Dissecting molecular mechanisms implicated in age- and osteoarthritis-related decline in anabolism in articular cartilage

摘要 人类关节软骨的修复能力通常被认为很低或可以忽略不计，而这 内在能力随着年龄的增长而降低。因此，关节软骨损伤往往会导致不可逆转的损伤。 导致骨性关节炎(OA)。我们和其他人最近定义了关节软骨细胞的异质性 无论是分子水平还是细胞水平。在小鼠和其他哺乳动物身上的研究表明，细胞的一个子集 关节软骨表层是再生能力的来源；到目前为止，这些发现还没有 已经扩展到人类个体发育中特定群体的软骨细胞。我们之前的研究表明 与成人软骨细胞不同的是，胎儿软骨细胞具有高度的增殖和迁移能力，并表现出较高的碱性。 磷酸化信号转导和转录激活子3(PSTAT3)的水平。我们的初步数据也 最多表达整合素α4和骨形态发生蛋白受体的细胞 人关节软骨中的未成熟软骨细胞在整个人类发育过程中。此外，我们已经展示了 在分子水平上，ITGA4+BMPR1B+细胞富含活性STAT3(PSTAT3)，这是已知的 促进细胞增殖、合成代谢，保持分化潜能。重要的是，成年的ITGA4+BMPR1B+细胞 定位于表层，也表达最高水平的SOX9，这与 骨软骨前体细胞的鉴定和合成代谢；事实上，ITGA4+BMPR1B+细胞是强健的软骨细胞-和 体外成骨。ITGA4+BMPR1B+细胞百分率与pSTAT3水平密切相关 生物学年龄，从关节发育中的20%-30%下降到成年健康软骨的1%-2%。我们 假设活性STAT3在未成熟的关节软骨细胞中表达，是一种允许因子 未成熟细胞合成代谢和分化所必需的，以响应特定的指示信号在小生境。 我们建议在人关节软骨细胞中确定STAT3的直接转录靶点 在类似于IL-6家族细胞因子驱动的促炎状态下的个体发生阶段 骨关节炎。为了解决IL-6家族细胞因子如何在上下文中驱动不同的生物学和功能结果- 具体方式，我们将利用纳米蛋白质组学和定向突变来确定后 核心IL-6家族细胞因子受体gp130在胎儿和成人软骨细胞中的翻译修饰不同 受IL-6家族细胞因子刺激。最后，我们将应用单细胞RNA-Seq来进一步提纯分子 未成熟关节软骨细胞的细胞表型。同时，我们将评估分子和 关节软骨细胞中STAT3的获得和功能丧失的功能后果。我们建议细胞 随着pSTAT3水平的升高，将证明由于变化而在体内具有更广泛的分化潜力 染色质构象中由STAT3介导。这项高度创新的研究的总体影响是定义 未成熟关节软骨细胞在整个个体发育过程中的细胞和分子表型及其联系 这与老化过程中软骨修复和/或再生的潜力有关。

ABSTRACT The reparative capacity in human articular cartilage is generally considered to be low or negligible, and this intrinsic capacity decreases with age. As a result, articular cartilage injuries often result in irreversible damage leading to osteoarthritis (OA). We and others have recently defined heterogeneity in articular chondrocytes at both the molecular and cellular levels. Work in mice and other mammals has implicated a subset of cells in the superficial layer of articular cartilage as the source of regenerative capacity; to date, these findings have not been extended to a specific population of chondrocytes in human ontogeny. Our previous studies have shown that unlike adult chondrocytes, fetal chondrocytes are highly proliferative and migratory, and exhibit high basal levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Our preliminary data also nominate cells expressing integrin α4 (ITGA4) and bone morphogenetic protein receptor (BMPR1B) as the most immature chondrocytes in human articular cartilage throughout human development. Moreover, we have shown at the molecular level that ITGA4+BMPR1B+ cells are enriched for active STAT3 (pSTAT3), which are known to drive proliferation, anabolism and preserve differentiation potential. Importantly, adult ITGA4+BMPR1B+ cells are localized to the superficial layer and also express the highest levels of SOX9, which is strongly identified with osteochondral progenitor identity and anabolism; indeed, ITGA4+BMPR1B+ cells are robustly chondro- and osteogenic in vitro. The percentage of ITGA4+BMPR1B+ cells and levels of pSTAT3 tightly correlate with biological age, decreasing from 20-30% in developing joints down to 1-2% in aged adult healthy cartilage. We hypothesize that active STAT3 is expressed in immature articular chondrocytes and is a permissive factor required for immature cell anabolism and differentiation in response to specific instructive signals in the niche. We propose to define the direct transcriptional targets of STAT3 in human articular chondrocytes at different ontogenic stages and under conditions similar to the pro-inflammatory state driven by IL-6 family cytokines in OA. To address how IL-6 family cytokines can drive varied biological and functional outcomes in a context- specific manner, we will employ nanoproteomics and targeted mutagenesis to determine how specific post- translational modifications in the core IL-6 family cytokine receptor gp130 differ in fetal vs. adult chondrocytes stimulated with IL-6 family cytokines. Finally, we will apply single cell RNA-Seq to further refine the molecular and cellular phenotype of immature articular chondrocytes. In parallel, we will assess the molecular and functional consequences of STAT3 gain and loss of function in articular chondrocytes. We propose that cells with higher levels of pSTAT3 will evidence broader differentiation potential in vivo, resulting from changes mediated by STAT3 in chromatin conformation. The overall impact of this highly innovative study is to define the cellular and molecular phenotype of immature articular chondrocytes throughout human ontogeny and to link this to the potential for cartilage repair and/or regeneration during aging.