Thyrotropin Receptor, Thyrotropin and Mechanisms of Bone Loss

项目摘要 在2003年，我们发现垂体前叶激素促甲状腺激素（又名促甲状腺激素），促甲状腺激素），迄今已知促进 甲状腺激素分泌是骨量的有效直接调节剂（Abe等人，Cell，2003，PMID：14567913）。 这一发现强调了低循环TSH水平在引起骨丢失中的潜在作用， 在甲状腺功能亢进症患者中认识到这一点已有一个多世纪2，并且传统上仅归因于 甲状腺激素过多相反，我们发现Tsh受体缺陷的Tshr-/-小鼠患有严重的骨质疏松症， 即使甲状腺功能正常1。重要的是，我们最近发现，Tshr-/-小鼠的骨丢失使 甲状腺功能亢进症显著超过野生型甲状腺功能亢进症小鼠（Baliram等，J Clin Invest，2012， PMID：22996689）3 -这一发现不仅证实了Tshr缺乏对骨的直接容许作用， 也支持了多项临床研究，显示低TSH与低TSH之间存在紧密且高度可重复的相关性。 水平，骨质流失和高骨折风险在世界范围内的甲亢患者队列4 -24。而且我们 发现破骨细胞生成细胞因子TNFα在Tshr-/-小鼠中显著升高，并且其遗传性 缺失挽救了Tshr缺陷的骨骼表型（Hase等，PNAS，2006，PMID：16908863; Sun等， 等人，PNAS，2013，PMID：23716650）25，26。这就引出了一个问题：哪种细胞--成骨细胞或破骨细胞--驱动 作用，以及两种Tnf受体，Tnfrsf 1a或Tnfrsf 1b，介导Tnfα在Tshr中的作用 缺陷？具体目标1将研究在成骨细胞中选择性缺失Tshr的小鼠， 破骨细胞，以及Tshr和Tnfrsf 1a或Tnfrsf 1b缺失的双突变体。 互补的共培养实验将确定成骨细胞TNFα是否介导骨细胞的过度吸收。 Tshr-/-小鼠。第二组数据，得到其他研究组的证实27 -33，表明促甲状腺激素既表现出抗甲状腺激素， 再吸收和合成代谢作用1，34-37。例如，间歇性低剂量TSH注射恢复了丢失的骨 卵巢切除术后7个月，重要的是没有升高T4水平（Sun等人，PNAS，2008，PMID： 37.第三十七章一个后续的问题由此产生：Tshr是一个药物靶点吗？为了找到答案，我们 将利用遗传学和药理学方法。在具体目标2中，我们将检查是否高 促甲状腺激素水平是使用小鼠合成代谢的，其中显性阴性Trβ 337在促甲状腺细胞中表达， 将促甲状腺激素抑制在高30倍的循环水平。在具体目标3中，我们将研究一个小的 Tshr的分子激活剂，MS 438，我们已经发现，它选择性地结合Tshr，并且具有纳摩尔浓度。 亲和力（Bisaf等人，Thyroid，2015，PMID：25333622）38.我们还发现，MS 438显示促成骨细胞， 体外抗骨质疏松作用，不升高血清T4。因此，我们将向小鼠注射MS 438 立即（“预防”）或卵巢切除术后7个月（“恢复”），以确定它是否可以防止骨 丢失和/或恢复丢失的骨。总之，这些研究不仅应该让我们深入了解 Tsh对骨骼的作用，而且还为靶向骨骼Tsh的新方法提供了概念验证。

PROJECT SUMMARY In 2003, we showed that the anterior pituitary hormone thyrotropin (a.k.a. TSH), hitherto known to promote thyroid hormone secretion, is a potent direct regulator of bone mass (Abe et al, Cell, 2003, PMID: 14567913)1. This finding underscored a potential role for low circulating TSH levels in causing the bone loss that has been recognized in patients with hyperthyroidism for over a century2, and, by tradition, has been attributed solely to thyroid hormone excess. We found instead that Tsh receptor-deficient Tshr-/- mice had profound osteoporosis, even when rendered euthyroid1. Importantly, we showed more recently that bone loss in Tshr-/- mice rendered hyperthyroid significantly exceeded that in wild type hyperthyroid mice (Baliram et al, J Clin Invest, 2012, PMID: 22996689)3 – this finding not only confirmed a direct permissive action of Tshr deficiency on bone, but also buttressed multiple clinical studies showing a tight and highly reproducible correlation between low TSH levels, bone loss, and a high fracture risk in cohorts of hyperthyroid patients worldwide4-24. Furthermore, we found that the osteoclastogenic cytokine, Tnfα, was grossly elevated in Tshr-/- mice, and that its genetic deletion rescued the skeletal phenotype of Tshr deficiency (Hase et al, PNAS, 2006, PMID: 16908863; Sun et al, PNAS, 2013, PMID: 23716650)25,26. This led to the question: which cell – osteoblast or osteoclast – drives the effect, and which of the two Tnf receptors, Tnfrsf1a or Tnfrsf1b, mediate the action of Tnfα in Tshr deficiency? Specific Aim 1 will study mice in which the Tshr is deleted selectively in osteoblasts or osteoclasts, as well as double mutants in which both the Tshr and either Tnfrsf1a or Tnfrsf1b are deleted. Complementary co-culture experiments will determine if osteoblastic Tnfα mediates the hyper-resorption in Tshr-/- mice. A second corpus of data, confirmed by other groups27-33, showed that Tsh displays both anti- resorptive and anabolic actions1,34-37. For example, intermittent low dose Tsh injections restored the lost bone 7 months post-ovariectomy, importantly without elevating T4 levels (Sun et al, PNAS, 2008, PMID: 18332426)37. A follow-up question thus arises: is the Tshr a druggable target? Towards finding an answer, we will utilize both genetic and pharmacological approaches. In Specific Aim 2, we will examine whether high Tsh levels are anabolic using mice in which the expression of dominant-negative Trβ337 in the thyrotrope clamps Tsh at ~30-fold higher circulating levels. In Specific Aim 3, we will study the effects of a small molecule activator of the Tshr, MS438, which, we have found, binds Tshrs selectively and with a nanomolar affinity (Latif et al, Thyroid, 2015, PMID: 25333622)38. We also find that MS438 displays pro-osteoblastic and anti-osteoclastic actions in vitro, and does not elevate serum T4. We will thus inject mice with MS438 immediately (‘prevention’) or 7-months following (‘restoration’) ovariectomy to determine if it can prevent bone loss and/or restore the lost bone. Together, these studies should not only allow an in-depth understanding of Tsh action on bone, but also provide proof-of-concept for a new approach that targets the skeletal Tshr.