Pancreatic-duodenal homeobox 1 regulates expression of liver receptor homolog 1 during pancreas development

Abstract

Liver receptor homolog 1 (LRH-1) and pancreatic-duodenal homeobox 1 (PDX-1) are coexpressed in the pancreas during mouse embryonic development. Analysis of the regulatory region of the human LRH-1 gene demonstrated the presence of three functional binding sites for PDX-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed that PDX-1 bound to the LRH-1 promoter, both in cultured cells in vitro and during pancreatic development in vivo. Retroviral expression of PDX-1 in pancreatic cells induced the transcription of LRH-1, whereas reduced PDX-1 levels by RNA interference attenuated its expression. Consistent with direct regulation of LRH-1 expression by PDX-1, PDX-1(-/-) mice expressed smaller amounts of LRH-1 mRNA in the embryonic pancreas. Taken together, our data indicate that PDX-1 controls LRH-1 expression and identify LRH-1 as a novel downstream target in the PDX-1 regulatory cascade governing pancreatic development, differentiation, and function.

FIG. 1.

LRH-1 and PDX-1 are coexpressed in the developing pancreas. (A) LRH-1 hybridization of Northern blots containing polyadenylated mRNA from E7, E11, E13, and E17 mouse embryos. Murine LRH-1 mRNA expression was detected at stage E7. (B) In situ hybridizations on adjacent sections of E7.5, E9.5, E13.5, and E16.5 mouse embryos. Coexpression of LRH-1 and PDX-1 was observed in the pancreas (panc) and in the intestine (int). LRH-1 was also detected in the yolk sac endoderm (YSE), the embryonic endoderm (E), the neural ectoderm (NEc), the amnion (A), the chorion (Ch), the parietal endodermal cells attached to the Reichert's membrane (PE), the liver (liv), the salivary gland (sg), and the spinal cord (sc). Counterstaining with toluidine blue is depicted on the left panel.

FIG. 2.

Identification of PDX-1 binding sites in the human LRH-1 promoter. (A) Computational analysis of a ±2-kb fragment of the regulatory region of the human LRH-1 gene demonstrating the presence of five potential binding sites for PDX-1 (Pdx 1 to 5) homologous to the PDX-1 consensus binding site present in the rat insulin promoter. In the 293T and MiaPaca-2 cell lines, cotransfection of an expression vector encoding murine PDX-1 and a luciferase reporter containing ±2 kb of the human LRH-1 promoter induces human LRH-1 promoter activity compared to an empty expression vector. (B) Alignment of ±500 bp of the human, mouse, and rat LRH-1 promoters. The highly conserved site 5 is indicated in gray. Sequence differences are boxed. Transcription initiation sites are indicated by arrows.

FIG. 3.

Human LRH-1 promoter is responsive to PDX-1 in vitro. (A) EMSA showing PDX-1 binding to three out of the five potential PDX-1 binding sites, Pdx 2, 3, and 5. Binding of PDX-1 to the PDX-1 consensus site in the insulin promoter (Ins) is shown as a positive control. (B) EMSA showing binding of in vitro-synthesized PDX-1 (IVT PDX-1, lanes 2, 6, and 10) and NIT-1 insulinoma nuclear extracts (NIT-1 NE, lanes 3, 7, and 11) to the Pdx 2, 3, and 5 sites of the human LRH-1 promoter. Incubation of NIT-1 nuclear extracts with an anti-PDX-1 antibody decreased the intensity of the retarded band, demonstrating the specificity of the binding (lanes 4, 8, and 12). No binding could be observed with nonprogrammed reticulocyte lysate (lanes 1, 5, and 9). (C) EMSA with the Pdx 2, 3, and 5 sites of the human LRH-1 promoter as probes. Increasing amounts of unlabeled wild-type oligonucleotides competed for binding with the radiolabeled Pdx 2, 3, and 5 oligonucleotides (lanes 5 and 6), whereas competition with the respective mutated oligonucleotides (mutations in sequences are indicated in panel D) did not displace the protein-DNA complex (lane 7). Incubation with a PDX-1 antibody abrogated the formation of the retarded band (α-PDX-1, lane 9), whereas no modification in PDX-1 binding was observed with a preimmune serum (PI, lane 8). No binding of PDX-1 to radiolabeled Pdx 2, 3s and 5 mutated oligonucleotides was observed (Pdxmut, lane 10). (D) Activity generated from the pGL3-LRH-1-2260 reporter cotransfected in 293T cells with an empty vector or an expression vector encoding murine PDX-1. Various unilateral deletion mutations as well as site-directed mutations in the putative PDX-1 sites of the human LRH-1 promoter indicated that the DNA region located between −2260 and −1394 confers responsiveness to PDX-1.

FIG. 4.

PDX-1 binds to the LRH-1 promoter in pancreatic cell lines. (A) RT-PCR experiment showing coexpression of PDX-1 and LRH-1 in two human pancreatic cell lines, MiaPaca-2 (MiaP) and Panc-1. A mock PCR was performed with water as the template and is shown as a negative control for PCR amplification of PDX-1 (lane 5) and LRH-1 (lane 6). (B) Immunoblot demonstrating coexpression of PDX-1 and LRH-1 proteins in the MiaPaca-2 (lane 1) and Panc-1 (lane 2) pancreatic cell lines. (C) Schematic representation of the 5′ region of the human LRH-1 gene. The Pdx 2, 3, and 5 binding sites are indicated. Transcription (arrow, TXN) and translation (ATG) initiation sites are shown. Amplimers are highlighted by the arrows, whereas the black line depicts the 320-bp PCR product. (D) ChIP assay demonstrating binding of endogenous PDX-1 to the human LRH-1 promoter. Cross-linked chromatin from Panc-1 and MiaPaca-2 cells was incubated with antibodies against PDX-1 (lane 3), acetylated histone H3 (lane 4), acetylated histone H4 (lane 5), or preimmune serum (lane 2, PI). Immunoprecipitates were analyzed by PCR with primers specific for the human LRH-1 promoter, as shown in panel B. A positive (input chromatin, lane 1) and a negative (with water as a template for PCR, lane 6) control are shown. (E) ChIP assay demonstrating increased binding of the LRH-1 promoter by PDX-1 in the murine LTPA pancreatic cell line stably overexpressing PDX-1. Cross-linked chromatin from LTPA cells transduced with an empty (−) or PDX-1-expressing (PDX-1) retroviral vector were analyzed by ChIP as described for panel C. The murine LRH-1 promoter bound more PDX-1 (lane 3) and acetylated histones H3 (lane 4) and H4 (lane 5) in PDX-1-expressing LTPA cells relative to control cells.

FIG. 5.

PDX-1 binds to the LRH-1 promoter during pancreatic development. (A) Schematic representation of the 5′ region of the mouse LRH-1 gene. The Pdx 5 binding site is indicated. Transcription (arrow, TXN) and translation (ATG) initiation sites are shown. Amplimers are highlighted by the arrows, whereas the black line depicts the 360-bp PCR product. (B) Scheme depicting the microdissection of the gut and pancreas used for the in vivo ChIP on E13.5 mouse embryos. (C) In vivo ChIP assay demonstrating binding of endogenous PDX-1 to the murine LRH-1 promoter in the gut and pancreas from E13.5 embryos. Chromatin from gut and pancreas was incubated with antibodies against PDX-1 (lane 3), acetylated histone H3 (lane 4), or preimmune serum (lane 2). Immunoprecipitates were analyzed by PCR with primers specific for the mouse LRH-1 promoter. A negative control with water as the template for PCR is shown in lane 5. Input chromatin was used as a positive control in lane 1. (D) Schematic representation showing the microdissection of the isolated organs used for in vivo ChIP on E16.5 (E) and E17.5 (F) embryos. (E) In vivo ChIP assay demonstrating occupancy of the murine LRH-1 promoter by PDX-1 in pancreas but not gut or liver of E16.5 embryos. Cross-linked chromatin from pancreas, gut, and liver was prepared for the ChIP assay as described for panel B. Immunoprecipitates were analyzed by PCR with primers specific for the murine LRH-1 and murine GAPDH (positive control) promoters. (F) PDX-1 does not occupy the LRH-1 promoter in endodermal tissues dissected from E17.5 mouse embryos. Cross-linked chromatin from the pancreas, gut, and liver of E17.5 embryos was analyzed by ChIP as described for panel D.

FIG. 6.

PDX-1 regulates the expression of LRH-1 in vivo. (A) Expression of PDX-1 and LRH-1 analyzed by RT-PCR (mRNA) and immunoblotting (protein) in LTPA cells overexpressing PDX-1 (PDX-1) or not (−) by retroviral infection. Reverse transcription reactions were performed with (RT+) or without (RT−, negative control) reverse transcriptase. Subsequent PCR was performed with primers specific for PDX-1, LRH-1, and 36B4. Immunoblotting was performed with specific antibodies raised against PDX-1, LRH-1, and β-actin. The intensity of the signals was quantitated by phosphorimager analysis, and the induction was determined after normalization to β-actin signals. (B) Decreasing PDX-1 levels by RNAi reduces LRH-1 expression. Immunoblotting of protein isolated from control LTPA cells (−), LTPA cells retrovirally overexpressing PDX-1 (PDX-1), or MiaPaca-2 cells, all transfected with an empty RNAi vector (−, lanes 1, 3, and 5) or an RNAi vector targeting the PDX-1 mRNA (RNAi, lanes 2, 4, and 6). The induction was calculated as described for panel A. (C) Expression of LRH-1 in transversal (upper panel) and sagittal (lower panel) sections of PDX-1^+/+ and PDX-1^−/− mouse embryos from stage E9.5 analyzed by in situ hybridization. LRH-1 expression was significantly stronger in pancreas (panc) and liver (liv) of 2 PDX-1^+/+ relative to PDX-1^−/− tissues.

FIG. 7.

Position of LRH-1 as a central part of a transcriptional network in pancreatic development. Scheme depicting the transcription factors involved in pancreatic development acting up- and downstream of LRH-1. Arrows and stop symbols show positive and negative regulation, respectively.