Osx is the only bone-specific transcription factor identified so far which is required for bone formation. Osx knockout is lethal. Heterozygous Osx mutant mice were normal and fertile. Homozygous Osx mutant mice had difficulty in breathing, and died within 15 min of birth [18]. Osteoblast marker genes such as ALP and OC are undetectable in Osx-null mutant. There is no bone formation without Osx. On the other hand, it was shown that Osx was sufficient to induce the expression of osteoblast marker OC in mesenchymal stem cells in vitro [18]. Osx controls osteogenesis as a downstream gene of Runx2 [18]. Runx2 is required for bone formation; however, Runx2 is expressed in different cells and tissues, including osteoblasts, chondrocytes, epithelial cells, glioma cells, brain tissues, and different tumor tissues [23]. Unlike Runx2, Osx is unique and bone-specific in that it is specifically expressed in osteoblasts and at low levels in prehypertrophic chondrocytes [18]. Osx is not only critical for embryonic bone formation but also essential in postnatal bone growth and in bone homeostasis using the conditional knockout approach [24].

Despite the discovery of its significance in skeletal physiology a decade ago [18], relatively little is known about direct target genes for Osx and molecular mechanisms through which Osx controls gene transcription. Recently, our research laboratory has identified several downstream bone-related target genes of Osx during bone formation, including Satb2, VDR, VEGF, SOST, DKK1, and MMP13 [25–30]. Identification of VEGF as a downstream direct target of Osx also indicates that Osx plays an important role in coordinating osteogenesis and angiogenesis [27]. These Osx downstream targets each play important roles during bone formation (Fig. 3.2), supporting the notion that Osx, as a bone-specific transcription factor, is a master gene for osteoblast differentiation and bone formation.

Identification of anabolic agents that can stimulate bone formation to treat osteoporosis has been recognized as a priority in the bone biology field. Recent genome-wide association studies have shown that Osx are associated with bone mineral density in both children and adults, suggesting that Osx may contribute to the cause of osteoporosis [31, 32]. Because of the tissue specificity and the critical role of Osx in bone formation, Osx can be considered an ideal novel target for the development of a therapeutic strategy to induce the anabolic pathway of bone synthesis. At present, no pharmacological approach to target Osx in osteoblasts has been identified. Focusing on novel target Osx that are responsible for driving osteoblast differentiation and bone formation increases the likelihood of discovering mechanism-based agents that are more effective and less toxic than drugs of the previous era. Therefore, HTS assay must be performed to identify compounds promoting osteoblast differentiation and bone formation through Osx. In this case, this mechanism-based approach aims to identify potential anabolic agents by targeting a bone-specific factor Osx. Protocols for assay development and HTS execution are shown in Fig. 3.3. Parameters and controls (positive, and neutral) for the proposed primary assay must be developed, refined, and validated such that it is robust (Z′ values ≥ 0.45 over many assays and experimental days) [33], tolerant of effects from DMSO, free from systematic effects (e.g., plating artifacts, liquid handling errors), simple (most assays have less than three liquid additions and are endpoint assays), and efficient in use of reagents and resources. Secondary assays will be developed in parallel to the primary assay and meet similar criteria. Candidate small molecules identified by the process will be validated and characterized for their osteogenic activities using in vitro assays. The role of candidate small molecules in osteogenesis in vivo will be explored in osteoporosis animal models.