Global energy balance in mammals is controlled by the actions of circulating hormones that coordinate fuel production and utilization in metabolically active tissues. Bone-derived osteocalcin, in its undercarboxylated, hormonal form, regulates fat deposition and is a potent insulin secretagogue. Here, we show that insulin receptor (IR) signaling in osteoblasts controls osteoblast development and osteocalcin expression by suppressing the Runx2 inhibitor Twist2. Mice lacking IR in osteoblasts have low circulating undercarboxylated osteocalcin and reduced bone acquisition due to decreased bone formation and deficient numbers of osteoblasts. With age, these mice develop marked peripheral adiposity and hyperglycemia accompanied by severe glucose intolerance and insulin resistance. The metabolic abnormalities in these mice are improved by infusion of exogenous undercarboxylated osteocalcin. These results indicate the existence of a bone-pancreas endocrine loop through which insulin signaling in the osteoblast ensures osteoblast differentiation and stimulates osteocalcin production, which in turn regulates insulin sensitivity and pancreatic insulin secretion to control glucose homeostasis.

Key Points• Deletion of Gs␣ in osteocytes induces severe osteopenia and a dramatic expansion of cells of the myeloid lineage.• Osteocytes regulate hematopoiesis and specifically contribute to myelopoiesis by secreting proliferative factors such as G-CSF.

Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSC) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM)1, and may be the cause of relapse following chemotherapy.2 Targeting the niche is a novel strategy to eliminate persistent and drug-resistant LSC. CD443,4 and IL-65 have been implicated previously in the LSC niche. Transforming growth factor (TGF)-β1 is released during bone remodeling6 and plays a role in maintenance of CML LSCs7, but a role for TGF-β1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell-specific activation of the parathyroid hormone (PTH) receptor8,9 attenuates BCR-ABL1-induced CML-like myeloproliferative neoplasia (MPN)10 but enhances MLL-AF9-induced AML11 in mouse transplantation models, possibly through opposing effects of increased TGF-β1 on the respective LSC. PTH treatment caused a 15-fold decrease in LSCs in wildtype mice with CML-like MPN, and reduced engraftment of immune deficient mice with primary human CML cells. These results demonstrate that LSC niches in chronic and acute myeloid leukemias are distinct, and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSC, a prerequisite for the cure of CML.

Background: Osteocytes, the most abundant cells in adult bone, express PPR. Results: Mice with constitutive PPR deletion in osteocytes demonstrate blunted anabolic and catabolic bone responses and the inability to recruit osteoblasts and osteoclasts upon PTH administration. Conclusion: PPR in osteocytes is needed for a full skeletal response to PTH administration. Significance: PPR signaling in osteocytes is necessary for PTH-driven anabolic effects during osteoporosis therapy.

The heterotrimeric G protein subunit G s α stimulates cAMP-dependent signaling downstream of G proteincoupled receptors. In this study, we set out to determine the role of G s α signaling in cells of the early osteoblast lineage in vivo by conditionally deleting G s α from osterix-expressing cells. This led to severe osteoporosis with fractures at birth, a phenotype that was found to be the consequence of impaired bone formation rather than increased resorption. Osteoblast number was markedly decreased and osteogenic differentiation was accelerated, resulting in the formation of woven bone. Rapid differentiation of mature osteoblasts into matrixembedded osteocytes likely contributed to depletion of the osteoblast pool. In addition, the number of committed osteoblast progenitors was diminished in both bone marrow stromal cells (BMSCs) and calvarial cells of mutant mice. In the absence of G s α, expression of sclerostin and dickkopf1 (Dkk1), inhibitors of canonical Wnt signaling, was markedly increased; this was accompanied by reduced Wnt signaling in the osteoblast lineage. In summary, we have shown that G s α regulates bone formation by at least two distinct mechanisms: facilitating the commitment of mesenchymal progenitors to the osteoblast lineage in association with enhanced Wnt signaling; and restraining the differentiation of committed osteoblasts to enable production of bone of optimal mass, quality, and strength.

Ca2؉ /calmodulin-dependent protein kinase II␣ (␣-CaMKII) was once thought to be exclusively expressed in neuronal tissue, but it is becoming increasingly evident that CaMKII is also expressed in various extraneural cells. CaMKII plays a critical role in regulating various signaling pathways leading to modulation of several aspects of cellular functions, including proliferation, differentiation, cytoskeletal structure, and gene expression. The purpose of this study was to examine the expression of CaMKII in osteoblast-like cells (MC4) and to elucidate its role in osteoblast differentiation. We demonstrated that CaMKII, specifically the ␣ isoform, is expressed in osteoblasts both in vitro and in vivo.

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulinlike growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (⌬IGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In ⌬IGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, ⌬IGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-3 H]-deoxy-D-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of ⌬IGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.Insulin and insulin-like growth factor 1 (IGF-1) 2 are related signaling molecules that evolved from a common ancestor pathway originally involved in sensing and integrating signals arising from nutrient and growth factor availability. During evolution, this primitive receptor pathway diverged into two distinct hormonal systems that in mammals serve different but overlapping developmental and metabolic functions (1, 2). These receptors belong to the family of ligand-activated receptor kinases and are abundantly expressed in osteoblasts (3, 4). Unlike other receptor tyrosine kinases, these proteins exist at the cell surface as homodimers composed of two identical ␣/␤ (␣ 2 /␤ 2 ) monomers or as heterodimers (discussed below) composed of two different receptor monomers (e.g. IR ␣ ␤/IGF-1R ␣ ␤). Upon ligand binding, they undergo a conformational change that facilitates binding to ATP and autophosphorylation (5, 6). Autophosphorylation increases the kinase activity of IR-type receptors by 3 orders of magnitude, enabling them to phosphorylate a number of substrate proteins and engender growth or metabolic responses (7). IRS proteins act as mediators of insulin, IGF, and cytokine signaling in a variety of cell types. These adaptor molecules recruit and activate downstream signaling cascades su...

Cells of the osteoblast lineage are increasingly identified as participants in whole-body metabolism by primarily targeting pancreatic insulin secretion or consuming energy. Osteocytes, the most abundant bone cells, secrete a Wnt-signaling inhibitor called sclerostin. Here we examined three mouse models expressing high sclerostin levels, achieved through constitutive or inducible loss of the stimulatory subunit of G-proteins (Gsα in mature osteoblasts and/or osteocytes). These mice showed progressive loss of white adipose tissue (WAT) with tendency toward increased energy expenditure but no changes in glucose or insulin metabolism. Interestingly beige adipocytes were increased extensively in both gonadal and inguinal WAT and had reduced canonical β-catenin signaling. To determine if sclerostin directly contributes to the increased beige adipogenesis, we engineered an osteocytic cell line lacking Gsα which has high sclerostin secretion. Conditioned media from these cells significantly increased expression of UCP1 in primary adipocytes, and this effect was partially reduced after depletion of sclerostin from the conditioned media. Similarly, treatment of Gsα-deficient animals with sclerostin-neutralizing antibody partially reduced the increased UCP1 expression in WAT. Moreover, direct treatment of sclerostin to wild-type mice significantly increased UCP1 expression in WAT. These results show that osteocytes and/or osteoblasts secrete factors regulating beige adipogenesis, at least in part, through the Wnt-signaling inhibitor sclerostin. Further studies are needed to assess metabolic effects of sclerostin on adipocytes and other metabolic tissues.