Retinoic acid repression of bone morphogenetic protein 4 in inner ear development - PubMed (original) (raw)

Retinoic acid repression of bone morphogenetic protein 4 in inner ear development

Deborah L Thompson et al. Mol Cell Biol. 2003 Apr.

Abstract

Bone morphogenetic protein 4 (BMP4) and retinoic acid are important for normal development of the inner ear, but whether they are linked mechanistically is not known. BMP4 antagonists disrupt semicircular canal formation, as does exposure to retinoic acid. We demonstrate that retinoic acid directly down-regulates BMP4 transcription in a mouse inner ear-derived cell line, and we identify a novel promoter in the second intron of the BMP4 gene that is a target of this regulation both in the cell line and in the mouse embryonic inner ear in vivo. The importance of this down-regulation is demonstrated in chicken embryos by showing that the retinoic acid effect on semicircular canal development can be overcome by exogenous BMP4.

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Figures

FIG. 1.

FIG. 1.

RAR expression. (A) Analysis of RARα and RARγ expression in 2B1 cells by Western blot analysis. (B) Schematic diagram of a prototypical RAR. Three RAR genes (α, β, and γ) encode receptors that are unrelated in their amino-terminal A and B domains but highly similar in their DNA and ligand binding domains (DBD and LBD, respectively).

FIG. 2.

FIG. 2.

TTNPB down-regulates BMP4 transcription in 2B1 cells. (A) RPA of BMP4 expression. 2B1 cells were cultured in the presence of the RAR-specific ligand TTNPB or the vehicle for up to 48 h. RNA was isolated, and expression of BMP4 RNA was quantified by RPA and phosphorimager analysis (normalized to cyclophilin as a control). Incubation with yeast RNA yielded no protected bands. The full-length (unprotected) probes are in the far right lane. Similar results were obtained in a second experiment. (B) Nuclear run-on assay of BMP4 transcription. 2B1 cells were cultured in the presence of the RAR-specific ligand TTNPB or the vehicle for 6 h. Nuclei were isolated, and the rate of BMP4 transcription was quantified in triplicate by nuclear run-on assay and phosphorimager analysis. Similar results were obtained in a second experiment. (C) TTNPB does not decrease the half-life of BMP4 RNA. 2B1 cells were cultured in the presence of the RAR-specific ligand TTNPB or the vehicle for 3 h, at which point actinomycin D was added. Cells were harvested at various time points (time zero represents the point of actinomycin D addition). The amount of BMP4 RNA was assessed by RPA with cyclophilin as a control. Incubation with yeast RNA yielded no protected bands. The full-length (unprotected) probes are in the far right lanes. (D) Semilog plot of the data in panel C (BMP4 normalized to cyclophilin) quantified by phosphorimager analysis. nt, nucleotides.

FIG. 3.

FIG. 3.

The TTNPB-mediated decrease in BMP4 expression does not require new protein synthesis. (A) Cycloheximide or the vehicle was added to cultures of 2B1 cells. Thirty minutes later, TTNPB or the vehicle was added and the cells were cultured for an additional 3 or 6 h. The amount of BMP4 RNA was assessed by RPA with cyclophilin as a control. (B) Graph of the data in panel A (BMP4 normalized to cyclophilin) quantified by phosphorimager analysis.

FIG. 4.

FIG. 4.

BMP4 transcription is driven by the 1B promoter and a novel promoter in intron 2. (A) Schematic diagram of the mouse BMP4 gene. Exon 1B is shown as a box with a dashed line near the 5′ end because, in 2B1 cells, this exon can be 21 or 279 bp. The diagram also indicates the locations of nested 5′ RACE primers complementary to exon 3 (primers 3.1R and 3.2R), as well as PCR primers used to detect transcripts driven by the 1A or 1B promoter (1AF plus 3.2R and 1BF plus 3.2R, respectively), a novel promoter located within intron 2 (i2F plus 3.2R), or all transcripts containing common coding exons 3 and 4 (3F plus 4R). (B) BMP4 promoter usage in 2B1 cells and whole-mouse embryo RNA analyzed by RT-PCR. RNA was isolated from 2B1 cells or an E9.5 whole-mouse embryo (WME). RT-PCR was performed with the intron-spanning primer pairs shown in panel A. No products were formed when the RT step was omitted (data not shown). (C) BMP4 promoter usage in 2B1 cells analyzed by RPA. 2B1 cells were cultured in the presence of TTNPB or the vehicle for 6 h. RNA was isolated, and expression of transcripts driven by the 1A, 1B, or intron 2 promoter was analyzed. Probes also were used to distinguish between the long and short versions of exon 1B-containing transcripts, and a probe for common coding exon 4 also was used. Cyclophilin served as a control. RNA from vehicle-treated cells was analyzed in lanes 1 to 5, and RNA from TTNPB-treated cells was analyzed in lanes 6 to 10. The full-length (unprotected) probes are in lanes 11 to 16.

FIG. 5.

FIG. 5.

TTNPB down-regulates the intron 2 and 1B promoters in transfected 2B1 cells. 2B1 cells were transfected with reporter plasmids in which luciferase is driven by 0.9 kb of the intron 2 promoter (top panel) or 1.8 kb of the 1B promoter (middle panel), along with an Rluc plasmid (as an internal control). Cells also received an RAR expression vector (or the empty vector [v]), as indicated. The cells were cultured with or without TTNPB for 2 days and then harvested for luciferase assays. Rluc was unaffected by TTNPB. Data are expressed as fold repression, which is defined as luciferase/Rluc for cells cultured without TTNPB divided by luciferase/Rluc for cells cultured with TTNPB. Results are the mean ± the standard error of the mean (n = 3). In the bottom panel, the transfection was as described for the upper two panels except that the reporter plasmid was 2xPal, in which the luciferase reporter is driven by a positive RARE. Data are expressed as fold induction, which is defined as luciferase/Rluc for cells cultured with TTNPB divided by luciferase/Rluc for cells cultured without TTNPB. Results are the mean ± the standard error of the mean (n = 3).

FIG. 6.

FIG. 6.

Retinoic acid (RA) down-regulates BMP4 transcripts driven by the intron 2 promoter in E10.5 mouse embryo otocysts. Day 10.5 postcoitus pregnant mice received at-RA (100 mg/kg) or the vehicle by gavage. Six hours later, the embryos were removed and the otocysts were microdissected. (A) Otocyst RNA was isolated, and a real-time RT-PCR was used to study BMP4 expression driven by the intron 2 promoter. β-Actin served as a control. The assay was run in triplicate with the higher dose (5 ng) of input RNA and in quadruplicate with the lower dose (1.25 ng). The graph shows the mean fluorescence ± the standard error of the mean at each cycle number. (B) Quantification of the data in panel A expressed as BMP4 normalized to β-actin per nanogram of input RNA, with the mean expression level from vehicle-treated mice defined as 1.0. Similar results were obtained in a second experiment with 25 mg of at-RA per kg.

FIG. 7.

FIG. 7.

Retinoic acid (RA) inhibits the development of SCCs in chicken embryo otocysts, and exogenous BMP4 overcomes this effect. (A) A small window was cut into White Leghorn chicken egg shells, and two beads (one bead soaked in at-RA or the vehicle and one bead adsorbed with either BMP4-secreting CHO cells or control CHO cells) were placed in the center of the otic vesicle of stage 16 to 17 embryos. Embryos were harvested at stages 34 to 35. Shown is a paint fill analysis of chicken inner ears illustrating representative abnormal phenotypes following exposure to at-RA. Inner ears were categorized by phenotype as follows: A, normal inner ear representing a type 0 phenotype (D, dorsal; A, anterior; L, lateral); B, type 1 inner ear with a single canal missing (ssc); C, type 2 inner ear with two missing SCCs (lsc and ssc); D, type 3 inner ear with no SCCs. Bar, 100 μm. Abbreviations: cd, cochlea; cc, common crus; es, endolymphatic sac; la, lateral ampulla; lsc, lateral SCC; pa, posterior ampulla; psc, posterior SCC; sa, superior ampulla; ssc, superior SCC. (B) Graph summarizing phenotypic abnormalities in ears treated with at-RA beads plus control CHO cell beads or at-RA beads plus BMP4 CHO cell beads.

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