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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.