ERK Signaling in Inflammatory Bone Loss

描述（由申请人提供）：我们的长期目标是开发炎症性骨丢失的预防性治疗方法，这些炎症性骨丢失表现为诸如假体松动（脓毒性或无菌性）和牙周感染等病理学。M-SCF和RANKL是两种必需的促破骨细胞生成和促炎细胞因子，其刺激巨噬细胞形成破骨细胞。我们的初步数据表明，MAP激酶/ERK 1/2是一个重要的信号转导负责RANKL和MCSF基因的表达。本A1提案的目的是通过进行体内和体外机制实验来确定ERK信号传导在成骨细胞介导的先天免疫中对生物材料的反应中的作用。我们的中心假设是ERK信号通过调节促破骨细胞生成细胞因子的表达和炎性骨质溶解来介导骨对生物材料的天然免疫反应。我们将使用临床相关的啮齿动物股骨植入物模型和成骨细胞特异性ERK 1/2功能障碍（“ERK def小鼠”）进行机制研究。ERK途径抑制剂的使用在临床上是可行的，因为此类抑制剂目前正用于治疗具有升高水平的ERK活化的癌症的临床试验中。ERK靶向的优势在于ERK是上游和下游炎症信号交叉的中心信号站。具体目标如下：1。探讨ERK是否通过调节成骨细胞中M-CSF和RANKL基因表达介导炎性骨丢失。我们将使用成骨细胞特异性ERKdef小鼠在体外和体内进行LPS和UHMWPE刺激后负责M-CSF和RANKL表达的机械细胞信号传导途径的实验。具体目标2。确定ERK信号阻断在临床相关股骨骨质溶解模型中的治疗效果。我们将通过定量microCT和生物力学拔出测试来检查ERK信号传导的药理学抑制对UHMWPE或LPS诱导的大鼠股骨骨溶解的影响。具体目标3。研究在生物材料和植入物污染物（如细菌毒素）的背景下，ERK 1/2信号传导阻断如何影响成骨细胞和破骨细胞之间的相互作用。我们将通过共培养ERKdef小鼠细胞和巨噬细胞来检查破骨细胞生成。我们将在存在和不存在ERK信号传导阻断的情况下测量破骨细胞生成、细胞因子表达和骨吸收。具体目标4。确定ERK和NF：B P50协同调节成骨细胞对颗粒生物材料和LPS的M-CSF基因诱导的具体机制。虽然ERK/ATF 4/RANKL通路已经建立，但在M-CSF的转录调控方面存在知识空白。我们将研究ERK和NF-κ B对MCSF基因诱导的协同调节。我们期望验证成骨细胞先天免疫的新概念，并确定ERK信号在生物材料和相关毒素引起的炎性骨丢失中的作用。公共卫生相关性：本研究的临床基本原理是，我们可以通过使用特定的局部或全身抑制剂靶向ERK介导的炎症途径来预防或治疗临床重要的炎性骨丢失。

DESCRIPTION (provided by applicant): Our long-term goal is to develop preventative treatments for inflammatory bone loss as manifested in pathologies such as prosthetic loosening (septic or aseptic) and periodontal infection. M-SCF and RANKL are two essential pro-osteoclastogenic and pro-inflammatory cytokines that stimulate macrophages to form osteoclasts. Our preliminary data showed that MAP Kinase/ ERK1/2 is an important signal transducer responsible for RANKL and MCSF gene expression. The objective of this A1 proposal is to define the role of ERK signaling in osteoblast-mediated innate immunity in response to biomaterials by conducting in vivo and in vitro mechanistic experiments. Our central hypothesis that ERK signaling mediates bone innate immunity in response to biomaterials by regulating pro-osteoclastogenic cytokine expression and inflammatory osteolysis. We will conduct mechanistic studies using a clinically relevant rodent femoral implant model and osteoblast-specific ERK1/2 dysfunction ("ERKdef mice"). The use of ERK pathway inhibitors is clinically feasible in that such inhibitors are currently being used in clinical trials for treatment of cancers with heightened levels of ERK activation. The advantage of ERK targeting lies in the fact that ERK is a central signaling station where upstream and downstream inflammatory signals cross. Specific Aims are as follows; Specific Aim 1. To determine whether ERK mediates inflammatory bone loss by regulating M-CSF and RANKL gene expression in osteoblasts. We will conduct experiments on mechanistic cellular signaling pathways responsible for M-CSF and RANKL expression after stimulation with LPS and UHMWPE in vitro and in vivo using osteoblast specific ERKdef mice. Specific Aim 2. To determine the therapeutic effect of ERK signal blockage in a clinically relevant femoral osteolysis model. We will examine the effect of pharmacologic inhibition of ERK signaling on UHMWPE or LPS-induced osteolysis in rat femora by quantitative microCT and biomechanical pullout testing. Specific Aim 3. To examine how ERK 1/2 signaling blockage affects interactions between osteoblasts and osteoclasts in the context of biomaterials and implant contaminants such as bacterial toxin. We will examine osteoclastogenesis by co-culturing ERKdef mice cells and macrophages. We will measure osteoclastogenesis, cytokine expression and bone resorption in the presence and absence of ERK signaling blockage. Specific Aim 4. To determine the specific mechanism by which ERK and NF:B P50 co-regulate M-CSF gene induction in osteoblasts in response to particulate biomaterials and LPS. While the ERK/ATF4/RANKL pathway is well established, there is a knowledge gap in the transcriptional regulation of M-CSF. We will examine the cooperative regulation of MCSF gene induction by ERK and NFkb We expect to verify a novel concept of competent osteoblastic innate immunity and define the role of ERK signaling in inflammatory bone loss in response to biomaterials and related toxins. PUBLIC HEALTH RELEVANCE: The clinical rationale underlying this study is that we can prevent or treat clinically important inflammatory bone loss by targeting an ERK-mediated inflammatory pathway with the use of specific topical or systemic inhibitors.