ERK Signaling in Inflammatory Bone Loss

描述（由申请人提供）：骨质疏松症、脆性骨折和癌症引起的病理性骨吸收对医疗费用和工资损失造成了重大的经济负担。这些溶骨性疾病具有相似的病理生理，因此了解骨吸收的机制有助于寻找治疗方案。开发具有最小副作用的治疗方案的需求是强烈的，但需要更深入地阐明炎症性骨吸收的机制。我们之前证实了pERK1/2在促破骨细胞因子如RANKL、MCSF、COX-2和IL-1b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发生的目标，IL-1b，一种与病理性骨溶解有关的炎症细胞因子。IL-1b转换酶（ICE，或Caspase-1）抑制剂显示出对破骨细胞发生的显著抑制作用。然而，caspase -1抑制剂-/-破骨细胞前体并没有显示出成骨细胞生成受损，这意味着这种骨溶解抑制机制的脱靶成分。随后，我们的蛋白质组学筛选工作将钙调节蛋白（CRG）作为我们的破骨细胞生成抑制重点。随后的体外和体内研究取得了巨大成功，重组人钙调节蛋白（rhCRG）在体外抑制破骨细胞前体巨噬细胞的破骨生成，并抑制lps诱导的体内溶骨。rhCRG抑制几种破骨细胞转录因子中的NFATc1。这种竞争性更新的新方向是测试钙调节蛋白在体外抑制破骨细胞生成和体内炎症相关破骨细胞生成的新假设。因此，我们的目标是采用3个平行目标来确定体外和体内的机制和治疗翻译。目的1将确定rhCRG在体外干扰NFATc1等关键破骨细胞生成途径的机制。我们将确定rhCRG是否干扰破骨细胞基因表达、钙振荡、NFATc1活性和骨吸收。此外，我们将研究rhCRG/NFATc1是否与6个候选蛋白在功能上相互作用，这些候选蛋白是我们通过His-CRG拉下蛋白的液相色谱/质谱法鉴定的。目的2将在体外和体内研究CRG是否调节炎症性溶骨过程中巨噬细胞的活化。巨噬细胞激活M1或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M1抑制中的作用，我们将建立另一种机制。目的3将建立局部rhCRG在体内炎症性骨溶解中的治疗作用。我们将使用临床相关的RANKL、TNF和LPS诱导骨溶解，以确定rhCRG是否可以预防或治疗炎症相关的破骨细胞生成和骨吸收。我们将通过组织蛋白酶K分子成像、动态骨组织形态测量和TRAP染色来测量rhCRG的骨保护作用。总体影响是高的，因为我们期望揭示新的抗破骨细胞机制和rhCRG在炎症性骨溶解中的治疗前景。

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.