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Laboratoire de Génétique Moléculaire Institut de Recherches Cliniques de Montréal Montréal Québec Canada H2W 1R7
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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GSU (glycoprotein hormone -subunit gene)
and ßFSH promoters. Synergism between these two classes of factors is
reminiscent of the interaction between the products of the
Drosophila genes Ftz (fushi tarazu) and Ftz-F1. Antisense
RNA experiments performed in T3–1 cells that express the GSU
gene showed that expression of endogenous GSU is highly dependent on
Ptx1 whereas many other genes are not affected. Interestingly, the only
other gene found to be highly dependent on Ptx1 for expression was the
gene for the Lim3/Lhx3 transcription factor. Thus, these experiments
place Ptx1 upstream of Lim3/Lhx3 in a cascade of regulators that appear
to work in a combinatorial code to direct pituitary-, lineage-, and
promoter-specific transcription. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit gene (GSU) is the first hormone
subunit to be expressed in the developing mouse pituitary on embryonic
day 11.5 (e11.5), followed by anterior pituitary POMC on e12, TSH on
e14, intermediate lobe POMC for MSH synthesis on e14.5, GH and PRL on
e15.5, LH on e16.5, and FSH on e17.5 (2, 3, 4, 5). During early development, the pituitary anlage, Rathke’s pouch, develops from a placode of the stomodeum, which itself is derived from the cephalic ectoderm of the anterior neural ridge (6, 7). Rathke’s pouch is first identified in mouse at e8.5 as an invagination of the oral epithelium that is in contact with the floor of the diencephalon (8). The posterior lobe of the pituitary arises simultaneously from a downward evagination of the diencephalic neuroectoderm, the infundibulum (8). Contact between Rathke’s pouch and the ventral diencephalon is crucial for further pituitary development (9, 10, 11, 12). For example, the TTF-1 (Nkx2.1,T/ebp) gene is essential for pituitary development although not expressed in Rathke’s pouch (12). Rather, TTF-1 is expressed in the neuroepithelium that will later give rise to the hypothalamus and to the infundibulum (12). Mice lacking this gene not only fail to develop the posterior lobe but also the anterior and intermediate pituitary lobes (12), confirming the importance of the interaction between diencephalon and Rathke’s pouch for proper pituitary development. Around e12.5, Rathke’s pouch pinches off from the oral ectoderm, and intense cell proliferation (e12.5-e14) triggers the formation of the anterior pituitary gland (8, 13).
The factors involved in the early events of pituitary development are just beginning to be identified. We previously cloned a homeoprotein, Ptx1 (pituitary homeobox 1), through its ability to bind and activate the POMC gene (14). Ptx1 expression precedes Rathke’s pouch formation as it is expressed in the stomodeum from its first appearance (15) and later maintained in all stomodeal derivatives, including Rathke’s pouch and the pituitary. Another recently reported Ptx family member, Ptx2 (Rieg) (16, 17, 18) is also expressed at this early stage of pituitary development (18). Thus, Ptx1 and Ptx2 represent the earliest known genetic markers for pituitary development.
The homeobox gene Rpx (Hesx1) is transiently expressed in the developing pituitary from e9 to e14.5 (19, 20). The precise function of Rpx remains unknown, since no target gene has yet been identified (19). However, Rpx can heterodimerize with Prop-1 (see below) and thus interfere with Prop-1-dependent activation of the Pit1 gene (21). Transgenic mice continuously expressing Rpx have hypoplasic pituitaries suggesting that the extinction of Rpx is essential for proper pituitary development (K. Mahon, personal communication). Lim3/Lhx3, a lim-homeodomain protein, which is expressed from e9.5 onward (22, 23) has recently been shown to be required for normal pituitary development since targeted ablation of its gene results in blockade of cell proliferation or survival at the Rathke’s pouch stage and prevents subsequent lineage specification (24). In these animals, Rpx gene expression is prematurely decreased implying that Lim3/Lhx3 is required for maintenance of Rpx gene expression (24). The Lim3/Lhx3 transcription factor may also take part in expression of pituitary hormone-coding genes (22).
Prop-1 is a recently identified homeoprotein that is transiently expressed during pituitary development (e10–10.5 to e14.5) where it stimulates the Pit1 gene, a member of the POU family of transcription factors (21). Insufficient Pit1 gene expression, caused by a mutation in the Prop-1 gene, is responsible for the Ames dwarf phenotype in which there is severe depletion of three Pit1-dependent lineages: the somatotropes, lactotropes, and thyrotropes (25). Moreover, Prop-1 seems to be required for extinction of the Rpx gene since Rpx expression persists through e18.5 in Prop-1-deficient mice (21, 26). Pit1 is first detected at e14 in the developing mouse pituitary (27). As indicated above, it is required for differentiation and maintenance of thyrotrope, somatotrope, and lactotrope cell lineages (28, 29, 30). Pit1 is an important transcription factor required for the expression of the GH, ßTSH, and PRL genes, and it also activates its own expression (29, 31, 32, 33, 34).
In the present study, we have defined the role of Ptx1 in
pituitary-specific transcription and its position in the regulatory
cascade of genes that direct pituitary development. Indeed, we show
that Ptx1 is essential for expression of the GSU and Lim3/Lhx3
genes, thus identifying Ptx1 as the earliest regulator of pituitary
transcription.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, a single Ptx1 RNA band of about 2.5 kb
was revealed in AtT-20 cells, a corticotrope cell line that expresses
POMC and from which we had cloned Ptx1 (14). Ptx1 mRNA was also
detected in several pituitary-derived cell lines including T3–1
(gonadotrope precursor), TSH (thyrotrope precursor), GHFT1.5
(somatolactotrope precursor), and GH3 and
GH4C1 (somatolactotropes), as well as in the
thyrotrope tumor TtT-97 and in adult mouse pituitary. Interestingly,
Ptx1 mRNA levels were much higher in T3–1, TSH, and GHFT1.5 than
in AtT-20 cells. These results confirm the presence of Ptx1 mRNA in
cells other than corticotropes.
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, the Ptx1 and Ptx2 genes are abundantly transcribed in pituitary cells
whereas Ptx3 is not. In some cell lines, such as AtT-20, T3–1, and
GHFT1.5, Ptx1 is the major mRNA species, whereas in others such as GC
and MMQ, Ptx2 mRNA predominates (Fig. 1).
The presence of Ptx1 mRNA does not necessarily imply synthesis of Ptx1
protein, as Pit1 mRNA, for example, is detectable in more pituitary
cells than those that express the protein (3, 27). To determine whether
Ptx1 protein was present in all pituitary-derived cell lines, we
performed Western blot analysis with a specific antiserum raised
against Ptx1 amino acids 24–56. As shown in Fig. 2, all cell lines tested, as well as the
adult mouse pituitary, contain Ptx1 protein. Overall, there is a good
correlation between the level of mRNA and amount of protein (Figs. 1 and 2), although some discrepancies are noted and discussed below.
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, several pituitary-specific promoters
or enhancers contain at least one putative Ptx1-binding site. These
sites were identified by comparison to the Ptx1-binding site of the CE3
element of the POMC promoter and by comparison to binding studies with
bicoid-related homeoproteins (36, 37, 38). To test the ability
of Ptx1 to activate pituitary-specific promoters, a Ptx1 expression
vector was transfected along with various reporter constructs in CV-1
cells. As shown in Fig. 3, Ptx1
significantly activates several pituitary promoters, including those
for GSU and the ß-subunits of LH (ßLH), FSH (ßFSH), and TSH
(ßTSH), GnRH receptor (GnRH-R), GH, the Pit1 enhancer but not its
promoter, as well as the POMC promoter (14). The rous sarcoma virus
(RSV) promoter, which was used as a negative control, was not activated
by Ptx1. Similarly, the thymidine kinase promoter was insensitive to
Ptx1 (data not shown).
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GSU and ßLH
PromotersGSU and ßLH
promoters and of its high expression in GSU cells (Fig. 2 and
below), we performed deletion analyses on the GSU as well as on the
ßLH promoters to identify Ptx1-responsive sequences. As shown in
Table 1, the GSU and ßLH promoters contain several putative
Ptx1-binding sites. A short (-120 bp) GSU promoter that contains
only one putative Ptx1-binding site was still activated by Ptx1,
whereas a deletion to -65 bp, which removes this site, was no longer
significantly activated (Fig. 4). Similar
results were obtained with the ßLH promoter (Fig. 4) where removal of
the most proximal putative Ptx1-binding site led to a loss of Ptx1
activation (Fig. 4). Taken together, these data suggest that
transactivation of both the GSU and ßLH promoters by Ptx1 is
likely to be mediated by the most proximal Ptx1-binding site. These
sites are conserved across many species (Table 1). We cannot exclude
the possibility that more distal sites may also contribute to promoter
activity in an in vivo context or in association with other
transcription factors.
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Previous studies have reported the role of the orphan nuclear receptor
SF-1 in activation of the ßLH promoter (40, 41). In the pituitary,
this nuclear receptor is only expressed in gonadotrope cells (Refs. 42
and 43 and data not shown). As shown in Fig. 5A, SF-1 and Ptx1 can each individually
activate the ßLH promoter. Coexpression of both factors resulted in a
strong synergistic activation of the ßLH (-776 bp) promoter (Fig. 5A). The SF-1/Ptx1 synergism was lost when the SF-1-binding site
(located at -120 bp) was deleted from the promoter as in the -104-bp
ßLH promoter construct (Fig. 5A); the SF-1- and Ptx1-binding sites
are 20 bp apart in the promoter. It was also suggested that SF-1 might
be implicated in expression of the GSU and ßFSH genes (42, 44) and
Ptx1 can activate both promoters (Fig. 3). No synergy, however, was
observed between Ptx1 and SF-1 on these two promoters (Fig. 5A). The
combination of Ptx1, SF-1, and Lim3/Lhx3 did not result in a stronger
synergy on the ßLH promoter (data not shown).
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), can be synergistically activated
by Ptx1 and Pit1 (Fig. 5B). The same was true for the GH promoter
although to a lesser extent (Fig. 5B). This interaction between Ptx1
and Pit1 was not observed on all Pit1-dependent promoters since the
ßTSH promoter and Pit1 promoter/enhancer were not synergistically
activated by the two factors (Fig. 5B). Taken together, these
results indicate that Ptx1 exerts promoter-specific effects by
synergism with cell type-restricted transcription factors.
Ptx1 Protein Is Expressed at High Level in GSU-Expressing
Cells
To correlate Ptx1 expression in cell lines derived from various
pituitary lineages (Figs. 1 and 2) with normal pituitary cells, we used
double-labeling immunohistochemistry to analyze Ptx1 expression in the
adult pituitary gland. As shown in Fig. 6, the Ptx1 protein can be detected in
the nuclei of all pituitary cells. The nuclear signal was not detected
with preimmune serum, and it was competed by addition of
maltose-binding protein (MBP)-Ptx1 but not with MBP-ßGal (data not
shown). Interestingly, all cells do not express Ptx1 at the same level,
as was observed previously for Ptx1 mRNA (14). Many strongly positive
cells for Ptx1 were identified as GSU-expressing cells by
double-labeling immunohistochemistry (Fig. 6). This result correlates
with expression in pituitary-derived cell lines (Fig. 2). In
addition, high Ptx1 expression colocalized with GSU-expressing
cells from the onset of GSU expression during pituitary development
(C. Lanctôt and J. Drouin, in preparation).
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GSU-Expressing
T3–1 Cells and 6), T3–1 cells as
well as mouse pituitary GSU-positive cells contain high levels of
Ptx1 protein. Ptx2 mRNA was also detected in the developing pituitary
and in some pituitary-derived cell lines including T3–1 cells (Fig. 1 and Ref.16). We do not yet know whether these cells contain Ptx2
protein. To determine the relative importance of Ptx1 protein in
T3–1 cells, we performed supershift experiments using nuclear
extracts from T3–1 cells. The Ptx1 antibody used in our experiments
(Fig. 2) is specific for Ptx1 since it did not recognize Ptx2 in
Western blot (Fig. 7A, lane 3) or in gel
shift assays (Fig. 7B, lane 4). The Ptx-binding activity present in
T3–1 nuclear extracts (Fig. 7C, lane 2) was almost completely
supershifted by saturating amounts of the Ptx1-specific antibody (Fig. 7C, lane 3). Taken together, these data clearly demonstrate that Ptx1
is by far the most abundant member of the Ptx family in T3–1
cells.
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GSU and Lim3/Lhx3 Gene ExpressionT3–1 cells have been considered as a model of
gonadotrope precursors because they express the GSU and GnRH-R genes
but none of the ß-subunit genes (46). This cell line contains the
highest level of Ptx1 mRNA and protein (Figs. 1, 2, and 7). Moreover,
Ptx1 strongly activated the GSU promoter (Figs. 4 and 5). To further
confirm the role of Ptx1 in GSU gene expression, we generated Ptx1
knockdown cell lines by stably transfecting a Ptx1 antisense RNA
expression vector in T3–1 cells. Three independent
neomycin-resistant clones expressing the Ptx1 antisense RNA were
analyzed. As control, clones stably transfected with the same vector
without the Ptx1 cDNA (empty vector) were generated, and one was chosen
as negative control (clone Ctl) along with the wild-type T3–1 cells
(WT). In the three Ptx1 antisense clones (8, 9, 13), endogenous
Ptx1 mRNA (Fig. 8A, upper
panel) and protein as assessed by DNA-binding assay (Fig. 8A, lower panel) were markedly decreased, whereas other
transcription factors such as Pan1 and Oct1 were not significantly
affected, neither at the mRNA (Fig. 8B, upper panel) nor at
the protein (DNA binding in electrophoretic mobility shift assay)
levels (Fig. 8B, lower panel). GATA DNA-binding activity
might be slightly decreased (Fig. 8B). The fact that Ptx DNA-binding
activity was almost undetectable in the antisense clones (Fig. 8A) also
suggests that the Ptx2 gene product was not up-regulated in response to
the decrease in Ptx1. These Ptx1 null cell lines were then used to
analyze the level of GSU gene expression. As shown in Fig. 9A (top panel), no or little
GSU mRNA was detected in the antisense clones. This clearly
indicates that the endogenous GSU gene was almost silent in these
cells. Further, the activity of a transfected GSU-luciferase
reporter is considerably lower in the antisense clones than in control
cells (data not shown). Thus, Ptx1 is essential for GSU gene
expression.
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T3–1 cells. The GnRH-R,
another differentiation marker of the gonadotrope lineage, was also
decreased in these cells, although much less so than GSU. There
might be a very small decrease of Six3 mRNA levels in the antisense
clones compared with the control, but the fold reduction was even less
than for the GnRH-R. The gonadotrope-restricted transcription factor
SF-1 and the lim factor LH-2 were not affected in the Ptx1-antisense
clones (Fig. 9A). Similarly, neither the low-level Ptx2 expression nor
Pax6 (47) mRNA levels were altered in the antisense clones (Fig. 9B).
These data do not support any cross-regulation between Ptx1 and Ptx2
gene expression. The Rathke’s pouch marker Rpx was not detected in the
antisense or control cells (data not shown). Strikingly, the Lim3/Lhx3
mRNA was basically undetectable in the antisense clones, suggesting
that, as for the GSU gene, Ptx1 is essential for Lim3/Lhx3 gene
expression. These experiments clearly indicate an essential role of
Ptx1 in control of Lim3/Lhx3 transcription and place Ptx1 upstream of
Lim3/Lhx3 in the regulatory cascade for pituitary development (Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Differential Expression of Ptx1 and Ptx2 in Pituitary Lineages
The Ptx1 and Ptx2 genes have an overlapping pattern of expression
in the stomodeum and in some of its derivatives with differences of
expression in craniofacial mesenchyme (15, 17, 18). Both are also
expressed in the pituitary primordium, Rathke’s pouch, and we have
shown in the present work that both are expressed in the adult mouse
pituitary gland as well as in a panel of pituitary-derived cell lines.
Taken together, these lines are representative of many pituitary
lineages captured at specific moments of their differentiation pathway
(48). We have shown that all these cell lines but one, the
POMC-expressing AtT-20 cell, expressed both Ptx1 and Ptx2 mRNA (Figs. 1 and 2). Although, in general, there is good correlation between Ptx1
mRNA and protein levels, one exception is GHFT1.5 cells in which Ptx1
mRNA levels are similar to those of T3–1 cells, whereas protein
levels are much lower (compare Figs. 1 and 2). Another discrepancy may
exist in T3–1 cells that have both Ptx1 and Ptx2 mRNA but the bulk
of Ptx protein appears to be Ptx1 (Fig. 7). The remainder of the
Ptx-related DNA-binding activity in those cells may be a N-terminal
variant of Ptx1, Ptx1b, that is not recognized by the antiserum used in
these experiments (J. J. Tremblay and J. Drouin, in preparation).
This discrepancy in mRNA and protein levels may be explained by
posttranscriptional regulation. At present, we do not know of any other
Ptx family member expressed in the pituitary and, as shown, the Ptx3
gene (35), which has an almost identical homeodomain, is not expressed
in this tissue (Fig. 1 and data not shown). Thus, Ptx1 appears to be
the only Ptx family member expressed in corticotropes and the
predominant one in gonadotropes.
A pan-Pituitary Regulator of Transcription
As a marker of the stomodeum (15), the most anterior segment of
the body plan, Ptx1 may be recruited as a tissue-specific regulator of
transcription in many epithelial derivatives of the stomodeum, as has
been shown in the present work for the pituitary. This recruitment
would be consistent with a combinatorial model for cell-specific gene
expression (Fig. 10) in which genes encoding transcription factors are
activated at specific times and places during development to control
organogenesis, cell differentiation, and gene transcription. All the
pituitary-specific promoters found to be activated by Ptx1 in the
present work (Fig. 3) have putative Ptx1-binding sites (Table 1) except
the Pit1 gene, which does not have a site in its promoter but does in
its enhancer. Only one Ptx1-binding site appears to be necessary for
transcriptional activation as we have shown for POMC (14), ßTSH
(Table 1 and Fig. 3), ßLH, and GSU (Fig. 4). These sites appear to
bind Ptx1 monomers (14), and their sequence is consistent with the
documented DNA-binding specificity of bicoid-related
homeoproteins (36, 37, 38).
Despite their great conservation in DNA-binding specificity, the various bicoid-related homeoproteins have different transcriptional properties (35). The homeobox transcription factors most closely related to Ptx1 are the Otx1 and Otx2, which are specifically expressed in the brain (49), but not at all in the pituitary (Otx2) or at very low levels postnatally (Otx1) (D. Acampora, S. Mazan, F. Tuorto, V. Avantaggiato, J. J. Tremblay, D. Lazzaro, A. di Carlo, A. Mariano, P. E. Macchia, V. Macchia, J. Drouin, P. Brûlet, and A. Simeone, in preparation). In striking contrast to Ptx1, Otx1 has no effect on POMC, and it does not synergize with SF-1 on the ßLH promoter (data not shown). Thus, in addition to their complementary expression patterns during development of head structures, the ability of these homeobox factors to synergize with specific partners for control of transcription may account for the specificity of their roles during development.
Promoter-Specific Synergism
While Ptx1 may contribute to mechanisms for
pituitary-specific transcription as we had originally shown for POMC
(14), it is clearly not the sole determinant for lineage-specific
transcription of either POMC or any other pituitary hormone-coding
gene. For this reason, the transcriptional interaction with other
transcription factors for cell-specific activity is of great
significance. Prior work has shown the importance of the bHLH factor
NeuroD1 for corticotrope-specific transcription of POMC (39, 50), thus
defining one partner of Ptx1 in a code for cell- and promoter-specific
control of transcription. Another Ptx1 partner is Pit1, which
specifically acts in synergy with Ptx1 to stimulate PRL gene
expression, and less so on the GH promoter (Fig. 5 and Ref.45).
The current work has extended the model by showing marked synergism
between Ptx1 and SF-1, an orphan nuclear receptor transcription factor
(Fig. 5). This synergism is specifically exerted on the ßLH promoter
but not on the promoters of other genes specific to the gonadotrope
lineage such as GSU, ßFSH, or GnRH-R (Fig. 5 and data not shown).
Both ßLH (40, 41) and GSU (44) promoters contain SF-1-binding
sites. The SF-1-binding site of the ßLH promoter was shown to be
essential for promoter activity (Refs. 40 and 41) and Fig. 5) but less
data support the role of SF-1 in GSU promoter activity. The only
supporting data rested on the activity of oligomerized synthetic GSU
SF-1- binding sites inserted upstream of the thymidine kinase promoter
(44). Inactivation of the SF-1 gene also suggested a predominant role
in ßLH expression. Indeed, both ßLH and ßFSH transcripts were
undetectable in SF-1-/- mice while GSU transcripts
were only decreased (42), and expression of both ß-subunit genes was
restored by injection of GnRH (51). However, in their discussion, Ikeda
et al. (51) indicate that one third of the
SF-1-/- mice had detectable ßFSH mRNA by in
situ hybridization but never ßLH or GnRH-R, and they suggested
that transcription of ßFSH may be under more indirect SF-1 control
than ßLH. Our observation (Fig. 5) of SF-1 synergism on the ßLH,
but not on the ßFSH, promoter is entirely consistent with their
hypothesis. It is noteworthy that T3–1 cells that express Ptx1 and
SF-1 (Fig. 9) do not express the ßLH gene (46): it is therefore
likely that other factor(s) are involved in further differentiation of
the gonadotrope lineage and activation or derepression of the ßLH
gene.
The Drosophila homolog of SF-1, Ftz-F1, was recently shown
to interact directly with an homeodomain transcription factor, fushi
tarazu (Ftz) to activate transcription synergistically (52, 53). Our
observations (Fig. 5) constitute the first example of similar synergism
between a mammalian nuclear receptor and a homeobox factor. The domain
of Ftz that interacts with Ftz-F1 (53) is not conserved in Ptx1 such
that it is not possible, at the molecular level, to extend the
comparison with the synergism between Ptx1 and SF-1. Nonetheless, it
appears that synergism between these classes of transcription factor
may be a conserved mechanism for tissue specificity during
development.
The promoter-specific action of factors that synergize with Ptx1
correlates with their cell-restricted pattern of expression. Indeed,
NeuroD1 appears to be predominantly expressed in corticotrope cells
(39). Pit1 is expressed in GH, PRL, and TSH cells and its synergism
with Ptx1 is observed on the PRL and less so on the GH promoters.
Similarly, SF-1 is specifically expressed in the gonadotrope lineage
(42, 43), and its Ptx1 synergism is restricted to only one promoter
which is specific to this lineage. Taken together, these data are
consistent with a model in which progressive differentiation of the
different pituitary lineages is accomplished by the sequential
activation of regulatory genes during organogenesis (Fig. 10).
Regulatory Cascade during Pituitary Development
The hierarchy of action of different factors involved in pituitary
development could be inferred from the timing of their initial
expression during development. As summarized in Fig. 10, Ptx1 appears
to be the earliest factor in this cascade as it is already expressed in
the stomodeum before development of Rathke’s pouch (15). It is
followed by Rpx at the early pouch stage (19) and by Lim3/Lhx3 soon
after (22, 23). As Rpx is expressed transiently in the pituitary, it
may be involved in activation of downstream genes but certainly not in
their maintenance (19, 24). In contrast, Ptx1 and Lim3/Lhx3 expression
is maintained throughout development in adult pituitary and in
pituitary-derived cell lines. The availability of these cells has
allowed us to demonstrate a strict dependence on Ptx1 for Lim3/Lhx3
expression (Fig. 9) in at least one cellular model: this contrasts with
other regulators of the gonadotrope function such as LH-2 and SF-1. If
extrapolated to development in vivo, this dependence would
be consistent with a model in which activation of the Lim3/Lhx3 gene by
Ptx1 is required for differentiation of all pituitary lineages, except
for corticotropes, as indicated in Fig. 10. The absence of Lim3/Lhx3
expression in AtT-20 cells is consistent with this as is the presence
of POMC-positive cells in the Lim3/Lhx3-/- mice (24).
The results of our knockdown experiments suggest that Ptx1 is essential
for the sustained expression of Lim3/Lhx3 and GSU (Fig. 9A).
Although Ptx1 directly activates the GSU promoter (Figs. 3 and 4),
we cannot exclude that part of the Ptx1 knockdown effect on GSU
might also be mediated through depletion of Lim3/Lhx3. Indeed, it was
suggested that Lim3/Lhx3 might stimulate GSU promoter activity
directly (22), but we have been unable to reproduce this finding (data
not shown). Ptx1, and consequently Lim3/Lhx3, appears to be dispensable
for expression of other gonadotrope marker genes such as SF-1 and,
thus, not all gonadotrope-specific functions require the continued
expression of Ptx1 and/or Lim3/Lhx3.
In summary, we have shown the importance of Ptx1 expression for
the maintenance of cell-specific transcription in two pituitary
lineages that either express Ptx1 exclusively (corticotropes) or
predominantly (gonadotropes). Indeed, we have previously documented the
importance of Ptx1 for POMC expression (14), and in the current work,
we show the importance of Ptx1 for Lim3/Lhx3 and GSU expression
(Fig. 9). Thus, Ptx1 may be the most upstream factor in the cascade of
regulators for pituitary gene expression. Its recruitment for
pituitary-specific transcription of most hormone-coding genes is
consistent with this role. Toward the establishment of a lineage- and
promoter-specific code for transcription, Ptx1 synergizes with
cell-restricted factors such as NeuroD1 in corticotropes (POMC) (39),
Pit1 in somatolactotropes (PRL, GH) (Fig. 5 and Ref.45) and with SF-1
in gonadotropes (ßLH, Fig. 5).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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GSU (63). As a loading
control, all Northern blots were stripped and rehybridized with a
32P-labeled oligonucleotide
(5'-ACGGTATCTGATCGTCTTCGAACC-3') specific for 18S ribosomal RNA.
Nuclear Extracts and Gel Retardation Assays
Nuclear microextracts from cell lines used in the present study
were prepared as described previously (50). Ptx1 gel retardation assays
and supershift experiments were performed as outlined by Lamonerie
et al. (14) whereas GATA gel retardation assays were done
according to Grépin et al. (64).
Cell Culture and Transfection Assays
Murine T3–1, TSH, AtT-20, GHFT1.5, MMQ, L, and rat
GH4C1, GH3, GC, and African green
monkey kidney CV-1 cells were grown in DMEM supplemented with 10% FCS.
CV-1 and L cells were transfected by the calcium phosphate method as
described previously (14). T3–1 cells were transfected using the
LipofectAMINE Reagent, as described (65). Data are presented as the
means ± SEM of three to eight experiments each
performed in duplicate.
For stably transfected T3–1 cells, the evening before transfection,
cells were seeded at 400,000/90-mm petri dish and transfected the next
morning with 10 µg of control vector (pCDNA3, Invitrogen, San Diego,
CA) or antisense Ptx1 vector (containing the full-length Ptx1 cDNA in
reverse orientation) mixed with 600 µl of serum-free DMEM and added
to a solution containing 27 µl of LipofectAMINE Reagent and 600 µl
of serum-free DMEM, incubated for 30–45 min, and applied on cells for
5 h before rinsing with FCS-supplemented DMEM. Stable
transfectants were selected 24 h later for resistance to neomycin
(300 µg/ml), and individual clones were picked and subsequently
cultured in medium containing 50 µg/ml neomycin.
Western Blot Analysis
Thirty microgram whole cell extracts from transfected CV-1 cells
and 60 µg nuclear extracts from AtT-20, T3–1, TSH, GHFT1.5,
GH3, GH4C1 cells and adult mouse
pituitary were denatured before electrophoresis by boiling the samples
for 3 min in loading buffer containing 1% SDS, 1%
ß-mercaptoethanol, and 100 mM dithiothreitol. Samples
were loaded on denaturing 12% polyacrylamide gel containing 0,1% SDS.
The gel was migrated at 200 V for 75 min at room temperature using the
Bio-Rad Mini-Protean II electrophoresis apparatus (Bio-Rad, Richmond,
CA). Proteins were transferred to polyvinylidene fluoride membranes
(Amersham Canada) by electroblotting at 100 mA for 2 h at 4 C in
transfer buffer [25 mM Tris-HCl, 192 mM
glycine, 20% methanol (vol/vol), pH 8.4] using the Bio-Rad Mini
Trans-Blot apparatus. Polyvinylidene fluoride membranes were blocked
for 16 h at 4 C and then for 30 min at room temperature in 20
mM Tris-HCl, 0.9% NaCl (wt/vol) (TBS) and 15% powdered
milk (wt/vol). Membranes were incubated in TBS containing 0.2%
Tween-20 (vol/vol) (TBST) and 5% powdered milk (wt/vol) and a 1:20
dilution of an affinity-purified Ptx1-specific antiserum for 90 min at
RT. The rabbit antiserum was raised against a MBP-Ptx1 fusion protein
containing amino acids 24–56 of Ptx1. After the incubation, membranes
were washed three times for 5 min each in TBST at room temperature and
then incubated for 1 h at room temperature in TBST containing a
1:2000 dilution of a biotinylated anti-rabbit IgG (Vector Laboratories,
Burlingame, CA). Meanwhile, an avidin-biotin complex was prepared using
a 1:500 dilution of Avidin-D and a 1:1000 dilution of
biotinylated-horseradish peroxidase (Vector Laboratories) and kept on
ice for 1 h. The membranes were washed as described above and
incubated with the avidin-biotin complex for 1 h at RT. Finally,
the membranes were washed and immune complexes were visualized using
0.8 mM diaminobenzamine as substrate in the presence of 0.3
mM nickel chloride and 0.009% hydrogen peroxide at RT for
10 min (66).
Plasmids and Oligonucleotides
The SF-1 expression vector was generously provided by Dr. Keith
L. Parker. Mouse -6 kb ßTSH-luciferase and GSU-luciferase (-1.7
kb, -0.48 kb, -0.381 kb, and -0.297 kb) reporter plasmids were
kindly provided by Dr. David F. Gordon. GSU promoter deletions to
-0.212 kb, -0.113 kb, and -0.065 kb were generated by PCR. Bovine
-0.776 kb ßLH-luciferase was kindly provided by Dr. John Nilson.
Deletion to -0.104 kb ßLH-luciferase was obtained by cutting the
-0.776 kb plasmid with SmaI and religating, and to -0.033
kb by cutting the -0.776 kb plasmid with XhoI and
PstI, blunting both extremities with T4 DNA
polymerase, and religating. Ptx1 expression vector was constructed by
cloning a NcoI-KpnI fragment of Ptx1 cDNA in the
corresponding sites of a RSV-driven expression vector. This vector was
derived from RSV-Luc reporter by replacing the
HindIII-KpnI luciferase fragment by the multiple
cloning site of Bluescript KS- and by changing the pBR322 backbone to
Bluescript SK+ to increase copy number in bacteria.
The Ptx2 cDNA was obtained by RT-PCR from mouse pituitary first-strand
cDNA using forward (5'-TCCTCTAGACGATAACCGGGAATGGAG-3') and reverse
(5'-CAGGATCCTCAGTCTTTCTGGGGCAGA-3') primers and subsequently subcloned
in Bluescript KS- and the RSV expression vector. WT and
mutant (M1) Ptx1 oligonucleotides, as well as DE2A and GATA probes used
in the gel retardation assays, were described previously (14, 64).
Oligonucleotides were synthesized with an Applied Biosystem (Foster
City, CA) synthesizer.
|
ACKNOWLEDGMENTS |
|---|
GSU,
ßTSH), Michael Karin (GH), Kathy Mahon (Pit1 promoter and enhancer),
Richard Maurer (ßFSH), John Nilson (ßLH), and Michael Rosenfeld
(PRL) for their reporter constructs. We also thank Keith Parker and
Michael Karin for the SF-1 and Pit1 expression vectors, respectively.
Pan-1, Oct1, Lim3, Rpx, LH-2, GnRH-R, Pax6, and Six3 probes were
provided by Chris Nelson, Hans Schöler, Nabil Seidah, Kathy
Mahon, Richard Maurer, Kevin Catt, Tom Glaser, and Peter Gruss,
respectively. We thank Michel Chamberland for oligonucleotide
synthesis. The efficient secretarial assistance of Lise Laroche was
much appreciated. |
FOOTNOTES |
|---|
J. J. Tremblay was recipient of a studentship from the Cancer Research Society Inc. and C. L. Lanctôt was a Research Student of the National Cancer Institute of Canada. This work was funded by the National Cancer Institute of Canada supported with funds provided by the Canadian Cancer Society.
Received for publication October 2, 1997. Revision received November 14, 1997. Accepted for publication December 8, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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J. J. Tremblay and R. S. Viger GATA Factors Differentially Activate Multiple Gonadal Promoters through Conserved GATA Regulatory Elements Endocrinology, March 1, 2001; 142(3): 977 - 986. [Abstract] [Full Text] [PDF]
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B. Andersen and M. G. Rosenfeld POU Domain Factors in the Neuroendocrine System: Lessons from Developmental Biology Provide Insights into Human Disease Endocr. Rev., February 1, 2001; 22(1): 2 - 35. [Abstract] [Full Text]
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H. Pincas, K. Amoyel, R. Counis, and J.-N. Laverrière Proximal cis-Acting Elements, Including Steroidogenic Factor 1, Mediate the Efficiency of a Distal Enhancer in the Promoter of the Rat Gonadotropin-Releasing Hormone Receptor Gene Mol. Endocrinol., February 1, 2001; 15(2): 319 - 337. [Abstract] [Full Text]
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S. Lopez, M.-L. Island, J. Drouin, M.-T. Bandu, N. Christeff, N. Darracq, R. Barbey, J. Doly, D. Thomas, and S. Navarro Repression of Virus-Induced Interferon A Promoters by Homeodomain Transcription Factor Ptx1 Mol. Cell. Biol., October 15, 2000; 20(20): 7527 - 7540. [Abstract] [Full Text]
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U. B. Kaiser, L. M. Halvorson, and M. T. Chen Sp1, Steroidogenic Factor 1 (SF-1), and Early Growth Response Protein 1 (Egr-1) Binding Sites Form a Tripartite Gonadotropin-Releasing Hormone Response Element in the Rat Luteinizing Hormone-{beta} Gene Promoter: an Integral Role for SF-1 Mol. Endocrinol., August 1, 2000; 14(8): 1235 - 1245. [Abstract] [Full Text]
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G. Poulin, M. Lebel, M. Chamberland, F. W. Paradis, and J. Drouin Specific Protein-Protein Interaction between Basic Helix-Loop-Helix Transcription Factors and Homeoproteins of the Pitx Family Mol. Cell. Biol., July 1, 2000; 20(13): 4826 - 4837. [Abstract] [Full Text]
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R. A. Keri, D. J. Bachmann, A. Behrooz, B. D. Herr, R. K. Ameduri, C. C. Quirk, and J. H. Nilson An NF-Y Binding Site Is Important for Basal, but Not Gonadotropin-releasing Hormone-stimulated, Expression of the Luteinizing Hormone beta Subunit Gene J. Biol. Chem., April 21, 2000; 275(17): 13082 - 13088. [Abstract] [Full Text] [PDF]
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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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I. Pellegrini-Bouiller, C. Manrique, G. Gunz, M. Grino, A. J. Zamora, D. Figarella-Branger, F. Grisoli, P. Jaquet, and A. Enjalbert Expression of the Members of the Ptx Family of Transcription Factors in Human Pituitary Adenomas J. Clin. Endocrinol. Metab., June 1, 1999; 84(6): 2212 - 2220. [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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P. L. M. Dahia and A. B. Grossman The Molecular Pathogenesis of Corticotroph Tumors Endocr. Rev., April 1, 1999; 20(2): 136 - 155. [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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J.-J. Lareyre, T. Z. Thomas, W.-L. Zheng, S. Kasper, D. E. Ong, M.-C. Orgebin-Crist, and R. J. Matusik A 5-Kilobase Pair Promoter Fragment of the Murine Epididymal Retinoic Acid-binding Protein Gene Drives the Tissue-specific, Cell-specific, and Androgen-regulated Expression of a Foreign Gene in the Epididymis of Transgenic Mice J. Biol. Chem., March 19, 1999; 274(12): 8282 - 8290. [Abstract] [Full Text] [PDF]
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C. Lanctôt, Y. Gauthier, and J. Drouin Pituitary Homeobox 1 (Ptx1) Is Differentially Expressed during Pituitary Development Endocrinology, March 1, 1999; 140(3): 1416 - 1422. [Abstract] [Full Text]
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E. J. Lee, L. M. Anderson, B. Thimmapaya, and J. L. Jameson Targeted Expression of Toxic Genes Directed by Pituitary Hormone Promoters: A Potential Strategy for Adenovirus-Mediated Gene Therapy of Pituitary Tumors J. Clin. Endocrinol. Metab., February 1, 1999; 84(2): 786 - 794. [Abstract] [Full Text]
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P. Gage, H Suh, and S. Camper Dosage requirement of Pitx2 for development of multiple organs Development, January 10, 1999; 126(20): 4643 - 4651. [Abstract] [PDF]
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C Lanctot, A Moreau, M Chamberland, M. Tremblay, and J Drouin Hindlimb patterning and mandible development require the Ptx1 gene Development, January 5, 1999; 126(9): 1805 - 1810. [Abstract] [PDF]
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H. Kaji, S. Tai, Y. Okimura, G. Iguchi, Y. Takahashi, H. Abe, and K. Chihara Cloning and Characterization of the 5'-Flanking Region of the Human Growth Hormone Secretagogue Receptor Gene J. Biol. Chem., December 18, 1998; 273(51): 33885 - 33888. [Abstract] [Full Text] [PDF]
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S. L. Asa and S. Ezzat The Cytogenesis and Pathogenesis of Pituitary Adenomas Endocr. Rev., December 1, 1998; 19(6): 798 - 827. [Abstract] [Full Text]
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A Duggan, C Ma, and M Chalfie Regulation of touch receptor differentiation by the Caenorhabditis elegans mec-3 and unc-86 genes Development, January 10, 1998; 125(20): 4107 - 4119. [Abstract] [PDF]
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