C4ST-1 Overexpression Suppresses Osteoblast Differentiation via a Wnt/β-Catenin-p53 Axis

Disruption of osteoblast differentiation can lead to severe bone diseases, such as osteoporosis and osteosclerosis. Our previous study showed that a reduced 4S/6S ratio promotes osteoblast differentiation, linking this specific chondroitin sulfate (CS) modification to bone pathology. This study investigated the effect of forced elevation of the 4S/6S ratio on differentiation. C4ST-1 was found to exhibit overexpression, increasing the 4S/6S ratio, significantly suppressing osteoblast differentiation, as evidenced by reduced Akp2 gene expression and ALP activity. This inhibition was far more potent than that caused by the enzymatic removal of CS. Notably, treating C4ST-1-overexpressing cells with chondroitinase reduced differentiation inhibition to a level similar to that in mock cells treated with chondroitinase. These results suggest that the strong inhibition was due to the excessive 4-sulfated CS produced by C4ST-1, implying a mechanism distinct from the inhibition caused by a lack of CS. To elucidate this mechanism, we investigated the potential feedback loop. The increase in 4-sulfated CS from C4ST-1 overexpression enhanced Wnt3a expression, which upregulated p53 expression. Pharmacological inhibition of β-catenin and p53 partially restored Akp2 expression, confirming their roles as key mediators. We propose that a high 4S/6S ratio activates a Wnt/β-catenin-p53 axis, where p53 acts as a brake on differentiation. Our findings highlight the critical role of CS sulfation patterns in fine-tuning the osteoblast fate.