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

揭示 Pyk2 抑制对骨量的双重合成代谢和抗分解代谢作用

• 批准号: 10688085
• 负责人: ANGELA BRUZZANITI
• 金额: $ 51.33万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-22 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10688085
• 关键词: Acceleration; Actins; Address; Adverse event; Age; Aging; Alzheimer' s Disease; Architecture; Arm Bones; Biological Markers; Bone Diseases; Bone Tissue; Bone remodeling; C57BL/6 Mouse; Cardiovascular Diseases; Categories; Chemicals; Clinical; Clinical Trials; Collagen; Data; Deposition; Deterioration; Dimerization; Dual-Energy X-Ray Absorptiometry; Effectiveness; Elderly man; Elderly woman; Enzyme-Linked Immunosorbent Assay; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogen Receptors; Exhibits; Female; Femur; Focal Adhesion Kinase 1; Focal Adhesions; Fracture; Functional disorder; Genetic; Genotype; Goals; Gonadal Steroid Hormones; Hindlimb Suspension; In Vitro; Investigation; Knowledge; LoxP-flanked allele; Mechanical Stimulation; Menopause; Minerals; Modeling; Mus; Osteoblasts; Osteoclasts; Osteocytes; Osteogenesis; Osteoporosis; Osteoporosis prevention; Osteoporotic; Phenotype; Property; Protein Tyrosine Kinase; Proteins; Public Health; Raloxifene; Raman Spectrum Analysis; Reporting; Repression; Role; Selective Estrogen Receptor Modulators; Serum; Sex Differences; Signal Transduction; Testing; Thick; Thinness; Tissues; Ubiquitin; Woman; antagonist; bone; bone cell; bone loss; bone mass; bone quality; bone strength; cathepsin K; clinical effect; clinical translation; clinically relevant; human female; improved; in vitro activity; in vivo; inhibitor; kinase inhibitor; male; mechanical properties; men; middle age; mineralization; multicatalytic endopeptidase complex; novel strategies; novel therapeutics; osteogenic; preservation; prevent; response; seal; sex; skeletal; substantia spongiosa; translational impact; translational potential; ubiquitin mediated proteasome degradation

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.

概括。更年期女性骨质流失加速，男性和女性更普遍 老化。目前的抗骨吸收和骨合成代谢疗法通常仅在明显的骨质疏松后进行 丢失，所有这些都可能产生不良的临床影响。具有骨合成代谢和抗骨吸收双重作用的药物 可能提供增加骨量的新方法。 Pyk2 酪氨酸激酶具有双重作用，既能降低 成骨细胞（OB）活性和增加破骨细胞（OC）吸收。尽管皮质也有类似的增加 在雄性和雌性 Pyk2-/- 小鼠的微结构中，只有雌性表现出松质骨量增加，并且 机械特性，表明组织水平特性的性别特异性差异。在初步研究中，我们 发现补充 17β-雌二醇 (E2) 的去卵巢 Pyk2-/- 雌性小鼠的骨质含量更高 质量比 WT 更大，并且尺骨负载 Pyk2-/- 的男性（女性未测试）表现出更高的骨形成率， 表明体内成骨反应增强。 Pyk2-/- OB 具有增强的体外矿化活性， 17β-雌二醇 (E2) 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）。 Pyk2-/- OB 由于其泛素蛋白酶体而表现出雌激素受体 ERα 水平降低 降解，我们发现 ERβ 拮抗剂可以阻止 E2 刺激的 Pyk2-/- OB 活性增加， 表明 E2-ERβ 信号传导增强。另一方面，Pyk2-/- OCs 表现出有缺陷的 OC 吸收 活性，并且在 E2 存在的情况下，与 WT OC 相比，OC 的形成和存活率降低。我们的具体 目标将测试 Pyk2 通过不同的 E2-ERα/ERβ 调节机制发挥双重作用的假设 抑制 OB 活性并促进 OC 吸收的调节作用。在目标 1 中，我们将展示角色 通过检查 4、12 和 21 个月，了解 Pyk2 在骨重塑周期的合成代谢与分解代谢臂中的作用 Pyk2 缺失靶向具有 1a 型胶原蛋白、2.3-Cre (Pyk2Col2.3) 的 OB 或成熟 OC 的小鼠 与组织蛋白酶 K-Cre (Pyk2CatK)。此外，Pyk2Col2.3 小鼠将承受胫骨合成代谢负荷，而 Pyk2CatK 将在后肢卸载引起的骨质流失后对小鼠进行检查。在目标 2 中，我们将确定骨量是否为 4 个月大的雄性/雌性 Pyk2Col2.3 小鼠的性类固醇消耗（性腺切除术）后保留（由于升高 成骨）和 Pyk2CatK 小鼠（由于吸收减少），以及 E2 或雷洛昔芬是否会额外改善骨质 大量的。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 OB 和体外 OB 中的二聚化和 E2-ERβ 信号传导。在目标 3 中，我们将建立 Pyk2 选择性抑制剂可预防/逆转与年龄相关的骨量和机械性能下降 与广泛作用的粘着斑激酶抑制剂相比，在 12 个月大的 C57BL/6 雌性小鼠中， 雷洛昔芬。将使用男性/女性制备的 OB 和 OC 来证明临床转化影响 体外人类捐赠者。这些研究将共同​​探讨 Pyk2 抑制作为双重骨合成代谢和抗骨代谢药物的作用。 骨质疏松症和衰老过程中逆转骨质流失并改善骨质量/强度的再吸收方法。