Endoplasmic Reticulum stress and thyroid cell death

ABSTRACT Efficient generation of the thyroid hormone thyroxine (T4) requires the iodination of thyroglobulin (Tg), which is synthesized in the endoplasmic reticulum (ER) and undergoes folding and trafficking to the thyroid follicle lumen wherein iodination takes place. Endogenous T4 is 100% produced by thyrocytes, and the formation of T4 in Tg

has been conserved in evolution for ≥ 500 million years. To date, hundreds of different pathogenic mutations of the TG gene have been found to cause congenital hypothyroidism in humans. Genetic hypothyroidism from homozygous (or compound heterozygous) TG mutation is rare, but the frequency of a single pathogenic TG

allele in the human population is very common (≥ 1:200 individuals). All Tg mutants studied to date are misfolded proteins trapped in the ER, causing ER stress. We recently reported that in untreated TG homozygotes, endogenous thyroid hormone synthesis still occurs despite failure of Tg export from the ER. The mechanism

involves thyroid epithelial cell death, with extrusion of dead thyrocytes into the lumen of thyroid follicles, leading to the disintegration and iodination of those cells in the follicle lumen. Massive expression of Tg protein in thyrocytes, and the extremely high frequency of heterozygous TG mutations in the population, now leads us to

ask whether thyroid epithelial cell death may also be a (heretofore unsuspected) widespread feature across individuals who are simple heterozygotes for mutant TG. This can be easily tested in animal models. Additionally, in homozygous rdw/rdw (Tg-G2298R) rats, thyroid cell death has long been recognized as a factor

blocking goiter growth. Remarkably, we now find that in the congenital goiter mouse (cog/cog, Tg-L2263P), there is also widespread thyroid cell death (that is nevertheless outpaced by thyroid growth). In the current proposal, 1) we have engineered a rdw/rdw knockin mouse and will directly compare cell growth and cell death to that

seen in cog/cog mice. 2) We will examine thyroid cell death in simple heterozygotes of these two models, which have perfectly normal serum T4 levels but exhibit thyrocyte ER stress. 3) As heterozygous mutant Tg is misfolded in the ER, it may be degraded at least in part by ER-associated protein degradation (ERAD). We find that

defective ERAD of Tg leads to a thyroiditis with infiltration of inflammatory cells and follicle involuion. 4) Extrusion of thyrocytes into the follicle lumen may expose non-apical portions of the cell to iodination by surrounding follicular cells as a contributing mechanism of cytotoxicity. To test this, we will examine a TG-KO mouse model

in which TSH-stimulated overgrowth (and not ER stress) drives thyrocytes into the follicle lumen, exposing the sensitive basolateral plasma membrane to the oxidative / iodination environment of the lumen, where it may trigger thyroid cell death. In summary, all of the Aims of this proposal will highlight questions of ER stress-

dependent and independent mechanisms of thyrocyte death.