Core binding factor beta (Cbfβ), the partner protein of Runx family transcription factors, enhances Runx function by increasing the binding of Runx to DNA. Null mutations of Cbfb result in embryonic death, which can be rescued by restoring fetal hematopoiesis but only until birth, where bone formation is still nearly absent. Here, we address a direct role of Cbfβ in skeletal homeostasis by generating osteoblast‐specific Cbfβ‐deficient mice (Cbfb^Δob/Δob) from Cbfb‐floxed mice crossed with mice expressing Cre from the Col1a1 promoter. Cbfb^Δob/Δob mice showed normal growth and development but exhibited reduced bone mass, particularly of cortical bone. The reduction of bone mass in Cbfb^Δob/Δob mice is similar to the phenotype of mice with haploinsufficiency of Runx2. Although the number of osteoblasts remained unchanged, the number of active osteoblasts decreased in Cbfb^Δob/Δob mice and resulted in lower mineral apposition rate. Immunohistochemical and quantitative real‐time PCR analyses showed that the expression of osteogenic markers, including Runx2, osterix, osteocalcin, and osteopontin, was significantly repressed in Cbfb^Δob/Δob mice compared with wild‐type mice. Cbfβ deficiency also reduced Runx2 protein levels in osteoblasts. The mechanism was revealed by forced expression of Cbfβ, which increased Runx2 protein levels in vitro by inhibiting polyubiquitination‐mediated proteosomal degradation. Collectively, these findings indicate that Cbfβ stabilizes Runx2 in osteoblasts by forming a complex and thus facilitates the proper maintenance of bone mass, particularly cortical bone. © 2015 American Society for Bone and Mineral Research.

The transdifferentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells has been implicated in the context of vascular calcification. We investigated the roles of vitamin D receptor (Vdr) and runt-related transcription factor 2 (Runx2) in the osteoblastic differentiation of VSMCs in response to vitamin D₃ using in vitro VSMCs cultures and in vivo in Vdr knockout (Vdr^-/-) and Runx2 carboxy-terminus truncated heterozygous (Runx2^+/ΔC) mice. Treatment of VSMCs with active vitamin D₃ promoted matrix mineral deposition, and increased the expressions of Vdr, Runx2, and of osteoblastic genes but decreased the expression of smooth muscle myosin heavy chain in primary VSMCs cultures. Immunoprecipitation experiments suggested an interaction between Vdr and Runx2. Furthermore, silencing Vdr or Runx2 attenuated the procalcific effects of vitamin D₃. Functional cooperation between Vdr and Runx2 in vascular calcification was also confirmed in in vivo mouse models. Vascular calcification induced by high-dose vitamin D₃ was completely inhibited in Vdr^-/- or Runx2^+/ΔC mice, despite elevated levels of serum calcium or alkaline phosphatase. Collectively, these findings suggest that functional cooperation between Vdr and Runx2 is necessary for vascular calcification in response to vitamin D₃.

Dual Ig domain-containing adhesion molecule (DICAM), a protein with homology to the junctional adhesion molecule family, has been demonstrated to interact with integrin αVβ3 that plays a critical role in angiogenesis. Here, we determined the role of DICAM during angiogenesis and the molecular mechanisms involved in the inhibition of angiogenesis. DICAM was expressed on the endothelial cells of large vessels to small capillaries. In human umbilical vein endothelial cells (HUVECs), DICAM was up-regulated by vascular endothelial growth factor (VEGF) through the MEK/ERK and PI3K/AKT pathways. Furthermore, the exogenous expression of DICAM in HUVECs suppressed angiogenesis in vitro Matrigel and in vivo plug assays, and conversely, DICAM knockdown enhanced angiogenesis. In addition, DICAM inhibited HUVEC migration and accelerated apoptosis via down-regulation of Bcl-2, but did not affect viability or proliferation of HUVEC. Mechanistically, the exogenous expression of DICAM suppressed VEGF-induced phosphorylarion of AKT and p38 MAP kinase. When integrin signalling was activated by vitronectin, a forced expression of DICAM attenuated integrin β3/FAK signalling and downstream AKT and p38 MAP kinase signalling in HUVECs. Collectively, DICAM suppressed angiogenesis by attenuating AKT and p38 MAP kinase signalling, which suggests that DICAM may be a novel negative regulator of angiogenesis.

Dual immunoglobulin (Ig) domain-containing adhesion molecule (DICAM) is involved in cell–cell adhesion through a heterophilic interaction with αVβ3 integrin, which suggests that DICAM may participate in osteoclast differentiation. DICAM was localized in the plasma membrane of RAW264.7 and THP-1 cells, and its expression gradually increased during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) treated with receptor activator of nuclear factor κ-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Forced expression of DICAM in BMMs and RAW264.7 cells blocked the generation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. Conversely, knockdown of DICAM by small hairpin RNA (shRNA) increased osteoclast formation in RAW264.7 cells. DICAM-mediated suppression of osteoclast differentiation was in part due to the inhibition of the p38 mitogen-activated protein (MAP) kinase pathway, which was corroborated by a decrease in the expression of c-Fos and nuclear factor of activated T cells (NFAT)c1. Mechanistically, DICAM directly interacted with integrin β3, which inhibited heterodimerization between integrin αV and β3. Exogenous expression of integrin β3 or high-dose M-CSF rescued DICAM-mediated inhibition of osteoclastogenesis, suggesting crosstalk between the integrin β3 and c-Fms pathways. Finally, recombinant DICAM ectodomain suppressed the RANKL- and M-CSF–induced osteoclastogenesis of BMMs. Collectively, these results indicate that DICAM acts as a negative regulator of osteoclast differentiation by suppressing the integrin αVβ3 pathway. © 2012 American Society for Bone and Mineral Research.