Thyrocyte cell survival and adaptation to chronic endoplasmic reticulum stress due to misfolded thyroglobulin

Abstract

The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the “tunicamycin transporter” (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho–AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho–S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

Thyroid hormone synthesis continues despite biallelic thyroglobulin mutation with cell death

“…The synthesis of misfolded Tg is accompanied by ER stress (38,(48)(49)(50)(51). Ongoing ER stress in the thyroids of TG cog/cog mice was demonstrable (Fig.…”

Cell death–associated lipid droplet protein CIDE-A is a noncanonical marker of endoplasmic reticulum stress

“…PCCL3 cells that ultimately adapt to chronic TUN exhibit a disproportionately lower relative mRNA level of Cidea compared to that of classical ER stress markers. PCCL3 cells grown for up to one-year in stepwise increasing doses of TUN have been described, and the upper two graphs in panel A show BiP and Chop mRNA levels in the surviving, growing cells, respectively (for comparison), reproduced from the previous work (3). The lower graph of panel A shows Cidea mRNA levels in the same samples, from the same cells, over the same time course.…”

“…Misfolding of mutant TG in the ER is an established cause of congenital hypothyroidism ( 2 ), which, via endocrine feedback, triggers an increase in circulating levels of thyroid-stimulating hormone (TSH) released from the anterior pituitary. In this disease (in both humans and animal models), thyroid follicular (thyrocyte) cell growth ( 3 ) and thyrocyte cell death ( 4 ) take place simultaneously in different cells of the same thyroid gland. This behavior suggests that affected cells in the same tissue exhibit a range of (successful and unsuccessful) attempts in adaptive response to chronic ER stress.…”

The Probable, Possible, and Novel Functions of ERp29

“…Manifestations of these mutations include congenital goiter, low serum Tg, high serum TSH, and hypothyroidism (Targovnik et al, 2010). The most severe Tg-mutant disorders are associated with Tg misfolding and sequestration within the ER (Citterio et al, 2013;Morishita et al, 2020), including some Tg mutations that cause defects in the export of Tg from the ER (Kim et al, 1996;Rubio and Medeiros-Neto, 2009;Targovnik et al, 2010). While the precise location of ERp29 binding on Tg is not known, Tg has several -(F,Y)-X-(F,Y) and -(F,Y)-(F,Y)-motifs that are consistent with other ERp29 clients (Barak et al, 2009), such as CFTR and ENaC.…”