Systemic lupus erythematosus (SLE) is an autoimmune disease that occurs primarily in women during their childbearing years. The effects of SLE are physically and emotionally debilitating and can be life threatening due to the involvement of a variety of organs including the renal and central nervous systems. Estrogen is a female sex hormone that acts on target cells though specific receptor proteins. Estrogen receptors (ER) are ligand activated transcription factors that bind to unique DNA sequences of target genes and alter the rates of transcription. In lupus T cells, estrogen increases the expression of calcineurin, a protein phosphatase involved in T cell activation. The proposed research will investigate the molecular mechanisms by which estrogen, acting through the ER, significantly increases calcineurin, activates nuclear factor of activated T cells (NFAT), and augments CD4O ligand (CD4OL) expression in the T cells from female lupus patients but not in T cells from healthy individuals. The molecular mechanisms responsible for increased calcineurin expression in lupus T cells will be investigated using assays that distinguish transcriptional and posttranscriptional gene regulation. To determine if the differential control of calcineurin in lupus T cells is due to differences in the ratio of ER subtypes (ERa versus ERB), reverse transcription and quantitative polymerase chain amplification will be used to determine the amount of ER subtype transcripts. Altered ER binding to DNA regulatory elements will be assessed by electrophoretic mobility shift assays (EMSA). To investigate if the estrogen-dependent increase in calcineurin hyperactivates the transcription factor NFAT, the duration of NFAT dephosphorylation will be analyzed and the ratio of phosphorylated and dephosphorylated NFAT will be compared between T cells treated without and with estradiol. Biological activity over the same time points will be measured by comparing the amount of NEAT binding to consensus DNA sequences of the CD4OL promoter and by measuring changes in CD4OL expression in response to estradiol. The knowledge gained by completion of this study will identify potential sites in human lupus T cells at which to block signal transduction and decrease the abnormal synthesis of cytokines that promote inflammation and B cell activation.
