TNFα-mediated activation of NF-κB downregulates sodium-iodide symporter expression in thyroid cells

Abstract

The sodium-iodide symporter (NIS) mediates transport of iodide across the basolateral membrane of thyroid cells. NIS expression in thyroid cancer (TC) cells allows the use of radioactive iodine (RAI) as a diagnostic and therapeutic tool, being RAI therapy the systemic treatment of choice for metastatic disease. Still, a significant proportion of patients with advanced TC lose the ability to respond to RAI therapy and no effective alternative therapies are available. Defective NIS expression is the main reason for impaired iodide uptake in TC and NIS downregulation has been associated with several pathways linked to malignant transformation. NF-κB signaling is one of the pathways associated with TC. Interestingly, NIS expression can be negatively regulated by TNF-α, a bona fide activator of NF-κB with a central role in thyroid autoimmunity. This prompted us to clarify NF-kB's role in this process. We confirmed that TNF-α leads to downregulation of TSH-induced NIS expression in non-neoplastic thyroid follicular cell-derived models. Notably, a similar effect was observed when NF-κB activation was triggered independently of ligand-receptor specificity, using phorbol-myristate-acetate (PMA). TNF-α and PMA downregulation of NIS expression was reverted when NF-κB-dependent transcription was blocked, demonstrating the requirement for NF-kB activity. Additionally, TNF-α and PMA were shown to have a negative impact on TSH-induced iodide uptake, consistent with the observed transcriptional downregulation of NIS. Our data support the involvement of NF-κB-directed transcription in the modulation of NIS expression, where up- or down-regulation of NIS depends on the combined output to NF-κB of several converging pathways. A better understanding of the mechanisms underlying NIS expression in the context of normal thyroid physiology may guide the development of pharmacological strategies to increase the efficiency of iodide uptake. Such strategies would be extremely useful in improving the response to RAI therapy in refractory-TC.

Fig 1. Effect of TNF-α and PMA on NIS transcriptional expression in non-neoplastic, TSH-responsive thyroid follicular cell lines.

NIS mRNA levels were assessed by RT-qPCR and correspond to arbitrary units representing fold differences relative to a reference sample, corrected to HPRT levels used as endogenous control gene. Impact on NIS transcript levels of TNFα (A) and PMA (B) stimuli. FRTL5 and PCCL3 were subjected to a 96h starvation period followed by stimulation with TSH (1 mU/mL for 24h), in the presence of either TNFα (10 ng/mL; 1h and 24h) or PMA (10 ng/mL; 1h and 24h). As controls, non-stimulated cells and cells stimulated with TSH for 24h in the absence of TNFα and PMA were included. Plotted values are the mean ±SD (error bars) of three independent assays. Comparisons of TNF-α and PMA stimulation to non-stimulated conditions were made using two-tailed Student’s t-tests (*p≤0.05; **p ≤0.01).

Fig 2. NF-κB impact on TNF-α- and PMA-mediated downregulation of NIS transcriptional expression.

NIS (A) and IKBα (B) mRNA levels were quantified by RT-qPCR. TSH-stimulated cells were treated for 24h with TNF-α and PMA either in the absence (vehicle) or presence of the NF-κB inhibitor BMS-345541 (10 μM; 6h). Plotted values are the mean ±SD (error bars) of three independent assays, compared with the group treated with only TSH. Comparisons were made using two-tailed Student’s t-tests (*p≤0.05; **p ≤0.01; *** p ≤0.001).

Fig 3. Effect of TNF-α and PMA on TSH-induced iodide uptake and NIS protein levels in Y-PCCL3 cells.

Cells PCCL3 cells stably expressing the YFP-halide sensor were subjected to a 24h starvation period and then treated with TSH for 96h (TSH ctrl), in the presence or absence of either TNF-α or PMA Additional iodide influx assays were performed in the presence of both (24h treatment), and subjected to iodide influx assays and Western blot. TSH and ClO4- (1 mM, 10 min), a competitive inhibitor of iodide uptake by NIS. Representative HS-YFP fluorescence decay traces (A) recorded continuously for 600 seconds, acquiring an image every 10 s, after exposure to 1mM NaI (as described in [30]). Fluorescence (F) was plotted over time as percentage of fluorescence at time 0 (F0). Iodide influx rates (B) calculated by fitting the curves to the exponential decay function to derive the maximal slope that corresponds to initial influx of I⁻ into the cells. Endogenous NIS protein expression upon TSH, TNF-α or PMA treatment was monitored Western Blot using anti-NIS primary antibody. Endogenous PCNA expression was used as loading control. Data are means ± SEM of three independent assays. Comparisons were made using one-tailed Student’s t-tests (*p≤0.05; **p ≤0.01; ***p ≤0.001).