stimulation of T3–1 cells for 24 h with PACAP, GnRH, or
vasoactive intestinal peptide (VIP) resulted in a time- and
concentration-dependent increase in -promoter transcription at 100
nM for GnRH (17.5-fold, P < 0.001),
PACAP (12.7-fold, P < 0.01), and VIP (4.1-fold,
P < 0.05). Incubation of T3–1 cells in
calcium-depleted medium suggested that the transcriptional response to
PACAP was less dependent on changes in intracellular calcium
concentration, in contrast to the results seen with GnRH or VIP, where
-subunit transcription was significantly reduced. Transfection of an
-promoter construct containing a mutant cAMP response element (CRE)
suggested that the CRE region is involved in PACAP and VIP
responsiveness, with stimulatory effects on the mutant construct by
PACAP (11.1-fold) and VIP (7.6-fold) being significantly
(P < 0.001) reduced, compared with their
stimulatory effects (PACAP: 25.6-fold, VIP: 23.1-fold) on the native
-promoter. In the same experiment, the transcriptional response of
the mutant CRE construct and the native CRE construct to GnRH was not
significantly different. Both PACAP and VIP enhanced GnRH-stimulated
-subunit gene transcription, but this additive effect was lost when
their combined effects on the mutant CRE were examined. Deletion
analysis indicated that sequences between -244 and -195 bp were
involved in mediating the response to PACAP, with a dramatic reduction
in fold-stimulation by PACAP (2.0-fold) of the -195-bp construct,
compared with the -244-bp construct (15.8-fold). Constructs containing
only upstream -promoter sequences from -517 bp to -98 bp, fused to
the heterologous thymidine kinase promoter, exhibited a similar loss of
responsiveness to PACAP below -298 bp. Thus, our studies show that,
unlike GnRH, PACAP stimulation of -subunit gene transcription in
T3–1 cells is less dependent on changes in intracellular calcium
concentration; and full transcriptional activation of the -subunit
by PACAP requires an intact CRE. PACAP responsiveness involves
sequences between -244 and -195 bp of the -promoter. These
sequences have been implicated also in GnRH-responsiveness and may thus
provide a mechanism for coordinated regulation of the -subunit gene
by PACAP and GnRH in T3–1 cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit, common to both of
these hormones, are examined. Thus, PACAP has been shown to stimulate
-subunit synthesis and release from both primary rat pituitary cells
(3, 4, 6) and the clonal gonadotrope cell line T3–1 (5, 6).
The intracellular signaling pathways that might mediate these
stimulatory and synergistic actions of PACAP on gonadotropin
transcription are poorly understood. At least three subtypes of PACAP
receptor (PVR1, PVR2, and PVR3) exist that are capable of activating
both adenylate cyclase (AC) and phospholipase C (PLC) signaling
pathways (7). These receptor subtypes differ in their specificity of
binding to PACAP and the structurally related vasoactive intestinal
peptide (VIP), and also in their activation of the AC and PLC pathways.
It seems that normal rat gonadotropes express the PVR1 subtype (8),
whereas clonal T3–1 cells have been shown to express the mRNA for
both PVR1 and PVR3 (9). Studies on the effects of PACAP on
intracellular signaling pathways in T3–1 cells seem to confirm the
receptor subtype findings. Thus, PACAP stimulates inositol phosphate
turnover with a potency approximately 1000-fold higher than VIP (6, 9)
and stimulates cAMP production 100-fold more potently than VIP (6, 9).
In addition, PACAP has been shown to increase cytosolic free
Ca2+ concentration through an inositol
trisphosphate-dependent mechanism (10).
In this study, we have used T3–1 cells to examine the effects of
PACAP on -subunit promoter transcription. We have shown previously
that extracellular calcium influx can stimulate -subunit
transcription (11), and we were interested to see whether PACAP and
GnRH might employ overlapping pathways to regulate expression of the
-subunit gene. We have therefore used deletion, mutation, and
heterologous constructs of the -subunit promoter to define DNA
sequences responsive to PACAP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-846 -Luciferase (LUC) contains 846 bp of the 5' flanking
sequence and 44 bp of exon 1 of the human -gene linked to the
luciferase (LUC) reporter gene in the plasmid pA3LUC (12). Deletions of
the 846-bp 5' flanking sequence linked to a LUC reporter gene and
termed -517, -420, -346, -244, -195, -156, and -132 LUC have
been previously characterized and described (11). The internal control
plasmid BOS-ßGal contains the promoter of the human elongation factor
1 gene driving expression of ß-galactosidase (13). The control
plasmid TKLUC contains the promoter of the herpes simplex virus
thymidine kinase (TK) gene linked to the LUC reporter gene in the
plasmid pA3LUC. For analysis of specific -subunit promoter elements,
constructs containing only upstream -promoter sequences from -517
to -98 bp, fused to the heterologous TK promoter and termed -517,
-398, -298, and -195 TKLUC were used (11). The plasmid 
cAMP
response element (CRE) C 
G CAT is a previously characterized
mutant construct of the -846-bp -subunit promoter containing only a
single copy of a variant CRE, in which there is a C G substitution
corresponding to a mutation that has been shown to affect the ability
of CREB to bind to the CRE (14) (Fig. 1
).
This construct was obtained from Dr. V. K. K. Chatterjee
(University of Cambridge, Cambridge, UK), and subcloned into the
HindIII site of pA3LUC, and was termed -846 C G LUC.
All constructs were verified for orientation and correct sequence by
restriction endonuclease digests and the dideoxy DNA-sequencing method.
Large-scale preparation and purification of plasmids was performed by
alkaline lysis and resin purification (Qiagen Ltd, Dorking, UK).
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T3–1 cells (from Dr. P. Mellon, University of California,
San Diego, CA) were grown in monolayer culture in DMEM (Gibco BRL,
Paisley, Scotland, UK) supplemented with 10% (vol/vol) FCS (Gibco),
penicillin (48 mg/liter), streptomycin (125 mg/liter), and fungizone
(125 mg/liter), hereafter referred to as culture medium. Cells were
incubated at 37 C in a humidified atmosphere of 95% air-5%
CO2. Before transfection, cells were washed with PBS and
harvested by treatment with 0.05% (vol/vol) trypsin in 0.5
mM EDTA. Cells were stained with trypan blue, counted in a
hemocytometer, and subcultured into 24-well culture dishes in aliquots
of 1.0 x 106 cells per well in 2 ml culture medium.
Cells were incubated overnight and subsequently transfected. For
experiments studying the effects of calcium depletion, cells were
incubated in Ham’s F10 medium (Gibco) with a measured total calcium
concentration of 0.7 mM and subsequently referred to as
calcium-depleted medium (CDM). Calcium concentration in the medium was
measured by a standard spectrophotometric technique using the selective
calcium binding dye, o-cresolphthalein complexone. Results were
compared with cells incubated in Ham’s F10 medium supplemented with
additional calcium to a final measured concentration of 1.7
mM and referred to as calcium-supplemented media (CSM).
Both media contained penicillin (48 mg/liter), streptomycin (125
mg/liter), fungizone (125 mg/liter), and 10% vol/vol FCS. Cells were transfected with 5 µg LUC plasmid by the calcium phosphate technique (15) without glycerol shock. Plasmid (5 µg) containing a ß-galactosidase reporter gene (BOS-ßGal) was cotransfected to normalize transfection efficiencies in some experiments. After 4 h, the cells were washed, and fresh culture medium was added, with or without the appropriate treatment, as indicated in the text and figure legends. After incubation, cellular extracts were prepared, and 100 µl cell lysate was assayed for LUC activity, as described previously (11). Light emission was measured using a Berthold LB953 luminometer (Berthold, St. Albans, UK). ß-galactosidase activity was assayed in the same cell extracts using a spectrophotometric microtiter plate assay with D-nitrophenyl-ß-o-galactopyranoside as substrate. Absorbance was measured at 405 nm and was compared with a standard curve prepared using known concentrations of enzyme. Protein content of the cell extracts was determined using the Bradford protein assay (Bio-Rad, Hemel Hempstead, UK) and LUC activity expressed as arbitrary light units (ALU) per mg of cellular protein. LUC data from independent experiments of duplicate or triplicate transfections were pooled by normalizing the data to BOS-ßGal.
Statistical analysis
Data are expressed as the mean ± SEM for each
experiment, and statistical analysis of the data were performed by
ANOVA; when the F statistic was significant (P <
0.05), the analysis was continued using Fischer’s multiple-comparisons
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit promoter activity-subunit promoter to
PACAP, VIP, and GnRH-A was ascertained in T3–1 cells transfected
with 5 µg of the -846 LUC plasmid. Cells were incubated for
24 h post transfection, in the presence of increasing
concentrations of these three hormones, and results are expressed as
mean-fold increase in LUC expression, relative to the activity, with no
hormone added (Fig. 2). The TKLUC fusion
gene was transfected in parallel for comparison. The maximal
transcriptional response to PACAP and VIP occurred with a concentration
of 100 nM, whereas GnRH-A exerted a maximal effect at 10
nM. Higher concentrations resulted in no further
stimulation of -subunit promoter activity. At this peak
concentration of 100 nM, treatment of transfected T3–1
cells with PACAP resulted in a 12.7-fold increase in LUC activity,
relative to control value (P < 0.01). VIP and GnRH-A
stimulated -subunit promoter expression 4.1-fold (P
< 0.05) and 17.5-fold (P < 0.001), respectively, with
VIP being the least potent stimulator at this concentration. LUC
activity from the TK promoter was unchanged by any of these treatments
(data not shown).
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-subunit
promoter activity, as reflected by LUC expression, is summarized in
Fig. 3. In these experiments, T3–1
cells were transfected with -846 LUC and were subsequently
incubated for 24 h, but some cultures were incubated for 2, 4, 8,
or 24 h in the presence of PACAP (100 nM), VIP (100
nM), or GnRH-A (100 nM). Thus, the time
reflects the hours of exposure of the transfected T3–1 cells to the
hormones during the 24-h period post transfection. Addition of PACAP
induced a time-dependent response, which reached significance, compared
with control, at 2 h with a 2.1-fold rise (P <
0.05), increasing to 4.5-fold at 4 h (P < 0.01)
and a peak 9.3-fold at 8 h (P < 0.001), then
falling to 4.5-fold at 24 h (P < 0.01). A similar
time-dependent response was seen with GnRH-A and VIP, with the peak
transcriptional response to both compounds again occurring at 8
h.
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-subunit transcription by
calcium-subunit gene
expression may be influenced by changes in intracellular calcium (11).
The actions of PACAP on T3–1 cells are known to affect cytosolic
Ca2+ concentrations (6, 10), and we therefore examined the
dependence of PACAP-stimulated -subunit promoter activity on changes
in intracellular Ca2+ using the following approach. Before
transfection, T3–1 cells were incubated in CDM (calcium
concentration = 0.7 mM) for 24 h. This has been
shown previously to cause depletion of the intracellular calcium stores
in a nonpharmacological manner (16). After transfection with -846
LUC, T3–1 cells were incubated for 8 h in media containing
either 0.7 mM or 1.7 mM calcium in the presence
or absence of PACAP (100 nM), VIP (100 nM) or
GnRH-A (100 nM). The effects of this protocol on the
stimulation of -subunit promoter activity are shown in Fig. 4. As we have shown previously, GnRH-A
stimulation of -subunit transcription is dramatically reduced from
1150 ± 187 ALU to 226 ± 46 ALU (P < 0.001)
in cells depleted of intracellular calcium. Incubation in CDM also
significantly (P < 0.01) reduced the ability of 100
nM VIP to stimulate -subunit gene expression at 8h. In
contrast, there was no apparent effect on the ability of PACAP to
stimulate -subunit transcription. The results of our experiment
suggest that, unlike GnRH, the transcriptional response of the
-subunit promoter to PACAP is less dependent on changes in
intracellular calcium concentration.
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-subunit promoter is involved in
PACAP responsiveness-subunit gene transcription, the mutant CRE construct -846 C G
LUC or native -846 LUC was transiently transfected into T3–1
cells, together with the control plasmid BOS ß-Gal. After
transfection, cells were treated for 8 h with either no additions,
PACAP (100 nM), VIP (100 nM), or the native
GnRH peptide (100 nM) or a combination of GnRH and PACAP or
GnRH and VIP. The results are shown in Fig. 5. As shown previously, GnRH, PACAP, and
VIP all caused a significant stimulation in LUC activity from the -846
LUC construct, compared with control. Interestingly, the combination
of GnRH with either PACAP or VIP caused a significantly greater
stimulation of LUC activity than any of these treatments alone, despite
using a concentration (100 nM) shown previously to produce
the maximal transcriptional response for each compound. The stimulatory
effects of PACAP, VIP, and the cAMP analog, 8-bromo-cAMP, on the -846
C G LUC construct were significantly (P <
0.001) reduced, compared with their stimulatory effects on -846
LUC. The effect of GnRH on the mutant CRE construct was also less
marked (12.0-fold, compared with 18.3-fold), but this did not achieve
significance (P = 0.09). The combined effects of GnRH
with PACAP or VIP were also dramatically reduced on the -846 C G
LUC construct with the combined stimulatory effect being decreased
to that of GnRH alone. The results from these experiments suggest that
converting the palindromic CRE in the human -subunit promoter to a
mutant CRE reduces both PACAP and VIP responsiveness, whereas GnRH
responsiveness is preserved. The marked additive effect seen with the
combination of GnRH with either PACAP or VIP was also abolished in
transfections using the -846 C G LUC construct.
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-subunit sequences required for response to
PACAP-subunit promoter that might also be
involved in the transcriptional response to PACAP, 5'-deletion mutants
with 5'-termini at -420, -346, -244, -195, and -156 were used.
These deletion mutants were transiently transfected into T3–1 cells
together with the control plasmid BOS-ßGal. Post transfection, cells
were treated for 8 h with either no additions or PACAP (100
nM). The TKLUC plasmid was also transfected in parallel,
and LUC activity in all cell extracts was measured 8 h later (Fig. 6). LUC activity from the TK promoter was
unchanged by PACAP over the 8-h time period (data not shown). As
described previously (11), a stepwise loss of basal LUC expression was
seen with progressive truncations of the -subunit promoter from
-420 bp to -195 bp. However, the -420, -346, and -244 LUC
constructs were each stimulated to a similar extent (10- to 15-fold) by
PACAP, but the transcriptional response to PACAP was dramatically and
significantly decreased (P < 0.001) with deletion to
-195 bp. Thus, our data would suggest that sequences between -244 and
-195 are important for PACAP-mediated expression of the human
-subunit promoter in T3–1 cells, in addition to the CRE.
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-subunit promoter that
confers PACAP responsiveness to the TK promoter-subunit promoter
are able to mediate PACAP responsiveness, even though the CRE sites are
downstream of -195 bp. To determine whether this upstream region could
function as a PACAP-responsive unit when linked to a heterologous
promoter, sequences between -517 and -98 bp of the -subunit
promoter were subcloned upstream of a minimal TK promoter in a LUC
reporter vector. Transcriptional activity of these heterologous
TKLUC constructs transfected into T3–1 cells was then examined
in the presence of PACAP (100 nM) for 8 h. As shown
with the native promoter, the -517, -398, and -298 TKLUC
constructs showed a similar and significant 8- to 10-fold stimulation
of LUC activity with PACAP (Fig. 7).
Again, responsiveness was lost after deletion of the sequence -298 to
-195 bp, supporting the earlier data with the native constructs (that
one or more PACAP-responsive elements were contained in this region).
Thus, our results using overlapping native and heterologous
-promoter constructs strongly suggest that a PACAP-responsive region
lies between -244 and -195 bp upstream of the transcription start
site in the human -subunit promoter.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit mRNA concentrations and
-subunit gene transcription in pituitary cell cultures and T3–1
gonadotropes (4, 5, 6). The present studies confirm that PACAP
regulates expression of the -subunit gene at the transcriptional
level. Treatment of T3–1 cells with PACAP caused a dose-dependent
increase of -subunit promoter-directed LUC expression with maximal
effects occurring at 100 nM. The stimulation of -subunit
promoter activity occurred in a time-dependent manner, with significant
increases observed as early as 2 h and maximal values obtained at
8 h. We are only aware of one previous study using human
-subunit promoter LUC fusion genes, where a similar response to
PACAP was observed with a 6- to 8-fold increase in LUC activity
occurring at 20 h (6). Kinetic studies of PACAP stimulated
-subunit mRNA levels (2- to 3-fold increases) also suggest that
maximum values occur between 6–12 h at a concentration of 100
nM (5, 6). We have compared the effects of PACAP and VIP on
-subunit promoter activity and found that both peptides increased
-subunit gene transcription, with the effects of PACAP being
consistently greater than those of VIP, suggesting that these
transcriptional effects may be mediated via the PVR1 receptor in
T3–1 cells.
In T3–1 cells, mRNA for both the PVR1 and PVR3 subtypes of the
PACAP/VIP receptor has been demonstrated (9). PACAP has a higher
binding affinity than VIP for the PVR1 receptor subtype, which has been
shown to activate both the AC and PLC pathways (17). The lower-affinity
PVR3 receptor subtype has approximately equal affinity for PACAP and
VIP, and in anterior pituitary cells, it apparently couples to the
activation of AC, but not PLC (7). PACAP has been shown to stimulate
cAMP production and inositol phospholipid turnover and to increase
cytosolic free Ca2+ concentration in T3–1 cells (5, 8, 9, 10), and thus, it has been suggested that these actions could be
mediated via the PVR1 receptor (6, 7). Because -subunit gene
expression and transcription can be stimulated by cAMP, phorbol esters,
and changes in cytosolic Ca2+ (11, 18), these effects could
all be mediated by the PVR1 receptor. The -subunit transcriptional
response to PACAP was preserved in T3–1 cells incubated in CDM.
Preincubation of cells in CDM has been shown to deplete intracellular
calcium stores (16), and our results suggest, therefore, that
mobilization of intracellular calcium is not required for the
transcriptional response. Schomerus et al. (5) have shown
that cytosolic Ca2+ is elevated by PACAP, even when
T3–1 cells are incubated in Ca2+-free medium, implying
that mobilization of intracellular Ca2+ occurs via an
inositol trisphosphate-dependent mechanism, an observation confirmed by
Rawlings et al. (10). Interestingly, previous studies have
shown that in T3–1 cells depleted of PKC activity by prolonged
treatment with a PKC activator, PACAP was still able to stimulate
-subunit mRNA levels (6). In addition, PACAP effects on -subunit
gene expression can be reproduced by cAMP analogs (4). Thus, our
results, and those previously published, suggest that in contrast to
GnRH, the PKC/calcium pathway is relatively unimportant for the
transcriptional response of the -subunit to PACAP. Transcriptional
effects occurring through the AC pathway could be mediated by binding
to the PVR3 receptor. However, this receptor does not discriminate
between PACAP and VIP; and we, and others (6), have shown that the
effects of PACAP on -subunit transcription are consistently greater
than VIP, arguing against the involvement of the PVR3 receptor in this
response. Interestingly, -subunit promoter activity was
significantly less in calcium-depleted cells incubated with VIP,
suggesting that the mechanisms by which VIP stimulates -subunit gene
expression may be partially dependent on mobilization of intracellular
calcium.
In the placenta, the palindromic CRE in the human -subunit promoter
has been shown to be important for both basal and cAMP-regulated
expression of the -gene. Block mutational studies of the -subunit
promoter in T3–1 cells suggest that the CRE may also play a role in
basal expression in the pituitary (19). We found that both PACAP and
VIP responsiveness was significantly reduced after conversion of the
palindromic CRE, in the context of the human -846 -subunit
promoter, to a single CRE containing a point mutation, suggesting that
the transcriptional response to PACAP and VIP requires an intact CRE
site. However, some residual responsiveness of the variant construct to
PACAP and VIP was retained. This construct has been used previously in
JEG-3 cells (a human placental cell line) and in GH3 cells
(a rat pituitary cell line), where markedly reduced basal activity was
observed, although full responsiveness to 8-bromo-cAMP was retained
(14). This construct has not been transfected previously into T3–1
cells, and we found that responsiveness to 8-bromo-cAMP was lost in
these clonal gonadotropes, although partial responsiveness to PACAP and
VIP was retained. This could be caused by the ability of this construct
to interact with other transcription factors in this cell type.
Interestingly, no significant alterations in GnRH responsiveness were
seen using this construct, again suggesting that GnRH does not mediate
its effects on the -promoter via the cAMP/PKA signaling system or
the CRE.
Transient transfections of the -846 -promoter construct and the
-846 C G LUC, incubated with combinations of GnRH and PACAP or
GnRH and VIP, demonstrated that both PACAP and VIP had additive effects
to increase GnRH-stimulated -subunit transcription. Previous studies
have shown that PACAP is able to increase GnRH-stimulated -subunit
release from both rat gonadotropes and T3–1 cells (4, 5).
Similarly, PACAP has been shown to produce a statistically significant
enhancement of GnRH-stimulated -subunit mRNA expression (4). The
ability of two factors to cause additive or synergistic effects is
thought to occur by the activation of different intracellular
mechanisms. Because the additive effect on -subunit gene
transcription was reduced to that of GnRH alone when the variant CRE
construct was used, it suggests that the cAMP-PKA pathway mediates this
action of PACAP.
Other studies have investigated possible mechanisms by which GnRH and
PACAP might interact to influence signaling pathways in T3–1 cells.
Over short incubation periods (45–60 min) PACAP has been shown to
amplify GnRH-stimulated inositol phosphate accumulation (5), and this
interaction may explain the enhancement by PACAP of acute
GnRH-stimulated gonadotropin release. This could also be the mechanism
for PACAP enhancement of GnRH-stimulated -subunit transcription, but
our data would suggest this is unlikely because the effect is lost when
a mutant CRE is used. Again, over short time periods, GnRH has been
shown to inhibit the effect of PACAP on cAMP production (20), but we
could find no evidence of a functional consequence of this action at
8 h, when looking at -subunit gene transcription.
Further insight into the interaction between these two peptides on gene
transcription is gained by examining the DNA elements in the
-promoter, which mediate transcriptional responsiveness. We used
deletion constructs to define regions of the -subunit gene necessary
for regulation by PACAP. As we have shown previously (11), a stepwise
loss in basal activity of the human promoter occurred with progressive
truncations from -420 bp. Despite this, there was no change in the 10-
to 14-fold stimulation of -subunit transcription by PACAP, until
deletion of the sequence between -244 and -195 bp, when a dramatic
loss in PACAP responsiveness occurred. Our studies, using -sequences
linked to a heterologous promoter, also confirmed that a
PACAP-responsive region lay between -298 and -195 bp of the
-promoter. This sequence of the human -subunit promoter includes
the gonadotrope-specific element or GSE, which binds to the adrenal
transcription factor steroidogenic factor-1 (SF-1) (21). The GSE is an
8-bp sequence including a single consensus nuclear hormone receptor
half-site (Fig. 8). This site was
originally identified as having a role in basal SU gene expression
(21), but we and others have shown that this region of the gene is also
involved in mediating GnRH-responsiveness (11, 22). Our study,
therefore, raises the possibility that in T3–1 cells, at least, the
GSE is involved in mediating both the GnRH and PACAP-response.
Surprisingly, stimulation by PACAP of the -195 bp construct could not
be detected, even though this construct contains the palindromic CRE
site. Our earlier data, using a mutant CRE construct in the context of
a full-length promoter, suggested that an intact CRE site was required
to mediate full PACAP-responsiveness. The combined data leads us to
speculate that a cooperative interaction between the transcription
factors binding to these two promoter regions may be involved in
PACAP-mediated transcriptional activation of the -subunit. It is
thus of interest to note recent reports that demonstrated
phosphorylation of SF-1 by cAMP-dependent protein kinase in
vitro (23) and SF-1 mediating a cAMP response in adrenal and
luteal cells (24). Further studies are necessary to confirm that such
interactions occur in gonadotropes.
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-subunit
gene transcription. Because, at present, there are no stable human cell
lines of gonadotrope origin available for studying regulation of the
human -subunit promoter, species-specific effects cannot be
excluded. However, we have shown that in T3–1 cells, the
transcriptional response of the human -subunit gene response seems
to be calcium-independent and requires an intact CRE-site in the
promoter. The -244 to -195 bp region of the human -subunit gene
seems to be sufficient to permit PACAP-responsiveness. The data suggest
that some interaction between this site and the CRE may occur, but
further studies are required to explore this possibility.
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Acknowledgments |
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T3–1 cells, and Dr.
V. K. K. Chatterjee for the -846 C G CAT
construct. |
Footnotes |
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Received September 25, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cell line. Endocrinology 134:315–323-subunit mRNA transcripts by pituitary adenylate cyclase-activating
polypeptide (PACAP) in pituitary cell cultures and T3–1 cells. Mol
Cell Endocrinol 113:123–130[CrossRef][Medline]
-subunit gene expression and secretion in
T3–1 gonadotropes. Mol Endocrinol 10:1308–1317-subunit gene to pituitary and placenta. J Biol Chem 270:26497–26504T3–1 cells.
Endocrinology 134:2599–2605-subunit gene in pituitary gonadotropes. Mol Endocrinol 8:878–885-subunit promoter. Mol Endocrinol 6:1767–1773This article has been cited by other articles:
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K K Sidhu, R C Fowkes, R H Skelly, and J M Burrin Exogenous expression of glucagon-like peptide 1 receptor and human insulin in AtT-20 corticotrophs confers cAMP-mediated gene transcription and insulin secretion J. Endocrinol., December 1, 2005; 187(3): 419 - 427. [Abstract] [Full Text] [PDF]
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J. Xie, S. P. Bliss, T. M. Nett, B. J. Ebersole, S. C. Sealfon, and M. S. Roberson Transcript Profiling of Immediate Early Genes Reveals a Unique Role for Activating Transcription Factor 3 in Mediating Activation of the Glycoprotein Hormone {alpha}-Subunit Promoter by Gonadotropin-Releasing Hormone Mol. Endocrinol., October 1, 2005; 19(10): 2624 - 2638. [Abstract] [Full Text] [PDF]
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R. C. Fowkes, M. Desclozeaux, M. V. Patel, S. J. B. Aylwin, P. King, H. A. Ingraham, and J. M. Burrin Steroidogenic Factor-1 and The Gonadotrope-Specific Element Enhance Basal and Pituitary Adenylate Cyclase-Activating Polypeptide-Stimulated Transcription of the Human Glycoprotein Hormone {alpha}-Subunit Gene in Gonadotropes Mol. Endocrinol., November 1, 2003; 17(11): 2177 - 2188. [Abstract] [Full Text] [PDF]
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H. Kanasaki, T. Yonehara, Y. Yamada, K. Takahashi, K. Hata, R. Fujiwaki, H. Yamamoto, Y. Takeuchi, K. Fukunaga, E. Miyamoto, et al. Regulation of Gonadotropin {alpha} Subunit Gene Expression by Dopamine D2 Receptor Agonist in Clonal Mouse Gonadotroph {alpha}T3-1 Cells Biol Reprod, October 1, 2002; 67(4): 1218 - 1224. [Abstract] [Full Text] [PDF]
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R. C. Fowkes, P. King, and J. M. Burrin Regulation of Human Glycoprotein Hormone {alpha}-Subunit Gene Transcription in L{beta}T2 Gonadotropes by Protein Kinase C and Extracellular Signal-Regulated Kinase 1/2 Biol Reprod, September 1, 2002; 67(3): 725 - 734. [Abstract] [Full Text] [PDF]
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 A. Siddiqi, M. P. Parsons, J. L. Lewis, J. P. Monson, G. R. Williams, and J. M. Burrin TR Expression and Function in Human Bone Marrow Stromal and Osteoblast-Like Cells J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 906 - 914. [Abstract] [Full Text] [PDF]
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D. A. Schreihofer, E. M. Resnick, V. Y. Lin, and M. A. Shupnik Ligand-Independent Activation of Pituitary ER: Dependence on PKA-Stimulated Pathways Endocrinology, August 1, 2001; 142(8): 3361 - 3368. [Abstract] [Full Text] [PDF]
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 J. J. Evans Modulation of Gonadotropin Levels by Peptides Acting at the Anterior Pituitary Gland Endocr. Rev., February 1, 1999; 20(1): 46 - 67. [Abstract] [Full Text]
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 H. Pincas, J.-N. Laverriere, and R. Counis Pituitary Adenylate Cyclase-activating Polypeptide and Cyclic Adenosine 3',5'-Monophosphate Stimulate the Promoter Activity of the Rat Gonadotropin-releasing Hormone Receptor Gene via a Bipartite Response Element in Gonadotrope-derived Cells J. Biol. Chem., June 22, 2001; 276(26): 23562 - 23571. [Abstract] [Full Text] [PDF]
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, 10 
,
100 
, 1000 
nM). Cells were harvested and assayed for
LUC activity at 24 h. Results are expressed as mean-fold
stimulation, with respect to control ± SEM.
Determinations are from two independent experiments, including at least
duplicate transfections for each treatment. Statistical significance is
designated as: *, P < 0.05; **,
P < 0.01; or ***, P < 0.001,
compared with control.
