title: Transcription of cathepsin B in glioma cells: regulation by an E-box adjacent to the transcription initiation site.
abstract: We have previously isolated the human cathepsin B promoter and shown that Sp1 and Ets factors are involved in the regulation of cathepsin B expression. Using mutagenesis, transient transfection and electrophoretic mobility shift assays (EMSAs), we further identified regulatory factors that mediate cathepsin B transcription in U87 human glioblastoma cells. An E-box element (CACGTG) adjacent to the transcription initiation site (at nucleotides -7 to -2) was found to be indispensable for cathepsin B promoter activity. Mutation of this E-box element in both pSCB2, a promoter construct with high promoter activity, and pSCB6, a construct with basal promoter activity, led to a 90% decrease in promoter activity in U87 cells. EMSAs demonstrated that upstream stimulatory factor 1 (USF-1) and upstream stimulatory factor 2 (USF-2) bound to the E-box as a heterodimer. Chromatin immunoprecipitation assays revealed that both USF-1 and USF-2 were associated with the cathepsin B promoter. The roles of USF-1 and USF-2 in the regulation of cathepsin B expression were demonstrated by (i) co-transfection experiments showing that USF-1 or USF-2 increased promoter activity by 2.5-fold individually and by 3.4-fold together; (ii) co-transfection of pSCB6 with pUSF-2deltaN (a dominant negative USF-2 expression plasmid) resulting in an 80% decrease in promoter activity; and (iii) mutation of the E-box element (from 5'-CACGTG to 5'-CGCGTT in the pSCB6 basal promoter construct) abolishing transactivation of cathepsin B by USF-1 and USF-2. These results collectively indicate that an E-box at nucleotides -7 to -2 of the cathepsin B promoter is critical to the expression of cathepsin B and that binding of USF-1 and USF-2 to this E-box can regulate cathepsin B promoter activity.
authors.affiliation: Department of Pharmacology, Wayne State University, 540 E. Canfield Ave., Detroit, MI 48201, USA.
authors.forename: Shiqing
authors.initials: S
authors.lastname: Yan
authors.forename: Derek T
authors.initials: DT
authors.lastname: Jane
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Dufresne
authors.forename: Bonnie F
authors.initials: BF
authors.lastname: Sloane
mesh_terms: D002401:Cathepsin B / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024721:E-Box Elements / Q000235:genetics / Q000502:physiology*; D005910:Glioma; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D051720:Upstream Stimulatory Factors
chemical_list: D004268:DNA-Binding Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; D004247:DNA; D002401:Cathepsin B