title: dickkopf-3-related gene regulates the expression of zebrafish myf5 gene through phosphorylated p38a-dependent Smad4 activity.
abstract: Myf5 is a myogenic regulatory factor that functions in myogenesis. An intronic microRNA, miR-In300, located within zebrafish myf5 intron I, has been reported to silence myf5 through the targeting of dickkopf-3-related gene (dkk3r). However, the molecular mechanism underlying the control of myf5 expression by dkk3r is unknown. By injecting dkk3r-specific morpholino-oligonucleotide (dkk3r-MO) to knock down Dkk3r, we found that the phosphorylated p38a protein was reduced. Knockdown of p38a resulted in malformed somites and reduced myf5 transcripts, which photocopied the defects induced by injection of dkk3r-MO. To block the MAPK pathway, phosphorylation of p38 was inhibited by introduction of SB203580, which caused the down-regulation of myf5 expression. The GFP signal was dramatically decreased in somites when we injected p38a-MO into embryos derived from transgenic line Tg(myf5(80K):GFP), in which the GFP was driven by the myf5 promoter. Although these p38a-MO-induced defects were rescued by co-injection with p38a mRNA, they were not rescued with p38a mRNA containing a mutation at the phosphorylation domain. Moreover, overexpression of Smad2 or Smad3a enhanced myf5 expression, but the defects induced by the dominant negative form of either Smad2 or Smad3a equaled those of embryos injected with either dkk3r-MO or p38a-MO. These results support the involvement of Smad2·Smad3a in p38a mediation. Overexpression of Smad4 enabled the rescue of myf5 defects in the dkk3r-MO-injected embryos, but knockdown of either dkk3r or p38a caused Smad4 protein to lose stability. Therefore, we concluded that Dkk3r regulates p38a phosphorylation to maintain Smad4 stability, in turn enabling the Smad2·Smad3a·Smad4 complex to form and activate the myf5 promoter.
authors.affiliation: Institute of Molecular and Cellular Biology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan.
authors.forename: Ren-Jun
authors.initials: RJ
authors.lastname: Hsu
authors.forename: Chiu-Chun
authors.initials: CC
authors.lastname: Lin
authors.forename: Ying-Fang
authors.initials: YF
authors.lastname: Su
authors.forename: Huai-Jen
authors.initials: HJ
authors.lastname: Tsai
mesh_terms: D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology*; D055785:Gene Knockdown Techniques; D007093:Imidazoles / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D051787:Myogenic Regulatory Factor 5 / Q000096:biosynthesis* / Q000235:genetics; D009841:Oligonucleotides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D055550:Protein Stability; D011725:Pyridines / Q000494:pharmacology; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D007093:Imidazoles; D036341:Intercellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; D051787:Myogenic Regulatory Factor 5; D009841:Oligonucleotides; D011725:Pyridines; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D029961:Zebrafish Proteins; C497786:smad2 protein, zebrafish; C497792:smad3a protein, zebrafish; C490809:smad4a protein, zebrafish; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580