title: Hexosamine biosynthesis in keratinocytes: roles of GFAT and GNPDA enzymes in the maintenance of UDP-GlcNAc content and hyaluronan synthesis.
abstract: UDP-N-acetylglucosamine (UDP-GlcNAc) is a glucose metabolite with pivotal functions as a key substrate for the synthesis of glycoconjugates like hyaluronan, and as a metabolic sensor that controls cell functions through O-GlcNAc modification of intracellular proteins. However, little is known about the regulation of hexosamine biosynthesis that controls UDP-GlcNAc content. Four enzymes can catalyze the crucial starting point of the pathway, conversion of fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P): glutamine-fructose-6-phosphate aminotransferases (GFAT1 and 2) and glucosamine-6-phosphate deaminases (GNPDA1 and 2). Using siRNA silencing, we studied the contributions of these enzymes to UDP-GlcNAc content and hyaluronan synthesis in human keratinocytes. Depletion of GFAT1 reduced the cellular pool of UDP-GlcNAc and hyaluronan synthesis, while simultaneous blocking of both GNPDA1 and GDPDA2 exerted opposite effects, indicating that in standard culture conditions keratinocyte GNPDAs mainly catalyzed the reaction from GlcN6P back to Fru6P. However, when hexosamine biosynthesis was blocked by GFAT1 siRNA, the effect by GNPDAs was reversed, now catalyzing Fru6P towards GlcN6P, likely in an attempt to maintain UDP-GlcNAc content. Silencing of these enzymes also changed the gene expression of related enzymes: GNPDA1 siRNA induced GFAT2 which was hardly measurable in these cells under standard culture conditions, GNPDA2 siRNA increased GFAT1, and GFAT1 siRNA increased the expression of hyaluronan synthase 2 (HAS2). Silencing of GFAT1 stimulated GNPDA1 and GDPDA2, and inhibited cell migration. The multiple delicate adjustments of these reactions demonstrate the importance of hexosamine biosynthesis in cellular homeostasis, known to be deranged in diseases like diabetes and cancer.
authors.affiliation: Institutes of Biomedicine Department of Dentistry, University of Eastern Finland, Yliopistonranta 1E, PO Box 1627, Kuopio 70211, Finland sanna.oikari@uef.fi.
authors.forename: Sanna
authors.initials: S
authors.lastname: Oikari
authors.affiliation: Institutes of Biomedicine Department of Dentistry, University of Eastern Finland, Yliopistonranta 1E, PO Box 1627, Kuopio 70211, Finland.
authors.forename: Katri
authors.initials: K
authors.lastname: Makkonen
authors.affiliation: Institutes of Biomedicine.
authors.forename: Ashik Jawahar
authors.initials: AJ
authors.lastname: Deen
authors.affiliation: Institutes of Biomedicine.
authors.forename: Ilari
authors.initials: I
authors.lastname: Tyni
authors.affiliation: Institutes of Biomedicine.
authors.forename: Riikka
authors.initials: R
authors.lastname: Kärnä
authors.affiliation: Institutes of Biomedicine.
authors.forename: Raija H
authors.initials: RH
authors.lastname: Tammi
authors.affiliation: Institutes of Biomedicine.
authors.forename: Markku I
authors.initials: MI
authors.lastname: Tammi
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000037:antagonists & inhibitors / Q000235:genetics*; D002465:Cell Movement / Q000235:genetics; D005636:Fructosephosphates / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors / Q000235:genetics*; D006595:Hexosamines / Q000096:biosynthesis*; D006801:Humans; D000076002:Hyaluronan Synthases / Q000235:genetics*; D006820:Hyaluronic Acid / Q000096:biosynthesis; D015603:Keratinocytes / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000235:genetics / Q000378:metabolism*
chemical_list: D005636:Fructosephosphates; D006595:Hexosamines; D034741:RNA, Small Interfering; C001293:glucosamine 6-phosphate; D014537:Uridine Diphosphate N-Acetylglucosamine; D019298:Glucose-6-Phosphate; C027618:fructose-6-phosphate; D006820:Hyaluronic Acid; C101754:HAS2 protein, human; D000076002:Hyaluronan Synthases; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); C020582:glucosamine-6-phosphate isomerase; D019747:Aldose-Ketose Isomerases; D005947:Glucose; D005944:Glucosamine
keywords: GFAT; GNPDA; UDP-N-acetylglucosamine; hexosamine biosynthesis; hyaluronan