The Mechanism of Ascorbic Acid - Induced Differentiation of ATDC 5 Chondrogenic Cells 1

25 The ATDC5 cell line exhibits a multistep process of chondrogenic differentiation analogous to 26 that observed during endochondral bone formation. Previous investigators have induced 27 ATDC5 cells to differentiate by exposing them to insulin at high concentrations. We have 28 observed spontaneous differentiation of ATDC5 cells maintained in ascorbic acid-containing 29 αMEM. A comparison of the differentiation events in response to high dose insulin versus 30 ascorbic acid showed similar expression patterns of key genes, including collagen II, Runx2, 31 Sox9, Indian Hedgehog and collagen X. We took advantage of the action of ascorbic acid to 32 examine signaling events associated with differentiation. In contrast to high dose insulin, which 33 downregulates both IGF-1 and insulin receptors, there were only minimal changes in the 34 abundance of these receptors during ascorbic acid-induced differentiation. Furthermore, 35 ascorbic acid exposure was associated with Erk activation, and Erk inhibition attenuated 36 ascorbic acid-induced differentiation. This was in contrast to the inhibitory effect of Erk 37 activation during IGF-1-induced differentiation. Inhibition of collagen formation with a proline 38 analog markedly attenuated the differentiating effect of ascorbic acid on ATDC5 cells. When 39 plates were conditioned with ATDC5 cells exposed to ascorbic acid, ATDC5 cells were able to 40 differentiate in the absence of ascorbic acid. Our results indicate that matrix formation early in 41 the differentiation process is essential for ascorbic acid-induced ATDC5 differentiation. We 42 conclude that ascorbic acid can promote the differentiation of ATDC5 cells by promoting the 43 formation of collagenous matrix and that matrix formation mediates activation of the Erk 44 signaling pathway, which promotes the differentiation program. 45