Background/Purpose: Treg cells play a central role in the tolerance of self-antigens and prevention of autoimmune disease. FoxP3 is a Treg-specific transcription factor that plays a critical role in Treg differentiation and function. The vitamin A metabolite all-trans retinoic acid (atRA) has a wide range of biological activity including cellular differentiation and proliferation. Previous studies have shown that atRA regulates Treg stability under inflammatory conditions and promotes plasma-derived Treg development in-part through regulating FoxP3 gene expression. Although these transcriptional effects have been characterized, post-transcriptional effects of atRA on FoxP3 protein stability have not been investigated. The aim of the present study was to test the hypothesis that atRA suppresses FoxP3 protein degradation under pro-inflammatory conditions through altering FoxP3 protein-protein interactions.

Methods: To examine the effect of atRA on FoxP3 protein levels in the absence of FoxP3 transcriptional regulation, FoxP3 was genetically fused to an N-terminal HaloTag reporter protein (Promega) and expressed in the immortalized T lymphocyte jurkat cell line under control of the human cytomegalovirus (CMV) promoter. cDNAs encoding HaloTag and FoxP3 or HaloTag alone were assembled into a lentiviral expression vector via In-Fusion cloning technology (Takara) and transfected into 293T cells to produce VSV-G pseudotyped virus for cell transductions. Halo-FoxP3 and Halo-control expressing cells were then selected with puromycin, creating stable cell lines for experiments. FoxP3 protein levels were assessed in the presence or absence of atRA with IL-6 or IL-1β alone or in combination by Western blot. To characterize the effect of atRA on FoxP3 protein interactions, Halo-FoxP3 was isolated and purified from cell lysates with HaloLink resin (Promega). FoxP3-interacting proteins were then eluted from the resin, digested with trypsin and profiled by nano LC-MS/MS with a Waters M-Class HPLC system interfaced to a ThermoFisher Fusion Lumos mass spectrometer. The resulting Mascot DAT files were parsed into Scaffold (Proteome Software) for validation and filtering to create non-redundant FoxP3-interacting protein lists for each sample.

Results: atRA increased Halo-FoxP3 protein levels in the Halo-FoxP3 expressing cells. In contrast, both IL-6 and IL-1β alone or in combination induced Halo-FoxP3 protein degradation without affecting Halo levels in the Halo-control cells. However, concomitant treatment with atRA abrogated IL-6/IL-1β-induced FoxP3 degradation. Profiling of FoxP3-interacting proteins in cell lysates from Halo-FoxP3 expressing cells by LC/MS-MS indicated that atRA treatment had extensive effects on FoxP3 protein-protein interactions, which included suppression of IL-6/IL-1β-induced interaction with the 26S proteasome and a novel FoxP3-interacting E3 ubiquitin ligase, RNF213.

Conclusion: These results suggest that in addition to previously characterized transcriptional mechanisms, atRA may suppress FoxP3 degradation under inflammatory conditions through preventing interaction with RNF213, a novel FoxP3-interacting E3 ubiquitin ligase.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/all-trans-retinoic-acid-atra-enhances-foxp3-protein-stability-under-inflammatory-conditions-through-altering-the-foxp3-protein-interactome/