Uncovering the dual anabolic and anti-catabolic effects of Pyk2 inhibition on bone mass

总结。更年期女性的骨质流失会加速，更普遍的是，在患有 衰老。目前的抗骨吸收和骨合成代谢治疗一般只在明显的骨量增加后才能开出。 损失，所有这些都会对临床产生不利的影响。兼具骨合成代谢和抗吸收作用的双重作用药物 可能会提供增加骨量的新方法。Pyk2酪氨酸激酶是双重作用的，两者都在减少 成骨细胞(OB)活性和破骨细胞(OC)吸收增加。尽管大脑皮层也有类似的增加 在雄性和雌性PYK2-/-小鼠中，只有雌性表现出松质骨量增加和 力学特性，表明组织水平特性的性别差异。在初步研究中，我们 发现去卵巢的PYK2-/-雌性小鼠补充17β-雌二醇(E_2)后骨量增加 体重高于WT，尺骨负荷的PYK2-/-雄性(未测试的雌性)表现出更高的骨形成率， 提示体内的成骨反应增强。PYK2-/-OBS在体外具有增强矿化活性， 17β-雌二醇(E_2)和雷洛昔芬(一种临床批准的选择性雌激素受体)进一步刺激 调制器(SERM)。由于其泛素蛋白酶体，PYK2-/-OBS显示雌激素受体ERα水平降低 降解，我们发现ERβ拮抗剂阻止了E2刺激的PYK2-/-OB活性的增加， 提示E2-ERβ信号转导增加。另一方面，PYK2-/-OCs表现出有缺陷的OC吸收 与WT OCS相比，在E2存在的情况下，OC的形成和存活率降低。我们的特定 AIMS将检验这一假设，即PYK2通过不同的E2-ERα/ERβ调节机制发挥作用，以施加双重- 抑制OB活性和促进OC吸收的调节作用。在目标1中，我们将演示该角色 通过4个月、12个月和21个月的检查，在骨重建周期的合成代谢臂和分解代谢臂中检测Pyk2的表达 以具有1a，2.3-Cre(Pyk2Col2.3)型胶原的OBS或成熟OCS为靶点的PYK2缺失小鼠 用组织蛋白酶K-Cre(Pyk2CatK)。此外，Pyk2Col2.3小鼠将受到胫骨合成代谢负荷的影响，而Pyk2CatK 对后肢卸载所致骨丢失小鼠进行检查。在目标2中，我们将确定骨量是否 4个月龄雄性/雌性Pyk2Col2.3小鼠性类固醇耗竭(性腺切除)后保存(由于升高 成骨)和Pyk2CatK小鼠(由于吸收减少)，以及如果E2或雷洛昔芬将额外改善骨骼 质量。我们将剖析调节ERα降解/稳定性的PYK2机制，以及对ERα/ERβ的影响 OBS和OBS中的二聚化和E2-ERβ信号转导。在目标3中，我们将建立一个非劣势的 PYK2选择性抑制剂预防/逆转增龄性骨量和力学性能下降 在12个月龄的C57BL/6雌性小鼠中，与广泛作用的粘着斑激酶抑制剂相比， 雷洛昔芬。将使用从男性/女性准备的OBS和OCS来展示临床翻译效果 体外人类捐献者。总之，这些研究将调查作为一种双重的，骨合成代谢和抗PYK2抑制 在骨质疏松症和衰老中逆转骨丢失和改善骨质量/强度的吸收方法。

SUMMARY. Bone loss is accelerated in menopausal women, and more generally in both men and women with aging. Current anti-resorptive and osteoanabolic therapies are generally prescribed only after pronounced bone loss, and all can have adverse clinical effects. Dual-acting agents that are both osteoanabolic and anti-resorptive may offer novel approaches to increase bone mass. The Pyk2 tyrosine kinase is dual-acting, both decreasing osteoblast (OB) activity and increasing osteoclast (OC) resorption. Despite a similar increase in cortical microarchitecture in male and female Pyk2-/- mice, only females exhibit increased cancellous bone mass and mechanical properties, suggesting sex-specific differences in tissue level properties. In preliminary studies, we found that ovariectomized Pyk2-/- female mice supplemented with 17β-estradiol (E2) have additively higher bone mass than WT, and that ulnar loaded Pyk2-/- males (females not tested) exhibit higher bone formation rate, suggesting enhanced osteogenic responses in vivo. Pyk2-/- OBs have enhanced mineralizing activity in vitro, which is further stimulated by 17β-estradiol (E2) and raloxifene, a clinically-approved selective estrogen receptor modulator (SERM). Pyk2-/- OBs exhibit reduced levels of estrogen receptor, ERα, due to its ubiquitin-proteasome degradation, and we found that ERβ antagonists prevent the E2-stimulated increase in Pyk2-/- OB activity, suggesting an increase in E2-ERβ signaling. On the other hand, Pyk2-/- OCs exhibit defective OC resorption activity, and in the presence of E2, OC formation and survival are reduced compared to WT OCs. Our Specific Aims will test the hypothesis that Pyk2 acts via distinct E2-ERα/ERβ-regulated mechanisms to exert dual- modulating effects that repress OB activity and promote OC resorption. In Aim 1, we will demonstrate the role of Pyk2 in the anabolic versus catabolic arms of the bone remodeling cycle by examining 4-, 12- and 21-month mice in which Pyk2-deletion is targeted to either OBs with collagen type 1a, 2.3-Cre (Pyk2Col2.3) or mature OCs with cathepsin K-Cre (Pyk2CatK). In addition, Pyk2Col2.3 mice will be subject to tibial anabolic loading, while Pyk2CatK mice will be examined after hindlimb unloading-induced bone loss. In Aim 2, we will determine if bone mass is preserved after sex steroid depletion (gonadectomy) of 4-month male/female Pyk2Col2.3 mice (due to elevated osteogenesis) and Pyk2CatK mice (due to reduced resorption), and if E2 or raloxifene will additively improve bone mass. We will dissect the Pyk2 mechanisms that regulate ERα degradation/stability, and effects on ERα/ERβ dimerization, and E2-ERβ signaling in OBs and OBs in vitro. In Aim 3, we will establish the non-inferiority of a Pyk2-selective inhibitor in preventing/reversing age-associated decline of bone mass and mechanical properties in 12-month C57BL/6 female mice, compared to a broad-acting inhibitor of focal adhesion kinases, and raloxifene. Clinical translational impact will be demonstrated using OBs and OCs prepared from male/female human donors in vitro. Together, these studies will investigate Pyk2 inhibition as a dual, osteoanabolic and anti- resorptive approach to reverse bone loss and improve bone quality/strength in osteoporosis and aging.