Runx2 is essential for osteoblast differentiation and chondrocyte maturation. During osteoblast differentiation, Runx2 is weakly expressed in uncommitted mesenchymal cells, and its expression is upregulated in preosteoblasts, reaches the maximal level in immature osteoblasts, and is down-regulated in mature osteoblasts. Runx2 enhances the proliferation of osteoblast progenitors by directly regulating Fgfr2 and Fgfr3. Runx2 enhances the proliferation of suture mesenchymal cells and induces their commitment into osteoblast lineage cells through the direct regulation of hedgehog (Ihh, Gli1, and Ptch1), Fgf (Fgfr2 and Fgfr3), Wnt (Tcf7, Wnt10b, and Wnt1), and Pthlh (Pthr1) signaling pathway genes, and Dlx5. Runx2 heterozygous mutation causes open fontanelle and sutures because more than half of the Runx2 gene dosage is required for the induction of these genes in suture mesenchymal cells. Runx2 regulates the proliferation of osteoblast progenitors and their differentiation into osteoblasts via reciprocal regulation with hedgehog, Fgf, Wnt, and Pthlh signaling molecules, and transcription factors, including Dlx5 and Sp7. Runx2 induces the expression of major bone matrix protein genes, including Col1a1, Spp1, Ibsp, Bglap2, and Fn1, in vitro. However, the functions of Runx2 in differentiated osteoblasts in the expression of these genes in vivo require further investigation.

Osteocalcin (Ocn), which is specifically produced by osteoblasts, and is the most abundant non-collagenous protein in bone, was demonstrated to inhibit bone formation and function as a hormone, which regulates glucose metabolism in the pancreas, testosterone synthesis in the testis, and muscle mass, based on the phenotype of Ocn^−/− mice by Karsenty’s group. Recently, Ocn^−/− mice were newly generated by two groups independently. Bone strength is determined by bone quantity and quality. The new Ocn^−/− mice revealed that Ocn is not involved in the regulation of bone formation and bone quantity, but that Ocn regulates bone quality by aligning biological apatite (BAp) parallel to the collagen fibrils. Moreover, glucose metabolism, testosterone synthesis and spermatogenesis, and muscle mass were normal in the new Ocn^−/− mice. Thus, the function of Ocn is the adjustment of growth orientation of BAp parallel to the collagen fibrils, which is important for bone strength to the loading direction of the long bone. However, Ocn does not play a role as a hormone in the pancreas, testis, and muscle. Clinically, serum Ocn is a marker for bone formation, and exercise increases bone formation and improves glucose metabolism, making a connection between Ocn and glucose metabolism.