Thyroid hormone synthesis continues despite biallelic thyroglobulin mutation with cell death.

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.

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

Secretory protein misfolding has been linked to ER stress and cell death. We expressed a TGrdw transgene encoding TG-G(2298)R, a misfolded mutant thyroglobulin reported to be linked to thyroid cell death. When the TGrdw transgene was expressed at low level in thyrocytes of TGcog/cog mice that experienced severe ER stress, we observed increased thyrocyte cell death and increased expression of CIDE-A (cell death-inducing DFFA-like effector-A, a protein of lipid droplets) in whole thyroid gland. Here we demonstrate that acute ER stress in cultured PCCL3 thyrocytes increases Cidea mRNA levels, maintained at least in part by increased mRNA stability, while being negatively regulated by activating transcription factor 6 - with similar observations that ER stress increases Cidea mRNA levels in other cell types. CIDE-A protein is sensitive to proteasomal degradation yet is stabilized by ER stress, and elevated expression levels accompany increased cell death. Unlike acute ER stress, PCCL3 cells adapted and surviving chronic ER stress maintained a disproportionately lower relative mRNA level of Cidea compared with that of other, classical ER stress markers, as well as a blunted Cidea mRNA response to a new, unrelated acute ER stress challenge. We suggest that CIDE-A is a novel marker linked to a noncanonical ER stress response program, with implications for cell death and survival.

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

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.

Transient covalent interactions of newly synthesized thyroglobulin with oxidoreductases of the endoplasmic reticulum.

Newly synthesized thyroglobulin (Tg), the thyroid prohormone, forms detectable high molecular weight mixed disulfide adducts: until now, only Tg "adduct B" was identified as primarily engaging the endoplasmic reticulum oxidoreductases ERp57 and protein disulfide isomerase. Here, we demonstrate that the faster migrating Tg adduct C primarily engages the CaBP1/P5 oxidoreductase, whereas the slower migrating Tg adduct A primarily engages ERp72. Upon siRNA-mediated knockdown of CaBP1/P5 or ERp72, adducts C or A, respectively, are decreased. Within the three Tg adduct bands that do not exhibit a precursor-product relationship, Tg exhibits distinct oxidation patterns. We present evidence suggesting that disulfide maturation occurs within Tg monomers engaged in each of the adduct bands. Moreover, the same Tg substrate molecules can form simultaneous mixed disulfides with both CaBP1/P5 and protein disulfide isomerase, although these are generally viewed as components of distinct oxidoreductase-chaperone protein complexes. Such substrate-oxidoreductase combinations offer Tg the potential for simultaneous oxidative maturation along different parallel tracks leading to the native state.

Dominant protein interactions that influence the pathogenesis of conformational diseases.

Misfolding of exportable proteins can trigger endocrinopathies. For example, misfolding of insulin can result in autosomal dominant mutant INS gene-induced diabetes of youth, and misfolding of thyroglobulin can result in autosomal recessive congenital hypothyroidism with deficient thyroglobulin. Both proinsulin and thyroglobulin normally form homodimers; the mutant versions of both proteins misfold in the ER, triggering ER stress, and, in both cases, heterozygosity creates potential for cross-dimerization between mutant and WT gene products. Here, we investigated these two ER-retained mutant secretory proteins and the selectivity of their interactions with their respective WT counterparts. In both cases and in animal models of these diseases, we found that conditions favoring an increased stoichiometry of mutant gene product dominantly inhibited export of the WT partner, while increased relative level of the WT gene product helped to rescue secretion of the mutant partner. Surprisingly, the bidirectional consequences of secretory blockade and rescue occur simultaneously in the same cells. Thus, in the context of heterozygosity, expression level and stability of WT subunits may be a critical factor influencing the effect of protein misfolding on clinical phenotype. These results offer new insight into dominant as well as recessive inheritance of conformational diseases and offer opportunities for the development of new therapies.

Effects of cyclooxygenase-2 gene inactivation on cardiac autonomic and left ventricular function in experimental diabetes.

Glucose-mediated oxidative stress and the upregulation of cyclooxygenase (COX)-2 pathway activity have been implicated in the pathogenesis of several vascular complications of diabetes including diabetic neuropathy. However, in nondiabetic subjects, the cardiovascular safety of selective COX-2 inhibition is controversial. The aim of this study was to explore the links between hyperglycemia, oxidative stress, activation of the COX-2 pathway, cardiac sympathetic integrity, and the development of left ventricular (LV) dysfunction in experimental diabetes. R wave-to-R wave interval (R-R interval) and parameters of LV function measured by echocardiography using 1% isoflurane, LV sympathetic nerve fiber density, LV collagen content, and markers of myocardial oxidative stress, inflammation, and PG content were assessed after 6 mo in control and diabetic COX-2-deficient (COX-2(-/-)) and littermate, wild-type (COX-2(+/+)) mice. There were no differences in blood glucose, LV echocardiographic measures, collagen content, sympathetic nerve fiber density, and markers of oxidative stress and inflammation between nondiabetic (ND) COX-2(+/+) and COX-2(-/-) mice at baseline and thereafter. After 6 mo, diabetic COX-2(+/+) mice developed significant deteriorations in the R-R interval and signs of LV dysfunction. These were associated with a loss of LV sympathetic nerve fiber density, increased LV collagen content, and a significant increase in myocardial oxidative stress and inflammation compared with those of ND mice. Diabetic COX-2(-/-) mice were protected against all these biochemical, structural, and functional deficits. These data suggest that in experimental diabetes, selective COX-2 inactivation confers protection against sympathetic denervation and LV dysfunction by reducing intramyocardial oxidative stress, inflammation, and myocardial fibrosis.

Cyclooxygenase-2 pathway as a potential therapeutic target in diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is the most common diabetic complication and is the leading cause of diabetes-related hospital admissions and non-traumatic amputations. DPN is also associated with a poor quality of life and high economic costs for both type 1 and type 2 diabetic patients. An effective treatment for DPN, besides tight glycemic control, is not yet available. The pathogenesis of DPN is complex and involves an intertwined array of mechanisms. Glucose-mediated alteration of cyclooxygenase (COX) pathway activity with subsequent impaired production and function of prostaglandins (PGs) is one mechanism that is implicated in the pathogenesis of DPN. COX-2, the inducible COX isoform, is upregulated in a variety of pathophysiological conditions including diabetes. COX-2 upregulation has tissue-specific consequences and is associated with activation of downstream inflammatory reactions. We have previously reported that COX-2 is upregulated in the peripheral nerves and dorsal root ganglia neurons in experimental diabetes and that COX-2 gene inactivation and/or selective COX-2 inhibition provides protection against various DPN deficits. This review will summarize current evidence supporting the role of COX-2 activation in inducing diabetic neurovascular dysfunction and that modulation of the COX-2 pathway is a potential therapeutic target for DPN.

Protective effects of cyclooxygenase-2 gene inactivation against peripheral nerve dysfunction and intraepidermal nerve fiber loss in experimental diabetes.

OBJECTIVE: Activation of the cyclooxygenase (COX) pathway with secondary neurovascular deficits are implicated in the pathogenesis of experimental diabetic peripheral neuropathy (DPN). The aim of this study was to explore the interrelationships between hyperglycemia, activation of the COX-2 pathway, and oxidative stress and inflammation in mediating peripheral nerve dysfunction and whether COX-2 gene inactivation attenuates nerve fiber loss in long-term experimental diabetes. RESEARCH DESIGN AND METHODS: Motor and sensory digital nerve conduction velocities, sciatic nerve indexes of oxidative stress, prostaglandin content, markers of inflammation, and intraepidermal nerve fiber (IENF) density were measured after 6 months in control and diabetic COX-2-deficient (COX-2(-/-)) and littermate wild-type (COX-2(+/+)) mice. The effects of a selective COX-2 inhibitor, celecoxib, on these markers were also investigated in diabetic rats. RESULTS: Under normal conditions, there were no differences in blood glucose, peripheral nerve electrophysiology, markers of oxidative stress, inflammation, and IENF density between COX-2(+/+) and COX-2(-/-) mice. After 6 months, diabetic COX-2(+/+) mice experienced significant deterioration in nerve conduction velocities and IENF density and developed important signs of increased oxidative stress and inflammation compared with nondiabetic mice. Diabetic COX-2(-/-) mice were protected against functional and biochemical deficits of experimental DPN and against nerve fiber loss. In diabetic rats, selective COX-2 inhibition replicated this protection. CONCLUSIONS: These data suggest that selective COX-2 inhibition may be useful for preventing or delaying DPN.

Peripheral nerve dysfunction in experimental diabetes is mediated by cyclooxygenase-2 and oxidative stress.

Glucose-mediated oxidative stress and alterations in cyclooxygenase (COX) pathway activity with secondary deficits of endoneurial perfusion have been implicated in the pathogenesis of experimental diabetic neuropathy (EDN). We have previously reported that activation of the COX-2 pathway is an important mediator of neurochemical and neurovascular defects in EDN in a rat model. Considering that chemical COX inhibition may exert other pharmacological effects in addition to inhibition of COX activity, the aim of this study was to explore the role of COX-2 in experimental diabetic neuropathy, using a COX-2 knockout mouse model. Here we provide evidence that COX-2 inactivation had a protective effect against diabetes-induced motor and sensory nerve conduction slowing and impaired nerve antioxidative defense that were clearly manifest in the wild-type (COX-2(+/+)) diabetic mice. These preliminary data support the role of the activation of the COX-2 pathway in mediating sensory and motor nerve conduction velocity deficits in EDN. These findings also suggest that the COX-2 pathway seems to be an important modulator of oxidative stress in EDN.