title: Amino acids regulate transgene expression in MDCK cells.
abstract: Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway.
authors.affiliation: Institute of Physiology and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
authors.forename: Marta
authors.initials: M
authors.lastname: Torrente
authors.affiliation: Institute of Physiology and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
authors.forename: Adriano
authors.initials: A
authors.lastname: Guetg
authors.affiliation: Division of Clinical Chemistry & Biochemistry, University Children's Hospital, Zurich, Zurich, Switzerland.
authors.forename: Jörn Oliver
authors.initials: JO
authors.lastname: Sass
authors.affiliation: Institute of Physiology and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
authors.forename: Lisa
authors.initials: L
authors.lastname: Arps
authors.affiliation: Institute of Physiology and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
authors.forename: Lisa
authors.initials: L
authors.lastname: Ruckstuhl
authors.affiliation: Institute of Physiology and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
authors.forename: Simone M R
authors.initials: SM
authors.lastname: Camargo
authors.affiliation: Institute of Physiology and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
authors.forename: François
authors.initials: F
authors.lastname: Verrey
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D003470:Culture Media; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D008562:Membrane Glycoproteins / Q000235:genetics*; D009174:Mycoplasma / Q000378:metabolism; D010766:Phosphorylation; D060888:Real-Time Polymerase Chain Reaction; D019076:Transgenes
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; C429534:CLTRN protein, human; D003470:Culture Media; D008562:Membrane Glycoproteins; C491436:SLC6A19 protein, human; D001120:Arginine