The role of fetuin-a in endochondral ossification

The hepatic serum glycoprotein fetuin-A plays a role in mineralized matrix metabolism. It prevents unwanted ectopic calcification by stabilizing the amorphous phase of calcium phosphate and facilitating excess mineral clearance. Fetuin-A is one of the most highly abundant non-collagenous proteins in bone. As Ahsg-/- mice have shortened femoral bones, fetuin-A plays an important role in endochondral ossification. However, the underlying mechanisms are yet unknown. Earlier studies explained the shortened femora as a lack of TGF-β antagonization or as premature growth plate mineralization.In this study, I first hypothesized that fetuin-A regulates endochondral ossification through retinoic acid signaling, as retinoic acid intoxication leads to growth plate closure, especially in the femur. Using a postnatal bone culture system, the effect of fetuin-A and all-trans-retinoic acid (ATRA) on bone growth was investigated. Treatment of bones with ATRA alone led to growth plate cartilage degradation; a combination of ATRA and fetuin-A led to an increased degradation to the point of cartilage detachment. This indicated that fetuin-A might play a role in cartilage degradation, but it was not clear how, if at all, fetuin-A regulated retinoic acid signaling in cartilage.In histological sections from cultured bones, I discovered that bones from fetuin-A deficient mice had elongated hypertrophic zones in their growth plates. Histological sections of eight weeks old mice showed that growth plates of Ahsg-/- mice were grossly disordered and deformed. In order to gain three-dimensional information of growth plate morphology, bones were measured using μCT. Three-dimensional reconstructions of bones from eight weeks old mice revealed that the distal femora were displaced and deformed and the growth plates showed an angular deformation.Histological examination of bones from mice of different ages showed that, starting from three weeks of age, growth plates of both homo- and heterozygous fetuin-A deficient mice had lesions and were invaded by foreign cells. At eight weeks of age, lesions in the growth plates of Ahsg-/- mice had healed, partially by bone bridge formation, and growth plate architecture was distorted. Interestingly, in heterozygous Ahsg+/- mice, growth plates were either inconspicuous, or contained cavities which were lined with cellular infiltrate, although Ahsg+/- mice never developed the dysplasia.A microarray analysis of growth plates from 13 days old mice revealed a strong immune response in Ahsg-/- mice. A comparison to the Interferome database showed, that a huge amount of the upregulated genes were regulated through either a type I or a type II interferon response.I therefore hypothesize that fetuin-A plays an anti-inflammatory role in endochondral ossification and the dysplasia in Ahsg-/- mice is a consequence of an inflammatory response in the growth plate, which leads to cellular infiltration, mechanical weakening of growth plate cartilage and angular deformation of the distal femoral growth plate