Iwamoto M, Golden E B, Adams S L, Noji S, Pacifici M
Department of Anatomy-Histology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6003.
Exp Cell Res. 1993 Apr;205(2):213-24. doi: 10.1006/excr.1993.1079.
We previously showed that retinoic acid (RA) participates in the regulation of chondrocyte maturation during endochondral ossification, a process involving multiple developmental stages. To assess whether the responsiveness to RA treatment changes during chondrocyte maturation, immature chondrocytes were isolated from the caudal portion of Day 18-19 chick embryo sterna, a portion that remains cartilaginous through early postnatal life but ossifies with age. The immature cells were allowed to reach different stages of maturation by growth for different time in culture. Progression by the cells toward the mature phenotype during culture was confirmed by increases in average cell diameter, proteoglycan synthesis, and alkaline phosphatase (APase) activity. When developmentally immature passage 0 (PO) cultures were treated with RA (10-100 nM) for 72 h, the cells readily became fibroblastic, reduced drastically their proteoglycan synthesis, and failed to activate type X collagen gene expression. When older cultures (P1 and P2) were treated with RA, the cells acquired a characteristic epithelioid shape and increased their APase activity. Moreover, 5-10% of P1 cells and 20-25% of P2 cells activated type X collagen synthesis in response to RA. RA treatment markedly induced expression of the gene encoding the beta isoform of retinoic acid receptor (RAR beta) and also provoked a moderate 2.5-fold increase in RAR alpha gene expression. A similar change in responsiveness to RA was observed during maturation in vivo. Chondrocytes were isolated from the cephalic portion of Day 10, 11, 13, and 16 chick embryo sterna, and were treated with different doses of RA (10-100 nM) for 72 h. The cells from the Day 10 sternum failed to activate type X collagen gene expression in response to RA. In contrast, with increasing age of the embryos, an increasing fraction of cells induced type X collagen gene expression in response to RA. We conclude that responsiveness to RA changes during the early stages of chondrocyte maturation and that maturation depends on interactions between exogenous retinoids and the endogenous developmental program of chondrocytes.
我们之前的研究表明，视黄酸（RA）参与软骨内骨化过程中软骨细胞成熟的调控，这一过程涉及多个发育阶段。为了评估软骨细胞成熟过程中对RA处理的反应性是否发生变化，从未成熟的软骨细胞取自第18 - 19天鸡胚胸骨的尾部，该部位在出生后早期仍为软骨状态，但会随着年龄增长而骨化。将未成熟细胞在培养中生长不同时间，使其达到不同的成熟阶段。通过平均细胞直径、蛋白聚糖合成和碱性磷酸酶（APase）活性的增加，证实了细胞在培养过程中向成熟表型的进展。当处于发育未成熟的第0代（P0）培养物用RA（10 - 100 nM）处理72小时时，细胞很容易变成成纤维细胞样，其蛋白聚糖合成急剧减少，并且未能激活X型胶原基因表达。当用RA处理较老的培养物（P1和P2）时，细胞获得了特征性的上皮样形态并增加了其APase活性。此外，5 - 10%的P1细胞和20 - 25%的P2细胞在RA作用下激活了X型胶原合成。RA处理显著诱导了视黄酸受体β亚型（RARβ）编码基因的表达，并且还使RARα基因表达适度增加了2.5倍。在体内成熟过程中也观察到了对RA反应性的类似变化。从第10、11、13和16天鸡胚胸骨的头部分离软骨细胞，并用不同剂量的RA（10 - 100 nM）处理72小时。来自第10天胸骨的细胞在RA作用下未能激活X型胶原基因表达。相反，随着胚胎年龄的增加，响应RA而诱导X型胶原基因表达的细胞比例增加。我们得出结论，在软骨细胞成熟的早期阶段，对RA的反应性会发生变化，并且成熟取决于外源性类视黄醇与软骨细胞内源性发育程序之间的相互作用。
