title: Nedd4 E3 ubiquitin ligase promotes cell proliferation and autophagy.
abstract: OBJECTIVE
  Nedd4 (neural precursor cell expressed developmentally down-regulated protein 4) is a member of the HECT E3 ubiquitin ligases, and is elevated in prostate, bladder and colorectal cancers, and promotes colonic cell population expansion. Up to now, molecular mechanisms of how Nedd4 functions, have not been well understood.
  
  
  METHODS
  In this study, shRNA was used to reduce expression of Nedd4 in the human prostate carcinoma cell line DU145. To analyse effects of Nedd4 on cell proliferation, MTT and colony formation assays were performed. DAPI staining and FACS analysis were used to investigate outcomes of Nedd4 activity, on apoptosis. Results of Nedd4 expression on lysosomal membrane permeabilization and autophagy were further investigated using acridine orange (AO) staining, immunofluorescence and western blot analysis.
  
  
  RESULTS
  We found that in HeLa cells, expression of Nedd4 promoted cell proliferation, whereas its knockdown inhibited colony formation and induced apoptosis in DU145 cells. Furthermore, down-regulation of Nedd4 in DU145 cells promoted lysosomal membrane permeabilization. We also found that down-regulation of Nedd4 inhibited autophagy in both DU145 and A549 cells. Investigation into mechanisms involved revealed that knockdown of endogenous Nedd4 expression notably increased activated mTOR (p-mTOR) levels, which suggests that mTOR signalling was involved in the Nedd4-mediated autophagy.
  
  
  CONCLUSIONS
  Our results indicate that expression of Nedd4 promoted cell proliferation and colony formation but prevented apoptosis. Moreover, Nedd4 promoted autophagy and was associated with the mTOR signalling pathway.
authors: School of Biotechnology, Tianjin University of Science and Technology, Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin, 300457, China.
authors: Yuyin
authors: Y
authors: Li
authors: Li
authors: L
authors: Zhang
authors: Jie
authors: J
authors: Zhou
authors: Shenheng
authors: S
authors: Luo
authors: Rui
authors: R
authors: Huang
authors: Changcai
authors: C
authors: Zhao
authors: Aipo
authors: A
authors: Diao
mesh_terms: D000165:Acridine Orange / Q000737:chemistry; D017209:Apoptosis; D001343:Autophagy / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D056827:Endosomal Sorting Complexes Required for Transport / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D000075702:Nedd4 Ubiquitin Protein Ligases; D010539:Permeability; D011467:Prostate / Q000378:metabolism* / Q000473:pathology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D034741:RNA, Small Interfering; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D000165:Acridine Orange