Skeletal cell YAP and TAZ combinatorially promote bone development

The functions of the paralogous transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in bone are controversial. Each has been observed to promote or inhibit osteogenesis in vitro, with reports of both equivalent and divergent functions. Their combinatorial roles in bone physiology are unknown. We report that combinatorial YAP/TAZ deletion from skeletal lineage cells, using Osterix-Cre, caused an osteogenesis imperfecta-like phenotype with severity dependent on allele dose and greater phenotypic expressivity with homozygous TAZ vs. YAP ablation. YAP/TAZ deletion decreased bone accrual and reduced intrinsic bone material properties through impaired collagen content and organization. These structural and material defects produced spontaneous fractures, particularly in mice with homozygous TAZ deletion and caused neonatal lethality in dual homozygous knockouts. At the cellular level in vivo, YAP/TAZ ablation reduced osteoblast activity and increased osteoclast activity, in an allele dose-dependent manner, impairing bone accrual and remodeling. Transcriptionally, YAP/TAZ deletion and small-molecule inhibition of YAP/TAZ interaction with the transcriptional coeffector TEAD reduced osteogenic and collagen-related gene expression, both in vivo and in vitro. These data demonstrate that YAP and TAZ combinatorially promote bone development through regulation of osteoblast activity, matrix quality, and osteoclastic remodeling.—Kegelman, C. D., Mason, D. E., Dawahare, J. H., Horan, D. J., Vigil, G. D., Howard, S. S., Robling, A. G., Bellido, T. M., Boerckel, J. D. Skeletal cell YAP and TAZ combinatorially promote bone development. Bone is a living hierarchical composite, with form and function dependent not only on tissue structure but also on matrix composition and organization. Each of these components is controlled during development by skeletal cell lineage progression and by dynamic regulation of bone deposition and remodeling. Various genetic, hormonal, or environmental abnormalities can impair these processes, leading to debilitating diseases including osteoporosis and osteogenesis imperfecta (OI). However, the molecular mechanisms that govern cell fate and matrix production in bone remain poorly understood, limiting therapeutic intervention. Several transcriptional programs have been described as essential regulators of bone development, but current understanding is insufficient to fully explain the heterogeneity found in congenital and acquired bone diseases (1–3). In this study, we sought to define the functions of the paralogous transcriptional coactivators yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in bone development. YAP/TAZ functional diversity YAP and TAZ (also known as WWTR1) display either equivalent or divergent functions, depending on cell type and context (4). YAP and TAZ possess transcription activation domains, but they lack DNA-binding domains and require interaction with cofactors for transcriptional activity (5). Their most potent and well-studied interactions are with the transcriptional enhancer activator domain (TEAD) family proteins, which themselves lack activation domains, providing specificity for YAP/TAZ–TEAD signaling (6). However, other coeffectors are also known, including runt-related transcription factor (Runx)-2 (7), β-catenin (8–10), and Smad2/3 (11, 12), each of which contributes to bone development and osteoprogenitor lineage progression (13–17). Thus, independent pathways that regulate coincident activation of these various binding partners could provide additional layers of contextual specificity in bone. Further, as paralogues, the YAP and TAZ proteins also possess structural differences (reviewed in Ref. 18) that enable distinct protein interactions to confer unique physiologic functions of YAP vs. TAZ. Notably, global YAP deletion in mice is embryonic lethal [embryonic day (E)8.5] because of impaired–yolk sac vasculogenesis (19), whereas the global TAZ knockout lives to maturity with modest skeletal defects and polycystic kidney disease (20), demonstrating conclusive gene-specific functions. However, in other contexts, they exhibit clear functional homology, with either protein capable of compensation for the other (21, 22). YAP and TAZ function in bone: conflicting evidence Roles for YAP and TAZ in osteogenesis were first described in 2004 and 2005, respectively (23, 24). YAP was reported to suppress osteoblastic differentiation through sequestration and transcriptional repression of Runx2 (23), whereas TAZ was identified as a Runx2 coactivator and an inhibitor of the adipogenic nuclear receptor, peroxisome proliferator-activated receptor-γ (24, 25). A subsequent study found that overexpression of a constitutively active YAP mutant in marrow stromal cells (MSCs) promoted osteogenic differentiation, even under conditions more favorable for adipogenesis (26). In contrast, another report found that YAP overexpression inhibits osteogenesis in MSCs by suppressing activation of wingless-type (WNT) target genes (27). The role of TAZ in osteogenic differentiation in vitro is similarly complicated, with reports demonstrating both inhibition (28) and induction (29) of osteogenic differentiation by modulating the canonical WNT pathway. In vivo, osteoblast-specific overexpression of TAZ promotes bone formation with higher expression levels of Runx2 expression (30), whereas YAP overexpression in chondrocytes impairs cartilage template formation during endochondral bone development (31). Together, these observations suggest the importance of YAP and TAZ in bone, but the conflicting evidence remains unresolved and their combinatorial roles in bone physiology remain unknown. To address these questions, we implemented a combinatorial deletion approach in vivo to evaluate the influence of allele dose-dependent YAP/TAZ deletion on bone development.