Activation of Liver X Receptors Prevents Statin-induced Death of 3T3-L1 Preadipocytes

Lise Madsen,Rasmus K Petersen,Knut R Steffensen,Lone M Pedersen,Philip Hallenborg,Tao Ma,Livar Frøyland,Stein Ove Døskeland,Jan-Åke Gustafsson,Karsten Kristiansen

doi:10.1074/jbc.m800720200

Abstract

The biological functions of liver X receptors (LXRs) alpha and beta have primarily been linked to pathways involved in fatty acid and cholesterol homeostasis. Here we report a novel role of LXR activation in protecting cells from statin-induced death. When 3T3-L1 preadipocytes were induced to differentiate by standard isobutylmethylxanthine/dexamethasone/insulin treatment in the presence of statins, they failed to differentiate and underwent massive apoptosis. The simultaneous addition of selective LXR agonists prevented the statin-induced apoptosis. By using mouse embryo fibroblasts from wild-type (LXRalpha+/+/LXRbeta+/+), LXRalpha knock-out mice (LXRalpha(-/-)/LXRbeta+/+), LXRbeta knock-out mice (LXRalpha+/-/LXRbeta(-/-)), and LXR double knock-out mice (LXRalpha(-/-)/LXRbeta(-/-)) as well as 3T3-L1 cells transduced with retroviruses expressing either wild-type LXRalpha or a dominant negative version of LXRalpha, we demonstrate that the response to LXR agonists is LXR-dependent. Interestingly, LXR-mediated rescue of statin-induced apoptosis was not related to up-regulation of genes previously shown to be involved in the antiapoptotic action of LXR. Furthermore, forced expression of Bcl-2 did not prevent statin-induced apoptosis; nor did LXR action depend on protein kinase B, whose activation by insulin was impaired in statin-treated cells. Rather, LXR-dependent rescue of statin-induced apoptosis in 3T3-L1 preadipocytes required NF-kappaB activity, since expression of a dominant negative version of IkappaBalpha prevented LXR agonist-dependent rescue of statin-induced apoptosis. Thus, the results presented in this paper provide novel insight into the action of statins on and LXR-dependent inhibition of apoptosis.

Highlights

Used in the treatment of hypercholesterolemia [1, 2]
By using mouse embryo fibroblasts from wild-type (LXR␣؉/؉/liver X receptor (LXR)␤؉/؉), LXR␣ knock-out mice (LXR␣؊/؊/LXR␤؉/؉), LXR␤ knock-out mice (LXR␣؉/؊/LXR␤؊/؊), and LXR double knock-out mice (LXR␣؊/؊/LXR␤؊/؊) as well as 3T3-L1 cells transduced with retroviruses expressing either wild-type LXR␣ or a dominant negative version of LXR␣, we demonstrate that the response to LXR agonists is LXR-dependent
LXR Agonists Rescue Statin-induced Cell Death—When 2 days postconfluent 3T3-L1 preadipocytes are treated with isobutylmethylxanthine, dexamethasone, and insulin, they synchronously reenter the cell cycle and undergo two sequential rounds of mitosis in a process known as mitotic clonal expansion and subsequently express genes that control the adipocyte phenotype [64]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Differentiation—3T3-L1 cells were cultured to confluence in DMEM supplemented with 10% calf serum. Two days postconfluent (designated day 0) cells were induced to differentiate with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 ␮M dexamethasone (Sigma), 0.5 mM isobutylmethylxanthine (Sigma), and 1 ␮g/ml insulin (Roche Applied Science). 2-day postconfluent cells (day 0) were treated with growth medium containing 1 ␮M dexamethasone (Sigma), 0.5 mM isobutylmethylxanthine, and 5 ␮g/ml insulin for 2 days. 6 h after transfection, the medium was changed, and cells were incubated for 20 h with tumor necrosis factor-␣ or vehicle (Me2SO) as indicated. Two days post-transfection, the virus-containing media were collected by centrifugation and immediately used to infect 30 – 40% confluent 3T3-L1 cells by mixing viral supernatant 1:1 with DMEM supplemented with 10% calf serum. TCGTCC and 5Ј-CCCACAGACACTGCACAG; LXR␤, 5ЈTACATCGTGGTCATCTTAGAG and 5Ј-GGCACAGCTCATGGC; ABCA1, 5Ј-TGGTGTCTGTACCGCAAGC and 5Ј-CCCCATTACATAACACATGCC; ABCG1, 5Ј-CTGTGAGCCTCAGTTTCCCC and 5Ј-GGTAAGTCCAATTCGGACCC; Nip3, 5Ј-TTAATCAGGATCCAAAGCTGCTTA and 5Ј-GGCAGGCTTTAAAACCATGCT; CIDE-A, 5Ј-CCGAGTACTGGGCGATACAGA and 5Ј-GGTTACATGAACCAGCCTTTGG; CIDE-B, 5Ј-GGATGTTGTCATACGGCCTAGG and 5Ј-AAGGTGATGCGGGCGA; Aatk, 5Ј-GCCCAGTTGGAGTGCAGC and 5Ј-GTGGAACGGAGGTCAGCG; TIA1, 5Ј-GGATGGGACCCAATTACAGTGT and 5Ј-GCAGGCTGACTAGGCAACATG; Bax, 5Ј-CACGGACTCCCCCCGA and 5Ј-GAAGTTGCCATCAGCAAACATG; p53, 5ЈTGCACAAGCGCCTCTCC and 5Ј-CGCGGATCTTGAGGGTGA; Bcl2l2, 5Ј-ACCTTCTCTGACCTGGCCG and 5Ј-GAAACCTGGGTGAAGCGTTG; Mcl-1 5Ј-GACGGCCTTCCAGGGC and 5Ј-CGAGAAAAAGATTTAACATCGCC; Bad, 5Ј-CTCCGAAGGATGAGCGATGA and 5Ј-GCGAGGAAGTCCCTTGAAGG; Bcl-2, 5Ј-CTGGGATGCCTTTGTGGAAC and 5Ј-GAGACAGCCAGGAGAAATCAAAC; CARD14, 5Ј-TGGACAAAGCCGCTGTCAG and 5Ј-AGCACACGCTAGGACCACCT; Bcl-xL, 5Ј-GCGGCTGGGACACTTTTGT and 5Ј-TCTCGGCTGCTGCATTGTT; AIM/SP␣, 5Ј-CCAGACAGTGACCTCCTCTTCAT and 5Ј-ACCCTGGCAGTGCCCC; CARD9, 5Ј-GCATCTTTCACTGACCCATGTAAG and 5Ј-CCAGGGCTCTTGGGAACC; SREBP1c, 5Ј-GGAGCCATGGATTGCACATT and GCTTCCAGAGAGGAGGCCAG; SREBP2, 5Ј-GCGTTCTGGAGACCATGG and 5Ј-ACAAAGTTGCTCTGAAAACAAATC

RESULTS

Involved in Apoptosis and Survival by Simvastatin and the LXR Agonist

DISCUSSION