Regulation of stimulus-induced interleukin-8 gene transcription in human adrenocortical carcinoma cells - Role of AP-1 and NF-κB

Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated G alpha q-coupled designer receptor. H295R cells stimulated with either interleukin-1 beta (IL-1 beta) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1 beta-treated H295R cells required NF-kappa B while the phorbol ester TPA used both the AP-1 and NF-kappa B sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NE-kappa B-responsive reporter genes. While stimulation of G alpha q-coupled designer receptors increased the AP-1 activity in the cells, IL-1 beta specifically stimulated NF-kappa B-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-kappa B activities. We conclude that stimulation of Gaq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-kappa B. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.