Highlights

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Free cholesterol is constant during differentiation of dental follilce cells.
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A reduction of free cholesterol impairs alkaline phosphatase (ALP) activity.
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Induced protein kinase C (PKC) activity after inhibition of cholesterol synthesis.
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PKC inhibitor restores ALP activity after inhibition of cholesterol synthesis.

Abstract
During the osteogenic differentiation of dental follicle cells (DFCs) the concentration of free cholesterol remains constant during differentiation. In less agreement with this result, several studies have shown that inhibition of cholesterol synthesis promotes differentiation of osteogenic progenitor cells. This study therefore investigated the effect of cholesterol synthesis inhibition on the osteogenic differentiation of DFCs. The induction of osteogenic differentiation showed a significant increase in free cholesterol concentration. Inhibition of cholesterol synthesis decreased both free cholesterol concentration and the alkaline phosphatase activity, a marker of osteogenic differentiation. However, a lower concentration of the inhibitor, which does not affect the concentration of free cholesterol, did not inhibit the activity of alkaline phosphatase (ALP). Previous studies have shown that the differentiation of DFCs is controlled by classical protein kinases C (PKC) and the induction of protein kinase B (AKT). In correlation with the reduced concentration of free cholesterol, AKT was also downregulated and the activity of classical PKCs was upregulated. Although inhibition of AKT was shown to downregulate the ALP activity, induction of AKT could not compensate for the reduced differentiation following inhibition of cholesterol. However, inhibition of PKC induced ALP-activity and suppressed the inhibitory effect of low free cholesterol concentration on the ALP activity. In conclusion, our study suggests that a reduced concentration of free cholesterol has an inhibitory effect on osteogenic differentiation through activation of PKC.