title: UVB upregulates the bax promoter in immortalized human keratinocytes via ROS induction of Id3.
abstract: Id3 belongs to the inhibitor of differentiation family of helix-loop-helix transcription factors, important in proliferation, differentiation and apoptosis. We showed that Id3, but not Id2 or Id1, mediates the UVB-sensitization of immortalized keratinocytes by inducing caspase 9-dependent apoptosis. In this study, quantitative PCR analysis revealed a time-dependent increase in Id3 mRNA induced by UVB, dependent on reactive oxygen species. UVB upregulated promoter activity of Id3, but not Id2, at early time points, as shown by reporter assays and also stabilized Id3 mRNA, increasing its half-life from 10 to approximately 60 min. We next examined downstream events related to UVB-induced Id3 upregulation and investigated the effects of UVB or ectopic expression of Id3 on bax promoter activity. Regulatory elements in the bax promoter that mediate transcriptional activation by UVB and Id3, in the absence of p53, were identified. Bax promoter deletion analysis revealed that transcriptional activation by UVB involves a 738-bp region upstream from the transcription start site of bax. Mimicking the effects of UVB, ectopic expression of Id3 also upregulated bax mRNA and activated this 738-bp fragment. Mutational analysis of the transcription binding sites further showed that point mutations of the E-box region found in the 738-bp fragment, but not in a 174-bp fragment, completely abolished Id3- and UVB-inducible bax promoter activity, thus confirming the importance of Id3 and UVB-mediated Id3 upregulation in activating the bax promoter. These results suggest a mechanism whereby reactive oxygen species upregulation of Id3 relieves repression of bax via E-box-binding factors.
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA.
authors.forename: Valerie Anne
authors.initials: VA
authors.lastname: Trabosh
authors.forename: Ahmad
authors.initials: A
authors.lastname: Daher
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Divito
authors.forename: Karishma
authors.initials: K
authors.lastname: Amin
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Simbulan-Rosenthal
authors.forename: Dean S
authors.initials: DS
authors.lastname: Rosenthal
mesh_terms: D017209:Apoptosis / Q000502:physiology; D002478:Cells, Cultured; D024721:E-Box Elements / Q000502:physiology; D006801:Humans; D051796:Inhibitor of Differentiation Proteins / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects; D009363:Neoplasm Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000528:radiation effects*; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014466:Ultraviolet Rays; D015854:Up-Regulation / Q000528:radiation effects*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D051796:Inhibitor of Differentiation Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; C075640:ID3 protein, human