Defining thein vivorole of mTORC1 in thyrocytes by studying the TSC2 conditional knockout mouse model

Abstract

The serine-threonine protein kinase mechanistic target of rapamycin complex 1 (mTORC1) plays a pivotal role in regulating cellular metabolism, growth, and proliferation. The thyroid gland is a frequent site of abnormal epithelial cell growth and proliferation that can lead to thyroid dysfunction. In the thyroid gland, the PI3K-Akt-mTOR pathway is abnormally overactive in tumors and has been linked to increased aggressiveness in thyroid cancer. However, our understanding of mTORC1’s role in thyrocytes is primarily based onin vitrostudies, leaving itsin vivofunction unclear. To bridge this knowledge gap, we developed a novel mouse model of mTOR gain of function specifically in thyrocytes by crossing the mouse with thyroperoxidase (TPO)-driven cre recombinase expression with the Tuberous Sclerosis Complex 2 (TSC2)-floxed mouse. TSC2 acts as an intracellular inhibitor of mTORC1 and its conditional disruption leads to overactivation of mTOR activity. In TPO-TSC2KOmice, thyroid glands exhibited 70-80% less TSC2 protein and mRNA levels. Concomitantly, the TSC2 knockout in thyrocytes led to increased protein expression of mTORC1 target pS6 and decreased expression of mTORC2 target Akt phosphorylated in serine 473. Thyroid gland size was bigger in the TPO-TSC2KOmice as early as 1-month of age and continued to grow throughout life in both genders. Microscopic examination revealed significant heterogeneity among thyroid follicles in the TPO-TSC2KOmice, and their thyroids present larger follicles, colloid and epithelium. Overactivation of mTORC1 resulted in increased thyrocyte proliferation, as indicated by Ki67 staining, and elevated Cyclin D3 levels. Chronic mTOR overactivation suppressed mRNA expression of key genes involved in thyroid hormone biosynthesis such as thyroglobulin, thyroperoxidase and sodium-iodide symporter in both male and female mice. As a result, TPO-TSC2KOmice exhibited slight hypothyroidism with 20% less plasma thyroxine and 2-fold increase in TSH levels. Systemic treatment with rapamycin, an mTORC1 inhibitor, prevented thyroid mass expansion and gene expression alterations in TPO-TSC2KOmice. Finally, TPO-TSC2KOmice aged 12 months or older developed aberrant thyroid conditions including follicular hyperplasia, inflammation and thyroid tumors. In summary, our study demonstrates that mTORC1 gain of function specifically in thyrocytes leads to alterations in follicle morphology and impairs thyroid gland function.