Endoplasmic reticulum stress as a novel mechanism in amiodarone-induced destructive thyroiditis

Abstract

Context: Amiodarone (AMIO) is one of the most effective antiarrhythmic drugs available; however, its use is limited by a serious side effect profile, including thyroiditis. The mechanisms underlying AMIO thyroid toxicity have been elusive; thus, identification of novel approaches in order to prevent thyroiditis is essential in patients treated with AMIO.

Objective: Our aim was to evaluate whether AMIO treatment could induce endoplasmic reticulum (ER) stress in human thyroid cells and the possible implications of this effect in AMIO-induced destructive thyroiditis.

Results: Here we report that AMIO, but not iodine, significantly induced the expression of ER stress markers including Ig heavy chain-binding protein (BiP), phosphoeukaryotic translation initiation factor 2α (eIF2α), CCAAT/enhancer-binding protein homologous protein (CHOP) and spliced X-box binding protein-1 (XBP-1) in human thyroid ML-1 cells and human primary thyrocytes. In both experimental systems AMIO down-regulated thyroglobulin (Tg) protein but had little effect on Tg mRNA levels, suggesting a mechanism involving Tg protein degradation. Indeed, pretreatment with the specific proteasome inhibitor MG132 reversed AMIO-induced down-regulation of Tg protein levels, confirming a proteasome-dependent degradation of Tg protein. Corroborating our findings, pretreatment of ML-1 cells and human primary thyrocytes with the chemical chaperone 4-phenylbutyric acid completely prevented the effect of AMIO on both ER stress induction and Tg down-regulation.

Conclusions: We identified ER stress as a novel mechanism contributing to AMIO-induced destructive thyroiditis. Our data establish that AMIO-induced ER stress impairs Tg expression via proteasome activation, providing a valuable therapeutic avenue for the treatment of AMIO-induced destructive thyroiditis.

Figure 1.

AMIO, but not NaI, triggers BiP induction in ML-1 cells. A and B, ML-1 cells were treated with 0.5 μM THAP or with the indicated concentrations of AMIO or NaI for 24 hours, and the levels of BiP mRNA were determined by real-time RT-PCR analysis of total RNA using GAPDH as internal standard. The mRNA levels in treated cells are relative to those in vehicle-treated cells (CTRL). B, Cells were solubilized and an equal amount of proteins (50 μg per sample) were analyzed by immunoblotting. Filters were probed with antibodies against BiP and β-actin and band intensities were quantified by densitometry using ImageJ software (C) (National Institutes of Health, Bethesda, Maryland). D, Time course of BiP mRNA in ML-1 cells cultured with 5 μM AMIO for the indicated times. Bars represent means ± SEM from four to five independent experiments. *, P < .001; ***, P < .001 compared with CTRL cells.

Figure 2.

Chemical chaperones prevent AMIO-induced BiP up-regulation in ML-1 cells. A and B, ML-1 cells were pretreated or not for 24 hours with 5 mM TUDCA or with 7.5 mM PBA and then cultured in presence of 5 μM AMIO for 24 hours. A, Real-time RT-PCR analysis of BiP mRNA levels using the total RNAs from ML-1 cells treated as indicated, with GAPDH as internal standard. mRNA levels in treated cells are relative to those in vehicle-treated cells (CTRL). B, Immunoblot using an equal amount of proteins (50 μg per sample) and the indicated antibodies. C, Densitometric quantification of ML-1 cells treated as described; band fluorescence intensity was assessed with ImageJ software. Experiments were repeated three times, and values are mean ± SEM. *, P < .001; ***, P < .001 compared with CTRL cells.

Figure 3.

AMIO induces UPR activation in ML-1 cells. A and C, Patterns of expression of phospho-eIF2α and CHOP in ML-1 cells as determined by immunostaining. ML-1 cells were grown on glass coverslips for 24 hours and then pretreated or not with 7.5 mM PBA for 24 hours followed by 24 hours in medium with 5 μM AMIO. Immunofluorescence staining was performed using phospho-eIF2α antibody (green) or CHOP antibody (red). Nuclei were counterstained with DAPI (blue). B and D, Quantification of fluorescence intensity for phospho-eIF2α or CHOP in cultured ML-1 cells treated as described. E and F, The levels of mRNAs for CHOP, XBP-1, and active XBP-1 spliced mRNA were determined by real-time RT-PCR analysis of total RNA from ML-1 cells treated as above. GAPDH was used as an internal standard. Each bar represents the mean ± SEM of four independent experiments, each performed in triplicate. *, P < .05; **, P < .01, ***, P < .001 compared with vehicle-treated cells (CTRL).

Figure 4.

AMIO up-regulates BiP in human primary thyrocytes. A–C, Human primary thyrocytes were pretreated or not for 24 hours with 7.5 mM PBA or 5 mM TUDCA followed by a treatment with 5 μM AMIO for 24 hours. A, BiP mRNA was determined by real-time RT-PCR analysis of total RNA isolated from human primary thyrocytes, using GAPDH as internal standard. mRNA levels in treated cells are relative to those in vehicle-treated cells (CTRL). B, Human primary thyrocytes were solubilized and cell lysates were analyzed by Western blotting with anti BiP antibody using β-actin as a loading control. C, Densitometric quantification of human primary thyrocytes treated as described; band fluorescence intensity was assessed with ImageJ software. These results have been replicated three times. All data are shown as mean ± SEM. *, P < .001, **, P < .01 compared with CTRL cells.

Figure 5.

Effect of ER stress on Tg expression in human primary thyrocytes and ML-1 cells. A–D, Human primary thyrocytes and ML-1 cells were pretreated or not for 24 hours with 7.5 mM PBA and then cultured in the presence of 5 μM AMIO for 72 hours. Immunoblot analysis and quantification of total protein extracts from human primary thyrocytes (A and B) and ML-1 cells (C and D) were performed using specific antibodies against Tg and β-actin. E, Real-time RT-PCR analysis from human primary thyrocytes and ML-1 cells vehicle-treated (CTRL) or treated with 5 μM AMIO for 72 hours. Tg mRNA levels in treated cells are relative to those in control cells. GAPDH was used as internal standard. Experiments were repeated three times, and values are mean ± SEM. *, P < .001, ***, P < .001 compared with CTRL cells.

Figure 6.

Proteasome activation is involved in ER-stress induced Tg protein down-regulation. A–D, Human primary thyrocytes and ML-1 cells were grown on glass coverslips for 24 hours and subsequently pretreated or not overnight with 0.8 μM MG132 followed by 72 hours incubation in medium with 5 μM AMIO. Human primary thyrocytes (A) and ML-1 cells (C) were stained with a specific antibody against Tg (green). Nuclei were counterstained with DAPI (blue). B, Quantification of fluorescence intensity for Tg in human primary thyrocytes and cultured ML-1 cells (D) treated as described. The data in this figure are representative of four replicate experiments. Asterisks indicate statistically significant differences (*, P < .05, ***, P < .001 compared with CTRL cells).