Differential regulation of RANTES and IL-8 expression in lung adenocarcinoma cells.
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.
Henriquet
Corinne
C
Institut National de la Santé et de la Recherche Médicale U454-IFR3, 34295 Montpellier cedex 5, France.
Gougat
Claire
C
Combes
Audrey
A
Lazennec
Gwendal
G
Mathieu
Marc
M
D000230:Adenocarcinoma / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D002273:Carcinogens / Q000494:pharmacology; D045744:Cell Line, Tumor; D018946:Chemokine CCL5 / Q000096:biosynthesis*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis*; D008175:Lung Neoplasms / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects / Q000378:metabolism
D002273:Carcinogens; D018946:Chemokine CCL5; D016209:Interleukin-8; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate