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Division of Genetics (U.B.K., W.W.C.), Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; Oregon Regional Primate Research Center (P.M.C.), Beaverton, Oregon 97006; and Oregon Health Sciences University, Portland, Oregon 97201-3098
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Abstract |
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 Top Abstract I. IntroductionII. GnRHR Structure AnalysisIII. Studies of GnRH...IV. Studies of GnRH...V. Studies of GnRH...VI. Future DirectionsReferences |
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T3-1 Cells T3-1 cells T3-1 cells -Subunit gene expression T3-1 cells -, LHß-,
and FSHß-subunit genes) -subunit gene -, LHß-, and FSHß-subunit genes |
I. Introduction |
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TopAbstractI. IntroductionII. GnRHR Structure AnalysisIII. Studies of GnRH...IV. Studies of GnRH...V. Studies of GnRH...VI. Future DirectionsReferences |
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Research into the neuroendocrine control of reproductive function by GnRH has undergone an explosion in the past 25 yr, marked first by the isolation and chemical characterization of GnRH (3, 4, 5). This led to the development of both agonist and antagonist analogs, resulting in rapid advances in our basic understanding as well as clinical applications to the treatment of disorders such as prostate cancer, endometriosis, precocious puberty, and infertility (6, 7). More recently, the molecular cloning of cDNAs encoding receptors for GnRH (GnRHR)2 was achieved, first in mouse (8, 9) and subsequently in human, rat, cow, and sheep (10, 11, 12, 13, 14, 15, 16, 17). The availability of the GnRHR cDNA has allowed studies leading to further understanding of the mechanisms of GnRH action.
Primary anterior pituitary cells are comprised of a heterogeneous population of well differentiated, secretory cell types. These include somatotropes, which secrete GH; lactotropes, which secrete PRL; corticotropes, which secrete ACTH as well as other hormones derived from the peptide precursor, POMC, including MSH, lipotropins, endorphins, and enkephalin; thyrotropes, which secrete TSH; and gonadotropes, which secrete LH and/or FSH (18, 19). Several anterior pituitary cell types produce more than one of the anterior pituitary hormones; for example, LH and FSH are often colocalized to the same cell, as are GH and PRL. More recently, there has been evidence of colocalization of GH with LH or FSH (20) .
A major hindrance to progress in our understanding of the mechanisms of neuroendocrine control of reproduction at the hypothalamo-pituitary level is the lack of an ideal cell model for these studies. Historically, such studies have been performed in vivo in a variety of animal models and in vitro in dispersed primary pituitary cell cultures. These studies are limited by the heterogeneity of anterior pituitary cell types; gonadotropes make up only 6–15% of anterior pituitary secretory cells in adult animals (21). In addition, anterior pituitary cells cannot be propagated in culture systems, thus limiting the feasibility of many studies. Recently, a number of immortalized pituitary cell lines have been used as models for studies of the mechanisms of action of GnRH and its receptor.
Several aspects of the GnRHR and its signaling properties have been reviewed previously (22, 23, 24, 25, 26, 27, 28, 29, 30). Past reviews have focused on the molecular mechanisms of action of GnRH and the signaling properties of the GnRHR in primary pituitary cells. In this review, we will focus on studies of GnRH action using GnRHR-expressing pituitary cell lines as model systems. The results of these studies will be compared with what is known about GnRH signaling in primary pituitary cells. In addition, we will focus on the role of the GnRHR pathway in the regulation of gene expression.
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II. GnRHR Structure Analysis |
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TopAbstractI. IntroductionII. GnRHR Structure AnalysisIII. Studies of GnRH...IV. Studies of GnRH...V. Studies of GnRH...VI. Future DirectionsReferences |
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) (31). Interestingly,
the GnRHR lacks the typical intracellular carboxyl terminus, making it
one of the smallest receptors with the seven-transmembrane segment
motif. The lack of a carboxyl-terminal tail domain is a unique feature
of the GnRHR among G protein-coupled receptors.
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T3-1
(described below) (8, 9, 32). mRNAs of similar sizes are present in
other species as well. An additional mRNA approximately 5.0–5.5 kb in
size is present in rat and sheep pituitaries, and a smaller 1.3-kb mRNA
is also detected in sheep pituitaries (13, 16). It is not clear whether
the differences between T3-1 cells and rat and sheep pituitaries
reflect species differences or differences between primary gonadotropes
and an immortalized cell line. The presence of these multiple
transcripts raises the possibility that alternative functional forms of
the GnRHR may exist.
Cloning of the mouse and human GnRHR genes reveals the presence of two
introns (Fig. 2) (33, 34). The introns in the mouse gene
occur in the sequences encoding the fourth transmembrane helix and the
third intracellular loop. The human gene has the same structure, with
the introns interrupting the coding sequences at the same locations,
although the introns appear to vary in size. Both the human and the
mouse appear to have only a single GnRHR gene, as determined by
Southern blot analysis. Analysis of multiple cDNA clones obtained from
T3-1 cells revealed the presence of at least four alternative
transcripts, derived largely by alternate splicing (34). It is possible
that these alternative transcripts account for some of the additional
bands seen on Northern blot analysis. However, these alternative
transcripts are less abundant than the original cDNA clone and appear
to encode nonfunctional, truncated GnRHRs.
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T3-1 cells. Some expression also occurred in the
rat somatolactotropic GH3 pituitary cell line, whereas only
low levels of expression occurred in a placental cell line, JEG-3, and
in a kidney fibroblast cell line, CV-1. These data suggest that this
region of the GnRHR gene confers pituitary-specific, and, to a large
extent, gonadotrope-specific expression. 5'-Deletion analyses indicate
the presence of sequences between -500 and -400 relative to the
translational start site that appear to activate GnRHR gene expression
in the T3-1 cell line (36). The 5'-flanking region of the human GnRHR gene has also been cloned and sequenced (37). Five consensus TATA boxes were identified, distributed within a 700-nucleotide region, and multiple transcriptional start sites were detected associated with these TATA sequences. These transcriptional start sites reside further upstream than the major transcriptional start site identified in the mouse, although the mouse 5'-flanking sequence also reveals several putative TATA boxes. These findings raise the possibility of species-specific or tissue-specific transcription initiation sites. The 3'-end of the human GnRHR gene has also been sequenced, revealing five classical polyadenylation signals (37). The large 3'-untranslated sequence likely accounts for the greatest portion of the major mRNA species observed by Northern blot analysis.
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III. Studies of GnRH Action in T3-1 Cells
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TopAbstractI. IntroductionII. GnRHR Structure AnalysisIII. Studies of GnRH...IV. Studies of GnRH...V. Studies of GnRH...VI. Future DirectionsReferences |
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T3-1 cells-subunit gene linked to the
protein-coding sequences of the simian virus-40 (SV-40) T antigen
oncogene was used to generate transgenic mice. Mice carrying this
fusion gene developed tumors of the anterior pituitary. The T3-1
cell line was derived from a pituitary tumor in such a mouse. Cells
from this tumor were dispersed and maintained in monolayer culture.
Stable cultures were established, and monoclonal cell lines were
derived and characterized (38). These cells have provided a continuous
cell model system for the study of the GnRHR and GnRH action, as well
as for cell-specific expression of the -subunit; indeed, the
availability of T3-1 cells was critical for the molecular cloning of
cDNAs encoding the GnRHR (8, 9).
B. Characterization of T3-1 cells
T3-1 cells express -subunit mRNA. In addition, -subunit
protein is synthesized and secreted by these cells. The cells do not
express TSHß, GH, PRL, or POMC genes, the hormones expressed in
other, nongonadotrope anterior pituitary cell types. However, neither
LHß nor FSHß subunit mRNA, expressed in primary pituitary
gonadotropes, is expressed in the T3-1 cells. The cells respond to
GnRH with an increase in -subunit mRNA levels, whereas levels remain
unchanged after exposure to TRH. The GnRH response is time- and
dose-dependent and blocked by a GnRH antagonist, consistent with action
through the GnRHR (38). Furthermore, GnRH binding and expression of
GnRHR mRNA in T3-1 cells have been shown (39). T3-1 cells also
bind activin A and express mRNAs for the activin receptor types I, II,
and IIB, as well as for the inhibin ßB-subunit (40, 41). The
expression of the gonadotropin -subunit and GnRHR in T3-1 cells
is consistent with their derivation from the gonadotrope lineage;
however, they fail to express the full complement of
gonadotrope-specific proteins, specifically the LHß and FSHß
subunits. This suggests that T3-1 cells are derived from precursor
cells that were not fully differentiated into gonadotropes. This is
supported by observations that -subunit expression occurs early in
ontogeny before LHß or FSHß (42, 43). The presence of GnRH
responsiveness indicates that these cells likely arose after the
expression of GnRHR; GnRH-binding sites have been reported to appear,
albeit at very low levels, several days earlier in development than the
ß-subunits (44).
C. GnRH binding
Specific, high-affinity binding sites for GnRH have been
identified in T3-1 cell membrane preparations (39). A GnRH analog
binds to these sites with a dissociation constant of 0.50
nM, similar to that measured in normal mouse (0.51
nM) and rat (0.20 nM) anterior pituitary. The
total number of binding sites for GnRH is 1.6 pmol/mg protein, about 5
times higher than in normal mouse (0.33 pmol/mg) and rat (0.31 pmol/mg)
anterior pituitary (Table 1) (39). However, one must
take into account that T3-1 cells represent a homogeneous cell
population, in which all the cells express the GnRHR and bind the GnRH
analog, whereas anterior pituitary cells are a heterogeneous cell
population, in which only approximately 10% of the cells, the
gonadotropes, express the GnRHR. Therefore, the estimated number of
GnRH-binding sites on T3-1 cells is approximately 50% of the number
on primary gonadotropes.
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T3-1 cells to 10-10
or 10-8 M GnRH for 20 min has been shown to
induce a 50% increase in the number of GnRHRs 24 h later, as
determined by GnRH-binding studies (32). This appears to occur at a
posttranscriptional level, as GnRHR mRNA levels were unchanged.
Interestingly, treated T3-1 cells with increased GnRH-binding
capacity showed a corresponding increase in cellular GnRHR mRNA
"activity." That is, T3-1 RNA was injected into
Xenopus oocytes, and the GnRH-stimulated Cl-
current was quantitated by voltage clamp recording of the response to
GnRH. The evoked current, a measure of the levels of functional GnRHR
translated from the injected mRNA, was almost 2-fold higher in oocytes
injected with RNA from treated T3-1 cells compared with controls.
These data suggest that GnRH regulates GnRHR numbers in T3-1 cells
by altering GnRHR mRNA translational efficiency. Similarly, prolonged
exposure of T3-1 cells to continuous high concentrations of GnRH, 1
µM for 24 h, resulted in a decrease in GnRH-binding
sites to 25% of control levels, no change in GnRHR mRNA levels, but a
decrease in GnRH-induced currents in oocytes injected with RNA isolated
from the down-regulated cells (48). The changes in GnRH binding in
response to GnRH are qualitatively similar to those seen in primary
pituitary cells, but this novel mechanism has not yet been shown to
occur in primary gonadotropes; indeed, it has been shown that GnRH can
regulate GnRHR mRNA levels in primary pituitary cells (49). Hence, it
is unclear whether this mechanism of modulation of GnRHR mRNA
translational efficiency is unique to T3-1 cells or is a generalized
phenomenon. Alarid and Mellon (50) also found no change in GnRHR mRNA
levels in T3-1 cells in response to continuous exposure to a GnRH
agonist for 1–24 h. In contrast, Catt and co-workers (51) showed that
exposure of T3-1 cells to GnRH or a GnRH agonist resulted in a time-
and dose-dependent reduction in the level of GnRHR mRNA. Nevertheless,
the reductions in mRNA levels were less pronounced than the decreases
in receptor number, consistent with the involvement of additional,
posttranscriptional mechanisms.
2. Regulation by gonadal steroid hormones. Estradiol has been
shown to reduce GnRHR number in T3-1 cells, as determined by
GnRH-binding studies, without significantly altering the dissociation
constant (Kd) (52). This inhibitory effect of estradiol is
dose- and time-dependent. A reduction in GnRHR number was measurable
after 24 h of exposure to estradiol and was maximal after 4–5
days. The EC50 of the estradiol effect was approximately
10-11 M. In primary cultures of rat pituitary
cells, estradiol can both increase (chronic exposure) and decrease
(short-term exposure) GnRH binding (53, 54). In ovine pituitary
cultures, estradiol increased GnRH binding by 10 h, and this
increase was maintained up to 48 h (55). Thus, there appear to be
some differences in the responses of T3-1 cells and primary
gonadotropes to estradiol. These discrepancies may be attributable to
differences between physiological cellular responses of T3-1 cells
and primary gonadotropes; alternatively, the up-regulation of GnRHR
number seen in primary cultures may occur indirectly, involving steroid
hormone effects on cells other than gonadotropes.
3. Regulation by gonadal peptides. Activin A increases GnRHR
mRNA levels in T3-1 cells in a time- and dose-dependent fashion,
with maximal stimulation occurring after 24–48 h of exposure (40).
This stimulation of GnRHR mRNA levels by activin A occurs at the
transcriptional level, as indicated by nuclear run-off and transient
transfection experiments. Furthermore, pretreatment of T3-1 cells
with activin A is able to enhance GnRH-induced activation of the
gonadotropin -subunit promoter, suggesting that activin A may have a
functional role in modulating the responsiveness of the gonadotrope to
GnRH by increasing the expression of the GnRHR. Follistatin is able
to block the effects of activin on the GnRHR gene, possibly by binding
to and inactivating activin. These data are consistent with data in
primary pituitary cells, demonstrating stimulation of the synthetic
rate of GnRHRs by activin A (56). In contrast, recent data demonstrated
that activin A blocked the stimulatory effect of GnRH on -subunit
promoter activity in T3-1 cells; whether this was a receptor or
postreceptor effect was not determined (57) .
4. Regulation by second messenger activators. In an
attempt to identify possible regulators of GnRHR, T3-1 cells were
treated with the second messenger activators, phorbol myristic acid
(PMA) and forskolin (50). These agents activate the signal transduction
pathways of a multitude of potential effectors that might regulate
GnRHR. PMA, a phorbol ester that activates protein kinase C (PKC), had
no effects on GnRHR mRNA levels in T3-1 cells. However, forskolin,
which activates adenylyl cyclase, leading to increases in intracellular
cAMP levels and hence activation of protein kinase A (PKA), decreased
GnRHR mRNA levels by up to 6-fold. This effect was maximal after 8
h, but was transient, with GnRHR mRNA levels returning to control
levels by 24 h after treatment. Correlation with GnRH binding is
not yet known. Thus, in T3-1 cells, factors that activate the PKA
pathway may decrease GnRHR mRNA levels, whereas activation of the PKC
pathway appears to have no effect. In contrast, activation of PKC
appears to play a role in mediating up-regulation of the GnRHR by GnRH
in primary rat pituitary cells (27, 58, 59).
E. Intracellular second messengers
Studies of signal transduction pathways activated by GnRH in
T3-1 cells have included studies of G protein coupling, generation
of inositol phosphates, stimulation of increases in intracellular
calcium concentration, activation of PKC, generation of cAMP, and
activation of mitogen-activated protein kinases. The majority of
studies have observed the responses to a single pulse of GnRH or to
continuous GnRH; the responses to pulsatile administration of GnRH have
not yet been described.
1. G protein coupling. Activation of the GnRHR by GnRH has
long been known to result in the activation of heterotrimeric
GTP-binding (G) proteins. Therefore, when the GnRHR cDNA was cloned, it
was no surprise to find that it encoded a protein predicted to be a
member of the family of cell surface, seven-transmembrane domain, G
protein-coupled receptors (31). Because GnRH actions are generally not
affected by cholera or pertussis toxin, a novel G protein
(Gp) was suggested to mediate receptor activation. Using an
antibody to the common Gq/G11
carboxy-terminal sequence, it has been shown that GnRH activation of
phospholipase C (PLC) in T3-1 cells requires GnRHR coupling to
Gq, G11, or both (60). Sustained exposure of
T3-1 cells to a GnRH agonist results in the specific down-regulation
of cellular levels of both Gq and G11
(Fig. 3) (61, 62, 63). This was
attributable to enhanced proteolysis of the activated G proteins; there
was no change in Gq or G11 mRNA levels
(64). Sustained activation of PKC with the phorbol ester, PMA, was
unable to mimic the GnRH agonist-mediated down-regulation of
Gq and G11, and inhibition of PKC with
the selective inhibitor chelerythrine did not prevent this effect of
GnRH, suggesting that the down-regulation of the G protein -subunits
is a direct result of activation of the G protein, and does not require
activation of a downstream second messenger-activated protein kinase.
Interestingly, the rate of decay of
Gq/G11 in the presence of GnRH agonist
had two components: an initial rapid rate and a slower secondary phase.
It is possible that the initial fast decay rate occurring upon receptor
occupancy is reduced to a lower rate with desensitization of the
receptor response; alternatively, the fast decay rate may be dependent
on the fraction of the cellular G protein that becomes activated upon
occupancy of the GnRHR, whereas the lower decay rate depends on the
residual G protein pool. The down-regulation of Gq and
G11 may, in turn, be a component of the desensitization
of the cellular response to GnRH upon sustained exposure to GnRH or to
an agonist.
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T3-1 cells to a GnRH agonist and continued
to accumulate, reaching a maximum after 20 min (Fig. 4)
(39, 61). The IP responses were pertussis toxin-insensitive. Levels of
inositol 1,4,5-trisphosphate, the immediate product of the cleavage of
phosphatidylinositol 4,5-bisphosphate (the major substrate of PLCß),
were rapidly but transiently stimulated after exposure of T3-1 cells
to GnRH. Levels increased within 10 sec, reached a maximum after 30
sec, and returned to basal values after 60 sec. The accumulation of IPs
in response to GnRH was inhibited by estradiol. The maximum levels of
IPs attained were decreased, and estradiol caused a rightward shift in
the dose-response relationship for GnRH-stimulated IP accumulation.
This suggests that estradiol reduces GnRHR number and also reduces the
efficiency with which the residual receptors are able to activate PLC
(52). Estradiol has been shown to regulate levels of G proteins in rat
pituitaries; hence, down-regulation of Gq and
G11 levels may contribute to this effect (65).
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T3-1 cells
after exposure to GnRH. [Ca2+]i started to
increase by 5 sec following GnRH exposure, with the majority of cells
showing a maximal response within 15 sec. Thereafter,
[Ca2+]i decreased, although there was a
prolonged secondary phase of the GnRH-induced calcium response, with
levels increased up to 11 min after the addition of GnRH (Fig. 5) (61, 66). Thus, GnRH augments calcium currents in
T3-1 cells, with a functionally similar response to that reported in
primary gonadotropes. Primary gonadotropes have at least two types of
voltage-sensitive calcium channels, resembling T- and L-type calcium
channels and giving transient and sustained currents, respectively
(67). Like T-type current, the transient current in T3-1 cells was
activated by low voltage and rapidly inactivated, and, like L-type
current, the sustained current was activated by high voltage and
dihydropyridine-sensitive (39, 68). Precise measurements of
[Ca2+]i have been done in single,
fura-2-loaded T3-1 cells by dual wavelength fluorescence microscopy,
as well as in cell suspension by spectrofluorometric analysis, and in
single indo-1 AM-loaded cells (66, 69). These studies revealed a
biphasic rise in [Ca2+]i in response to
10-8 to 10-7 M GnRH. The initial
calcium response was complete within seconds and involved primarily an
IP3-mediated rise in cytosolic calcium due to release from
intracellular stores. Importantly, the peak elevation in
[Ca2+]i was around 500 nM, above
the threshold for activation of exocytosis (24). The smaller secondary
plateau phase lasted several minutes and primarily involved the influx
of extracellular calcium through specific, dihydropyridine-sensitive,
L-type, PKC-activated channels. The biphasic nature and duration of the
calcium response in T3-1 cells is similar to the response obtained
in studies using enriched gonadotrope preparations. In single T3-1
cells exposed to increasing doses of GnRH, from 10-10 to
10-6 M, amplitude-modulated calcium responses
were elicited, with no indication of [Ca2+]i
oscillations or frequency modulation. This finding contrasts with
observations in primary pituitary gonadotropes, in which GnRH induces
prominent [Ca2+]i oscillations and
frequency-modulated calcium signaling (Fig. 6) (25, 70).
An additional difference observed in the calcium response of T3-1
cells compared with primary gonadotropes is that activation of PKC
exerts only a negative feedback effect on calcium entry in T3-1
cells, whereas in cultured primary pituitary gonadotropes, PKC
activators cause transient activation of calcium entry, followed by an
inactivation phase (69, 70). This effect in T3-1 cells is similar to
that observed in the rat somatolactotropic GH3 cell line
(71). Although the reasons for these differences between the
immortalized cell lines and primary pituitary cells are not known, it
is possible that calcium channels in T3-1 cells and GH3
cells are spontaneously active and undergo inactivation in a
Ca2+- and PKC-dependent manner.
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T3-1 cells (39). PMA, a potent activator of PKC, caused an even more
pronounced translocation of the enzyme. The effects of GnRH on PKC in
T3-1 cells is similar to that observed in primary pituitary cells
in vivo and in vitro (72, 73). T3-1 cells
contain PKC -, 
-, and 
-isoforms, as detected by immunostaining
(74). By Northern blot analysis, mRNAs for PKC and -ß, but not
-
, were detected. Exposure of T3-1 cells to a GnRH agonist
resulted in a dose-dependent increase in PKCß, but not PKC, mRNA
levels. This response was mimicked by PMA. The calcium ionophore,
ionomycin, stimulated the expression of both PKC and PKCß mRNA
levels. Removal of intra- or extracellular calcium or inhibition of PKC
abolished the effect of GnRH, indicating that GnRH-induced PKCß gene
expression is Ca2+-dependent and autoregulated by PKC (75).
5. cAMP. No significant change in cAMP levels could be
detected in T3-1 cells after treatment with a GnRH agonist, even in
the presence of a phosphodiesterase inhibitor to prevent the
degradation of cAMP (39). This is in contrast to the rise in cAMP
levels that has been observed in whole pituitaries (76). This
difference may lie in the possible need for the presence of
testosterone for this response; the GnRH-induced rise in cAMP levels
was observed in intact male rats only (77). Others have not been able
to detect significant changes in cAMP levels after GnRH treatment of
primary gonadotropes (78).
6. Mitogen-activated protein kinases (MAPKs). MAPKs, also
known as extracellular signal-related kinases (ERKs), are a family of
serine/threonine protein kinases that are rapidly activated in response
to a wide variety of stimuli (Fig. 7) (79, 80, 81, 82, 83). Several
members of the MAPK family have been identified, including
p42mapk (ERK2) and p44mapk (ERK1). Stimuli for
their activation include growth factors, many of which have receptors
with intrinsic protein tyrosine kinase activity. MAPKs are involved in
transmitting extracellular growth and differentiation signals into the
cell nucleus, resulting in an array of transcriptional and mitogenic
effects. Recent evidence indicates that some G protein-coupled
receptors can activate the MAPK family of enzymes and that MAPKs may
also be involved in nonproliferative signaling cascades (84, 85, 86, 87). G
protein-coupled receptors appear to activate MAPK through Ras-dependent
and -independent pathways, and both G- and Gß-subunits appear
to be variably involved. These findings have led several investigators
to study the ability of the GnRHR to activate MAPK and the role of MAPK
in mediating cellular effects of GnRH (88, 89, 90, 91, 92, 93).
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T3-1 cells with GnRH resulted in phosphorylation of
both ERK1 and ERK2, and rapid and sustained activation of both, as
assayed by their ability to phosphorylate myelin basic protein (91, 92, 94). Stimulation of enzyme activity was detected within 5 min after the
addition of GnRH and remained elevated for 60 min. A maximal activation
of 4- to 5-fold was achieved, at a GnRH concentration of 100
nM. Activation of ERK1 and ERK2 was blocked by treatment of
T3-1 cells with a GnRHR antagonist, Antide, demonstrating that
activation of the MAPK signal transduction cascade by GnRH is
receptor-mediated (92). Activation of MAPK by GnRH was comparable to
that observed in response to PMA. Furthermore, PMA pretreatment for
24 h to deplete phorbol ester-sensitive forms of PKC blocked the
activation of ERK1 by GnRH. These data suggest that the activation of
MAPK by GnRH may involve activation of PKC (91). MAPK activity was also
stimulated, although to a lesser extent, by GnRH in primary cultures of
male rat pituitary cells. The lower level of activation probably
reflects the heterogeneity of the pituitary cell population. Thus, it
appears that the MAPK signal transduction pathway is activated by GnRH
in both T3-1 cells and primary pituitary gonadotropes.
Interestingly, treatment of T3-1 cells with pertussis toxin blocked
GnRH-induced MAPK activation, suggesting that this signaling pathway is
coupled to the pertussis toxin-sensitive Gi or
Go pathway. This provides evidence for
Gi/Go-mediated signal transduction by GnRHR in
addition to Gq-mediated signal transduction (88, 90).
F. -Subunit gene expression
1. Cell-specific expression. T3-1 cells have proven to be a
useful cell model for the isolation and characterization of
transcription factors that appear to be involved in mediating
gonadotrope-specific expression of the -subunit gene (Fig. 8). Some of these factors may be involved in mediating
stimulation of -subunit gene expression by GnRH as well. However,
because these factors appear to be more important for basal or
tissue-specific -subunit gene expression rather than GnRH-stimulated
expression, they will be mentioned only briefly here.
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-subunit promoter that has been best
characterized as a basal, tissue-specific enhancer is the
gonadotrope-specific element (GSE). The GSE sequence, TGACCTTG, occurs
upstream of the placenta-specific elements, at positions -215/-208 in
the mouse -subunit gene, and is highly conserved among mouse, human,
rat, cow, and horse species (95). The GSE is bound by a 54-kDa protein,
steroidogenic factor-1 (SF-1) (96). SF-1 was first identified by its
ability to bind to and coordinately regulate the expression of genes
encoding enzymes in the corticosteroid biosynthetic pathway (97, 98).
Subsequently, it has also been shown to bind to and regulate the
aromatase and Mullerian-inhibiting substance genes in gonadal tissues
(99, 100). Disruption of the gene encoding SF-1 in mice precludes
adrenal and gonadal development and also results in the selective loss
of expression of gonadotrope-specific markers, including LHß, FSHß,
and GnRHR mRNAs, and a reduction in -subunit mRNA levels (101, 102).
Thus, SF-1 appears to be important for function of the reproductive
axis at multiple levels. Treatment of SF-1-deficient mice with
exogenous GnRH stimulates expression of LHß and FSHß, suggesting
that SF-1 is not necessary for GnRH stimulation of gonadotropin gene
expression (103) .
An additional putative basal enhancer, referred to as the pituitary
glycoprotein hormone basal element (PGBE), has been identified at
-344/-300 of the mouse -subunit gene (104). The PGBE is able to
direct expression of the -subunit promoter to cells of both
gonadotrope and thyrotrope lineages, but not to placenta. A member of
the LIM (lin-11, isl-1, mec-3)-homeodomain family of transcription
factors, LH-2, binds to a 14-bp imperfect palindrome within the PGBE
domain in vitro (105). This element and factor are discussed
further below.
Other elements that have been identified to play a role in expression
of the -subunit gene in T3-1 cells include a GATA element, bound
by GATA-binding proteins (106), and two E boxes, which bind members of
the family of basic-helix-loop-helix-zipper proteins (107). The optimum
level of -subunit gene expression in gonadotropes is probably
determined by the combined actions of widely expressed,
pituitary-restricted, and gonadotrope-specific transcriptional
activators that act in combination and synergistically.
2. GnRH-stimulated expression. Although a number of factors
that may be necessary for maintenance of basal levels of
gonadotrope-specific gene expression have been identified in T3-1
cells, the identification of mechanisms for GnRH-stimulated expression
have been less forthcoming. Windle et al. (38) have
demonstrated that T3-1 cells respond to GnRH by elevating
-subunit gene expression. A similar increase of -subunit mRNA
levels was observed in response to PMA, and this increase was not
additive with GnRH, suggesting that PKC may play a role in transducing
the GnRH signal to the nucleus (39). The calcium ionophore, ionomycin,
also stimulates -subunit mRNA levels. In contrast, an inhibitor of
cAMP-dependent protein kinase did not affect the ability of GnRH or PMA
to stimulate expression of an -subunit promoter/luciferase reporter
gene (LUC), indicating that cAMP-dependent protein kinase is not
required for transcriptional activation by GnRH (104).
The increase in -subunit mRNA levels in response to GnRH was maximal
at 12–24 h and maintained for a further 24 h (Fig. 9) (108). The observed increase in mRNA levels appears
to be mediated by both an increase in -subunit gene transcription
and mRNA stability. Nuclear run-off assays demonstrated an increase in
-subunit gene transcription of 2- to 3-fold within 1 h after
exposure to GnRH but returned to baseline by 12 h. GnRH also
stimulated the activity of LUC, apparent after 1 h, maximal
after 4–6 h, but back to baseline by 24 h of GnRH treatment (Fig. 9). Thus, GnRH appears to stimulate a burst of -subunit gene
transcription lasting less than 4–6 h. The persistent elevation of
-subunit mRNA levels for at least 48 h suggests that the mRNA
has a long half-life and/or that GnRH stabilizes the mRNA in addition
to its transcriptional effects. Indeed, pulse-chase experiments showed
that the half-life of the -subunit mRNA increased from 1.2 h in
the absence of GnRH to 8 h in the presence of GnRH in T3-1
cells. Whether this mechanism also occurs in primary gonadotropes is
unclear, as the half-life of -subunit mRNA in primary pituitary
cultures is 6.5 h; however, in this case both gonadotropes and
thyrotropes contribute to -subunit mRNA levels (109). Interestingly,
while the stimulatory effects of GnRH on -subunit gene transcription
and mRNA levels were evident very rapidly, within 1 h after
exposure to GnRH, GnRH-induced -subunit release was detected only
after a lag of 4 h of incubation (110). Thus, there appears to be
dissociation between the stimulation of gene expression and exocytosis.
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T3-1 cells with mouse or human
LUC have been used to determine DNA sequences of the -subunit
gene that mediate transcriptional responses to GnRH. Deletion analyses
indicated that deletion of sequences between -507 and -205 of the
mouse -subunit gene resulted in a decrease in responsiveness to
GnRH, as well as to PMA and to cAMP (104). This region, when linked to
a heterologous promoter, was capable of supporting responses to GnRH,
PMA, and cAMP. Further mutational analysis revealed that mutations at
positions -406/-399 and -337/-330 resulted in a decrease in the
response to GnRH. Multimers of -416/-385, when linked to a minimal
promoter upstream of the luciferase gene, responded to GnRH with a
stimulation of luciferase activity (Fig. 10). In
contrast, multimers of -344/-300 enhanced basal transcription but did
not respond further to GnRH. These data suggest that GnRH
responsiveness requires the cooperative interaction of two distinct
sequences, an upstream GnRH-responsive element (GnRH-RE) at
-416/-385, and a downstream element at -344/-300, corresponding to
the location of the PGBE described above. The upstream GnRH-RE was also
responsive to PMA, further supporting the role of the PKC pathway in
mediating the effects of GnRH on expression of the -subunit gene.
The need for a complex response unit for the mediation of GnRH
stimulation may provide a mechanism for the maintenance of appropriate,
tissue-specific expression and regulation of the -subunit gene. The
involvement of a tissue-specific basal element may restrict -subunit
gene expression to the appropriate cell type, and the involvement of
two elements in mediating GnRH responses may prevent the -subunit
gene from responding to activation of the PKC-signaling pathway in
nongonadotrope cells and tissues.
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) (105).
LIM-homeodomain proteins contain both a zinc finger (the LIM domain)
and a homeodomain (111). The homeodomain of these factors is sufficient
for specific DNA binding; the LIM domains appear not to be DNA-binding
domains, but rather may function as protein-protein interaction domains
to facilitate homo- or heterodimer formation. LH-2 has a restricted
tissue distribution, being most abundant in T3-1 and TSH cells,
cell lines of gonadotropic and thyrotropic origin, respectively, and in
mouse brain; less abundant in whole rat pituitaries, corticotropic
AtT20 cells, and somatolactotropic GH3 cells; and
undetectable in placental JEG-3 cells and in mouse liver.
Cotransfection of LH-2 into COS cells showed that LH-2 is able to
activate specifically the -subunit promoter 2-fold and a 3XPGBE
reporter construct 5- to 6-fold. These studies suggest that the
LIM-homeodomain protein LH-2 is an activator of the glycoprotein
hormone -subunit gene in gonadotropes and thyrotropes. It is
possible that another transcription factor, binding to the upstream
GnRH-RE, may interact with LH-2 bound to the PGBE to mediate
GnRH-induced expression of the -subunit gene.
Another candidate factor for a role in mediating -subunit gene
expression by binding to the PGBE is mLim-3, a related member of the
family of LIM-homeodomain proteins. mLim-3, also known as P-Lim or
Lhx3, is a mouse gene expressed in the pituitary throughout development
and in the adult, as well as transiently in the spinal cord, pons, and
medulla oblongata, but with no detectable expression elsewhere. mLim-3
expression was detected in cell lines of pituitary origin,
including cells representative of somato-lactotropes
(GH3, GH4C1, GC), thyrotropes (TSH),
gonadotropes (T3), and corticotropes (AtT-20), but not in cell lines
derived from peripheral, other endocrine, or neural tissues (112, 113).
mLim-3 is able to bind to the PGBE sequence in vitro and is
a strong activator of transcriptional activity of the -subunit
promoter, as well as the PRL, TSHß, and Pit-1 promoters (112).
Interestingly, it was recently reported that targeted disruption of the
mLim-3 gene in mice leads to failure of growth and differentiation of
the anterior and intermediate lobes of the pituitary (114). The
development of all pituitary cell lineages, except the corticotropes,
was affected. This suggests that mLim-3 plays an important role not
only in -subunit gene expression, but in differentiation and
proliferation of nearly all the pituitary cell lineages.
Further studies of the putative GnRH-RE in the mouse -subunit
promoter have identified a core Ets factor (a family of transcription
factors that have been implicated in mediating transcriptional
responses to MAPK activation) binding site within the GnRH-RE, which
appears to be important in mediating GnRH stimulation of -subunit
gene expression (Fig. 8) (92). Recent evidence that GnRH activates the
MAPK signal transduction pathway, as discussed above, is relevant in
terms of the mechanisms of transcriptional stimulation of the
-subunit gene by GnRH. Activation of the MAPK cascade by a
constitutively active form of Raf kinase in T3-1 cells leads to
stimulation of the -subunit promoter. Furthermore, inhibition of
MAPK activity by kinase-defective ERK1 or ERK2, or overexpression of
MAPK phosphatase 2, which dephosphorylates and inactivates MAPK, leads
to the attenuation of GnRH-induced activation of the -subunit
promoter. The DNA-binding domain of Ets-2 was able to bind specifically
to a site within the GnRH-RE, and a dominant negative Ets-2 expression
vector reduced the ability of GnRH to stimulate expression of LUC.
These findings suggest that the Ets factor-binding site in the GnRH-RE
may contribute to transcriptional stimulation of the -subunit gene
by GnRH, via activation of the MAPK pathway. In contrast, however,
Sundaresan et al. (91) found that dominant negative mutant
forms of Ras, ERK1, and ERK2 reduced basal expression of a human LUC
but had no effect on GnRH-stimulated expression. The reasons for the
differences between these two studies are not clear, although Roberson
et al. (92) used the mouse -subunit promoter, whereas
Sundaresan et al. used the human gene.
In addition to the studies characterizing GnRH-responsive DNA sequences
in the mouse -subunit gene using T3-1 cells as described above, a
GnRH-responsive region in the human gene was identified by transfection
analyses in primary rat pituitary cell cultures (115). Deletion
analyses suggested that one or more GnRH-responsive sequences reside
between -346 and -244 in the human -subunit promoter. This
GnRH-responsive region does not include the GnRH-RE defined in the
mouse -subunit promoter. In contrast to the findings with the mouse
-subunit gene in T3-1 cells, the regions of the human -subunit
gene that are important for the GnRH response appear to be distinct
from those required for basal activity. Basal expression appeared to be
primarily mediated through the proximal promoter and cAMP-responsive
regions. These differences may reflect different mechanisms of GnRH
stimulation of the human vs. the mouse -subunit gene or
differences in the mechanisms of regulation in T3-1 cells
vs. primary pituitary gonadotropes.
G. Desensitization
GnRH is secreted from the hypothalamus in a pulsatile fashion, and
pulsatile GnRH stimulates LH and FSH biosynthesis and secretion (116).
In contrast to the stimulatory effects of pulsatile GnRH, sustained
exposure to high concentrations of GnRH reduces the response of
gonadotropes to subsequent stimulation with GnRH (homologous
desensitization), leading to suppression of gonadotropin secretion
(117). This homologous desensitization to GnRH can occur rapidly,
within the time frame of endogenous GnRH pulses (118). The mechanism of
this desensitization is not known, and both receptor (119) and
postreceptor (120, 121) mechanisms have been proposed. For a number of
other G protein-coupled receptors, early desensitization events are
thought to involve the uncoupling of the receptor from its regulatory G
protein, with loss of downstream-signaling events (122). Rapid
desensitization appears to involve phosphorylation by specific
intracellular kinases of the third intracellular loop or the C-terminal
tail (123, 124). However, the GnRHR lacks the C-terminal cytoplasmic
tail as well as the third intracellular loop sequences implicated in
the desensitization of other receptors (31).
T3-1 cells have been used as a model for the study of mechanisms of
desensitization to GnRH. Stimulation of LUC activity in transfected
T3-1 cells was maximal 4–6 h after exposure to GnRH but thereafter
declined, returning to levels in unstimulated control cells by 12–24
h. LUC activity was also stimulated by a PKC activator, PMA, a
calcium channel agonist, BAY K 8644, and an activator of the PKA
pathway, 8-bromo-cAMP. Maximal responses to these agents also occurred
after 4–6 h of exposure, although the maximal levels of activity were
less than those observed in response to GnRH. A decline in LUC
activity over time with continuous exposure to these agents was
particularly marked for PMA, but was also seen with BAY K 8644, whereas
stimulation by 8-bromo-cAMP was maintained for at least 24 h.
Pretreatment of T3-1 cells with GnRH blocked subsequent stimulation
of LUC activity by either GnRH or PMA. In contrast, both
8-bromo-cAMP and BAY K 8644 were still able to stimulate LUC
activity after pretreatment with GnRH. These data suggest that the
transcriptional stimulation of the -subunit gene by GnRH is mediated
by the PKC pathway, and that this pathway can be desensitized in
T3-1 cells by continuous exposure to GnRH. The kinetics of
desensitization are difficult to infer from these studies; exposure to
GnRH may incite a short burst of transcriptional activity of the
-subunit promoter, which then leads to a more gradual accumulation
of the luciferase product. However, the addition of the GnRH
antagonist, Antide, after treatment of the cells with GnRH resulted in
a reduction of luciferase activity compared with exposure to GnRH
alone, even when Antide was added up to 6 h after GnRH, indicating
that some stimulation of the -subunit promoter by GnRH was still
occurring, i.e. the cells were not fully desensitized to
GnRH. Continuous exposure of primary pituitary cells to GnRH causes
rapid desensitization at the secretory level for free -subunit as
well as intact LH and FSH, evident within 15 min (125). The differences
in kinetics for transcriptional and secretory desensitization may
reflect different cellular mechanisms or differences between the
T3-1 cell line and primary gonadotropes.
Regulation of -subunit gene transcription is a relatively downstream
endpoint for the study of homologous GnRH desensitization. Measurements
of second messengers may lead to insights into early or short-term
desensitization events. GnRH treatment led to a linear increase in
total IP production in T3-1 cells over 0–15 min (126, 127, 128).
Furthermore, GnRH pretreatment for 5 min did not alter subsequent
stimulation of IP3 production by GnRH 15 min later. These
data indicate a lack of desensitization of the rapid GnRH-induced
IP3 response in T3-1 cells. Pretreatment with GnRH for
1 h did reduce subsequent cellular IP accumulation in response to
GnRH, but this may be attributable to a reduction in GnRHR numbers.
GnRH pretreatment of T3-1 cells for short times (5–15 min) had no
effect on GnRHR number; however, treatment for 1 h with
10-7 M GnRH reduced GnRHR number by 48%. The
affinity for GnRH was not altered. Desensitization of both the
extracellular Ca2+-dependent and -independent phases of the
Ca2+ response to GnRH were observed after pretreatment with
10-7 M GnRH for 1 h (128). Thus, one
mechanism of intermediate desensitization to GnRH may be receptor loss.
However, this does not account for rapid or early desensitization or
the degree of desensitization of the Ca2+ response. An
additional uncoupling event may occur during the pretreatment, which
reduces the ability of the agonist-occupied GnRHR to elevate
intracellular Ca2+. Treatment of T3-1 cells with 5-min
pulses of GnRH every 15 min resulted in desensitization of the
Ca2+ response after the first pulse in a dose-dependent
manner, being evident at GnRH concentrations greater than 2 x
10-9 M (126). The mechanisms underlying this
desensitization are not known but could include loss of IP3
receptors, depletion of intracellular Ca2+ stores, and
inactivation of Ca2+ channels, as has been suggested in
studies of primary pituitary cells (120). The dissociation of IP
production and calcium stimulation suggests that desensitization of
GnRH-induced calcium mobilization is a postreceptor phenomenon
occurring distal to PLC activation. The lack of the C-terminal
cytoplasmic tail, implicated in the desensitization of other G
protein-coupled receptors, in the GnRHR therefore appears to correlate
with a lack of receptor desensitization; rather, desensitization to
GnRH appears to be primarily a postreceptor phenomenon. Alternatively,
T3-1 cells may be lacking a factor(s) necessary for mediating rapid
receptor desensitization in primary gonadotropes.
H. Summary of GnRH action in T3-1 cells
The development of the T3-1 gonadotropic cell line has enabled
significant advances in our understanding of gonadotrope function and
gonadotropin regulation, particularly in the areas of -subunit gene
expression and GnRHR structure and function. T3-1 cells were
critical for the initial cloning of GnRHR cDNAs as well as for
elucidation of the GnRHR gene structure, confirming previous findings
in primary pituitary cells which suggested that the GnRHR was a member
of the G protein-coupled receptor family. The absence of a
carboxy-terminal intracellular tail on the receptor was a surprising
finding, which makes questions about the mechanisms of
gonadotrope desensitization to GnRH all the more intriguing.
T3-1 cells have been used to elucidate a number of components of the
GnRH signal transduction pathway (Fig. 11). The GnRHR
in T3-1 cells is coupled to G proteins of the
Gq/G11 family, leading to production of IPs and
increases in intracellular calcium levels, which, in turn, leads to
activation of PKC. While cAMP has, in some studies, been suggested to
be activated by GnRH, and has been shown to lead to increases in
expression of the -subunit gene, there is no evidence for increases
in cAMP levels in response to GnRH in T3-1 cells. Furthermore, there
is now evidence that the MAPK pathway is activated by GnRH in T3-1
cells and may be important in the stimulation of -subunit gene
expression by GnRH.
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T3-1 cells have proven to be invaluable for the study of GnRH
action, there are some differences between T3-1 cells and primary
pituitary gonadotropes. The regulation of the GnRHR in T3-1 cells is
different from primary gonadotropes; in particular, the receptor does
not appear to be markedly regulated by GnRH itself in T3-1 cells,
especially at the level of gene expression, whereas it is markedly
regulated in primary pituitary cells. In addition, detailed studies of
intracellular calcium profiles in response to GnRH reveal that
amplitude-modulated intracellular calcium responses occur in T3-1
cells, in contrast to primary gonadotropes, in which GnRH induces
calcium oscillations and frequency-modulated calcium signaling. A major
difference between T3-1 cells and primary gonadotropes, however, is
the lack of expression of the gonadotropin ß-subunit genes by T3-1
cells. |
IV. Studies of GnRH Action in GH3 Cells Transfected with the GnRH Receptor (GGH3 Cells) |
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TopAbstractI. IntroductionII. GnRHR Structure AnalysisIII. Studies of GnRH...IV. Studies of GnRH...V. Studies of GnRH...VI. Future DirectionsReferences |
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B. Characterization of GH3 cells
These cells are somatolactotropic in origin. They secrete
large amounts of GH into culture medium and stimulate body weight gain
and growth after injection into normal or hypophysectomized rats (129, 130). They express PRL and GH genes and also secrete PRL and GH in a
regulated fashion. GH3 cells express TRH receptors (TRHR)
and respond to TRH with an increase in PRL biosynthesis and secretion,
and a reduction in GH production (131, 132). GH3 cells do
not express -subunit, TSHß, LHß, FSHß, and POMC genes,
hormones expressed in other, nonsomatolactotropic anterior pituitary
cell types. However, they are capable of supporting the expression of
exogenous - and TSHß-subunit genes, introduced into the cells by
transient transfection (133, 134, 135, 136, 137, 138). In addition to TRHR, GH3
cells also express somatostatin, galanin, and pituitary adenylate
cyclase-activating polypeptide receptors (139, 140, 141), as well as
activin types I and II receptors (142). However, GH3
cells appear to lack functional dopamine receptors (143).
C. Derivation of GH3 cells transfected with the GnRHR
(GGH3 cells)
GH3 cells stably expressing the rat GnRHR
(GGH3 cells) were developed by cotransfecting
GH3 cells with a rat GnRHR expression vector (pcDNA1-GnRHR)
and a pSVneo plasmid, which expresses the neomycin resistance gene, by
electroporation (144). Control cells were transfected with pSVneo and
pcDNA1 vector. The cells were grown in the presence of neomycin, to
select for neomycin-resistant cell clones, and monoclonal cells were
then expanded and tested for binding of GnRH and GnRH responsiveness.
The general morphology of the stably transfected GGH3 cell
lines is indistinguishable from that of the parent GH3
cells and of control, vector-transfected cells (145).
D. GnRH binding
Specific, high-affinity binding sites for GnRH and GnRH analogs
are present on GGH3 cells but not on the parental
GH3 cell line (127, 144, 146). Scatchard analysis of the
binding of [125I]Buserelin, a metabolically stable analog
of GnRH, shows a Kd of 4.1 ± 1.0 x
10-8 M using GGH3-1' cells, one of
the clonal strains of GH3 cells that have been stably
transfected with the rat GnRHR cDNA. Each GGH3-1' cell has
11,000 ± 2,800 receptors. Both GnRH agonists and antagonists are
able to displace binding of [125I]Buserelin to
GGH3-1' cells (Table 2), whereas chemically
unrelated peptides such as PRL, GH, and TRH do not significantly
displace binding, even at high concentrations. The relative affinities
of the GnRH agonists and antagonists are similar to those observed in
cell cultures derived from rat pituitaries, suggesting that this
receptor is similar to those expressed in primary gonadotropes,
although the absolute affinities are slightly lower. Another clonal
strain of stably transfected GH3 cells, GGH3-2
cells, had 13,000 ± 1,000 binding sites for
[125I]Buserelin per cell. In comparison, this cell line
had 64,800 ± 3,700 specific [3H]MeTRH binding sites
per cell, representing binding to the TRHR, which is expressed
endogenously in these cells.
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E. GnRHR regulation
The expression of the GnRHR in GGH3 cells is driven by
a cytomegalovirus promoter, which is not regulated by GnRH (144). The
concentration of GnRHR is therefore unaffected at the transcriptional
level by hormonal manipulation. This cell model may be useful,
therefore, for distinguishing transcriptional from posttranscriptional
regulation of cell surface GnRHR concentrations. Many G protein-coupled
receptors are down-regulated by their ligands (123). Receptors are
sequestered from the plasma membrane and internalized, followed by
proteolytic degradation. This leads to a reduction in receptor number
(homologous down-regulation) over a period of hours. In addition, the
receptor may be down-regulated at the level of gene expression as well,
leading to a reduction in receptor number over a period of several
hours to days. Because GnRH will not affect the transcriptional rate of
the GnRHR gene in GGH3 cells, changes in cell surface GnRHR
numbers would be expected to reflect translational or posttranslational
regulation of the receptor by GnRH.
Indeed, the GnRHR in GGH3 cells does undergo homologous down-regulation followed by recovery after continuous exposure to 10 nM GnRH, as determined by GnRH-binding studies (145). Down-regulation of the GnRHR was evident by 1 h of GnRH treatment, reached a nadir of 50–80% by 2–5 h, and returned to baseline levels by 7 h. This biphasic regulation of GnRHR is similar in time course and extent to that reported in primary pituitary cells (119). The ability of the receptor to be down-regulated in GGH3 cells suggests that down-regulation does not require cell-specific components other than the receptor itself, does not involve transcriptional down-regulation (which may occur in primary gonadotropes, but over a longer time course), and does not require an intracellular C-terminal region. The mechanisms of this down-regulation of the GnRHR in GGH3 cells remain to be elucidated. It will be interesting to determine whether the GnRHR is internalized and degraded or recycled after exposure to its ligand, as is the case for other receptors of this family, such as the TRHR (150, 151) and the ß-adrenergic receptor (152).
F. Intracellular second messengers
1. G protein coupling. Direct studies of G protein coupling of
the GnRHR in GGH3 cells, as were done in T3-1 cells,
have not been reported. However, the TRHR, which is expressed
endogenously by GH3 cells, has been shown to be directly
coupled to G proteins of the Gq/11 family (60, 153).
The ß-subunits of a G protein can mediate signals as well as the
-subunit (154, 155). This confers to any G protein the potential for
dual signaling. This has been best described for the Gi
protein, i.e. the -subunit inhibits adenylyl cyclase
activity, whereas at higher concentrations the ß-subunits can
stimulate the activity of some adenylyl cyclase subtypes (156, 157).
Similarly, G protein ß-subunits have been shown to be capable of
stimulating the ß2-isoform of PLC (158). Recent data suggest that the
ß-complex of G proteins is involved in mediating GnRH effects. A
125-amino acid pleckstrin homology domain within the carboxyl terminus
of ß-adrenergic receptor kinase (ßARK1-495/689) binds to
Gß (159). When transiently transfected and expressed
in cell culture, this polypeptide has been shown to inhibit
ß-mediated signal transduction (160). Expression of this ßARK1
fragment in GGH3 cells inhibits basal and GnRH-stimulated
IP production, cAMP release, and PRL release (161). The numbers and
GnRH binding affinity of the GnRHR were unaffected by the expression of
ßARK1-495/689, indicating that the changes in signal transduction and
PRL release are not due to a change in receptor expression or affinity.
These data suggest that the ß-complex of G proteins may play a
role in mediating GnRH-stimulated signal transduction in
GGH3 cells.
2. Inositol phosphates. IP production was stimulated by a GnRH
agonist (Buserelin) in GGH3 clonal cell lines in a
dose-dependent manner (145, 162). The EC50 was
approximately 10-10 M GnRH. Production of IPs
is an early response of GGH3 cells to Buserelin, measurable
at 15–30 min, maximal at 60 min, and maintained for at least 120 min
after treatment (Fig. 12). GGH3 cells also
respond to TRH with an increase in IP production. Interestingly, the
production of IPs by a GnRH agonist and by TRH were additive when
maximal concentrations of both peptides were present. This suggests
that GnRH and TRH may provoke IP production in GGH3 cells
by different means. This conclusion is supported by the finding that
cholera toxin and pertussis toxin inhibit IP production in response to
TRH, whereas they augment Buserelin-stimulated IP production.
Alternatively, receptor number may be limiting the maximal IP
production in response to either ligand. As is the case for T3-1
cells, studies in GGH3 cells have observed the responses to
a single pulse of GnRH or to continuous GnRH; the responses to
pulsatile administration of GnRH have not yet been described.
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). The EC50
was approximately 10-8 M, about 100-fold
higher than for IP generation. The stimulation of cAMP production by
GnRH suggests that the GnRHR may be able to couple to Gs as
well as Gq and is analogous to reports that GnRH can
increase cAMP production in primary pituitary cells (76, 77).
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G. Regulation of secretion
1. PRL. The TRHR and GnRHR are both members of the family of G
protein-coupled receptors, and both are coupled to G proteins of the
Gq/11 family (60, 153). The cellular responses to TRHR or
GnRHR activation include enhanced phosphoinositide turnover, calcium
mobilization, and PKC activation (24, 164, 165). Thus, the effects of
TRH and GnRH appear to be mediated through the same or similar
intracellular signal transduction pathways. Therefore, one might expect
that GnRH would be able to stimulate PRL release in GGH3
cells. Indeed, a GnRH agonist is able to stimulate PRL release from
GGH3 cells, whereas it does not in the parental
GH3 cells or in control cells, confirming that this
response is mediated by the GnRHR, rather than by cross-activation of
the TRHR by GnRH (145, 146, 162). The release of PRL in response to
exposure of GGH3 cells to a GnRH agonist was nearly linear
to 96 h and was dose-dependent with an EC50 of
approximately 10-8 M (Fig. 14). Cycloheximide inhibited the release of PRL in
response to GnRH, indicating that this response is dependent on protein
synthesis. This inhibition, and the slow time course for PRL release
after GnRH stimulation, suggest that GnRH-stimulated PRL release is
regulated at the level of PRL synthesis, rather than by release of
stored hormone. The secretion of PRL in response to GnRH in these cells
indicates that all of the components needed for coupling of the GnRHR
to the activation of secretory events are present in the
GGH3 cell and suggests that no gonadotrope-specific
components other than the secretory proteins themselves are necessary.
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The regulation of PRL by GnRH in GGH3 cells could be interpreted to suggest that, at least in some instances, the hormonal regulation of gene expression in given pituitary cell subtypes is determined by the presence or absence of receptors for a particular hormone in that cell type, rather than or in addition to other tissue-specific intracellular factors or second messengers. A similar situation has, in fact, been observed in vivo. While LH and FSH are not normally regulated by TRH, in the case of gonadotrope adenomas secreting LH or FSH, their secretion is often stimulated by TRH (166, 167). Presumably, these adenomas express the TRHR, enabling a response to TRH, while normal gonadotropes do not.
2. LH and FSH (in GH3 cells transfected with the -,
LHß-, and FSHß-subunit genes). GH3 cells are able
to secrete proteins through both constitutive and regulated pathways
(168). To compare the mechanisms of storage and release of LH and FSH,
expression vectors carrying the human LHß or FSHß gene in
combination with the common gonadotropin -subunit gene were
transfected into GH3 cells (169, 170). After transit and
processing in the Golgi, the majority (>85%) of LH was retained
intracellularly in Golgi/post-Golgi compartments, and decreased
gradually with a t1/2 of about 13 h. In contrast, FSH
did not accumulate in GH3 cells to the same extent as LH
and was secreted with a t1/2 of about 6 h. KCl or
forskolin was able to stimulate LH secretion by 4- to 5-fold compared
with unstimulated (basal) release, supporting the observation that LH
enters a regulated secretory pathway in GH3 cells and is
stored in a secretagogue-releasable pool. FSH secretion was also
stimulated by KCl or forskolin, but to a lesser extent (2.5-fold),
possibly reflecting the smaller size of the stored,
secretagogue-releasable pool. These studies indicate that in
GH3 cells, LH is secreted primarily through a regulated
pathway, whereas the majority of FSH is released constitutively. These
findings are consistent with observations in primary gonadotropes that
FSH secretion is more tightly coupled to FSH biosynthesis, and that the
magnitude of FSH secretion in response to secretagogues is smaller than
that of LH (171, 172, 173). The effects of GnRH on LH and FSH secretion in
this system, using cells in which the GnRHR is coexpressed, have not
yet been studied.
3. Secretogranin-II. Secretogranin-II, an acidic glycoprotein that is a marker for the regulated pathway of secretion and a component of secretory granules, is synthesized in GGH3 cells (174). It is released in a time- and dose-dependent manner in response to GnRH agonists, as well as to a cAMP analog. Release is inhibited by cycloheximide, an inhibitor of protein synthesis, and by actinomycin-D, an RNA synthesis inhibitor. Interestingly, while secretogranin-II is generally thought to be a marker for the regulated pathway of secretion, it appears to be secreted in a constitutive manner in GGH3 cells.
H. Regulation of PRL mRNA
In addition to stimulating PRL secretion, TRH also stimulates PRL
gene transcription and mRNA levels, leading to an increase in PRL
biosynthesis (175, 176). Analogously, PRL mRNA levels are stimulated in
GGH3 cells by GnRH (144). Treatment of GGH3
cells with 100 nM of a GnRH agonist for 24 h resulted
in the stimulation of PRL mRNA levels by 3- to 4-fold, whereas PRL mRNA
levels were unchanged in response to the GnRH agonist in the parental
GH3 cells or in control cells. In the same cell line, TRH
stimulated PRL mRNA levels by approximately 3-fold.
I. Regulation of expression of transiently expressed reporter genes
1. PRL vs. -subunit gene. TRH stimulates PRL mRNA
levels in GH3 cells by increasing gene transcription, as
determined by nuclear run-off assays (176). Time course experiments
indicate that transcriptional activation is maximal 2 h after
treatment with TRH. We therefore hypothesized that TRH and GnRH would
stimulate the activity of a fusion gene comprised of the PRL promoter
fused to a luciferase reporter gene (PRLLUC) in GGH3 cells.
Indeed, when PRLLUC was transiently transfected into GGH3
cells, luciferase activity was stimulated by treatment with either TRH
or a GnRH agonist in a dose- and time-dependent manner (144). In the
parental GH3 cells and in the control cell line,
pcGH3-1 (stably transfected with the pcDNA1 vector),
luciferase activity was stimulated by TRH, but not by the GnRH agonist
(Fig. 15). Time course studies using maximal doses of
TRH and a GnRH agonist showed an increase in luciferase activity in
response to both TRH and GnRH agonist by 2 h; maximal stimulation
occurred at 4–6 h in both cases. The difference in the time courses
for TRH response in nuclear run-off experiments and luciferase
activities likely reflects the time needed for translation and
accumulation of the luciferase enzyme in the cells after
transcriptional activation. When a human LUC was transiently
transfected into GGH3 cells, luciferase activity was also
stimulated by treatment with either TRH or a GnRH agonist, whereas
pcGH3-1 cells and GH3 cells responded only to
TRH (144). Dose-response and time course studies for LUC were
similar to those for PRLLUC.
|
LUC by both TRH and GnRH agonist was no
greater than with either one alone (144). If TRH and GnRH share the
identical signal transduction pathway for the stimulation of gene
expression, it would be expected that the induction of gene expression
by maximally effective concentrations of one hormone would prevent any
further stimulation by the other, as is the case for the -subunit
gene. Therefore, the signal transduction pathways used by TRH and GnRH
in the regulation of PRL gene expression in GGH3 cells may
have some differences, particularly in the response-limiting step(s).
These results also imply that the regulation of -subunit and PRL
gene expression by TRH and GnRH may not occur by identical pathways.
The additivity of TRH and GnRH effects on PRL gene expression could
also be accounted for if receptor number was the limiting factor in the
pathway for either ligand. However, if this were the case, one might
expect TRH and GnRH to be additive on -subunit gene expression as
well.
In time course studies, luciferase activity declined by 18–24 h of
exposure to TRH or GnRH, similar to studies of LUC in T3-1 cells,
suggesting that the signal transduction pathways became desensitized.
To study this further, GGH3 cells were pretreated with
either TRH or a GnRH agonist, and then the responses of PRLLUC and
LUC to TRH and the GnRH agonist were tested. These studies show that
homologous desensitization occurs, i.e. pretreatment with
the GnRH agonist blocks subsequent stimulation of PRLLUC and LUC
activity by the agonist, and pretreatment with TRH blocks subsequent
stimulation of PRLLUC and LUC activity by TRH. In the case of the
-subunit promoter, heterologous desensitization also occurs.
Stimulation of LUC activity by TRH was blunted by pretreatment with
a GnRH agonist, and conversely, there was loss of responsiveness to
GnRH agonist after pretreatment with TRH. In contrast, heterologous
desensitization of the PRL promoter did not occur (144). These data
provide additional support that TRH and GnRH stimulate the -subunit
promoter by the same mechanism, whereas the mechanisms of
transcriptional regulation of PRL by TRH and GnRH are not identical. It
would be interesting to know whether similar effects would be observed
in T3-1 cells transfected with the TRHR; however, these studies have
not been performed.
2. -, LHß-, and FSHß-subunit genes. The gonadotropin
subunit genes, , LHß, and FSHß, are not expressed endogenously
in the somatolactotropic GH3 cell line. As previously
reported and discussed above, GH3 cells are capable of
supporting expression of transiently transfected luciferase reporter
constructs driven by the rat, mouse, or human -subunit promoter
(133, 138). Furthermore, in GGH3 cells, the -subunit is
stimulated in response to GnRH. Similarly, studies have shown that the
TSHß-subunit gene promoter, normally active in thyrotropes, can be
expressed in GH3 cells (134, 135). The rat LHß gene
promoter, fused to a chloramphenicol acetyltransferase (CAT) gene, was
expressed at low levels when transiently transfected into
GH3 cells (177). Transcription was initiated correctly at
the same LHß transcriptional start site used in primary pituitary
gonadotropes, as determined by RNase analysis. CAT activity was
consistently induced by forskolin and by cAMP analogs, suggesting the
presence of a cAMP-responsive cis-acting domain in the LHß
5'-flanking region.
GH3 cells transiently transfected with reporter genes in
which regulatory regions of the human -subunit, rat LHß, and rat
FSHß genes are fused to the luciferase gene (LUC, LHßLUC, and
FSHßLUC, respectively) yield expression levels 250-fold higher than
those of the promoterless luciferase vector for LUC, 5-fold higher
for LHßLUC, and 12-fold higher for FSHßLUC (178). Cotransfection of
the GnRHR cDNA results in the additional specific stimulation of
luciferase activity by a GnRH agonist, 10-fold for LUC, 8-fold for
LHßLUC, and 4-fold for FSHßLUC. TRH is also able to stimulate the
expression of the gonadotropin subunit gene reporter constructs in
these transfected cells, although to a lesser degree than the GnRH
agonist (Fig. 16).
|
LUC or LHßLUC and progressively increasing
amounts of GnRHR cDNA, the degree of stimulation of luciferase activity
by GnRH is increased in proportion to the numbers of GnRHR. In
contrast, FSHßLUC activity is optimally stimulated when relatively
low numbers of GnRHR are expressed; at higher concentrations of
cell surface GnRHR, the magnitude of stimulation of FSHßLUC by GnRH
is decreased (178).
Observations in vivo indicate that cell surface GnRHR
numbers are regulated by varying GnRH pulse frequencies (47, 49, 179).
The highest concentrations of cell surface GnRHR, as reflected by
GnRH-binding activity, occur at a GnRH pulse frequency of every 30 min,
which has also been shown to stimulate preferentially LH biosynthesis
and secretion in vivo (180, 181, 182, 183, 184). Lower cell surface GnRHR
numbers occur at a GnRH pulse frequency of every 2 h, which, in
turn, has been shown to stimulate preferentially FSH biosynthesis and
secretion. GnRHR concentration in primary pituitary cells is
approximately 2- to 3-fold higher when the GnRH pulse frequency is
every 30 min, compared with that at a frequency of every 2 h
(179), a magnitude of change in GnRHR concentration similar to that
in the GGH3 cells which resulted in the maximal differences
in LHß and FSHß gene expression. These observations support the
hypothesis that varying GnRH pulse frequencies regulate differentially
LH and FSH biosynthesis and secretion in vivo by regulating
pituitary GnRHR numbers (Fig. 17). These data suggest
that the mechanisms by which GnRH regulates and LHß gene
expression are distinct from those by which FSHß gene expression is
regulated. Furthermore, the signal transduction pathways activated by
GnRH may be different at low vs. high GnRH receptor numbers.
The details of the different intracellular signaling pathways activated
by GnRH at low vs. high GnRHR numbers remain to be
elucidated. It is possible that the GnRHR may couple to different G
proteins at low vs. high cellular receptor numbers or that
different signaling pathways are activated by the - and
ß-subunits of a single G protein.
|
T3-1 cells, GnRH
appears to increase cAMP production as well as stimulating IP
production and intracellular calcium levels in GGH3 cells.
The GnRHR may be coupled to more than one G protein in GGH3
cells; additionally, there is evidence to suggest a role for
Gß-subunits in mediating GnRH responses as well. GnRH is able to
stimulate gene expression as well as hormone release in
GGH3 cells. GH3 cells have both advantages and disadvantages for the study of GnRH action. Because the parental GH3 cell line does not express the GnRHR gene, these cells are useful for structure-function studies of the GnRHR. Wild type and mutant receptors can be transfected into GH3 cells and their function studied without interference from endogenously expressed receptors. Such studies to date have been largely done in nonpituitary cell lines, such as COS cells (148, 149). However, unlike the GGH3 cells, COS cells do not support gonadotropin subunit promoter activity, so that domains of the GnRHR important for mediating signals for transcriptional stimulation cannot be defined. The transfected GnRHR is driven by a heterologous viral promoter, which is not regulated by GnRH. This means that GGH3 cells cannot be used for studies of GnRHR gene expression. However, it allows effects of GnRHR concentration on GnRH action and effects of GnRH on GnRHR desensitization to be studied independently of regulatory effects at the level of gene expression. Finally, GH3 cells support the expression of transfected reporter genes driven by regulatory regions of the LHß- or FSHß-subunit genes, making them the first homogeneous, immortalized cell population available for the study of gonadotropin ß-subunit gene expression and regulation by GnRH.
Nonetheless, an important caveat to studies performed in GGH3 cells is that this is a heterologous cell expression system, and the signal transduction pathways used in these cells may differ from those used in primary gonadotropes as well as in thyrotropes. It must be remembered that GH3 cells, while they are pituitary in origin, are derived from somatolactotropes rather than from gonadotropes, and do not express the gonadotropin subunit genes endogenously. They may, therefore, lack gonadotrope-specific factors that may be important for cell-specific and/or regulated expression of gonadotropin subunit genes. Observations made using such a cell model should be confirmed in primary pituitary cells or in vivo before physiological relevance is assigned to such observations.
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V. Studies of GnRH Action in Other Pituitary Cell Lines |
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TopAbstractI. IntroductionII. GnRHR Structure AnalysisIII. Studies of GnRH...IV. Studies of GnRH...V. Studies of GnRH...VI. Future DirectionsReferences |
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T3-1
cells and GGH3 cells in that the FSHß- and LHß-subunit
genes are expressed endogenously. However, a disadvantage of the
RC-4B/C cell line is that the cells are a heterogeneous population, so
only a fraction of the cells express the gonadotropin subunit genes.
Furthermore, it has not yet been shown that the gonadotropins are
synthesized and secreted in a regulated manner analogous to that which
occurs in vivo. Keri et al. (187) were unable to
obtain activity greater than that of a promoterless reporter for a
fusion gene in which the bovine LHß promoter was fused upstream of
the CAT gene. Activity remained low even after the upstream addition of
a Rous sarcoma virus enhancer. Also, surprisingly, although the
proportion of cells staining for LHß and/or FSHß are higher than in
normal pituitary, the FSH and LH content of RC-4B/C cells was 70- and
800-fold lower, respectively, than that of the normal male rat
pituitary gland. Thus, the validity of this cell line as a
physiological model has yet to be determined.
B. LßT2 cells
Recently, targeted expression of the SV40 T antigen with the rat
LHß-subunit gene regulatory region was used to generate transgenic
mice. An immortalized cell line (LßT2 cells) was derived from a tumor
generated in a LHß-Tag mouse, in a manner analogous to the
preparation of the T3-1 cells (188). These cells express both the
- and ß-subunits of LH as well as GnRHR, estrogen receptors, and
estrogen-inducible progesterone receptors. However, the FSHß-subunit
is not expressed. LßT2 cells probably arose later in ontogeny than
T3-1 cells and thus represent a more mature gonadotrope precursor
than do the T3-1 cells.
LßT2 cells have been used to study the effects of GnRH on intracellular Ca2+ concentrations. Cells stimulated with 1 µM GnRH responded with an increase in intracellular Ca levels and also had a secretory response, as measured by changes in plasma membrane capacitance (189). Furthermore, LßT2 cells responded to a 15-min pulse of 1–100 nM GnRH with a dose-dependent increase in LH secretion (188, 190). Exposure of the cells to four, 15-min, 10 nM GnRH pulses every 90 min for 4 days led to an increase in LH secretion in response to the initial GnRH pulse on each succeeding day, independent of cell number. The stimulation of LH secretion by GnRH was enhanced by steroids: the LH-secretory response to GnRH by day 4 was 4-fold in the absence of added steroids, 7-fold in the presence of 0.2 nM estrogen (E2), 14-fold in the presence of 20 nM dexamethasone, and 15-fold in the presence of both E2 and dexamethasone. These changes in responsiveness to GnRH appeared to be due in part to changes in GnRHR number and in part to changes in the LH synthesis/secretory pathway independent of changes in the GnRHR.
Studies of [Ca2+]i and exocytosis in
individual LßT2 cells have been performed using the whole-cell
perforated patch clamp technique to measure plasma membrane capacitance
(191). These studies show that GnRH evokes dose-dependent increases in
[Ca2+]i and secretion. The
[Ca2+]i responses to GnRH are biphasic, as in
T3-1 cells and primary gonadotropes. However, the
[Ca2+]i oscillations observed in primary
gonadotropes at low GnRH concentrations are not observed in the LßT2
cells. The extent of the changes in [Ca2+]i
and exocytosis in response to GnRH were dependent on the steroid
hormone background. E2 and dexamethasone caused an increase
in the peak [Ca2+]i stimulated by GnRH as
well as a shift toward increased sensitivity of the Ca2+
dependency of exocytosis. The increased
[Ca2+]i response may be due to an increase in
GnRHR numbers (see below). The increase in GnRH-induced secretion may
be due to both an increase in [Ca2+]i and an
increase in the sensitivity of the secretory apparatus to
[Ca2+]i, which may, in turn, be due to
effects of other second messenger pathways activated by GnRH and/or
steroids.
Studies of mRNA levels indicate that LßT2 cells respond to pulsatile
GnRH administration with an increase in GnRHR mRNA levels (190).
However, this increase is only approximately 2-fold, compared with the
much greater increase observed in primary pituitary cells (49). This
difference may be due to differences in the experimental paradigm of
pulsatile GnRH delivery. GnRHR mRNA levels are also increased in LßT2
cells by estradiol and dexamethasone. LHß mRNA levels are markedly
induced by pulsatile GnRH in LßT2 cells, with no effect of steroids,
and -subunit mRNA levels are unaffected by either steroids or GnRH.
The LßT2 cell line thus exhibits some characteristics consistent with
those of pituitary gonadotropes, including the expression of the ,
LHß, and GnRHR genes, the biphasic stimulation of
[Ca2+]i by GnRH, the secretion of LH via a
regulated pathway, and the regulation of LHß and GnRHR mRNA levels in
response to GnRH and steroid hormones. These cells may be useful for
studying the molecular and cellular mechanisms involved in the
regulation of LH subunit gene expression and LH secretion.
|
VI. Future Directions |
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|
TopAbstractI. IntroductionII. GnRHR Structure AnalysisIII. Studies of GnRH...IV. Studies of GnRH...V. Studies of GnRH...VI. Future DirectionsReferences |
|---|
-subunit gene in
particular, and to some extent the gonadotropin ß-subunit genes as
well. Nonetheless, further studies to clarify the molecular mechanisms
by which GnRH regulates LH and FSH subunit gene expression, hormone
biosynthesis, and secretion are needed. In addition, the mechanisms of
the unique responses of gonadotropes to varying GnRH pulse frequencies
and amplitudes are not well understood. For these studies, we eagerly
await the development and availability of a novel, immortalized
gonadotrope cell line that expresses the LHß and FSHß-subunit genes
as well as the -subunit and the GnRHR and that has the unique
responses to different modes of administration of GnRH in a manner
reflecting that which occurs in primary pituitary gonadotropes.
|
Footnotes |
|---|
1 This work was supported by NIH HD-19938 (to W.W.C.), HD-19899 (MERIT;
to P.M.C.), and HD-33001 (to U.B.K.), an American Society for
Reproductive Medicine–Serono Research Grant (to U.B.K.), and a Medical
Research Council of Canada Clinician-Scientist Award (to U.B.K.). 
2 Abbreviations used: GnRHR, GnRH receptor; TRHR,
TRH receptor; SV40, simian virus-40; PMA, phorbol myristic acid; PKC,
protein kinase C; PKA, protein kinase A; PLC, phospholipase C; IP,
inositol phosphate; [Ca2+]i, ionized
intracellular calcium concentration; MAPK, mitogen-activated protein
kinase; ERK, extracellular signal-related kinase; GSE,
gonadotrope-specific element; SF-1, steroidogenic factor-1; MIS,
Mullerian inhibiting substance; PGBE, pituitary glycoprotein hormone
basal element; GnRH-RE, GnRH-responsive element; LUC, -subunit
gene promoter/luciferase reporter fusion gene; PRLLUC, PRL gene
promoter/luciferase reporter fusion gene; LHßLUC, LHß subunit gene
promoter/luciferase reporter fusion gene; FSHßLUC, FSHß subunit
gene promoter/luciferase reporter fusion gene; CAT, chloramphenicol
acetyltransferase; E2, estradiol.
|
References |
|---|
|
TopAbstractI. IntroductionII. GnRHR Structure AnalysisIII. Studies of GnRH...IV. Studies of GnRH...V. Studies of GnRH...VI. Future DirectionsReferences |
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V. Rochira, L. Zirilli, A. D Genazzani, A. Balestrieri, C. Aranda, B. Fabre, P. Antunez, C. Diazzi, C. Carani, and L. Maffei Hypothalamic-pituitary-gonadal axis in two men with aromatase deficiency: evidence that circulating estrogens are required at the hypothalamic level for the integrity of gonadotropin negative feedback. Eur. J. Endocrinol., October 1, 2006; 155(4): 513 - 522. [Abstract] [Full Text] [PDF]
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V. G. Thackray, S. M. McGillivray, and P. L. Mellon Androgens, Progestins, and Glucocorticoids Induce Follicle-Stimulating Hormone {beta}-Subunit Gene Expression at the Level of the Gonadotrope Mol. Endocrinol., September 1, 2006; 20(9): 2062 - 2079. [Abstract] [Full Text] [PDF]
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 B.-S. An, D. M. Selva, G. L. Hammond, A. Rivero-Muller, N. Rahman, and P. C. K. Leung Steroid Receptor Coactivator-3 Is Required for Progesterone Receptor Trans-activation of Target Genes in Response to Gonadotropin-releasing Hormone Treatment of Pituitary Cells J. Biol. Chem., July 28, 2006; 281(30): 20817 - 20824. [Abstract] [Full Text] [PDF]
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N. J. Westphal and A. F. Seasholtz Gonadotropin-Releasing Hormone (GnRH) Positively Regulates Corticotropin-Releasing Hormone-Binding Protein Expression via Multiple Intracellular Signaling Pathways and a Multipartite GnRH Response Element in {alpha}T3-1 Cells Mol. Endocrinol., November 1, 2005; 19(11): 2780 - 2797. [Abstract] [Full Text] [PDF]
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F. Liu, M. S. Ruiz, D. A. Austin, and N. J. G. Webster Constitutively Active Gq Impairs Gonadotropin-Releasing Hormone-Induced Intracellular Signaling and Luteinizing Hormone Secretion in L{beta}T2 Cells Mol. Endocrinol., August 1, 2005; 19(8): 2074 - 2085. [Abstract] [Full Text] [PDF]
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D.J. Haisenleder, L.L. Burger, K.W. Aylor, A.C. Dalkin, H.E. Walsh, M.A. Shupnik, and J.C. Marshall Testosterone Stimulates Follicle-Stimulating Hormone {beta} Transcription via Activation of Extracellular Signal-Regulated Kinase: Evidence in Rat Pituitary Cells Biol Reprod, March 1, 2005; 72(3): 523 - 529. [Abstract] [Full Text] [PDF]
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K.-Y. Kam, K.-H. Jeong, E. R. Norwitz, E. M. Jorgensen, and U. B. Kaiser Oct-1 and Nuclear Factor Y Bind to the SURG-1 Element to Direct Basal and Gonadotropin-Releasing Hormone (GnRH)-Stimulated Mouse GnRH Receptor Gene Transcription Mol. Endocrinol., January 1, 2005; 19(1): 148 - 162. [Abstract] [Full Text] [PDF]
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 L L Burger, D J Haisenleder, A C Dalkin, and J C Marshall Regulation of gonadotropin subunit gene transcription J. Mol. Endocrinol., December 1, 2004; 33(3): 559 - 584. [Abstract] [Full Text] [PDF]
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K.-H. Jeong, W. W. Chin, and U. B. Kaiser Essential Role of the Homeodomain for Pituitary Homeobox 1 Activation of Mouse Gonadotropin-Releasing Hormone Receptor Gene Expression through Interactions with c-Jun and DNA Mol. Cell. Biol., July 15, 2004; 24(14): 6127 - 6139. [Abstract] [Full Text] [PDF]
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J.L. Crawford, J.R. McNeilly, and A.S. McNeilly No Evidence for Pituitary Priming to Gonadotropin-Releasing Hormone in Relation to Luteinizing Hormone (LH) Secretion Prior to the Preovulatory LH Surge in Ewes Biol Reprod, July 1, 2004; 71(1): 224 - 235. [Abstract] [Full Text] [PDF]
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 R. P. Millar, Z.-L. Lu, A. J. Pawson, C. A. Flanagan, K. Morgan, and S. R. Maudsley Gonadotropin-Releasing Hormone Receptors Endocr. Rev., April 1, 2004; 25(2): 235 - 275. [Abstract] [Full Text] [PDF]
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D. Coss, S. B. R. Jacobs, C. E. Bender, and P. L. Mellon A Novel AP-1 Site Is Critical for Maximal Induction of the Follicle-stimulating Hormone {beta} Gene by Gonadotropin-releasing Hormone J. Biol. Chem., January 2, 2004; 279(1): 152 - 162. [Abstract] [Full Text] [PDF]
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S. Roelle, R. Grosse, A. Aigner, H. W. Krell, F. Czubayko, and T. Gudermann Matrix Metalloproteinases 2 and 9 Mediate Epidermal Growth Factor Receptor Transactivation by Gonadotropin-releasing Hormone J. Biol. Chem., November 21, 2003; 278(47): 47307 - 47318. [Abstract] [Full Text] [PDF]
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F. Liu, D. A. Austin, and N. J. G. Webster Gonadotropin-Releasing Hormone-Desensitized L{beta}T2 Gonadotrope Cells Are Refractory to Acute Protein Kinase C, Cyclic AMP, and Calcium-Dependent Signaling Endocrinology, October 1, 2003; 144(10): 4354 - 4365. [Abstract] [Full Text] [PDF]
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A. M. Navratil, S. P. Bliss, K. A. Berghorn, J. M. Haughian, T. A. Farmerie, J. K. Graham, C. M. Clay, and M. S. Roberson Constitutive Localization of the Gonadotropin-releasing Hormone (GnRH) Receptor to Low Density Membrane Microdomains Is Necessary for GnRH Signaling to ERK J. Biol. Chem., August 22, 2003; 278(34): 31593 - 31602. [Abstract] [Full Text] [PDF]
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D. J. Haisenleder, L. L. Burger, K. W. Aylor, A. C. Dalkin, and J. C. Marshall Gonadotropin-Releasing Hormone Stimulation of Gonadotropin Subunit Transcription: Evidence for the Involvement of Calcium/Calmodulin-Dependent Kinase II (Ca/CAMK II) Activation in Rat Pituitaries Endocrinology, July 1, 2003; 144(7): 2768 - 2774. [Abstract] [Full Text] [PDF]
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D. J. Haisenleder, H. A. Ferris, and M. A. Shupnik The Calcium Component of Gonadotropin-Releasing Hormone-Stimulated Luteinizing Hormone Subunit Gene Transcription Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase Type II Endocrinology, June 1, 2003; 144(6): 2409 - 2416. [Abstract] [Full Text] [PDF]
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G. Y. Bedecarrats and U. B. Kaiser Differential Regulation of Gonadotropin Subunit Gene Promoter Activity by Pulsatile Gonadotropin-Releasing Hormone (GnRH) in Perifused L{beta}T2 Cells: Role of GnRH Receptor Concentration Endocrinology, May 1, 2003; 144(5): 1802 - 1811. [Abstract] [Full Text] [PDF]
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 K. L. Anderes, D. R. Luthin, R. Castillo, E. A. Kraynov, M. Castro, K. Nared-Hood, M. L. Gregory, V. P. Pathak, L. C. Christie, G. Paderes, et al. Biological Characterization of a Novel, Orally Active Small Molecule Gonadotropin-Releasing Hormone (GnRH) Antagonist Using Castrated and Intact Rats J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 688 - 695. [Abstract] [Full Text] [PDF]
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W.-H. Yang, M. Wieczorck, M. C. Allen, and T. M. Nett Cytotoxic Activity of Gonadotropin-Releasing Hormone (GnRH)-Pokeweed Antiviral Protein Conjugates in Cell Lines Expressing GnRH Receptors Endocrinology, April 1, 2003; 144(4): 1456 - 1463. [Abstract] [Full Text] [PDF]
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B. S. Ellsworth, B. R. White, A. T. Burns, B. D. Cherrington, A. M. Otis, and C. M. Clay c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in {alpha}T3-1 Cells Endocrinology, March 1, 2003; 144(3): 839 - 849. [Abstract] [Full Text] [PDF]
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B. H. Shah, J.-W. Soh, and K. J. Catt Dependence of Gonadotropin-releasing Hormone-induced Neuronal MAPK Signaling on Epidermal Growth Factor Receptor Transactivation J. Biol. Chem., January 24, 2003; 278(5): 2866 - 2875. [Abstract] [Full Text] [PDF]
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 P. M. Conn, A. Leanos-Miranda, and J. A. Janovick Protein Origami: Therapeutic Rescue of Misfolded Gene Products Mol. Interv., September 1, 2002; 2(5): 308 - 316. [Abstract] [Full Text] [PDF]
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V. V. Vasilyev, M. A. Lawson, D. Dipaolo, N. J. G. Webster, and P. L. Mellon Different Signaling Pathways Control Acute Induction versus Long-Term Repression of LH{beta} Transcription by GnRH Endocrinology, September 1, 2002; 143(9): 3414 - 3426. [Abstract] [Full Text] [PDF]
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F. Liu, I. Usui, L. G. Evans, D. A. Austin, P. L. Mellon, J. M. Olefsky, and N. J. G. Webster Involvement of Both Gq/11 and Gs Proteins in Gonadotropin-releasing Hormone Receptor-mediated Signaling in Lbeta T2 Cells J. Biol. Chem., August 23, 2002; 277(35): 32099 - 32108. [Abstract] [Full Text] [PDF]
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J. A. Janovick, G. Maya-Nunez, and P. M. Conn Rescue of Hypogonadotropic Hypogonadism-Causing and Manufactured GnRH Receptor Mutants by a Specific Protein-Folding Template: Misrouted Proteins as a Novel Disease Etiology and Therapeutic Target J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3255 - 3262. [Abstract] [Full Text] [PDF]
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G. Maya-Nunez, J. A. Janovick, A. Ulloa-Aguirre, D. Soderlund, P. M. Conn, and J. P. Mendez Molecular Basis of Hypogonadotropic Hypogonadism: Restoration of Mutant (E90K) GnRH Receptor Function by a Deletion at a Distant Site J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2144 - 2149. [Abstract] [Full Text] [PDF]
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S. Gill, J. L. Sharpless, K. Rado, and J. E. Hall Evidence That GnRH Decreases with Gonadal Steroid Feedback but Increases with Age in Postmenopausal Women J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2290 - 2296. [Abstract] [Full Text] [PDF]
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F. Liu, D. A. Austin, P. L. Mellon, J. M. Olefsky, and N. J. G. Webster GnRH Activates ERK1/2 Leading to the Induction of c-fos and LH{beta} Protein Expression in L{beta}T2 Cells Mol. Endocrinol., March 1, 2002; 16(3): 419 - 434. [Abstract] [Full Text] [PDF]
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J. S. Jorgensen and J. H. Nilson AR Suppresses Transcription of the LH{beta} Subunit by Interacting with Steroidogenic Factor-1 Mol. Endocrinol., September 1, 2001; 15(9): 1505 - 1516. [Abstract] [Full Text] [PDF]
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G. Maya-Nunez and P. Michael Conn Cyclic Adenosine 3',5'-Monophosphate (cAMP) and cAMP Responsive Element-Binding Protein Are Involved in the Transcriptional Regulation of Gonadotropin-Releasing Hormone (GnRH) Receptor by GnRH and Mitogen-Activated Protein Kinase Signal Transduction Pathway in GGH3 Cells Biol Reprod, August 1, 2001; 65(2): 561 - 567. [Abstract] [Full Text] [PDF]
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N. B. Schwartz Perspective: Reproductive Endocrinology and Human Health in the 20th Century--A Personal Retrospective Endocrinology, June 1, 2001; 142(6): 2163 - 2166. [Full Text] [PDF]
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R. D. Horvat, D. A. Roess, S. E. Nelson, B. G. Barisas, and C. M. Clay Binding of Agonist but Not Antagonist Leads to Fluorescence Resonance Energy Transfer between Intrinsically Fluorescent Gonadotropin-Releasing Hormone Receptors Mol. Endocrinol., May 1, 2001; 15(5): 695 - 703. [Abstract] [Full Text]
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J. N. Hislop, M. T. Madziva, H. M. Everest, T. Harding, J. B. Uney, G. B. Willars, R. P. Millar, B. E. Troskie, J. S. Davidson, and C. A. McArdle Desensitization and Internalization of Human and XenopusGonadotropin-Releasing Hormone Receptors Expressed in {{alpha}}T4 Pituitary Cells Using Recombinant Adenovirus Endocrinology, December 1, 2000; 141(12): 4564 - 4575. [Abstract] [Full Text] [PDF]
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J. Schwartz Intercellular Communication in the Anterior Pituitary Endocr. Rev., October 1, 2000; 21(5): 488 - 513. [Abstract] [Full Text]
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K. W. Cheng, E. S. W. Ngan, S. K. Kang, B. K. C. Chow, and P. C. K. Leung Transcriptional Down-Regulation of Human Gonadotropin-Releasing Hormone (GnRH) Receptor Gene by GnRH: Role of Protein Kinase C and Activating Protein 1 Endocrinology, October 1, 2000; 141(10): 3611 - 3622. [Abstract] [Full Text] [PDF]
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D. Vanderschueren and R. Bouillon Estrogen Deficiency in Men Is a Challenge for Both the Hypothalamus and Pituitary J. Clin. Endocrinol. Metab., September 1, 2000; 85(9): 3024 - 3026. [Full Text]
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 F. E. M. Rebers, P. T. Bosma, W. van Dijk, H. J. T. Goos, and R. W. Schulz GnRH stimulates LH release directly via inositol phosphate and indirectly via cAMP in African catfish Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2000; 278(6): R1572 - R1578. [Abstract] [Full Text] [PDF]
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R. Grosse, S. Roelle, A. Herrlich, J. Hohn, and T. Gudermann Epidermal Growth Factor Receptor Tyrosine Kinase Mediates Ras Activation by Gonadotropin-releasing Hormone J. Biol. Chem., April 14, 2000; 275(16): 12251 - 12260. [Abstract] [Full Text] [PDF]
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R. Grosse, A. Schmid, T. Schoneberg, A. Herrlich, P. Muhn, G. Schultz, and T. Gudermann Gonadotropin-releasing Hormone Receptor Initiates Multiple Signaling Pathways by Exclusively Coupling to Gq/11 Proteins J. Biol. Chem., March 24, 2000; 275(13): 9193 - 9200. [Abstract] [Full Text] [PDF]
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 J. J. Blum, M. C. Reed, J. A. Janovick, and P. M. Conn A mathematical model quantifying GnRH-induced LH secretion from gonadotropes Am J Physiol Endocrinol Metab, February 1, 2000; 278(2): E263 - E272. [Abstract] [Full Text] [PDF]
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S. K. Kang, K.-C. Choi, K. W. Cheng, P. S. Nathwani, N. Auersperg, and P. C. K. Leung Role of Gonadotropin-Releasing Hormone as an Autocrine Growth Factor in Human Ovarian Surface Epithelium Endocrinology, January 1, 2000; 141(1): 72 - 80. [Abstract] [Full Text] [PDF]
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G. Maya-Núñez and P. M. Conn Transcriptional Regulation of the Gonadotropin-Releasing Hormone Receptor Gene Is Mediated in Part by a Putative Repressor Element and by the Cyclic Adenosine 3',5'-Monophosphate Response Element Endocrinology, August 1, 1999; 140(8): 3452 - 3458. [Abstract] [Full Text]
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 E. R. Norwitz, K.-H. Jeong, and W. W. Chin Molecular Mechanisms of Gonadotropin-Releasing Hormone Receptor Gene Regulation Reproductive Sciences, July 1, 1999; 6(4): 169 - 178. [Abstract] [PDF]
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J. D. Neill, L. C. Musgrove, L. W. Duck, and J. C. Sellers High Efficiency Method for Gene Transfer in Normal Pituitary Gonadotropes: Adenoviral-Mediated Expression of G Protein-Coupled Receptor Kinase 2 Suppresses Luteinizing Hormone Secretion Endocrinology, June 1, 1999; 140(6): 2562 - 2569. [Abstract] [Full Text]
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C. Dorn, Q. Ou, J. Svaren, P. A. Crawford, and Y. Sadovsky Activation of Luteinizing Hormone beta Gene by Gonadotropin-releasing Hormone Requires the Synergy of Early Growth Response-1 and Steroidogenic Factor-1 J. Biol. Chem., May 14, 1999; 274(20): 13870 - 13876. [Abstract] [Full Text] [PDF]
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X.-b. Han and P. M. Conn The Role of Protein Kinases A and C Pathways in the Regulation of Mitogen-Activated Protein Kinase Activation in Response to Gonadotropin-Releasing Hormone Receptor Activation Endocrinology, May 1, 1999; 140(5): 2241 - 2251. [Abstract] [Full Text]
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J. J. Tremblay and J. Drouin Egr-1 Is a Downstream Effector of GnRH and Synergizes by Direct Interaction with Ptx1 and SF-1 To Enhance Luteinizing Hormone beta Gene Transcription Mol. Cell. Biol., April 1, 1999; 19(4): 2567 - 2576. [Abstract] [Full Text] [PDF]
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B. R. White, D. L. Duval, J. M. Mulvaney, M. S. Roberson, and C. M. Clay Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene Is Partially Mediated by Protein Kinase C Activation of an Activator Protein-1 Element Mol. Endocrinol., April 1, 1999; 13(4): 566 - 577. [Abstract] [Full Text]
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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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S. Nelson, R. D. Horvat, J. Malvey, D. A. Roess, B. G. Barisas, and C. M. Clay Characterization of an Intrinsically Fluorescent Gonadotropin-Releasing Hormone Receptor and Effects of Ligand Binding on Receptor Lateral Diffusion Endocrinology, February 1, 1999; 140(2): 950 - 957. [Abstract] [Full Text]
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X. Lin and P. M. Conn Transcriptional Activation of Gonadotropin-Releasing Hormone (GnRH) Receptor Gene by GnRH: Involvement of Multiple Signal Transduction Pathways Endocrinology, January 1, 1999; 140(1): 358 - 364. [Abstract] [Full Text]
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F. Tejada, A. Cremades, M. Aviles, M. T. Castells, and R. Penafiel Hypokalemia alters sex hormone and gonadotropin levels: evidence that FSH may be required for luteinization Am J Physiol Endocrinol Metab, December 1, 1998; 275(6): E1037 - E1045. [Abstract] [Full Text] [PDF]
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X. Lin, J. A. Janovick, and P. M. Conn Mutations at the Consensus Phosphorylation Sites in the Third Intracellular Loop of the Rat Gonadotropin-Releasing Hormone Receptor: Effects on Receptor Ligand Binding and Signal Transduction Biol Reprod, December 1, 1998; 59(6): 1470 - 1476. [Abstract] [Full Text]
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B. D. Strahl, H.-J. Huang, J. Sebastian, B. R. Ghosh, and W. L. Miller Transcriptional Activation of the Ovine Follicle-Stimulating Hormone {beta}-Subunit Gene by Gonadotropin-Releasing Hormone: Involvement of Two Activating Protein-1-Binding Sites and Protein Kinase C Endocrinology, November 1, 1998; 139(11): 4455 - 4465. [Abstract] [Full Text] [PDF]
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X. Lin and P. M. Conn Transcriptional Activation of Gonadotropin-Releasing Hormone (GnRH) Receptor Gene by GnRH and Cyclic Adenosine Monophosphate Endocrinology, September 1, 1998; 139(9): 3896 - 3902. [Abstract] [Full Text] [PDF]
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J. Weck, P. C. Fallest, L. K. Pitt, and M. A. Shupnik Differential Gonadotropin-Releasing Hormone Stimulation of Rat Luteinizing Hormone Subunit Gene Transcription by Calcium Influx and Mitogen-Activated Protein Kinase-Signaling Pathways Mol. Endocrinol., March 1, 1998; 12(3): 451 - 457. [Abstract] [Full Text]
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X. Lin, J. A. Janovick, S. Brothers, M. Blömenrohr, J. Bogerd, and P. M. Conn Addition of Catfish Gonadotropin-Releasing Hormone (GnRH) Receptor Intracellular Carboxyl-Terminal Tail to Rat GnRH Receptor Alters Receptor Expression and Regulation Mol. Endocrinol., February 1, 1998; 12(2): 161 - 171. [Abstract] [Full Text]
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M. Thibonnier, J. A. Preston, N. Dulin, P. L. Wilkins, L. N. Berti-Mattera, and R. Mattera The Human V3 Pituitary Vasopressin Receptor: Ligand Binding Profile and Density-Dependent Signaling Pathways Endocrinology, October 1, 1997; 138(10): 4109 - 4122. [Abstract] [Full Text] [PDF]
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E. J. Kaftan, T. Xu, R. F. Abercrombie, and B. Hille Mitochondria Shape Hormonally Induced Cytoplasmic Calcium Oscillations and Modulate Exocytosis J. Biol. Chem., August 11, 2000; 275(33): 25465 - 25470. [Abstract] [Full Text] [PDF]
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,
Control; •, LHRH-E treated. C, LHRH-E treatment does not alter the
turnover of Gi2
, Control; 
, LHRH-E treated. LHRH-E,
des-Gly10-[D-Ala6] LH-releasing hormone
ethylamide. (The term LHRH used in this figure is synonymous with GnRH
used elsewhere.) [Reprinted with permission from B. H. Shah et
al: Proc Natl Acad Sci USA 92:1886–1890, 1995
(64) (Fig. 3).].
) or cells stimulated with either GnRH (GnRH; 10
µmol/liter (•) or the GnRH agonist, buserelin (10 nmol/liter;
(
