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: 10522662
• 负责人: ANGELA BRUZZANITI
• 金额: $ 50.08万
• 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/10522662
• 关键词: Actins; Address; Adverse event; 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; Focal Adhesion Kinase 1; Focal Adhesions; Fracture; Functional disorder; Genetic; Genotype; Goals; Gonadal Steroid Hormones; Hindlimb Suspension; In Vitro; Investigation; Knowledge; Lead; 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; 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; clinically relevant; falls; human female; improved; in vitro activity; in vivo; inhibitor; kinase inhibitor; male; mechanical properties; men; middle age; 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.

摘要绝经期妇女的骨丢失加速，更普遍的是， 衰老目前的抗骨吸收和骨合成代谢疗法通常仅在骨吸收明显后才开处方。 所有这些都可能产生不良的临床影响。骨合成代谢和抗吸收双重作用药物 可能提供增加骨量的新方法。Pyk 2酪氨酸激酶具有双重作用， 成骨细胞（OB）活性和增加破骨细胞（OC）再吸收。尽管大脑皮层的 在雄性和雌性Pyk 2-/-小鼠的微结构中，仅雌性表现出松质骨质量增加， 机械性能，表明性别特异性差异的组织水平的属性。在初步研究中，我们 发现卵巢切除的Pyk 2-/-雌性小鼠补充17β-雌二醇（E2）后， 重量高于WT，并且尺骨负载的Pyk 2-/-雄性（未测试的雌性）表现出更高的骨形成率， 表明体内成骨反应增强。Pyk 2-/- OBs在体外具有增强的矿化活性， 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）。Pyk 2-/- OBs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拮抗剂阻止E2刺激的Pyk 2-/- OB活性增加， 提示E2-ERβ信号传导增加。另一方面，Pyk 2-/-OC表现出OC吸收缺陷 活性，并且在E2存在下，与WT OC相比，OC形成和存活减少。我们的具体 本研究旨在验证Pyk 2通过不同的E2-ERα/ERβ调节机制发挥双重作用的假设。 抑制OB活性和促进OC再吸收的调节作用。在目标1中，我们将展示 通过检查4个月、12个月和21个月的Pyk 2在骨重建周期的合成代谢与分解代谢臂中的表达， 其中Pyk 2缺失靶向具有1a型胶原2.3-Cre的OB（Pyk2Col2.3）或成熟OC的小鼠 组织蛋白酶K-Cre（Pyk 2CatK）。此外，Pyk2Col2.3小鼠将经受胫骨合成代谢负荷，而Pyk 2CatK小鼠将经受胫骨合成代谢负荷。 在后肢卸载诱导的骨损失后检查小鼠。在目标2中，我们将确定骨量是否 在4个月雄性/雌性Pyk2Col2.3小鼠的性类固醇耗竭（性腺切除术）后保存（由于升高的 骨生成）和Pyk 2CatK小鼠（由于吸收减少），以及E2或雷洛昔芬是否会额外改善骨 马萨诸塞州我们将剖析Pyk 2调节ERα降解/稳定性的机制，以及对ERα/ERβ的影响。 体外OB和OB中的E2-ERβ信号传导。在目标3中，我们将确定a的非劣效性 Pyk 2选择性抑制剂预防/逆转与年龄相关的骨量和力学性能下降 在12个月C57 BL/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。将使用由男性/女性制备的OB和OC证明临床转化影响 体外的人类捐赠者总之，这些研究将研究Pyk 2抑制作为一种双重的，骨合成代谢和抗- 骨吸收的方法，以扭转骨丢失和改善骨质疏松症和衰老的骨质量/强度。