Noriaki Ono

Osterix Marks Distinct Waves of Primitive and Definitive Stromal Progenitors during Bone Marrow Development

Mesenchymal stem and progenitor cells (MSPCs) contribute to bone marrow (BM) homeostasis by generating multiple types of stromal cells. MSPCs can be labeled in the adult BM by Nestin-GFP, whereas committed osteoblast progenitors are marked by Osterix expression. However, the developmental origin and hierarchical relationship of stromal cells remain largely unknown. Here, by using a lineage-tracing system, we describe three distinct waves of contributions of Osterix(+) cells in the BM. First, Osterix(+) progenitors in the fetal BM contribute to nascent bone tissues and transient stromal cells that are replaced in the adult marrow. Second, Osterix-expressing cells perinatally contribute to osteolineages and long-lived BM stroma, which have characteristics of Nestin-GFP(+) MSPCs. Third, Osterix labeling in the adult marrow is osteolineage-restricted, devoid of stromal contribution. These results uncover a broad expression profile of Osterix and raise the intriguing possibility that distinct waves of stromal cells, primitive and definitive, may organize the developing BM.

A subset of chondrogenic cells provides early mesenchymal progenitors in growing bones

The hallmark of endochondral bone development is the presence of cartilaginous templates, in which osteoblasts and stromal cells are generated to form mineralized matrix and support bone marrow haematopoiesis. However, the ultimate source of these mesenchymal cells and the relationship between bone progenitors in fetal life and those in later life are unknown. Fate-mapping studies revealed that cells expressing cre-recombinases driven by the collagen II (Col2) promoter/enhancer and their descendants contributed to, in addition to chondrocytes, early perichondrial precursors before Runx2 expression and, subsequently, to a majority of osteoblasts, Cxcl12 (chemokine (C–X–C motif) ligand 12)-abundant stromal cells and bone marrow stromal/mesenchymal progenitor cells in postnatal life. Lineage-tracing experiments using a tamoxifen-inducible creER system further revealed that early postnatal cells marked by Col2–creER, as well as Sox9–creER and aggrecan (Acan)–creER, progressively contributed to multiple mesenchymal lineages and continued to provide descendants for over a year. These cells are distinct from adult mesenchymal progenitors and thus provide opportunities for regulating the explosive growth that occurs uniquely in growing mammals.

Loss of wnt/β-catenin signaling causes cell fate shift of preosteoblasts from osteoblasts to adipocytes.

Wnt signaling is essential for osteogenesis and also functions as an adipogenic switch, but it is not known if interrupting wnt signaling via knockout of β‐catenin from osteoblasts would cause bone marrow adiposity. Here, we determined whether postnatal deletion of β‐catenin in preosteoblasts, through conditional cre expression driven by the osterix promoter, causes bone marrow adiposity. Postnatal disruption of β‐catenin in the preosteoblasts led to extensive bone marrow adiposity and low bone mass in adult mice. In cultured bone marrow–derived cells isolated from the knockout mice, adipogenic differentiation was dramatically increased, whereas osteogenic differentiation was significantly decreased. As myoblasts, in the absence of wnt/β‐catenin signaling, can be reprogrammed into the adipocyte lineage, we sought to determine whether the increased adipogenesis we observed partly resulted from a cell‐fate shift of preosteoblasts that had to express osterix (lineage‐committed early osteoblasts), from the osteoblastic to the adipocyte lineage. Using lineage tracing both in vivo and in vitro we showed that the loss of β‐catenin from preosteoblasts caused a cell‐fate shift of these cells from osteoblasts to adipocytes, a shift that may at least partly contribute to the bone marrow adiposity and low bone mass in the knockout mice. These novel findings indicate that wnt/β‐catenin signaling exerts control over the fate of lineage‐committed early osteoblasts, with respect to their differentiation into osteoblastic versus adipocytic populations in bone, and thus offers potential insight into the origin of bone marrow adiposity.

Vasculature-Associated Cells Expressing Nestin in Developing Bones Encompass Early Cells in the Osteoblast and Endothelial Lineage

Nestin-positive (Nes(+)) cells are important hematopoiesis-supporting constituents in adult bone marrow. However, how these cells originate during endochondral bone development is unknown. Studies using mice expressing GFP under the direction of nestin promoter/enhancer (Nes-GFP) revealed distinct endothelial and nonendothelial Nes(+) cells in the embryonic perichondrium; the latter were early cells of the osteoblast lineage immediately descended from their progenitors upon Indian hedgehog action and Runx2 expression. During vascular invasion and formation of ossification centers, these Nes(+) cells were closely associated with each other and increased in number progressively. Interestingly, cells targeted by tamoxifen-inducible cre recombinase driven by nestin enhancer (Nes-creER) in developing bone marrow were predominantly endothelial cells. Furthermore, Nes(+) cells in postnatal bones were heterogeneous populations, including a range of cells in the osteoblast and endothelial lineage. These findings reveal an emerging complexity of stromal populations, accommodating Nes(+) cells as vasculature-associated early cells in the osteoblast and endothelial lineage.

Identification of a Prg4-expressing articular cartilage progenitor cell population in mice.

To generate knockin mice that express a tamoxifen‐inducible Cre recombinase from the Prg4 locus (Prg4GFPCreERt2 mice) and to use these animals to fate‐map the progeny of Prg4‐positive articular cartilage cells at various ages.

Osteopontin Negatively Regulates Parathyroid Hormone Receptor Signaling in Osteoblasts

Systemic hormonal control exerts its effect through the regulation of local target tissues, which in turn regulate upstream signals in a feedback loop. The parathyroid hormone (PTH) axis is a well defined hormonal signaling system that regulates calcium levels and bone metabolism. To understand the interplay between systemic and local signaling in bone, we examined the effects of deficiency of the bone matrix protein osteopontin (OPN) on the systemic effects of PTH specifically within osteoblastic cell lineages. Parathyroid hormone receptor (PPR) transgenic mice expressing a constitutively active form of the receptor (caPPR) specifically in cells of the osteoblast lineage have a high bone mass phenotype. In these mice, OPN deficiency further increased bone mass. This increase was associated with conversion of the major intertrabecular cell population from hematopoietic cells to stromal/osteoblastic cells and parallel elevations in histomorphometric and biochemical parameters of bone formation and resorption. Treatment with small interfering RNA (siRNA) for osteopontin enhanced H223R mutant caPPR-induced cAMP-response element (CRE) activity levels by about 10-fold. Thus, in addition to the well known calcemic feedback system for PTH, local feedback regulation by the bone matrix protein OPN also plays a significant role in the regulation of PTH actions.

Loss of Gsα early in the osteoblast lineage favors adipogenic differentiation of mesenchymal progenitors and committed osteoblast precursors

In humans, aging and glucocorticoid treatment are associated with reduced bone mass and increased marrow adiposity, suggesting that the differentiation of osteoblasts and adipocytes may be coordinately regulated. Within the bone marrow, both osteoblasts and adipocytes are derived from mesenchymal progenitor cells, but the mechanisms guiding the commitment of mesenchymal progenitors into osteoblast versus adipocyte lineages are not fully defined. The heterotrimeric G protein subunit Gsα activates protein kinase A signaling downstream of several G protein‐coupled receptors including the parathyroid hormone receptor, and plays a crucial role in regulating bone mass. Here, we show that targeted ablation of Gsα in early osteoblast precursors, but not in differentiated osteocytes, results in a dramatic increase in bone marrow adipocytes. Mutant mice have reduced numbers of mesenchymal progenitors overall, with an increase in the proportion of progenitors committed to the adipocyte lineage. Furthermore, cells committed to the osteoblast lineage retain adipogenic potential both in vitro and in vivo. These findings have clinical implications for developing therapeutic approaches to direct the commitment of mesenchymal progenitors into the osteoblast lineage.

Constitutively active parathyroid hormone receptor signaling in cells in osteoblastic lineage suppresses mechanical unloading-induced bone resorption.

Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity.

A novel population of cells expressing both hematopoietic and mesenchymal markers is present in the normal adult bone marrow and is augmented in a murine model of marrow fibrosis

Bone marrow (BM) fibrosis is a feature of severe hyperparathyroidism. Consistent with this observation, mice expressing constitutively active parathyroid hormone (PTH)/PTH-related peptide receptors (PPR) in osteoblasts (PPR*Tg) display BM fibrosis. To obtain insight into the nature of BM fibrosis in such a model, a double-mutant mouse expressing constitutively active PPR and green fluorescent protein (GFP) under the control of the type I collagen promoter (PPR*Tg/GFP) was generated. Confocal microscopy and flow cytometry revealed the presence of a cell population expressing GFP (GFP(+)) that was also positive for the hematopoietic marker CD45 in the BM of both PPR*Tg/GFP and control animals. This cell population was expanded in PPR*Tg/GFP. The existence of cells expressing both type I collagen and CD45 in the adult BM was confirmed by IHC and fluorescence-activated cell sorting. An analysis of total RNA extracted from sorted GFP(+)CD45(+) cells showed that these cells produced type I collagen and PTH/PTH-related peptide receptor and receptor activator for NF-κB mRNAs, further supporting their features of being both mesenchymal and hematopoietic lineages. Similar cells, known as fibrocytes, are also present in pathological fibroses. Our findings, thus, indicate that the BM is a permissive microenvironment for the differentiation of fibrocyte-like cells and raise the possibility that these cells could contribute to the pathogenesis of BM fibrosis.

Constitutively Active PTH/PTHrP Receptor Specifically Expressed in Osteoblasts Enhances Bone Formation Induced by Bone Marrow Ablation

Bone is maintained by continuous bone formation by osteoblasts provided by proliferation and differentiation of osteoprogenitors. Parathyroid hormone (PTH) activates bone formation, but because of the complexity of cells in the osteoblast lineage, how these osteoprogenitors are regulated by PTH in vivo is incompletely understood. To elucidate how signals by PTH in differentiated osteoblasts regulate osteoprogenitors in vivo, we conducted bone marrow ablation using Col1a1‐constitutively active PTH/PTHrP receptor (caPPR) transgenic mice. These mice express caPPR specifically in osteoblasts by using 2.3 kb Col1a1 promoter and showed higher trabecular bone volume under steady‐state conditions. In contrast, after bone marrow ablation, stromal cells recruited from bone surface extensively proliferated in the marrow cavity in transgenic mice, compared to limited proliferation in wild‐type mice. Whereas de novo bone formation was restricted to the ablated area in wild‐type mice, the entire marrow cavity, including not only ablated area but also outside the ablated area, was filled with newly formed bone in transgenic mice. Bone mineral density was significantly increased after ablation in transgenic mice. Bone marrow cell culture in osteogenic medium revealed that alkaline phosphatase‐positive area was markedly increased in the cells obtained from transgenic mice. Furthermore, mRNA expression of Wnt‐signaling molecules such as LRP5, Wnt7b, and Wnt10b were upregulated after marrow ablation in bone marrow cells of transgenic mice. These results indicate that constitutive activation of PTH/PTHrP receptor in differentiated osteoblasts enhances bone marrow ablation‐induced recruitment, proliferation, and differentiation of osteoprogenitors. J. Cell. Physiol. 227: 408–415, 2012.