Hagizawa Hiroki, Koyamatsu Saeko, Okada Seiji, Kaito Takashi, Tsumaki Noriyuki
Department of Tissue Biochemistry, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan.
Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan.
Front Cell Dev Biol. 2023 May 15;11:1151947. doi: 10.3389/fcell.2023.1151947. eCollection 2023.
The nucleus pulposus (NP) comprises notochordal NP cells (NCs) and chondrocyte-like NP cells (CLCs). Although morphological similarities between CLCs and chondrocytes have been reported, interactions between CLCs and NCs remain unclear. In this study, we aimed to clarify regulatory mechanisms of cells in the NP and chondrocytes. We performed single-cell RNA sequencing (scRNA-seq) analysis of the articular cartilage (AC) and NP of three-year-old cynomolgus monkeys in which NCs were present. We then performed immunohistochemical analysis of NP and distal femur. We added sonic hedgehog (SHH) to primary chondrocyte culture. The scRNA-seq analysis revealed that CLCs and some articular chondrocytes had similar gene expression profiles, particularly related to GLI1, the nuclear mediator of the hedgehog pathway. In the NP, cell-cell interaction analysis revealed SHH expression in NCs, resulting in hedgehog signaling to CLCs. In contrast, no hedgehog ligands were expressed by chondrocytes in AC samples. Immunohistochemical analysis of the distal end of femur indicated that SHH and Indian hedgehog (IHH) were expressed around the subchondral bone that was excluded from our scRNA-seq sample. scRNA-seq data analysis and treatment of primary chondrocytes with SHH revealed that hedgehog proteins mediated an increase in hypoxia-inducible factor 1-alpha (HIF-1α) levels. CLCs and some articular chondrocytes have similar transcriptional profiles, regulated by paracrine hedgehog proteins secreted from NCs in the NP and from the subchondral bone in the AC to promote the HIF-1α pathway.
髓核（NP）由脊索样髓核细胞（NCs）和软骨样髓核细胞（CLCs）组成。尽管已有报道称CLCs与软骨细胞在形态上有相似之处，但CLCs与NCs之间的相互作用仍不清楚。在本研究中，我们旨在阐明NP细胞和软骨细胞的调控机制。我们对存在NCs的三岁食蟹猴的关节软骨（AC）和NP进行了单细胞RNA测序（scRNA-seq）分析。然后我们对NP和股骨远端进行了免疫组织化学分析。我们在原代软骨细胞培养中添加了音猬因子（SHH）。scRNA-seq分析显示，CLCs和一些关节软骨细胞具有相似的基因表达谱，特别是与刺猬信号通路的核介质GLI1相关。在NP中，细胞间相互作用分析显示NCs中有SHH表达，从而导致向CLCs发出刺猬信号。相比之下，AC样本中的软骨细胞未表达刺猬配体。股骨远端免疫组织化学分析表明，SHH和印度刺猬因子（IHH）在我们scRNA-seq样本未包含的软骨下骨周围表达。scRNA-seq数据分析以及用SHH处理原代软骨细胞显示，刺猬蛋白介导缺氧诱导因子1-α（HIF-1α）水平升高。CLCs和一些关节软骨细胞具有相似的转录谱，受NP中的NCs和AC中的软骨下骨分泌的旁分泌刺猬蛋白调节，以促进HIF-1α通路。
