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Laboratoire Interactions Cellulaires Neuroendocriniennes, Unité Mixte de Recherche 6544, Centre National de la Recherche Scientifique, Université de la Méditerranée, Institut Fédératif Jean Roche, Faculté de Médecine Nord, Marseille 13916, France
Address all correspondence and requests for reprints to: Dr. Isabelle Pellegrini, Laboratoire Interactions Cellulaires Neuroendocriniennes, Unité Mixte de Recherche 6544, Centre National de la Recherche Scientifique-Université de la Méditerranée, Institut Fédératif Jean Roche, Faculté de Médecine Nord, Bd. P. Dramard, 13916 Marseille cedex 20, France. E-mail: . pellegrini.i{at}jean-roche.univ-mrs.fr
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Abstract |
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Introduction |
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The Pitx2 gene, expressed from embryonic d 8.5 in the rat, is one of the early markers of the developing anterior pituitary. Its expression is maintained throughout development in Rathke’s pouch and adult pituitary, suggesting a role in both early events and terminal differentiation of anterior pituitary cells (5). Also named Ptx2, Rieg, Otlx2, Brx1, and ARP1 (9, 10, 11, 12), this gene consists of four exons and encodes a homeodomain protein with a lysine residue at position 50, characteristic of the bicoid-related homeodomain proteins, which are major developmental transcription factors (9, 10, 13). Three isoforms that differ in their N-terminal part but possess identical C terminus and homeodomain were described for Pitx2 (12). Multiple members of this Pitx gene family with overlapping and distinctive expression patterns have been identified in vertebrates including amphibians, fish, birds, and mammals (13). In contrast to other factors that are pituitary specific, Pitx2 is expressed during mouse development in many tissues, including derivatives of the first branchial arch, eye, brain, mandible, heart, limbs, and left plate mesoderm (10, 14). Recent experiments have indicated a role for Pitx2 downstream of Sonic hedgehog and nodal in a genetic pathway regulating laterality of heart, gut, and other asymmetric organs (15). The Pitx2 gene-deleted mice exhibit defective body-wall closure, right pulmonary isomerism, altered cardiac position and development, and a late arrest in turning (16, 17, 18, 19). Another feature of -/- mice is the early arrest of pituitary development at the committed Rathke’s pouch stage. In these mice a reduction of the pituitary gland is observed because of a decrease of cell proliferation, and by embryonic d 14.5, only some corticotroph cells are detectable, indicating that Pitx2 is required for pituitary morphogenesis (16, 17, 18, 19). These data set Pitx2 as an early regulator of pituitary ontogeny that acts with Hesx1 and Lhx3 before the action of Prop-1 and Pit-1 to advance development of the committed Rathke’s pouch.
The aim of this work was to better understand the involvement of Pitx2 in the regulation of the specific functions of the pituitary somatolactotroph cells, which secrete GH and PRL and are known to express the Pitx2 transcription factor (5). Previous reports gave evidence to the role of Pitx2 during the development (15, 16, 17, 18, 19), but only few data exist concerning its function in the differentiated pituitary somatolactotroph cells. For this purpose, we have used five physiological disruptive mutant forms of Pitx2 to approach the involvement of Pitx2 in maintenance of pituitary functions. The five mutations named L54Q, T68P, R69H, R84W, and R91P, each characterized by a missense mutation located in the homeodomain, were previously reported in patients with iris hypoplasia (20), iridogoniodysgenesis syndrome (21), or Axenfeld-Rieger syndrome (ARS), an autosomal dominant disorder in which anterior segment dysgenesis of the eye is accompanied by facial, dental, and ombilical anomalies (14, 22, 23). Pituitary abnormalities (24) and growth retardation (25) can also be associated with ARS, although no genetic linkage has been clearly established yet between these abnormalities and a mutation in Pitx2. In the present study, the impact of the mutations on the functional properties of Pitx2 were investigated in vitro using PRL, GH, and Pit-1 promoters as targets in pituitary somatolactotroph and nonpituitary cell lines. We report that the five mutations lead to either inactive or dominant negative proteins in transactivation of pituitary target genes. These results, which support a critical role for this homeodomain factor not only during embryogenesis but in the differentiated functions of the pituitary somatolactotroph cells, too, might also form the basis for future experiments because the R91P dominant negative mutant might represent an interesting tool to further study Pitx2 involvement in cellular functions such as proliferation and differentiation.
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Materials and Methods |
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Cell culture and transfection
African green monkey kidney fibroblast-like CV-1 cells were grown in DMEM supplemented with 10% fetal calf serum (SVF). CV-1 cells were transfected by the calcium phosphate method with the MBS mammalian transfection kit (Stratagene) according to the manufacturer’s instruction. Briefly, cells were plated at 100,000 cells/well in 6-well plates 24 h before transfection. Transfection were carried out in triplicate wells using 3 µg reporter plasmid, 0.1–1 µg effector plasmid(s), and 0.3 µg CMV-ß-galactosidase (ß-gal) as internal control for transfection efficiency. GH4C1 somatolactotroph pituitary cells were grown in HamF10 medium supplemented with 15% horse serum and 2.5% SVF. Cells were transfected in triplicate wells by lipofection in serum-free medium using Transfast transfection kit (Promega Corp., Madison, WI) according to the manufacturer’s instruction. Briefly, cells were plated at 200,000 cells/well in 12-well plates 24 h before transfection and transfected with 1 µg DNA (0.3 µg reporter plasmid, 0.1–1 µg effector plasmid(s), and 0.2 µg CMV-ß-gal plasmid). Cells were incubated with the DNA/liposome complexes for 1 h and then supplemented with 1.5 ml complete medium. Cos-7 cells were grown in DMEM 10% SVF. Transfections were carried out in 100-mm-diameter dishes using the liposome-based transfection kit Polyfect (QIAGEN, Valencia, CA) and 5 µg wild-type or mutant Pitx2 expression vectors. In all transfections, total DNA was kept constant, and nonspecific effects of viral promoters were controlled by using the appropriate empty vectors.
Luc and ß-gal assays
CV-1 and GH4C1 cells were harvested 48 h after transfection and lysed in 200 µl reporter lysis buffer (Promega Corp.). After three sequential freeze-thaw cycles, cell debris was pelleted by centrifugation at 10,000 x g for 2 min at 4 C, and 20-µl aliquots of the supernatant were used for subsequent Luc (Luciferase assay system, Promega Corp.) and ß-gal assays. For each control, the total Luc activity normalized against ß-gal activity was taken as 1, and results were expressed as fold activation over control. Data are presented as the mean ±SE of three to five independent experiments using different plasmid preparations of each construct. Statistical significance was determined by Wilcoxon nonparametric paired test. Significance was declared at P < 0.05.
Total and nuclear cell extracts
Total cell extracts were prepared from confluent somatolactotroph pituitary GH4C1 cells grown in 60-mm dishes. Three hundred microliters ice-cold lysis buffer [25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P40, 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin] were added to each plate and gently stirred for 30 min at 4 C. Nonsolubilized material was removed by centrifugation at 10,000 x g for 10 min at 4 C. Nuclear proteins were extracted from Cos-7 cells 48 h following transfection. Cells were scraped into 1 ml PBS and centrifuged at 2000 rpm for 2 min. Four hundred microliters hypotonic buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) were added to the pellet and left on ice for 5 min. Following the addition of 25 µl 10% Nonidet P-40, nonsolubilized material was removed by centrifugation at maximum speed for 30 sec. The pellet was resuspended in 100 µl binding buffer 20 mM HEPES, 400 mM KCl, 20% glycerol, 2 mM dithiothreitol, 0.5 mM PMSF. After one freeze-thaw cycle, protein solution was centrifugated for 30 min at 14,000 rpm. Protein contents in the lysates were measured colorimetrically using Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA).
In vitro transcription/translation
TNT T7 coupled reticulocyte lysate system (Promega Corp.) was used for in vitro transcription/translation. Reactions were carried out in a total volume of 50 µl with reticulocyte lysate, 1 µg plasmid DNA, 1 mM amino acid mixture, RNasin (40 U/µl, Life Technologies, Inc.), T7 RNA polymerase in the presence, or absence, of 35S-Met (10 mCi/ml, NEN Life Science Products, Boston, MA). 35S-Met-radiolabeled translation products were separated by SDS-PAGE and exposed to autoradiographic (ARG) film.
Antibody generation and Western blot analysis
Polyclonal antibody against Pitx2 was produced by Eurogentec (Liège, Belgium) by immunization of rabbits with a 13-residue peptide located upstream of the Pitx2 homeodomain (QGKNEDVGAEDPPSKC), coupled to KLH-MBS. This sequence in exon 4 is present in the known isoforms of Pitx2 and is fully conserved among rat, mouse, and human sequences but markedly diverges from the corresponding Pitx1 sequence. IgG fraction was purified by chromatography on a protein A-Sepharose column. Whole-cell protein extracts (80 µg) or in vitro-translated products (5 µl) were resolved on a 12% SDS-PAGE using the Laemmli buffer system. After transfer to PVDF membrane (DuPont de Nemours, France), immunodetection of Pitx2 was performed using rabbit polyclonal Pitx2 IgG (4 µg/ml) and alkaline-phosphatase-conjugated goat antirabbit IgG (Tropix, Bedford, MA). Membranes were developed with the Western-Star TM immunodetection system (Tropix). The specificity of the antibody was tested by Western analysis and EMSA of extracts of Cos-7 cells expressing full-length Pitx2 (see Results section).
Northern blot analysis
Total RNA was extracted and purified from pituitary cells using the Rneasy kit (QIAGEN). Twenty micrograms total RNA were run on a 1% agarose/formaldehyde gel, transferred to nylon membrane, and hybridized with a SalI/NotI 723-bp cDNA fragment located in rat Ptx2 3' untranslated region (UTR) (gift of Dr. D. Jacoby, DIACRIN, Inc., Charlestown, MA). Prehybridization was performed at 42 C in 50% formamide, 6x saline sodium citrate, 5x Denhardt’s solution, 0.5% SDS, and 100 µg/ml denatured salmon sperm DNA. Hybridization with the dCTP
32P-labeled cDNA probes was performed in the same buffer (2 x 106 cpm/ml) for 16 h at 42 C. Blots were washed under stringent conditions, placed on a phosphor screen (molecular imager, Bio-Rad Laboratories, Inc.), and subsequently rehybridized with an 18S specific cDNA probe.
EMSA
Gel shifts were carried out using wild-type and mutant Pitx2 and the 32P-labeled double-stranded oligonucleotide: 5'-ACCAGGATGCTAAGCCTGTGTC-3', containing the CE3 Pitx-specific binding site (in bold) of the proopiomelanocortin (POMC) promoter (26). One hundred nanograms annealed double-stranded DNA were 5' end labeled with T4 polynucleotide kinase (Life Technologies, Inc.) and 2 µl 32P-
ATP and purified over a G-25 Sepharose column. Variable amounts of nuclear protein extracts or in vitro translated proteins were incubated on ice for 15 min in a 20-µl reaction of 1x binding buffer containing 1 µg poly(dI-dC) and 20,000 cpm of the radiolabeled probe. In some cases, 1 µl Pitx2 polyclonal antiserum, or 15- to 100-fold excess of competitor oligonucleotides (B1: 5'-GCAAAGGTTTATAAAGCCAATGC-3'; B2: 5'-GCATTATGGGGGTAATCTCAATGC-3') were added to the reactions before addition of the probe. The bound proteins were separated from the free probe on an 8% polyacrylamide gel in 0.5% Tris-borate EDTA by PAGE at 180 V for 3 h at 4 C before exposure to ARG film.
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Results |
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). The antibody was first characterized using in vitro-translated proteins. In vitro translation of Pitx2 and the related Pitx1 proteins in the presence of 35S-Met yielded radiolabeled bands of the expected size (30 and 35 kDa, respectively) and equal intensity (Fig. 1B, left panel). The translation products of reactions performed in parallel and in which 35S-Met was omitted were subjected to Western blot analysis using the anti-Pitx2 polyclonal serum. As shown in the central panel of Fig. 1B, a 30-kDa band was observed in Pitx2 in vitro translation reactions and not in control reactions. The Pitx1 protein was not recognized by the antibody. Furthermore, the Pitx2-specific band disappeared when the antibody was preincubated with an excess of the peptide used for immunization (Fig. 1B, right panel).
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, left panel). Some level of nonspecific binding was, however, observed in Cos-7 cells because a signal at higher molecular mass (about 60 kDa) was also detected and remained present when the adsorbed antibody was assayed in parallel (Fig. 1C, right panel). Western blot analysis of Cos-7 cells transfected with the L54Q, T68P, R69H, R84P, and R91P mutants revealed a band of 30 kDa and equivalent intensity to that of wild-type Pitx2 (Fig. 1D), indicating that the level of expression of the mutants in mammalian cells is comparable with that of wild-type Pitx2.
Pitx2 mutants retain DNA-binding capacity
Wild-type and mutant Pitx2, as well as Pitx1, were produced in in vitro translation reactions in the presence of 35S-Met. Similar amounts of wild-type and mutant Pitx2 were generated in this assay as shown by direct visualization of the radiolabeled proteins by SDS-PAGE (Fig. 2A) as well as by Western blot analysis (data not shown). Accordingly, equal amounts of recombinant wild-type and mutant Pitx2 proteins were subsequently used to compare their DNA-binding activities. In agreement with other studies, wild-type Pitx2 protein obtained from in vitro translation reactions formed a specific complex with the known bicoid CE3 sequence of the POMC promoter (Fig. 2B, lane 2). Pitx2 binding was competed by CE3 itself (lanes 3–7) but not by the unrelated Sp1-like double-stranded oligonucleotide (lanes 8 and 9) and was totally abolished by addition of the anti-Pitx2 antiserum (lane 11), whereas the preimmune serum had no effect (lane 12). For comparison, the antiserum had no effect on Pitx1 binding (lanes 13–15). Under the same conditions, the five Pitx2 mutant proteins demonstrated binding to the CE3 probe, which was also prevented by preincubation with the Pitx2 antiserum (Fig. 3A). Increasing volumes (1–10 µl) of the in vitro translated proteins were then used to further compare the binding properties of the five mutants. As shown in Fig. 3B, no binding could be detected in any cases with less than 5 µl of the translation reactions. In the case of wild-type Pitx2, as well as with the L54Q and R84W mutants, a complex was detected with 5 µl translated products, and the signal intensity increased at 7 and 10 µl. Binding of the T68P and R91P mutants was observed with 7–10 µl translation products, and 10 µl were necessary to achieve detectable binding of the R69H. Superimposable variations in the binding capacities of the different mutants were obtained when nuclear extracts from transfected Cos-7 cells were used for EMSAs (data not shown). Altogether these results show that although ponctual mutations in the bicoid homeodomain of Pitx2 can lead to variability in binding efficiency, all Pitx2 mutants keep their capability to bind the CE3 target.
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, binding of wild-type Pitx2 and also of the five mutants to the CE3 probe were completed by addition of a 100-fold molar excess of oligonucleotide containing the Pitx elements of the PRL promoter B1 and B2 but not by the unrelated Sp1 oligonucleotide. These results indicated that the Pitx2 wild-type and mutant forms were able to bind the Pitx elements of the hPRL promoter.
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). In these experiments, wild-type Pitx2 was able to activate the promoter by about 7-fold. In contrast, when the mutant L54Q, T68P, R69H, R84W, and R91P Pitx2 expression vectors were transfected, no transactivation of the promoter construct was observed. Similarly, the five mutants were inactive in transactivating two other human pituitary gene targets, the hGH, and hPit-1 promoters. By comparison, wild-type Pitx2 efficiently transactivated these constructs (3- and 11-fold, respectively) and had no significant effect on a thymidine kinase promoter (Fig. 5).
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). When the five Pitx2 mutants were assessed for their cooperativity with Pit-1 on the hPRL promoter, however, none further increased transactivation, compared with the level of induction observed with the Pit-1 expression vector alone (Fig. 6B).
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). In contrast, the R91P mutant almost abolished the wild-type-Pitx2-induced activation of the hPRL promoter because 70% of the wild-type-Pitx2 effect was reversed by cotransfection of an equal ratio of the R91P construct (Fig. 7B). This decrease was not due to saturation of the Pitx2-binding sites on the target genes because when double amounts of wild-type-Pitx2 construct alone were transfected, a significant 2-fold increase of the Luc activity was observed (Fig. 7B). Furthermore, an inhibitory effect of the mutant was also observed on the Pitx2/Pit-1 synergistic activation of the hPRL-164/Luc. Addition of the Pitx2 R91P mutant to the combination of Pit-1 and Pitx2 indeed prevented the synergistic activation of Luc activity (Fig. 7C).
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, cotransfection experiments of the hPRL-164/Luc construct along with Pitx1 and the R91P mutant also led to a reverse effect of Pitx1 alone, indicating that R91P also acts as a dominant inhibitor of Pitx1. Finally, transfection of R91P also inhibited the Pitx2-induced activation of the GH- and Pit-1/Luc constructs but not that of the pTKLuc construct (data not shown).
The R91P mutant inhibits pituitary promoters basal activities in GH4C1 cells
We next asked whether the transactivating properties of the Pitx2 R91P mutant are similar in the context of cells endogenously expressing Pitx2. To address this question, we used as a model the pituitary somatolactotroph GH4C1 cell line. The expression of the Pitx2 gene and protein was assessed by Northern and Western blot analysis. The use of a probe located in the 3'-UTR region of the rat Pitx2 gene, common to the known three isoforms of Pitx2, revealed a band of about 2.2 kb in GH4C1 cells (Fig. 8A). Gonadotroph T3 cells were also positive. Northern blot analysis was not sensitive enough to clearly evidence Pitx2 transcripts in AtT20 cells (Fig. 8A), but in agreement with other report (18), a low level of Pitx2 gene expression could be detected in these cells by RT-PCR (data not shown). Besides the 60-kDa nonspecific band similar to that previously observed in Cos-7 cells, the Pitx2 antibody revealed in GH4C1 three specific bands, with a Mr of 30–36 kDa, which most probably correspond to the three Pitx2 known isoforms (Fig. 8B). These bands, which disappeared when the antibody was preincubated with the Pitx2 immunizing peptide, were also present in T3 cells but not in AtT20 cells (Fig. 8B).
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). In these cells, which express the PRL, GH, and Pit-1 genes, the basal activities of the respective promoter constructs were high. Although the L54Q, T68P, R69H, and R84W mutants did not produce any significant variation of the various promoter basal activities (Fig. 9A), expression of the R91P mutant significantly reduced basal activities of the hPRL, hGH, and hPit-1/luc constructs by 30–50%. This inhibition was dose dependent, as demonstrated in panel B for the hPRL/Luc construct by transfecting increasing amounts of R91P mutant plasmid DNA. These results were not due to overexpression of the mutant vs. endogenous Pitx2 protein because a similar 40–50% reduction was observed when an equal ratio of wild-type to mutant constructs DNA was cotransfected in GH4C1 cells, as shown in Fig. 9C. Finally, R91P did not produce any significant effect on a pTKLuc construct (data not shown). Altogether these results indicate that R91P behaves as a dominant negative factor with respect to the three pituitary-specific promoters and significantly reduces their basal activities in GH4C1, probably by interfering with the action of endogenous factors.
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Discussion |
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-glycoprotein subunit, LHß, FSHß, PRL, and GH, was shown to be activated by both Pitx2 and Pitx1, suggesting a role for these Pitx factors in regulating pituitary differentiated functions. Transcription factor mutations include both loss-of-function mutations (which can be either recessive or dominant, depending on whether one functional copy of the gene can encode sufficient active protein to induce transcription) and dominant negative mutations (those in which the mutant protein interferes with the action of the wild-type protein). In the case of Pitx2, mutations are found sporadically or are inherited as autosomal-dominant traits (14). Our data clearly showed that the five Pitx2 mutations are functionally defective with respect to the three pituitary target genes studied. Indeed all five mutants were totally unable to transactivate human PRL, GH, and Pit-1 promoter constructs. This impaired transactivation could not be explained by differential levels of protein expression because Western blot analysis of transfected mammalian cells confirmed equivalent expression of wild-type and mutant proteins. The loss of function of the Pitx2 mutants does not appear to result from total loss of binding either, as shown by EMSA, but rather from a failure to interact with the transcriptional machinery and activate transcription.
These results also indicate that the DNA-binding and transcriptional activities are distinct within the homeodomain of this transcription factor. The Leu54 located at the position 16 in the homeodomain stands in the first helix, which does not make any contacts with the DNA. It is highly conserved among bicoid homeodomains (34), and its replacement by a Gln might be critical for the correct folding of the protein. The Arg84 (at position 46) is on the third helix, known to be the DNA recognition helix, but comparison with the three- dimensional DNA complexed crystal structure of the related engrailed homeodomain (35) gave no indication about a critical role of this residue in the recognition of the target site or in the stabilization of the protein-DNA complex. Our results are in accordance with these data because we observed an equivalent level of binding with L54Q, R84W, and Pitx2. On the other hand, Arg91 at position 53 of the homeodomain occurs in every one of the higher eukaryotic homeodomains and stands on the hydrophilic face of the third helix, which fits directly into the major groove (35). Replacement of Arg91 on the hydrophilic face of helix three with a nonconservative hydrophobic proline residue represents a substitution highly disruptive for the -helix, which may interfere with stability of the protein-DNA complex. Similarly, the Arg69, at position 31 of the homeodomain is the only residue in the second helix that provides a DNA contact and might be also critical in the stabilization of the folded protein-DNA complex. This explains that although R91P and R69H conserved DNA-binding activity, higher protein amounts were necessary to achieve binding comparable with that of wild-type Pitx2. Finally, the Pro68 lies at position 30 on the second helix. Several amino acids can be located at this position without affecting the specificity in homeodomain protein (23).
Two studies with slightly different results have previously addressed the question of the functional activity of Pitx2 mutants. Our results corroborate the initial report of Amendt et al. (23) who showed that the T68P mutant was transcriptionally inactive despite reduced but conserved DNA-binding capacity. In contrast, Kozlowski and Walter (36), using a CE3-pGL3 synthetic promoter construct as target gene, were able to detect some residual transactivation activities for the Pitx2 mutants in the context of HeLa cells, with the R91P mutant retaining the lowest amount of residual activity relative to wild-type Pitx2. They concluded that although they were stable in mammalian cells, the Pitx2 mutants had lost most, if not all, of their DNA-binding activities (36). Although the origin of these quantitative differences in EMSA cannot totally be clarified, they are likely related to differences in binding conditions, protein preparation, and/or sequence of the probe.
In our conditions, however, in both CV-1 and GH4C1 cells, the L54Q, T68P, R69H, and R84W mutants were transcriptionally inert, indicating that the probable action of these loss-of-function mutations was through haplo insufficiency in vivo, i.e. a condition in which the amount of functional protein produced from a single functional copy of the gene is not enough to achieve proper activation of the target. This hypothesis is consistent with the mice models of Gage et al. (18), establishing that heterozygotes for hypomorphic allele for Pitx2 is sufficient to disturb the development of some organs in the mice and mimic some features of ARS phenotype. Of note, the arrest of pituitary development at the committed Rathke’s pouch stage reported in Pitx2 gene targeting studies was observed only in -/- embryos, i.e. an embryonic lethal phenotype (16, 18, 19).
The R91P mutation leads to an interesting Pitx2 mutant, which dominantly inhibited the function of the wild-type factor despite conserved DNA binding. Because homeoproteins are expected to interact with other transcription factors, the potential disruption of the helix 3 structure in the R91P mutant may affect the interaction of the factor with a limiting cofactor when bound to DNA. Gel shift experiments using the CE3 element and the B1 and B2 elements of the PRL promoter as probes or competitors demonstrated conserved DNA binding for all five mutants to the Pitx elements of the PRL promoter, yet only the R91P mutant inhibited the function of the wild-type factor on this promoter. We took precaution in cotransfection assays to use relatively small amounts of Pitx2 expression vectors, to prevent promoter competition and saturation of binding sites. This probably explains why, although they bind DNA, the four loss-of-function mutants do not show competition for wild-type factor on the PRL gene promoter. In contrast, under the same conditions, the R91P inhibited in a dominant manner the effect of the wild-type factor. Moreover, the R91P dominant negative effects were also observed on the Pitx2/Pit-1synergistic activation of the hPRL promoter and on the transactivation by Pitx1, which is known to share with Pitx2 DNA-binding and transactivating characteristics (29). The functional significance of these results was further demonstrated in transfection experiments performed in the somatolactotroph GH4C1 pituitary cell line, which endogenously express Pitx2 as well as Pitx1 and Pit-1. In accordance with the expression of the these factors, the basal activities of the hPRL, hGH, and hPit-1 promoters were high in these cells, and the R91P mutant reduced the activity of these promoters in a dose-dependent manner. Therefore, although we do not provide direct evidence for impaired interaction of Pitx2 with cofactors, the analysis of the binding and transactivation characteristics of the Pitx2 mutants on natural promoters favor the hypothesis of altered association with cofactors of the transcriptional complex rather than a competition for binding sites. Depending on the conformational changes induced by the mutations, the corresponding mutant proteins could either functionally block partners (dominant negative mutant) or be unable to recruit the transcriptional machinery (loss-of-function mutants).
In the past few years, several mutations of pituitary transcription factors affecting not the binding but rather interactions with cofactors of the transcriptional complex have been described. Thus, in the case of Pitx2, two other dominant mutations of Pitx2 have been described. A new mutation (K88E) in Pitx2 homeodomain was reported in a patient with ARS and was characterized in vitro using nonpituitary cell lines and pituitary target genes (37). Similar to R91P, this mutant acts in a dominant manner to repress wild-type Pitx2 activity, although probably through a different mechanism. Indeed, the K88E was totally defective in binding DNA, and its dominant negative effects were observed only in the presence of Pit-1 (37). Priston et al. (38) identified a ponctual mutation of Pitx2 (V45L) that retained almost all its DNA-binding capacity and showed an increased transactivation activity, compared with wild-type Pitx2. In the past few years, mutations have been described in several other genes encoding pituitary transcription factors such as Pit-1 (39), Prop1 (40), Lhx3 (41), or Tpit (7, 8). We recently described a Pit-1-recessive mutation, F135C, located in the hydrophobic region of the POU-specific domain (42), for which molecular modelization studies supported the hypothesis of altered interactions with cofactors of the transcriptional complex (43). In contrast, an heterozygous mutation of Pit-1, L216G, had no effect on DNA binding, and it did not directly interfere with GH or PRL gene expression. Instead it blocked retinoic acid signaling on the Pit-1 gene enhancer as a result of impaired interaction between Pit-1 and the retinoic acid receptor on DNA (44). Study and comparison of interactions of wild-type and dominant negative forms of these pituitary transcription factors with others proteins could be interesting to give precision to the involvement of each of these interactions in the differentiated somatolactotroph cells.
In conclusion, the functional analysis of Pitx2 mutants in a pituitary context using pituitary target genes revealed that both loss-of-function and dominant negative effects could mediate the disruptive effects of the mutants. Furthermore, our results obtained in the GH4C1 cell line confirm that Pitx2 participates in the regulation of the specific functions of the somatolactotroph cells such as transactivation of pituitary hormone gene promoters. The development of the pituitary gland has provided a particularly useful model for exploring the complex transcriptional mechanisms underlying the specification and maintenance of differentiated cells types in mammalian organogenesis. Recent data have underlined the key role of combinatorial interactions of Pitx2 with pituitary transcription factors. Further studies using dominant negative mutants of Pitx2 as tools in this instructive model system should help in understanding the detailed mechanisms mediating Pitx2 functions and its role in the differentiated cell.
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Acknowledgments |
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Footnotes |
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Abbreviations: ARG, Autoradiographic; ARS, Axenfeld-Rieger syndrome; CMV, cytomegalovirus; ß-gal, ß-galactosidase; h, human; Luc, luciferase; PMSF, phenylmethylsulfonyl fluoride; POMC, proopiomelanocortin; PRL, prolactin; SVF, fetal calf serum; UTR, untranslated region.
Received November 26, 2001.
Accepted for publication April 19, 2002.
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 M. P. Smidt, S. M. Smits, H. Bouwmeester, F. P. T. Hamers, A. J. A. van der Linden, A. J. C. G. M. Hellemons, J. Graw, and J. P. H. Burbach Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3 Development, March 1, 2004; 131(5): 1145 - 1155. [Abstract] [Full Text] [PDF]
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D. Schausi, C. Tiffoche, and M.-L. Thieulant Regulation of the Intronic Promoter of Rat Estrogen Receptor {alpha} Gene, Responsible for Truncated Estrogen Receptor Product-1 Expression Endocrinology, July 1, 2003; 144(7): 2845 - 2855. [Abstract] [Full Text] [PDF]
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 I. Saadi, A. Kuburas, J. J. Engle, and A. F. Russo Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome Mol. Cell. Biol., March 15, 2003; 23(6): 1968 - 1982. [Abstract] [Full Text] [PDF]
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 C. E. Keegan and S. A. Camper Mouse knockout solves endocrine puzzle and promotes new pituitary lineage model Genes & Dev., March 15, 2003; 17(6): 677 - 682. [Full Text] [PDF]
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 M.-H. Quentien, I. Manfroid, D. Moncet, G. Gunz, M. Muller, M. Grino, A. Enjalbert, and I. Pellegrini Pitx Factors Are Involved in Basal and Hormone-regulated Activity of the Human Prolactin Promoter J. Biol. Chem., November 8, 2002; 277(46): 44408 - 44416. [Abstract] [Full Text] [PDF]
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T. C. Voss and R. N. Day Editorial: Pitx-2 Mutants and Somatolactotroph Gene Regulation--Deciphering the Combinatorial Code Endocrinology, August 1, 2002; 143(8): 2836 - 2838. [Full Text] [PDF]
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