1. Academic Validation
  2. HEPES activates a MiT/TFE-dependent lysosomal-autophagic gene network in cultured cells: A call for caution

HEPES activates a MiT/TFE-dependent lysosomal-autophagic gene network in cultured cells: A call for caution

  • Autophagy. 2018;14(3):437-449. doi: 10.1080/15548627.2017.1419118.
Marc J Tol 1 2 Martijn J C van der Lienden 3 Tanit L Gabriel 1 Jacob J Hagen 4 Saskia Scheij 1 Tineke Veenendaal 5 Judith Klumperman 5 Wilma E Donker-Koopman 1 Arthur J Verhoeven 1 Hermen Overkleeft 3 Johannes M Aerts 3 Carmen A Argmann 1 4 Marco van Eijk 1 3
Affiliations

Affiliations

  • 1 a Department of Medical Biochemistry , University of Amsterdam , Academic Medical Centre , The Netherlands.
  • 2 b Department of Pathology and Laboratory Medicine , UCLA , Los Angeles , CA , USA.
  • 3 c Leiden Institute of Chemistry , Leiden University , The Netherlands.
  • 4 d Department of Genetics and Genomic Sciences , Icahn Institute for Genomics and Multiscale Biology , Icahn School of Medicine at Mount Sinai , New York , NY , USA.
  • 5 e Department of Cell Biology , University Medical Centre Utrecht , The Netherlands.
Abstract

In recent years, the lysosome has emerged as a highly dynamic, transcriptionally regulated organelle that is integral to nutrient-sensing and metabolic rewiring. This is coordinated by a lysosome-to-nucleus signaling nexus in which mTORC1 controls the subcellular distribution of the microphthalmia-transcription factor E (MiT/TFE) family of "master lysosomal regulators". Yet, despite the importance of the lysosome in cellular metabolism, the impact of traditional in vitro culture media on lysosomal dynamics and/or MiT/TFE localization has not been fully appreciated. Here, we identify HEPES, a chemical buffering agent that is broadly applied in Cell Culture, as a potent inducer of lysosome biogenesis. Supplementation of HEPES to cell growth media is sufficient to decouple the MiT/TFE family members-TFEB, TFE3 and MITF-from regulatory mechanisms that control their cytosolic retention. Increased MiT/TFE nuclear import in turn drives the expression of a global network of lysosomal-autophagic and innate host-immune response genes, altering lysosomal dynamics, proteolytic capacity, autophagic flux, and inflammatory signaling. In addition, siRNA-mediated MiT/TFE knockdown effectively blunted HEPES-induced lysosome biogenesis and gene expression profiles. Mechanistically, we show that MiT/TFE activation in response to HEPES requires its macropinocytic ingestion and aberrant lysosomal storage/pH, but is independent of mTORC1 signaling. Altogether, our data underscore the cautionary use of chemical buffering agents in Cell Culture media due to their potentially confounding effects on experimental results.

Keywords

Autophagy, cell culture; HEPES; MTOR; MiT/TFE; lysosome; metabolism.

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