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.
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Abbreviations
- AARE:
-
amino acid responsive element
- Act D:
-
actinomycin D
- CHX:
-
cyclohexamide
- Cys:
-
cysteine
- CySS:
-
cystine
- Eh:
-
redox potential
- GSH:
-
reduced glutathione
- GSSG:
-
oxidized glutathione
- NAC:
-
N-acetyl-l-cysteine
References
Gahl WA, Thoene JG, Schneider JA . Cystinosis. N Engl J Med 347: 111–121 2002
Theodoropoulos DS, Krasnewich D, Kaiser-Kupfer MI, Gahl WA 1993 Classic nephropathic cystinosis as an adult disease. JAMA 270: 2200–2204
Town M, Jean G, Cherqui S, Attard M, Forestier L, Whitmore SA, Callen DF, Gribouval O, Broyer M, Bates GP, van't Hoff W, Antignac C 1998 A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis. Nat Genet 18: 319–324
Taranta A, Petrini S, Palma A, Mannucci L, Wilmer MJ, De Luca V, Diomedi-Camassei F, Corallini S, Bellomo F, van den Heuvel LP, Levtchenko EN, Emma F 2008 Identification and subcellular localization of a new cystinosin isoform. Am J Physiol Renal Physiol 294: F1101–F1108
Taranta A, Wilmer MJ, van den Heuvel LP, Bencivenga P, Bellomo F, Levtchenko EN, Emma F 2010 Analysis of CTNS gene transcripts in nephropathic cystinosis. Pediatr Nephrol 25: 1263–1267
Phornphutkul C, Anikster Y, Huizing M, Braun P, Brodie C, Chou JY, Gahl WA 2001 The promoter of a lysosomal membrane transporter gene, CTNS, binds Sp-1, shares sequences with the promoter of an adjacent gene, CARKL, and causes cystinosis if mutated in a critical region. Am J Hum Genet 69: 712–721
Hyde R, Taylor PM, Hundal HS 2003 Amino acid transporters: roles in amino acid sensing and signalling in animal cells. Biochem J 373: 1–18
Fafournoux P, Bruhat A, Jousse C 2000 Amino acid regulation of gene expression. Biochem J 351: 1–12
Ling R, Bridges CC, Sugawara M, Fujita T, Leibach FH, Prasad PD, Ganapathy V 2001 Involvement of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A. Biochim Biophys Acta 1512: 15–21
Campbell WA, Sah DE, Medina MM, Albina JE, Coleman WB, Thompson NL 2000 TA1/LAT-1/CD98 light chain and system L activity, but not 4F2/CD98 heavy chain, respond to arginine availability in rat hepatic cells. Loss of response in tumor cells. J Biol Chem 275: 5347–5354
Hyatt SL, Aulak KS, Malandro M, Kilberg MS, Hatzoglou M 1997 Adaptive regulation of the cationic amino acid transporter-1 (Cat-1) in Fao cells. J Biol Chem 272: 19951–19957
Sato H, Nomura S, Maebara K, Sato K, Tamba M, Bannai S 2004 Transcriptional control of cystine/glutamate transporter gene by amino acid deprivation. Biochem Biophys Res Commun 325: 109–116
Palii SS, Chen H, Kilberg MS 2004 Transcriptional control of the human sodium-coupled neutral amino acid transporter system A gene by amino acid availability is mediated by an intronic element. J Biol Chem 279: 3463–3471
Bellomo F, Corallini S, Pastore A, Palma A, Laurenzi C, Emma F, Taranta A 2010 Modulation of CTNS gene expression by intracellular thiols. Free Radic Biol Med 48: 865–872
Pastore A, Massoud R, Motti C, Lo Russo A, Fucci G, Cortese C, Federici G 1998 Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine. Clin Chem 44: 825–832
Jones DP, Carlson JL, Mody VC, Cai J, Lynn MJ, Sternberg P 2000 Redox state of glutathione in human plasma. Free Radic Biol Med 28: 625–635
Levtchenko E, de Graaf-Hess A, Wilmer M, van den Heuvel L, Monnens L, Blom H 2005 Altered status of glutathione and its metabolites in cystinotic cells. Nephrol Dial Transplant 20: 1828–1832
Mannucci L, Pastore A, Rizzo C, Piemonte F, Rizzoni G, Emma F 2006 Impaired activity of the gamma-glutamyl cycle in nephropathic cystinosis fibroblasts. Pediatr Res 59: 332–335
Laube GF, Shah V, Stewart VC, Hargreaves IP, Haq MR, Heales SJ, van't Hoff WG 2006 Glutathione depletion and increased apoptosis rate in human cystinotic proximal tubular cells. Pediatr Nephrol 21: 503–509
Park MA, Pejovic V, Kerisit KG, Junius S, Thoene JG 2006 Increased apoptosis in cystinotic fibroblasts and renal proximal tubule epithelial cells results from cysteinylation of protein kinase C delta. J Am Soc Nephrol 17: 3167–3175
Sansanwal P, Yen B, Gahl WA, Ma YW, Ying LH, Wong LJ, Sarwal MM 2010 Mitochondrial autophagy promotes cellular injury in nephropathic cystinosis. J Am Soc Nephrol 21: 272–283
Jousse C, Bruhat A, Ferrara M, Fafournoux P 2000 Evidence for multiple signaling pathways in the regulation of gene expression by amino acids in human cell lines. J Nutr 130: 1555–1560
Lee JI, Dominy JE, Sikalidis AK, Hirschberger LL, Wang W, Stipanuk MH 2008 HepG2/C3A cells respond to cysteine deprivation by induction of the amino acid deprivation/integrated stress response pathway. Physiol Genomics 33: 218–229
Bruhat A, Jousse C, Carraro V, Reimold AM, Ferrara M, Fafournoux P 2000 Amino acids control mammalian gene transcription: activating transcription factor 2 is essential for the amino acid responsiveness of the CHOP promoter. Mol Cell Biol 20: 7192–7204
Guerrini L, Gong SS, Mangasarian K, Basilico C 1993 Cis- and trans-acting elements involved in amino acid regulation of asparagine synthetase gene expression. Mol Cell Biol 13: 3202–3212
Tan NY, Khachigian LM 2009 Sp1 phosphorylation and its regulation of gene transcription. Mol Cell Biol 29: 2483–2488
Casey JL, Koeller DM, Ramin VC, Klausner RD, Harford JB 1989 Iron regulation of transferrin receptor mRNA levels requires iron-responsive elements and a rapid turnover determinant in the 3′ untranslated region of the mRNA. EMBO J 8: 3693–3699
Seiser C, Posch M, Thompson N, Kuhn LC 1995 Effect of transcription inhibitors on the iron-dependent degradation of transferrin receptor mRNA. J Biol Chem 270: 29400–29406
Belasco JG 2010 All things must pass: contrasts and commonalities in eukaryotic and bacterial mRNA decay. Nat Rev Mol Cell Biol 11: 467–478
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Supported by a grant from Cystinosis Research Foundation and in part by a grant from the Cystinosis Research Network.
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Corallini, S., Taranta, A., Bellomo, F. et al. Transcriptional and Posttranscriptional Regulation of the CTNS Gene. Pediatr Res 70, 130–135 (2011). https://doi.org/10.1203/PDR.0b013e3182200187
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DOI: https://doi.org/10.1203/PDR.0b013e3182200187
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