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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

Immunoprecipitation and Western blot analysis. a. Immunoprecipitation: lung extract from E15.5 wild-type embryos was used for immunoprecipitation using Smad2 and p53 antibodies. The Western blots were probed with pSmad2, Fbox11 and p53 antibodies. b. Western blot analysis: protein lysates from wild-type, heterozygote and homozygote E15.5 palates. Equal amounts of protein were subjected to tris-acetate PAGE, transferred and probed with pSmad2, Fbox11, p53 antibodies and actin antibody for loading control. c. Graphical representation of p53 levels normalized against actin in wild-type, heterozygote and homozygote E15.5 palates, showing approximately threefold reduction of p53 in homozygote palate compared with wild type.
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Figure 7: Immunoprecipitation and Western blot analysis. a. Immunoprecipitation: lung extract from E15.5 wild-type embryos was used for immunoprecipitation using Smad2 and p53 antibodies. The Western blots were probed with pSmad2, Fbox11 and p53 antibodies. b. Western blot analysis: protein lysates from wild-type, heterozygote and homozygote E15.5 palates. Equal amounts of protein were subjected to tris-acetate PAGE, transferred and probed with pSmad2, Fbox11, p53 antibodies and actin antibody for loading control. c. Graphical representation of p53 levels normalized against actin in wild-type, heterozygote and homozygote E15.5 palates, showing approximately threefold reduction of p53 in homozygote palate compared with wild type.

Mentions: We explored further the genetic interaction between Fbxo11 and Smad2 by performing immunoprecipitations to test whether these two proteins interact. We used a cross-linking agent to improve the likelihood of detecting the interactions. However, immunoprecipitations with Smad2 antibodies failed to reveal any interaction with Fbxo11 (Figure 7a), suggesting that Smad2 or pSmad2 is not a substrate for ubiquitination by Fbxo11. However, Smad2 did immunoprecipitate p53, confirming the known interaction between these two proteins (Figure 7a).


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)

Immunoprecipitation and Western blot analysis. a. Immunoprecipitation: lung extract from E15.5 wild-type embryos was used for immunoprecipitation using Smad2 and p53 antibodies. The Western blots were probed with pSmad2, Fbox11 and p53 antibodies. b. Western blot analysis: protein lysates from wild-type, heterozygote and homozygote E15.5 palates. Equal amounts of protein were subjected to tris-acetate PAGE, transferred and probed with pSmad2, Fbox11, p53 antibodies and actin antibody for loading control. c. Graphical representation of p53 levels normalized against actin in wild-type, heterozygote and homozygote E15.5 palates, showing approximately threefold reduction of p53 in homozygote palate compared with wild type.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Immunoprecipitation and Western blot analysis. a. Immunoprecipitation: lung extract from E15.5 wild-type embryos was used for immunoprecipitation using Smad2 and p53 antibodies. The Western blots were probed with pSmad2, Fbox11 and p53 antibodies. b. Western blot analysis: protein lysates from wild-type, heterozygote and homozygote E15.5 palates. Equal amounts of protein were subjected to tris-acetate PAGE, transferred and probed with pSmad2, Fbox11, p53 antibodies and actin antibody for loading control. c. Graphical representation of p53 levels normalized against actin in wild-type, heterozygote and homozygote E15.5 palates, showing approximately threefold reduction of p53 in homozygote palate compared with wild type.
Mentions: We explored further the genetic interaction between Fbxo11 and Smad2 by performing immunoprecipitations to test whether these two proteins interact. We used a cross-linking agent to improve the likelihood of detecting the interactions. However, immunoprecipitations with Smad2 antibodies failed to reveal any interaction with Fbxo11 (Figure 7a), suggesting that Smad2 or pSmad2 is not a substrate for ubiquitination by Fbxo11. However, Smad2 did immunoprecipitate p53, confirming the known interaction between these two proteins (Figure 7a).

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