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Int. J. Mol. Sci. 2019, 20(2), 234; https://doi.org/10.3390/ijms20020234

Silencing of Transcription Factor Sp1 Promotes SN1 Transporter Regulation by Ammonia in Mouse Cortical Astrocytes

Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawińskiego Str., 02-106 Warsaw, Poland
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Received: 9 November 2018 / Revised: 27 December 2018 / Accepted: 27 December 2018 / Published: 9 January 2019
(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)
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Abstract

The involvement of the astrocytic SN1 (SNAT3) transporter in ammonia-induced l-glutamine retention was recently documented in mouse-cultured astrocytes. Here we investigated the involvement of specificity protein 1 (Sp1) transcription factor in SN1 regulation in ammonium chloride (“ammonia”)-treated astrocytes. Sp1 expression and its cellular localization were determined using real-time qPCR, Western blot, and confocal microscopy. Sp1 binding to Snat3 promoter was analyzed by chromatin immunoprecipitation. The role of Sp1 in SN1 expression and SN1-mediated [3H]glutamine uptake in ammonia-treated astrocytes was verified using siRNA and mithramycin A. The involvement of protein kinase C (PKC) isoforms in Sp1 level/phosphorylation status was verified using siRNA technology. Sp1 translocation to the nuclei and its enhanced binding to the Snat3 promoter, along with Sp1 dependence of system N-mediated [3H]glutamine uptake, were observed in astrocytes upon ammonia exposure. Ammonia decreased the level of phosphorylated Sp1, and the effect was reinforced by long-term incubation with PKC modulator, phorbol 12-myristate 13-acetate, which is a treatment likely to dephosphorylate Sp1. Furthermore, silencing of the PKCδ isoform appears to enhance the ammonia effect on the Sp1 level. Collectively, the results demonstrate the regulatory role of Sp1 in regulation of SN1 expression and activity in ammonia-treated astrocytes and implicate altered Sp1 phosphorylation status in this capacity. View Full-Text
Keywords: astrocytes; glutamine; SN1 (SNAT3), Sp1; protein kinase C; ammonia astrocytes; glutamine; SN1 (SNAT3), Sp1; protein kinase C; ammonia
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Dąbrowska, K.; Zielińska, M. Silencing of Transcription Factor Sp1 Promotes SN1 Transporter Regulation by Ammonia in Mouse Cortical Astrocytes. Int. J. Mol. Sci. 2019, 20, 234.

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