Abstract
Estrogen can significantly influence CD16 expression and alter monocytic cytokine release upon CD16 receptor activation. However, the function of the estrogen receptor (ER) alpha and beta in this response is unclear. To test whether estrogen binds ERalpha and/or ERbeta to affect CD16 expression, monocytic cells were treated with and without physiological levels of 17beta-estradiol and various doses of the ERalpha and ERbeta antagonist fulvestrant followed by measurement of CD16 transcript levels. To determine how estrogen induced changes in TNF-alpha and IL-1beta release due to CD16 activation we quantitated the amount of cytokines after treatment with estrogen, fulvestrant and antibodies that specifically bind and activate the CD16 receptor. Interaction of ERalpha and the CD16 promoter was then determined by chromatin immunoprecipitation. Furthermore, specific promoter elements utilized by estrogen to control CD16 expression were mutated and expression from a luciferase reporter quantitated after transfection. Using the luciferase reporter construct containing a wild type CD16 promoter, the role of ERalpha and ERbeta in the estrogen response was tested by treating transfected monocytes with an ERalpha specific agonist or an ERbeta specific agonist and measuring expression. Our results show that CD16 transcript levels significantly decreased in monocytic cells due to estrogen and that the observed decrease in message was blocked by the antagonist fulvestrant. Estrogen reduced CD16 expression and decreased TNF-alpha and IL-1beta release upon CD16 activation but the administration of fulvestrant blocked this decrease. ERalpha was found to interact with a region 5' of the CD16 gene in the presence of estrogen, and site-directed mutational analysis of this region indicated the necessity for an estrogen response element in modulating estrogen effects on CD16 expression. Moreover, both an ERalpha and an ERbeta agonist reduced expression of the CD16 reporter construct suggesting both receptors can play a role in CD16 regulation. In conclusion, CD16 expression can be altered by the activity of ERalpha or ERbeta and our results also show that ERalpha can associate with a region within the CD16 promoter that is important in production of transcript.