Department of Pediatrics, Division of Pediatric Endocrinology and Metabolism, Rhode Island Hospital and Brown University, 593 Eddy Street, Providence, RI 02903, USA.
Am J Physiol Endocrinol Metab. 2010 Aug;299(2):E325-34. doi: 10.1152/ajpendo.00145.2010. Epub 2010 Jun 8.
The ATDC5 cell line exhibits a multistep process of chondrogenic differentiation analogous to that observed during endochondral bone formation. Previous investigators have induced ATDC5 cells to differentiate by exposing them to insulin at high concentrations. We have observed spontaneous differentiation of ATDC5 cells maintained in ascorbic acid-containing alpha-MEM. A comparison of the differentiation events in response to high-dose insulin vs. ascorbic acid showed similar expression patterns of key genes, including collagen II, Runx2, Sox9, Indian hedgehog, and collagen X. We took advantage of the action of ascorbic acid to examine signaling events associated with differentiation. In contrast to high-dose insulin, which downregulates both IGF-I and insulin receptors, there were only minimal changes in the abundance of these receptors during ascorbic acid-induced differentiation. Furthermore, ascorbic acid exposure was associated with ERK activation, and ERK inhibition attenuated ascorbic acid-induced differentiation. This was in contrast to the inhibitory effect of ERK activation during IGF-I-induced differentiation. Inhibition of collagen formation with a proline analog markedly attenuated the differentiating effect of ascorbic acid on ATDC5 cells. When plates were conditioned with ATDC5 cells exposed to ascorbic acid, ATDC5 cells were able to differentiate in the absence of ascorbic acid. Our results indicate that matrix formation early in the differentiation process is essential for ascorbic acid-induced ATDC5 differentiation. We conclude that ascorbic acid can promote the differentiation of ATDC5 cells by promoting the formation of collagenous matrix and that matrix formation mediates activation of the ERK signaling pathway, which promotes the differentiation program.
ATDC5 细胞系表现出类似于骺软骨内骨形成过程的多步骤软骨分化过程。先前的研究人员通过将 ATDC5 细胞暴露在高浓度的胰岛素中诱导其分化。我们观察到在含有抗坏血酸的 α-MEM 中维持的 ATDC5 细胞的自发分化。高剂量胰岛素与抗坏血酸诱导的分化相比，关键基因的表达模式相似，包括胶原 II、Runx2、Sox9、Indian hedgehog 和胶原 X。我们利用抗坏血酸的作用来研究与分化相关的信号事件。与下调 IGF-I 和胰岛素受体的高剂量胰岛素相反，在抗坏血酸诱导的分化过程中，这些受体的丰度只有很小的变化。此外，抗坏血酸暴露与 ERK 激活相关，ERK 抑制减弱了抗坏血酸诱导的分化。这与 IGF-I 诱导的分化过程中 ERK 激活的抑制作用形成对比。用脯氨酸类似物抑制胶原形成显著减弱了抗坏血酸对 ATDC5 细胞的分化作用。当用暴露于抗坏血酸的 ATDC5 细胞处理平板时，即使没有抗坏血酸，ATDC5 细胞也能够分化。我们的结果表明，在分化过程的早期，基质形成对于抗坏血酸诱导的 ATDC5 分化是必不可少的。我们得出结论，抗坏血酸可以通过促进胶原基质的形成来促进 ATDC5 细胞的分化，并且基质形成介导 ERK 信号通路的激活，从而促进分化程序。
