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

摘要 人关节软骨中的修复能力通常被认为是低的或可忽略的，并且这 内在能力随着年龄的增长而下降。因此，关节软骨损伤往往导致不可逆的损害 导致骨关节炎（OA）。我们和其他人最近定义了关节软骨细胞的异质性， 分子和细胞水平。在小鼠和其他哺乳动物中的研究表明， 关节软骨浅层作为再生能力的来源;迄今为止，这些发现还没有 已经扩展到人类个体发育中的特定软骨细胞群体。我们之前的研究表明 与成人软骨细胞不同，胎儿软骨细胞具有高度增殖性和迁移性， 磷酸化信号转导子和转录激活子3（pSTAT 3）的水平。我们的初步数据还 将表达整合素α4（ITGA 4）和骨形态发生蛋白受体（BMPR 1B）的细胞提名为最多 未成熟软骨细胞在整个人类发育过程中的作用。此外，我们还表明， 在分子水平上，ITGA 4 + BMPR 1B+细胞富含活性STAT 3（pSTAT 3），已知其 促进增殖、抑制并保持分化潜能。重要的是，成年ITGA 4 + BMPR 1B+细胞是 定位于浅表层，也表达最高水平的SOX 9，这是强烈确定与 骨软骨细胞祖细胞的身份和无骨软骨细胞;事实上，ITGA 4 + BMPR 1B+细胞是强健的软骨细胞， 体外成骨。ITGA 4 + BMPR 1B+细胞的百分比和pSTAT 3水平与 生物年龄，从发育中关节的20-30%下降到老年健康软骨的1-2%。我们 假设活性STAT 3在未成熟的关节软骨细胞中表达，是一种允许因子， 所需的未成熟细胞的贴壁和分化，以响应特定的指导信号在小生境。 我们建议在不同的条件下确定人关节软骨细胞中STAT 3的直接转录靶点。 个体发育阶段和在类似于由IL-6家族细胞因子驱动的促炎状态的条件下， OA。为了解决IL-6家族细胞因子如何在一定背景下驱动不同的生物学和功能结果， 具体的方式，我们将采用纳米蛋白质组学和靶向诱变，以确定如何具体后， 胎儿与成人软骨细胞中核心IL-6家族细胞因子受体gp 130的翻译修饰不同 用IL-6家族细胞因子刺激。最后，我们将应用单细胞RNA-Seq来进一步完善分子水平。 和未成熟关节软骨细胞的细胞表型。同时，我们将评估分子和 STAT 3在关节软骨细胞中获得和丧失功能的功能后果。我们认为细胞 具有较高水平的pSTAT 3将证明体内更广泛的分化潜力， 在染色质构象中由STAT 3介导。这项高度创新的研究的总体影响是定义 细胞和分子表型的未成熟关节软骨细胞在整个人类个体发育和连接 这对老化过程中软骨修复和/或再生的潜力有影响。

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.