P450 oxidoreductase expressed in rat chondrocytes modulates chondrogenesis via cholesterol- and Indian Hedgehog-dependent mechanisms.

Cytochrome P450 oxidoreductase (POR) is the electron donor for microsomal cytochrome P450 enzymes and other non-P450 enzymes. Targeted deletion of POR expression in mice leads to a variety of embryonic defects, including bone abnormalities. In addition, POR mutations in humans are associated with impaired steroidogenesis and skeletal malformations. Yet, little is known on the mechanisms underlying the skeletal abnormalities secondary to impaired POR activity. In our study, rat chondrocytes transfected with POR-specific short interfering RNAs exhibited decreased cell proliferation and differentiation and induced apoptosis. In addition, the reduced expression of POR in chondrocytes caused decreased intracellular cholesterol content. The addition of cholesterol in the culture medium prevented the POR small interfering RNA (siRNA)-mediated effects on chondrocyte proliferation, differentiation, and apoptosis. Because cholesterol is required for normal activity of the hedgehog proteins, we evaluated the effects of POR siRNAs on the expression of Indian hedgehog (Ihh), an important regulator of chondrogenesis. POR siRNA-transfected chondrocytes exhibited reduced Ihh expression, with such effect being neutralized by cholesterol. Lastly, recombinant human/mouse Ihh prevented the POR siRNA-mediated effects on chondrocyte proliferation, differentiation, and apoptosis. Our findings suggest that the bone malformations associated with defective POR activity are due to reduced cholesterol synthesis and, in turn, reduced Ihh expression in chondrocytes.