The pan-Pituitary Activator of Transcription, Ptx1 (Pituitary Homeobox 1), Acts in Synergy with SF-1 and Pit1 and Is an Upstream Regulator of the Lim-Homeodomain Gene Lim3/Lhx3

Jacques J. Tremblay, Christian Lanctôt and Jacques Drouin

Laboratoire de Génétique Moléculaire Institut de Recherches Cliniques de Montréal Montréal Québec Canada H2W 1R7


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Ptx1 (pituitary homeobox 1) homeobox transcription factor was isolated as a transcription factor of the pituitary POMC gene. In corticotrope cells that express POMC, cell-specific transcription is conferred in part by the synergistic action of Ptx1 with the basic helix-loop-helix factor NeuroD1. Since Ptx1 expression precedes pituitary development and differentiation, we investigated its expression and function in other pituitary lineages. Ptx1 is expressed in most pituitary-derived cell lines and as is the related Ptx2 (Rieger) gene. However, Ptx1 appears to be the only Ptx protein in corticotropes and the predominant one in gonadotrope cells. Most pituitary hormone-coding gene promoters are activated by Ptx1. Thus, Ptx1 appears to be a general regulator of pituitary-specific transcription. In addition, Ptx1 action is synergized by cell-restricted transcription factors to confer promoter-specific expression. Indeed, in the somatolactotrope lineage, synergism between Ptx1 and Pit1 is observed on the PRL promoter, and strong synergism between Ptx1 and SF-1 is observed in gonadotrope cells on the ßLH promoter but not on the {alpha}GSU (glycoprotein hormone {alpha}-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 {alpha}T3–1 cells that express the {alpha}GSU gene showed that expression of endogenous {alpha}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pituitary gland has been a fruitful model with which to identify factors involved in cell-specific transcription and in the regulation of cell fate during development. The mature pituitary is composed of six different cell types, five in the anterior lobe and one in the intermediate lobe, that arise sequentially during development and are easily distinguishable by the hormone they secrete (1). The glycoprotein hormone {alpha}-subunit gene ({alpha}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 {alpha}GSU and Lim3/Lhx3 genes, thus identifying Ptx1 as the earliest regulator of pituitary transcription.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ptx1 Is Expressed in All Pituitary Cell Types
We showed previously that Ptx1 is expressed in corticotrope cells of the pituitary where it activates the POMC gene. In situ hybridization analysis had suggested that Ptx1 mRNA was also present in other pituitary cell lineages (14). To investigate the expression pattern of Ptx1, we performed Northern blot analysis on RNA obtained from a panel of cell lines. As shown in Fig. 1Go, 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 {alpha}T3–1 (gonadotrope precursor), {alpha}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 {alpha}T3–1, {alpha}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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Figure 1. Ptx1 and Ptx2, but Not Ptx3, Are Expressed in Several Pituitary-Derived Cell Lines

Northern blot analysis of 20 µg total RNA from multiple pituitary-derived cell lines and mouse pituitary was used to determine the expression pattern of the three Ptx family members. The blots were subsequently probed with an 18S ribosomal RNA probe to ensure integrity and loading of the RNA. Note the difference in exposure time for the three blots.

 
Two Ptx1-related cDNAs have been identified recently: they are Ptx2 (RIEG) (16, 17, 18), hereafter referred to as Ptx2, and Ptx3 (35). Ptx2 was shown to be expressed in the pituitary by RT-PCR and in situ hybridization (16, 18). However, no data are presently available concerning its expression relative to Ptx1. Thus, we compared the expression of Ptx1, 2, and 3 in pituitary-derived cell lines by Northern blot analysis using gene-specific probes. As shown in Fig. 1Go, the Ptx1 and Ptx2 genes are abundantly transcribed in pituitary cells whereas Ptx3 is not. In some cell lines, such as AtT-20, {alpha}T3–1, and GHFT1.5, Ptx1 is the major mRNA species, whereas in others such as GC and MMQ, Ptx2 mRNA predominates (Fig. 1Go).

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. 2Go, 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. 1Go and 2Go), although some discrepancies are noted and discussed below.



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Figure 2. The Ptx1 Protein Is Present in Several Pituitary-Derived Cell Lines

The level of Ptx1 protein was assessed by Western blot analysis. Aliquots of 40 µg whole cell extracts (overexpressing cells, lanes 1 and 2) or 80 µg nuclear extracts (cell lines and adult mouse pituitary) were subjected to immunoblotting using a Ptx1-specific antiserum as described in Materials and Methods. Protein molecular size standards are indicated on the left.

 
Ptx1 Activates Several Pituitary-Specific Promoters
We previously showed that Ptx1 is an important determinant for expression of the POMC gene in AtT-20 cells (14). Ptx1 may play a similar role for other pituitary genes. As shown in Table 1Go, 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. 3Go, Ptx1 significantly activates several pituitary promoters, including those for {alpha}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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Table 1. Putative Ptx1 Binding Sites Present in Pituitary-Specific Promoters

 


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Figure 3. Ptx1 Transactivates Several Pituitary-Specific Promoters

The effect of Ptx1 was tested on various pituitary promoters including -1700 bp {alpha}GSU, -776 bp ßLH, -2400 bp ßFSH, -1200 bp GnRH-R, -320 bp GH, -422 bp PRL, -220 bp Pit1, -220 bp Pit1+700 bp enhancer, -6000 bp ßTSH, and -480 bp POMC. Each construct was cotransfected in CV-1 cells with a control plasmid (empty RSV expression vector, open bars) or a RSV-Ptx1 expression (14) vector (solid bars). A viral promoter, RSV, was used as negative control. Results are shown as fold activation (± SEM).

 
Mapping of Ptx1-Responsive Elements in the {alpha}GSU and ßLH Promoters
In view of the large effect of Ptx1 on the {alpha}GSU and ßLH promoters and of its high expression in {alpha}GSU cells (Fig. 2Go and below), we performed deletion analyses on the {alpha}GSU as well as on the ßLH promoters to identify Ptx1-responsive sequences. As shown in Table 1Go, the {alpha}GSU and ßLH promoters contain several putative Ptx1-binding sites. A short (-120 bp) {alpha}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. 4Go). Similar results were obtained with the ßLH promoter (Fig. 4Go) where removal of the most proximal putative Ptx1-binding site led to a loss of Ptx1 activation (Fig. 4Go). Taken together, these data suggest that transactivation of both the {alpha}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 1Go). 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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Figure 4. Mapping of Ptx1-Responsive Elements in the {alpha}GSU and ßLH Promoters

CV-1 cells were cotransfected with various 5'-deletion constructs of the {alpha}GSU (left panel) and ßLH (right panel) promoters with either a control vector (open bars) or a Ptx1 expression vector (solid bars). Results are shown as fold activation (± SEM). The sequence of the putative Ptx1-responsive element is shown under the graph for each promoter.

 
Ptx1 Acts in Synergy with SF-1 and Pit1
To define the role of Ptx1 in lineage- and/or promoter-specific expression, we tested its ability to stimulate promoter activity in synergy with cell-type restricted transcription factors. Consistent with this hypothesis, we have shown that Ptx1 specifically synergizes with basic helix-loop-helix (bHLH) heterodimers containing NeuroD1 for activation of POMC transcription (39).

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. 5AGo, 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. 5AGo). 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. 5AGo); 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 {alpha}GSU and ßFSH genes (42, 44) and Ptx1 can activate both promoters (Fig. 3Go). No synergy, however, was observed between Ptx1 and SF-1 on these two promoters (Fig. 5AGo). 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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Figure 5. Ptx1 Can Synergize with SF-1 and Pit1

A, Synergistic transactivation by Ptx1 and SF-1. The effect of Ptx1 and SF-1 alone or in combination was tested on the -776 bp and -104 bp ßLH, -2400 bp ßFSH, and -341 bp {alpha}GSU promoters. The -104 bp ßLH promoter no longer contains the SF-1-binding site present at -120 bp (40, 41). B, Ptx1 synergizes with Pit1. Transactivation by either Ptx1, Pit1, or both was tested on four Pit1-dependent promoters: -422 bp PRL, -320 bp GH, -6000 bp ßTSH, and the Pit1 promoter/enhancer. Promoter constructs were cotransfected with the indicated expression plasmids in CV-1 cells. Results are shown as fold activation (± SEM).

 
Ptx1 can also synergistically activate transcription with another cell-specific factor, Pit1 (45). The PRL promoter, which was only slightly activated by Ptx1 (Fig. 3Go), can be synergistically activated by Ptx1 and Pit1 (Fig. 5BGo). The same was true for the GH promoter although to a lesser extent (Fig. 5BGo). 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. 5BGo). 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 {alpha}GSU-Expressing Cells
To correlate Ptx1 expression in cell lines derived from various pituitary lineages (Figs. 1Go and 2Go) with normal pituitary cells, we used double-labeling immunohistochemistry to analyze Ptx1 expression in the adult pituitary gland. As shown in Fig. 6Go, 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 {alpha}GSU-expressing cells by double-labeling immunohistochemistry (Fig. 6Go). This result correlates with expression in pituitary-derived cell lines (Fig. 2Go). In addition, high Ptx1 expression colocalized with {alpha}GSU-expressing cells from the onset of {alpha}GSU expression during pituitary development (C. Lanctôt and J. Drouin, in preparation).



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Figure 6. Ptx1 Is Strongly Expressed in {alpha}GSU-Expressing Cells

Immunohistochemistry was used to detect {alpha}GSU and Ptx1 expression in adult mouse pituitary. To detect {alpha}GSU-positive cells (blue cytoplasm), a monoclonal antibody coupled with an alkaline phosphatase reaction was used while Ptx1-expressing cells (brown nuclear staining) were detected with a Ptx1 polyclonal affinity-purified antiserum and peroxidase-coupled anti-rabbit IgG.

 
Ptx1 Is the Major Ptx Protein Expressed in {alpha}GSU-Expressing {alpha}T3–1 Cells
As shown in the present study (Figs. 2Go and 6Go), {alpha}T3–1 cells as well as mouse pituitary {alpha}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 {alpha}T3–1 cells (Fig. 1Go and Ref.16). We do not yet know whether these cells contain Ptx2 protein. To determine the relative importance of Ptx1 protein in {alpha}T3–1 cells, we performed supershift experiments using nuclear extracts from {alpha}T3–1 cells. The Ptx1 antibody used in our experiments (Fig. 2Go) is specific for Ptx1 since it did not recognize Ptx2 in Western blot (Fig. 7AGo, lane 3) or in gel shift assays (Fig. 7BGo, lane 4). The Ptx-binding activity present in {alpha}T3–1 nuclear extracts (Fig. 7CGo, lane 2) was almost completely supershifted by saturating amounts of the Ptx1-specific antibody (Fig. 7CGo, lane 3). Taken together, these data clearly demonstrate that Ptx1 is by far the most abundant member of the Ptx family in {alpha}T3–1 cells.



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Figure 7. {alpha}T3–1 Cells Contain Almost Exclusively Ptx1

A, Western blot analysis of extracts from L cells overexpressing Ptx1 or Ptx2 using a Ptx1-directed antiserum. B, Gel retardation assay using the CE3 element of the POMC gene (14) as probe and extracts from L cells transfected with either Ptx1 or Ptx2. A supershift is only observed with the Ptx1-binding activity when using a Ptx1-specific antibody ({alpha}Ptx1). C, Gel retardation assay using the CE3 element as probe and extracts from {alpha}T3–1 cells. The binding activity is almost completely supershifted by the Ptx1-specific antiserum.

 
Ptx1 Is Essential for {alpha}GSU and Lim3/Lhx3 Gene Expression
{alpha}T3–1 cells have been considered as a model of gonadotrope precursors because they express the {alpha}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. 1Go, 2Go, and 7Go). Moreover, Ptx1 strongly activated the {alpha}GSU promoter (Figs. 4Go and 5Go). To further confirm the role of Ptx1 in {alpha}GSU gene expression, we generated Ptx1 knockdown cell lines by stably transfecting a Ptx1 antisense RNA expression vector in {alpha}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 {alpha}T3–1 cells (WT). In the three Ptx1 antisense clones (8, 9, 13), endogenous Ptx1 mRNA (Fig. 8AGo, upper panel) and protein as assessed by DNA-binding assay (Fig. 8AGo, lower panel) were markedly decreased, whereas other transcription factors such as Pan1 and Oct1 were not significantly affected, neither at the mRNA (Fig. 8BGo, upper panel) nor at the protein (DNA binding in electrophoretic mobility shift assay) levels (Fig. 8BGo, lower panel). GATA DNA-binding activity might be slightly decreased (Fig. 8BGo). The fact that Ptx DNA-binding activity was almost undetectable in the antisense clones (Fig. 8AGo) 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 {alpha}GSU gene expression. As shown in Fig. 9AGo (top panel), no or little {alpha}GSU mRNA was detected in the antisense clones. This clearly indicates that the endogenous {alpha}GSU gene was almost silent in these cells. Further, the activity of a transfected {alpha}GSU-luciferase reporter is considerably lower in the antisense clones than in control cells (data not shown). Thus, Ptx1 is essential for {alpha}GSU gene expression.



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Figure 8. Endogenous Ptx1 Activity Is Decreased in Ptx1 Antisense Clones

{alpha}T3–1 cells were stably transfected with a Ptx1 antisense RNA expression vector as described in Materials and Methods. A, Levels of Ptx1 mRNA (upper panel) and protein (lower panel) in wild-type {alpha}T3–1 cells (WT), one control clone (Ctl), and three independent antisense clones (8, 9, and 13) were monitored by Northern blot analysis and gel retardation assay, respectively. B, Other transcription factors are not affected in the Ptx1 antisense clones. The Northern blot used in panel A was successively rehybridized with Pan1 and Oct1a probes (upper panel), and the quality of the nuclear extracts was tested by gel retardation assay for Oct1- and GATA-binding activity (lower panel).

 


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Figure 9. Ptx1 Knockdown Cells Fail to Express the {alpha}GSU and Lim3/Lhx3 Genes

A, Northern blot analysis of several genes normally expressed in {alpha}T3–1 cells using RNA from the control clone (Ctl) and three independent Ptx1-antisense clones (8, 9, and 13). Blots were successively hybridized with probes for {alpha}GSU, Lim3/Lhx3, LH-2, SF-1, GnRH-R, and Six3. A ribosomal 18S RNA probe was used to verify RNA loading. B, Similar blot hybridized with {alpha}GSU, Ptx2, Pax6, and 18S RNA probes.

 
Using the Ptx1 knockdown cell lines, we investigated expression of other genes normally expressed in {alpha}T3–1 cells. The GnRH-R, another differentiation marker of the gonadotrope lineage, was also decreased in these cells, although much less so than {alpha}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. 9AGo). Similarly, neither the low-level Ptx2 expression nor Pax6 (47) mRNA levels were altered in the antisense clones (Fig. 9BGo). 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 {alpha}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. 10Go).



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Figure 10. Regulatory Factors during Pituitary Development

Summary of expression patterns for transcription factors involved in pituitary gene expression and development. Top, Representation of putative cellular intermediates during pituitary differentiation indicating transcription factors expressed at each stage. For terminally differentiated cells, factors shown previously or in the present work to activate transcription synergistically with Ptx1 are shown in bold. Bottom, Timing of onset and extinction (where appropriate) for pituitary transcription factors. References for the data are provided in the text.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The present work supports the model that Ptx1 is a pan-pituitary regulator of transcription and that it contributes to promoter-specific transcription by synergism with cell-restricted factors such as NeuroD1, SF-1, and Pit1. This broad regulatory function is consistent with the early activation of Ptx1 in the stomodeum, the ectodermal epithelium from which Rathke’s pouch develops. Further, Ptx1 appears to be an upstream regulator in a cascade of transcription factors that control pituitary-specific transcription. Indeed, Ptx1 is required for Lim3/Lhx3 gene expression in at least one pituitary cell model. This regulatory hierarchy may also operate in vivo during pituitary organogenesis.

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. 1Go and 2Go). 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 {alpha}T3–1 cells, whereas protein levels are much lower (compare Figs. 1Go and 2Go). Another discrepancy may exist in {alpha}T3–1 cells that have both Ptx1 and Ptx2 mRNA but the bulk of Ptx protein appears to be Ptx1 (Fig. 7Go). 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. 1Go 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. 10Go) 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. 3Go) have putative Ptx1-binding sites (Table 1Go) 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 1Go and Fig. 3Go), ßLH, and {alpha}GSU (Fig. 4Go). 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. 5Go 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. 5Go). This synergism is specifically exerted on the ßLH promoter but not on the promoters of other genes specific to the gonadotrope lineage such as {alpha}GSU, ßFSH, or GnRH-R (Fig. 5Go and data not shown). Both ßLH (40, 41) and {alpha}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. 5Go) but less data support the role of SF-1 in {alpha}GSU promoter activity. The only supporting data rested on the activity of oligomerized synthetic {alpha}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 {alpha}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. 5Go) of SF-1 synergism on the ßLH, but not on the ßFSH, promoter is entirely consistent with their hypothesis. It is noteworthy that {alpha}T3–1 cells that express Ptx1 and SF-1 (Fig. 9Go) 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. 5Go) 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. 10Go).

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. 10Go, 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. 9Go) 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. 10Go. 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 {alpha}GSU (Fig. 9AGo). Although Ptx1 directly activates the {alpha}GSU promoter (Figs. 3Go and 4Go), we cannot exclude that part of the Ptx1 knockdown effect on {alpha}GSU might also be mediated through depletion of Lim3/Lhx3. Indeed, it was suggested that Lim3/Lhx3 might stimulate {alpha}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 {alpha}GSU expression (Fig. 9Go). 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. 5Go and Ref.45) and with SF-1 in gonadotropes (ßLH, Fig. 5Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RNA Extraction and Analysis
Total RNA was extracted by the guanidium thiocyanate-phenol-chloroform method (54) and analyzed by Northern blot analysis (55). Ten or 20 µg of RNA were separated by agarose-formaldehyde gel electrophoresis and then transferred to nylon membrane (Hybond-N, Amersham Canada, Oakville, Ontario, Canada). Membrane hybridizations with 32P-labeled cDNA probes were done in 1 M NaCl, 1% SDS, 10% dextran sulfate, and 200 µg/ml of denatured salmon sperm DNA at 65 C. Blots were washed under stringent conditions: 1x saline sodium citrate, 0.1% SDS for 30 min at 65 C and 0.1x saline sodium citrate, 0.1% SDS for 30 min at 65 C. DNA probes were cDNA fragments specific for Ptx1 (14), Six3 (56), Ptx2 (16), Rpx (19), Ptx3 (35), Pax6 (57), Pan1 (58), LH-2 (59), SF-1 (60), Lim3/Lhx3 (23), GnRH-R (61), Oct1a (62), and {alpha}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 {alpha}T3–1, {alpha}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). {alpha}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 {alpha}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, {alpha}T3–1, {alpha}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 {alpha}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. {alpha}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
 
We are grateful to Pamela Mellon for her pituitary-derived cell lines and for the GnRH-R-Luc reporter, to David Gordon ({alpha}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
 
Address requests for reprints to: Dr. Jacques Drouin, Institut de Recherches Cliniques de Montréal, 110 des Pins Ouest, Montréal Québec Canada H2W 1R7. e-mail: drouinj@ircm.umontreal.ca.

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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