Differential regulation of RANTES and IL-8 expression in lung adenocarcinoma cells

Summary 

In lung adenocarcinoma, expression of Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) is a predictor of survival while that of interleukin (IL)-8 is associated with a poor prognosis. In several models, tumorigenesis is abolished by RANTES, while it is facilitated by IL-8. We studied the regulation of RANTES and IL-8 expression in A549 lung adenocarcinoma cells. The effects of tumor necrosis factor (TNF)-α and regulators of protein kinases C (PKC)α/β were tested because these have been shown to modulate cancer development and progression. TNF-α stimulated expression of both chemokines, while the PKCα/β activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced only expression of IL-8 and inhibited TNF-α-induced RANTES expression. The PKCα/β inhibitor Gö 6976 increased TNF-α-induced RANTES production and prevented its down-regulation by TPA. In contrast, it decreased TNF-α or TPA-induced IL-8 release. The differential regulation of RANTES and IL-8 expression was further analyzed. Site-directed mutagenesis indicated that regulation of RANTES promoter activity required two nuclear factor (NF)-κB response elements but not its activator protein (AP)-1 binding sites. An AP-1 and a NF-κB recognition sites were necessary for full induction of IL-8 promoter activity by TNF-α and TPA. Moreover, electrophoretic mobility shift assays demonstrated that NF-κB response elements from the RANTES promoter were of lower affinity than that from the IL-8 promoter. Immunoblotting experiments showed that TPA was more potent than TNF-α to induce in a PKCα/β dependent manner the p44/p42 mitogen-activated protein kinases (MAPK) signaling cascade which controls AP-1 activity. Conversely, TPA inhibited TNF-α-induced NF-κB signaling and was a weak activator of this pathway. Thus, TPA did not sufficiently activate NF-κB to increase transcription through the low affinity NF-κB binding sites on RANTES promoter and its inhibitory effect on TNF-α-induced NF-κB signaling resulted in a reduced transcription rate. On IL-8 promoter, increased transcription through the high affinity NF-κB binding site occurred even with poorly activated NF-κB and the functional AP-1 response element compensated any loss of transcription rate. These data provide a mechanistic insight into the differential regulation of IL-8 and RANTES expression by PKCα/β in lung adenocarcinoma cells.

Keywords: Adenocarcinoma, Bronchiolo-alveolar, Chemokines, Interleukin-8, NF-kappaB, Protein kinase C, RANTES, Transcription factor AP-1