fushi tarazu factor 1 (FTZ-F1, NR5A3) is a member of the nuclear receptor superfamily that regulates the transcription of the homeobox fushi tarazu (ftz
) gene during early development (30
). The best-characterized FTZ-F1 homologue is steroidogenic factor-1 (SF-1, NR5A1), a nuclear receptor expressed in particular regions of the steroidogenic organs, the hypothalamus, and the pituitary gonadotropic cells, where it controls critical developmental and physiological processes (48
). Liver receptor homolog 1 (LRH-1, NR5A2) has been characterized as a paralogue of SF-1 in tissues of endodermal origin, such as liver, pancreas, and intestine. LRH-1 was first cloned in the mouse (GenBank accession number M81385 ), and several orthologues have subsequently been cloned in different species, including Xenopus laevis
(FTZ-F1-related receptor xFF1) (15
), chicken (orphan receptor 2.0) (29
), rat (fetoprotein transcription factor) (19
), zebrafish (FTZ-F1-related receptor zFF1) (35
), frog (Rana rugosa
), and human (pancreas homologue receptor 1) (4
); human fetoprotein transcription factor (18
); human B1-binding factor (34
); and Cyp7a promoter binding factor (43
In the adult animal, LRH-1 has a critical function in diverse pathways controlling cholesterol homeostasis, as evidenced by its role in the control of the expression of cholesterol 7α-hydroxylase (Cyp7A1), the rate-limiting enzyme of the bile acid biosynthesis pathway (37
), sterol 12α-hydroxylase (Cyp8B1), involved in cholic acid synthesis (9
), multidrug resistance protein 3, implicated in the enterohepatic circulation of bile salts (24
), and the cholesteryl ester transfer protein (38
) and scavenger receptor class B type I (50
), two key players in reverse cholesterol transport.
Besides its role in metabolism, LRH-1 also controls the expression of a number of developmental genes, such as α-fetoprotein, a marker of early liver development (19
), and the transcription factors HNF-3β (49
), HNF-4α, and HNF-1α (47
), which coordinate hepatic developmental gene expression. Conversely, the expression of the mouse LRH-1 gene is under the control of the transcription factors GATA, Nkx, basic helix-loop-helix factors, and HNF-4α (47
) whereas the human LRH-1 gene is regulated by HNF-3β and HNF-1 (65
), all transcription factors involved in developmental control of gene expression. These findings establish a critical role for LRH-1 in hepatic development and homeostasis, yet the contribution of LRH-1 to the formation and function of the pancreas, a tissue in which LRH-1 is abundantly expressed, is still poorly understood.
PDX-1 is first detected in mouse embryos at embryonic day 8.5 (E8.5), and its expression is localized throughout the pancreas during embryonic development (45
). During adulthood, PDX-1 is predominantly expressed in the β-cells of the islets, where it regulates directly or indirectly the expression of genes such as insulin (45
), glucokinase (62
), islet amyloid polypeptide (52
), and the glucose transporter type 2 (61
). This transcriptional regulation occurs via the binding of monomeric PDX-1 through a GTAATC consensus site. PDX-1 is also found in pancreatic ductal and acinar cells, where it controls the expression of elastase I as a heterodimeric complex with two homeodomain proteins, PBX-1b and MRG1 (58
In this study, we characterized the developmental regulation of the LRH-1 gene. Most importantly, we show here that LRH-1 and PDX-1 are coexpressed during pancreatic development and that LRH-1 expression is regulated by PDX-1, both in vitro and in vivo. Altogether, our data suggest that LRH-1 is a major player in pancreatic development and homeostasis.
MATERIALS AND METHODS
The anti-PDX-1 polyclonal rabbit antibody was raised against amino acids 269 to 284 (SPQPSSIAPLRPQEPR) of the murine PDX-1 protein. Validation of the antibody was performed with the immunogenic peptide as a competitor in electrophoretic mobility shift assays (EMSAs) and immunoprecipitation (data not shown). Anti-acetylated H3 and H4 antibodies were purchased from Euromedex (Souffelweyersheim, France). Anti-LRH-1 monoclonal antibody H2325 was produced with the baculovirus gp64 display system (Invitrogen, Carlsbad, Calif.). The human LRH-1 cDNA encoding amino acids 161 to 280 was amplified by PCR and ligated into the surface glycoprotein gp64 gene of Autographa californica
multiple nuclear polyhedrosis virus to create a fusion protein expressed on the viral surface. The monoclonal antibody was produced exactly as described previously (59
In situ hybridizations.
Wild-type embryos from stages E7.5 to E16.5 were directly embedded in Cryomatrix (Shandon, Pittsburgh, Pa.). In situ hybridizations were performed on 10-μm cryosections with 35
S-labeled antisense RNA probes as described previously (10
). Negative controls were performed in parallel with sense RNA probes for murine LRH-1 and murine PDX-1 (data not shown). PDX-1−/−
mice were genotyped as described previously (26
Cloning of human LRH-1 promoter and mutagenesis.
The human LRH-1 promoter was cloned with the Genome Walker kit (Clontech Laboratories Inc., Palo Alto, Calif.). PCR amplifications were performed according to the manufacturer's instructions with primers AP-1 (5′-GTAATACGACTCACTATAGGGC-3′) and JSA 55L (5′-GTGCAGCTTGTCAAATTTCGTGGCCTTGGG-3′) and primers AP-2 (nested to AP-1, 5′-ACTATAGGGCACGCGTGT-3′) and JSA 54L (nested to JSA 55L, 5′-CCCTGGACTCTGTACTTTTTCCAACATTAG-3′). Four major bands (600 bp, 1.2 kb, 1.5 kb, and 2.4 kb), differing from their 5′ ends, were purified on agarose gels with the Qiaquick gel extraction kit (Qiagen, Hilden, Germany) and subcloned into the T/A cloning vector pTAdv (Clontech). Positive clones were used to PCR amplify the promoter fragment with AP-1 (MluI site) and JSA 74L (BglII site, 5′-GAAGATCTTCCATGATGGTTTCTAATCAGA-3′), and PCR products were then purified on a 1% agarose gel and cloned into the pGL3-basic vector (Promega Life Science, Madison, Wis.) digested with MluI/BglII.
The different pGL3-human LRH-1 promoter constructs were sequenced and used in transient transfections. The promoter region was analyzed on the web site http://transfac.gbf.de/
with programs designed for promoter and transcription binding site predictions (Matinspector Transfac version 2.2). PDX-1 binding sites were identified and mutations were performed with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) and the following forward primers: mutPdx2, 5′-CCCTAAATAAACCGG
TAGACCGTAAATTC-3′; mutPdx3, 5′-CAACCTGCATTTACTTGGCC
TAAAAGGAG-3′; and mutPdx5, 5′-TCACTTAAAGAGCGC
ATGTCTGCCAATGTTATC-3′ (italicized bases are mutated).
Cell culture and transient transfection assays.
Panc-1 (human pancreatic carcinoma), MiaPaca-2 (human pancreatic carcinoma), LTPA (murine pancreatic carcinoma), NIT-1 (mouse insulinoma), and 293gp and 293T (human embryonic kidney) cells were maintained according to the suppliers' instructions (American Type Culture Collection, Manassas, Va.). Cells were transfected according to the CaCl2
precipitation technique, and luciferase assays were carried out as described previously (50
). Expression plasmid pBKCMV (Stratagene) containing mouse PDX-1 cDNA was described previously (36
). Luciferase activity measurements were normalized for β-galactosidase activity to correct for differences in transfection efficiency, and graphed values represent the means of three independent experiments.
Electrophoretic mobility shift assays.
Double-stranded oligonucleotides containing the PDX-1 binding site described in the insulin promoter (12
) (consensus: Ins, 5′-GACCTTAATGGGCCAAACAGCA-3′) or present in the human LRH-1 promoter were labeled with T4 polynucleotide kinase, and EMSA binding reactions were performed as described previously (50
). For competition experiments, increasing amounts (from 50- to 200-fold molar excess) of unlabeled wild-type or mutated double-stranded oligonucleotide (Pdx 2, 5′-CTAAATAAATTAATAGACCGTA-3′; mutPdx 2, 5′-CTAAATAAACCGG
TAGACCGTA-3′; Pdx 3, 5′-TGCATTTACTTAATTTAAAAGG-3′; mutPdx 3, 5′-TGCATTTACTTGGCC
TAAAAGG-3′; Pdx 5, 5′-CTTAAAGAATTAATGTCTGCCA-3′; and mutPdx 5, 5′-CTTAAAGAGCGC
ATGTCTGCCA-3′) were included just before adding labeled wild-type PDX-1 oligonucleotide. Preimmune serum (negative control) or anti-PDX-1 serum was incubated for 30 min with in vitro-translated PDX-1 proteins or NIT-1 nuclear extracts before adding radioactive probes. DNA-protein complexes were separated by electrophoresis on a 4% polyacrylamide gel in 0.25× TBE (Tris-borate-EDTA) buffer at 4°C.
RNA extraction, Northern blotting, and RT-PCR.
RNA extraction and Northern blot analysis were performed as described previously (51
P-labeled full-length murine LRH-1 cDNA (accession number M81385 ) was used as a probe and hybridized to the mouse embryo multiple tissue Northern blot (Clontech). Reverse transcription of mRNA was performed at 37°C for 1 h with the Moloney murine leukemia virus reverse transcriptase (Life Technologies, Burlington, Canada) and random hexanucleotides, followed by a 15-min inactivation at 70°C.
Reverse transcription (RT)-PCR was performed with oligonucleotides specific to hPDX-1 cDNA (5′-GGCGCACCTTCACCACCACCTC-3′ and 5′-GCCGCCGCGCTTCTTGTCCT-3′), human LRH-1 cDNA (5′-TCAATGCCGCCCTGCTGGACTACACAATG-3′ and 5′-CTTCTTCCCCCTCCCCACTCCCCCAATCT-3′), murine PDX-1 cDNA (5′-CGACGACCCGGCTGGCGCTCACCTC-3′ and 5′-CTTCGCCCCCACCGCCCCCACTCG-3′), murine LRH-1 cDNA (5′-CTAGTTTGGATACTGGAGATTTTCA-3′ and 5′-ATAGGAGTAATTCACCATTTTAAAT-3′), human and murine acidic ribosomal phosphoprotein 36B4 (5′-ATGTGAAGTCACTGTGCCAG-3′ and 5′-GTGTAATCCGTCTCCACAGA-3′), and murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5′-GCTCACTGGCATGGCCTTCCGTGT-3′ and 5′-TGGAAGAGTGGGAGTTGCTGTTGA-3′) under the following conditions: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and a final extension step at 72°C for 10 min. Amplification products were loaded on a 1% agarose gel.
For in vitro chromatin immunoprecipitation (ChIP), Panc-1, MiaPaca-2, and retrovirus-infected LTPA cells were used. For in vivo ChIP, gut/pancreas combined, pancreas, liver, and gastrointestinal tracts were microdissected from 20 e13.5, E16.5, and E17.5 embryos. Cells and organs were fixed for 10 min and 16 h, respectively, in phosphate-buffered saline containing 1% formaldehyde and protease inhibitor cocktail and subsequently rinsed five times in phosphate-buffered saline. Cells were collected and centrifuged at 4°C for 5 min at 2,000 × g and resuspended in lysis buffer (50 mM HEPES [pH 7.5], 140 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail). After 30 min of lysis, three cycles of sonication (three pulses of 9 s, 20% amplitude for cells, 80% amplitude for organs) were performed to prepare DNA fragments ranging in size from 200 bp to 1,000 bp, followed by centrifugation for 10 min.
Supernatants were collected and cleared by incubation with protein A-Sepharose (2.5 mg), sonicated salmon sperm DNA (2 μg), and 10 μl of preimmune serum for 2 h at 4°C. Then 20 μl of supernatant was collected and used as the input. Immunoprecipitation was carried out overnight at 4°C with preimmune serum (negative control) or antibodies raised against PDX-1, acetylated histone H3 (dilution 1:200), or acetylated histone H4 (dilution 1:200). After centrifugations, washing, and elution, the cross-linking was reversed by heating the samples at 65°C overnight. DNA was then purified with the Qiagen PCR purification kit, and PCR was performed with primers 5′-GATTGCTTAAGTCCATGAGTCTGAGGTT-3′ and 5′-CAAAGTGCTGGAATTATAGGCGTGAG-3′, 5′-CAAGCAAGCTAATGCCTCTTCTAAC-3′ and 5′-CTGCAGCCCAGAGTGTGGAAAGTTG-3′, and 5′-AGTGCCAGCCTCGTCCCGTAGACAAAATG-3′ and 5′-AAGTGGGCCCCGGCCTTCTCCAT-3′ to amplify the human LRH-1, the murine LRH-1, and the murine GAPDH promoters, respectively. The ChIP assay was performed at least twice for each condition.
Virus production and infection were performed as described previously (50
). For retroviral infection specifically, the 293gp packaging cell line was transfected with Lipofectamine (Life Technologies), with 15 μg of empty control retroviral vector or retroviral vector containing the cDNA for murine PDX-1, and with 5 μg of the ecotropic vector SV-E-MLV-env, containing the Moloney murine leukemia virus envelope cDNA downstream of the simian virus 40 promoter-enhancer.
RNAi and immnuoblotting.
The RNA interference (RNAi) experiment was performed with the pSUPER RNAi system, following the manufacturer's instructions (DNAengine, Seattle, Wash.). Briefly, a double-stranded oligonucleotide targeting nucleotides 709 to 727 (5′-GATCCCCCCCGAGGAAAACAAGAGGATTCAAGAGATCCTCTTGTTTTCCTCGGGTTTTTGGAA-3′) of murine PDX-1 mRNA was cloned in the Bgl
II and Hin
dIII sites of the pSUPER vector. Transient transfection was carried out in 60-mm plates containing mock- and PDX-infected LTPA cells, with Lipofectamine (Life Technologies) and 10 μg of the empty pSUPER or PDX-1 RNAi vector. After 48 h, cells were harvested for whole-cell protein extracts, and Western blotting was performed as described previously (17
). RNAi experiments were repeated three times. Intensities of the LRH-1 and PDX-1 protein signals were quantified by phosphorimager analysis, and the induction was calculated after normalization to β-actin protein levels.
Temporally and spatially controlled gene expression patterns are the basis of tissue development and differentiation. Since LRH-1 is expressed during early pancreatic development, this orphan nuclear receptor could constitute an important chain in the gene regulatory cascade leading to pancreatic development. To identify the players involved in the regulation of LRH-1, we cloned and characterized the regulatory region of the human LRH-1 gene. Computational analysis of the human LRH-1, murine LRH-1, and rat LRH-1 promoters revealed the presence of consensus sequences for several ubiquitous transcription factors, such as AP-1, GATA-1, USF, and Sp1, but also highlighted the existence of binding sites for tissue-specific transcription factors, such as HNF-3β, C/EBPβ, and PDX-1.
Since LRH-1 and PDX-1 are coexpressed throughout pancreatic development, we evaluated whether PDX-1 could be a transcriptional regulator of LRH-1. PDX-1 was shown to activate the human LRH-1 promoter in transient transfections and to bind to specific DNA sequences in the LRH-1 promoter, both in vitro and in vivo. Changes in PDX-1 levels in cells, by either retroviral overexpression or RNAi-mediated inhibition, induced concomitant changes in murine LRH-1 expression. In vivo ChIP analysis furthermore demonstrated that PDX-1 regulates pancreatic LRH-1 expression until E16.5 of mouse pancreatic development. Although LRH-1 and PDX-1 are also coexpressed in the small intestine, PDX-1 is not bound to the murine LRH-1 promoter in this tissue, demonstrating that this regulation occurs only in a temporally and tissue-restricted fashion, i.e., in a well-defined time window (E8.5 to E16.5) during pancreatic development, but not in the rest of the gut.
PDX-1 null embryos (E9.5) were also examined in order to investigate the relevance of the role of PDX-1 in LRH-1 expression. Compared to wild-type embryos, PDX-1 null embryos show a decrease in LRH-1 expression in the pancreatic bud. Interestingly, we also observed lower levels of LRH-1 in the liver, a tissue where PDX-1 is not supposed to be expressed. Since the expression of PDX-1 begins at stage e8.5, it is possible that non-cell-autonomous effects might affect the expression of a number of genes critical in tissues that arise from the gut endoderm, such as the liver. The absence of PDX-1 could in fact alter the expression of a morphogen in the gut anlagen and affect the expression of a number of genes in the liver, as we observed here for LRH-1. Although the in vivo data go in the same direction as the in vitro data, they should be interpreted with caution in view of the complexity of the regulatory systems specifying development, the potential interference by other regulatory factors, and the difficulty in quantifying in situ hybridization experiments.
PDX-1 is a critical developmental transcriptional regulator in the pancreas and is itself controlled by a number of endodermal transcription factors. PDX-1 expression is under the control of Foxa2/HNF-3β, as shown by the characterization of HNF-3β−/−
differentiated embryoid bodies and of β-cell-specific HNF-3β conditional knock-out mice (11
). Mice homozygous for a null mutation of HNF-3β have severe problems in gastrulation, and mutant embryos do not develop beyond E8.5, suggesting that HNF-3β is a key factor for development (3
). However, conditional deletion of HNF-3β in adult hepatocytes shows that this factor is dispensable for normal adult liver function, demonstrating different roles in embryos and adults (57
Interestingly, LRH-1, which we show to be a PDX-1 target gene, regulates HNF-3β in vitro (49
), highlighting a potential autoregulatory loop involving HNF-3β, PDX-1, and LRH-1 (Fig. 7
). Although the relevance of the regulation of HNF-3β by LRH-1 needs to be demonstrated in vivo, regulatory loops involving transcriptional cascades could play important roles in establishing control circuits that govern pancreatic development. More generally, these data also suggest a role for LRH-1 as an eventual more global regulator of endodermal development, given the fact that LRH-1 controls HNF-3β expression in vitro and LRH-1−/−
embryos die at E7 (47
; J.-S. Annicotte, K. Schoonjans, and J. Auwerx, unpublished data). It is also interesting that the basic helix-loop-helix transcription factor Ptf1a, which was first described as an exocrine-specific protein involved in exocrine pancreatic development and islet architecture (28
), was recently shown to be crucial to orient undifferentiated foregut endoderm into the pancreatic fate (27
). This raises the possibility that LRH-1, which, like Ptf1a, is expressed in an exocrine-specific manner in the adult, might participate in the early steps of the pancreatic differentiation program.
The central position of LRH-1 in the transcriptional cascade controlling pancreatic development suggests that LRH-1 could also play an important role in homeostatic regulation during adulthood, as described previously for these other transcription factors that control endodermal development (reviewed in reference 55
). Several reports demonstrate that PDX-1 is required not only for pancreas development but also for normal β-cell function and insulin secretion (2
). Also, the homeodomain transcription factor HNF-1α, which regulates expression of PDX-1, LRH-1, Beta2/NeuroD, and HNF-4α, has been reported to control insulin secretion (32
). Mutations in the human HNF-1α gene are linked to maturity-onset diabetes of the young type 3 (8
). Moreover, in HNF-1α−/−
mice, PDX-1 and Beta-2/NeuroD levels are downregulated in islets in newborns and adults, confirming the importance of these factors in insulin production (54
). A recent study with compound heterozygous mutations for PDX-1+/−
further underscores the importance of PDX-1, HNF-1α, and HNF-3β in controlling glucose tolerance, glucose-stimulated insulin secretion, and islet architecture (53
Another player in this regulatory network is HNF-4α, whose promoter is under the control of LRH-1, HNF-1α, and PDX-1 (5
). Mutations in the human HNF-4α gene have been linked to maturity-onset diabetes of the young type 1 (64
). Furthermore, a mutation of the PDX-1 binding site in the HNF-4α P2 alternative promoter was identified in a large family with maturity-onset diabetes of the young (60
). This mutation cosegregates with diabetes and leads to decreased PDX-1 binding and transcriptional activation, suggesting that a transcriptional deregulation of PDX-1 target genes could be linked to maturity-onset diabetes of the young. The atypical nuclear receptor SHP has also been demonstrated to be a direct target gene of both LRH-1 and HNF-4α (22
). Interestingly, mutations in the SHP gene were again linked to diabetes (42
). Since all these transcription factors, which function either upstream or downstream of LRH-1, as highlighted in Fig. 7
, affect glucose homeostasis, it is tempting to speculate that LRH-1 could also participate in this process and alludes to the existence of similar transcriptional regulatory cascades in developing and adult pancreas.
One problem in concluding that LRH-1 also plays a role in the endocrine pancreas is the fact that, unlike the other transcription factors, LRH-1 is not expressed in β-cells but is confined to the exocrine pancreas in the adult mouse (no data exist on LRH-1 expression in human islets). LRH-1 could, however, still take part in β-cell differentiation and homeostasis, a phenomenon linked to its expression in ductal epithelial cells in adult pancreas (49
). Ductal cells are known to possess characteristics of pancreatic progenitor cells through their capacity to proliferate or to differentiate into endocrine β-cells (6
). Our observation might reinforce a potential link between LRH-1 in the exocrine pancreatic compartment and β-cell neogenesis. The exact role of LRH-1 in ductal cells hence warrants further investigation, and modulation of its activity might eventually open novel therapeutic strategies for the treatment of diabetes by in vitro differentiation of ductal cells into β-cells.
In conclusion, we demonstrate here that the expression of LRH-1 is controlled by the transcription factor PDX-1. LRH-1 might be a component of a transcriptional network involving PDX-1, HNF-1α, HNF-4α, and HNF-3β that determines pancreatic development and could also play a role in pancreas homeostasis in the adult. These data hence underscore the requirement for careful mechanistic and genetic studies to define whether LRH-1 contributes to diseases linked to pancreatic dysfunction, such as diabetes.