Nemours Biomedical Research, Nemours/Alfred I. duPont Hospital for Children, 1701 Rockland Rd, Wilmington, DE, 19803, USA.
Rheumatology, Johns Hopkins All Children's Hospital, St. Petersburg, FL, USA.
Pediatr Rheumatol Online J. 2021 May 12;19(1):72. doi: 10.1186/s12969-021-00556-8.
We examined influences of conditioned media from chondrocytes (Ch) on juvenile idiopathic arthritis synovial fibroblasts (JFLS) and potential for JFLS to undergo endochondral bone formation (EBF).
Primary cells from three control fibroblast-like synoviocytes (CFLS) and three JFLS were cultured in Ch-conditioned media and compared with untreated fibroblast-like synoviocytes (FLS). RNA was analyzed by ClariomS microarray. FLS cells cultured in conditioned media were exposed to either TGFBR1 inhibitor LY3200882 or exogenous BMP4 and compared with FLS cultured in conditioned media from Ch (JFLS-Ch). Media supernatants were analyzed by ELISA.
In culture, JFLS downregulate BMP2 and its receptor BMPR1a while upregulating BMP antagonists (NOG and CHRD) and express genes (MMP9, PCNA, MMP12) and proteins (COL2, COLX, COMP) associated with chondrocytes. Important TGFβ superfamily member gene expression (TGFBI, MMP9, COL1A1, SOX6, and MMP2) is downregulated when JFLS are cultured in Ch-conditioned media. COL2, COLX and COMP protein expression decreases in JFLS-Ch. BMP antagonist protein (NOG, CHRD, GREM, and FST) secretion is significantly increased in JFLS-Ch. Protein phosphorylation increases in JFLS-Ch exposed to exogenous BMP4, and chondrocyte-like phenotype is restored in BMP4 presence, evidenced by increased secretion of COL2 and COLX. Inhibition of TGFBR1 in JFLS-Ch results in overexpression of COL2.
JFLS are chondrocyte-like, and Ch-conditioned media can abrogate this phenotype. The addition of exogenous BMP4 causes JFLS-Ch to restore this chondrocyte-like phenotype, suggesting that JFLS create a microenvironment favorable for endochondral bone formation, thereby contributing to joint growth disturbances in juvenile idiopathic arthritis.
我们研究了软骨细胞（Ch）条件培养基对幼年特发性关节炎滑膜成纤维细胞（JFLS）的影响，以及 JFLS 发生软骨内骨形成（EBF）的潜力。
从三个对照成纤维细胞样滑膜细胞（CFLS）和三个 JFLS 中分离原代细胞，在 Ch 条件培养基中培养，并与未经处理的成纤维细胞样滑膜细胞（FLS）进行比较。通过 ClariomS 微阵列分析 RNA。将在条件培养基中培养的 FLS 细胞暴露于 TGFBR1 抑制剂 LY3200882 或外源性 BMP4，并与在 Ch 条件培养基中培养的 FLS（JFLS-Ch）进行比较。通过 ELISA 分析培养基上清液。
在培养过程中，JFLS 下调 BMP2 及其受体 BMPR1a，而上调 BMP 拮抗剂（NOG 和 CHRD），并表达与软骨细胞相关的基因（MMP9、PCNA、MMP12）和蛋白（COL2、COLX、COMP）。当 JFLS 在 Ch 条件培养基中培养时，重要的 TGFβ 超家族成员基因表达（TGFBI、MMP9、COL1A1、SOX6 和 MMP2）下调。COL2、COLX 和 COMP 蛋白表达在 JFLS-Ch 中减少。JFLS-Ch 中 BMP 拮抗剂蛋白（NOG、CHRD、GREM 和 FST）分泌显著增加。在存在外源性 BMP4 的情况下，JFLS-Ch 中的蛋白磷酸化增加，并且恢复了软骨细胞样表型，表现为 COL2 和 COLX 的分泌增加。在 JFLS-Ch 中抑制 TGFBR1 导致 COL2 的过度表达。
JFLS 具有软骨细胞样特征，而 Ch 条件培养基可以消除这种表型。添加外源性 BMP4 可使 JFLS-Ch 恢复这种软骨细胞样表型，表明 JFLS 创造了有利于软骨内骨形成的微环境，从而导致幼年特发性关节炎关节生长障碍。
