Mechanobiological Mechanism for Inflammaory Bone Loss

描述（由申请人提供）：目前的治疗剂如双膦酸盐或合成代谢剂并不总是有效地预防或治疗骨质疏松症和类风湿性关节炎、牙周炎和植入物松动中的炎性骨丢失。因此，开发有效的治疗方法来保存骨存在障碍。在为期4年的母基金资助期间（2007-2011），我们在炎症性骨质溶解背景下描绘分子通路（如NFATc 1和pERK 1/2）方面取得了重大进展。为了将我们的发现转化为临床前竞技场，我们筛选了几种候选药物并将PTH（1-34）鉴定为新型抗炎剂。现在，我们正在为我们的A1竞争性更新提案设定一个新的方向，该提案旨在揭示PTH（1-34）在炎性破骨细胞生成和骨质溶解背景下的抗炎功能。LPS与骨感染和植入物相关的骨丢失有关。LPS已被普遍用于建立一个新的治疗概念。当局部递送PTH（1-34）时，我们观察到PTH（1-34）令人惊讶地抑制体内LPS诱导的破骨细胞生成。随后进行了实验来研究这一现象。我们观察到PTH（1-34）不通过破骨细胞前体抑制RANKL，但在成骨细胞中抑制促破骨细胞生成细胞因子如MCSF。LPS诱导的ERK 1/2磷酸化，一种常见的炎性骨质溶解信号转导，也通过LPS治疗抑制。我们已经优化了PTH（1-34）剂量和递送方法用于所提出的实验。因此，我们提出了一个治疗创新的假设，即局部给予低剂量PTH（1- 34）通过抑制成骨谱系细胞中破骨细胞因子的产生来抑制炎性骨丢失。我们试图通过两个平行的目的来研究这一假设，并最终建立PTH（1-34）的新的抗破骨细胞生成功能。在具体目标1中，我们将在体内骨质溶解的背景下建立PTH（1-34）的新型抗炎作用。我们将 确定水凝胶中局部应用的低剂量PTH（1-34）是否可预防响应临床相关刺激（如RANKL、LPS和髋关节模拟器产生的CoCr磨损颗粒）的炎性骨质溶解。破骨细胞生成将通过组织蛋白酶K光信号和破骨细胞数量计数来测量。动态骨组织形态计量学将使我们能够检查骨转换。在具体目标2中，我们将定义PTH（1-34）在成骨细胞中表现出抗炎作用的机制。我们将研究pERK/细胞因子表达和两个不同的PTH信号通路（Gs/腺苷酸环化酶/cAMP和Gq/11-磷脂酶C）之间的功能相互作用。我们将使用通路特异性类似物、我们的体内骨质溶解和体外巨噬细胞-骨祖细胞共培养模型进一步描述每个通路的功能重要性。总之，我们将提供新的治疗和机制的见解，具体的抗炎功能的水凝胶为基础的低剂量PTH的交付在炎症性骨质溶解的设置药物开发。

DESCRIPTION (provided by applicant): Current therapeutics such as bisphosphonates or anabolic agents do not always effectively prevent or treat osteoporosis and inflammatory bone loss in rheumatoid arthritis, periodontitis, and implant loosening. Therefore, there is a barrier t developing effective therapeutics to preserve bone. During the 4-year parent grant award period (2007-2011), we made significant progress in delineating molecular pathways such as NFATc1 and pERK1/2 in the context of inflammatory osteolysis. In order to translate our findings into a preclinical arena, we screened several drug candidates and identified PTH(1-34) as a novel anti-inflammatory agent. Now, we are setting a new direction for our A1 competitive renewal proposal that seeks to unravel, an as yet unknown, anti-inflammatory function of PTH(1-34) in the context of inflammatory osteoclastogenesis and osteolysis. LPS has been implicated in bone infection and implant-related bone loss. LPS has been commonly used to establish a new therapeutic concept. When PTH(1-34) was delivered topically, we observed that PTH(1-34) surprisingly inhibited LPS-induced osteoclastogenesis in vivo. Subsequent experiments were conducted to investigate this phenomenon. We observed that PTH(1-34) was not inhibiting RANKL through osteoclast precursors, but was inhibiting pro-osteoclastogenic cytokines like MCSF in osteoblast cells. LPS induced phosphorylation of ERK1/2, a common inflammatory osteolysis signal transducer, was also inhibited via LPS treatment. We have optimized PTH(1-34) doses and delivery methods for the proposed experiments. Therefore, we developed a therapeutically innovative hypothesis that regionally administered low-dose PTH(1- 34) inhibits inflammatory bone loss by suppressing osteoclastogenic cytokine production in osteogenic lineage cells. We seek to investigate this hypothesis through two parallel Aims and ultimately, establish a novel anti- osteoclastogenic function of PTH(1-34). In Specific Aim 1, we will establish a novel anti-inflammatory role of PTH(1-34) in the context of osteolysis in vivo. We will determine whether regionally applied low-dose PTH(1-34) in a hydrogel prevents inflammatory osteolysis in response to clinically relevant stimuli such as RANKL, LPS and hip joint simulator generated CoCr wear particles. Osteoclastogenesis will be measured by cathepsin K optical signals and by counting osteoclast numbers. Dynamic bone histomorphometry will enable us to examine bone turnover. In Specific Aim 2, we will define the mechanism by which PTH(1-34) exhibits an anti- inflammatory effect in osteoblasts. We will examine the functional interactions between pERK/cytokine expression and two diverging PTH signaling pathways (Gs/adenylate cyclase/cAMP and Gq/11-phospholipase C). We will further delineate the functional importance of each pathway using pathway-specific analogs, our in vivo osteolysis and in vitro macrophage-osteoprogenitor co-culture models. In summary, we will provide novel therapeutic and mechanistic insights into specific anti-inflammatory function of hydrogel-based delivery of low- dose PTH in the setting of inflammatory osteolysis for drug development.