FoxO transcription factors in joint aging and osteoarthritis pathogenesis

摘要 骨关节炎（OA）是最常见的关节疾病。尽管衰老是人类最重要的 OA的危险因素，导致与年龄相关的软骨退变的机制仍有待确定。 我们报道了细胞内稳态机制，如自噬和氧化防御， 在老化和OA影响的软骨中受损。在我们最近的研究中，我们研究了FoxO转录 因子，其调节自噬蛋白的表达和自噬激活。特别是FoxO 3a， 被称为细胞衰老的分子守门人。我们观察到FoxO mRNA和蛋白的减少， 在人类和小鼠的衰老和OA影响的软骨中表达。 这些发现支持了这样的假设，即衰老相关的FoxO表达减少损害了保护作用。 细胞内稳态机制，损害软骨细胞存活和生物合成能力，并导致 加速关节老化和引发OA发病机制。这一假设将在以下目标中得到检验。 目的1：FoxO在软骨细胞中的表达和功能调控。我们将检查细胞外 调节因子和FoxO抑制或激活的机制，并确定负责任的顺式元件， FoxO发起人。在正常人和OA人中使用FoxO敲低和过表达的体外模型 从FoxO缺陷小鼠的软骨细胞和软骨外植体中，我们将研究FoxO在调节 软骨细胞功能 目的2：软骨中FoxO信号网络。FoxO靶基因是组织和环境特异性的。我们将 利用RNA-seq、ChIP-seq和 ChIP-seq用于组蛋白标记。对这些数据集的综合分析将确定途径和信号传导 由FoxO调节的机制。 目的3：FoxO在软骨稳态、衰老和实验性OA中的作用。我们的初步研究显示 条件性软骨特异性缺失FoxO 1导致出生后软骨生长异常， 6个月时自发OA样降解。我们将产生出生后的三重和单敲除小鼠 使用Acan-CreER，并测试它们的老化相关变化和实验性OA的严重程度。 目的4：FoxO过表达和激活的保护作用 我们将产生在软骨中过表达活性形式FoxO 1或FoxO 3的小鼠，以平衡OA。 相关的抑制，并确定结果方面的稳态机制和OA的严重程度， 衰老和手术模型。 该项目将确定衰老相关的FoxO表达减少是OA的早期和关键事件 发病机制这将为旨在调节FoxO的治疗方法提供基础。 表达和/或活性，以预防与年龄相关的和损伤诱导的OA的发作和进展。 !

ABSTRACT Osteoarthritis (OA) is the most prevalent joint disease. Although aging represents one of the most important risk factors for OA, mechanisms leading to the aging-related cartilage degeneration remain to be determined. We reported that cellular homeostasis mechanisms such as autophagy and oxidant defenses are compromised in aging and OA-affected cartilage. In our recent studies we investigated FoxO transcription factors, which regulate expression of autophagy proteins and autophagy activation. FoxO3a in particular is known as molecular gatekeeper of cellular aging. We observed a reduction in FoxO mRNA and protein expression in aging and OA-affected cartilage in humans and mice. These findings support the hypothesis that ‘Aging-related reduction of FoxO expression impairs protective cellular homeostasis mechanisms, compromises chondrocyte survival and biosynthetic capacity and leads to accelerated joint aging and initiation of OA pathogenesis’. This hypothesis will be tested in the following aims. Aim 1: Regulation of FoxO expression and function in chondrocytes. We will examine the extracellular regulators and mechanisms of FoxO suppression or activation and identify the responsible cis-elements in the FoxO promoters. Using in vitro models of FoxO knock down and overexpression in normal and OA human chondrocytes and cartilage explants from FoxO deficient mice we will examine the role of FoxO in regulating chondrocyte functions. Aim 2: The FoxO signaling network in cartilage. FoxO target genes are tissue and context specific. We will establish FoxO target genes in mouse cartilage and human chondrocytes by using RNA-seq, ChIP-seq and ChIP-seq for histone marks. Integrative analysis of these datasets will identify pathways and signaling mechanisms that are regulated by FoxO. Aim 3: Role of FoxO in cartilage homeostasis, aging and experimental OA. Our preliminary studies show that conditional cartilage specific deletion of FoxO1 leads to abnormal cartilage growth postnatally and spontaneous OA-like degradation by 6 months. We will generate postnatal triple and single knock out mice using Acan-CreER and test them for aging related changes and severity of experimental OA. Aim 4: Protective effects of FoxO overexpression and activation We will generate mice that overexpress active forms of FoxO1 or FoxO3 in cartilage to balance the OA associated suppression and determine outcomes with respect to homeostasis mechanisms and OA severity in the aging and surgical models. This project will establish that aging-related reduction in FoxO expression is an early and critical event in OA pathogenesis. This will provide the foundation for therapeutic approaches aimed at modulating FoxO expression and/or activity to prevent age-related and injury-induced onset and progression of OA. !