title: Transcriptional and posttranscriptional regulation of the CTNS gene.
abstract: Cell cysteine (Cys) levels and/or the [Cys/CySS] redox potential have been shown to regulate mRNA levels of the CTNS gene, which encodes for a lysosomal cystine (CySS) carrier that is defective in cystinosis. To investigate the mechanisms involved CTNS mRNA regulation, different portions of the CTNS promotor were cloned into a luciferase vector and transfected in HK2 cells. A 1.5-2.4-fold increase in luciferase activity was observed when cells were incubated in culture medium containing low CySS concentrations. Conversely, CTNS mRNA levels decreased by 47-56% in the presence of N-acetyl-L-cysteine (NAC). Chase experiments with actinomycin D (ActD) demonstrated a 3-fold stabilization of the CTNS mRNA when cells were cultured in low CySS medium for 48 h. Treatment of control cells with cyclohexamide (CHX) increased CTNS mRNA levels, suggesting that CHX blocked the synthesis of proteins involved in mRNA degradation or in repression of the CTNS gene. Finally, in vitro binding assays showed increased binding (30-110%) of the Sp-1 transcription factor to two regions of the CTNS promotor when cells were incubated in low CySS medium. These results indicate that the CTNS gene is actively regulated at the transcriptional and posttranscriptional levels and suggest that CTNS plays a pivotal role in regulating cell thiol concentrations.
authors.affiliation: Division of Nephrology and Dialysis, Bambino Gesù Children's Hospital and Research Institute, 00165 Rome, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Corallini
authors.forename: Anna
authors.initials: A
authors.lastname: Taranta
authors.forename: Francesco
authors.initials: F
authors.lastname: Bellomo
authors.forename: Alessia
authors.initials: A
authors.lastname: Palma
authors.forename: Anna
authors.initials: A
authors.lastname: Pastore
authors.forename: Francesco
authors.initials: F
authors.lastname: Emma
mesh_terms: D000111:Acetylcysteine; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003553:Cystine / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D003609:Dactinomycin; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018709:Statistics, Nonparametric; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014162:Transfection
chemical_list: D026921:Amino Acid Transport Systems, Neutral; C111445:CTNS protein, human; D017931:DNA Primers; D012333:RNA, Messenger; D013438:Sulfhydryl Compounds; D003609:Dactinomycin; D003553:Cystine; D008156:Luciferases; D000111:Acetylcysteine