Abstract
Endoplasmic reticulum (ER) stress is characterized by the accumulation of misfolded proteins due to an impairment of ER quality control pathways leading to the activation of a defense system, called unfolded protein response (UPR). While thyrocytes are supposed to be highly susceptible to environmental conditions that cause ER stress due to the synthesis of large amounts of secretory proteins required for thyroid hormone synthesis, systematic investigations on the effect of ER stress on expression of key genes of thyroid hormone synthesis and their transcriptional regulators are lacking. Since the aim of the ER stress-induced UPR is to restore ER homeostasis and to facilitate cell survival through transient shutdown of ribosomal protein translation, we hypothesized that the expression of genes involved in thyroid hormone synthesis and their transcriptional regulators, all of which are not essential for cell survival, are down-regulated in thyrocytes during ER stress, while sterol regulatory element-binding proteins (SREBPs) are activated during ER stress in thyrocytes. Treatment of FRTL-5 thyrocytes with the ER stress inducer tunicamycin (TM) dose-dependently increased the mRNA and/or protein levels of known UPR target genes, stimulated phosphorylation of the ER stress sensor protein kinase RNA-like ER kinase (PERK) and of the PERK target protein eukaryotic initiation factor 2α (eIF2α) and caused splicing of the ER stress-sensitive transcription factor X-box binding protein (XBP-1) (P < 0.05). The mRNA levels and/or protein levels of genes involved in thyroid hormone synthesis, sodium/iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), their transcriptional regulators and thyrotropin (TSH) receptor and the uptake of Na125I were reduced at the highest concentration of TM tested (0.1 μg/mL; P < 0.05). Proteolytic activation of the SREBP-1c pathway was not observed in FRTL-5 cells treated with TM, whereas TM reduced proteolytic activation of the SREBP-2 pathway at 0.1 μg TM/mL (P < 0.05). In conclusion, the expression of key genes involved in thyroid hormone synthesis and their critical regulators and of the TSH receptor as well as the uptake of iodide is attenuated in thyrocytes during mild ER stress. Down-regulation of NIS, TPO and TG during ER stress is likely the consequence of impaired TSH/TSHR signaling in concert with reduced expression of critical transcriptional regulators of these genes.
Highlights
One major function of the endoplasmic reticulum (ER) is to maintain the integrity of newly synthesized proteins by partitioning proteins between ER folding, trafficking and degradation pathways in a process called ER quality control [1]
We have recently reported that sterol regulatory element-binding proteins (SREBP)-1c and SREBP-2, which have been initially identified as master regulators of genes involved in lipid synthesis and uptake such as fatty acid synthase (FASN), glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and LDL receptor (LDLR) [26], act as transcriptional regulators of NIS, thyroid peroxidase (TPO) and TG and are under the control of TSH in thyrocytes [27,28,29]
We cannot explain the lack of proteolytic activation of SREBP-1 in Fisher rat thyroid cell line-5 (FRTL-5) thyrocytes under ER stress conditions, but it may be speculated that this is due to the impairment of TSH/TSH receptor (TSHR) signaling because we have recently shown that TSH stimulates proteolytic processing of pSREBP-1 in FRTL-5 cells [27]
Summary
One major function of the endoplasmic reticulum (ER) is to maintain the integrity of newly synthesized proteins by partitioning proteins between ER folding, trafficking and degradation pathways in a process called ER quality control [1]. One important effect of the UPR, which is mediated by PERK-dependent phosphorylation/inactivation of eukaryotic initiation factor 2α (eIF2α), is transient attenuation of new protein synthesis leading to a decrease of newly synthesized proteins entering the ER, thereby reducing the load of ER folding and degradation pathways and enabling the ER to better cope with misfolded proteins accumulating during ER stress [5,6]. A further important effect mediated by IRE1 is unconventional mRNA splicing of X-box binding protein (XBP)-1, which leads to the production of the active transcription factor sXBP-1 inducing genes involved in chaperoning, disulfide isomerization, N-glycosylation and vesicular trafficking [8]. ER quality control capacity is improved as a consequence of the UPR due to activation of ATF6, a transcription factor inducing genes involved in protein folding and degradation [9]
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