Regulable Expression of Inhibin A in Wild-Type and Inhibin
Null Mice
Tyler Mark Pierson,
Yaolin Wang,
Francesco J. DeMayo,
Martin M. Matzuk,
Sophia Y. Tsai and
Bert W. OMalley
Department of Molecular and Cellular Biology (T.M.P., F.J.D.,
M.M.M., S.Y.T., B.W.O.) Department of Pathology (M.M.M.), and
Department of Molecular and Human Genetics (M.M.M.) Baylor College
of Medicine Houston, Texas 77030
Schering-Plough Corp. Research Institute
(Y.W.) Kenilworth, New Jersey
07033
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ABSTRACT
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Exogenous regulation of protein expression creates
the potential to examine the consequences of homeostatic Dysregulation
in many physiological systems and, when used in transgenic mice,
provides the capability of restoring a gene product to its knockout
background without antigenicity issues. In this study, we used a
mifeprisone-inducible system (the GeneSwitch system) to regulate the
expression of inhibin A from the liver of mice. Inhibin is a
heterodimeric protein (
/ß) wherein one of its subunits (ß) is
capable of homodimerizing to form its physiological antagonist, activin
(ß/ß). Inhibin is also expressed in two forms, A and B, as
determined by the subtype of ß-subunit that dimerizes with the
-subunit (
/ßA or
/ßB). To utilize the GeneSwitch system,
transgenic transactivator mice with liver-specific expression of a
mifepristone-activated chimeric nuclear receptor (GLVP) were
crossed with transgenic target mice containing a GVLP-responsive
promoter upstream of poliovirus IRES (internal ribosome entry
site)-linked sequences coding for the
- and ß-subunits of inhibin
A. This intercross produced "bigenic" mice capable of regulable
expression of inhibin A from the liver. Overexpression of inhibin A in
wild-type mice produced a phenotype wherein males had decreased testis
size and females had a block in folliculogenesis at the early antral
stage, findings similar to activin type IIA receptor (ActRIIA) null
mice. These phenotypes were most likely due to suppressed serum FSH,
confirming that the liver-derived inhibin A was secreted into the serum
to down-regulate pituitary FSH levels. Furthermore, the generation of
bigenic mice in the inhibin
null background allowed for the
induction of inhibin A in inhibin
null male mice with subsequent
rescue of these mice from their gonadal tumor-induced lethal phenotype.
This work demonstrates the in vivo production of a
heterodimeric hormone from a single inducible promoter to study its
therapeutic and physiological effects. In addition, these studies are
the first example of an inducible system being used to prevent a lethal
knockout phenotype in an animal model.
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INTRODUCTION
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Inhibins function to negatively regulate the production and
secretion of FSH from the anterior pituitary, regulate intragonadal
events including follicle development and steroidogenesis, and act as
tumor suppressors in the gonads and adrenal cortex (1 2 3 4 5 ). At the level
of the pituitary, inhibins function to down-regulate the production and
the secretion of FSH in a cycle-dependent manner in females and in a
tonic pattern in males (5 ). Interestingly, the expression patterns of
the two inhibins, inhibin A or inhibin B, are sexually, spatially, and
temporally dimorphic (5 ) and have been shown to be differentially
regulated by various hormonal stimuli in vitro (6 ). When the
inhibin
-subunit is deleted in mice, inhibin-deficient mice form
gonadal sex cord-stromal tumors with 100% penetrance as early as 4
weeks of age (3 ). Soon after the development of these tumors, the mice
acquire a cancer cachexia-like syndrome and die (4 ). This syndrome, the
result of large amounts of activins secreted by the tumors (7 ),
consists of a block in gastric epithelial cell differentiation, severe
weight loss, hepatocellular degeneration due to apoptosis, and anemia
(4 7 8 ).
Inhibin and activin physiology are interwoven at several levels.
Molecularly, activins are homodimers of the ß-subunit, while inhibins
consist of
/ß-subunit heterodimers. In order for a cell to produce
the inhibin heterodimer without concurrent activin production, the
-subunit must be produced in excess of the ß-subunit (creating
a situation wherein the
-subunit monomer is also secreted in
substantial quantities from an inhibin-producing cell) (9 ). Inhibins
and activins also have been shown to have overlapping binding sites in
a number of tissues (10 11 ). This overlapping pattern and in
vitro data indicate that the inhibins, and in particular inhibin
A, may have a dominant-negative effect upon activin signaling via the
activin type II receptor (12 13 14 ). However, inhibin-specific binding
sites in the gonads, pituitary, and gonadal-tumor tissue of inhibin
null mice also have been observed (10 15 16 17 ). An especially
intriguing aspect in the study of inhibin function is that an
inhibin-specific receptor has not been elucidated in great detail
and so the mechanism by which inhibin transduces its signal and/or
antagonizes activins signal remains unknown (18 ).
The use of ligand-inducible systems has been very productive in the
study of protein function and the generation of animal models of
disease or disease therapy (19 20 21 22 23 ). One ligand-inducible system, which
can be used to address in vivo questions, is the GeneSwitch
system. This system utilizes a regulator protein, GLVP (a chimeric
VP16-GAL4-PR
42), whose gene is expressed from one promoter, that is
able to induce (in the presence of its activator, mifepristone) the
expression of another gene via binding to an upstream GAL4 (promoter)
response element. We successfully used this bigenic GeneSwitch system
to regulably express inhibin A from the livers of either wild-type mice
or mice with a knockout of the inhibin
gene. By using the liver, we
could determine the efficacy of our system in the delivery of hormones
into the bloodstream from "exogenous" tissues (i.e. a
tissue that does not normally express inhibins) and the potential use
of these "exogenous" tissues in future gene therapy protocols
involving the GeneSwitch system. In this manuscript, we show that we
can induce the production of inhibin A into the circulation from a
source (i.e., the liver) that normally does not synthesize
inhibin. In addition, we demonstrate an endocrine effect of the inhibin
A at the level of the pituitary (in wild-type mice) and at the level of
the testes (in inhibin
knockout mice). In the latter case,
regulable production of inhibin A blocks the development of testicular
tumors in the inhibin
knockout, confirming previous studies that
inhibin is a secreted tumor suppressor protein. The successful
generation of mice capable of producing bioactive inhibin A from the
liver in response to mifepristone treatment has broad implications in
the study of reproductive physiology and tumorigenesis, the development
of novel contraceptive technologies, and the further use of similar
inducible systems for gene therapy.
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RESULTS
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Development of an Inducible System for the Study of Inhibin
Function
To study the in vivo consequences of overexpression of
inhibin A in mice, we used the bigenic GeneSwitch system. The
GeneSwitch system consists of a chimeric regulator (GLVP) composed of a
mutated progesterone receptor ligand-binding domain (PR
42) that
binds and is activated by mifepristone (MFP), but not by endogenous
steroids (24 ) (Fig. 1
). The DNA binding
domain of the yeast GAL4 transcription factor and the transcriptional
activation domain of the herpes virus VP16 protein are fused to the
mutant progesterone receptor ligand-binding domain. The GLVP is
responsive to mifepristone and activates transcription from a target
locus consisting of GAL4 response elements (17 mer sequences)
positioned upstream from a target gene or genes (19 24 25 26 27 ) (in our
case, there were four copies of the 17 mer sequence; see below). The
production of transgenic mice expressing the GLVP specifically in the
liver has been previously reported (glvp transactivator
line) (26 ).
To produce the GLVP-responsive inhibin A target locus used in the
present study, an inhibin A target (transgene) construct
(X3-inh) was constructed with four copies of the 17 mer-Gal4
responsive elements upstream of a minimal thymidine kinase promoter.
Downstream of this promoter is an intron containing SV40 splice donor
and acceptor sites followed by the cDNA for the murine inhibin
-subunit, the poliovirus internal ribosome entry site (IRES), the
cDNA for the murine inhibin/activin ßA subunit, and an SV40 polyA
addition consensus sequence (Fig. 1
). When activated, this locus would
produce a bicistronic mRNA, in which the poliovirus IRES links the
inhibin
and ßA subunit cDNAs (28 ). This strategy would permit a
relative excess production of the inhibin
subunit in comparison
with the inhibin/activin ßA subunit, since the inhibin
subunit is
translated by a more efficient cap-dependent translational mechanism
whereas the inhibin/activin ßA subunit would be translated by a
less-efficient IRES-dependent mechanism. By biasing our translational
efficiency in this manner, our goal was to produce a ratio of the two
subunits (i.e.,
>> ßA) that would favor the formation
of inhibin A (
/ßA heterodimer) over activin A (ßA:ßA
homodimer) (9 28 ).
Mifepristone-Inducible Inhibin A Production in Tissue Culture and
in Vivo
Before induction of inhibin A from the liver of mice, transient
transfection studies in HepG2 cells were performed to test the
inducibility of the X3-inh transgene in a hepatocyte cell
line. In these assays we used another transactivator, GLp65, which
differs from GLVP in that the transactivation domain is derived from
the p65 protein (a member of the NF-
B family) (27 ). With all
functional assays the GLp65 works similarly to GLVP with the exception
that in transient transfections the GLp65 has a much lower basal
activity than the GLVP. The X3-inh was transfected into
HepG2 cells in the presence or absence of the GLp65 encoding plasmid.
Cells were subsequently treated with or without mifepristone. The
X3-inh-transfected cells expressed inhibin A only when both
the GLp65 transactivator and MFP were present. In contrast, there was
no basal level of inhibin A expression in the absence of MFP (Fig. 2A
). Thus, our bigenic system was
functional in transient transfection assays.

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Figure 2. Inhibin A Is Induced in Vitro and
in Vivo
A, Inducible expression of inhibin A in HepG2 cells. Target vector DNA
(0.3 µg pX3) was transiently transfected into HepG2 cells with or
without transactivator plasmid (0.2 µg pSBC-1 or pSBC-GLp65). Either
MFP or vehicle was administered to cells. Only cells transfected with
the target and transactivator constructs and treated with MFP expressed
inhibin A (155.7 ± 7.6 pg/ml). B, Dosage-dependent expression of
inhibin A in inh/glvp mice. Serum inhibin A levels were
measured before (0 h) and after (12 h) ip injection of either 250
µg/kg or 500 µg/kg of MFP (n = 3). C, Effects of
overexpression of inhibin A on serum FSH levels. Male
inh/glvp mice were given 500 µg/kg MFP for 7 days, and
serum was collected pre- and posttreatment. Inhibin A and FSH levels
were measured from the serum of each mouse. Inhibin A levels were induced whereas FSH levels were significantly
reduced in male inh/glvp mice before and after MFP
treatment (423.7 ± 113.5 vs. 161.4 ± 48.7 ng/ml;
respectively, P < 0.04, paired one-tailed
Students t test, n = 3).
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Based on the above findings in the hepatocyte cell line, the
X3-inh transgene was subsequently microinjected into mouse
embryos, and nine founder target lines (X3 mice) were generated. These
founder target lines were then crossed with transactivator mice
(glvp mice) to produce bigenic mice (X3/glvp
mice). Since inhibin A is not expressed at detectable levels in the
serum of adult male rodents (5 ), serum samples from the males were
assayed to avoid any confounding endogenous inhibin signal. Male
X3/glvp mice generated from the nine X3 founder mouse lines
were subsequently tested for MFP-induced inhibin A secretion. One of
the X3 founder lines (line 3065, hereafter referred to as the
inh transgenic mouse line) was capable of expressing inhibin
A at high levels in the presence of MFP and the GLVP. When male
inh/glvp mice from this line were ip injected with MFP (250
and 500 µg/kg dosages), inhibin A expression was dependent on the MFP
dose and expressed at either physiological (436 ± 155 pg/nl when
250 µg/kg MFP was injected) or supraphysiological concentrations
(1,210 ± 232 pg/nl when 500 µg/kg MFP was injected) relative to
inhibin B levels in the male rat (5 ) (Fig. 2B
). To confirm that the
immunoreactive inhibin A secreted from the liver was also bioactive,
male inh/glvp mice were injected with high-dose MFP for 7
days, and the effect of the induced inhibin A on serum FSH levels was
determined. These bigenic males expressing the induced inhibin A had a
2.6-fold reduction in serum FSH levels (Fig. 2C
). Monogenic mice
(inh or glvp mice) treated with MFP did not have
altered serum levels of FSH (data not shown).
The need to perform long-term studies facilitated the use of
mifepristone-containing time-release pellets. These pellets were
designed to release MFP in measured dosages over a 60-day period. Male
inh/glvp mice receiving pellets that released appropriately
6 µg MFP a day had inhibin A levels of 1,803 ± 258 pg/ml after
1 week of treatment. Eight weeks after treatment, serum FSH levels in
MFP-treated inh/glvp male mice were significantly reduced in
comparison to placebo-treated inh/glvp males [126.3 ±
22.1 (n = 4) vs. 197.8 ± 16.0 ng/ml,
(respectively; n = 5, P<0.02)]. These findings
demonstrate that inhibin A (
:ßA) secretion from the liver must
predominate over the activin A (ßA:ßA) levels (see below and Fig. 5C
). Thus, the GS system can regulate the expression of inhibin A in
cell culture and in vivo, and the induced inhibin A is
bioactive and capable of reducing serum FSH levels.

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Figure 5. Male inh/glvp Mice Expressing
Inhibin A in the Inhibin Null Background Do Not Undergo Gonadal
Tumorigenesis
A, Testicular weights for inhibin null males. Male
inh/glvp -/- mice treated
with MFP exhibited testis weights that were similar to monogenic
inhibin wild-type males treated with placebo (data not shown). Male
inh/glvp -/- mice treated
with MFP had significantly smaller testes in comparison to male
inh/glvp -/- mice treated
with placebo (135 ± 20 vs. 381 ± 78 mg,
respectively; P < 0.001, one-tailed Students
t test, n = 6) or
monogenic -/- controls. B, Gross and
histological analysis of testes from inhibin null males. Male
inh/glvp -/- mice treated
with MFP had testes that grossly and histologically (bottom
right panel) appeared tumor free. Male
inh/glvp -/- mice treated
with placebo exhibited hemorrhagic and tumorous testes (top
panel, left; bottom left panel) similar to monogenic null
controls. Spermatozoa were evident in the seminiferous tubules of male
inh/glvp -/- mice treated
with MFP (bottom right panel). C, Serum activin levels
in inhibin null males. Male
inh/glvp -/- mice treated
with MFP did not express any detectable serum activin A (n = 3),
while male inh/glvp -/- mice
treated with placebo (127.7 ± 20.5 pg/ml, n = 3) had serum
activin A levels that were high similar to the
monogenic -/- controls. D, Body weights of
male inh/glvp -/- mice
during and after MFP treatment (vertical line at 11
weeks represents the end of the MFP treatment); 18- to 21-day-old male
inh/glvp -/- mice were given
60-day time-release pellets delivering 12 µg of MFP a day
(open circle) or placebo (closed
circles). These mice continued to maintain body weights similar to
monogenic inhibin wild-type controls (closed
diamonds) for up to 12 weeks after removal of MFP (n = 6).
Male inh/glvp -/- mice
treated with placebo (closed circles) lost weight in a
manner similar to studies presented in Fig. 3A .
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Effects of Overexpression of Inhibin A in Wild-Type Mice
By overexpressing inhibin A in bigenic mice, a greater
understanding of inhibin signaling and physiology can be achieved.
Long-term studies were initiated by implanting timed-release MFP (6
µg/day) or placebo pellets into 18- to 21-day-old bigenic and
monogenic mice of both sexes. These mice were weighed weekly and
killed 8 weeks after pellet placement. Blood, livers, and gonads were
harvested. Male inh/glvp mice that received MFP were found
to have significantly reduced testis weights as compared with
inh/glvp males treated with placebo or monogenic males
receiving MFP or placebo (monogenic controls) (Fig. 3A
). These mice also had a decrease in
seminiferous tubule volume and diameter as compared with bigenic males
receiving placebo pellets and monogenic controls (Fig. 3B
). There were
no significant differences in body weight over time between the
treatment groups (data not shown). Although the mifepristone-treated
inh/glvp males had smaller testes, spermatozoa were present
in the lumens of the seminiferous tubules and, when paired with
females, these mice were found to be fertile. Female
inh/glvp mice receiving MFP also had defects in the gonads,
demonstrating an arrest in folliculogenesis at the early antral
follicle stage and a lack of corpora lutea (Fig. 3C
). In contrast,
female inh/glvp mice receiving placebo and monogenic female
controls receiving MFP or placebo had ovaries that underwent normal
folliculogenesis and contained numerous corpora lutea; this indicated
that the MFP dosage did not antagonize oogenesis, folliculogenesis, and
ovulation (Fig. 3C
). Thus, overexpression of inhibin A leading to
reduced serum FSH levels in male and female mice resulted in gonadal
defects reminiscent of activin receptor type II knockout mice, which
also have reduced FSH levels (see Discussion) (30 ).

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Figure 3. Inhibin A Overexpression Mimics the
Activin Type IIA Receptor Null Phenotype
A, Testis size in bigenic and monogenic mice ( 11 weeks of age).
Testis size was significantly reduced in male inh/glvp
mice treated with 6 µg/day MFP as compared with male
inh/glvp mice treated with placebo [68.8 ± 5.4 mg
vs. 102.5 ± 6.2 mg, respectively,
P < 0.006, one-tailed Students t
test] or monogenic male controls. B, Gross and microscopic analysis of
testes from placebo (left) or MFP-treated
(right) male inh/glvp mice. Male
inh/glvp mice receiving MFP had smaller testes that
contained seminiferous tubules with a narrower cross-sectional diameter
as compared with male inh/glvp mice receiving placebo or
monogenic controls. Mature spermatozoa were evident in the tubules of
all mice. C, Microscopic analysis of ovaries from female
inh/glvp mice. The ovaries of the majority of female
inh/glvp mice treated with MFP (right
panels) had a block in folliculogenesis at the early antral
follicle stage, had a number of atretic follicles, and did not contain
corpora lutea (4 of 7), whereas ovaries from female
inh/glvp mice treated with placebo (left
panel) underwent normal folliculogenesis and contained numerous
corpora lutea (*) (8 of 8). Monogenic female controls treated with MFP
also underwent normal folliculogenesis and contained corpora lutea (8
of 8 each) (data not shown).
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Regulable Rescue of Male Inhibin
Null Mice
Inhibin
knockout mice develop gonadal sex cord-stromal tumors
as early as 4 weeks of age (3 ). More than 95% of the
inhibin
homozygote knockout male and female mice will die by 12 and 17 weeks of
age, respectively, of a cancer cachexia-like syndrome due to high
levels of tumor-secreted activins signaling through activin receptor
type IIA in the liver and glandular stomach (4 7 8 ). Because of the
earlier death of male inhibin
null mice, we addressed whether
overproduction of inhibin A can rescue the male inhibin
null lethal
phenotype. Bigenic
(inh/glvp
-/-
mice) and monogenic
(inh
-/- or
glvp
-/- mice; called
monogenic
-/- controls
below) 18- to 21 day-old inhibin
knockout males were treated with
MFP or placebo timed-release pellets, which should be active over an
8-week period. Bigenic inhibin
null male mice treated with MFP did
not exhibit cachexia or tumors when examined up to 11 weeks of age.
Body weights for
inh/glvp
-/-
male mice given MFP were comparable to monogenic inhibin
heterozygote mice treated with placebo (Fig. 4A
). In contrast, the
inh/glvp
-/-
male mice given placebo underwent weight loss, and many of these mice
died in a manner similar to monogenic inhibin
null male mice
receiving MFP or placebo
(monogenic
-/-
controls) (Fig. 4A
). Thus, overexpression of inhibin A can prevent the
inhibin knockout mice from dying of the cachexia-like syndrome.
To determine how overexpression of inhibin A was rescuing the inhibin
knockout mice, morphological and histological analysis was
performed. Special attention was paid to the livers of these mice since
activin-induced hepatocellular necrosis is a major finding in the
inhibin
knockout mice (4 ). Livers from
inh/glvp
-/-
male mice treated with MFP were normal in appearance and weight in
contrast to the small, pale livers of
inh/glvp
-/-
male mice treated with placebo or
monogenic
-/- controls
(Fig. 4
, B and C). Bigenic male
inh/glvp
-/-
mice treated with MFP exhibited normal liver architecture, while
inh/glvp
-/-
males treated with placebo had pale livers with extensive lymphocytic
infiltration and hepatocellular degeneration around the central vein
similar to monogenic null controls (Fig. 4C
). Thus, overexpression of
inhibin A prevents the liver pathology seen in the inhibin
knockout
mice.
We next determined the long-term effect of overexpression of inhibin A
on testicular tumorigenesis in the inhibin
knockout mice. Testes
from
inh/glvp
-/-
male mice treated with MFP appeared grossly tumor free in contrast to
inh/glvp
-/-
males treated with placebo or
monogenic
-/- controls,
both of which exhibited grossly hemorrhagic tumors (Fig. 5
, A and B). Male
inh/glvp
-/-
mice treated with MFP exhibited normal testicular cytoarchitecture and
had spermatozoa in the lumen of their seminiferous tubules (Fig. 5B
)
consistent with the normal fertility of these mice (see below). In
contrast,
inh/glvp
-/-
males treated with placebo and monogenic
-/- controls had testes
that had disorganized testicular cytoarchitecture, hemorrhage, and
tumor cells of sex cord-stromal origin (Fig. 5B
). Furthermore, male
inh/glvp
-/-
mice treated with MFP were fertile, while
inh/glvp
-/-
males given placebo and monogenic null controls were infertile. Thus,
high level production of inhibin A is sufficient to prevent testicular
tumor development in our inhibin
knockout mice.
In inhibin
null mice, high activin secretion from the testicular
tumors directly causes the cachexia-like syndrome (4 7 8 ). Consistent
with this data, serum activin A levels were undetectable in
inh/glvp
-/-
males treated with MFP and lacking testicular tumors, while
placebo-treated
inh/glvp
-/-
males and monogenic
-/-
controls with testicular tumors had similar elevated serum activin
levels (Fig. 5C
). These results confirm that gonadal tumorigenesis is
required for the elevation of serum activins in inhibin
null mice,
while also verifying that our system does not produce significant
amounts of activin A as a by product of the inducible inhibin A
expression.
Rescue of Inhibin
Null Male Mice Is Reversible
To determine whether the preventative effects of overexpression of
inhibin A on the tumorigenesis process is reversible, we analyzed the
inh/glvp
-/- after
cessation of MFP treatment. When the
inh/glvp
-/-
males are treated for 60 days with MFP, with subsequent cessation of
MFP treatment, the testicular tumors and the cancer cachexia-like
syndrome did not occur within the first 12 weeks after MFP removal.
However, by the 13th week after the MFP removal, many of these
inh/glvp
-/-
males began to exhibit the phenotypic characteristics of the cancer
cachexia-like syndrome and demonstrated obvious testicular tumors (Fig. 5D
). Thus, high level inhibin A from 311 weeks of age does not
preclude the eventual development of testicular cancer in these mice.
The reason why the tumorigenesis and the cancer cachexia-like syndrome
developed so slowly after MFP withdrawal is not clear (see below).
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DISCUSSION
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In this study, we used the GeneSwitch system to study the
overexpression of a target gene and to rescue a knockout mouse from its
lethal phenotype. This regulable gene expression system was capable of
the tonic expression of inhibin A, while also allowing for a precise
control of temporal activation and deactivation of inhibin A expression
that would not be possible using constitutive promoters or daily
injections. The use of a regulable transgenic inhibin A expression
system in inhibin-deficient mice would also allow these mice to be
exposed to inhibin A without activation of the immune system. Thus, the
regulable expression of inhibin A in mice allows for several aspects of
inhibin physiology to be investigated. For example, in wild-type mice,
we tested the effects of increased levels of circulating inhibin on the
pituitary physiology without any of its normal homeostatic feedback
responses to FSH and steroids. This system also allowed for inhibin A
production to be initiated just before the onset of tumorigenesis in
inhibin-deficient mice with the potential of inhibiting the formation
of the sex cord-stromal tumors in a regulable manner. The system also
allowed us to determine whether the gonadal cells could initiate the
oncogenic process when the inhibin A secretion was "turned off."
Thus, the use of a regulable gene expression system was predicted to
provide enough flexibility to alter the exposure strategy to more
thoroughly investigate inhibins role in the onset of tumorigenesis
and cancer cachexia-like syndrome in these mice.
In our studies, the poliovirus IRES was shown to be functional for the
dual translation of a bicistronic mRNA in transgenic mice. This
translation resulted in the production of the two subunits favoring the
formation of heterodimeric inhibin instead of homodimeric activin. The
inh/glvp male and female mice treated with MFP and
overexpressing inhibin A phenocopy the activin receptor type IIA
(ActRIIA) null mice (30 ). Both the ActRIIA null mice and the inhibin
A-overexpressing mice have reduced serum FSH levels, which is the most
likely cause of both phenotypes [i.e. both mouse models are
reminiscent of a theoretical FSH hypomorph since the FSH null mice have
a similar, but slightly more dramatic phenotype (31 )]. Alternatively,
the gonadal phenotypic similarity of the inhibin A-overexpressing mice
to ActRIIA null mice could indicate that inhibin A is functioning to
antagonize signaling through this receptor and/or other receptors that
interact with the ActRIIA. Unfortunately, the lack of adult phenotypes,
due to embryonic or neonatal lethality, for mice deficient in other
"activin" receptors [ActRIIB(32 ), ActRIA/ALK2 (33 ), and
ActRIB/ALK4(34 )] or both activin ligands (35 ) does not allow for a
comparison of these mice to the inhibin A-overexpressing mice. The
phenotypic similarity of the ActRIIA null mice and the inhibin
A-overexpressing mice could also be the result of a combination of
effects at the level of the pituitary (decreased FSH secretion) and
gonad (inhibin A antagonism of signaling via the activin and/or
putative inhibin receptors).
Overexpression of inhibin A rescued the inhibin
null male mice from
their lethal phenotype. These results are relevant to the fields of
physiology, oncology, and gene therapy for several reasons. The induced
inhibin A was not expressed in the inhibin
null mice until 1821
days after birth and yet prevented testicular tumorigenesis. In
addition, when the inducing mifepristone is removed, these mice formed
gonadal tumors (albeit at a slower rate than untreated inhibin
null
mice). These results indicate that a lack of inhibin A in either
pubertal or adult male mice sets the testis on the pathway to
tumorigenesis. In contrast, exposure of inhibin
null mice to
inhibin A at 1821 days of age inhibits the formation of sex
cord-stromal tumors in these mice for a period coinciding with inhibin
A expression. The results also indicate that the system is reversible
and that early inhibin A exposure is not "curative" for gonadal
tumorigenesis in the inhibin
null background (i.e., loss
of inhibin A expression permits subsequent initiation of gonadal
tumorigenesis). This ability to delay the formation of the tumors could
be of specific and general value in the study of tumorigenesis. In our
case, factors affecting the progression of the gonads into
tumorigenesis can now be manipulated and evaluated in this background
to determine which factors regulate the rate of tumorigenesis after
inhibin A expression is terminated.
This study indicates the utility of inducible systems for the
expression of therapeutic proteins. Even with a complicated scenario
requiring production of a heterodimeric protein (each subunit of which
must also be processed from precursors), the GeneSwitch system was
capable of producing adequate amounts of the therapeutic protein to
rescue a genetic disease. The regulable system was also capable of
producing inhibin A in supraphysiological amounts so that
concentrations in the gonad could approach normal local tissue
concentrations to simulate the effects of the higher concentrations
observed in paracrine and autocrine interactions. Because gene therapy
vectors may not be capable of transducing certain cell types, the
ability to express a protein under an inducible promoter in a different
cell type, such as hepatocytes, may prove to be a common method of
delivery and therapy. In summary, these results indicate the value of
inducible gene systems in future in vivo physiology,
tumorigenesis, and gene therapy research.
 |
MATERIALS AND METHODS
|
---|
Plasmid Construction
Vectors were constructed using standard cloning procedures.
Construction of the pTTR-GLVP transgene-containing plasmid has been
described previously (26 ). The pX3 target transgene-containing plasmid
was constructed using a poliovirus IRES cloning system (28 ). The SV40
promoter/enhancer in the pSBC-1 vector was removed, and a fragment
containing the 17mer x 4 tk promoter sequences was inserted into
the promotorless pSBC-1 vector (p17mer x 4 tk BC-1). A fragment
containing the murine inhibin
cDNA was isolated and subcloned into
the p17mer x 4 tk BC-1 construct (p17mer x 4 tk
). A
fragment containing the murine inhibin/activin ßA cDNA was cloned
into the pSBC-2 vector (pSBC2-ßA). p17mer x 4 tk
and
pSBC2-ßA were then combined to produce the X3-inh
transgene-containing plasmid. The GLp65 has been described previously
(27 ) and was placed into the pSBC-1 vector (pSBC-GLp65).
Transient Transfection of HepG2 Cells and Assay of Inhibin A
HepG2 cells (600,000/well) were cotransfected with 0.3 µg of
the pX3-inh transgene-containing plasmid and 0.2 µg of
pSBC-1 or pSBC-GLp65 using SuperFect reagents (QIAGEN
Inc., Valencia, CA). Sixteen hours later the media were removed, and
cells were exposed to DMEM (10% FBS) containing either 70% ethanol
vehicle or 10-8 M
mifepristone (Rousell-Uclaf, Paris, France) for 24 h. Conditioned
media were then collected and stored at -70 C. One milliliter of
conditioned media was then concentrated 10-fold with Ultrafree-4
Centrifugal Filter Devices (Millipore Corp., Bedford, MA)
and reconstituted to a 2-fold dilution with FBS. Inhibin A assays were
performed using a human inhibin A enzyme-linked immunosorbent assay
(ELISA) kit (Serotec Inc., Raleigh, NC).
Experimental Animals
All mouse studies were conducted in accord with the principles
and procedures outlined by Molecular Endocrinologys
instructions to authors under "Guidelines for the Care and Use of
Experimental Animals."
Generation of Target Transgenic Mice and Bigenic Mice
The glvp transactivator mice were produced and
screened as described previously (26 ). The X3 transgene was restriction
endonuclease released from pX3 and a 4.3-kb fragment isolated and
microinjected into the pronuclei of mouse embryos. Injected embryos
were transferred into pseudopregnant female mice and allowed to develop
to term. Mice were screened at 2 weeks of age by PCR using genomic tail
DNA. PCR analysis of tail DNA to detect the X3 transgene was carried
out with primers TT1 (5-CAAAGTGCAGTGTCTTCCTGGCTGTGC-3') and TT2
(5'-CGGAACCGACTACTTTGGGTGTC-3'). Nine founder X3 lines were generated,
with only one having inducible inhibin A expression (line
3065/inh) when crossed with glvp mice and treated
with MFP.
Generation and Analysis of inh/glvp Mice and
inh/glvp
-/-
Mice
Transactivator and target mice were mated, and bigenic
mice containing both transgenes were genotyped by PCR analysis.
Heterozygote inhibin
null mice were bred with both transactivator
and target mice to produce monogenic glvp or inh
inhibin
heterozygous mice. Crossing of these monogenic inhibin
heterozygotes would produce monogenic or bigenic inhibin
null mice.
PCR analysis was also used for determining the genotype at the inhibin
locus. PCR analysis of tail DNA using the primers E22
(5'-GGTCTCCTGCGGCTTTGCGC-3'), E23 (5'-GGCCTCCCGAGGAACCCGCTG-3'), HPRT
(5'-GGATATGCCCTTGACTATAATG-3'), and Intron
(5'-CCTGGGTGGAGCAGGATATGG-3') determined whether the mouse was wild
type, heterozygous, or homozygous null at the inhibin
locus.
Male inh/glvp mice had MFP or vehicle injected ip at the
specified dosages or released from sc 60-day timed release pellets
(Innovative Research of America, Sarasota, FL).
Retroorbital or cardiac blood was collected at the specified time
intervals before or after treatment and incubated at room temperature
for 30 min before isolation of serum in Microtainer tubes (Becton Dickinson and Co., Franklin Lakes, NJ) and stored at -70 C.
Inhibin A levels were determined by using a human inhibin A ELISA assay
kit. FSH levels were determined with a rat FSH EIA assay kit
(Amersham Pharmacia Biotech Inc., Piscataway, NJ).
Inhibin Long-Term Overexpression Studies and Rescue of Inhibin
Null Male Mice
Male and female inh/glvp mice(1821 days old) were
implanted with 60-day timed-release MFP or placebo pellets. In the
rescue experiments, bigenic and monogenic 18- to 21-day-old male
inhibin
null mice were implanted with 60-day timed-release MFP or
placebo pellets. MFP pellets released 6 or 12 µg MFP/day for 60 days
as specified. Mice were weighed weekly and killed after 8 weeks, and
blood, livers, and gonads were harvested. Blood was treated as stated
previously, while livers were weighed, sectioned, and stained with
hematoxylin and eosin. Testes were weighed, placed into 10% buffered
formalin, processed for histology, and stained as described (30 ).
Ovaries were treated similarly to the livers but not weighed. Tumorous
testes were sectioned and stained with hematoxylin and eosin. Inhibin A
levels were determined by using a human inhibin A ELISA assay kit.
Activin A levels were determined by using a human activin A ELISA assay
kit (Serotec Inc., Raleigh, NC).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. J. C. Smith for the generous gift of the
inhibin
cDNA; Drs. Mark M. Burcin, Sherry C. Cipriano, and Simona
Varani for helpful comments and excellent review of the manuscript;
Drs. T. Rajendra Kumar, Jason Yovandich, Dorit Elberg, and Steven Chua
for helpful comments and technical assistance; Blake Abbot, Mei-jin
Chu, Lei Gong, and Elizabeth Hopkins for technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Bert W. OMalley, M.D., Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.
These studies were supported by NIH Grant HL-59314 to S.Y.T. M.M.M.
acknowledges the support of his NIH Grant (CA-60651). T.M.P. is funded
in part by the Edward and Josephine Hudson Scholarship through the
M.D./Ph.D. Program at Baylor College of Medicine.
Received for publication February 2, 2000.
Revision received March 14, 2000.
Accepted for publication March 15, 2000.
 |
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