Background/Purpose:

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with multifactorial etiopathogenesis that primarily afflicts women in the childbearing years. Hormones especially estrogen are implicated in the pathogenesis of disease, however the precise molecular events regulated by estrogen in immune cells are not well understood. T cells play a key role in SLE pathogenesis and display abnormalities in gene expression and function. We previously identified a novel role for the multifunctional protein serine arginine rich splicing factor 1 (SRSF1) in T cell gene regulation and function. We showed that SRSF1 promotes IL-2 production in normal T cells, and its overexpression into SLE T cells rescues IL-2 production. SRSF1 expression is decreased in SLE T cells, and associates with severe disease activity. We recently generated a T cell specific Srsf1-conditional knockout mouse, which exhibits defects in T cell function and develops autoimmune disease. These results suggest that the deficiency of SRSF1 is an important molecular defect in immune-mediated disease. However, not much is known about the regulation of SRSF1 in T cells, and if or how hormones may contribute to its expression. Here we asked whether estrogen regulates the expression of SRSF1 in human T cells.

Methods:

Peripheral blood was collected from healthy women in the follicular phase of the menstrual cycle. T cells were isolated by negative selection from peripheral blood. T cells were cultured (in RPMI without phenol red supplemented with charcoal-stripped serum), with increasing concentrations (0, 1nM, 10nM, 100nM) of beta-estradiol for 18 – 24 hours. Total protein and RNA were isolated and the expression of SRSF1 assessed by Western blotting and RT-qPCR. To assess mRNA stability, transcription was blocked with actinomycin D, and cells collected at 0, 0.5, 1, 1.5 and 2 hours, and Srsf1 expression assessed by RT-qPCR. To assess posttranslational protein degradation mechanisms, the proteasome inhibitor MG132 or the lysosome inhibitor Bafilomycin A1 were added to cultures.

Results:

We found that exposure to beta-estradiol led to a dose dependent increase in Srsf1 mRNA expression, but a decrease in protein levels in T cells. This discrepancy between mRNA and protein expression suggests that estrogen may downregulate protein levels via post-transcriptional mechanisms such as mRNA decay, translation or protein degradation. The discrepancy between high mRNA versus low protein levels was not due to reduced mRNA stability because the rate of Srsf1 mRNA decay in control versus estrogen-treated T cells did not show significant differences. Culturing estrogen-treated T cells in the presence of inhibitors of the lysosome or proteasome showed that the proteasome but not the lysosome is involved in the degradation of SRSF1. In silico analysis of the Srsf1 3`UTR revealed microRNA (miR) target sites, of which miR-21-5p, miR-27, and miR-200b-3p are known to be regulated by estrogen and to be dysregulated in SLE.

Conclusion:

Our results suggest that estrogen can modulate the expression of SRSF1 via posttranscriptional mechanisms in human T lymphocytes, thus revealing a potential molecular link between hormones, immune cells and autoimmune disease.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/estrogen-downregulates-the-expression-of-serine-arginine-splicing-factor-1-srsf1-in-human-t-lymphocytes/