The Serine/Threonine Transmembrane Receptor ALK2 Mediates Müllerian Inhibiting Substance Signaling
Jenny A. Visser,
Robert Olaso,
Miriam Verhoef-Post,
Piet Kramer,
Axel P. N. Themmen and
Holly A. Ingraham
Department of Physiology (J.A.V., R.O., H.A.I.) Graduate
Programs in Biomedical Sciences (H.A.I.) and Developmental Biology
(H.A.I.) University of California, San Francisco San Francisco,
California 94143-0444
Department of Endocrinology and
Reproduction (M.V.P., P.K., A.P.N.T.) Erasmus
University Rotterdam, The Netherlands
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ABSTRACT
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Müllerian inhibiting substance (MIS or
anti-Müllerian hormone) is a member of the transforming
growth factor-ß family and plays a pivotal role in proper male sexual
differentiation. Members of this family signal by the assembly of two
related serine/threonine kinase receptors, referred to as type I or
type II receptors, and downstream cytoplasmic Smad effector proteins.
Although the MIS type II receptor (MISRII) has been identified, the
identity of the type I receptor is unclear. Here we report that MIS
activates a bone morphogenetic protein-like signaling pathway, which is
solely dependent on the presence of the MISRII and bioactive MIS
ligand. Among the multiple type I candidates tested, only ALK2 resulted
in significant enhancement of the MIS signaling response. Furthermore,
dominant-negative and antisense strategies showed that ALK2 is
essential for MIS-induced signaling in two independent assays, the
cellular Tlx-2 reporter gene assay and the Müllerian
duct regression organ culture assay. In contrast, ALK6, the other
candidate MIS type I receptor, was not required. Expression analyses
revealed that ALK2 is present in all MIS target tissues including the
mesenchyme surrounding the epithelial Müllerian duct.
Collectively, we conclude that MIS employs a bone morphogenetic
protein-like signaling pathway and uses ALK2 as its type I receptor.
The use of this ubiquitously expressed type I receptor underscores the
role of the MIS ligand and the MIS type II receptor in establishing the
specificity of the MIS signaling cascade.
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INTRODUCTION
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Müllerian inhibiting substance (MIS), also known as
anti-Müllerian hormone (AMH), is a member of the large
transforming growth factor-ß (TGFß) superfamily that includes
TGFßs, activins, and the bone morphogenetic proteins (BMPs) (1).
While many TGFß factors exert diverse effects in multiple tissues,
the principle function of MIS is to induce regression of the
Müllerian ducts during vertebrate male sexual differentiation (2, 3). Biologically active MIS is secreted from embryonic Sertoli cells of
the testis and through unknown mechanisms is delivered to the
paramesonephric region in the genital ridge. There, it binds the MIS
type II receptor that is expressed in the mesenchyme surrounding the
epithelial Müllerian duct. MIS signaling results in cell death of
the Müllerian duct, thereby eliminating the female reproductive
tract in males (4, 5, 6). In addition to this well defined role during
male sexual differentiation, more subtle but important roles for MIS in
somatic cell proliferation and gonadal germ cell maturation have been
revealed by genetic experiments in mice (7, 8). For example, loss of
function studies show that male MIS null mice develop Leydig cell
hyperplasia (8). Careful analyses of adult MIS -/- ovaries also
suggest that MIS-deficient ovaries exhibit enhanced primordial follicle
recruitment eventually leading to premature ovarian failure (9).
Finally, gain-of-function studies in mice in which MIS is overexpressed
suggest a putative role for MIS in attenuating steroid production (7, 10). Presently, it is uncertain whether the same molecular machinery
mediates all biological effects of MIS in both the embryo and adult.
However, it is generally accepted that there is only one type II
receptor for MIS in mediating Müllerian duct regression based
on data obtained from both mouse and human mutants (11, 12).
All members of the TGFß family presumably mediate their biological
effects by two related transmembrane serine/threonine kinase receptors,
type II and type I. Extensive studies with some members of the TGFß
family have established that upon ligand binding to type II receptors,
such as the MISRII, type I receptors are recruited to form a
heteromeric receptor complex. The type I receptors are then
transphosphorylated by the type II receptor in the GS box, which is
located close to the transmembrane region in the cytoplasm. Activation
of the type I receptor leads to phosphorylation of receptor-specific
Smad proteins. Phosphorylated Smads associate with the common Smad4 and
are then translocated to the nucleus where they participate in
regulating gene expression (13, 14). Selective recruitment of Smad
proteins by ligand- receptor complexes suggests that two major
signaling pathways exist for members of the TGFß superfamily. In the
first pathway, Smad2 or Smad3 are recruited by both TGFß and activin
through their respective type I receptors, ALK5 (TßRI) and ALK4
(ActRIB). In the second, Smad1, Smad5, or Smad8 are recruited by BMPs
through ALK2, ALK3, and ALK6 or so-called BMP type I receptors (15, 16). Thus far, almost all TGFß-like factors examined appear to use
these two distinct intracellular signaling pathways.
The precise delineation of the MIS signaling pathway has been hampered
because bona fide MIS target genes and a partner type I receptor for
MIS have not been identified. One previous report proposed that ALK6
could serve as the MIS type I receptor based on in vitro
data (17). However, mice deficient in ALK6 survive into adulthood and
appear to show normal Müllerian duct regression (18, 19).
Unfortunately, mouse knockouts of other type I receptors are
uninformative because of embryonic lethality; the early developmental
delays observed in ALK2, ALK3, and ALK4 knockouts (12, 20, 21, 22) preclude
examination of MIS-triggered events, such as Müllerian duct
regression. To address the molecular nature of the MIS signaling
pathway, we surveyed different cell types and potential reporters that
might be useful in dissecting the components of the MIS signaling
pathway. These studies suggested that the mouse embryonic carcinoma
cell line P19 and the mouse Tlx-2 homeobox gene promoter
fused to a reporter gene would be useful in identifying type I receptor
candidates. Here we report that nanomolar concentrations of bioactive
MIS ligand induce a BMP-like signal through Smad1 and Smad5.
Furthermore, our collective data obtained from cell studies, expression
analyses, and antisense studies done in both cells and organ culture
all strongly support the hypothesis that ALK2 is the type I receptor
for MIS signaling in Müllerian duct regression and possibly in
adult gonads.
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RESULTS
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A BMP Response Is Induced by Bioactive MIS Ligand and the
MISRII
Attempts to identify a MIS type I receptor have been hampered by
the lack of known MIS target genes. Moreover, MIS does not activate
commonly used TGFß-responsive reporter genes, such as 3TP-Lux or
PAI-Luc, suggesting that MIS does not signal via the classic TGFß
pathway. To overcome these limitations, we investigated whether MIS
induced a response in P19 mouse embryonic carcinoma cells, which have
been shown to contain both BMP and TGFß/activin signaling pathways.
Two reporters were tested; the BMP responsive mouse Tlx-2
promoter reporter (TLX2-Lux) and the activin responsive Xenopus
Mix.2 gene (A3-Lux) (23, 24). We observed significant induction
(6-fold) of the Tlx-2 promoter after transiently
transfecting P19 cells with the MISRII and MIS stimulation; this
MIS-induced response was completely dependent on transfection of the
MISRII (Fig. 1A
). By contrast, MIS did
not induce the activin-responsive A3-Lux reporter (Fig. 1A
), whereas
activin did (data not shown). MIS induction of the Tlx-2
promoter required the MISRII, whereas introduction of other type II
receptors, such as BMPRII and ActRIIB or TßRII, failed to elicit
MIS-dependent reporter activity (Fig. 1A
). Transfection of a
kinase-inactive MISRII (MISRII-KR), either alone or in the presence of
the wild-type MISRII, completely inhibited MIS-induced Tlx-2
activation (Fig. 1B
). Thus, when coexpressed with wild-type MISRII, the
MISRII-KR mutant served as a dominant-negative mutant receptor. Our
findings are consistent with a previous report showing a 2-fold
activation of another BMP-type promoter (Xvent-Luc) in P19 cells upon
MISRII transfection and MIS stimulation (17).

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Figure 1. MIS Induces MISRII-Dependent Transcription of the
Tlx2 Reporter Gene in P19 Cells
A, P19 cells were transiently transfected with an activin-responsive
(A3-Lux) or BMP-responsive (TLX2-Lux) reporter gene together with the
indicated type II receptors. Cells were incubated overnight in the
presence of conditioned medium (CM, open bars) or
bioactive MIS (closed bars). The relative luciferase
activity was measured in cell lysates and normalized to
ß-galactosidase activity. Data are given as mean ±
SD of triplicates and are representative of at least three
independent experiments. B, P19 cells were transiently transfected with
TLX2-Lux reporter and wild type-MISRII, kinase-inactive MISRII
(MISRII-KR) or a combination of wild-type and mutant MISRII in a 1:1
ratio of expression plamids.
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Using the TLX2-Lux reporter assay in P19 cells, we then
determined the effective concentration of bioactive MIS ligand needed
to activate this reporter. A saturable and dose-dependent profile of
MIS-induced Tlx-2 reporter activity was observed with significant
activity observed at subnanomolar levels. The estimated
ED50 (effective dose) for recombinant bioactive
MIS (MIS-RARR) was determined to be approximately 18 nM MIS
(Fig. 2
); this value corresponds with the
necessary concentration required for full Müllerian duct
regression in organ explant culture assays of 1520 nM
(25). Taken together, these results demonstrate that MIS is capable of
generating a BMP-like signal in P19 cells that does not require
addition of type I receptors, but is solely dependent on the presence
of the MISRII.

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Figure 2. Dose-Dependent Activation of TLX2-Lux by MIS
P19 cells were transiently transfected with TLX2-Lux reporter and
MISRII. Cells were incubated overnight with increasing concentrations
of bioactive MIS ligand (refer to Materials and Methods
for quantitation). To control for potential activating factors present
in conditioned medium, each assay point was normalized with control
conditioned medium. Luciferase activity was determined in triplicate.
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Dominant Negative Smad5, but not Smad2, Disrupts MIS Signaling
MIS-induced activation of a BMP-like signal suggests that MIS
signaling is mediated through BMP receptor-specific Smads, such as
Smad5, Smad1, or Smad8. Therefore, we investigated the effects of
overexpressing representative Smads of either the BMP or TGFß/activin
signaling pathways. Increased reporter activity was observed after
cotransfection of Smad5 or Smad1 and the MISRII (Fig. 3A
and data not shown). In contrast,
Smad2 failed to enhance MIS signaling above the baseline observed with
transfection of only MISRII (Fig. 3A
). To examine this issue further,
we made use of a dominant-negative strategy to block endogenous Smad
recruitment in P19 cells. Mutations of the carboxy-terminal
phosphorylation sites of Smad proteins prevent dissociation from the
activated receptor complex, thereby inhibiting activation
(phosphorylation) of endogenous Smad proteins (26, 27, 28). We found that
elevation of mutant Smad5 (Smad52SA), but not mutant Smad2
(Smad22SA), attenuated MIS-induced activation of TLX2-Lux reporter
(Fig. 3B
). Collectively, these data are consistent with the hypothesis
that MIS signaling uses a BMP-like pathway.

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Figure 3. Smad5 Is Involved in MIS Signaling
A, The TLX2-Lux reporter construct and MISRII expression vector were
transfected into P19 cells with (+) or without (-) wild-type Smad5,
wild-type Smad2. Transcriptional activity was measured after overnight
incubation with conditioned medium (open bars) or
bioactive MIS (closed bars), and normalized for
transfection efficiency. In each transfection, equal amounts of DNA
were transfected by adjusting with empty expression vector. Data are
presented as mean ± SD of triplicates. B, MIS-induced
activity of the TLX2-Lux reporter was measured with (+) or without (-)
MISRII in increasing amounts of mutant Smad52SA, or mutant Smad22SA
(10, 50, 100 ng/well). Data are expressed as fold induction of
luciferase activity measured in the presence of bioactive MIS over
activity measured in the presence of conditioned medium.
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ALK2 Is Involved in MIS Signaling
The results described above suggest that P19 cells contain an
endogenous type I receptor capable of transducing MIS signaling when
the MISRII is present. To test whether one of the previously identified
type I receptors functions as a MIS type I receptor, multiple type I
receptors (ALK16) were cotransfected with TLX2-Lux in P19 cells using
lower concentrations of MISRII (1 ng/well) to observe additional
activation. Under these conditions the strongest activation of the
TLX2-Lux reporter was observed with a MISRII/ALK2 combination.
Surprisingly, other BMP type I receptors known to signal through Smad5,
such as ALK1, ALK3, and ALK6, failed to enhance the MIS response
significantly, and ALK1 and ALK3 actually diminished MIS activation of
the reporter (Fig. 4A
). Western blot
analysis revealed that each type I receptor was expressed at nearly
equivalent levels (data not shown). That ALK2 participates in MIS
signaling was further supported by using a dominant-negative or
kinase-inactive ALK2 mutant receptor (ALK2-KR). Indeed,
increasing amounts of ALK2-KR severely attenuated MIS-induced
activation of TLX2-Lux (Fig. 4B
). Although the dominant-negative
ALK6-KR was previously reported to inhibit Xvent- reporter
activity, in our hands we failed to observe inhibition of MIS-induced
signaling with transfection of this mutant type I receptor (Fig. 4B
and
Ref. 17). Finally, increased activation of the MIS signaling by ALK2
was dependent on addition of biologically active MIS ligand and was not
observed with either conditioned medium or inactive noncleavable MIS
(MIS-RAGA, Fig. 4C
).

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Figure 4. ALK2 Transduces MIS Responses
A, P19 cells were transiently transfected with TLX2-Lux reporter and
MISRII expression vector, either in the absence or presence of type I
receptors (ALK16). Data are expressed as a percent of the maximum
induction observed with transfection of MISRII alone, in the presence
of MIS. The fold induction (+MIS over -MIS) is indicated
above every bar. B, TLX2-Lux was
transfected into P19 cells with MISRII, or MISRII together with
increasing concentrations (10, 50, 100 ng/well) of kinase-inactive ALK2
(ALK2-KR) or kinase-inactive ALK6 (ALK6-KR). Data are expressed as fold
induction as described in legend to Fig. 3 . C, TLX2-Lux was transfected
together with MISRII, ALK2 or MISRII, and ALK2 expression vectors.
Cells were stimulated overnight with conditioned medium (CM;
open bars), inactive (noncleavable) MIS (MIS-RAGA;
gray bars) or bioactive MIS (MIS-RARR; closed
bars). The relative luciferase activity was measured in cell
lysates and normalized to ß-galactosidase activity.
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ALK2 Is Expressed in All the MIS Target Tissues
Although our cell transfection studies suggested that ALK2
functions as a MIS type I receptor, supportive genetic data have been
uninformative due to the early embryonic lethality observed in ALK2
null mice (21, 22). Therefore, we determined whether the expression
profile of ALK2 coincides with the established expression patterns of
MISRII. Based on previous studies regarding expression of the MISRII,
we would expect ALK2 to be present in periductal mesenchymal cells
surrounding the Müllerian duct as well as in both fetal and adult
gonads. Indeed, we noted prominent expression of ALK2 in isolated
urogenital ridges and gonads at several stages of embryonic development
(Fig. 5
, A and B). Interestingly, both
ALK2 and MISRII levels diminished during the window of Müllerian
duct regression, which commences at about E13 in male mice.
Furthermore, similar to MISRII expression, ALK2 was easily detected in
fetal gonads of both sexes, albeit at lower levels in fetal ovaries. By
contrast, the other candidate MIS type I receptor, ALK6, is expressed
at very low levels in both male and female urogenital ridges (Fig. 5A
)
and is barely detectable in embryonic gonads of both sexes (Fig. 5B
).
In situ hybridization analysis of E13 mouse lower body
sections showed more precisely that ALK2 is expressed in mesenchymal
cells adjacent to the concentric Müllerian duct (Fig. 5C
). This
result agrees with the expression pattern of the rat ALK2 (R1) in the
genital ridge reported by Donahoe and co-workers (29). We also noted
prominent ALK2 expression in the embryonic adrenal and liver (Fig. 5C
).
Given that the MISRII is also expressed in postnatal gonads (30, 31, 32),
we determined the transcript levels of both ALK2 and ALK6 in these
tissues. ALK6 mRNA expression was restricted to the ovary with
virtually no signal observed in the testes; whereas ALK2 mRNA is
expressed in both female and male gonads, albeit at a lower level in
the testes (Fig. 5D
). Taken together, our expression studies of ALK2 in
both adult and embryonic reproductive tissues strongly support ALK2s
role as a MIS type I receptor.

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Figure 5. ALK2 Is Expressed in All MIS Target Tissues
Levels of ALK2, ALK6, and MISRII mRNAs were measured in embryonic mouse
urogenital ridges (A) and gonads (B) at various fetal ages by RNase
protection assay. For staging of fetuses, refer to Materials and
Methods. C, In situ hybridization analysis shows
expression and localization of ALK2 mRNA in the urogenital ridge in an
E13 male mouse. A transversal hematoxylin-eosin-stained section of the
lower body is shown (left panel) along with a darkfield
view (right panel). Sites of ALK2 expression are
indicated by white arrows. Abbreviations for structures
are as follows: A, adrenal; L, liver; T, testis, and M, the
Müllerian duct as outlined by a white broken
circle in left panel. The scale bar
in the left panel equals 100 µm. D, Adult testis (T)
and adult ovary (O) levels of ALK2, ALK6, and MISRII mRNAs were
measured by RNase protection assay. GAPD mRNA measurements were
included as a control for RNA loading.
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Antisense ALK2 Blocks MIS Signaling in Cells and in Organ
Cultures
To determine whether endogenous ALK2 in P19 cells is capable of
conferring MIS-induced activity in the presence of the MISRII, an
antisense approach was undertaken to disrupt ALK2 protein expression.
P19 cells were treated with morpholino antisense oligomers 24 h
before cotransfection with TLX2-Lux and MISRII. The efficiency of
morpholino oligomers resides in their prolonged stability and the
efficient delivery system [ethoxylated polyethylenimine solution
(EPEI)] in which almost 100% of the cells transfected take up these
morpholino oligomers (Ref. 33 and Fig. 6A
). Treatment of cells with an antisense
ALK2 oligomer greatly diminished MIS-induced activation of TLX2-Lux to
near basal levels (Fig. 6B
). Incubation of cells with the delivery
solution without antisense oligomers or with a control antisense ALK2
oligomer showed no inhibition of MIS-induced activity. The ALK2 control
oligomer was identical to ALK2 except for four nucleotide mismatches.
In contrast to the marked inhibition observed with the antisense ALK2
oligo, no inhibition of TLX2-Lux was observed using an ALK6 antisense
oligomer (Fig. 6B
). However, treatment of P19 cells with the ALK6
antisense oligomer decreased BMP4 signaling by approximately 50% (data
not shown). Collectively, our findings suggest that MIS signaling is
mediated through an endogenous ALK2 type I receptor present in P19
cells.

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Figure 6. Antisense ALK2 Oligomers Abrogate MIS Signaling in
P19 Cells and in Urogenital Ridges
A, P19 cells were treated for 3 h with morpholino antisense
oligomers using the special delivery system (EPEI). Cells were
photographed with normal light (left panel) and with
fluorescent light (right panel). Note that nearly all
cells are fluorescent, indicating the efficient uptake of the
fluorescent oligomer. B, Cells were transiently transfected with
TLX2-Lux reporter and MISRII expression vector 24 h after
treatment with antisense ALK2 oligomer (as-ALK2), mispaired ALK2
oligomer (4 m-ALK2), antisense ALK6 oligomer (as-ALK6), or delivery
solution (EPEI). Luciferase activity was measured after treatment with
conditioned medium (CM, black bars) or bioactive MIS
(green bars), and normalized for transfection
efficiency. C, Representative urogenital organ explants are shown after
3 days of culture in the presence of MIS after treatment with either an
antisense ALK2 oligomer (as-ALK2), a control antisense ALK2 oligomer (4
m-ALK2), an antisense ALK6 oligomer (as-ALK6), or the delivery solution
(EPEI); a persistent Müllerian duct (MD) was observed with
antisense ALK2 oligomer and MIS treatment (left panel).
Each condition was carried out in four independent experiments with an
n = 4 or more. Also shown is a urogenital organ culture treated
with ALK2 antisense and cultured for 3 days in absence of MIS
(right panel). Arrows indicate the
Müllerian duct (MD) and Wolffian duct (WD) in each panel and the
ovary (Ov) is indicated in the far left panel. The
aterisk indicates residual Müllerian duct.
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To assess whether ALK2 mediates MIS signaling in vivo, a
similar antisense approach was used in the Müllerian duct
regression assay. In the absence of MIS, female urogenital ridges
exhibit both the Müllerian (MD) and Wolffian (WD) ducts. In the
presence of MIS, regression of the Müllerian duct occurs leaving
only a visible Wolffian duct. Treatment of urogenital ridges with the
antisense ALK2 oligomer partially or fully blocked MIS-induced
regression (Fig. 6
, left panel, as-ALK2). In contrast,
treatment of the urogenital ridges with the control antisense ALK2
oligomer or the antisense ALK6 oligomer did not block MIS- induced
regression (Fig. 6
, m4-ALK2, as-ALK6). Furthermore, MIS-induced
regression was unaffected by the special delivery reagent as judged by
the normal loss of the Müllerian duct after incubation with MIS
and the EPEI solution (Fig. 6
, EPEI). In all cases, addition of
morpholino oligomers alone had no effect on duct morphology (Fig. 6
, right panel, no MIS, as-ALK2). These results strongly suggest that
ALK2 mediates MIS-induced signaling in Müllerian duct
regression.
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DISCUSSION
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The molecular signaling mechanisms of some TGFß superfamily
members have been studied in great detail, and it is now established
that at least two distinct signaling pathways exist. The first of these
is a Smad2 and 3 signaling pathway shared by TGFß and activin, and
the second is a Smad1 and 5 signaling pathway shared by the BMP family
(14). Here, we report that MIS activates a BMP-like pathway using Smad1
or Smad5 and requires bioactive MIS and the MIS type II receptor.
Moreover, our cellular studies and antisense experiments indicate that
the type I receptor that bridges the MISRII and Smad intracellular
signaling is ALK2.
The best known function of MIS is induction of Müllerian duct
regression in male fetuses (2, 3) where MIS induces apoptosis in the
epithelial layer of the Müllerian duct by activating a signaling
cascade in the mesenchymal layer (4). Although Müllerian ducts
normally differentiate in female mice because MIS synthesis is
restricted to embryonic testes, female Müllerian ducts are
competent to respond to MIS in vitro and in vivo
(8, 25). Indeed, several groups have established expression of MISRII
in the mesenchyme surrounding the Müllerian duct of both sexes
(4, 5, 6, 32). Here we report that the expression of ALK2 correlates well
with the known expression pattern of the MISRII and is found in the
urogenital ridge of male and female fetuses. In situ
hybridization analysis revealed expression of ALK2 in the mesenchymal
cells surrounding the Müllerian ducts. Interestingly, Smad5 is
also expressed at a relatively high level in the mesenchymal cells
surrounding the Müllerian duct (34). Most important, we believe
that ALK2 plays an important role in Müllerian duct regression
based on the blockage of duct regression after ALK2 antisense treatment
in organ cultures. However, it is clear from our study and previous
reports that ALK2 is not restricted to reproductive tissues. Despite
the role of ALK2 in MIS signaling, the specificity and propagation of
this signaling pathway are conferred by the MIS ligand and the MIS
type II receptor and their subsequent association.
Our studies demonstrate that MIS signaling in P19 cells is mediated
through an endogenous type I receptor. Interestingly, P19 cells express
multiple endogenous BMP type I receptors, such as ALK2, ALK3, and ALK6,
as well as endogenous activin type I receptor (ALK4) (35). In these
cells, the identity of this endogenous BMP type I receptor involved in
MIS signaling is likely to be ALK2. Consistent with this hypothesis,
MIS signaling increased after cotransfection of ALK2 and was decreased
in either the presence of a dominant-negative ALK2 or after treatment
with antisense ALK2 oligomers. Although we found that other type I
receptors were unable to significantly enhance MIS responsiveness in
P19 cells, we noted that ALK2, ALK5, and ALK6 are all capable of
forming a receptor complex with MISRII in vitro (data not
shown). Interestingly, formation of these receptor complexes was
ligand-independent, and addition of ligand showed no further increases
in type I receptor recruitment by the MISRII. The lack of
ligand-induced ALK2 recruitment could suggest that very few MISRII/ALK2
receptor complexes are needed for signal transduction and thus these
low levels would fall below the limits of detection. Alternatively, the
MIS ligand may induce a conformational change in the MISRII/ALK2
receptor complex rather than simply increasing their association.
Whether all of these MISRII-type I receptor complexes are biologically
relevant or merely reflect overexpression in a heterologous system
remains uncertain. Nonetheless, our functional data suggest strongly
that a MISRII/ALK2 receptor complex is capable of transducing the MIS
signal.
It is plausible that additional type I receptors or other plasma
membrane proteins contribute to MIS signal transduction by their
association with the MISRII/ALK2 receptor complex. For instance, ALK6
may indeed be part of the MIS signal transduction complex, as
previously suggested, but at least in our system, ALK2 appears to be
the essential signaling type I receptor. In contrast to a previous
study, which suggested that MIS signals by recruiting ALK6 (17), we
failed to observe any attenuation of MIS-induced signaling after
overexpression of a dominant-negative ALK6-KR or treatment with
antisense ALK6 oligomers. Potential reasons for this discrepancy may
rest with the reporter used or the amounts of mutant type I receptor
used in each study. In our hands, significant inhibition of the
Tlx-2 reporter was noted at a ratio of 1:10, MISRII:ALK2-KR.
Collectively our data suggest that a role for ALK6 in the MIS signaling
cascade will be less straightforward than previously proposed.
While a clearly defined role for MIS in adult reproduction remains to
be established, the apparent modulation of gonadal cell function by MIS
infers the presence of an active signaling pathway(s) in these tissues.
In addition to a developmental role for MIS in Leydig cell
proliferation, several groups also report that MIS dampens
steroidogenesis in the ovary and testis by repressing transcription of
key steroidogenic enzymes (7, 10, 36, 37). Whether steroidogenic
enzymes are directly downstream of MIS signaling is still uncertain,
given that responses in steroidogenic cell lines are not robust and
require MIS treatment for a minimum of 2 days (Ref. 36 and our
unpublished results). Nevertheless, gonadal expression of both MISRII
and ALK2 in the embryo and adult suggests that, similar to the
urogenital ridge, a competent MISRII/ALK2-receptor complex is able to
function in these reproductive tissues. Likewise, the strong ALK6
expression in the adult ovary (Fig. 5D
) raises the possibility that a
MISRII/ALK6 complex could function in the adult female. Clearly,
identification of MIS target genes may elucidate the full physiological
relevance of MIS in gonadal function and may also reveal its potential
interplay with additional TGFß signaling pathways in reproductive
function.
Our findings imply that MIS signaling activates a BMP-like pathway
through ALK2 and Smad5. Presently, our studies are unable to exclude
the possibility that novel type I receptors play a role in MIS
signaling. The ability to conditionally ablate ALK2 in mice
in the genital ridge would circumvent the embryonic lethality at E9.5
during the early stages of gastrulation (21, 22) and should provide
definitive data as to its role in mediating MIS signaling during
Müllerian duct regression. Obviously, an ideal promoter to
execute this in vivo genetic strategy is the MISRII
promoter. It will also be of interest to determine whether similar
factors known to modulate ligand availability and ligand binding
(i.e. follistatin, inhibin binding proteins) also function
in the MIS signaling pathway. Continued investigation of this signaling
cascade should provide new insights into the molecular mechanisms of
developmental and adult reproductive physiology.
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MATERIALS AND METHODS
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Plasmids
The full-length rat MISRII cDNA was isolated from E14 genital
ridges by RT-PCR using the following primers: sense,
5'-GACGAATTCCTTTAGTAGGATGCTG-3'; antisense, 5'-CC G
GTCGACGGACTTAGAGCCAGAGCC-3'. The sequence of this MISRII cDNA was
verified by comparison to the published sequence (6) and subcloned into
EcoRI/SalI sites of pRK5 containing a
carboxy-terminal Flag epitope tag. Type I receptor expression vectors
were provided by Dr. J. Massagué (Sloan Kettering, New York, NY;
human ALK1, mouse ALK3, and mouse ALK6), Dr. R. Derynck (University of
California, San Francisco, CA; mouse ALK2 and rat ALK5), and Dr. L.
Mathews (University of Michigan, Ann Arbor, MI; human ALK4) (38, 39, 40).
Kinase inactive mutants, MISRII-K228R, ALK2-K235R, and ALK6-K230R, were
generated by PCR-based mutagenesis strategy. TLX2-Lux and A3-Lux
reporter genes and human Smad1 and human Smad2 expression vectors were
provided by Dr. J. Wrana (Samuel Lunenfeld Research Institute, Toronto,
Ontario, Canada) (23, 24, 26, 41). Mouse Smad5 expression vector was
provided by Dr. X. Wang (Duke University, Durham, NC) (42), and human
Smad2-SA and mouse Smad52SA constructs were provided by Dr. P. ten
Dijke (The Netherlands Cancer Institute, Amsterdam, The Netherlands)
(28, 43). The human BMPRII, mouse ActRIIB, and human TßRII were
provided by Dr. M. Kawabata (Cancer Institute, Tokyo, Japan) (44), Dr.
L. Attisano (University of Toronto, Ontario, Canada) (45), and Dr. R.
Derynck (46), respectively.
Cell Culture, Transfections, and Luciferase Assays
P19 cells were cultured in
-MEM containing 7.5% calf serum
and 2.5% FBS. For MIS-induced luciferase assays, P19 cells were seeded
at 20% confluency in 12-well plates and transfected with TLX2-Lux or
A3-Lux reporter plasmid (100 ng/well) and the indicated receptor
expression vectors (25 ng/well) using Fugene 6 transfection reagent
(Roche Molecular Biochemicals, Indianapolis, IN).
Twenty-four hours after transfection, cells were cultured for 2 h
in medium containing 0.2% serum followed by 16 h treatment with
the MIS ligand (15 nM). Luciferase was measured using the
luciferase assay system (PharMingen, San Diego, CA) in a
Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). In all transfections,
ß-galactosidase expression plasmid (pCMV5-ßGal) served as internal
control to normalize for transfection efficiency.
MIS Preparation and Enzyme-Linked Immunosorbent Assay (ELISA)
Recombinant bioactive (MIS-RARR) and inactive (MIS-RAGA)
conditioned medium were obtained from stably transfected HEK-293S cells
expressing these rat MIS cDNAs as described previously (25).
Conditioned medium of wild-type HEK-293S cells served as the control.
Collected media were concentrated approximately 30-fold using a
Centriprep system (Millipore Corp., Bedford, MA). The
integrity of recombinant MIS protein was determined by Western blot
analysis using purified antirat MIS antibody (25), and the amount of
MIS was measured by an in-house ELISA assay. The standard curve was
generated using purified MIS at concentrations ranging from 0.0251
µg/ml. Purified MIS or MIS- conditioned medium were serially
diluted in coating buffer (0.015 M
Na2CO3, 0.035 M
NaHCO3, pH 9.6) overnight at 4 C in a 96-well
Immulon plate (Nunc, Rochester, NY), followed by incubation with
blocking buffer (3% BSA, 0.02% NaN3 in PBS) for
2 h at room temperature. Plates were incubated overnight at 4 C
with a polyclonal MIS rabbit antibody (1:250 dilution) (25). The
following day, wells were washed with PBST (0.1% Tween in PBS) and
incubated with biotinylated conjugated donkey antirabbit IgG
(Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA) (1:500 dilution) for 1 h at room temperature. After
extensive washing with PBST, Streptavidin-peroxidase conjugate
(Caltag Laboratories, Inc. Burlingame, CA) was added
(1:500 dilution) for 30 min at room temperature. Wells were washed with
PBST and incubated with BM blue (Roche Molecular Biochemicals) to visualize the complex. The reaction was stopped
by adding 100 µl 1 M
H2SO4, and the optical
density was measured at 490 nm on a reader plate (BioTek,
Winooski, VT). The molar concentrations of our recombinant HIS-tagged
MIS were calculated based on an apparent molecular mass of 155
kDa as judged by SDS-PAGE in the absence of ß-mercaptoethanol.
Morpholino Antisense Oligo Treatment and Regression
Assays
Special delivery morpholino antisense oligos (33, 47) were
designed for ALK2 (as-ALK2: 5'-CTCCATCGACCATTGTATAACC-3') and ALK6
(as-ALK6: 5'-ATTTTCCAGAGC-TTCGTAAGAGCAT-3') with guidance by Dr. Paul
Marcos (Gene Tools LLC, Corvallis, OR). To create a control oligo, 4
mispairs were introduced into the antisense ALK2 oligo (4 m-ALK2:
5'-CTgCATgGACCATTGaATAtCC-3'). Antisense morpholino oligos were mixed
with a delivery reagent, EPEI, according to the manufacturers
directions (Gene Tools) for 20 min incubation at room temperature. A
complete delivery solution containing 1.4 µM of oligo was
added to P19 cells for 3 h and medium was replaced with fresh
medium. Transfection and ligand treatment were performed (as described
above) 24 h after antisense oligo treatment. Female rat urogenital
ridges were dissected from E14.5 embryos and incubated for 3 h
with the delivery solution containing 1.4 µM of
morpholino antisense oligos (as described above). Treated organs were
cultured on MilliCell-CM biopore membranes (Millipore Corp.) floated in 0.2 ml medium as previously described (4, 25),
in the presence or absence of MIS (32 nM) for 3 days.
RNAse Protection Assay and in Situ Hybridization
To obtain embryos for RNA protection assays and in
situ hybridization studies, pregnant FVB mice were killed by
cervical dislocation on embryonic (E) day 13, E14, E15, or E18; vaginal
plug detection was considered to be E0. Fetal tissues were isolated and
snap frozen in liquid nitrogen and stored at -80 C. Fetal sex was
determined by PCR using placental genomic DNA, and total RNA was
isolated as previously described (48). An
EcoRI-HindIII fragment containing 1472 bp of
the mouse ALK2 cDNA, and a HpaI-ApaI fragment
containing 79551 bp of the mouse ALK6, both encoding the
extracellular domain, were subcloned in pBKS and used to generate
[32P]-UTP-labeled antisense RNA probes. Mouse
MISRII and control glyceraldehyde 3-phosphate dehydrogenase (GAPD)
antisense RNA probes were generated (6) and used in RNase protection
assays using 10 µg of total RNA as described previously (48).
In situ hybridization analyses was performed as described
previously (6) on tissue sections fixed overnight in Bouins fixative,
embedded in paraffin, and sectioned transversally at 8 µm. The same
EcoRI-HindIII fragment of mouse ALK2 cDNA was
used to generate sense and antisense
[35S]-UTP-labeled transcripts. Mice and rats
were housed in accordance with NIH guidelines.
Note Added in Proof
Similar findings showing the use of ALK2 in the MIS signaling
pathway are reported in the following article (pp. 946959) by
Clarke et al. (19A ).
 |
ACKNOWLEDGMENTS
|
---|
We are especially grateful to all investigators for providing
plasmids as noted in Materials and Methods. We also wish to
thank Dr. Carol Basbaum for technical advice on aspects of this
project.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Holly A. Ingraham, Department of Physiology, University of California, San Francisco, 513 Parnassus, San Francisco, California 94143-0444. E-mail: hollyi{at}itsa.ucsf.edu
This investigation was supported by Californian Division-American
Cancer Society Fellowship 21-00 (to J.A.V.), The French ARC
Foundation (Postdoctoral Fellowship to R.O.), and NIH-NICHD (Grant RO1,
RCDA to H.A.I.).
Received for publication December 29, 2000.
Revision received February 2, 2001.
Accepted for publication February 9, 2001.
 |
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