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Digoxin, an Overlooked Agonist of ROR?/ROR?T.

ABSTRACT: Digoxin was one of the first identified ROR?T receptor inverse agonists inhibiting the differentiation of Th17 cells. However, this compound exhibits inhibitory activity at relatively high concentrations that mediate cytotoxic effects. We previously identified several cardenolides that are structurally similar to digoxin that were able to induce ROR?/ROR?T-dependent transcription. These observations encouraged us to reanalyze the effects of digoxin on ROR?/ROR?T-dependent transcription at low, noncytotoxic concentrations. Digoxin induced ROR?/ROR?T-dependent transcription in HepG2 and Th17 cells. Furthermore, analysis of the transcriptomes of Th17 cells cultured in the presence of digoxin revealed the induction of the expression of numerous Th17-specific genes, including IL17A/F, IL21, IL22, IL23R, CCR4, and CCR6. Thus, our study, which includes data obtained from intact cells, indicates that digoxin, similar to other cardenolides, is a potent ROR?/ROR?T receptor activator and that its structure may serve as a starting point for the design of dedicated molecules that can be used in the development of adoptive cell therapy (ACT).

SUBMITTER: Karas K

PROVIDER: S-EPMC6330298 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Digoxin, an Overlooked Agonist of RORγ/RORγT.

Karaś Kaja K Sałkowska Anna A Sobalska-Kwapis Marta M Walczak-Drzewiecka Aurelia A Strapagiel Dominik D Dastych Jarosław J Bachorz Rafał A RA Ratajewski Marcin M

Frontiers in pharmacology 20190107

Digoxin was one of the first identified RORγT receptor inverse agonists inhibiting the differentiation of Th17 cells. However, this compound exhibits inhibitory activity at relatively high concentrations that mediate cytotoxic effects. We previously identified several cardenolides that are structurally similar to digoxin that were able to induce RORγ/RORγT-dependent transcription. These observations encouraged us to reanalyze the effects of digoxin on RORγ/RORγT-dependent transcription at low, n ...[more]

PMID: 30666196

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Project description:CD4+ T helper lymphocytes that express interleukin-17 (Th17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in Th17 cell differentiation and function with susceptibility to Crohn’s disease, rheumatoid arthritis, and psoriasis1-3. Thus, the pathway towards differentiation of Th17 cells and, perhaps, of related innate lymphoid cells with similar effector functions4, 5, is an attractive target for therapeutic applications. Mouse and human Th17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORγt, which is required for induction of IL-17 transcription and for the manifestation of Th17-dependent autoimmune disease in mice6. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORγt transcriptional activity. Digoxin inhibited murine Th17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives, 20,22-dihydrodigoxin-21,23-diol (Dig(dhd)) and digoxin-21-salicylidene (Dig(sal)), specifically inhibited induction of IL-17 in human CD4+ T cells. Using these small molecule compounds, we demonstrated that RORγt is imporant for the maintenance of IL-17 expression in mouse and human effector T cells. These data suggest that derivatives of digoxin can be used as chemical probes for development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease. We performed gene expression profiling with total RNA samples isolated from DMSO- or digoxin-treated wildtype or RORγt-deficient cells cultured in Th17 conditions. Total RNA was extracted with TRIzol. RNA was labeled and hybridized to GeneChip Mouse Genome 430 2.0 arrays following the Affymetrix protocols. Data were analyzed in GeneSpring GX11.5.

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Digoxin, an Overlooked Agonist of ROR?/ROR?T.

Publications

Digoxin, an Overlooked Agonist of RORγ/RORγT.

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