Dickkopf 3 (Dkk3) regulates the expression of zebrafish myf5 promoter via phosphorylated p38a-dependent Smad4 stability
Date Issued
2009
Date
2009
Author(s)
Lin, Chiu-Chun
Abstract
Myf5, one of the myogenic regulatory factors, plays roles in the specification and differentiation of muscular cells during myogenesis. An intronic microRNA, miR-In300, located within zebrafish myf5 intron I, has been reported to silence myf5 through targeting dkk3. However, the detailed molecular mechanism underlying how the secreted Dkk3 controls the myf5 expression is totally unknown. In this study, we found that knockdown of dkk3 reduced the protein level of the phosphorylated p38a. Injection of p38a-specific morphorlino (MO), which inhibits the translation of p38a mRNA, resulted that the malformed somites and the reduced myf5 transcripts, which photocopied the defects induced by injection of dkk3-MO. These phenotypes were also observed in the embryos injected with the dominant-negative form of p38a. Blocking the MAPK pathway through interfering the phosphorylation of p38 by introducing SB203580 caused the down-regulation of myf5 expression. Furthermore, the GFP fluorescent signal was dramatically decreased when we injected p38a-MO into the embryos derived from transgenic line Tg(myf5(80K):GFP), in which the GFP was driven by myf5 promoter. This p38a-MO-induced defects were rescued by co-injection with p38a mRNA, but were not rescued with the mutated p38a mRNA containing a mutation at phosphorylation domain. This line of evidences suggested that the phosphorylation of p38a is required for activating of myf5 promoter activity. Moreover, the defective phenotypes induced by injection the dominant-negative form of either Smad2 or Smad3 were as same as those of embryos injected with either dkk3- or p38a-MO. Using luciferase assay and injection of samd2/3a/4 mRNA in the embryos derived from Tg(myf5(80K):GFP), we found that overexpression of Smad2 and Smad3a did enhance the myf5 promoter activity. Furthermore, we proved that Smad3a directly bound at the upstream regulatory region of myf5 using chromatin immunoprecipitation assay. Interestingly, knockdown of either dkk3 or p38a resulted in the decrease of Smad4 protein level, but did not change the Smad3 protein level. Injection of smad4 mRNA enabled embryos to rescue the down-regulation of myf5 in the dkk3-MO-injected embryos. Taken together, we concluded that Dkk3 regulates the phosphorylation of p38a to maintain the stability of Smad4 protein, which, in turn, to allow the complex of Smad2/3a/4 to activate the zebrafish myf5 promoter activity.
Subjects
zebrafish
myogenesis
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