The mechanism of ascorbic acid-induced differentiation of ATDC5 chondrogenic cells

Abstract

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.

Chondrogenic ATDC5 cells: An optimised model for rapid and physiological matrix mineralisation

“…This reduces the proliferation phase of the cells and promotes their differentiation (6,16). Ascorbic acid has also been shown to promote the hypertrophic differentiation of cultured primary chick chondrocytes (17).…”

The Roles and Mechanisms of Actions of Vitamin C in Bone: New Developments

“…In terms of mechanisms by which AA induces chondrocyte differentiation, studies using ATDC5 chondrogenic cells have shown evidence for involvement of ERK signaling. AA treatment induced ERK activation, while ERK inhibition attenuated vitamin C-induced chondrocyte differentiation (63) . These data, taken together with the in vivo data, imply that AA is an important regulator of chondrocyte fate determination.…”

“…Vitamin C treatment upregulates sonic hedgehog (Shh) signaling by promoting expression of Gli1 and Ptc1, target genes of the Shh pathway in MC3T3-E1 osteoblasts (81) . Similarly, AA modulates MapK/ERK signaling as well as Indian hedgehog (Ihh), bone morphogenetic protein 2 (BMP-2), and SOX9 expression via induction of a collagen matrix (63) . The issue of whether the changes in expression or actions of these growth factors are due to direct or indirect effects of AA in bone cells remain to be examined.…”

The Increase in O-Linked N-Acetylglucosamine Protein Modification Stimulates Chondrogenic Differentiation Both in Vitro and in Vivo

“…Ascorbic acid (AA) has been described previously as a differentiation agent in ATDC5 cells (28). Therefore, we analyzed whether a chondrogenic stimuli, nondirectly related to glucose metabolism, such as AA, would also be able to modify the amount of O-GlcNAcylated proteins in ATDC5 cells in the absence of insulin.…”