Natalie A. Sims

Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs.

In the bone marrow, hematopoietic stem cells (HSCs) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate the regulation of these endosteal niches, we studied the mobilization of HSCs into the bloodstream in response to granulocyte colony-stimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts, leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression, and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed, in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the: (1) loss of endosteal osteoblasts and (2) marked reduction of HSC-trophic cytokines at the endosteum, with (3) HSC mobilization into the blood, as observed during G-CSF administration. Together, these results establish that bone marrow macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSCs into the blood.

Rb Regulates Interactions between Hematopoietic Stem Cells and Their Bone Marrow Microenvironment

Hematopoiesis is maintained by stem cells (HSCs) that undergo fate decisions by integrating intrinsic and extrinsic signals, with the latter derived from the bone marrow (BM) microenvironment. Cell-cycle regulation can modulate stem cell fate, but it is unknown whether this represents an intrinsic or extrinsic effector of fate decisions. We have investigated the role of the retinoblastoma protein (RB), a central regulator of the cell cycle, in hematopoiesis. Widespread inactivation of RB in the murine hematopoietic system resulted in profound myeloproliferation. HSCs were lost from the BM due to mobilization to extramedullary sites and differentiation. This phenotype was not intrinsic to HSCs, but, rather, was the consequence of an RB-dependent interaction between myeloid-derived cells and the microenvironment. These findings demonstrate that myeloproliferation may result from perturbed interactions between hematopoietic cells and the niche. Therefore, RB extrinsically regulates HSCs by maintaining the capacity of the BM to support normal hematopoiesis and HSCs.

Activated parathyroid hormone/parathyroid hormone–related protein receptor in osteoblastic cells differentially affects cortical and trabecular bone

Parathyroid hormone (PTH), an important regulator of calcium homeostasis, targets most of its complex actions in bone to cells of the osteoblast lineage. Furthermore, PTH is known to stimulate osteoclastogenesis indirectly through activation of osteoblastic cells. To assess the role of the PTH/PTH-related protein receptor (PPR) in mediating the diverse actions of PTH on bone in vivo, we generated mice that express, in cells of the osteoblastic lineage, one of the constitutively active receptors described in Jansens metaphyseal chondrodysplasia. In these transgenic mice, osteoblastic function was increased in the trabecular and endosteal compartments, whereas it was decreased in the periosteum. In trabecular bone of the transgenic mice, there was an increase in osteoblast precursors, as well as in mature osteoblasts. Osteoblastic expression of the constitutively active PPR induced a dramatic increase in osteoclast number in both trabecular and compact bone in transgenic animals. The net effect of these actions was a substantial increase in trabecular bone volume and a decrease in cortical bone thickness of the long bones. These findings, for the first time to our knowledge, identify the PPR as a crucial mediator of both bone-forming and bone-resorbing actions of PTH, and they underline the complexity and heterogeneity of the osteoblast population and/or their regulatory microenvironment.

Deletion of estrogen receptors reveals a regulatory role for estrogen receptors-β in bone remodeling in females but not in males

To determine the contributions of estrogen receptor (ER)alpha and ERbeta in bone growth and remodeling in male and female mice, we generated and analyzed full knockouts for each receptor, and a double ER knockout. Although suppression of the ligand to the ERs (i.e., estradiol) after menopause or gonadectomy in females led to a catastrophic increase in bone turnover and concomitant bone loss, deletion of one or both ERs failed to show such an effect. Complete deletion of ERalpha led to a decrease, not an increase, in bone turnover and an increase, not a decrease, in trabecular bone volume in both male and female animals. Deletion of ERbeta led to different responses in males, where bone was unaffected, and in females, where bone resorption was decreased and trabecular bone volume increased. In contrast, deletion of both ERs led to a profound decrease in trabecular bone volume in females, which was associated with a decrease, not an increase, in bone turnover. Finally, deletion of ERalpha, but not ERbeta, led to major changes in circulating levels of estradiol and/or testosterone, indirectly affecting bone remodeling and bone mass. Thus, only ERalpha was shown to regulate bone remodeling in males, whereas in females both receptor subtypes influenced this process and could, at least under basal knockout conditions, compensate for each other.

Overexpression of ΔFosB transcription factor(s) increases boneformation and inhibits adipogenesis

Members of the AP-1 family of transcription factors participate in the regulation of bone cell proliferation and differentiation. We report here a potent AP-1-related regulator of osteoblast function: ΔFosB, a naturally occurring truncated form of FosB that arises from alternative splicing of the fosB transcript and is expressed in osteoblasts. Overexpression of ΔFosB in transgenic mice leads to increased bone formation throughout the skeleton and a continuous post-developmental increase in bone mass, leading to osteosclerosis. In contrast, ΔFosB inhibits adipogenesis both in vivo and in vitro, and downregulates the expression of early markers of adipocyte differentiation. Because osteoblasts and adipocytes are thought to share a common precursor, it is concluded that ΔFosB transcriptionally regulates osteoblastogenesis, possibly at the expense of adipogenesis.

Coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit

Coupling between bone formation and bone resorption refers to the process within basic multicellular units in which resorption by osteoclasts is met by the generation of osteoblasts from precursors, and their bone-forming activity, which needs to be sufficient to replace the bone lost. There are many sources of activities that contribute to coupling at remodeling sites, including growth factors released from the matrix, soluble and membrane products of osteoclasts and their precursors, signals from osteocytes and from immune cells and signaling taking place within the osteoblast lineage. Coupling is therefore a process that involves the interaction of a wide range of cell types and control mechanisms. As bone remodeling occurs at many sites asynchronously throughout the skeleton, locally generated activities comprise very important control mechanisms. In this review, we explore the potential roles of a number of these factors, including sphingosine-1-phosphate, semaphorins, ephrins, interleukin-6 (IL-6) family cytokines and marrow-derived factors. Their interactions achieve the essential tight control of coupling within individual remodeling units that is required for control of skeletal mass.

Osteoprotegerin Reduces Osteoclast Numbers and Prevents Bone Erosion in Collagen-Induced Arthritis

Rheumatoid arthritis is characterized by progressive synovial inflammation and joint destruction. While matrix metalloproteinases (MMPs) are implicated in the erosion of unmineralized cartilage, bone destruction involves osteoclasts, the specialized cells that resorb calcified bone matrix. RANK ligand (RANKL) expressed by stromal cells and T cells, and its cognate receptor, RANK, were identified as a critical ligand-receptor pair for osteoclast differentiation and survival. A decoy receptor for RANKL, osteoprotegerin, (OPG) impinges on this system and regulates osteoclast numbers and activity. RANKL is also expressed in collagen-induced arthritis (CIA) in which focal collections of osteoclasts are prominent at sites of bone destruction. To determine the role of RANK signaling events in the effector phase of CIA, we investigated effects of Fc-osteoprotegerin fusion protein (Fc-OPG) in CIA. After induction of CIA in Dark Agouti rats, test animals were treated with or without Fc-OPG (3 mg/kg/day) subcutaneously for 5 days, beginning at the onset of disease. Paraffin-embedded joints were then analyzed histologically and the adjacent bone assessed by histomorphometry. Osteoclasts were identified using TRAP staining and expression of the mRNA for OPG and RANKL was identified by in situ hybridization. The results indicated that short-term Fc-OPG effectively prevented joint destruction, even though it had no impact on the inflammatory aspects of CIA. In arthritic joints, Fc-OPG depleted osteoclast numbers by over 75% and diminished bone erosion scores by over 60%. Although cartilage loss was also reduced by Fc-OPG, the effects on cartilage were less striking than those on bone. In arthritic joints OPG mRNA was highly expressed and co-localized with RANK ligand, and treatment with Fc-OPG did not affect the expression of endogenous RANKL or OPG mRNA. These data demonstrate that short term Fc-OPG treatment has powerful anti-erosive effects, principally on bone, even though synovitis is not affected. These findings indicate the potential utility of disrupting RANK signaling to preserve skeletal integrity in inflammatory arthritis.

Bone homeostasis in growth hormone receptor–null mice is restored by IGF-I but independent of Stat5

Growth hormone (GH) regulates both bone growth and remodeling, but it is unclear whether these actions are mediated directly by the GH receptor (GHR) and/or IGF-I signaling. The actions of GH are transduced by the Jak/Stat signaling pathway via Stat5, which is thought to regulate IGF-I expression. To determine the respective roles of GHR and IGF-I in bone growth and remodeling, we examined bones of wild-type, GHR knockout (GHR(-/-)), Stat5ab(-/-), and GHR(-/-) mice treated with IGF-I. Reduced bone growth in GHR(-/-) mice, due to a premature reduction in chondrocyte proliferation and cortical bone growth, was detected after 2 weeks of age. Additionally, although trabecular bone volume was unchanged, bone turnover was significantly reduced in GHR(-/-) mice, indicating GH involvement in the high bone-turnover level during growth. IGF-I treatment almost completely rescued all effects of the GHR(-/-) on both bone growth and remodeling, supporting a direct effect of IGF-I on both osteoblasts and chondrocytes. Whereas bone length was reduced in Stat5ab(-/-) mice, there was no reduction in trabecular bone remodeling or growth-plate width as observed in GHR(-/-) mice, indicating that the effects of GH in bone may not involve Stat5 activation.

Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcoma, and targeted disruption accelerates osteosarcomagenesis in mice

Wnt signaling increases bone mass by stimulating osteoblast lineage commitment and expansion and forms the basis for novel anabolic therapeutic strategies being developed for osteoporosis. These strategies include derepression of Wnt signaling by targeting secreted Wnt pathway antagonists, such as sclerostin. However, such therapies are associated with safety concerns regarding an increased risk of osteosarcoma, the most common primary malignancy of bone. Here, we analyzed 5 human osteosarcoma cell lines in a high-throughput screen for epigenetically silenced tumor suppressor genes and identified Wnt inhibitory factor 1 (WIF1), which encodes an endogenous secreted Wnt pathway antagonist, as a candidate tumor suppressor gene. In vitro, WIF1 suppressed beta-catenin levels in human osteosarcoma cell lines, induced differentiation of human and mouse primary osteoblasts, and suppressed the growth of mouse and human osteosarcoma cell lines. Wif1 was highly expressed in the developing and mature mouse skeleton, and, although it was dispensable for normal development, targeted deletion of mouse Wif1 accelerated development of radiation-induced osteosarcomas in vivo. In primary human osteosarcomas, silencing of WIF1 by promoter hypermethylation was associated with loss of differentiation, increased beta-catenin levels, and increased proliferation. These data lead us to suggest that derepression of Wnt signaling by targeting secreted Wnt antagonists in osteoblasts may increase susceptibility to osteosarcoma.

Terminal Osteoblast Differentiation, Mediated by runx2 and p27KIP1, Is Disrupted in Osteosarcoma

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.