Valerie S. Salazar

β-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 β‐catenin signaling. We tested the hypothesis that β‐catenin is involved in mesenchymal lineage allocation to osteogenic cells using a β‐catenin mutant with constitutive transcriptional activity (ΔN151). Although this stable β‐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, ΔN151 and BMP‐2 synergistically stimulated new bone formation after subperiosteal injection in mouse calvaria in vivo. Conversely, ΔN151 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, β‐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.

BMP signalling in skeletal development, disease and repair

Since the identification in 1988 of bone morphogenetic protein 2 (BMP2) as a potent inducer of bone and cartilage formation, BMP superfamily signalling has become one of the most heavily investigated topics in vertebrate skeletal biology. Whereas a large part of this research has focused on the roles of BMP2, BMP4 and BMP7 in the formation and repair of endochondral bone, a large number of BMP superfamily molecules have now been implicated in almost all aspects of bone, cartilage and joint biology. As modulating BMP signalling is currently a major therapeutic target, our rapidly expanding knowledge of how BMP superfamily signalling affects most tissue types of the skeletal system creates enormous potential to translate basic research findings into successful clinical therapies that improve bone mass or quality, ameliorate diseases of skeletal overgrowth, and repair damage to bone and joints. This Review examines the genetic evidence implicating BMP superfamily signalling in vertebrate bone and joint development, discusses a selection of human skeletal disorders associated with altered BMP signalling and summarizes the status of modulating the BMP pathway as a therapeutic target for skeletal trauma and disease.

N-cadherin and cadherin 11 modulate postnatal bone growth and osteoblast differentiation by distinct mechanisms.

We have previously shown that targeted expression of a dominant-negative truncated form of N-cadherin (Cdh2) delays acquisition of peak bone mass in mice and retards osteoblast differentiation; whereas deletion of cadherin 11 (Cdh11), another osteoblast cadherin, leads to only modest osteopenia. To determine the specific roles of these two cadherins in the adult skeleton, we generated mice with an osteoblast/osteocyte specific Cdh2 ablation (cKO) and double Cdh2+/−;Cdh11−/− germline mutant mice. Age-dependent osteopenia and smaller diaphyses with decreased bone strength characterize cKO bones. By contrast, Cdh2+/−;Cdh11−/− exhibit severely reduced trabecular bone mass, decreased in vivo bone formation rate, smaller diaphyses and impaired bone strength relative to single Cdh11 null mice. The number of bone marrow immature precursors and osteoprogenitor cells is reduced in both cKO and Cdh2+/−;Cdh11−/− mice, suggesting that N-cadherin is involved in maintenance of the stromal cell precursor pool via the osteoblast. Although Cdh11 is dispensable for postnatal skeletal growth, it favors osteogenesis over adipogenesis. Deletion of either cadherin reduces β-catenin abundance and β-catenin-dependent gene expression, whereas N-cadherin loss disrupts cell-cell adhesion more severely than loss of cadherin 11. Thus, Cdh2 and Cdh11 are crucial regulators of postnatal skeletal growth and bone mass maintenance, serving overlapping, yet distinct, functions in the osteogenic lineage.

N-cadherin restrains PTH activation of Lrp6/β-catenin signaling and osteoanabolic action.

Interaction between parathyroid hormone/parathyroid hormone–related peptide receptor 1 (PTHR1) and low‐density lipoprotein receptor–related protein 6 (Lrp6) is important for parathyroid hormone (PTH) signaling and anabolic action. Because N‐cadherin has been shown to negatively regulate canonical Wnt/β‐catenin signaling, we asked whether N‐cadherin alters PTH signaling and stimulation of bone formation. Ablation of the N‐cadherin gene (Cdh2) in primary osteogenic lineage cells resulted in increased Lrp6/PTHR1 interaction in response to PTH1‐34, associated with enhanced PTH‐induced PKA signaling and PKA‐dependent β‐catenin C‐terminus phosphorylation, which promotes β‐catenin transcriptional activity. β‐catenin C‐terminus phosphorylation was abolished by Lrp6 knockdown. Accordingly, PTH1‐34 stimulation of Tcf/Lef target genes, Lef1 and Axin2, was also significantly enhanced in Cdh2‐deficient cells. This enhanced responsiveness to PTH extends to the osteo‐anabolic effect of PTH, as mice with a conditional Cdh2 deletion in Osx+ cells treated with intermittent doses of PTH1‐34 exhibited significantly larger gains in trabecular bone mass relative to control mice, the result of accentuated osteoblast activity. Therefore, N‐cadherin modulates Lrp6/PTHR1 interaction, restraining the intensity of PTH‐induced β‐catenin signaling, and ultimately influencing bone formation in response to intermittent PTH administration.

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

Summary To examine interactions between bone morphogenic protein (BMP) and canonical Wnt signaling during skeletal growth, we ablated Smad4, a key component of the TGF-&bgr;–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 &bgr;-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.

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

Summary Canonical Wnt (cWnt) signaling through &bgr;-catenin regulates osteoblast proliferation and differentiation to enhance bone formation. We previously reported that osteogenic action of &bgr;-catenin is dependent on BMP signaling. Here, we further examined interactions between cWnt and BMP in bone. In osteoprogenitors stimulated with BMP2, &bgr;-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 &bgr;-catenin, thus restraining cWnt-dependent proliferative signals while favoring the matrix synthesizing activity of osteoblasts.

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. Summary: Osteoblast specification requires regulatory inputs from BMP2 at the Runx2/Osx1 transition by a mechanism that is not compensated for by canonical Wnt signaling and involves Grhl3.

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

N‐cadherin inhibits osteogenic cell differentiation and canonical Wnt/β‐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 β‐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.