ERK Signaling in Inflammatory Bone Loss

炎症性骨丢失中的 ERK 信号传导

• 批准号: 8734714
• 负责人: Francis Young-In Lee
• 金额: $ 7.2万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-20 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8734714
• 关键词: Address; Adverse effects; Affect; Anti-Inflammatory Agents; Anti-inflammatory; Biology; Bone Resorption; CSF1 gene; Calcium Oscillations; Caspase; Caspase-1; Cells; Data; Disease; Dissociation; Economic Burden; Elderly; Exhibits; Fracture; Functional disorder; Future; Gene Expression; Genes; Goals; Healed; Health Care Costs; Human; Image; Immunoprecipitation; In Vitro; Inflammation; Inflammatory; Intervention; Knockout Mice; Knowledge; Macrophage Activation; Malignant Neoplasms; Measures; Mediating; Medical; Messenger RNA; Metastatic Neoplasm to the Bone; Methods; Molecular; Mus; Musculoskeletal Diseases; Osteoclasts; Osteolysis; Osteolytic; Osteoporosis; PTGS2 gene; Parents; Pathogenesis; Pathologic; Pathway interactions; Pharmacologic Substance; Play; Population; Process; Production; Proteins; Proteomics; Recombinants; Recruitment Activity; Research; Role; Signal Transduction; Site; Staining method; Stains; TNF gene; TNFSF11 gene; Testing; Therapeutic; Therapeutic Effect; Time; Tissues; Topical application; Translations; Treatment Efficacy; Wages; Work; base; bone; bone loss; bone mass; calreticulin; cathepsin K; clinical application; clinically relevant; cytokine; disability; healing; in vivo; in vivo Model; inflammatory bone resorption; inhibitor/antagonist; innovation; interest; interleukin-1beta-converting enzyme inhibitor; liquid chromatography mass spectrometry; mRNA Expression; macrophage; meetings; molecular imaging; monocyte; new therapeutic target; novel; osteoclastogenesis; pathologic bone resorption; prevent; protective effect; public health relevance; rapid growth; research study; screening; success; therapeutic target; transcription factor

DESCRIPTION (provided by applicant): Pathologic bone resorption due to osteoporosis, fragility fractures and cancers pose a significant economic burden to healthcare costs and lost wages. These osteolytic diseases share a similar pathophysiology, so understanding the mechanisms of bone resorption greatly aids the search for treatment options. The need to develop therapeutic options with minimal side effects is strong, but requires greater elucidation into the mechanisms of inflammatory bone resorption. We previously confirmed the role of pERK1/2 on the production of pro-osteoclastogenic cytokines such as RANKL, MCSF, COX-2 and IL-1b. Then, we focused on a seemingly overlooked target for inhibiting osteoclastogenesis, IL-1b, an inflammatory cytokine implicated in pathologic osteolysis. An IL-1b Converting Enzyme (ICE, or Caspase-1) inhibitor showed dramatic inhibition of osteoclastogenesis. However, Caspase-1inhibitor -/- osteoclast precursors did not demonstrate impaired osteclastogenesis, implying off-target components to this osteolysis-inhibition mechanism. Subsequently, our proteomic screening efforts yielded us Calregulin (CRG) as our osteoclastogenic-inhibition focus. Subsequent in vitro and in vivo studies met with great success as administration of recombinant human Calregulin (rhCRG) demonstrated inhibition of osteoclastogenesis in vitro in osteoclast-precursor macrophages and inhibition of LPS-induced osteolysis in vivo. rhCRG inhibited NFATc1 among several osteoclastogenic transcription factors. The new direction of this competitive renewal is to test a novel hypothesis that Calregulin inhibits osteoclastogenesis in vitro and inflammation-associated osteoclastogenesis in vivo. Therefore, our aims for this proposal employ 3 parallel Aims to determine mechanisms and therapeutic translation in vitro and in vivo. Aim 1 will determine mechanisms by which rhCRG interferes with key osteoclastogenic pathways such as NFATc1 in vitro. We will determine whether rhCRG interferes with osteoclastogenic gene expression, calcium oscillation, NFATc1 activity, and bone resorption. In addition, we will examine whether rhCRG/NFATc1 functionally interact with 6 candidate proteins which we identified on Liquid Chromatography/Mass Spectrometry of His-CRG pulled-down proteins. Aim 2 will determine whether CRG regulates macrophage activation in inflammatory osteolysis in vitro and in vivo. Macrophage activation of M1 or M2 plays pro- and anti-inflammatory roles, respectively. By determining the CRG's role in the polarization of M2 macrophages and inhibition of M1, we will establish an alternative mechanism. Aim 3 will establish a therapeutic role of topical rhCRG in inflammatory osteolysis in vivo. We will induce osteolysis with clinically relevant RANKL, TNF and LPS to determine whether rhCRG can prevent or treat inflammation-associated osteoclastogenesis and bone resorption. We will measure bone protective effects of rhCRG using Cathepsin K molecular imaging, dynamic bone histomorphometry and TRAP staining. Overall impact is high in that we expect to unravel novel anti-osteoclastogenic mechanisms and therapeutic promise of rhCRG in the context of inflammatory osteolysis.

描述(申请人提供)：骨质疏松症、脆性骨折和癌症引起的病理性骨吸收给医疗费用和工资损失带来了巨大的经济负担。这些溶骨性疾病有着相似的病理生理学，因此了解骨吸收的机制对寻找治疗方案有很大帮助。开发副作用最小的治疗方案的必要性很强，但需要更多地阐明炎症性骨吸收的机制。我们先前证实了pERK1/2在RANKL、MCSF、COX-2和IL-1b等促破骨细胞因子的产生中的作用。然后，我们专注于一个似乎被忽视的抑制破骨细胞生成的靶点，IL-1b，一种与病理性骨溶解有关的炎性细胞因子。一种IL-1b转换酶(ICE，或Caspase-1)抑制剂显示出对破骨细胞生成的显著抑制。然而，Caspase-1抑制剂/破骨细胞前体并没有显示出破骨细胞生成受损，这意味着非靶点成分参与了这种溶骨-抑制机制。随后，我们的蛋白质组筛选工作使我们的钙调蛋白(CRG)成为我们破骨细胞生成抑制的焦点。随后的体外和体内研究都取得了巨大的成功，重组人钙调蛋白(RhCRG)在体外抑制了破骨细胞前体巨噬细胞的破骨细胞生成，并在体内抑制了内毒素诱导的骨溶解。在多种破骨细胞转录因子中，rhCRG抑制NFATc1的表达。这一竞争性更新的新方向是测试一个新的假设，即CalRegin在体外抑制破骨细胞生成，在体内抑制炎症相关的破骨细胞生成。因此，我们对这一建议的目标是采用3个平行的目标来确定体外和体内的机制和治疗性翻译。目的1体外实验将确定重组人CRG干扰NFATc1等关键破骨细胞途径的机制。我们将确定重组人CRG是否干扰破骨细胞基因表达、钙振荡、NFATc1活性和骨吸收。此外，我们还将研究rh CRG/NFATc1是否与我们在His-CRG下拉蛋白的液相色谱/质谱学上鉴定的6个候选蛋白发生功能相互作用。目的研究CRG在体外和体内对炎性骨溶解中巨噬细胞活化的调节作用。巨噬细胞活化的M1和M2分别具有促炎和抗炎作用。通过确定CRG在M2巨噬细胞极化和抑制M1中的作用，我们将建立另一种机制。目的3建立局部应用重组人CRG在体内炎性骨溶解中的治疗作用。我们将用临床上相关的RANKL、肿瘤坏死因子和内毒素诱导骨溶解，以确定重组人CRG是否可以预防或治疗炎症相关的破骨细胞生成和骨吸收。我们将使用组织蛋白酶K分子成像、动态骨组织形态计量学和TRAP染色来检测重组人CRG的骨保护作用。总体影响很大，因为我们希望在炎症性骨溶解的背景下揭示新的抗骨破坏机制和重组人CRG的治疗前景。