ABSTRACT
Iodine deficiency (ID) induces TSH-independent microvascular activation in the thyroid via the reactive oxygen species/nitric oxide-hypoxia-inducible factor-1α/vascular endothelial growth factor (VEGF) pathway. We hypothesized the additional involvement of mammalian target of rapamycin (mTOR) as a positive regulator of this pathway and AMP-activated protein kinase (AMPK) as a negative feedback regulator to explain the transient nature of ID-induced microvascular changes under nonmalignant conditions. mTOR and AMPK involvement was investigated using an in vitro model (human thyrocytes in primary cultures) and 2 murine models of goitrogenesis (normal NMRI and RET-PTC mice [a papillary thyroid cancer model]). In NMRI mice, ID had no effect on the phosphorylation of ribosomal S6 kinase (p70S6K), a downstream target of mTOR. However, rapamycin inhibited ID-induced thyroid blood flow and VEGF protein expression. In the RET-PTC model, ID strongly increased the phosphorylation of p70S6K, whereas rapamycin completely inhibited the ID-induced increase in p70S6K phosphorylation, thyroid blood flow, and VEGF-A expression. In vitro, although ID increased p70S6K phosphorylation, the ID-stimulated hypoxia-inducible factor/VEGF pathway was inhibited by rapamycin. Activation of AMPK by metformin inhibited ID effects both in vivo and in vitro. In AMPK-α1 knockout mice, the ID-induced increase in thyroid blood flow and VEGF-A protein expression persisted throughout the treatment, whereas both parameters returned to control values in wild-type mice after 4 days of ID. In conclusion, mTOR is required for early ID-induced thyroid microvascular activation. AMPK negatively regulates this pathway, which may account for the transient nature of ID-induced TSH-independent vascular effects under benign conditions.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Iodine/deficiency , TOR Serine-Threonine Kinases/metabolism , Thyroid Gland/metabolism , AMP-Activated Protein Kinases/genetics , Animals , Cell Proliferation/genetics , Cell Proliferation/physiology , Female , Humans , In Vitro Techniques , Male , Metformin/metabolism , Mice , Mice, Knockout , Phosphorylation/drug effects , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/genetics , Thyroid Gland/pathology , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Iodine deficiency (ID) induces microvascular changes in the thyroid gland via a TSH-independent reactive oxygen species-hypoxia inducible factor (HIF)-1α-vascular endothelial growth factor (VEGF) pathway. The involvement of nitric oxide (NO) in this pathway and the role of calcium (Ca(2+)) and of ryanodine receptors (RYRs) in NO synthase 3 (NOS3) activation were investigated in a murine model of goitrogenesis and in 3 in vitro models of ID, including primary cultures of human thyrocytes. ID activated NOS3 and the production of NO in thyrocytes in vitro and increased the thyroid blood flow in vivo. Using bevacizumab (a blocking antibody against VEGF-A) in mice, it appeared that NOS3 is activated upstream of VEGF-A. L-nitroarginine methyl ester (a NOS inhibitor) blocked the ID-induced increase in thyroid blood flow in vivo and NO production in vitro, as well as ID-induced VEGF-A mRNA and HIF-1α expression in vitro, whereas S-nitroso-acetyl-penicillamine (a NO donor) did the opposite. Ca(2+) is involved in this pathway as intracellular Ca(2+) flux increased after ID, and thapsigargin activated NOS3 and increased VEGF-A mRNA expression. Two of the 3 known mammalian RYR isoforms (RYR1 and RYR2) were shown to be expressed in thyrocytes. RYR inhibition using ryanodine at 10µM decreased ID-induced NOS3 activation, HIF-1α, and VEGF-A expression, whereas RYR activation with ryanodine at 1nM increased NOS3 activation and VEGF-A mRNA expression. In conclusion, during the early phase of TSH-independent ID-induced microvascular activation, ID sequentially activates RYRs and NOS3, thereby supporting ID-induced activation of the NO/HIF-1α/VEGF-A pathway in thyrocytes.
