Narelle E. McGregor

Oncostatin M promotes bone formation independently of resorption when signaling through leukemia inhibitory factor receptor in mice

Effective osteoporosis therapy requires agents that increase the amount and/or quality of bone. Any modification of osteoclast-mediated bone resorption by disease or drug treatment, however, elicits a parallel change in osteoblast-mediated bone formation because the processes are tightly coupled. Anabolic approaches now focus on uncoupling osteoblast action from osteoclast formation, for example, by inhibiting sclerostin, an inhibitor of bone formation that does not influence osteoclast differentiation. Here, we report that oncostatin M (OSM) is produced by osteoblasts and osteocytes in mouse bone and that it has distinct effects when acting through 2 different receptors, OSM receptor (OSMR) and leukemia inhibitory factor receptor (LIFR). Specifically, mouse OSM (mOSM) inhibited sclerostin production in a stromal cell line and in primary murine osteoblast cultures by acting through LIFR. In contrast, when acting through OSMR, mOSM stimulated RANKL production and osteoclast formation. A key role for OSMR in bone turnover was confirmed by the osteopetrotic phenotype of mice lacking OSMR. Furthermore, in contrast to the accepted model, in which mOSM acts only through OSMR, mOSM inhibited sclerostin expression in Osmr-/- osteoblasts and enhanced bone formation in vivo. These data reveal what we believe to be a novel pathway by which bone formation can be stimulated independently of bone resorption and provide new insights into OSMR and LIFR signaling that are relevant to other medical conditions, including cardiovascular and neurodegenerative diseases and cancer.

Cardiotrophin‐1 Is an Osteoclast‐Derived Stimulus of Bone Formation Required for Normal Bone Remodeling

Cardiotrophin (CT‐1) signals through gp130 and the LIF receptor (LIFR) and plays a major role in cardiac, neurological, and liver biology. We report here that CT‐1 is also expressed within bone in osteoclasts and that CT‐1 is capable of increasing osteoblast activity and mineralization both in vitro and in vivo. Furthermore, CT‐1 stimulated CAAT/enhancer‐binding protein‐δ (C/EBPδ) expression and runt‐related transcription factor 2 (runx2) activation. In neonate CT‐1−/− mice, we detected low bone mass associated with reduced osteoblasts and many large osteoclasts, but increased cartilage remnants within the bone, suggesting impaired resorption. Cultured bone marrow (BM) from CT‐1−/− mice generated many oversized osteoclasts and mineralized poorly compared with wildtype BM. As the CT‐1−/− mice aged, the reduced osteoblast surface (ObS/BS) was no longer detected, but impaired bone resorption continued resulting in an osteopetrotic phenotype in adult bone. CT‐1 may now be classed as an essential osteoclast‐derived stimulus of both bone formation and resorption.

The Primary Function of gp130 Signaling in Osteoblasts Is To Maintain Bone Formation and Strength, Rather Than Promote Osteoclast Formation

Interleukin‐6 (IL‐6) family cytokines act via gp130 in the osteoblast lineage to stimulate the formation of osteoclasts (bone resorbing cells) and the activity of osteoblasts (bone forming cells), and to inhibit expression of the osteocyte protein, sclerostin. We report here that a profound reduction in trabecular bone mass occurs both when gp130 is deleted in the entire osteoblast lineage (Osx1Cre gp130 f/f) and when this deletion is restricted to osteocytes (DMP1Cre gp130 f/f). This was caused not by an alteration in osteoclastogenesis, but by a low level of bone formation specific to the trabecular compartment. In contrast, cortical diameter increased to maintain ultimate bone strength, despite a reduction in collagen type 1 production. We conclude that osteocytic gp130 signaling is required for normal trabecular bone mass and proper cortical bone composition.

Germline deletion of AMP-activated protein kinase β subunits reduces bone mass without altering osteoclast differentiation or function

Since AMP‐activated protein kinase (AMPK) plays important roles in modulating metabolism in response to diet and exercise, both of which influence bone mass, we examined the influence of AMPK on bone mass in mice. AMPK is an αβy hetero‐trimer where the β subunit anchors the a catalytic and y regulatory subunits. Germline deletion of either AMPK β 1orβ2 subunit isoforms resulted in reduced trabecular bone density and mass, but without effects on osteoclast (OC) or osteoblast (OB) numbers, as compared to wild‐type littermate controls. We tested whether activating AMPK in vivo would enhance bone density but found AICA‐riboside treatment caused a profound loss of trabecular bone volume (49.5%) and density and associated increased OC numbers. Consis‐tent with this, AICA‐riboside strongly stimulated OC differentiation in vitro, in an adenosine kinase‐depen‐dent manner. OCs and macrophages (unlike OBs) lacked AMPK β2 subunit expression, and when generated from AMPK β1_/_ mice displayed no detectable AMPK activity. Nevertheless, AICA‐riboside was equally effective at stimulating OC differentiation from wildtype or β1_/_ progenitors, indicating that AMPK is not essential for OC differentiation or the stimulatory action of AICA‐riboside. These results show that AMPK is required to maintain normal bone density, but not through bone cell differentiation, and does not mediate powerful osteolytic effects of AICA‐riboside.—Quinn, J. M. W., Tam, S., Sims, N. A., Saleh, H., McGregor, N. E., Poulton, I. J., Scott, J. W., Gillespie, M. T., Kemp, B. E., van Denderen, B. J. W. Germline deletion of AMP‐activated protein kinase β subunits reduces bone mass without altering osteoclast differentiation or function. FASEB J. 24, 275–285 (2010). www.fasebj.org

Ciliary Neurotrophic Factor Inhibits Bone Formation and Plays a Sex-Specific Role in Bone Growth and Remodeling

Ciliary neurotrophic factor (CNTF) receptor (CNTFR) expression has been described in osteoblast-like cells, suggesting a role for CNTF in bone metabolism. When bound to CNTF, neuropoietin (NP), or cardiotrophin-like-cytokine (CLC), CNTFR forms a signaling complex with gp130 and the leukemia inhibitory factor receptor, which both play critical roles in bone cell biology. This study aimed to determine the role of CNTFR-signaling cytokines in bone. Immunohistochemistry detected CNTF in osteoblasts, osteocytes, osteoclasts, and proliferating chondrocytes. CNTFR mRNA was detected in primary calvarial osteoblasts and was upregulated during osteoblast differentiation. Treatment of osteoblasts with CNTF or CLC, but not NP, significantly inhibited mineralization and osterix mRNA levels. Twelve-week-old male CNTF−/− mice demonstrated reduced femoral length, cortical thickness, and periosteal circumference; but femoral trabecular bone mineral density (Tb.BMD) and tibial trabecular bone volume (BV/TV) were not significantly different from wild-type, indicating a unique role for CNTF in bone growth in male mice. In contrast, female CNTF−/− femora were of normal width, but femoral Tb.BMD, tibial BV/TV, trabecular number, and trabecular thickness were all increased. Female CNTF−/− tibiae also demonstrated high osteoblast number and mineral apposition rate compared to wild-type littermates, and this was intrinsic to the osteoblast lineage. CNTF is expressed locally in bone and plays a unique role in female mice as an inhibitor of trabecular bone formation and in male mice as a stimulus of cortical growth.

Contrasting roles of leukemia inhibitory factor in murine bone development and remodeling involve region-specific changes in vascularization

We describe here distinct functions of leukemia inhibitory factor (LIF) in bone development/growth and adult skeletal homeostasis. In the growth plate and developing neonate bones, LIF deficiency enhanced vascular endothelial growth factor (VEGF) levels, enlarged blood vessel formation, and increased the formation of “giant” osteoclasts/chondroclasts that rapidly destroyed the mineralized regions of the growth plate and developing neonatal bone. Below this region, osteoblasts formed large quantities of woven bone. In contrast, in adult bone undergoing remodeling osteoclast formation was unaffected by LIF deficiency, whereas osteoblast formation and function were both significantly impaired, resulting in osteopenia. Consistent with LIF promoting osteoblast commitment, enhanced marrow adipocyte formation was also observed in adult LIF null mice, and adipocytic differentiation of murine stromal cells was delayed by LIF treatment. LIF, therefore, controls vascular size and osteoclast differentiation during the transition of cartilage to bone, whereas an anatomically separate LIF‐dependent pathway regulates osteoblast and adipocyte commitment in bone remodeling.

EphrinB2 signaling in osteoblasts promotes bone mineralization by preventing apoptosis

Cells that form bone (osteoblasts) express both ephrinB2 and EphB4, and previous work has shown that pharmacological inhibition of the eph‐rinB2/EphB4 interaction impairs osteoblast differentiation in vitro and in vivo. The purpose of this study was to determine the role of ephrinB2 signaling in the osteoblast lineage in the process of bone formation. Cultured osteoblasts from mice with osteoblast‐specific ablation of ephrinB2 showed delayed expression of osteoblast differentiation markers, a finding that was reproduced by ephrinB2, but not EphB4, RNA interference. Microcomputed tomography, histomorphometry, and mechanical testing of the mice lacking ephrinB2 in osteoblasts revealed a 2‐fold delay in bone mineralization, a significant reduction in bone stiffness, and a 50% reduction in osteoblast differentiation induced by anabolic parathyroid hormone (PTH) treatment, compared to littermate sex‐ and age‐matched controls. These defects were associated with significantly lower mRNA levels of late osteoblast differentiation markers and greater levels of osteoblast and osteocyte apoptosis, indicated by TUNEL staining and transmission electron microscopy of bone samples, and a 2‐fold increase in annexin V staining and 7‐fold increase in caspase 8 activation in cultured ephrinB2 deficient osteoblasts. We conclude that osteoblast differentiation and bone strength are maintained by antiapoptotic actions of ephrinB2 signaling within the osteoblast lineage.—Tonna, S., Takyar, F. M., Vrahnas, C., Crimeen‐Irwin, B., Ho, P. W. M., Poulton, I. J., Brennan, H. J., McGregor, N. E., Allan, E. H., Nguyen, H., Forwood, M. R., Tatarczuch, L., Mackie, E. J., Martin, T. J., Sims, N. A., EphrinB2 signaling in osteoblasts promotes bone mineralization by preventing apoptosis. FASEB J. 28, 4482–4496 (2014). www.fasebj.org

Sustained RANKL response to parathyroid hormone in oncostatin M receptor‐deficient osteoblasts converts anabolic treatment to a catabolic effect in vivo

Parathyroid hormone (PTH) is the only approved anabolic agent for osteoporosis treatment. It acts via osteoblasts to stimulate both osteoclast formation and bone formation, with the balance between these two activities determined by the mode of administration. Oncostatin M (OSM), a gp130‐dependent cytokine expressed by osteoblast lineage cells, has similar effects and similar gene targets in the osteoblast lineage. In this study, we investigated whether OSM might participate in anabolic effects of PTH. Microarray analysis and quantitative real‐time polymerase chain reaction (qPCR) of PTH‐treated murine stromal cells and primary calvarial osteoblasts identified significant regulation of gp130 and gp130‐dependent coreceptors and ligands, including a significant increase in OSM receptor (OSMR) expression. To determine whether OSMR signaling is required for PTH anabolic action, 6‐week‐old male Osmr−/− mice and wild‐type (WT) littermates were treated with hPTH(1–34) for 3 weeks. In WT mice, PTH increased trabecular bone volume and trabecular thickness. In contrast, the same treatment had a catabolic effect in Osmr−/− mice, reducing both trabecular bone volume and trabecular number. This was not explained by any alteration in the increased osteoblast formation and mineral apposition rate in response to PTH in Osmr−/− compared with WT mice. Rather, PTH treatment doubled osteoclast surface in Osmr−/− mice, an effect not observed in WT mice. Consistent with this finding, when osteoclast precursors were cultured in the presence of osteoblasts, more osteoclasts were formed in response to PTH when Osmr−/− osteoblasts were used. Neither PTH1R mRNA levels nor cAMP response to PTH were modified in Osmr−/− osteoblasts. However, RANKL induction in PTH‐treated Osmr−/− osteoblasts was sustained at least until 24 hours after PTH exposure, an effect not observed in WT osteoblasts. These data indicate that the transient RANKL induction by intermittent PTH administration, which is associated with its anabolic action, is changed to a prolonged induction in OSMR‐deficient osteoblasts, resulting in bone destruction.

Osteoclast Inhibitory Lectin, an Immune Cell Product That Is Required for Normal Bone Physiology in Vivo

Osteoclast inhibitory lectin (OCIL or clrb) is a member of the natural killer cell C-type lectins that have a described role mostly in autoimmune cell function. OCIL was originally identified as an osteoblast-derived inhibitor of osteoclast formation in vitro. To determine the physiological function(s) of OCIL, we generated ocil-/- mice. These mice appeared healthy and were fertile, with no apparent immune function defect, and phenotypic abnormalities were limited to bone. Histomorphometric analysis revealed a significantly lower tibial trabecular bone volume and trabecular number in the 10- and 16-week-old male ocil-/- mice compared with wild type mice. Furthermore, ocil-/- mice showed reduced bone formation rate in the 10-week-old females and 16-week-old males while Static markers of bone formation showed no significant changes in male or female ocil-/- mice. Examination of bone resorption markers in the long bones of ocil-/- mice indicated a transient increase in osteoclast number per unit bone perimeter. Enhanced osteoclast formation was also observed when either bone marrow or splenic cultures were generated in vitro from ocil-/- mice relative to wild type control cultures. Loss of ocil therefore resulted in osteopenia in adult mice primarily as a result of increased osteoclast formation and/or decreased bone formation. The enhanced osteoclastic activity led to elevated serum calcium levels, which resulted in the suppression of circulating parathyroid hormone in 10-week-old ocil-/- mice compared with wild type control mice. Collectively, our data suggest that OCIL is a physiological negative regulator of bone.

gp130 in late osteoblasts and osteocytes is required for PTH-induced osteoblast differentiation.

Parathyroid hormone (PTH) treatment stimulates osteoblast differentiation and bone formation, and is the only currently approved anabolic therapy for osteoporosis. In cells of the osteoblast lineage, PTH also stimulates the expression of members of the interleukin 6 (IL-6) cytokine superfamily. Although the similarity of gene targets regulated by these cytokines and PTH suggest cooperative action, the dependence of PTH anabolic action on IL-6 cytokine signaling is unknown. To determine whether cytokine signaling in the osteocyte through glycoprotein 130 (gp130), the common IL-6 superfamily receptor subunit, is required for PTH anabolic action, male mice with conditional gp130 deletion in osteocytes (Dmp1Cre.gp130(f/f)) and littermate controls (Dmp1Cre.gp130(w/w)) were treated with hPTH(1-34) (30 μg/kg 5× per week for 5 weeks). PTH dramatically increased bone formation in Dmp1Cre.gp130(w/w) mice, as indicated by elevated osteoblast number, osteoid surface, mineralizing surface, and increased serum N-terminal propeptide of type 1 collagen (P1NP). However, in mice with Dmp1Cre-directed deletion of gp130, PTH treatment changed none of these parameters. Impaired PTH anabolic action was associated with a 50% reduction in Pth1r mRNA levels in Dmp1Cre.gp130(f/f) femora compared with Dmp1Cre.gp130(w/w). Furthermore, lentiviral-Cre infection of gp130(f/f) primary osteoblasts also lowered Pth1r mRNA levels to 16% of that observed in infected C57/BL6 cells. In conclusion, osteocytic gp130 is required to maintain PTH1R expression in the osteoblast lineage, and for the stimulation of osteoblast differentiation that occurs in response to PTH.