YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulation in murine bone development.

Fetal bone development occurs through the conversion of avascular cartilage to vascularized bone at the growth plate. This requires coordinated mobilization of osteoblast precursors with blood vessels. In adult bone, vessel-adjacent osteoblast precursors are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Here, we show that the mechanoresponsive transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) spatially couple osteoblast precursor mobilization to angiogenesis, regulate vascular morphogenesis to control cartilage remodeling, and mediate mechanoregulation of embryonic murine osteogenesis. Mechanistically, YAP and TAZ regulate a subset of osteoblast-lineage cells, identified by single-cell RNA sequencing as vessel-associated osteoblast precursors, which regulate transcriptional programs that direct blood vessel invasion through collagen-integrin interactions and Cxcl12. Functionally, in 3D human cell co-culture, CXCL12 treatment rescues angiogenesis impaired by stromal cell YAP/TAZ depletion. Together, these data establish functions of the vessel-associated osteoblast precursors in bone development.

YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulated bone development.

Endochondral ossification requires coordinated mobilization of osteoblast precursors with blood vessels. During adult bone homeostasis, vessel adjacent osteoblast precursors respond to and are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Previously, we found that deletion of the mechanoresponsive transcriptional regulators, YAP and TAZ, from Osterix-expressing osteoblast precursors and their progeny caused perinatal lethality. Here, we show that embryonic YAP/TAZ signaling couples vessel-associated osteoblast precursor mobilization to angiogenesis in developing long bones. Osterix-conditional YAP/TAZ deletion impaired endochondral ossification in the primary ossification center but not intramembranous osteogenesis in the bone collar. Single-cell RNA sequencing revealed YAP/TAZ regulation of the angiogenic chemokine, Cxcl12, which was expressed uniquely in vessel-associated osteoblast precursors. YAP/TAZ signaling spatially coupled osteoblast precursors to blood vessels and regulated vascular morphogenesis and vessel barrier function. Further, YAP/TAZ signaling regulated vascular loop morphogenesis at the chondro-osseous junction to control hypertrophic growth plate remodeling. In human cells, mesenchymal stromal cell co-culture promoted 3D vascular network formation, which was impaired by stromal cell YAP/TAZ depletion, but rescued by recombinant CXCL12 treatment. Lastly, YAP and TAZ mediated mechanotransduction for load-induced osteogenesis in embryonic bone.

Tunnels in the rock: Dynamics of osteocyte morphogenesis.

Osteocytes are dynamic, bone matrix-remodeling cells that form an intricate network of interconnected projections through the bone matrix, called the lacunar-canalicular system. Osteocytes are the dominant mechanosensory cells in bone and their mechanosensory and mechanotransductive functions follow their morphological form. During osteocytogenesis and development of the osteocyte lacunar-canalicular network, osteocytes must dramatically remodel both their cytoskeleton and their extracellular matrix. In this review, we summarize our current understanding of the mechanisms that govern osteocyte differentiation, cytoskeletal morphogenesis, mechanotransduction, and matrix remodeling. We postulate that the physiologic activation of matrix remodeling in adult osteocytes, known as perilacunar/canalicular remodeling (PLR) represents a re-activation of the developmental program by which the osteocyte network is first established. While much of osteocyte biology remains unclear, new tools and approaches make the present moment a particularly fruitful and exciting time to study the development of these remarkable cells.

YAP and TAZ Promote Periosteal Osteoblast Precursor Expansion and Differentiation for Fracture Repair.

In response to bone fracture, periosteal progenitor cells proliferate, expand, and differentiate to form cartilage and bone in the fracture callus. These cellular functions require the coordinated activation of multiple transcriptional programs, and the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) regulate osteochondroprogenitor activation during endochondral bone development. However, recent observations raise important distinctions between the signaling mechanisms used to control bone morphogenesis and repair. Here, we tested the hypothesis that YAP and TAZ regulate osteochondroprogenitor activation during endochondral bone fracture healing in mice. Constitutive YAP and/or TAZ deletion from Osterix-expressing cells impaired both cartilage callus formation and subsequent mineralization. However, this could be explained either by direct defects in osteochondroprogenitor differentiation after fracture or by developmental deficiencies in the progenitor cell pool before fracture. Consistent with the second possibility, we found that developmental YAP/TAZ deletion produced long bones with impaired periosteal thickness and cellularity. Therefore, to remove the contributions of developmental history, we next generated adult onset-inducible knockout mice (using Osx-CretetOff ) in which YAP and TAZ were deleted before fracture but after normal development. Adult onset-induced YAP/TAZ deletion had no effect on cartilaginous callus formation but impaired bone formation at 14 days post-fracture (dpf). Earlier, at 4 dpf, adult onset-induced YAP/TAZ deletion impaired the proliferation and expansion of osteoblast precursor cells located in the shoulder of the callus. Further, activated periosteal cells isolated from this region at 4 dpf exhibited impaired osteogenic differentiation in vitro upon YAP/TAZ deletion. Finally, confirming the effects on osteoblast function in vivo, adult onset-induced YAP/TAZ deletion impaired bone formation in the callus shoulder at 7 dpf before the initiation of endochondral ossification. Together, these data show that YAP and TAZ promote the expansion and differentiation of periosteal osteoblast precursors to accelerate bone fracture healing. © 2020 American Society for Bone and Mineral Research (ASBMR).

Suppression of Notch Signaling in Osteoclasts Improves Bone Regeneration and Healing.

Owing to the central role of osteoclasts in bone physiology and remodeling, manipulation of their maturation process provides a potential therapeutic strategy for treating bone diseases. To investigate this, we genetically inhibited the Notch signaling pathway in the myeloid lineage, which includes osteoclast precursors, using a dominant negative form of MAML (dnMAML) that inhibits the transcriptional complex required for downstream Notch signaling. Osteoclasts derived from dnMAML mice showed no significant differences in early osteoclastic gene expression compared to the wild type. Further, these demonstrated significantly lowered resorption activity using bone surfaces while retaining their osteoblast stimulating ability using ex vivo techniques. Using in vivo approaches, we detected significantly higher bone formation rates and osteoblast gene expression in dnMAML cohorts. Further, these mice exhibited increased bone/tissue mineral density compared to wild type and larger bony calluses in later stages of fracture healing. These observations suggest that therapeutic suppression of osteoclast Notch signaling could reduce, but not eliminate, osteoclastic resorption without suppression of restorative bone remodeling and, therefore, presents a balanced paradigm for increasing bone formation, regeneration, and healing. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2089-2103, 2019.

Notch signaling inhibition protects against LPS mediated osteolysis.

Chronic inflammatory responses have profound effects on the differentiation and activity of both the bone-forming osteoblasts and bone-resorbing osteoclasts. Importantly, inflammatory bone diseases characterized by clinical osteolysis promote bone resorption and decrease bone formation by uncoupling the process in favor of excess resorption. Notch signaling regulates osteoclast development and thus its manipulation has the potential to suppress resorptive potential. Here, we have utilized a genetic model of Notch inhibition in osteoclasts by expression of dnMAML to prevent formation of transcriptional complex essential for downstream Notch signaling. Using this model and LPS as a tool for experimental inflammatory osteolysis, we have demonstrated that dnMAML-expressing osteoclasts exhibited significantly lower maturation and resorption/functional potential ex vivo using TRAP staining and calcium phosphate coated surfaces. Moreover, we observed that while LPS stimulated the formation of wildtype osteoclasts pre-treated with RANKL, dnMAML expression produced resistance to osteoclast maturation after LPS stimulation. Genetically, Notch-inhibited animals showed a significantly lower TRAP and CTX-1 levels in serum after LPS treatment compared to the control groups in addition to a marked reduction in osteoclast surfaces in calvaria sections. This report provides evidence for modulation of Notch signaling activity to protect against inflammatory osteolysis. Taken together, the findings of this study will help guide the development of Notch signaling-based therapeutic approaches to prevent bone loss.