Osteogenesis is a complex and still poorly understood biological process regulated by intrinsic cellular signals and extrinsic micro-environmental stimuli from the surrounding stem cell-niche. Some regulators of osteogenesis, including growth factors and transcriptional factors, have been already identified, although further studies to better elucidate the molecular basis of this biological process are required in order to develop new and more effective therapeutic strategies for the treatment of osteogenic diseases, such as osteoporosis, bone fractures and osteosarcoma.
Osteoblasts arise from mesenchymal stem cells (MSCs), which are adult multipotent stem cells that can be successfully isolated from several adult and neonatal tissues, are able to generate, following appropriate stimuli, several differentiated mesoderm-derived cell lineages, including adipocytes, osteoblasts, chondrocytes and myoblasts and, therefore, represent a promising cell source for regenerative medicine.
In order to identify genes involved in the commitment of MSCs to osteoblasts, we performed a screening with an RNA interference-based approach and silenced a large number of genes during osteoblast differentiation of a murine MSC-derived cell line (W20-17). With this procedure we identified several candidate genes potentially involved in osteogenesis; in fact, their silencing caused a reduction of mineralized deposits, as assessed by Alizarin Red staining. We then analyzed the Gene Ontology classification of candidate genes and we found a significant fraction (30%) of genes with unknown function at the time of the screening. Hence, we focused our attention on one of these genes that we named Osteoblasts Inducer (ObI-1), predicted to be a transcription factor. Indeed, Ensembl analysis showed that ObI-1 encodes for a protein containing several zinc-finger domains and a Kruppel-associated box (KRAB) domain, usually involved in transcriptional repression.
We were able to observe a significant impairment in osteoblast differentiation, resulting in reduction of both alkaline phosphatase expression and mineralization, as a result of ObI-1 silencing. In addition, we evaluated ObI-1 expression in our cell system as well as in several mouse tissues and organs. Interestingly, ObI1 expression increases during osteogenic differentiation of both W20-17 cell line and primary murine MSCs from bone marrow. We demonstrated also the nuclear localization of ObI-1 through immunofluorescence analysis, corroborating the hypothesis that this gene may encode for a transcription factor. Finally, we analyzed the effect of ObI-1 silencing on the expression of osteogenic markers during the differentiation process