(Received for publication, Febraury 21, 1996 )
From the
Analysis of luteinizing hormone (LH) subunit promoters
from a broad range of species including teleosts and humans revealed
strict conservation of a sequence homologous to the steroidogenic
factor-1 (SF-1) regulatory element of cytochrome P-450 steroid
hydroxylase genes. Interaction between SF-1 and this putative response
element in the bovine LH
promoter was confirmed by electrophoretic
mobility shift assays. Furthermore, cotransfection of
T3-1
cells with an expression vector encoding SF-1 induced binding
site-dependent transcription from the bovine LH
promoter.
Physiological significance of the LH
SF-1 consensus sequence was
established using transgenic mice containing either the wild type
bovine promoter or a promoter with a site-specific mutation of this
site. Mutation of the SF-1 binding site nearly eliminated promoter
activity, and the mutant transgene remained inactive following
induction of gonadotropin-releasing hormone accomplished by castrating
male and female mice. Thus, increases of gonadotropin-releasing hormone
within a physiological range did not compensate for the loss of the
SF-1 binding site. Together, these findings indicate that the SF-1
binding site is a key regulator of LH
promoter activity in
vivo and implicate SF-1 as at least one of the transcription
factors that acts through this site.
Gonadotropes within the anterior pituitary are defined, in part,
by their unique ability to synthesize and secrete luteinizing hormone
(LH) ()and follicle-stimulating hormone (FSH). These
glycoprotein hormones contain an identical
subunit that
heterodimerizes with unique
subunits(1) . Expression of
gonadotropin
and
subunit genes depends on secretion of
gonadotropin-releasing hormone (GnRH) from the
hypothalamus(2) . GnRH responsiveness, in turn, requires that
gonadotropes also express GnRH receptors(3) . Thus, functional
integrity and physiological control of mature gonadotropes relies on
regulated expression of at least four genes:
, LH
, FSH
,
and GnRH receptor.
While a complex array of regulatory elements
controls transcription of the subunit gene in
gonadotropes(4) , the elements regulating the genes encoding
LH
, FSH
, or GnRH receptor remain undefined. This lag is due
partly to weak activity of LH
and FSH
promoters when examined
in cell culture models(5, 6) . Moreover, the promoter
of the GnRH receptor gene has only recently been cloned(7) .
Nevertheless, there is a possibility that a single transcription factor
may contribute to regulated expression of all four genes.
One such candidate was revealed by gene-targeting studies directed at the Fushi tarazu factor 1 (FTZ-F1) gene that encodes steroidogenic factor-1 (SF-1) and embryonal long terminal repeat-binding protein (ELP)(8, 9, 10) . SF-1 is an orphan member of the nuclear receptor superfamily initially identified as a transcription factor that controls expression of the cytochrome P-450 steroid hydroxylase genes in gonads and adrenal cortex(8) . Targeted disruption of FTZ-F1 results in an absence of the adrenal cortex(9) , ovary(9) , testes(9) , ventromedial hypothalamic nucleus(11) , and gonadotropes (10) . This result was later attributed solely to the loss of the SF-1 encoding portion of the FTZ-F1 gene(12) .
Although all members of the
hypothalamic/pituitary/gonadal axis are influenced by removal of the
SF-1 gene, the mechanism responsible for gonadotrope deficiency remains
unclear. This could be explained by a direct effect of SF-1 on
gonadotrope development. Alternatively, if absence of the VMH
attenuates secretion of GnRH, then gonadotropes would fail to
develop(11) . A direct gonadotrope site of action of SF-1 is
suggested by studies with the subunit gene. All mammalian
subunit promoter regions examined to date contain a
gonadotrope-specific element (GSE) (13) that has strong
homology to SF-1 binding sites in steroid hydroxylase genes. This
element in the human
promoter binds to SF-1 (14) and
accounts for approximately 50% of its activity when analyzed after
transfection in the gonadotrope-lineage cell line,
T3-1(4, 13) . In contrast to evidence suggesting
a direct pituitary effect of SF-1, an indirect hypothalamic site of
action is supported by the recovery of detectable gonadotropin gene
expression in GnRH-treated, SF-1 ``knock-out''
mice(11) . The latter finding implies that restoration of GnRH
secretion may compensate for loss of SF-1 in gonadotropes. Thus, the
exact role SF-1 plays in regulating expression of the genes that define
the functional properties of gonadotropes remains to be determined.
Herein, we address the functional significance of the SF-1 binding
site found within the proximal 776-bp promoter-regulatory region of the
bovine LH gene. This promoter directs high level expression of
reporter genes specifically to gonadotropes in transgenic mice, is
fully penetrant, and is appropriately regulated by GnRH and gonadal
steroids(5) .
The intact/castrate/antide treatment paradigm was accomplished as follows. Male and female mice (6-11 weeks old) were divided into three groups for each sex. One group remained intact, received injections of antide vehicle (20% propylene glycol in normal saline) every 48 h, and animals were housed individually. The other two groups were gonadectomized under avertin anesthesia and treated with vehicle or 60 µg of antide every 48 h for 10 days(5) . On day 10, mice were killed and pituitaries and ventricular blood collected. CAT assays of 17.5 h duration were performed as above. All animal studies were approved by the Case Western Reserve University Institutional Animal Care and Use Committee.
Figure 1:
A consensus SF-1 binding site resides
within the 5` flanking regions of the genes encoding , LH
,
and GnRH receptor. Sequences from the
, LH
, and GnRH receptor
genes are compared to the SF-1 consensus from the steroid hydroxylase (15) genes, with mismatches in lower case. The SF-1 binding
site (GSE) from the human
(h
) subunit gene has
previously been characterized(14) . Numbers indicate
the position of the GSE homolog relative to the transcription start
site (+1). The LH
sequence was analyzed from: salmon (sGTHII
, (16) ), equine (PMSG
, (17) ), racine (rLH
, (18) ), bovine (bLH
, (19) ), and human (hLH
, (20) ). The bovine sequence is equivalent to that used in EMSA.
The GnRH receptor (mGnRHR) sequence was from
mouse(7) .
Figure 2:
The bovine LH GSE binds SF-1. EMSA
with
T3-1 nuclear extracts were performed with wild type (wt) or mutant (µ) GSE probes. Competitions were performed
with either wild type or mutant GSE oligodeoxynucleotides at 50-, 100-,
and 500-fold molar excess. Anti-SF-1 antisera or nonimmune serum (NS) were added to some reactions. The arrow denotes
the presence of an SF-1-containing complex.
As expected, activities of the wild type LH promoter
(bLH
) and a promoterless control were indistinguishable (data not
shown). In contrast, overexpression of SF-1 resulted in
5-fold
induction of bLH
promoter activity relative to that obtained in
the presence of the GH control (Fig. 3). Greater induction
(
8-fold; data not shown) was observed following addition of a
constitutively active form of SF-1(22) . Although the bLH
promoter containing an 8-bp GSE mutation was 2-fold more active than
the wild type promoter in the presence of GH, its response to SF-1 was
negligible and equivalent to that observed with the heterologous SV40
promoter. Thus, much of the stimulatory effect of SF-1 is mediated
through the GSE.
Figure 3:
Overexpression of SF-1 activates the
LH promoter in
T3-1 cells.
T3-1 cells were cotransfected
with various promoter/luciferase chimeric constructs and CMV expression
vectors encoding either growth hormone (CMVGH) or SF-1 (CMVSF-1). The
following reporter constructs were used:
(-776/+10)bLH
luc,
(-776/+10)µGSEbLH
luc, and pGL2Control, which
contains the SV40 promoter linked to luciferase. Values are means
± S.E. for four transfections, each containing three
plates.
While SF-1 stimulated LH promoter activity
only 5-fold, this was sufficient to establish a functional and
statistically significant correlation between binding of SF-1 and
transcriptional activation. A site-dependent effect of full-length SF-1
on other promoters has been difficult to establish. For example, Shen et al.(22) only observed activation of the
Müllerian inhibiting substance promoter with a
truncated form of SF-1 devoid of the putative ligand binding domain.
Similarly, the human
subunit promoter with an intact SF-1 binding
site is refractory to overexpression of both full-length and truncated
SF-1 (data not shown), and ablation of the SF-1 binding site in the
human
subunit promoter reduces basal activity by only
50%(4, 13) . This suggests that transcription effects
of SF-1 may be dependent on promoter context. The level of
SF-1-dependent activation observed with the LH
promoter is also
significant because overexpression of other proteins potentially
involved in the GnRH signal transduction pathway, including
constitutively active forms of Ras (23) or
G
(24) , had no effect (data not shown). Thus,
these transfection assays are the first to identify both a discrete
promoter element and a transcription factor that regulate activity of
the LH
promoter.
Transgenic mice were made with the 776-bp
LH promoter, containing the same GSE mutation used in the EMSA and
transfection assays, linked to CAT. Integrity of the transgene in seven
founders was confirmed by Southern blot analysis (data not shown), and
lines were derived from five to obviate integration site effects.
Activity of the µGSE-LH
promoter in pituitary was reduced at
least 10-fold when compared to activity in two lines of mice containing
the wild type promoter (Fig. 4). No ectopic expression was
observed in nine other tissues (data not shown).
Figure 4:
The
GSE is essential for LH promoter activity in transgenic mice.
Males (closed circles) and females (open circles)
from two lines of mice containing the wild type bLH
CAT transgene (wt), or six lines of mice containing the bLH
µGSECAT
transgene (denoted 1-6), were assessed for CAT activity
in pituitary. Each circle represents an individual mouse. All
mice were at least one generation from the founder animals except the
mouse from line 6, which was the founder. All assays were performed
with 10 µg of pituitary lysate for 1 h with CAT activity expressed
as percent conversion/µg of protein/h. Mouse pituitaries containing
the wild type or µGSE transgenes were assayed in
parallel.
Activity of the
wild type promoter tended to be higher in females than in males (Fig. 4). This trend continued with the mutant promoter, such
that the occasional mouse having detectable CAT was always female.
Thus, while mutation of the GSE has a substantial effect on activity of
the LH promoter in both sexes, it may be even greater in male
mice. From these results, we conclude that the SF-1 binding site (GSE)
is required for full bLH
promoter activity in pituitaries of
transgenic mice.
Figure 5:
GnRH cannot compensate for loss of the
GSE. Female (A) and male (B) mice from line 1
containing the bLHµGSECAT transgene remained intact or were
gonadectomized. The intact animals and a subset of gonadectomized mice
were treated with vehicle, while another subset received antide. At the
conclusion of the experiment, blood and pituitaries were collected for
LH and CAT assays, respectively. All CAT assays were performed with 10
µg of protein for 17.5 h. CAT activity is expressed as percent
conversion/100 µg of protein/h. Values are means ± S.E. for
5-6 animals/group. The shaded region in the lower
portion of the figure represents the mean activity of multiple liver
samples included in this assay (1.14 ± 0.14). Any values that
approach this level are considered non-expressing. The activity of wild
type mouse pituitaries was 49.8 ±
3.0.
In contrast to females, intact male
mice containing the mutant promoter lacked any detectable CAT activity (Fig. 5B). Castration also failed to induce expression,
whereas an antide-reversible increase in serum LH was observed. Thus,
changes in GnRH were unable to compensate for loss of the GSE in the
LH promoter in either sex. This result contrasts with those
reported by Ikeda et al.(11) , where GnRH treatment
of SF-1-deficient mice resulted in an unquantified activation of the
endogenous LH
gene. Several differences between the two studies
render valid comparison difficult. Pharmacological doses of GnRH were
used to treat the knock-out mice (11) , whereas the increase in
GnRH observed in our mice should more accurately approximate
physiological levels. In addition, the extent of disruption of the
reproductive axis in mice lacking SF-1 is unclear, as is its possible
impact on gonadotropin gene responsiveness to GnRH. Finally, it is
possible that additional transcription factors could compensate for
SF-1 at the level of the GSE. Removal of the GSE itself, which occurred
with the mutant LH
CAT mice, would negate any potential
compensatory mechanism. Precedence for such redundancy has been
observed with transcription factors that are members of larger
families, including MyoD (25) and cAMP response element-binding
protein (26) . Insertion of the mutant transgene into the
background of the SF-1-deficient mouse may resolve these differences.
In summary, the GSE within the bovine LH subunit promoter binds
SF-1 with high affinity, mediates activation of transcription by SF-1 in vitro and is considerably important for full activity in
gonadotropes of transgenic mice. This is the first complete
characterization of an element important for expression of the LH
subunit gene in gonadotropes and the first physiological demonstration
of a functional requirement for an SF-1 binding site within any gene.
Conservation of the SF-1 binding site in three of the genes that define
a mature gonadotrope (
, LH
, and GnRH receptor) may provide a
unifying mechanism for their coordinated onset and maintenance of
expression.