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

Abstract

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)-alpha and regulators of protein kinases C (PKC)alpha/beta were tested because these have been shown to modulate cancer development and progression. TNF-alpha stimulated expression of both chemokines, while the PKCalpha/beta activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced only expression of IL-8 and inhibited TNF-alpha-induced RANTES expression. The PKCalpha/beta inhibitor Gö 6976 increased TNF-alpha-induced RANTES production and prevented its down-regulation by TPA. In contrast, it decreased TNF-alpha 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)-kappaB response elements but not its activator protein (AP)-1 binding sites. An AP-1 and a NF-kappaB recognition sites were necessary for full induction of IL-8 promoter activity by TNF-alpha and TPA. Moreover, electrophoretic mobility shift assays demonstrated that NF-kappaB 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-alpha to induce in a PKCalpha/beta dependent manner the p44/p42 mitogen-activated protein kinases (MAPK) signaling cascade which controls AP-1 activity. Conversely, TPA inhibited TNF-alpha-induced NF-kappaB signaling and was a weak activator of this pathway. Thus, TPA did not sufficiently activate NF-kappaB to increase transcription through the low affinity NF-kappaB binding sites on RANTES promoter and its inhibitory effect on TNF-alpha-induced NF-kappaB signaling resulted in a reduced transcription rate. On IL-8 promoter, increased transcription through the high affinity NF-kappaB binding site occurred even with poorly activated NF-kappaB 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 PKCalpha/beta in lung adenocarcinoma cells.

Fig. 1. Regulation of RANTES and IL-8 expression

A, A549 cells were serum deprived overnight and stimulated for 4 h with TNF-α (10 ng/ml) and TPA (100 ng/ml) as indicated. Total RNA was analyzed by Northern blotting. The ribosomal protein S26 mRNA served as loading control. Approximate size of transcript was 1.9 kb for RANTES, 2.2 kb for IL-8, and 0.7 kb for S26. B and C, A549 cells were pretreated with Gö 6976 (1 microM) for 1 h and stimulated with TNF-α (10 ng/ml) or TPA (100 ng/ml) for 20 h as indicated. Concentrations of RANTES (B) and IL-8 (C) in supernatants were determined. Data (n = 6) are plotted as means ± SE.

Fig. 2. Regulation of RANTES and IL-8 promoter activity after site-directed mutagenesis

(A) A549 cells were transfected with intact or point mutated RANTES promoter-luciferase gene construct as indicated, and CMVβ-gal. These were treated for 4 h with TNF-α (10 ng/ml) or TPA (100 ng/ml) or the combination of both compounds, and lysed to determine relative luciferase activities after normalization for β-galactosidase activity. Data (n = 6) are shown as fold inductions over basal activity and are plotted as means ± SE. (B) A549 cells were transfected with intact or mutated IL-8 promoter-luciferase gene construct as indicated, and CMVβ-gal. These were then treated as in (A). Data (n = 6) are shown as fold inductions over basal activity and are plotted as means ± SE.

Fig. 3. Regulation of p44/42 MAPK signaling

A549 cells were pretreated with Gö 6976 (1 microM) for 60 min and exposed to TNF-α (10 ng/ml) and/or TPA (100 ng/ml) for 10 min. Cell extracts were analyzed by Western blotting for their content in total p44/42 and phosphorylated p44/42 (phospho-p44/42).

Fig. 4. Time-course analyses of NF-κB signaling

(A) A549 cells were exposed to TNF-α (10 ng/ml) for the indicated time. Cell extracts were analyzed by Western blotting for their content in p65, phosphorylated p65 (phospho-p65), IκBα and phosphorylated IκBα (phospho-IκBα). (B) A549 cells were exposed to TNF-α (10 ng/ml) or TPA (100 ng/ml) for the indicated time. Cell extracts were analyzed as in (A).

Fig. 5. Regulation of TNF-α-induced NF-κB signaling by PKCα/b

(A) A549 cells were pretreated with TPA (100 ng/ml) for the indicated time and further exposed for 5 min to TNF-α (10 ng/ml). Cell extracts were analyzed by Western blotting for their content in p65, phosphorylated p65 (phospho-p65), IκBα and phosphorylated IκBα (phospho-IκBα). (B) A549 cells were pretreated with Gö 6976 (1 μM) and TPA (100 ng/ml) for 60 min and 10 min, respectively. These were then treated with TNF-α (10 ng/ml) for 5 min. Cell extracts were analyzed as in A.

Fig. 6. Relative affinity of NF-κB response elements from the IL-8 and RANTES promoters

Nuclear proteins were extracted from A549 cells treated for 1 h with medium alone (first lane) or 10 ng/ml of TNF-α (+). EMSA was performed using as labeled probe a tandem repeat of the NF-κB response element from the IL-8 promoter (2xNF-κBRE IL-8; 15 nM) and as competitors 2xNF-κBRE IL-8 or the two NF-κB binding sites present in tandem on RANTES promoter (2xNF-κBRE RANTES) at increasing concentrations.