Session Information
Title: T-cell Biology and Targets in Autoimmune Disease: Signaling Pathways in T-cell Differentiation
Session Type: Abstract Submissions (ACR)
Background/Purpose: T cells from patients with systemic lupus erythematosus (SLE) are poor producers of the vital cytokine interleukin (IL)-2. We recently showed that SLE T cells exhibit reduced expression of the splicing factor 2/ Alternative splicing factor (SF2/ASF), more so in patients with worse disease as evidenced by higher SLE disease activity index (SLEDAI) scores compared to those with mild disease. SF2/ASF, a member of the serine arginine (SR) family of splicing proteins is not only a key regulator of alternative splicing via its RNA binding capacity, but also plays roles in transcription and translation by virtue of its protein-protein interaction properties. Increasing the expression of SF2/ASF in T cells from SLE patients restored their IL-2 production, indicating that SF2/ASF is an important regulator of T cell function, and its reduced expression is a potential molecular defect in SLE T cells. How the expression of SF2/ASF is controlled in T cells is not known. The goal of this study was to determine the mechanism/s controlling SF2/ASF expression in human T cells in resting state and during T cell activation.
Methods: T cells were isolated from peripheral blood of healthy volunteers. T cells were activated for various time points (3, 6, 24 hours) with either anti-CD3, anti-CD28 antibodies, or phorbol myristic acid (PMA) plus Ionomycin. mRNA expression was assessed by reverse transcription and real time quantitative PCR. Protein expression was studied by immunoblotting. Actinomycin D was used to block transcription and assess mRNA stability. To assess post translational mechanisms, the lysosome inhibitor Bafilomycin A1 and proteasome inhibitor MG132 were added during activation to block protein degradation.
Results: T cells in resting state expressed SF2/ASF mRNA and protein abundantly. T cell activation increased mRNA expression of SF2/ASF by 3- to 4- fold whereas protein expression did not change much, or even decreased. Stimulating T cells with PMA and Ionomycin similarly resulted in increased mRNA and reduced SF2/ASF protein expression. This discrepancy between mRNA and protein expression suggests that post-transcriptional and/or post-translational mechanisms regulate SF2/ASF expression during T cell activation. The discrepancy was not due to reduced transcript stability, as the rate of SF2/ASF mRNA decay in resting versus activated T cells showed no significant difference. Activating T cells in the presence of inhibitors of the lysosome or proteasome revealed that the proteasome but not lysosome is involved in the degradation of SF2/ASF.
Conclusion: Our results indicate that SF2/ASF mRNA and protein expression are differentially regulated during T cell activation, and that proteasome mediated degradation may represent an important physiologic mechanism to tightly control expression of this essential splicing factor. Our previous work showed that increased ubiquitination and proteasome degradation are important in the downregulation of the CD3 zeta signaling protein in SLE T cells. Therefore proteasome mediated SF2/ASF degradation may represent a molecular mechanism that contributes to its reduced expression in SLE T cells.
Disclosure:
V. R. Moulton,
None;
A. R. Gillooly,
None;
G. C. Tsokos,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/t-cell-activation-induces-increased-mrna-expression-of-the-splicing-factor-sf2asf-but-simultaneous-protein-downregulation-via-proteasome-mediated-degradation/