Endosteal Osteoblasts Research Articles

Osteocytes regulate bone anabolic response to mechanical loading in male mice via activation of integrin α5

Physical mechanical stimulation can maintain and even increase bone mass. Here, we report an important role of osteocytic integrin α5 in regulating the anabolic response of bone to mechanical loading using an Itga5 conditional gene knockout (cKO) mouse model. Integrin α5 gene deletion increased apoptotic osteocytes and reduced cortical anabolic responses to tibial compression including decreased endosteal osteoblasts and bone formation, and increased endosteal osteoclasts and bone resorption, contributing to the decreased bone area fraction and biomechanical properties, leading to an enlarged bone marrow area in cKO mice. Similar disruption of anabolic responses to mechanical loading was also detected in cKO trabecular bone. Moreover, integrin α5 deficiency impeded load-induced Cx43 hemichannel opening, and production and release of PGE2, an anabolic factor, resulting in attenuated effects of the loading on catabolic sclerostin (SOST) reduction and anabolic β-catenin increase. Together, this study shows an indispensable role of integrin α5 in osteocytes in the anabolic action of mechanical loading on skeletal tissue through activation of hemichannels and PGE2-evoked gene expression. Integrin α5 could act as a potential new therapeutic target for bone loss, especially in the elderly population with impeded mechanical sensitivity.

Connexin hemichannels with prostaglandin release in anabolic function of bone to mechanical loading.

Mechanical stimulation, such as physical exercise, is essential for bone formation and health. Here, we demonstrate the critical role of osteocytic Cx43 hemichannels in anabolic function of bone in response to mechanical loading. Two transgenic mouse models, R76W and Δ130-136, expressing dominant-negative Cx43 mutants in osteocytes were adopted. Mechanical loading of tibial bone increased cortical bone mass and mechanical properties in wild-type and gap junction-impaired R76W mice through increased PGE2, endosteal osteoblast activity, and decreased sclerostin. These anabolic responses were impeded in gap junction/hemichannel-impaired Δ130-136 mice and accompanied by increased endosteal osteoclast activity. Specific inhibition of Cx43 hemichannels by Cx43(M1) antibody suppressed PGE2 secretion and impeded loading-induced endosteal osteoblast activity, bone formation and anabolic gene expression. PGE2 administration rescued the osteogenic response to mechanical loading impeded by impaired hemichannels. Together, osteocytic Cx43 hemichannels could be a potential new therapeutic target for treating bone loss and osteoporosis.

Cod Liver Oil, but Not Retinoic Acid, Treatment Restores Bone Thickness in a Vitamin A-Deficient Rat.

Vitamin A plays a prominent role for maintaining optimal bone status, but its impact upon the bone in response to vitamin A deficiency is not well defined. The purpose of this study was to evaluate how replenishing vitamin A by either whole food cod liver oil (COD) or the active metabolite of vitamin A, retinoic acid (RA), altered bone thickness of vitamin A-deficient (VAD) rats. Weanling rats were administered a control diet (CTRL) or VAD diet for 9 weeks. This was followed by four weeks of treatment in which the VAD group was divided into the following 4 subgroups: (1) VAD (9 weeks)-VAD (4 weeks); (2) VAD-CTRL; (3) VAD-COD; and (4) VAD-RA. Compared to controls, VAD rats had thicker bones which showed marked dysplasia. VAD-rats treated with COD produced a thinner bone that was not significantly different from that of untreated rats. In contrast, RA did not significantly change the thicker bone, and also had significantly greater periosteal and endosteal osteoblast numbers compared to VAD-COD. Active osteoclasts were not detected in VAD rats, nor during the treatment period. These findings suggest that the abnormal bone thickness in VAD rats appears to be more effectively restored to bone thickness of untreated control rats when treated with COD.

Compound deletion of thrombospondin-1 and -2 results in a skeletal phenotype not predicted by the single gene knockouts.

The trimeric thrombospondin homologs, TSP1 and TSP2, are both components of bone tissue and contribute in redundant and distinct ways to skeletal physiology. TSP1-null mice display increased femoral cross-sectional area and thickness due to periosteal expansion, as well as diminished matrix quality and impaired osteoclast function. TSP2-null mice display increased femoral cross-sectional thickness and reduced marrow area due to increased endosteal osteoblast activity, with very little periosteal expansion. Osteoblast lineage cells are reduced in TSP2-null mice, but not in TSP1-null. The functional effects of combined TSP1 and TSP2 deficiency remain to be elucidated. Here, we examined the spectrum of detergent soluble proteins in diaphyseal cortical bone of growing (6-week old) male and female mice deficient in both thrombospondins (double knockout (DKO)). Of 3429 detected proteins, 195 were differentially abundant in both male and female DKO bones. Physiologically relevant annotation terms identified by Ingenuity Pathway Analysis included "ECM degradation" and "Quantity of Monocytes." Manual inspection revealed that a number of proteins with shared expression among osteoclasts and osteocytes were reduced in DKO bones. To associate changes in protein content with phenotype, we examined 12-week old male and female DKO and WT mice. DKO mice were smaller than WT and in male DKO, femoral cross section area was reduced. Some of the male DKO femora also had a flattened, less circular cross-section. Male DKO bones were less stiff in bending and they displayed reduced ultimate load. Displacements at yield load and at max load were both elevated in male DKO. However, the ratios of post-yield to pre-yield displacements significantly diminished in DKO suggesting proportionally reduced post-yield behavior. Male DKO mice also exhibited reductions in trabecular bone mass, which were surprisingly associated with equivalent osteoblast numbers and accordingly increased osteoblast surface. Marrow-derived colony forming unit-fibroblastic was reduced in male and female DKO mice. Together our data suggest that when both TSP1 and TSP2 are absent, a unique, sex-specific bone phenotype not predicted by the single knockouts, is manifested.

Mature Osteoblasts Are Critical Bone Marrow Niche Cells Regulating Multiple Myeloma Cell Growth, at Least in Part, By Affecting the Local Immune Microenvironment

Despite high response rates to new therapies, most multiple myeloma (MM) patients relapse and become refractory to treatment. This is likely because MM cells enter dormancy and reactivate only under permissive conditions. Cellular dormancy is ubiquitous in normal tissues as well as in malignant, non-malignant, and metastasizing tumors. Dormant tumor cells (DTC) are protected from therapies which can reactivate later at the time of disease progression, resulting in relapsed refractory MM (RRMM). The tumor microenvironment (TME) is a key determinant of tumor dormancy and reactivation. We have previously shown that inhibition of Activin A in SCID mice transplanted with human MM.1S increased osteoblast numbers and resulted in inhibition of MM growth (Vallet et al., PNAS). We hypothesize that MM-DTCs reside in osteoblastic niche in the BM TME. Our long-term goal is to identify factors regulating DTCs in MM and develop strategies for long term disease control.We have generated mice in which mature osteoblasts can be postnatally deleted in an inducible and reversible manner. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre). This led to the expression of DTR in mature osteoblasts (OC-Cre/iDTR) only. The control mice were littermates lacking the OC-Cre allele (iDTR). Until the treatment with diphtheria toxin (DT), the OC-Cre/iDTR mice were immunocompetent compared to iDTR mice, as they did not show any differences in immune cell populations by flow cytometry, and did not show any skeletal phenotype, as measured by micro-computed CT (micro-CT).We chose to induce mature osteoblast-deficiency at 8-weeks of age to allow skeletal maturation which is assumed to be completed by 6-7weeks of age. To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/kg DT once a week, beginning at 8-weeks of age. Micro-CT analysis showed a significant increase in cortical porosity within 1-week after DT injection. 8-weeks of DT treatment significantly reduced trabecular bone fraction (BV/TV), trabecular numbers (Tb.N), and bone mineral density (BMD) with a significant increase in trabecular spacing (Tb.Sp). Immunohistochemistry for osteocalcin showed rapid loss of mature and endosteal osteoblasts (N.Ob/T.Ar). This was accompanied with a marked decreased in serum sclerostin and serum osteocalcin levels suggesting reduced osteocytes and mature osteoblasts, respectively. Importantly, serum CTX levels or numbers of osteoclasts (N.Oc/T.Ar) were unchanged. To study MM engraftment and progression, 3x10 6 5TGM1 luciferase tdTomato positive (5TGM1-Luc-Tom) MM cells were injected into the tibia of OC-Cre/iDTR and control iDTR mice followed by weekly injection of DT for 8-weeks. Flow cytometry analysis showed a 4-fold increase in the 5TGM1-Tom cells in the BM from OC-Cre/iDTR mice compared to controls. Bioluminescence imaging (BLI) for 5TGM1-Luc-Tom cells at 4-weeks showed dramatic increase in the OC-Cre/iDTR mice compared to controls. Interestingly, by 8-weeks, the BLI imaging showed 5TGM1-Luc-Tom cells in other long-bones of OC-Cre/iDTR mice but not the controls. To identify local BM TME changes, we assessed the levels of 200 cytokines and growth factors in the BM from these mice. The levels of immune regulatory cytokines, such as IL-17F and IL-21, were found to be decreased in the OC-Cre/iDTR mice compared to control iDTR mice, both after 4-weeks from intratibial 5TGM1 cell injection. This was accompanied by significant decrease in the fraction of CD4+ T-helper and Th17+ cells in the OC-Cre/iDTR mice compared to controls, as assessed by flow cytometry.These data show that MM cells engraft and proliferate rapidly within the BM in the absence of mature osteoblasts and have a propensity to migrate to other long bones with time. This may be partially mediated by the altered immune cell profile in the BM in the absence of mature osteoblasts. These data further suggest that expanding the mature osteoblast niche may provide novel therapeutic avenues and reduce disease burden creating an environment for long term tumor control. Importantly, this model will allow us to understand the osteoblastic niche for MM and the mechanisms of activation and dormancy, at least in part, via a role of the immune cells. DisclosuresFulzele: Constellation Pharma: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Raje: Celgene, Amgen, Bluebird Bio, Janssen, Caribou, and BMS: Other.

The extracellular matrix protein Agrin is expressed by osteoblasts and contributes to their differentiation.

The extracellular matrix protein Agrin has been detected in chondrocytes and endosteal osteoblasts but its function in osteoblast differentiation has not been investigated yet. Thus, it is possible that Agrin contributes to osteoblast differentiation and, due to Agrin and wingless-related integration site (Wnt) sharing the same receptor, transmembrane low-density lipoprotein receptor-related protein 4 (Lrp4), and the crosstalk between Wnt and bone morphogenetic protein (BMP) signalling, both pathways could be involved in this Agrin-mediated osteoblast differentiation. Confirming this, Agrin and its receptors Lrp4 and α-dystroglycan (Dag1) were expressed during differentiation of osteoblasts from three different sources. Moreover, the disruption of Agrin impaired the expression of its receptors and osteoblast differentiation, and the treatment with recombinant Agrin slightly increase this process. In addition, whilst Agrin knockdown downregulated the expression of genes related to Wnt and BMP signalling pathways, the addition of Agrin had no effect on these genes. Altogether, these data uncover the contribution of Agrin to osteoblast differentiation and suggest that, at least in part, an Agrin-Wnt-BMP circuit is involved in this process. This makes Agrin a candidate as target for developing new therapeutic strategies to treat bone-related diseases and injuries.

PPARG in osteocytes controls sclerostin expression, bone mass, marrow adiposity and mediates TZD-induced bone loss.

The peroxisome proliferator activated receptor gamma (PPARG) nuclear receptor regulates energy metabolism and insulin sensitivity. In this study, we present novel evidence for an essential role of PPARG in the regulation of osteocyte function, and support for the emerging concept of the conjunction between regulation of energy metabolism and bone mass. We report that PPARG is essential for sclerostin production, a recently approved target to treat osteoporosis. Our mouse model of osteocyte-specific PPARG deletion (Dmp1CrePparγflfl or γOTKO) is characterized with increased bone mass and reduced bone marrow adiposity, which is consistent with upregulation of WNT signaling and increased bone forming activity of endosteal osteoblasts. An analysis of osteocytes derived from γOTKO and control mice showed an excellent correlation between PPARG and SOST/sclerostin at the transcript and protein levels. The 8kb sequence upstream of Sost gene transcription start site possesses multiple PPARG binding elements (PPREs) with at least two of them binding PPARG with dynamics reflecting its activation with full agonist rosiglitazone and correlating with increased levels of Sost transcript and sclerostin protein expression (Pearson's r=0.991, p=0.001). Older γOTKO female mice are largely protected from TZD-induced bone loss providing proof of concept that PPARG in osteocytes can be pharmacologically targeted. These findings demonstrate that transcriptional activities of PPARG are essential for sclerostin expression in osteocytes and support consideration of targeting PPARG activities with selective modulators to treat osteoporosis.

A Novel in-Vivo Model to Examine Homing of Multiple Myeloma Cells in Postnatal, Inducible, and Reversible Loss of Mature Osteoblasts

The initiation of Multiple myeloma (MM) coincides with a decrease in osteoblasts and an increase in osteoclasts and adipocytes. However, the contribution of osteoblasts to the initiation and progression of MM remains mostly unknown. In-vitro studies have shown that co-cultures with MC3T3 murine osteoblast cell-line induced quiescence in 5TGM1 murine myeloma cells. In SCID or C57BL/KaLwRijHsd mice, the fluorescently labeled MM cell lines, injected through tail-vein, co-localized with endosteal osteoblasts and were found to be quiescent. We previously demonstrated that increased osteoblast numbers using Activin A inhibition in SCID mice with human-origin MM.1S cells resulted in inhibition of MM growth (Vallet et al., PNAS 2010). Here we aimed to develop a genetic in-vivo model for a better understanding of the interactions between osteoblasts and MM cells. Osteolineage cells develop through the stages of pre-osteoblasts, committed osteoblasts, mature osteoblasts, and osteocytes; each stage governed by a distinct molecular signature and function. To delineate these differences, individual osteoblast populations were FACS-sorted from long bones of Osterix-GFP+ (Osx+) mice (pre-osteoblasts), Collagen 2.3-GFP+ (Col2.3+) mice (committed osteoblasts), and Osteocalcin-YFP+ (OCN+) mice (mature osteoblasts). Co-cultured of these osteoblast populations with 5TGM1 MM cells showed that the OCN+ mature osteoblasts suppressed MM proliferation the most. Therefore, we focused our studies on the role of mature osteoblasts in MM homing and engraftment. We next generated mice in which mature osteoblasts were postnatally deleted in an inducible and reversible manner to better understand the molecular mechanisms of engraftment, proliferation, and migration of 5TGM1 injected intra-tibialy. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre). This led to the expression of DTR in mature osteoblasts (OC-Cre/iDTR) only. The control mice were littermates lacking the OC-Cre allele (iDTR). The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). We chose to induce mature osteoblast-deficiency at 8-weeks to allow skeletal maturation which is assumed to be completed by 6-7weeks of age. To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/Kg DT once a week, beginning at 8-weeks of age. Micro-CT analysis showed a significant increase in cortical porosity within 1-week after DT injection. 8-weeks of DT treatment significantly reduced trabecular bone fraction (BV/TV), trabecular numbers (Tb.N), and bone mineral density (BMD) with a significant increase in trabecular spacing (Tb.Sp). Immunohistochemistry for osteocalcin showed rapid loss of mature and endosteal osteoblasts (N.Ob/T.Ar). This was accompanied with a marked decreased in serum sclerostin and serum osteocalcin levels suggesting reduced osteocytes and mature osteoblasts, respectively. Importantly, serum CTX levels or numbers of osteoclasts (N.Oc/T.Ar) were unchanged. To study MM engraftment and progression, 3x106 5TGM1 luciferase eGFP positive (5TGM1-Luc-GFP) MM cells were injected into the tibia of OC-Cre/iDTR and control iDTR mice followed by weekly injection of DT for 8-weeks. Flow cytometry analysis showed a 4-fold increase in the 5TGM1-GFP cells in the BM from OC-Cre/iDTR mice compared to controls. Bioluminescence imaging (BLI) for 5TGM1-Luc-GFP cells at 4-weeks showed 600-fold increase in the OC-Cre/iDTR mice compared to controls. Interestingly, by 8-weeks, the BLI imaging showed 5TGM1-Luc-GFP cells in other long-bones of OC-Cre/iDTR mice but not the controls. These data show that MM cells engraft and proliferate rapidly in the absence of mature osteoblasts in-vivo. Moreover, in the absence of mature osteoblasts, MM cells have a propensity to migrate to other long bones. These data further suggest that expanding the mature osteoblast niche may provide novel therapeutic avenues and reduce disease burden and create an environment for long term tumor control. Importantly, this model will allow us to follow and analyze the sequential engraftment, dormancy, reactivation, proliferation, and migration of myeloma cells, and evaluate the effects of osteoanabolic and anti-myeloma therapies. Disclosures Fulzele: FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Raje:BMS: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Astrazeneca: Consultancy; Bluebird, Bio: Consultancy, Research Funding; Takeda: Consultancy; Immuneel: Membership on an entity's Board of Directors or advisory committees; Caribou: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy.

Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells.

The periosteum is critical for bone maintenance and healing. However, the invivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible forlifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration invivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair.

Discovery of a periosteal stem cell mediating intramembranous bone formation

Bone consists of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells owing to physical separation by the bone cortex. The skeletal stem cell that gives rise to endosteal osteoblasts has been extensively studied; however, the identity of periosteal stem cells remains unclear1–5. Here we identify a periosteal stem cell (PSC) that is present in the long bones and calvarium of mice, displays clonal multipotency and self-renewal, and sits at the apex of a differentiation hierarchy. Single-cell and bulk transcriptional profiling show that PSCs display transcriptional signatures that are distinct from those of other skeletal stem cells and mature mesenchymal cells. Whereas other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway4, PSCs form bone via a direct intramembranous route, providing a cellular basis for the divergence between intramembranous versus endochondral developmental pathways. However, there is plasticity in this division, as PSCs acquire endochondral bone formation capacity in response to injury. Genetic blockade of the ability of PSCs to give rise to bone-forming osteoblasts results in selective impairments in cortical bone architecture and defects in fracture healing. A cell analogous to mouse PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. The identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions. A periosteal stem cell specialized in intramembranous bone formation has been identified and was found to be essential for normal bone development and fracture healing.

Mature Osteoblasts Provide a Protective Niche Against Multiple Myeloma Growth and Survival within the Tumor Microenvironment

Multiple myeloma (MM), a tumor of B-lymphocyte lineage cells, originates in the bone marrow (BM) and is highly influenced by the BM tumor microenvironment (TME). Among cells of the TME, osteoblasts are the most versatile regulators of many hematopoietic lineage cells through either direct cell-cell communication or secreted factors. Specifically relevant to MM, G-protein coupled receptor signaling in pre-osteoblasts is essential for the differentiation, maturation, and egress of B-cells (Panaroni et al., 2015). Despite these key roles, the contribution of osteoblasts to the initiation and progression of MM is not well understood. MM is characterized by osteolytic bone lesions partly due to decreased numbers of osteoblasts. Here, we hypothesize that osteoblasts provide niche support to maintain myeloma cells in a quiescent stage and that the loss of the osteoblastic niche leads to the progression of MM. As a proof of concept, we previously showed that increasing osteoblastogenesis by inhibiting Activin A led to inhibition of MM growth in an in vivo humanized myeloma model (Vallet et al., 2010).We generated mice in which mature osteoblasts could be postnatally deleted in an inducible and reversible manner. Diphtheria toxin receptor floxed mice were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter to generate OC-Cre/iDTR mice. Littermates heterozygous for DTR but lacking the OC-Cre expression were used as controls. The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/Kg DT beginning at 8-weeks of age. Osteocalcin immunohistochemistry of trabecular bone showed that the DT treated OC-Cre/iDTR mice were completely devoid of endosteal osteoblasts and young osteocytes. Consequently, serum levels of sclerostin were also significantly reduced in OC-Cre/iDTR mice compared to the controls.To study MM engraftment and progression, 3x106 5TGM1-Luciferase MM cells were inject into tibia of OC-Cre/iDTR and control mice followed by a weekly injection of DT for 8-weeks. Bioluminescence imaging (BLI) was used to assess tumor progression. Both the control and OC-Cre/iDTR mice started with similar BLI signal at 1-week. Interestingly, 4-weeks onwards only the OC-iDTR mice continued to express and increase the BLI signal indicating that the MM cells engrafted and continued to proliferate only in the OC-Cre/iDTR mice. This data suggests that under physiological conditions mature osteoblasts actively suppress MM engraftment and progression.To determine the direct effects of osteoblasts on MM cells, we established co-cultures of osteoblasts with MM cells. FACS sorting was used to isolate pre-osteoblasts from the long bones of Osterix-GFP+ (Osx+) mice, committed osteoblasts from Collagen 2.3-GFP+ (Col2.3+) mice, and mature osteoblasts from Osteocalcin-YFP+ (OCN+) mice. 5TGM1 MM cells were co-cultured along with 10,000 cells from each of the osteoblast populations. Although all three populations of osteoblasts significantly suppressed MM proliferation, the OCN+ mature osteoblasts suppressed MM proliferation the most.The mature osteoblastic niche may regulate MM cells via a) direct cell-to-cell contact, b) secreted factors, and/or c) other intermediary cells. To identify these possibilities, the BM TME was examined at 2-weeks following the 5TGM1 intratibia injection into the OC-Cre/iDTR and control mice. Quantitative protein antibody arrays analysis on the BM supernatant identified numerous key factors involved in cell-cell communication and immunomodulation in MM. These factors included BAFF-R, TACI, IL-33, IL-3, IL-21, and IL-17F. Gene expression analysis of the sorted 5TGM1 cells from the injected tibia indicated increased expression of genes involved in mitochondrial metabolism.Taken together, using in vivo and in vitro models, we show that mature osteoblasts offer specialized niches for MM cells where tumor cells are maintained in quiescence. The loss of the niche support allows the reactivation and progression of MM through the loss of cell-cell communication or through activation of immunomodulatory intermediary cells. Normalizing osteoblasts, such as through Activin A treatment, could provide novel avenues to reduce disease burden and long term tumor control. DisclosuresNo relevant conflicts of interest to declare.

Loss of Gsα in osteocytes leads to osteopenia due to sclerostin induced suppression of osteoblast activity.

The stimulatory subunit of G-protein, Gsα, acts as a secondary messenger of G-protein coupled receptors (GPCRs) that primarily activates cAMP-induced signaling. GPCRs, such as the parathyroid hormone receptor (PTHR), are critical regulators of bone formation as shown by number of genetic manipulation studies targeting early osteoblast lineage cells. In this study, we have examined the role of Gsα in osteocytes, the terminally differentiated and most abundant cells of the osteoblast lineage. Mice lacking the stimulatory subunit of G-proteins (Gsα) in osteocytes (DMP1-GsαKO) have significant decrease of both trabecular and cortical bone, as assessed by μCT. Histomorphometric analysis showed that the osteopenia was mostly driven by more than 90% decrease in osteoblast numbers and activity whereas osteoclasts were only slightly decreased. The decrease in osteoblast number was associated with a striking lack of endocortical osteoblasts. We have previously shown that loss of the stimulatory subunit of G-proteins (Gsα) in osteocytes in vitro or in vivo induces high expression of sclerostin. To determine if the increased sclerostin levels contributed to the decreased endosteal bone lining cells and osteopenia, we treated wild-type mice with recombinant sclerostin and the DMP1-GsαKO mice with anti-sclerostin antibody. Treatment of wild-type mice with 100 μg/kg sclerostin for 3-weeks significantly reduced the numbers of bone lining cells and led to osteopenia. Next, the DMP1-GsαKO and control littermates were treated with the anti-sclerostin antibody (25 mg/kg, 2 times per week) for 4-weeks. Upon the antibody treatment, the endocortical osteoblasts reappeared in the DMP1-GsαKO mice to a comparable level to that of the vehicle treated control littermates. In control mice, E11/gp38 positive osteocytes were observed in parallel with the endocortical osteoblasts with higher dendrite density towards the endocortical osteoblasts. In DMP1-GsαKO mice, E11/gp38 positive osteocytes were lacking dendrites and were randomly scattered throughout the bone matrix. After treatment with anti-sclerostin antibody, DMP1-GsαKO mice showed increased E11/gp38 positive osteocytes near the endosteal bone surface and endosteal osteoblasts. The anti-sclerostin antibody treatment proportionally increased the bone volume but it could not completely rescue the osteopenia in the DMP1-GsαKO mice. Taken together, this data suggests that Gsα signaling in osteocytes leads to osteopenia driven, at least in part, by increased secretion of sclerostin.

Discovery of a periosteal stem cell mediating intramembranous bone formation.

Bone is comprised of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells due to physical separation by the bone cortex. While the skeletal stem cell giving rise to endosteal osteoblasts has been extensively studied, the identification of a periosteal stem cell has been elusive1–5. Here, we identify a periosteal stem cell (PSC) present in the long bones and calvarium of mice that displays clonal multipotency, self-renewal and sits at the apex of a differentiation hierarchy. Single cell and bulk transcriptional profiling show that PSCs display distinct transcriptional signatures in comparison with both other skeletal stem cells and mature mesenchymal cells. While other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway4, PSCs form bone via a direct intramembranous route, providing a cellular basis for the divergence between intramembranous versus endochondral developmental pathways. However there is plasticity in this division, as PSCs acquire endochondral bone formation capacity in response to injury. Genetic blockade of the ability of PSCs to give rise to bone-forming osteoblasts results in selective impairments in cortical bone architecture and defects in fracture healing. A cell analogous to PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. Moreover, the identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions.

Identification of a murine CD45-F4/80lo HSC-derived marrow endosteal cell associated with donor stem cell engraftment.

Hematopoietic stem cells (HSCs) reside in specialized microenvironments within the marrow designated as stem cell niches, which function to support HSCs at homeostasis and promote HSC engraftment after radioablation. We previously identified marrow space remodeling after hematopoietic ablation, including osteoblast thickening, osteoblast proliferation, and megakaryocyte migration to the endosteum, which is critical for effective engraftment of donor HSCs. To further evaluate the impact of hematopoietic cells on marrow remodeling, we used a transgenic mouse model (CD45Cre/iDTR) to selectively deplete hematopoietic cells in situ. Depletion of hematopoietic cells immediately before radioablation and hematopoietic stem cell transplantation abrogated donor HSC engraftment and was associated with strikingly flattened endosteal osteoblasts with preserved osteoblast proliferation and megakaryocyte migration. Depletion of monocytes, macrophages, or megakaryocytes (the predominant hematopoietic cell populations that survive short-term after irradiation) did not lead to an alteration of osteoblast morphology, suggesting that a hematopoietic-derived cell outside these lineages regulates osteoblast morphologic adaptation after irradiation. Using 2 lineage-tracing strategies, we identified a novel CD45-F4/80lo HSC-derived cell that resides among osteoblasts along the endosteal marrow surface and, at least transiently, survives radioablation. This newly identified marrow cell may be an important regulator of HSC engraftment, possibly by influencing the shape and function of endosteal osteoblasts.

Fibroblast growth factor 2 supports osteoblastic niche cells during hematopoietic homeostasis recovery after bone marrow suppression

BackgroundHematopoietic stem cell (HSC) maintenance requires a specific microenvironment. HSC niches can be activated by tissue damaging chemotherapeutic drugs and various cell signaling molecules such as SDF-1 and FGF, which might also result in bone marrow stress. Recent research has insufficiently shown that endosteal osteolineage cells and other niche constituents recover after marrow injury.MethodsWe investigated the role of FGF2 in the osteoblastic niche cells during hematopoietic homeostasis recovery after bone marrow injury. Mice were treated with 5-fluorouracil (5FU) to eliminate actively cycling cells in the bone marrow. Primary osteoblasts were isolated and subjected to cell culture. Real-time PCR, western blot and immunohistochemical staining were performed to study niche-related genes, osteoblast markers, and FGF2 signaling. Proliferation rate were analyzed by marker gene Ki67 and colony formation assay. Also, osterix-positive osteoprogenitor cells were isolated by FACS from Osx-GFP-Cre mice after 5FU treatment, and subjected to RNA-sequencing and analyzed for Fgf receptors and niche markers.ResultsThe endosteal osteolineage cells isolated from 5FU-treated mice showed increased expression of the niche-related genes Sdf-1, Jagged-1, Scf, N-cad, Angpt1 and Vcam-1 and the osteoblast marker genes Osx, Opn, Runx2, and Alp, indicating that BM stress upon 5FU treatment activated the osteoblastic niche. Endosteal osteoblast expanded from a single layer to several layers 3 and 6 days after 5FU treatment. During the early recovery phase in 5FU-activated osteoblastic niches increased FGF2 expression and activated its downstream pERK. FGF2 treatment resulted in increased proliferation rate and the expression of niche marker genes in 5FU-activated osteoblastic niche cells. RNA-seq analysis in Osterix-positive osteoprogenitor cells isolated from 5FU-treated Osx-GFP mice showed significantly increased expression of Fgf receptors Fgfr1, 2 and 3. Although osteoblastic niche cells were damaged by 5FU treatment in the beginning, the increased number of OB layers in the recovery phase may be derived from resident osteoprogenitor cells by FGF2 activation under stress.ConclusionsTaken together, FGF2 signaling can regulate osteoblastic niche cells to support HSC homeostasis in response to bone marrow damage.

The role and clinical implications of the endosteal niche and osteoblasts in regulating leukemia

Osteoblasts are one among the critical components of the endosteal bone marrow (BM) niche. In addition to hematopoietic stem cell fate, their role in leukemogenesis as well as metastasis of a variety of cancers has been demonstrated in various studies. In this regard, endosteal niche can have a dual role as an initiator and protective role against leukemia. Knowledge of growth factors, chemokines and cytokines secreted by osteoblasts as well as their interaction with signaling pathways inform our understanding of the development, prognosis, recurrence and treatment of malignant BM diseases. Clinical progress in targeting the endosteal niche is also discussed.

Adaptation of Marrow Osteoblast Morphology Mediated By a Hematopoietic-Derived Endosteal Cell Is Critical for Donor HSC Engraftment after BMT

Hematopoietic stem cells reside in the bone marrow within specialized microenvironments designated the stem cell niche. The remarkable advances over the past decade have dramatically enhanced our perception of the niche; yet, the operative mechanisms after radioablation in preparation for bone marrow transplantation (BMT) remain poorly understood. We have previously described a profound remodeling of the bone marrow architecture after total body irradiation (TBI). This remodeling, comprised of enlarged, proliferating marrow osteoblasts and megakaryocyte migration from the central marrow space to the endosteal surface, is essential for efficient engraftment of donor cells after BMT; hence, marrow remodeling seems to represent an adaptation of the endosteal niche. To investigate whether hematopoietic cells regulate these changes, we sought to deplete all hematopoietic cells prior to TBI. We generated mice expressing the diphtheria toxin receptor (DTR) in all CD45-derived cells using the Cre/loxP model. To validate this strategy, we first crossed CD45Cre mice, where cre is expressed under the control of the endogenous promoter, with Z/RED mice which will then irreversibly express red fluorescent protein (RFP) in all cells that were derived from CD45-expressing progenitors. Surprisingly, we identified a population of RFP-expressing cells residing among osteoblasts along the endosteal and trabecular bone surfaces (designated red Bone Lining Cell, red BLC). By immunofluorescence staining, these cells lacked expression of CD45, lineage markers (Gr1, CD11b, F 4/80, CD3, B220, Ter119), and cathepsin K indicating it is not a hematopoietic cell, specifically not an osteal macrophage or osteoclast, but was unequivocally derived from CD45-expressing progenitors. We reproduced this fate map by crossing vav1Cre mice with Z/RED mice, confirming the identification and hematopoietic lineage of the red BLC. When crossed with Col2.3GFP transgenic mice, which express green fluorescent protein (GFP) in mature osteoblasts, red BLCs lacked GFP co-expression indicating it is not a generic osteoblast. Interestingly, after TBI, red BLCs markedly proliferate, but do not enlarge, in the metaphysis and epiphysis, but not in the diaphysis, coincident with the osteoblast proliferation suggesting a possible role in marrow remodeling. To pursue our original hypothesis that hematopoietic cells may regulate marrow remodeling, we treated mice expressing DTR in all CD45-derived cells and their non-expressing littermates (controls) with diphtheria toxin (DT) followed by TBI to induce marrow remodeling without the effect of CD45-derived cells. Marrow remodeling ensued; however, the characteristically enlarged endosteal osteoblasts adopted a strikingly flattened morphology (cell thickness, 8.45±0.31 vs. 3.42±0.11 μm, P<0.0001). We then used our competitive secondary transplantation assay to assess engraftment of long-term hematopoietic stem cells (HSCs) in primary recipients. Only 1 of 15 CD45-cell depleted mice engrafted HSCs compared to 10 of 15 control mice (P=0.0017) indicating a critical role of osteoblast morphology, governed by a CD45-derived cell, for donor stem cell engraftment in BMT. Megakaryocytes (Mks) and monocytes/macrophages (MMs) are the two marrow hematopoietic lineages that are recognized to survive short term after TBI and we have shown that the CD45-derived red BLC survives and proliferates after TBI. To determine if these cells regulate osteoblasts, we depleted Mks by treating Mk-specific DTR-expressing mice (generated with PF4Cre mice) with DT (>95%), and in separate cohort, MMs using clondronate (>95%). In each cohort, post-TBI marrow remodeling included the expected enlarged endosteal osteoblasts indistinguishable from controls, suggesting that neither Mks nor MMs direct the acquired osteoblast morphology. Collectively, our data indicate that enlarging of endosteal osteoblasts after marrow ablation is critical for donor cell engraftment, possibly due to altered adhesive properties for primitive hematopoietic cells. During post-TBI marrow remodeling, a CD45-derived cell that survives radioablation governs this osteoblast morphology. Our data implicate the red BLC as this key regulatory element. Understanding the red BLC will likely offer new insight into the niche and may lead to novel strategies to enhance HSC engraftment in BMT. DisclosuresNo relevant conflicts of interest to declare.

Targeting Bone Marrow Mesenchymal Stromal Cells Using Cre-Recombinase Transgenes

The bone marrow microenvironment contains hematopoietic niches that regulate the proliferation, differentiation, and trafficking of hematopoietic stem/progenitors cells (HSPCs). These hematopoietic niches are comprised of a heterogeneous population of stromal cells that include, endothelial cells, osteoblasts, CXCL12-abundant reticular (CAR) cells, mesenchymal stem cells (MSCs), arteriolar pericytes, and sympathetic nerves. Emerging data suggest that specific stromal populations may regulate distinct types of HPSCs. Thus, it is important to have validated approaches to interrogate and target specific stromal cell populations. Prior studies have shown that Prx1-Cre, Osx-Cre, Lepr-Cre, and Nes-Cre broadly target mesenchymal stromal cells in the bone marrow. Here, we rigorously define the stromal cell populations targeted by two Cre-transgenes that are commonly used to target osteolineage cells (Ocn-Cre, and Dmp1-Cre) and introduce a new Cre-transgene (Tagln-Cre) that efficiently targets bone marrow pericytes. For each Cre-transgene, we performed lineage mapping using ROSA26Ai9/Ai9 mice, in which cells that have undergone Cre-mediated recombination express tdTomato. In some cases, we further crossed these mice to introduce the Cxcl12gfp transgene, which can be used to define GFP-bright CAR cells. Immunostaining of bone sections and flow cytometry were used to define the target stromal cell population(s) in these mice. Osteocalcin (Bglap, Ocn) is primarily expressed in mature osteoblasts. Accordingly, Ocn-Cre is widely used to specifically target osteoblasts. However, our lineage mapping studies show that Ocn-Cre targets not only all osteoblasts, but also 72 ± 4.0% of CAR cells. Ocn-Cre also targets a subset of NG2+ arteriolar pericytes. Dentin matrix acidic phosphoprotein 1 (Dmp1) is expressed primarily in osteocytes, and Dmp1-Cre has been widely used to specifically target osteocytes. However, we show that Dmp1-Cre also efficiently targets endosteal osteoblasts and approximately 40% of CAR cells. To target bone marrow pericytes, we tested several Cre-transgenes, ultimately focusing on Tagln-Cre. Transgelin (Tagln, SM22a) is broadly expressed in pericytes, smooth muscle cells, and cardiomyocytes. Lineage-mapping studies show that Tagln-Cre targets all arteriolar and venous sinusoidal pericytes in the bone marrow. It also targets osteoblasts and 75 ± 5.2% of CAR cells. There are several recent studies that have ascribed specific functions to osteoblasts or osteocytes based on targeting using Ocn-Cre or Dmp1-Cre, respectively. In light of our data, these conclusions need to be re-evaluated. Ocn-Cre, Dmp1-Cre, and Tagln-Cre each target a subset of CAR cells. Studies are underway to determine whether these CAR subsets have unique expression profiles and functions. Finally, Talgn-Cre represents a new tool for investigators in the field to efficiently target bone marrow pericytes. Disclosures No relevant conflicts of interest to declare.

Abstract 365: Mobilizing prostate cancer cells from the endosteal niche by targeting the SDF-1/CXCR4 axis

Abstract Metastatic prostate cancer cells preferentially home to bone, and skeletal metastases are present in approximately 90% of patients upon autopsy. Our lab was the first to show that prostate cancer cells compete with hematopoietic stem cells (HSCs) for space within the endosteal niche. The HSC niche is located on the surface of endosteal osteoblasts, where HSCs are kept in a slow-cycling, protective state. The niche also provides prostate cancer cells with a chemoprotective environment in which they can remain dormant for an extended period of time, even decades. C-X-C chemokine receptor 4 (CXCR4) is required for HSC recruitment to the HSC niche. Osteoblasts secrete stromal cell derived factor 1 (SDF-1, also known as CXCL12), the ligand for CXCR4, which attracts HSCs to the niche via a chemotactic gradient. CXCR4 is also expressed by disseminated prostate cancer cells and is required for prostate cancer cells homing to bone. HSCs can be mobilized from the niche into the bloodstream physiologically via infection or stress and pharmacologically via proteins like granulocyte-colony stimulating factor (G-CSF) or small molecule inhibitors like AMD3100 (plerixafor). AMD3100, which antagonizes CXCR4, is capable of mobilizing both HSCs and prostate cancer cells from the niche. When combined with AMD3100, docetaxel (the standard of care for prostate cancer) is more effective at killing tumor cells than when given as a single agent. This indicates that mobilization of disseminated tumor cells (DTCs) from the endosteal niche by targeting the SDF-1/CXCR4 axis might cause these cells to be more sensitive to chemotherapy in patients. This strategy would theoretically kill DTCs, which, when they become metastatic lesions, lead to most of the morbidity and mortality that affects prostate cancer patients. We are engaged in a Phase 0 clinical trial in metastatic prostate cancer patients to test this strategy in human patients. To complement the human trial, we are testing several CXCR4 inhibitors combined with G-CSF and docetaxel. We are able to detect CTCs in human patients, as well as CTCs and DTCs in mice via several techniques, and will use this technology as a readout for the clinical trial, as well as the preclinical studies. Citation Format: Kenneth C. Valkenburg, Kenneth J. Pienta. Mobilizing prostate cancer cells from the endosteal niche by targeting the SDF-1/CXCR4 axis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2015-365

Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

AbstractHematopoietic stem cells (HSCs) reside in regulatory niches in the bone marrow (BM). Although HSC niches have been extensively characterized, the role of endosteal osteoblasts (OBs) in HSC regulation requires further clarification, and the role of OBs in regulating leukemic stem cells (LSCs) is not well studied. We used an OB visualization and ablation mouse model to study the role of OBs in regulating normal HSCs and chronic myelogenous leukemia (CML) LSCs. OB ablation resulted in increase in cells with a LSK Flt3−CD150+CD48− long-term HSC (LTHSC) phenotype but reduction of a more highly selected LSK Flt3−CD34−CD49b−CD229− LTHSC subpopulation. LTHSCs from OB-ablated mice demonstrated loss of quiescence and reduced long-term engraftment and self-renewal capacity. Ablation of OB in a transgenic CML mouse model resulted in accelerated leukemia development with reduced survival compared with control mice. The notch ligand Jagged-1 was overexpressed on CML OBs. Normal and CML LTHSCs cultured with Jagged-1 demonstrated reduced cell cycling, consistent with a possible role for loss of Jagged-1 signals in altered HSC and LSC function after OB ablation. These studies support an important role for OBs in regulating quiescence and self-renewal of LTHSCs and a previously unrecognized role in modulating leukemia development in CML.