Downregulation of SnoN expression in obstructive nephropathy is mediated by an enhanced ubiquitin-dependent degradation.
Smad transcriptional co-repressor SnoN acts as an antagonist that tightly controls the trans-activation of TGF-beta/Smad target genes. SnoN protein is reduced progressively in the fibrotic kidney after obstructive injury, suggesting that the loss of Smad antagonist is a critical event that leads to an uncontrolled fibrogenic signaling. However, the mechanism underlying SnoN downregulation remains unknown. This study investigated the regulation and mechanism of renal SnoN expression in vivo. Whereas SnoN protein was markedly diminished, its mRNA levels remained relatively constant in the obstructed kidney after ureteral ligation. An increased ubiquitination and proteasome-dependent degradation of SnoN was found in obstructed kidney, compared with sham controls. Smad ubiquitination regulatory factor-2, an E3 ubiquitin ligase, was induced and formed a complex with SnoN in vivo. In vitro, TGF-beta1 promoted SnoN protein degradation, which was mediated by ubiquitination and a proteasome-dependent mechanism. SnoN constitutively interacted with another Smad co-repressor, Ski, and they formed ternary complex with Smad2/3 upon TGF-beta1 stimulation. However, ectopic expression of Ski did not alter the degradation rate of SnoN. Blockage of SnoN degradation by proteasome inhibitor abolished TGF-beta1-mediated alpha-smooth muscle actin and fibronectin induction, suggesting that SnoN degradation could be necessary for TGF-beta1 to exert its fibrogenic action. Furthermore, knockdown of Smad ubiquitination regulatory factor-2 expression by small interfering RNA strategy led to an increase in SnoN abundance and inhibited the TGF-beta1-mediated gene transcription. These results indicate that downregulation of SnoN expression in the obstructed kidney is mediated by an enhanced ubiquitin-dependent degradation. Preservation of SnoN by inhibiting its degradation may be a novel strategy for targeting hyperactive Smad signaling in renal fibrotic diseases.
Tan
Ruoyun
R
Department of Pathology, University of Pittsburgh, S-405 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261, USA.
Zhang
Jinglan
J
Tan
Xiaoyue
X
Zhang
Xianghong
X
Yang
Junwei
J
Liu
Youhua
Y
D000199:Actins / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D005353:Fibronectins / Q000378:metabolism; D005355:Fibrosis; D005455:Fluorescent Antibody Technique; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D009130:Muscle, Smooth / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051899:Smad2 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014517:Ureteral Obstruction / Q000378:metabolism*
D000199:Actins; D004268:DNA-Binding Proteins; D005353:Fibronectins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; C512653:Ski protein, mouse; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D053773:Transforming Growth Factor beta1; D025801:Ubiquitin; C492381:Smurf1 protein, mouse; D044767:Ubiquitin-Protein Ligases