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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

Immunolocalization in eyelid tissue. a. Coronal sections through the eye of E16 wild-type (WT) and homozygote (Jf/Jf) embryos immunohistochemically stained with antibodies against Fbox11, TGF-β3, TGFβR-I, Smad3, Smad2 and Smad4. Scale bar 200 μm. Graph: comparison of the percentage of epithelial cells positive for Smad2 in E16 upper and lower eyelids. b. Coronal sections through the eyes of E15.5 (before fusion) and E16 (after fusion) in wild-type (WT) and homozygote (Jf/Jf) embryos stained with pSmad2 antibody (scale bar 200 μm). Epidermis (ep), basal cells (bc) and dermis (d). Graph: comparison of the percentage of epithelial cells with positive nuclear and cytoplasmic localization of pSmad2 in E16 upper and lower eyelids. c. Coronal sections through the eyes of E16 in wild-type (WT) and homozygote (Jf/Jf) embryos stained with Ki67 and cleaved caspase-3 antibodies. Scale bar 200 μm. Graph: comparison of the percentage of epithelial cell positive for Ki67 and cleaved caspase-3 in E16 upper and lower eyelids. P-values were determined using two-tailed T-test comparing each homozygote eyelid with the wild type.
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Figure 4: Immunolocalization in eyelid tissue. a. Coronal sections through the eye of E16 wild-type (WT) and homozygote (Jf/Jf) embryos immunohistochemically stained with antibodies against Fbox11, TGF-β3, TGFβR-I, Smad3, Smad2 and Smad4. Scale bar 200 μm. Graph: comparison of the percentage of epithelial cells positive for Smad2 in E16 upper and lower eyelids. b. Coronal sections through the eyes of E15.5 (before fusion) and E16 (after fusion) in wild-type (WT) and homozygote (Jf/Jf) embryos stained with pSmad2 antibody (scale bar 200 μm). Epidermis (ep), basal cells (bc) and dermis (d). Graph: comparison of the percentage of epithelial cells with positive nuclear and cytoplasmic localization of pSmad2 in E16 upper and lower eyelids. c. Coronal sections through the eyes of E16 in wild-type (WT) and homozygote (Jf/Jf) embryos stained with Ki67 and cleaved caspase-3 antibodies. Scale bar 200 μm. Graph: comparison of the percentage of epithelial cell positive for Ki67 and cleaved caspase-3 in E16 upper and lower eyelids. P-values were determined using two-tailed T-test comparing each homozygote eyelid with the wild type.

Mentions: Jeff homozygous mutant eyelids start to grow on time, but fail to fuse at the correct developmental stage and the mice have an EOB phenotype (Figures 3a and 3b). We have applied the same panel of antibodies used on the palates to sections of E16 eyelids from both wild-type and Jf/Jf mice. These studies revealed similar results to those observed on palates. The majority of localization of pSmad2 in wild-type E16 eyelids was cytoplasmic. The staining was confined to the epidermis only (Figure 4b). In Jf/Jf mice more cells are positive for pSmad2 than in wild type. Moreover, pSmad2 was present as a nuclear stain in the majority of the cells of the epidermis. In homozygote mice 66% of epithelial cells in the upper eyelids and 56% of epithelial cells in the lower eyelids showed nuclear localization of pSmad2, compared with 11% and 17% in the wild-type mice (P = 0.000333, P = 0.0000473) (Figure 4b). Staining was also observed in the basal layer and also in some cells from the dermis of Jf/Jf eyelids (Figure 4b). The expression pattern of TGF-β3, TGFβR-I, TGFβR-II, Smad2, Smad3, Smad4, Smurf2 and Fbox11 at the same stage E16 in both wild-type and Jf/Jf eyelids was similar (Figure 4a and Additional file 1). They were all localized to the cytoplasm of the epithelial cells of the epidermis (Figure 4a and Additional file 1). We examined also the distribution of pSmad2 at E15.5, before the closure. In both wild-type and Jf/Jf eyelids activated Smad2 was detectable as nuclear and cytoplasmic staining. In homozygote mice 27% of epithelial cells in the upper eyelids and 19% of epithelial cells in the lower eyelids showed nuclear localization of pSmad2, compared with 41% and 32% in the wild-type mice. Eleven per cent of epithelial cells in the homozygote upper eyelids and 9% of epithelial cells in the homozygote lower eyelids showed cytoplasmic localization of pSmad2, compared with 12% and 9% in the wild-type eyelids (data not shown) (Figure 4b).


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)

Immunolocalization in eyelid tissue. a. Coronal sections through the eye of E16 wild-type (WT) and homozygote (Jf/Jf) embryos immunohistochemically stained with antibodies against Fbox11, TGF-β3, TGFβR-I, Smad3, Smad2 and Smad4. Scale bar 200 μm. Graph: comparison of the percentage of epithelial cells positive for Smad2 in E16 upper and lower eyelids. b. Coronal sections through the eyes of E15.5 (before fusion) and E16 (after fusion) in wild-type (WT) and homozygote (Jf/Jf) embryos stained with pSmad2 antibody (scale bar 200 μm). Epidermis (ep), basal cells (bc) and dermis (d). Graph: comparison of the percentage of epithelial cells with positive nuclear and cytoplasmic localization of pSmad2 in E16 upper and lower eyelids. c. Coronal sections through the eyes of E16 in wild-type (WT) and homozygote (Jf/Jf) embryos stained with Ki67 and cleaved caspase-3 antibodies. Scale bar 200 μm. Graph: comparison of the percentage of epithelial cell positive for Ki67 and cleaved caspase-3 in E16 upper and lower eyelids. P-values were determined using two-tailed T-test comparing each homozygote eyelid with the wild type.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 4: Immunolocalization in eyelid tissue. a. Coronal sections through the eye of E16 wild-type (WT) and homozygote (Jf/Jf) embryos immunohistochemically stained with antibodies against Fbox11, TGF-β3, TGFβR-I, Smad3, Smad2 and Smad4. Scale bar 200 μm. Graph: comparison of the percentage of epithelial cells positive for Smad2 in E16 upper and lower eyelids. b. Coronal sections through the eyes of E15.5 (before fusion) and E16 (after fusion) in wild-type (WT) and homozygote (Jf/Jf) embryos stained with pSmad2 antibody (scale bar 200 μm). Epidermis (ep), basal cells (bc) and dermis (d). Graph: comparison of the percentage of epithelial cells with positive nuclear and cytoplasmic localization of pSmad2 in E16 upper and lower eyelids. c. Coronal sections through the eyes of E16 in wild-type (WT) and homozygote (Jf/Jf) embryos stained with Ki67 and cleaved caspase-3 antibodies. Scale bar 200 μm. Graph: comparison of the percentage of epithelial cell positive for Ki67 and cleaved caspase-3 in E16 upper and lower eyelids. P-values were determined using two-tailed T-test comparing each homozygote eyelid with the wild type.
Mentions: Jeff homozygous mutant eyelids start to grow on time, but fail to fuse at the correct developmental stage and the mice have an EOB phenotype (Figures 3a and 3b). We have applied the same panel of antibodies used on the palates to sections of E16 eyelids from both wild-type and Jf/Jf mice. These studies revealed similar results to those observed on palates. The majority of localization of pSmad2 in wild-type E16 eyelids was cytoplasmic. The staining was confined to the epidermis only (Figure 4b). In Jf/Jf mice more cells are positive for pSmad2 than in wild type. Moreover, pSmad2 was present as a nuclear stain in the majority of the cells of the epidermis. In homozygote mice 66% of epithelial cells in the upper eyelids and 56% of epithelial cells in the lower eyelids showed nuclear localization of pSmad2, compared with 11% and 17% in the wild-type mice (P = 0.000333, P = 0.0000473) (Figure 4b). Staining was also observed in the basal layer and also in some cells from the dermis of Jf/Jf eyelids (Figure 4b). The expression pattern of TGF-β3, TGFβR-I, TGFβR-II, Smad2, Smad3, Smad4, Smurf2 and Fbox11 at the same stage E16 in both wild-type and Jf/Jf eyelids was similar (Figure 4a and Additional file 1). They were all localized to the cytoplasm of the epithelial cells of the epidermis (Figure 4a and Additional file 1). We examined also the distribution of pSmad2 at E15.5, before the closure. In both wild-type and Jf/Jf eyelids activated Smad2 was detectable as nuclear and cytoplasmic staining. In homozygote mice 27% of epithelial cells in the upper eyelids and 19% of epithelial cells in the lower eyelids showed nuclear localization of pSmad2, compared with 41% and 32% in the wild-type mice. Eleven per cent of epithelial cells in the homozygote upper eyelids and 9% of epithelial cells in the homozygote lower eyelids showed cytoplasmic localization of pSmad2, compared with 12% and 9% in the wild-type eyelids (data not shown) (Figure 4b).

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