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Regulation of TGF-beta signalling by Fbxo11, the gene mutated in the Jeff otitis media mouse mutant.

Tateossian H, Hardisty-Hughes RE, Morse S, Romero MR, Hilton H, Dean C, Brown SD - Pathogenetics (2009)

Bottom Line: Phospho-Smad2 (pSmad2) is significantly upregulated in epithelia of Jeff homozygotes.However, tissue immunoprecipitations failed to detect any interaction between Fbxo11 and Smad2.Fbxo11 is known to neddylate p53, a co-factor of pSmad2, but we did not find any evidence of genetic interactions between Jeff and p53 mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Mammalian Genetics Unit, Harwell, OX11 0RD, UK. s.brown@har.mrc.ac.uk.

ABSTRACT

Background: Jeff is a dominant mouse mutant displaying chronic otitis media. The gene underlying Jeff is Fbxo11, a member of the large F-box family, which are specificity factors for the SCF E3 ubiquitin ligase complex. Jeff homozygotes die shortly after birth displaying a number of developmental abnormalities including cleft palate and eyes open at birth. TGF-beta signalling is involved in a number of epithelial developmental processes and we have investigated the impact of the Jeff mutation on the expression of this pathway.

Results: Phospho-Smad2 (pSmad2) is significantly upregulated in epithelia of Jeff homozygotes. Moreover, there was a significant increase in nuclear localization of pSmad2 in contrast to wild type. Mice heterozygous for both Jeff and Smad2 mutations recapitulate many of the features of the Jeff homozygous phenotype. However, tissue immunoprecipitations failed to detect any interaction between Fbxo11 and Smad2. Fbxo11 is known to neddylate p53, a co-factor of pSmad2, but we did not find any evidence of genetic interactions between Jeff and p53 mutants. Nevertheless, p53 levels are substantially reduced in Jeff mice suggesting that Fbxo11 plays a role in stabilizing p53.

Conclusion: Overall, our findings support a model whereby Fbxo11, possibly via stabilization of p53, is required to limit the accumulation of pSmad2 in the nucleus of epithelial cells of palatal shelves, eyelids and airways of the lungs. The finding that Fbxo11 impacts upon TGF-beta signalling has important implications for our understanding of the underlying disease mechanisms of middle ear inflammatory disease.

No MeSH data available.


Related in: MedlinePlus

Cleft palate phenotype. a. Coronal sections through the palate of E14.5 (before the fusion) and E15.5 (after the fusion) wild-type (WT) and homozygote (Jf/Jf) embryos, haematoxylin-eosin stained. Scale bars 200 μm. b. Cross-sections of heads showing secondary palate of a wild-type (WT) newborn mouse with fused palate and a homozygote (Jf/Jf) newborn mouse with a cleft (arrow).
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Figure 1: Cleft palate phenotype. a. Coronal sections through the palate of E14.5 (before the fusion) and E15.5 (after the fusion) wild-type (WT) and homozygote (Jf/Jf) embryos, haematoxylin-eosin stained. Scale bars 200 μm. b. Cross-sections of heads showing secondary palate of a wild-type (WT) newborn mouse with fused palate and a homozygote (Jf/Jf) newborn mouse with a cleft (arrow).

Mentions: Jeff homozygote palatal shelves start to grow and lift on time, but they fail to fuse at the right developmental stage (Figure 1a) and homozygote mice are born with cleft palate (Figure 1b). This is reminiscent of TGF-β3 mutant mice [29]. To characterize the distribution pattern of TGF-β ligands, TGF-β receptors and Smads in the developing palates, we examined embryonic heads from wild-type and homozygote Jeff (Jf/Jf) mice by IHC (Figure 2). The result revealed one major difference in the pattern and localization of pSmad2. In wild types at embryonic day 15.5 (E15.5), as the midline epithelial seam was disrupted and medial edge epithelium (MEE) disappeared, pSmad2 was largely confined to the oral/nasal triangle area as a nuclear and cytoplasmic stain (Figure 2b). In contrast, in Jf/Jf mice at E15.5 an increased number of epithelial cells are positive for pSmad2 in the palatal shelves concentrated to the tip of the palatal epithelium and the oral/nasal palatal epithelial cells (Figure 2b). Moreover, in Jf/Jf palates at E15.5 pSmad2 was present as a nuclear stain in the majority of the cells. In homozygote mice 57% of epithelial cells in the palate showed a nuclear localization of pSmad2, compared with 27% in wild-type mice (P = 0.000255). There was a commensurate significant decrease in cells showing a cytoplasmic localization in Jf/Jf mice compared with wild-type mice. TGF-β3, TGFβR-I, TGFβR-II, Smad2, Smad4, Smurf2 and also Fbox11, in both wild-type and Jf/Jf mice, were localized to the cytoplasm of the epithelial cells (Figure 2a and Additional file 1). There was no difference in the distribution of activated Smad2 in wild-type and homozygote palates before the fusion (E14.5) (Figure 2b).


Regulation of TGF-beta signalling by Fbxo11, the gene mutated in the Jeff otitis media mouse mutant.

Tateossian H, Hardisty-Hughes RE, Morse S, Romero MR, Hilton H, Dean C, Brown SD - Pathogenetics (2009)

Cleft palate phenotype. a. Coronal sections through the palate of E14.5 (before the fusion) and E15.5 (after the fusion) wild-type (WT) and homozygote (Jf/Jf) embryos, haematoxylin-eosin stained. Scale bars 200 μm. b. Cross-sections of heads showing secondary palate of a wild-type (WT) newborn mouse with fused palate and a homozygote (Jf/Jf) newborn mouse with a cleft (arrow).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2714483&req=5

Figure 1: Cleft palate phenotype. a. Coronal sections through the palate of E14.5 (before the fusion) and E15.5 (after the fusion) wild-type (WT) and homozygote (Jf/Jf) embryos, haematoxylin-eosin stained. Scale bars 200 μm. b. Cross-sections of heads showing secondary palate of a wild-type (WT) newborn mouse with fused palate and a homozygote (Jf/Jf) newborn mouse with a cleft (arrow).
Mentions: Jeff homozygote palatal shelves start to grow and lift on time, but they fail to fuse at the right developmental stage (Figure 1a) and homozygote mice are born with cleft palate (Figure 1b). This is reminiscent of TGF-β3 mutant mice [29]. To characterize the distribution pattern of TGF-β ligands, TGF-β receptors and Smads in the developing palates, we examined embryonic heads from wild-type and homozygote Jeff (Jf/Jf) mice by IHC (Figure 2). The result revealed one major difference in the pattern and localization of pSmad2. In wild types at embryonic day 15.5 (E15.5), as the midline epithelial seam was disrupted and medial edge epithelium (MEE) disappeared, pSmad2 was largely confined to the oral/nasal triangle area as a nuclear and cytoplasmic stain (Figure 2b). In contrast, in Jf/Jf mice at E15.5 an increased number of epithelial cells are positive for pSmad2 in the palatal shelves concentrated to the tip of the palatal epithelium and the oral/nasal palatal epithelial cells (Figure 2b). Moreover, in Jf/Jf palates at E15.5 pSmad2 was present as a nuclear stain in the majority of the cells. In homozygote mice 57% of epithelial cells in the palate showed a nuclear localization of pSmad2, compared with 27% in wild-type mice (P = 0.000255). There was a commensurate significant decrease in cells showing a cytoplasmic localization in Jf/Jf mice compared with wild-type mice. TGF-β3, TGFβR-I, TGFβR-II, Smad2, Smad4, Smurf2 and also Fbox11, in both wild-type and Jf/Jf mice, were localized to the cytoplasm of the epithelial cells (Figure 2a and Additional file 1). There was no difference in the distribution of activated Smad2 in wild-type and homozygote palates before the fusion (E14.5) (Figure 2b).

Bottom Line: Phospho-Smad2 (pSmad2) is significantly upregulated in epithelia of Jeff homozygotes.However, tissue immunoprecipitations failed to detect any interaction between Fbxo11 and Smad2.Fbxo11 is known to neddylate p53, a co-factor of pSmad2, but we did not find any evidence of genetic interactions between Jeff and p53 mutants.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Mammalian Genetics Unit, Harwell, OX11 0RD, UK. s.brown@har.mrc.ac.uk.

ABSTRACT

Background: Jeff is a dominant mouse mutant displaying chronic otitis media. The gene underlying Jeff is Fbxo11, a member of the large F-box family, which are specificity factors for the SCF E3 ubiquitin ligase complex. Jeff homozygotes die shortly after birth displaying a number of developmental abnormalities including cleft palate and eyes open at birth. TGF-beta signalling is involved in a number of epithelial developmental processes and we have investigated the impact of the Jeff mutation on the expression of this pathway.

Results: Phospho-Smad2 (pSmad2) is significantly upregulated in epithelia of Jeff homozygotes. Moreover, there was a significant increase in nuclear localization of pSmad2 in contrast to wild type. Mice heterozygous for both Jeff and Smad2 mutations recapitulate many of the features of the Jeff homozygous phenotype. However, tissue immunoprecipitations failed to detect any interaction between Fbxo11 and Smad2. Fbxo11 is known to neddylate p53, a co-factor of pSmad2, but we did not find any evidence of genetic interactions between Jeff and p53 mutants. Nevertheless, p53 levels are substantially reduced in Jeff mice suggesting that Fbxo11 plays a role in stabilizing p53.

Conclusion: Overall, our findings support a model whereby Fbxo11, possibly via stabilization of p53, is required to limit the accumulation of pSmad2 in the nucleus of epithelial cells of palatal shelves, eyelids and airways of the lungs. The finding that Fbxo11 impacts upon TGF-beta signalling has important implications for our understanding of the underlying disease mechanisms of middle ear inflammatory disease.

No MeSH data available.


Related in: MedlinePlus