Rexinoids Modulate Steroid and Xenobiotic Receptor Activity by Increasing Its Protein Turnover in a Calpain-dependent Manner

Filippa Pettersson,Nessrine Hanna,Marina Lagodich,Daphné Dupéré-Richer,Marie-Claude Couture,Catherine Choi,Wilson H Miller

doi:10.1074/jbc.m710358200

Filippa Pettersson, Nessrine Hanna + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m710358200

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Abstract

The steroid and xenobiotic receptor SXR (human pregnane X receptor) is a nuclear receptor that plays a key role in the body's detoxification response by regulating genes involved in drug metabolism and transport. SXR ligands include a wide range of compounds, which induce transcription of SXR target genes via activation of a heterodimeric transcription factor consisting of SXR and the related nuclear receptor retinoid X receptor (RXR). We investigated the effect of RXR-selective ligands, rexinoids, on SXR/RXR activity. In agreement with previous reports, we found that rexinoids are weak activators of SXR, but we also found that they can antagonize SXR activation by the potent SXR agonist rifampicin. This antagonism included suppression of rifampicin-induced expression of SXR target genes, as well as reduced binding of SXR/RXR to SXR response elements both in vivo and in vitro. Interestingly, two rexinoids, bexarotene (LGD1069/Targretin) and LG100268, caused a rapid and sustained decrease in the protein levels of both SXR and RXR. The decrease in SXR level was due to an enhanced rate of protein degradation and was dependent on calpain activity, as opposed to rexinoid-induced RXR degradation, which is mediated via the proteasome. Thus, we have demonstrated a novel, rexinoid-modulated mechanism regulating SXR protein stability, which may explain why rexinoids are only weak activators of SXR/RXR-mediated transcription, despite reports that they bind to SXR with high affinity. We suggest that the ability of rexinoids to induce degradation of both SXR and RXR, in combination with competition for binding to SXR, can also explain why rexinoids antagonize the activation of SXR by drugs like rifampicin.

Highlights

To test the effect of the rexinoid bexarotene on expression of endogenous SXR target genes, MDR1 expression was first assessed in LS180 cells treated with bexarotene, rifampicin, or both
Electrophoretic Mobility Shift Assay (EMSA) assessment of the ability of in vitro translated SXR/retinoid X receptor (RXR) to bind to the synthetic SXRE in the presence or absence of rifampicin and/or bexarotene showed no difference in binding affinity regardless of the drugs present (Fig. 2E), indicating that additional factors, present in nuclear extracts, are needed to alter receptor DNA binding
Rexinoids have been shown to bind to SXR with high affinity in competition binding assays, where bexarotene and LG100268 competed with radiolabeled SR12813 for binding to immobilized SXR more efficiently than rifampicin [8]

Summary

Introduction

EMSA assessment of the ability of in vitro translated SXR/RXR to bind to the synthetic SXRE in the presence or absence of rifampicin and/or bexarotene showed no difference in binding affinity regardless of the drugs present (Fig. 2E), indicating that additional factors, present in nuclear extracts, are needed to alter receptor DNA binding. Pulse-chase analysis of the degradation rate of 35S-labeled FLAG-SXR, transiently transfected into MCF-7 cells, was performed in the presence of DMSO, bexarotene, or LG100268, and we found that both rexinoids triggered a faster degradation rate (Fig. 4A).

Results

Conclusion