Mutations in the Wnt co-receptor LRP5 alter bone mass in humans, but the mechanisms responsible for Wnts actions in bone are unclear. To investigate the role of the classical Wnt signaling pathway in osteogenesis, we generated mice lacking the ␤-catenin or adenomatous polyposis coli (Apc) genes in osteoblasts. Loss of ␤-catenin produced severe osteopenia with striking increases in osteoclasts, whereas constitutive activation of ␤-catenin in the conditional Apc mutants resulted in dramatically increased bone deposition and a disappearance of osteoclasts. In vitro, osteoblasts lacking the ␤-catenin gene exhibited impaired maturation and mineralization with elevated expression of the osteoclast differentiation factor, receptor activated by nuclear factor-B ligand (RANKL), and diminished expression of the RANKL decoy receptor, osteoprotegerin. By contrast, Apc-deficient osteoblasts matured normally but demonstrated decreased expression of RANKL and increased osteoprotegerin. These findings suggest that Wnt/␤-catenin signaling in osteoblasts coordinates postnatal bone acquisition by controlling the differentiation and activity of both osteoblasts and osteoclasts.

Tumor necrosis factor-␣ (TNF) and the ligand for receptor activator of NF-B (RANKL) are abundant in sites of inflammatory bone erosion. Because these cytokines are potent osteoclastogenic factors and because their signaling pathways are considerably overlapping, we postulated that under pro-inflammatory conditions RANKL and TNF might synergistically orchestrate enhanced osteoclastogenesis via cooperative mechanisms. We found TNF, via TNF type 1 receptor (TNFr1), prompts robust osteoclastogenesis by osteoclast precursors pretreated with RANKL, and deletion of TNFr1 abrogates this response. Enhanced osteoclastogenesis is associated with high expression of otherwise TNF and RANKL-induced mediators, including c-Src, TRAF2, TRAF6, and MEKK-1, levels of which were notably reduced in TNFr1 knockouts. Recruitment of TRAFs and MEKK1 leads to activation of downstream pathways, primarily IB/NF-B, ERKs, and cJun/AP-1. Consistent with impaired osteoclastogenesis and reduced expression of TRAFs and MEKK1, we found that phosphorylation and activation of IB, NF-B, ERKs, and cJun/AP-1 are severely reduced in RANKL-treated TNFr1-null osteoclast precursors compared with wild type counterparts. Finally, we found that TNF and RANKL synergistically up-regulate RANK expression in wild type precursors, whereas basal and stimulated levels of RANK are significantly lower in TNFr1 knockout cells. Our data suggest that exuberant TNF-induced osteoclastogensis is the result of coupling between RANK and TNFr1 and is dependent upon signals transmitted by the latter receptor.

No abstract

Signaling through the IGF-I receptor by locally synthesized IGF-I or IGF-II is crucial for normal skeletal development and for bone remodeling. Osteogenesis is primarily regulated by bone morphogenetic proteins (BMPs), which activate gene expression programs driven by bone-specific transcription factors. In a mesenchymal stem cell model of osteoblast commitment and differentiation controlled by BMP2, we show that an inhibitor of PI3-kinase or a dominant-negative Akt were as potent in preventing osteoblast differentiation as the IGF binding protein IGFBP5, whereas a Mek inhibitor was ineffective. Conversely, an adenovirus encoding an inducible-active Akt was able to overcome the blockade of differentiation caused by IGFBP5 or the PI3-kinase inhibitor, and could restore normal osteogenesis. Inhibition of PI3-kinase or Akt did not block BMP2-mediated signaling, because the Smad-responsive genes Sox9 and JunB were induced normally under all experimental conditions. When activated during different stages of osteoblast maturation, dominant-negative Akt prevented accumulation of bone-specific alkaline phosphatase and reduced mineralization, and more significantly inhibited the longitudinal growth of metatarsal bones in primary culture by interfering with both chondrocyte and osteoblast development and function. We conclude that an intact IGF-induced PI3-kinase–Akt signaling cascade is essential for BMP2-activated osteoblast differentiation and maturation, bone development and growth, and suggest that manipulation of this pathway could facilitate bone remodeling and fracture repair.

Mesenchymal stem cells are essential for repair of bone and other supporting tissues. Bone morphogenetic proteins (BMPs) promote commitment of these progenitors toward an osteoblast fate via functional interactions with osteogenic transcription factors, including Dlx3, Dlx5, and Runx2, and also can direct their differentiation into bone-forming cells. BMP-2-stimulated osteoblast differentiation additionally requires continual signaling from insulin-like growth factor (IGF)-activated pathways. Here we identify Akt2 as a critical mediator of IGF-regulated osteogenesis. Targeted knockdown of Akt2 in mouse primary bone marrow stromal cells or in a mesenchymal stem cell line, or genetic knockout of Akt2, did not interfere with BMP-2-mediated signaling but resulted in inhibition of osteoblast differentiation at an early step that preceded production of Runx2. In contrast, Akt1-deficient cells differentiated normally. Complete biochemical and morphological osteoblast differentiation was restored in cells lacking Akt2 by adenoviral delivery of Runx2 or by a recombinant lentivirus encoding wild-type Akt2. In contrast, lentiviral Akt1 was ineffective. Taken together, these observations define a specific role for Akt2 as a gatekeeper of osteogenic differentiation through regulation of Runx2 gene expression and indicate that the closely related Akt1 and Akt2 exert distinct effects on the differentiation of mesenchymal precursors.Continual bone remodeling is necessary to maintain bone integrity and function throughout life. In the adult skeleton, optimal bone growth, remodeling, and repair require a balance between bone-forming osteoblasts and bone-resorbing osteoclasts (22,43,58). Osteoclasts are of hematopoietic origin, while osteoblasts are derived from pluripotent mesenchymal stem cells (43,48,50). With aging and under conditions such as osteoporosis, bone formation is diminished relative to the rate of resorption, leading to net bone loss and an increased risk of fractures (43,48). Under both normal and pathological conditions, multiple local and systemic signals derived from hormones, growth factors, and other agents control different aspects of bone remodeling (43, 58). Among key factors that promote bone formation are bone morphogenetic proteins (BMPs) and insulin-like growth factors (IGFs) (30,43,58).BMPs, members of the transforming growth factor ␤ (TGF-␤) superfamily, are potent inducers of osteoblast differentiation from mesenchymal progenitors (47) and have been used clinically to promote fracture repair (7,25). BMPs bind to specific transmembrane type I and type II receptors and, by sequentially inducing receptor serine kinase activity, stimulate the intracellular mediators, Smad1, -5, and -8 (16), which transmit the BMP signal into the nucleus to regulate target gene transcription (31). Among osteogenic genes induced by BMP-activated Smads are those encoding the osteoblast determination and differentiation factors Dlx3, Dlx5, and Runx2 (14,15,28,29). Runx2 in particular is a master regulator of osteoblast fate...

Growth hormone (GH) affects bone size and mass in part through stimulating insulin-like growth factor type 1 (IGF-1) production in liver and bone. Whether GH acts independent of IGF-1 in bone remains unclear. To define the mode of GH action in bone, we have used a Cre/loxP system in which the type 1 IGF-1 receptor (Igf1r) has been disrupted specifically in osteoblasts in vitro and in vivo. Calvarial osteoblasts from mice homozygous for the floxed IGF-1R allele (IGF-1R flox/flox ) were infected with adenoviral vectors expressing Cre. Disruption of IGF-1R mRNA (>90%) was accompanied by near elimination of IGF-1R protein but retention of GHR protein. GH-induced STAT5 activation was consistently greater in osteoblasts with an intact IGF-1R. Osteoblasts lacking IGF-1R retained GH-induced ERK and Akt phosphorylation and GH-stimulated IGF-1 and IGFBP-3 mRNA expression. GH-induced osteoblast proliferation was abolished by Cre-mediated disruption of the IGF-1R or co-incubation of cells with an IGF-1-neutralizing antibody. By contrast, GH inhibited apoptosis in osteoblasts lacking the IGF-1R. To examine the effects of GH on osteoblasts in vivo, mice wild type for the IGF-1R treated with GH subcutaneously for 7 days showed a doubling in the number of osteoblasts lining trabecular bone, whereas osteoblast numbers in similarly treated mice lacking the IGF-1R in osteoblasts were not significantly affected. These results indicate that although direct IGF-1R-independent actions of GH on osteoblast apoptosis can be demonstrated in vitro, IGF-1R is required for anabolic effects of GH in osteoblasts in vivo.The process of osteogenesis and remodeling of the skeleton is orchestrated by a constellation of local growth factors, cytokines, and systemic hormones (1, 2), of which growth hormone (GH) 2 and insulin-like growth factor type 1 (IGF-1) are key components. GH belongs to a family of cytokine peptides (3) and is produced and stored by somatotroph cells within the anterior pituitary. GH actions are mediated by binding to the transmembrane GHR, thereby triggering increased association with and activation of Janus kinases (JAKs) (4 -6) to activate signal transducers and activators of transcription (STATs) (7-15), phosphatidylinositol 3-kinase/Akt (16,17),. Growth hormone exerts many, but not all (21), of its effects by stimulation of IGF-1 from liver and peripheral tissues. IGF-1 is a small polypeptide with homology to pro-insulin that is produced by a number of cell types. IGF-1 signals via the type 1 IGF-1 receptor (IGF-1R), engaging ERK and phosphatidylinositol 3-kinase pathways through Src homology 2 domain-containing proteins and insulin receptor substrates-1 and 2 (22, 23). The effects of IGF-1 on bone have been well documented. IGF-1 has been shown to induce proliferation of MC3T3 osteoblast-like cells (24) and is an important survival factor for many mammalian cell types, including osteoblasts. IGF-1 production increases during the initial phases of fetal rat calvarial osteoblast differentiation in vitro and then declines wi...

Signaling through the IGF-I receptor by locally synthesized IGF-I or IGF-II is critical for normal skeletal development and for bone remodeling and repair throughout the lifespan. In most tissues, IGF actions are modulated by IGF-binding proteins (IGFBPs). IGFBP-5 is the most abundant IGFBP in bone, and previous studies have suggested that it may either enhance or inhibit osteoblast differentiation in culture and may facilitate or block bone growth in vivo. To resolve these contradictory observations and discern the mechanisms of action of IGFBP-5 in bone, we studied its effects in differentiating osteoblasts and in primary bone cultures. Purified wild-type (WT) mouse IGFBP-5 or a recombinant adenovirus expressing IGFBP-5WT each prevented osteogenic differentiation induced by the cytokine bone morphogenetic protein (BMP)-2 at its earliest stages without interfering with BMP-mediated signaling, whereas an analog with reduced IGF binding (N domain mutant) was ineffective. When added at later phases of bone cell maturation, IGFBP-5WT but not IGFBP-5N blocked mineralization, prevented longitudinal growth of mouse metatarsal bones in short-term primary culture, and inhibited their endochondral ossification. Because an IGF-I variant (R3IGF-I) with diminished affinity for IGFBPs promoted full osteogenic differentiation in the presence of IGFBP-5WT, our results show that IGFBP-5 interferes with IGF action in osteoblasts and provides a framework for discerning mechanisms of collaboration between signal transduction pathways activated by BMPs and IGFs in bone.

Maintaining optimal bone integrity, mass, and strength throughout adult life requires ongoing bone remodeling, which involves coordinated activity between actions of bone-resorbing osteoclasts and bone forming-osteoblasts. Osteoporosis is a disorder of remodeling in which bone resorption outstrips deposition, leading to diminished bone mass and an increased risk of fractures. Here we identify Akt1 as a unique signaling intermediate in osteoblasts that can control both osteoblast and osteoclast differentiation. Targeted knockdown of Akt1 in mouse primary bone marrow stromal cells or in a mesenchymal stem cell line or genetic knockout of Akt1 stimulated osteoblast differentiation secondary to increased expression of the osteogenic transcription factor Runx2. Despite enhanced osteoblast differentiation, coupled osteoclastogenesis in Akt1 deficiency was markedly inhibited, with reduced accumulation of specific osteoclast mRNAs and proteins and impaired fusion to form multinucleated osteoclasts, defects secondary to diminished production of receptor activator of NF-B ligand (RANKL) and macrophage colony-stimulating factor (m-CSF), critical osteoblast-derived osteoclast differentiation factors. Delivery of recombinant lentiviruses encoding Akt1 but not Akt2 to Akt1-deficient osteoblast progenitors reversed the increased osteoblast differentiation and, by boosting accumulation of RANKL and m-CSF, restored normal osteoclastogenesis, as did the addition of recombinant RANKL to conditioned culture medium from Akt1-deficient osteoblasts. Our results support the idea that targeted inhibition of Akt1 could lead to therapeutically useful net bone acquisition, and they indicate that closely related Akt1 and Akt2 exert distinct effects on cellular differentiation pathways.