BioProject

The onset of senile osteoporosis manifests itself in a growing imbalance of bone marrow adipo- and osteogenesis. Commonly used hormonal therapies ameliorate symptoms, but do not address the primary cause of senile osteoporosis onset. Several osteogenic cytokines and transcription factors (TFs) are known to be involved in the crosstalk of osteoblast and adipocyte differentiation. Yet, little is known about the overall dynamics and regulation of TF changes leading to the imbalance of osteoblast and adipocyte differentiation inside the bone marrow. Extrapolating from general TF network studies one would expect a high degree of inter-regulation with transient hubs of TFs or TF complexes at various stages of differentiations. To delineate the sequential changes of TFs activating and repressing downstream adipo- and/or osteogenic target genes we have determined temporal gene expression profiles using an in vitro cell differentiation system. Initial evaluation of six cell lines for dual differentiation capability revealed that the bone marrow-derived stromal cell line ST2 differentiated most efficiently into either osteoblasts or adipocytes. Using Affymetrix mouse gene chips, we aimed to identify clusters of TFs that are temporally co-regulated in one but not in another cluster. Of approximately 1,300 TFs, 329 genes were significantly up- or down-regulated during osteoblast and adipocyte differentiation compared to the non-induced control. Hierarchical clustering analysis of TF gene expression at 15/7 time points of osteoblast/adipocyte differentiation resulted in five clusters of sequentially expressed TFs during osteoblast differentiation and four clusters during adipocyte differentiation. The early phases of osteoblast (0-1h) and adipocyte (48h) differentiation showed the highest volatility in TF expression levels. Chi-square testing for over-representation of TFs in each differentiation cluster supported only six up-regulated bHLH family members (Id1, Id2, Id4, Npas4, Hes1 and Bhlhb2) of the early-phase osteoblast differentiation (0-1h) as significantly (p<0.01) enriched. Since Id4, Hes1 and Bhlhb2 showed also a two-fold or greater increase in expression during osteoblast differentiation as well as a similar decrease during adipocyte differentiation compared to the control, these TFs are likely to play a pivotal role in the regulation of osteoblast and adipocyte differentiation. Indeed, Hes1 and Bhlhb2 are known to be involved in both differentiation pathways, whereas Id4 has not yet been implicated in either differentiation pathways. Therefore we characterized Id4 function in osteoblast and adipocyte differentiation using siRNA knock-down in ST2 cells and Id4 knock-out mice.