No abstract

The mammalian Target Of Rapamycin (mTOR) is a nutrient-sensing protein kinase that regulates numerous cellular processes. Fetal rat metatarsal explants were used as a physiological model to study the effect of mTOR inhibition on chondrogenesis. Insulin significantly enhanced their growth. Rapamycin significantly diminished this response to insulin through a selective effect on the hypertrophic zone. Cell proliferation (bromodeoxyuridine incorporation) was unaffected by rapamycin. Similar observations were made when rapamycin was injected to embryonic day (E) 19 fetal rats in situ. In the ATDC5 chondrogenic cell line, rapamycin inhibited proteoglycan accumulation and collagen X expression. Rapamycin decreased content of Indian Hedgehog (Ihh), a regulator of chondrocyte differentiation. Addition of Ihh to culture medium reversed the effect of rapamycin. We conclude that modulation of mTOR signaling contributes to chondrocyte differentiation, perhaps through its ability to regulate Ihh. Our findings support the hypothesis that nutrients, acting through mTOR, directly influence chondrocyte differentiation and long bone growth. Developmental Dynamics 237:702-712, 2008.

Insulin-like growth factor-I (IGF-I) is a critical regulator of skeletal growth. While IGF-I has been shown to be a potent chondrocyte mitogen in vitro, its role in chondrocyte differentiation is less well characterized. We chose to study the action of IGF-I on an accepted model of chondrocyte differentiation, the ATDC5 cell line. Insulin concentrations sufficiently high to interact with the IGF-I receptor are routinely used to induce ATDC5 cells to differentiate. Therefore, we first examined the ability of IGF-I to promote chondrocyte differentiation at physiological concentrations. IGF-I could induce differentiation of these cells at concentrations below 10 nM. However, increasing IGF-I concentrations were less potent at inducing differentiation. We hypothesized that mitogenic effects of IGF-I might inhibit its differentiating effects. Indeed, the extracellular-signal-regulated kinase (ERK)-pathway inhibitor PD98059 inhibited ATDC5 cell DNA synthesis while enhancing differentiation. This suggested that the ability of IGF-I to promote both proliferation and differentiation might require that its signaling be modulated through the differentiation process. We therefore compared IGF-I-mediated ERK activation in proliferating and hypertrophic chondrocytes. IGF-I potently induced ERK activation in proliferating cells, but minimal ERK response was seen in hypertrophic cells. In contrast, IGF-I-mediated Akt activation was unchanged by differentiation, indicating intact upstream IGF-I receptor signaling. Similar findings were observed in the RCJ3·1C5·18 chondrogenic cell line and in primary chick chondrocytes. We conclude that IGF-I promotes both proliferation and differentiation of chondrocytes and that the differentiation effects of IGF-I may require uncoupling of signaling to the ERK pathway.

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.