Calcium Channel Blockers Activate the Interleukin-6 Gene Via the Transcription Factors NF-IL6 and NF-κB in Primary Human Vascular Smooth Muscle Cells

Abstract

Background —Calcium channel blockers (CCB) of all subclasses: the dihydropyridines, benzothiazepines, and phenylalkylamines, at nanomolar concentrations, have been shown to up-regulate interleukin-6 (IL-6) mRNA. We investigated the underlying molecular mechanism responsible for IL-6 induction in response to the CCB amlodipine, diltiazem, and verapamil in primary human vascular smooth muscle cells (VSMC). Methods and Results —All 3 CCB directly activated transcription of the human IL-6 gene in primary human VSMC in a time- and dose-dependent manner, as demonstrated by luciferase reporter gene assays using a 651-bp fragment of the human IL-6 gene promoter. Deletion analysis of the IL-6 promoter revealed that CCB inducible promoter activity was localized to a 160-bp fragment directly upstream of the transcriptional start site of the IL-6 gene. Known transcription factor consensus sequences within this fragment include a NF-IL6 and a NF-κB site. Site-directed mutagenesis suggested that both transcription factors had positive regulatory activity and cooperatively transmitted induction of the IL-6 gene by CCB. The data are confirmed by electrophoretic mobility shift analyses using nuclear extracts from CCB-stimulated and control primary VSMC. CCB of all subclasses increased DNA binding of NF-IL6 and NF-κB as early as 30 minutes after stimulation with the drugs. This effect was independent of intracellular calcium concentrations because calcium-free medium did not increase NF-IL6 or NF-κB activity. Conclusions —The results demonstrate that CCB of all 3 subclasses are capable of activating NF-IL6 and NF-κB. CCB may thus directly regulate cellular functions by affecting the activity of transcription factors independent of changes of intracellular calcium concentrations, an observation that is of interest considering the biological effects induced by CCB.