title: Identification of Nedd4 as a novel regulator in Hedgehog signaling.
abstract: BACKGROUND
  Hedgehog (Hh) signaling plays an important role in both embryonic development and postnatal tissue homeostasis. Aberrant Hh activation results in a large variety of cancers. This study was designed to discover novel modulators in Hh signaling pathway.
  
  
  METHODS
  We performed yeast-two-hybrid screening and immunoprecipitation to identify the interaction of Nedd4 and Smo. To verify whether Nedd4 is involved in the regulation of Hh signaling, we monitored the activation of Gli-luciferase reporter by overexpressing Nedd4 together with Gli-luciferase reporter. In order to examine the role of endogenous Nedd4 in regulating Hh signaling, we used a short hairpin RNA (shRNA) interference strategy to silence the Nedd4 expression, and then perform dual-luciferase reporter assay. Statistical comparisons were performed by Student's t tests.
  
  
  RESULTS
  We showed that Nedd4 binds to Smo in the transfected HEK293 cells. Overexpression of Nedd4 alone did not significantly activate the Gli reporter compared to pcDNA3 control (Nedd4 group: dimethyl sulfoxide (DMSO), relative luciferase unit (RLU) 1.87 ± 0.41). However, Smo agonist (SAG)-stimulated activation of Gli-luciferase reporter was markedly potentiated in Nedd4 transfected cells (Nedd4 group: SAG, RLU 13.49 ± 1.04, P < 0.05), indicating that overexpression of Nedd4 increases Gli luciferase reporter activity and Nedd4-induced activation of Hh signaling is activity dependent. In Nedd4 knockdown NIH 3T3 cells, the luciferase reporter activity was measured basally and after SAG treatment. In scrambled cells, compared to DMSO, SAG could activate reporter activity by (4.16 ± 0.84)-fold. In Nedd4 knockdown cells, the luciferase reporter activation by SAG was significantly inhibited (SAG, RLU 1.72 ± 0.24, P < 0.05); knockdown of Nedd4 did not change the basal activity of luciferase activity (DMSO, RLU 0.86 ± 0.11), suggesting that the loss of Nedd4 expression diminishes Gli-dependent activity in the Hh pathway and the regulation of Nedd4 in the Hh signaling pathway is activity-dependent.
  
  
  CONCLUSIONS
  Nedd4 positively regulates the Hh pathway and provides a potential target for inhibiting Hh signaling in cancer therapy.
authors.affiliation: Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China.
authors.forename: Qing-Feng
authors.initials: QF
authors.lastname: Luo
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Shu-Tian
authors.initials: ST
authors.lastname: Zhang
mesh_terms: D000818:Animals; D056827:Endosomal Sorting Complexes Required for Transport / Q000502:physiology*; D057809:HEK293 Cells; D053823:Hedgehog Proteins / Q000502:physiology*; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D000075702:Nedd4 Ubiquitin Protein Ligases; D043562:Receptors, G-Protein-Coupled / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D000072086:Smoothened Receptor; D014157:Transcription Factors / Q000502:physiology; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D000071676:Zinc Finger Protein GLI1
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; C400551:GLI1 protein, human; D053823:Hedgehog Proteins; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases