Abstract
Although bone morphogenic protein (BMP) signaling promotes chondrogenesis, it is not clear whether BMP-induced chondrocyte maturation is cell-autonomously terminated. Loss of function of Smpd3 in mice results in an increase in mature hypertrophic chondrocytes. Here, we report that in chondrocytes the Runx2-dependent expression of Smpd3 was increased by BMP-2 stimulation. Neutral sphingomyelinase 2 (nSMase2), encoded by the Smpd3 gene, was detected both in prehypertrophic and hypertrophic chondrocytes of mouse embryo bone cartilage. An siRNA for Smpd3, as well as the nSMase inhibitor GW4869, significantly enhanced BMP-2-induced differentiation and maturation of chondrocytes. Conversely, overexpression of Smpd3 or C2-ceramide, which mimics the function of nSMase2, inhibited chondrogenesis. Upon induction of Smpd3 siRNA or GW4869, phosphorylation of both Akt and S6 proteins was increased. The accelerated chondrogenesis induced by Smpd3 silencing was negated by application of the Akt inhibitor MK2206 or the mammalian target of rapamycin inhibitor rapamycin. Importantly, in mouse bone culture, GW4869 treatment significantly promoted BMP-2-induced hypertrophic maturation and calcification of chondrocytes, which subsequently was eliminated by C2-ceramide. Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes, probably as a result of decreased ceramide production. In addition, we found that expression of hyaluronan synthase 2 (Has2) was elevated by a loss of Smpd3, which was restored by MK2206. Indeed, expression of Has2 protein decreased in nSMase2-positive hypertrophic chondrocytes in the bones of mouse embryos. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has2 expression to control the rate of endochondral ossification as a negative feedback mechanism.
Highlights
It is not clear how bone morphogenic protein (BMP)-induced chondrocyte maturation is cell-autonomously terminated
We focused on the sphingomyelin phosphodiesterase 3 (Smpd3) gene, because it could be up-regulated by BMP-2 in C2C12 myoblasts of mesenchymal origin [39]; loss-of-function models for Smpd3 in mice showed an increased number of hypertrophic chondrocytes or retarded transition of proliferative chondrocytes into hypertrophic chondrocytes
To investigate the possible cellautonomous roles of Smpd3 in chondrocyte maturation, we mainly employed the clonal chondrogenic mouse cell line ATDC5 because it is an excellent in vitro model for skeletal development, which can be stimulated by BMP-2 [43, 44]
Summary
It is not clear how BMP-induced chondrocyte maturation is cell-autonomously terminated. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has expression to control the rate of endochondral ossification as a negative feedback mechanism. Over 95% of bone formation during the embryonic and developmental stages is achieved through endochondral ossification This process is primed by the condensation of mesenchymal progenitor cells expressing the chondrogenic master regulator Sox9 [1], after which cells further differentiate into proliferating chondrocytes that are able to express a specific marker, Col2a1, encoding type II collagen [2]. We report that Smpd is continuously up-regulated by BMP-2 stimulation of chondrocytes during the maturation stage to suppress the late differentiation step via the Akt pathway We found that this induction of Smpd by BMP-2 was Runx2-dependent and that the transcription factor is crucial for the hypertrophic maturation of chondrocytes. Application of an inhibitor compound for nSMase into the mouse bone organ culture system significantly increased the hypertrophic conversion and extracellular matrix calcification of cartilage
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