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.

Expression of tumor growth related genes in IRE1 knockdown U87 glioma cells: effect of hypoxia

“…Therefore, our results concerning strong down-regulation of COL6A1 gene expression in glioma cells upon inhibition of IRE1 completely agree with the functional role of this protein in tumor cells and with suppression of IRE1 knockdown glioma cell proliferation [7,22]. We have also shown that the expression of DEK, HOMER3, and GNPDA1 genes is downregulated and these results agree well with data concerning the functional role of DEK, HOMER3, and GNPDA1 proteins encoding by these genes [25][26][27]. Thus, DEK induces cell proliferation and its expression is required for tumorigenesis [25]; HOMER3 is overexpressed in some cancers [26], and GNPDA1 enhances cell proliferation through the synthesis of glycoconjugates and modification of intracellular proteins [27].…”

“…The suppression level of IRE1 and enzymatic activity in glioma cells that overexpress a dominantnegative construct of inositol requiring enzyme-1 was estimated previously [27,28] by determining the phosphorylation of IRE1 and the expression level of XBP1 alternative splice variant (XBP1s), a key transcription factor in IRE1 signaling, using cells treated by tunicamycin (0.01 mg/ml during 2 h). Moreover, the proliferation rate of glioma cells with mutated IRE1 is decreased 2 times [28].…”

“…Recently, it was shown that HOMER3 is overexpressed in some cancers and possibly related to tumor growth [26]. Glucosamine-6-phosphate deaminase (GNPDA1) catalyzes the reversible conversion of D-glucosamine-6-phosphate into D-fructose-6-phosphate and plays a key role in the maintenance of UDP-N-acetylglucosamine, which 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 modification of intracellular proteins [27].…”

High expression of GFAT1 predicts poor prognosis in patients with pancreatic cancer

“…It has been well recognized that changes in traditional N- and O-linked glycosylation is correlated with oncogenesis25. In addition, UDP-GlcNAc is also used as the substrate for O -linked β-N-actylglucosamine modification ( O -GlcNAcylation) on nucleocytoplasmic and mitochondrial proteins26. Hyper- O -GlcNAcylation was found in multiple types of human malignancies, and considered as a general feature of cancer cells.…”

An update on diabetic kidney disease, oxidative stress and antioxidant agents

“…Enzymatic antioxidant of GST converts reactive electrophilic species to hydrophilic forms then are conjugated by GSH and easily excreted. Non-enzymatic antioxidants like vitamins C and E and lipoic acid are involved in the termination of the lipid peroxidation process (13,14). …”