Annie Husson

Glutamine Stimulates Argininosuccinate Synthetase Gene Expression through Cytosolic O-Glycosylation of Sp1 in Caco-2 Cells

Glutamine stimulates the expression of the argininosuccinate synthetase (ASS) gene at both the level of enzyme activity and mRNA in Caco-2 cells. Searching to identify the pathway involved, we observed that (i) the stimulating effect of glutamine was totally mimicked by glucosamine addition, and (ii) its effect but not that of glucosamine was totally blocked by 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of amidotransferases, suggesting that the metabolism of glutamine to glucosamine 6-phosphate was required. Moreover, run-on assays revealed that glucosamine was acting at a transcriptional level. Because three functional GC boxes were identified on the ASS gene promoter (Anderson, G. M., and Freytag, S. O. (1991) Mol. Cell Biol. 11, 1935–1943), the potential involvement of Sp1 family members was studied. Electrophoretic mobility shift assays using either the Sp1 consensus sequence or an appropriate fragment of the ASS promoter sequence as a probe demonstrated that both glutamine and glucosamine increased Sp1 DNA binding. Immunoprecipitation-Western blot experiments demonstrated that both compounds increased O-glycosylation of Sp1 leading to its translocation into nucleus. Again, the effect of glutamine on Sp1 was inhibited by the addition of DON but not of glucosamine. Taken together, the results clearly demonstrate that the metabolism of glutamine through the hexosamine pathway leads to the cytosolic O-glycosylation of Sp1, which, in turn, translocates into nucleus and stimulates the ASS gene transcription. Collectively, the results constitute the first demonstration of a functional relationship between a regulating signal (glutamine), a transcription factor (Sp1), and the transcription of the ASS gene.

Control of mammalian gene expression by amino acids, especially glutamine

Molecular data rapidly accumulating on the regulation of gene expression by amino acids in mammalian cells highlight the large variety of mechanisms that are involved. Transcription factors, such as the basic‐leucine zipper factors, activating transcription factors and CCAAT/enhancer‐binding protein, as well as specific regulatory sequences, such as amino acid response element and nutrient‐sensing response element, have been shown to mediate the inhibitory effect of some amino acids. Moreover, amino acids exert a wide range of effects via the activation of different signalling pathways and various transcription factors, and a number of cis elements distinct from amino acid response element/nutrient‐sensing response element sequences were shown to respond to changes in amino acid concentration. Particular attention has been paid to the effects of glutamine, the most abundant amino acid, which at appropriate concentrations enhances a great number of cell functions via the activation of various transcription factors. The glutamine‐responsive genes and the transcription factors involved correspond tightly to the specific effects of the amino acid in the inflammatory response, cell proliferation, differentiation and survival, and metabolic functions. Indeed, in addition to the major role played by nuclear factor‐κB in the anti‐inflammatory action of glutamine, the stimulatory role of activating protein‐1 and the inhibitory role of C/EBP homology binding protein in growth‐promotion, and the role of c‐myc in cell survival, many other transcription factors are also involved in the action of glutamine to regulate apoptosis and intermediary metabolism in different cell types and tissues. The signalling pathways leading to the activation of transcription factors suggest that several kinases are involved, particularly mitogen‐activated protein kinases. In most cases, however, the precise pathways from the entrance of the amino acid into the cell to the activation of gene transcription remain elusive.

Glutamine and interleukin‐1β interact at the level of Sp1 and nuclear factor‐κB to regulate argininosuccinate synthetase gene expression

We previously demonstrated that the expression of the argininosuccinate synthetase (ASS) gene, a key step in nitric oxide production, is stimulated either by interleukin‐1β[Brasse‐Lagnel et al. (2005) Biochimie 87, 403–9] or by glutamine in Caco‐2 cells [Brasse‐Lagnel et al. (2003) J. Biol. Chem. 278, 52504–10], through the activation of transcription factors nuclear factor‐κB and Sp1, respectively. In these cells, the fact that glutamine stimulated the expression of a gene induced by pro‐inflammatory factors appeared paradoxical as the amino acid is known to exert anti‐inflammatory properties in intestinal cells. We therefore investigated the effect of simultaneous addition of both glutamine and interleukin‐1β on ASS gene expression in Caco‐2 cells. In the presence of both compounds for 4 h, the increases in ASS activity, protein amount and mRNA level were almost totally inhibited, implying a reciprocal inhibition between the amino acid and the cytokine. The inhibition was exerted at the level of the transcription factors Sp1 and nuclear‐κB: (a) interleukin‐1β inhibited the glutamine‐stimulated DNA‐binding of Sp1, which might be related to a decrease of its glutamine‐induced O‐glycosylation, and (b) glutamine induced per se a decrease in the amount of nuclear p65 protein without affecting the stimulating effect of interleukin‐1β on nuclear factor‐κB, which might be related to the metabolism of glutamine into glutamate. The present results constitute the first demonstration of a reciprocal inhibition between the effects of an amino acid and a cytokine on gene expression, and provide a molecular basis for the protective role of glutamine against inflammation in the intestine.

Glutamine accelerates interleukin-6 production by rat peritoneal macrophages in culture

The effect of glutamine on the production of interleukin‐6 (IL‐6) was studied in rat peritoneal macrophages in culture. A maximal production of IL‐6 was measured at 4 h in lipopolysaccharide (LPS)‐stimulated macrophages, and addition of glutamine (5 mM) anticipated this increase by 1 h without any increase in the IL‐6 mRNA level. The effect of glutamine required the presence of LPS. Thus, glutamine accelerates IL‐6 production from the pre‐existing mRNA. The effect of glutamine was not mediated by cell swelling since culture of macrophages in hypoosmotic condition decreased the production of IL‐6 in the culture medium with a corresponding decrease in the IL‐6 mRNA level.

Hypoosmolarity and glutamine increased the β-actin gene transcription in isolated rat hepatocytes

The mechanism of action of hydration state was studied on β‐actin gene expression in isolated hepatocytes. Results obtained with Northern blot analysis and run on transcription assays show that hypoosmolarity increased and hyperosmolarity decreased the β‐actin mRNA level through a corresponding modulation of the rate of the gene transcription. Glutamine, which is known to induce cell swelling, also increased the β‐actin mRNA level in a dose‐dependent manner and induced a stimulation of the β‐actin gene transcription. Thus, cell hydration state regulates gene expression in the liver through a transcriptional mechanism.

Amino acid regulation of mammalian gene expression in the intestine

Some amino acids exert a wide range of regulatory effects on gene expression via the activation of different signalling pathways and transcription factors, and a number of cis elements were shown to respond to changes in amino acid concentration. Particular attention has been paid to the effects of glutamine and arginine, which modulate a number of cell functions through the activation of various pathways in different tissues. In the intestine, appropriate concentrations of both arginine and/or glutamine contribute to facilitate cell proliferation, to limit the inflammatory response and apoptosis, and to modulate intermediary metabolism through specific transcription factors. Particularly, besides its role as a major fuel for enterocytes, the regulatory effects of glutamine have been extensively studied and the molecular mechanisms involved appear diversified and complex. Indeed, in addition to a major role of NF-kappaB in its anti-inflammatory action and a stimulatory role of AP-1 in its growth-promoting action and cell survival, the involvement of some other transcription factors, such as PPAR-gamma or HSF-1, was shown to maintain intestinal cell integrity. The signalling pathways leading to the activation of transcription factors imply several kinases, particularly MAP kinases in the effect of glutamine and p70 S6 kinase for those of arginine, but in most cases the precise pathways from the entrance of the aminoacid into the cell to the activation of gene transcription has remained elusive.

GLUTAMINE AND REGULATION OF GENE EXPRESSION IN RAT HEPATOCYTES : THE ROLE OF CELL SWELLING

Glutamine is able to regulate the expression of various genes in rat hepatocytes. This includes genes coding for proteins involved in glutamine utilization, such as argininosuccinate synthetase (ureagenesis) or phosphoenolpyruvate carboxykinase (gluconeogenesis). Moreover, glutamine is also able to stimulate the expression of genes involved in the acute-phase response, such as the alpha 2-macroglobulin gene. The effect of glutamine on the regulation of gene expression may be explained, at least in part, by the cell swelling due to its sodium-dependent transport. The physiological significance of the effect of glutamine is discussed.

Effets des glucocorticostéroïdes sur l'activité des enzymes du cycle de l'urée dans le foie foetal de rat

Summary The five urea cycle enzyme activities of rat liver are followed during late fetal period and the first day of life. All five enzymes exhibit relatively low activities in fœtal liver and a rapid postnatal increase. Lack of glucocorticosteroid (after hypophysectomy in utero) induces an important decrease of activity of three enzymes : carbamyl phosphate synthetase, ornithine transcarbamylase and argininosuccinate synthetase. Treatment with hydrocortisone acetate on decapitated fetuses results in a marked stimulation of the activity of four of the enzymes : carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase and arginase. Premature induction of carbamyl phosphate synthetase activity is obtained after intraperitoneal injection with hydrocortisone acetate at 16,5 days of gestation.

Glutamine and regulation of gene expression in mammmalian cells. Special reference to phosphoenolpyruvate carboxykinase (PEPCK)

The repertoire of the actions of specific amino acids on gene expression is relatively limited in mammalian cells. Glutamine constitutes the most studied amino acid and recent works intended to demonstrate its mechanism of action on two genes: the beta-actin and the phosphoenolpyruvate carboxykinase genes. From these studies, it appears that glutamine may regulate gene expression by, at least, two different mechanisms: one through the glutamine-induced cell swelling, and another through its intracellular metabolism. The involvement of phosphatidylinositol 3-kinase in the signaling pathway triggered by cell swelling is discussed.

Cell swelling increased the α2‐macroglobulin gene expression in cultured rat hepatocytes

The effect of cell swelling on the expression of the α2‐macroglobulin (α2M) gene was studied in hepatocytes in culture. Hypoosmolarity induced an increase (3‐fold increase) in the level of α2M mRNA through a corresponding stimulation of the rate of transcription of the α2M gene. The addition of raffinose (100 mM) corrected the effect of hypoosmolarity at both mRNA and transcriptional level, demonstrating that cell swelling per se was responsible for the observed effect on the expression of the α2M gene. Moreover, the effect of cell swelling was additive to that of interleukin 6, a major mediator of the acute‐phase response.