gms | German Medical Science

Objective: We have previously shown that apoE -/- mice display a high bone mass phenotype due to an increased bone formation rate. It remained unclear whether this phenotype was a secondary consequence of systemic influences onto osteoblasts or due to a cell autonomous effect of osteoblasts lacking apoE. To address this question, we hypothesized that apoE directly inhibits osteoblast differentiation.

Methods: Biochemical markers of osteoblast activity were measured with standard procedures. Osteogenic differentiation potential and in vitro mineralization were examined with primary murine calvarial osteoblast cultures. A human cell line (telomerase-immortalized mesenchymal stem cells, hMSC-tert) was used for shRNA-mediated apoE gene silencing and overexpression as well as for reporter assays of wnt3a-stimulated TCF/LEF transcriptional activity. TCF/LEF target gene expression was analyzed on mRNA and protein level.

Results and conclusions: Osteoblast activity markers were significantly higher in 3 and 8 month-old male and female apoE -/- animals than in wildtype (WT) littermates, indicating that age and sex were no major modifiers of the apoE effect on osteoblasts. Primary murine osteoblast cultures revealed a significant increase of osteoblast differentiation markers in apoE -/- cells. This difference was completely abolished by the reconstitution of exogenous apoE. In addition, the ability of apoE -/- osteoblasts to form mineralized extracellular bone matrix was markedly increased. shRNA-mediated apoE gene silencing resulted in a clear increase of differentiation markers, while the endogenous overexpression of apoE had an inhibitory effect. Reporter assays of wnt3a-stimulated TCF/LEF transcriptional activity revealed a dose-dependent decrease of activity in response to exogenous apoE, while activity was increased in shRNA apoE knock-down cells. The osteoprotegerin and RANK-ligand genes, which are TCF/LEF targets in osteoblasts, were regulated accordingly. Finally, western blot analysis showed increased ß-catenin accumulation in apoE -/- osteoblasts and a reduction of wnt3a-stimulated ß-catenin accumulation in response to apoE. Taken together, here we provide evidence that apoE directly inhibits osteoblast differentiation through suppression of canonical wnt signaling in osteoblasts.

This study provides an explanation for the so far molecularly unexplained high bone mass phenotype of apoE -/- mice. We conclude that due to the cell-autonomous defect of osteoblasts that lack apoE, an inhibitory modulator of the differentiation activating wnt/ß-catenin pathway is no longer present and a strong release of inhibition occurs. This study adds a further molecule to the complicated network of signals that govern osteoblast differentiation, opening an interesting perspective on the potential role of cell-surface bound lipoproteins in this context.