Expression of the interleukin-6 gene is constitutive and not regulated by estrogen in rat vascular smooth muscle cells in culture

Vascular smooth muscle cells (SMC) are major constituents of the medial lay er of blood vessels and are involved in the development of atherosclerotic plaque. SMC secrete copious IL-6 under basal conditions that can be increas ed by cytokines such as tumor necrosis factor-alpha and interleukin-1 beta (IL-1 beta). The goal of our studies was to define the role of estrogen in IL-6 production by SMC. In a first series of experiments, the expression of specific messenger RNAs as well as the production of IL-6 bioactivity by r at SMC in culture could be demonstrated in basal and IL-1-stimulated condit ions, but was unaffected by estrogen treatment. Different constructs contai ning deleted or mutated fragments of the human IL-6 promoter driving lucife rase or chloramphenicol acetyltransferase reporter gene were then transient ly transfected in these cells. A significant basal activity that was increa sed 2- to 4-fold after IL-1 beta stimulation was observed with the total IL -6 promoter. Deletion analysis indicated that the -158/+11 region containin g activator protein-1 and cAMP response element sites was apparently the mi nimal region of IL-6 promoter to confer both constitutive and IL-1-inducibl e activities. Site-directed mutagenesis experiments suggest that basal acti vity is dependent upon the promoter sequence -158 to -112 containing the nu clear factor (NF)-IL6(-153) and Sp1 sites, whereas IL-1 beta stimulation wo uld depend on the residual -112 nucleotides containing NF-IL6(-75) and NF-k appa B sites. in contrast to the down-regulation of IL-6 expression by estr ogen described in osteoblasts, ethinyl estradiol as well as 17 beta-estradi ol did not influence stimulated IL-6 activity in our experimental condition s whatever the construct tested, even when either estrogen receptor alpha o r beta was overexpressed. Thus, the atheroprotective properties of estrogen are probably not mediated through the regulation of IL-6 production by SMC .