title: Otubain 2 is a novel promoter of beta cell survival as revealed by siRNA high-throughput screens of human pancreatic islets.
abstract: OBJECTIVE
  Pro-inflammatory cytokines induce death of beta cells and hamper engraftment of transplanted islet mass. Our aim was to reveal novel genes involved in this process, as a platform for innovative therapeutic approaches.
  
  
  METHODS
  Small interfering RNA (siRNA) high-throughput screening (HTS) of primary human islets was employed to identify novel genes involved in cytokine-induced beta cell apoptosis. Dispersed human islets from nine human donors, treated with a combination of TNF-α, IL-1β and IFN-γ were transfected with ∼730 different siRNAs. Caspase-3/7 activity was measured, results were analysed and potential anti- and pro-apoptotic genes were identified.
  
  
  RESULTS
  Dispersed human pancreatic islets appeared to be suitable targets for performance of siRNA HTS. Using this methodology we found a number of potential pro- and anti-apoptotic target hits that have not been previously associated with pancreatic beta cell death. One such hit was the de-ubiquitinating enzyme otubain 2 (OTUB2). OTUB2 knockdown increased caspase-3/7 activity in MIN6 cells and primary human islets and inhibited insulin secretion and increased nuclear factor-κB (NF-κB) activity both under basal conditions and following cytokine treatment.
  
  
  CONCLUSIONS
  Use of dispersed human islets provides a new platform for functional HTS in a highly physiological system. Employing this technique enabled the identification of OTUB2 as a novel promoter of viability and insulin secretion in human beta cells. OTUB2 acts through the inhibition of NF-κB signalling, which is deleterious to beta cell survival. siRNA screens of human islets may therefore identify new targets, such as OTUB2, for therapeutic intervention in type 1 diabetes and islet transplantation.
authors.affiliation: Department of Molecular Cell Biology, Weizmann Institute of Science, 234 Herzl Street, Rehovot 76100, Israel.
authors.forename: A
authors.initials: A
authors.lastname: Beck
authors.forename: Y
authors.initials: Y
authors.lastname: Vinik
authors.forename: H
authors.initials: H
authors.lastname: Shatz-Azoulay
authors.forename: R
authors.initials: R
authors.lastname: Isaac
authors.forename: S
authors.initials: S
authors.lastname: Streim
authors.forename: G
authors.initials: G
authors.lastname: Jona
authors.forename: S
authors.initials: S
authors.lastname: Boura-Halfon
authors.forename: Y
authors.initials: Y
authors.lastname: Zick
mesh_terms: D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002470:Cell Survival; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000473:pathology; D007371:Interferon-gamma / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000378:metabolism*; D015398:Signal Transduction; D013869:Thiolester Hydrolases / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D053583:Interleukin-1beta; D016328:NF-kappa B; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; C496399:OTUB2 protein, human; D013869:Thiolester Hydrolases; D053148:Caspase 3; D053179:Caspase 7