Glutamine availability up-regulates expression of the amino acid transporter protein ASCT2 in HepG2 cells and stimulates the ASCT2 promoter.
Glutamine transport into the human hepatoma cell line HepG2 is catalysed primarily by an ASCT2-type transporter identical in sequence with that cloned previously from JAR cells. An antibody raised against the C-terminus of the ASCT2 protein was shown to recognize ASCT2 on Western blots. Using this antibody, it was found that variation in cell growth rate did not affect ASCT2 expression, but both growth rate and ASCT2 expression were significantly reduced by glutamine deprivation. Expression of a number of other proteins was shown to be unaffected under these conditions. The sequence of the 5'-flanking region of the ASCT2 gene was derived from the human genome database. A 907 bp fragment of this sequence was directionally ligated into a luciferase reporter vector and was shown to exhibit promoter activity when transfected into HepG2 cells. Promoter activity was greatly reduced when transfection was performed in glutamine-free medium and was restored when glutamine was added post-transfection. The absence of other essential amino acids did not affect promoter activity, and glutamine deprivation did not affect the MCT1 (monocarboxylate transporter 1) promoter. These results indicate that both ASCT2 promoter activity and ASCT2 protein expression in these cells are dependent on glutamine availability.
Bungard
Claire I
CI
Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK. C.I.Bungard@bristol.ac.uk
McGivan
John D
JD
D000595:Amino Acid Sequence; D026962:Amino Acid Transport System ASC / Q000235:genetics* / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D001693:Biological Transport, Active / Q000235:genetics; D019556:COS Cells / Q000737:chemistry / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D005973:Glutamine / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D015854:Up-Regulation / Q000235:genetics*
D026962:Amino Acid Transport System ASC; D000906:Antibodies; D015778:Minor Histocompatibility Antigens; D010455:Peptides; C420506:SLC1A5 protein, human; C498850:Slc1a5 protein, mouse; C498849:Slc1a5 protein, rat; D005973:Glutamine