Summary The molecular complexity of the bone marrow (BM) microenvironment and its response to stress are incompletely understood, despite its key role in the regulation of hematopoiesis. Here we map the transcriptional landscape of BM vascular, perivascular, and osteoblast niche populations at single-cell resolution at both homeostasis and under stress hematopoiesis. This analysis revealed a previously unappreciated level of cellular heterogeneity within the BM niche, identified novel cellular subsets, and resolved cellular sources of pro-hematopoietic growth factors, chemokines, and membrane-bound ligands. Under conditions of stress, our studies revealed a significant transcriptional remodeling of these niche elements, including an adipocytic skewing of the perivascular cells. Among the stress-induced changes, we observed that vascular Notch ligand delta-like ligands (Dll1,4) were downregulated. In the absence of vascular Dll4, hematopoietic stem cells (HSC) prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the BM niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress, and illustrate the utility of single cell transcriptomic data in systematically evaluating the regulation of hematopoiesis by discrete niche populations.

Both aging and loss of sex steroids have adverse effects on skeletal homeostasis, but whether and how they may influence each others negative impact on bone remains unknown. We report herein that both female and male C57BL/6 mice progressively lost strength (as determined by load-to-failure measurements) and bone mineral density in the spine and femur between the ages of 4 and 31 months. These changes were temporally associated with decreased rate of remodeling as evidenced by decreased osteoblast and osteoclast numbers and decreased bone formation rate; as well as increased osteoblast and osteocyte apoptosis, increased reactive oxygen species levels, and decreased glutathione reductase activity and a corresponding increase in the phosphorylation of p53 and p66 shc , two key components of a signaling cascade that are activated by reactive oxygen species and influences apoptosis and lifespan. Exactly the same changes in oxidative stress were acutely reproduced by gonadectomy in 5-month-old females or males and reversed by estrogens or androgens in vivo as well as in vitro. We conclude that the oxidative stress that underlies physiologic organismal aging in mice may be a pivotal pathogenetic mechanism of the age-related bone loss and strength. Loss of estrogens or androgens accelerates the effects of aging on bone by decreasing defense against oxidative stress.Age-related loss of bone mass and strength is an invariable feature of human biology, affecting women and men alike. Moreover, population-based studies demonstrate that substantial bone loss begins as early as the 20s in young adult women and men, long before any hormonal changes (1).3 The extent to which estrogen deficiency contributes to age-related bone loss and the slower rate of decline of bone mass and strength during the late postmenopausal years, and the molecular and cellular mechanisms of such putative interactions, are unknown.The universality of age-associated bone loss irrespective of sex steroid status notwithstanding, age is by far a more critical determinant of fracture risk than bone mass in humans indicating that age-related increase in fracture risk reflects a loss of bone strength that is only partly accounted for by loss of bone mass (2). Whereas an increased propensity to fall due to agerelated decline in neuromuscular function is a factor, there are also age-related changes in the bone itself. Such changes include disrupted architecture, altered composition of the bone mineral and matrix, delayed repair of fatigue microdamage, excessive turnover, and inadequate bone size (3-7). The most recently appreciated qualitative factor is loss of osteocytes (8, 9), former osteoblasts entombed into the mineralized matrix. Osteocyte death may influence the signals necessary for mechanical adaptation and repair and also lead to long term changes in bone hydration. The anti-apoptotic effect of sex steroids on osteocytes, which has been well documented in mice, rats, and humans (10 -12), may contribute to their anti-fracture efficacy independently of...

SummaryCells of the osteoblast lineage affect homing, 1, 2 number of long term repopulating hematopoietic stem cells (HSCs) 3, 4, HSC mobilization and lineage determination and B lymphopoiesis 5-8. More recently osteoblasts were implicated in pre-leukemic conditions in mice 9, 10. Yet, it has not been shown that a single genetic event taking place in osteoblasts can induce leukemogenesis. We show here that in mice, an activating mutation of β-catenin in osteoblasts alters the differentiation potential of myeloid and lymphoid progenitors leading to development of acute myeloid leukemia (AML) with common chromosomal aberrations and cell autonomous progression. Activated β-catenin stimulates expression of the Notch ligand Jagged-1 in osteoblasts. Subsequent activation of Notch signaling in HSC progenitors induces the malignant changes. Demonstrating the pathogenetic role of the Notch pathway, genetic or pharmacological inhibition of Notch signaling ameliorates AML. Nuclear accumulation and increased β-catenin signaling in osteoblasts was also identified in 38% of patients with MDS/AML. These patients showed increased Notch signaling in hematopoietic cells. These findings demonstrate that genetic alterations in osteoblasts can induce AML, identify molecular signals leading to this transformation and suggest a potential novel pharmacotherapeutic approach to AML.

Bone has recently emerged as a pleiotropic endocrine organ that secretes at least two hormones, FGF23 and osteocalcin, which regulate kidney function and glucose homeostasis, respectively. These findings have raised the question of whether other bone-derived hormones exist and what their potential functions are. Here we identify, through molecular and genetic analyses in mice, lipocalin 2 (LCN2) as an osteoblast-enriched, secreted protein. Loss- and gain-of-function experiments in mice demonstrate that osteoblast-derived LCN2 maintains glucose homeostasis by inducing insulin secretion and improves glucose tolerance and insulin sensitivity. In addition, osteoblast-derived LCN2 inhibits food intake. LCN2 crosses the blood–brain barrier, binds to the melanocortin 4 receptor (MC4R) in the paraventricular and ventromedial neurons of the hypothalamus and activates an MC4R-dependent anorexigenic (appetite-suppressing) pathway. These results identify LCN2 as a bone-derived hormone with metabolic regulatory effects, which suppresses appetite in a MC4R-dependent manner, and show that the control of appetite is an endocrine function of bone.

Summary Osteoporosis, a low bone mass disease, is associated with decreased osteoblast numbers and increased levels of oxidative stress in these cells. Since the FoxO family of transcription factors, confers stress resistance, we investigated their potential impact on skeletal integrity. We show here through cell-specific deletion and molecular analyses that, among the 3 FoxO proteins, only FoxO1 is required for proliferation and redox balance in osteoblasts, and as a result controls bone formation. FoxO1 regulation of osteoblast proliferation occurs because of its interaction with ATF4, a transcription factor regulating amino acid import; and of its regulation of a stress-dependent pathway influencing p53 signaling. Accordingly, decreasing oxidative stress levels or increasing protein intake normalizes bone formation and bone mass in mice lacking FoxO1 in osteoblasts only. These results identify FoxO1 as a crucial regulator of osteoblast physiology and provide a direct mechanistic link between oxidative stress and the regulation of bone remodeling.