Thyroid hormone synthesis continues despite biallelic thyroglobulin mutation with cell death

Abstract

Complete absence of thyroid hormone is incompatible with life in vertebrates. Thyroxine is synthesized within thyroid follicles upon iodination of thyroglobulin conveyed from the endoplasmic reticulum (ER), via the Golgi complex, to the extracellular follicular lumen. In congenital hypothyroidism from biallelic thyroglobulin mutation, thyroglobulin is misfolded and cannot advance from the ER, eliminating its secretion and triggering ER stress. Nevertheless, untreated patients somehow continue to synthesize sufficient thyroxine to yield measurable serum levels that sustain life. Here, we demonstrate that TGW2346R/W2346R humans, TGcog/cog mice, and TGrdw/rdw rats exhibited no detectable ER export of thyroglobulin, accompanied by severe thyroidal ER stress and thyroid cell death. Nevertheless, thyroxine was synthesized, and brief treatment of TGrdw/rdw rats with antithyroid drug was lethal to the animals. When untreated, remarkably, thyroxine was synthesized on the mutant thyroglobulin protein, delivered via dead thyrocytes that decompose within the follicle lumen, where they were iodinated and cannibalized by surrounding live thyrocytes. As the animals continued to grow goiters, circulating thyroxine increased. However, when TGrdw/rdw rats age, they cannot sustain goiter growth that provided the dying cells needed for ongoing thyroxine synthesis, resulting in profound hypothyroidism. These results establish a disease mechanism wherein dead thyrocytes support organismal survival.

Keywords: Endocrinology; Thyroid disease.

Figure 1. ER stress, cell death, and T4 synthesis in TG^cog/cog mice.

(A) Representative H&E-stained images of WT and TG^cog/cog thyroids (n = 6 animals/group), showing thyrocyte distention with apically displaced nuclei in TG^cog/cog mice compared with a thin monolayer of thyrocytes in WT (^+/+) mice. Scale bars: 20 μm. (B) Representative anti-Tg immunofluorescence in thyroid glands of WT and TG^cog/cog mice (n = 8 animals/group), with DAPI counterstain. Scale bars: 20 μm. (C) Representative immunofluorescence of T4-containing protein in thyroid follicles of WT or TG^cog/cog mice (n = 6 animals/group). Thyrocytes are highlighted by PAX8-positive nuclear transcription factor with DAPI counterstain. Scale bars: 10 μm. (D) Endoglycosidase H digest of thyroid homogenates before SDS-PAGE and Tg Western blotting from WT and TG^cog/cog mice (n = 3 animals/group; 2 of each kind shown). R, endoglycosidase H resistant; S, endoglycosidase H sensitive. (E) Top: Western blotting of BiP, p58ipk, and CHOP in the thyroids of TG^cog/cog mice (n = 3–4; each lane represents 1 animal). Bottom: Quantitation of bands (normalized to tubulin), shown as mean ± SD. ***P < 0.001 (unpaired 2-tailed Student’s t test). (F) Representative TUNEL staining and immunofluorescence of T4-containing protein with DAPI counterstain in thyroid sections of WT and TG^cog/cog mice (n = 7 animals/group). For clarity, in the merged image from TG^cog/cog mice, a dashed white line delimits the thyroid follicle lumen. Scale bars: 20 μm. (G) Thyroid homogenate from TG^cog/cog mice (n = 3) was immunoprecipitated with mAb anti-T4 in the presence or absence of T4 competitor, followed by either mock digest or Endo H digest and SDS-PAGE plus immunoblotting with mAb antibody that recognizes intact Tg. As a positive control, WT Tg secreted from PCCL3 (rat) thyrocytes was digested for Endo H resistance. The T4-containing Tg protein of TG^cog/cog mice was entirely Endo H sensitive. The position of the 250 kDa molecular weight marker is shown.

Figure 2. Tg and T4 synthesis in a homozygous patient bearing TG^{W2346R/W2346R}.

(A) Anti-Tg immunofluorescence (red) with DAPI counterstaining (blue) of human thyroid sections from a patient bearing TG^{W2346R/W2346R} and a representative unaffected (Control) individual (n = 3). The diseased thyroid gland shows abnormal accumulation of intracellular Tg but also shows Tg in a patchy distribution in the thyroid follicle lumen. Scale bars: 10 μm. (B) Anti-cleaved caspase-3 immunofluorescence (red) with DAPI counterstaining (blue) in the thyroid gland of the individuals from A. For clarity, a dashed white line delimits the thyroid follicle lumen in the control (in which cleaved caspase-3 is not seen). Scale bars: 10 μm. (C) Immunostaining of T4-containing protein (green) in thyroid follicles from the individuals in A. Thyrocyte identity is confirmed by PAX8-positive nuclei (red) with DAPI counterstain (blue). Scale bars: 10 μm.

Figure 3. Tg is entrapped in the ER, yet it reaches the thyroid follicle lumen in TG^rdw/rdw rats.

(A) Representative H&E-stained images of thyroid glands from WT (^+/+) and TG^rdw/rdw rats (n = 6 per group), showing abnormally heterogeneous eosinophilic content in the follicle lumen, surrounded by abnormally swollen thyrocytes in TG^rdw/rdw rats. Scale bars: 20 μm. (B) Representative distribution of aminopeptidase N by immunofluorescence (green) with DAPI counterstain (blue) in thyroid follicles of WT and TG^rdw/rdw rats (n = 4 per group). For clarity, a yellow dotted line highlights the outer boundary of the thyroid follicular cells. Scale bars: 10 μm. (C) Representative distribution of BiP by immunofluorescence (red) with DAPI counterstain (blue) in thyroid follicles of WT and TG^rdw/rdw rats (n = 4 per group). Scale bars: 10 μm. (D) Representative thyroid homogenates from WT and TG^rdw/rdw rats were either mock digested or digested with endoglycosidase H (Endo H), followed by SDS-PAGE and immunoblotting with anti-Tg (n = 4 animals per group). No Tg from TG^rdw/rdw rats was endo H resistant. (E) Representative distribution of Tg in the thyroid follicle lumen by immunofluorescence (green) with DAPI counterstain (blue) from WT and TG^rdw/rdw rats (n = 7 per group). Scale bars: 20 μm.

Figure 4. T4 synthesis in TG^rdw/rdw rats.

(A) Representative immunofluorescence of T4-containing protein (green) in the thyroid follicle lumen of WT (^+/+) and TG^rdw/rdw rats (n = 5 animals per group). Thyrocyte identity is confirmed by PAX8-positive nuclei (red) with DAPI counterstain (blue). Scale bars: 20 μm. (B) Representative immunofluorescence detection of T4-containing protein (green; with DAPI counterstain in blue) in the thyroids of WT and TG^rdw/rdw rats (n = 9 animals per group) was specifically blocked by addition of T4 competitor (1 μg/mL). For clarity, a dashed white line delimits the thyroid follicle lumen; a yellow dotted line highlights the outer boundary of the thyroid follicle. Scale bars: 10 μm.

Figure 5. ER stress, cell death, and T4 synthesis in TG^rdw/rdw rats.

(A) Left: BiP, p58ipk, and phospho-eIF2α Western blotting in thyroids of WT (^+/+) and TG^rdw/rdw rats (each lane represent 1 animal). Right: Quantification (BiP and p58ipk normalized to tubulin; phospho-eIF2α normalized to total eIF2α; mean ± SD). **P < 0.01, ***P < 0.001 (unpaired 2-tailed Student’s t test). (B) CHOP mRNA levels (normalized to YWHAZ) in the thyroid glands of WT and TG^rdw/rdw rats (n = 7–8 animals/group; each point represents 1 animal; mean ± SD). ***P < 0.001 (unpaired 2-tailed Student’s t test). (C) Top: Representative samples showing spliced and unspliced XBP1 mRNA in the thyroids of WT, TG^rdw/+, and TG^rdw/rdw rats (n = 3–6 animals/group; each lane represents 1 animal). Hprt1 was used as a loading control. Bottom: Quantitation of the fraction of spliced XBP1 (mean ± SD). **P < 0.01, ***P < 0.001 (1-way ANOVA, Bonferroni post hoc test). (D) Representative TUNEL staining and immunofluorescence of T4-containing protein with DAPI counterstain in the thyroids of WT and TG^rdw/rdw rats (n = 4 animals/group). Scale bars: 20 μm. (E) Representative immunofluorescence of cleaved caspase-3 with DAPI counterstain in thyroids of WT and TG^rdw/rdw rats (n = 5 animals/group). For clarity, a dashed white line delimits the thyroid follicle lumen in the WT rats (in which cleaved caspase-3 is not detectable). Scale bars: 20 μm. (F) Western blotting of PARP in thyroid glands from WT and TG^rdw/rdw rats (n = 3–4; each lane represents 1 animal). (G) Left: Representative Western blotting of T4-containing protein in thyroid homogenates of WT and TG^rdw/rdw rats (n = 5 animals/group) with or without soluble competitor T4 to block specific bands (left of dotted red line). Right: The same samples immunoblotted with mAb anti-Tg showing intentional overloading of the TG^rdw/rdw rat sample.

Figure 6. In congenital goiter with mutant TG, thyrocyte cell mass provides the dead-cell–derived substrate for T4 synthesis.

(A–H) Microscopy of WT and TG^rdw/rdw rat thyroid follicles. (A) WT rat thyroid. Cross-sections of several thyroid follicles are shown; each follicle lumen (F.L.) is acellular but filled with WT Tg protein (thin yellow arrows). Scale bar: 10 μm in 1.0 μm increments. (B) TG^rdw/rdw rat thyroid. Yellow arrows point to the follicle lumina; note the enlarged cytoplasm and abnormal, cellular contents of the follicle lumina. Scale bar: 10 μm in 1.0 μm increments. (C–H) Transmission EM survey of TG^rdw/rdw rat thyroid follicles. Scale bars: 2 μm. (C) Engorged ER vacuoles in the basal cytoplasm with apically displaced nuclei. (D–H) Dead-cell ghosts in various thyroid follicles, each at a different stage of cellular disintegration within the follicle lumen. (G) Living thyrocytes with abundant apical microvilli, which have internalized material from the follicle lumen into endo-lysosomes. (H) Until new dead cells enter the follicle lumen, there is progressive clearance of cellular debris from the luminal cavity. (I) Thyroid gland size (normalized to body weight) in a cohort of young versus older animals (open symbols represent rats at 8.9 ± 1.7 weeks of age; closed symbols represent rats at 33.4 ± 2.6 weeks of age; males are shown as squares and females as circles) (mean ± SD). ***P < 0.001 (2-way ANOVA, Bonferroni post hoc test). (J) Total T4 levels in serum of WT (^+/+) and TG^rdw/rdw rats as a function of age (males are shown as squares and females as circles) (mean ± SD). **P < 0.01, ***P < 0.001 (2-way ANOVA, Bonferroni post hoc test).