Metabolic reprogramming in skeletal cell differentiation

The human skeleton is a multifunctional organ made up of multiple cell types working in concert to maintain bone and mineral homeostasis and to perform critical mechanical and endocrine functions.From the beginning steps of chondrogenesis that prefigures most of the skeleton,to the rapid bone accrual during skeletal growth,followed by bone remodeling of the mature skeleton,cell differentiation is integral to skeletal health.

mTOR signaling in skeletal development and disease

The mammalian/mechanistic target of rapamycin(m TOR) is a serine/threonine protein kinase that integrates inputs from nutrients and growth factors to control many fundamental cellular processes through two distinct protein complexes m TORC1 and m TORC2.Recent mouse genetic studies have established that m TOR pathways play important roles in regulating multiple aspects of skeletal development and homeostasis. In addition, m TORC1 has emerged as a common effector mediating the bone anabolic effect of Igf1,Wnt and Bmp. Dysregulation of m TORC1 could contribute to various skeletal diseases including osteoarthritis and osteoporosis.Here we review the current understanding of m TOR signaling in skeletal development and bone homeostasis, as well as in the maintenance of articular cartilage. We speculate that targeting m TOR signaling may be a valuable approach for treating skeletal diseases.

Unintended targeting of Dmp1-Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice

Cre/loxP technology has been widely used to study cell type-specific functions of genes. Proper interpretation of such data critically depends on a clear understanding of the tissue specificity of Cre expression. The Dmpl- Cre mouse, expressing Cre from a 14-kb DNA fragment of the mouse Dmpl gene, has become a common tool for studying gene function in osteocytes, but the presumed cell specificity is yet to be fully established. By using the Ai9 reporter line that expresses a red fluorescent protein upon Cre recombination, we find that in 2-month-old mice, Dmpl-Cre targets not only osteocytes within the bone matrix but also osteoblasts on the bone surface and preosteoblasts at the metaphyseal chondro-osseous junction. In the bone marrow, Cre activity is evident in certain stromal cells adjacent to the blood vessels, but not in adipocytes. Outside the skeleton, Dmpl-Cre marks not only the skeletal muscle fibers, certain cells in the cerebellum and the hindbrain but also gastric and intestinal mesenchymal cells that express Pdgfra. Confirming the utility of Dmpl-Cre in the gastrointestinal mesenchyme, deletion of Bmprla with Dmpl-Cre causes numerous large polyps along the gastrointestinal tract, consistent with prior work involving inhibition of BMP signaling. Thus, caution needs to be exercised when using Dmpl-Cre because it targets not only the osteoblast lineage at an earlier stage than previously appreciated, but also a number of non-skeletal cell types.

Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

Bone morphogenetic proteins （Bmp） are well-known to induce bone formation following chondrogenesis, but the direct role of Bmp signaling in the osteoblast lineage is not completely understood. We have recently shown that deletion of the receptor Bmprla in the osteoblast lineage with Dmpl-Cre reduces osteoblast activity in general but stimulates proliferation of preosteoblasts specifically in the cancellous bone region, resulting in diminished periosteal bone growth juxtaposed with excessive cancellous bone formation. Because expression of sclerostin （SOST）, a secreted Wnt antagonist, is notably reduced in the Bmprla- deficient osteocytes, we have genetically tested the hypothesis that increased Wnt signaling might mediate the increase in cancellous bone formation in response to Bmprla deletion. Forced expression of human SOST from a Dmpl promoter fragment partially rescues preosteoblast hyperproliferation and cancellous bone overgrowth in the Bmprla mutant mice, demonstrating functional interaction between Bmp and Wnt signaling in the cancellous bone compat^a-tent. To test whether increased Wnt signaling can compensate for the defect in periosteal growth caused by Bmprla deletion, we have generated compound mutants harboring a hyperactive mutation （A214V） in the Wnt receptor Lrp5. However, the mutant Lrp5 does not restore periosteal bone growth in the Bmprla-deficient mice. Thus, Bmp signaling restricts cancellous bone accrual partly through induction of SOST that limits preosteoblast proliferation, but promotes periosteal bone growth apparently independently of Wnt activation.

Inducible expression of Wnt7b promotes bone formation in aged mice and enhances fracture healing

There remain unmet clinical needs for safe and effective bone anabolic therapies to treat aging-related osteoporosis and to improve fracture healing in cases of nonunion or delayed union. Wnt signaling has emerged as a promising target pathway for developing novel bone anabolic drugs. Although neutralizing antibodies against the Wnt antagonist sclerostin have been tested,Wnt ligands themselves have not been fully explored as a potential therapy. Previous work has demonstrated Wnt7b as an endogenous ligand upregulated during osteoblast differentiation, and that Wnt7b overexpression potently stimulates bone accrual in the mouse. The earlier studies however did not address whether Wnt7b could promote bone formation when specifically applied to aged or fractured bones. Here we have developed a doxycycline-inducible strategy where Wnt7b is temporally induced in the bones of aged mice or during fracture healing. We report that forced expression of Wnt7b for 1 month starting at 15 months of age greatly stimulated trabecular and endosteal bone formation, resulting in a marked increase in bone mass. We further tested the effect of Wnt7b on bone healing in a murine closed femur fracture model. Induced expression of Wnt7b at the onset of fracture did not affect the initial cartilage formation but promoted mineralization of the subsequent bone callus. Thus, targeted delivery of Wnt7b to aged bones or fracture sites may be explored as a potential therapy.

Wnt7b can replace Ihh to induce hypertrophic cartilage vascularization but not osteoblast differentiation during endochondral bone development

Indian hedgehog （Ihh） is an essential signal that regulates endochondral bone development. We have previously shown that Wnt7b promotes osteoblast differentiation during mouse embryogenesis, and that its expression in the perichondrium is dependent on Ihh signaling. To test the hypothesis that Wnt7b may mediate some aspects of Ihh function during endochondral bone development, we activated Wnt7b expression from the R26-Wnt7b allele with Col2-Cre in the Ihh-/- mouse. Artificial expression of Wnt7b rescued vascularization of the hypertrophic cartilage in the Ihh-/- mouse, but failed to restore orthotopic osteoblast differentiation in the perichondrium. Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh-/-; Gli3-/- embryo. Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area. Thus, Ihh-dependent expression of Wnt7b in the perichondrium may contribute to vascularization of the hypertrophic cartilage during endochondral bone development.