Expression of a Constitutively Active Form of in Chondrocytes Activates Wnt and Hedgehog Signaling Pathways, and Induces Chondrocyte Proliferation in Mice.

Runx2 is required for chondrocyte proliferation and maturation. In the search of Runx2 target genes in chondrocytes, we found that Runx2 up-regulated the expression of hematopoietic cell kinase (, which is a member of the Src tyrosine kinase family, in chondrocytes, that expression was high in cartilaginous limb skeletons of wild-type mice but low in those of mice, and that Runx2 bound the promoter region of . To investigate the functions of Hck in chondrocytes, transgenic mice expressing a constitutively active form of () were generated using the promoter/enhancer.

Antxr1, Which is a Target of Runx2, Regulates Chondrocyte Proliferation and Apoptosis.

Antxr1/Tem8 is highly expressed in tumor endothelial cells and is a receptor for anthrax toxin. Mutation of causes GAPO syndrome, which is characterized by growth retardation, alopecia, pseudo-anodontia, and optic atrophy. However, the mechanism underlying the growth retardation remains to be clarified.

Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation by regulating Fgfr2 and Fgfr3.

Runx2 and Sp7 are essential transcription factors for osteoblast differentiation. However, the molecular mechanisms responsible for the proliferation of osteoblast progenitors remain unclear. The early onset of Runx2 expression caused limb defects through the Fgfr1-3 regulation by Runx2.

Overexpression of Galnt3 in chondrocytes resulted in dwarfism due to the increase of mucin-type O-glycans and reduction of glycosaminoglycans.

Galnt3, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, transfers N-acetyl-D-galactosamine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2(-/-) cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3(-/-) mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer.

Galnt3 deficiency disrupts acrosome formation and leads to oligoasthenoteratozoospermia.

Galnt3 belongs to the GalNAc transferase gene family involved in the initiation of mucin-type O-glycosylation. Male Galnt3-deficient (Galnt3(-/-)) mice were infertile, as previously reported by Ichikawa et al. (2009).

SP7 inhibits osteoblast differentiation at a late stage in mice.

RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation.

Early onset of Runx2 expression caused craniosynostosis, ectopic bone formation, and limb defects.

RUNX2 is an essential transcription factor for osteoblast differentiation, because osteoblast differentiation is completely blocked in Runx2-deficient mice. However, it remains to be clarified whether RUNX2 is sufficient for osteoblast differentiation during embryogenesis. To address this issue, Runx2 transgenic mice were generated under the control of the Prrx1 promoter, which directs the transgene expression to mesenchymal cells before the onset of bone development.

Pyruvate dehydrogenase kinase 4 induces bone loss at unloading by promoting osteoclastogenesis.

Disuse osteoporosis, which occurs commonly in prolonged bed rest and immobilization, is becoming a major problem in modern societies; however, the molecular mechanisms underlying unloading-driven bone loss have not been fully elucidated. The osteocyte network is considered to be an ideal mechanosensor and mechanotransduction system. We searched for the molecules responsible for disuse osteoporosis using BCL2 transgenic mice, in which the osteocyte network was disrupted.

Regulation of Tcf7 by Runx2 in chondrocyte maturation and proliferation.

Runx2 plays important roles in the regulation of chondrocyte differentiation and proliferation; however, the Runx2 target molecules still remain to be investigated. We searched the genes upregulated by the introduction of Runx2 into Runx2(-/-) chondrocytes using microarray and found that Tcf7 is upregulated by Runx2. Thus, we examined the functions of Runx2 in the regulation of the Tcf/Lef family of transcription factors.

Akt regulates skeletal development through GSK3, mTOR, and FoxOs.

Although Akt plays key roles in various cellular processes, the functions of Akt and Akt downstream signaling pathways in the cellular processes of skeletal development remain to be clarified. By analyzing transgenic embryos that expressed constitutively active Akt (myrAkt) or dominant-negative Akt in chondrocytes, we found that Akt positively regulated the four processes of chondrocyte maturation, chondrocyte proliferation, cartilage matrix production, and cell growth in skeletal development. As phosphorylation of GSK3beta, S6K, and FoxO3a was enhanced in the growth plates of myrAkt transgenic mice, we examined the Akt downstream signaling pathways by organ culture.

Runx2 represses myocardin-mediated differentiation and facilitates osteogenic conversion of vascular smooth muscle cells.

Phenotypic plasticity and the switching of vascular smooth muscle cells (SMCs) play a critical role in atherosclerosis. Although Runx2, a key osteogenic transcription factor, is expressed in atherosclerotic plaques, the molecular mechanisms by which Runx2 regulates SMC differentiation remain unclear. Here we demonstrated that Runx2 repressed SMC differentiation induced by myocardin, which acts as a coactivator for serum response factor (SRF).

Runx2 determines bone maturity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency.

Runx2 is an essential transcription factor for osteoblast differentiation. However, the functions of Runx2 in postnatal bone development remain to be clarified. Introduction of dominant-negative (dn)-Runx2 did not inhibit Col1a1 and osteocalcin expression in mature osteoblastic cells.

Cbf beta regulates Runx2 function isoform-dependently in postnatal bone development.

Runx2 and Cbfbeta are essential for skeletal development during the embryonic stage. Runx2 has two isoforms with different N-termini. We examined the functions of the Runx2 isoforms and Cbfbeta in postnatal bone development.

Role of Runx proteins in chondrogenesis.

The mammalian RUNX protein family comprises three transcription factorsRUNX1, RUNX2, and RUNX3. RUNX1 is involved in hematopoiesis, RUNX2 has multiple roles in osteogenesis and RUNX3 is associated with neural and gut development. In addition, all RUNX proteins are expressed during chondrogenesis, the process by which cartilage is formed.

Runx2 and Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog.

The differentiation of mesenchymal cells into chondrocytes and chondrocyte proliferation and maturation are fundamental steps in skeletal development. Runx2 is essential for osteoblast differentiation and is involved in chondrocyte maturation. Although chondrocyte maturation is delayed in Runx2-deficient (Runx2(-/-)) mice, terminal differentiation of chondrocytes does occur, indicating that additional factors are involved in chondrocyte maturation.

Induction of osteoclast differentiation by Runx2 through receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin regulation and partial rescue of osteoclastogenesis in Runx2-/- mice by RANKL transgene.

Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2-/-) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished.

Core-binding factor beta interacts with Runx2 and is required for skeletal development.

Core-binding factor beta (CBFbeta, also called polyomavirus enhancer binding protein 2beta (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFbeta forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFbeta are essential for hematopoiesis.