N-cadherin Regulation of Bone Growth and Homeostasis Is Osteolineage Stage-Specific.

N-cadherin inhibits osteogenic cell differentiation and canonical Wnt/ß-catenin signaling in vitro. However, in vivo both conditional Cdh2 ablation and overexpression in osteoblasts lead to low bone mass. We tested the hypothesis that N-cadherin has different effects on osteolineage cells depending upon their differentiation stage. Embryonic conditional osteolineage Cdh2 deletion in mice results in defective growth, low bone mass, and reduced osteoprogenitor number. These abnormalities are prevented by delaying Cdh2 ablation until 1 month of age, thus targeting only committed and mature osteoblasts, suggesting they are the consequence of N-cadherin deficiency in osteoprogenitors. Indeed, diaphyseal trabecularization actually increases when Cdh2 is ablated postnatally. The sclerostin-insensitive Lrp5A214V mutant, associated with high bone mass, does not rescue the growth defect, but it overrides the low bone mass of embryonically Cdh2-deleted mice, suggesting N-cadherin interacts with Wnt signaling to control bone mass. Finally, bone accrual and ß-catenin accumulation after administration of an anti-Dkk1 antibody are enhanced in N-cadherin-deficient mice. Thus, although lack of N-cadherin in embryonic and perinatal age is detrimental to bone growth and bone accrual, in adult mice loss of N-cadherin in osteolineage cells favors bone formation. Hence, N-cadherin inhibition may widen the therapeutic window of osteoanabolic agents. © 2017 American Society for Bone and Mineral Research.

Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2.

Enhanced BMP or canonical Wnt (cWnt) signaling are therapeutic strategies employed to enhance bone formation and fracture repair, but the mechanisms each pathway utilizes to specify cell fate of bone-forming osteoblasts remain poorly understood. Among all BMPs expressed in bone, we find that singular deficiency of Bmp2 blocks the ability of cWnt signaling to specify osteoblasts from limb bud or bone marrow progenitors. When exposed to cWnts, Bmp2-deficient cells fail to progress through the Runx2/Osx1 checkpoint and thus do not upregulate multiple genes controlling mineral metabolism in osteoblasts. Cells lacking Bmp2 after induction of Osx1 differentiate normally in response to cWnts, suggesting that pre-Osx1+ osteoprogenitors are an essential source and a target of BMP2. Our analysis furthermore reveals Grainyhead-like 3 (Grhl3) as a transcription factor in the osteoblast gene regulatory network induced during bone development and bone repair, which acts upstream of Osx1 in a BMP2-dependent manner. The Runx2/Osx1 transition therefore receives crucial regulatory inputs from BMP2 that are not compensated for by cWnt signaling, and this is mediated at least in part by induction and activation of Grhl3.

Postnatal ablation of osteoblast Smad4 enhances proliferative responses to canonical Wnt signaling through interactions with ß-catenin.

Canonical Wnt (cWnt) signaling through ß-catenin regulates osteoblast proliferation and differentiation to enhance bone formation. We previously reported that osteogenic action of ß-catenin is dependent on BMP signaling. Here, we further examined interactions between cWnt and BMP in bone. In osteoprogenitors stimulated with BMP2, ß-catenin localizes to the nucleus, physically interacts with Smad4, and is recruited to DNA-binding transcription complexes containing Smad4, R-Smad1/5 and TCF4. Furthermore, Tcf/Lef-dependent transcription, Ccnd1 expression and proliferation all increase when Smad4, 1 or 5 levels are low, whereas TCF/Lef activities decrease when Smad4 expression is high. The ability of Smad4 to antagonize transcription of Ccnd1 is dependent on DNA-binding activity but Smad4-dependent transcription is not required. In mice, conditional deletion of Smad4 in osterix(+) cells increases mitosis of cells on trabecular bone surfaces as well as in primary osteoblast cultures from adult bone marrow and neonatal calvaria. By contrast, ablation of Smad4 delays differentiation and matrix mineralization by primary osteoblasts in response to Wnt3a, indicating that loss of Smad4 perturbs the balance between proliferation and differentiation in osteoprogenitors. We propose that Smad4 and Tcf/Lef transcription complexes compete for ß-catenin, thus restraining cWnt-dependent proliferative signals while favoring the matrix synthesizing activity of osteoblasts.

Embryonic ablation of osteoblast Smad4 interrupts matrix synthesis in response to canonical Wnt signaling and causes an osteogenesis-imperfecta-like phenotype.

To examine interactions between bone morphogenic protein (BMP) and canonical Wnt signaling during skeletal growth, we ablated Smad4, a key component of the TGF-ß-BMP pathway, in Osx1(+) cells in mice. We show that loss of Smad4 causes stunted growth, spontaneous fractures and a combination of features seen in osteogenesis imperfecta, cleidocranial dysplasia and Wnt-deficiency syndromes. Bones of Smad4 mutant mice exhibited markers of fully differentiated osteoblasts but lacked multiple collagen-processing enzymes, including lysyl oxidase (Lox), a BMP2-responsive gene regulated by Smad4 and Runx2. Accordingly, the collagen matrix in Smad4 mutants was disorganized, but also hypomineralized. Primary osteoblasts from these mutants did not mineralize in vitro in the presence of BMP2 or Wnt3a, and Smad4 mutant mice failed to accrue new bone following systemic inhibition of the Dickkopf homolog Dkk1. Consistent with impaired biological responses to canonical Wnt, ablation of Smad4 causes cleavage of ß-catenin and depletion of the low density lipoprotein receptor Lrp5, subsequent to increased caspase-3 activity and apoptosis. In summary, Smad4 regulates maturation of skeletal collagen and osteoblast survival, and is required for matrix-forming responses to both BMP2 and canonical Wnt.

Beta-catenin and BMP-2 synergize to promote osteoblast differentiation and new bone formation.

Mutations of critical components of the Wnt pathway profoundly affect skeletal development and maintenance, probably via modulation of beta-catenin signaling. We tested the hypothesis that beta-catenin is involved in mesenchymal lineage allocation to osteogenic cells using a beta-catenin mutant with constitutive transcriptional activity (DeltaN151). Although this stable beta-catenin had no effects by itself on osteogenic differentiation of multipotent embryonic cell lines, it synergized with bone morphogenetic protein-2 (BMP-2) resulting in dramatic stimulation of alkaline phosphatase activity, osteocalcin gene expression, and matrix mineralization. Likewise, DeltaN151 and BMP-2 synergistically stimulated new bone formation after subperiosteal injection in mouse calvaria in vivo. Conversely, DeltaN151 prevented adipogenic differentiation from pre-adipocytic or uncommitted mesenchymal cells in vitro. Intriguingly, the synergism with BMP-2 on gene transcription occurred without altering expression of Cbfa1/Runx2, suggesting actions independent or downstream of this osteoblast-specific transcription factor. Thus, beta-catenin directs osteogenic lineage allocation by enhancing mesenchymal cell responsiveness to osteogenic factors, such as BMP-2, in part via Tcf/Lef dependent mechanisms. In vivo, this synergism leads to increased new bone formation.

Cyclooxygenase-2 inhibition with celecoxib enhances antitumor efficacy and reduces diarrhea side effect of CPT-11.

Combining anticancer drugs with different mechanisms of action has the potential to enhance antitumor effect. CPT-11 (Camptosar, irinotecan), a topoisomerase I inhibitor, has been shown to be highly effective in the treatment of a variety of cancers. However, its clinical usage is often complicated by late diarrhea. A number of studies have shown that cyclooxygenase (COX)-2 is overexpressed in many forms of human tumors, suggesting that COX-2 inhibition may be useful in the treatment of cancer. In this study, we used two mouse tumor models (HT-29 and colon-26 cells) to evaluate the effect of combining CPT-11 with celecoxib on tumor growth. We also assessed the involvement of COX-2 in the pathogenesis of CPT-11-induced late diarrhea using a rat model. Results indicate that celecoxib enhances the antitumor effect of CPT-11 and reduces the severity of late diarrhea in a dose-dependent manner. The extended benefits of combining celecoxib with CPT-11 may significantly improve the outcome of cancer patients.