Elizabeth H. Allan

EphrinB2 regulation by PTH and PTHrP revealed by molecular profiling in differentiating osteoblasts.

With the aim of identifying new pathways and genes regulated by PTH(1–34) and PTH‐related protein 1–141 [PTHrP(1–141)] in osteoblasts, this study was carried out using a mouse marrow stromal cell line, Kusa 4b10, that acquires features of the osteoblastic phenotype in long‐term culture conditions. After the appearance of functional PTH receptor 1 (PTHR1) in Kusa 4b10 cells, they were treated with either PTH(1–34) or PTHrP(1–141), and RNA was subjected to Affymetrix whole mouse genome array. The microarray data were validated using quantitative real‐time RT‐PCR on independently prepared RNA samples from differentiated Kusa 4b10, UMR106 osteosarcoma cells, and primary mouse calvarial osteoblasts, as well as in vivo using RNA from metaphyseal bone after a single PTH injection to 3‐wk‐old and 6‐mo‐old ovariectomized rats. Of the 45,101 probes used on the microarray, 4675 were differentially expressed by ≥1.5 fold, with a false discovery rate <0.1. Among the regulated genes, ephrinB2 mRNA was upregulated in response to both PTH and PTHrP. This was confirmed by quantitative real‐time PCR in vitro and in vivo. Increased ephrinB2 protein was also shown in vitro by Western blotting, and immunostaining of femur sections showed ephrinB2 in both osteoclasts and osteoblasts. Production of ephrinB2, as well as other ephrins or Eph family members, did not change during differentiation of Kusa 4b10 cells. Blockade of ephrinB2/EphB4 interaction resulted in inhibition of mineralization of Kusa 4b10 cells. Together with the shown effect of ephrinB2 promoting osteoblast differentiation and bone formation through action on EphB4, the data raise the possibility that PTH or PTHrP might regulate ephrinB2 to act in a paracrine or autocrine manner on EphB4 or EphB2 in the osteoblast, contributing as a local event to the anabolic action of PTH or PTHrP.

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.

Expression of parathyroid hormone‐related protein in cells of osteoblast lineage

The expression of parathyroid hormone‐related protein (PTHrP) was studied in a range of cell cultures representative of the osteoblast lineage and in rat calvarial sections. Primary newborn rat calvarial cells, a rat preosteoblastic cell line (UMR 201), a mouse stromal cell line (ST 2), a mouse calvaria‐derived osteoblastic cell line (KS 4), and rat osteosarcoma cell lines (UMR 106‐01 and ‐06), all expressed PTHrP when examined by reverse transcription polymerase chain reaction (RT‐PCR). Using a radioimmunoassay we also demonstrated PTHrP in the conditioned medium of the cultured cells, with the exception of UMR 106‐01 and ‐06 cells. Treatment of UMR 201 cells with all‐trans‐retinoic acid which induces them to acquire a more differentiated phenotype, also led to a time‐dependent decrease in PTHrP mRNA levels as determined by RT‐PCR, Northern blot analysis, and in situ hybridization. Decreased PTHrP levels in the conditioned medium of the treated cells was also observed. These results suggested that PTHrP production might be greater in less mature osteoblasts. Examination of the populations obtained from newborn rat calvariae by sequential collagenase digestion revealed that the early digests exhibited low ALP activity, low expression of PTH/PTHrP receptor mRNA, and no adenylate cyclase response to PTHrP(1–34). These populations showed the highest level of mRNA and production of PTHrP. Cells from later digests, the “osteoblast‐rich” populations, had reduced PTHrP expression. Immunohistochemistry and in situ hybridization in sections of newborn rat calvariae showed PTHrp expression in cuboidal osteoblasts located adjacent to bone and in spindle‐shaped cells in the periosteal region. It is concluded that PTHrP is produced by cells of the osteoblast lineage, supporting the hypothesis that PTHrP may function physiologically as a paracrine factor in bone.

Differentiation potential of a mouse bone marrow stromal cell line.

In order to study osteoblast differentiation we subcloned a cell derived from a mouse a bone marrow stromal cell line, Kusa O, and obtained a number of clones representative of three different phenotypes. One that neither differentiated into osteoblasts nor into adipocytes, a second that differentiated into osteoblasts but not adipocytes, and a third that differentiated into both osteoblasts and adipocytes. Four subclones were selected for further characterization according to their ability to mineralize and/or differentiate into adipocytes. The non‐mineralizing clone had no detectable alkaline phosphatase activity although some alkaline phosphatase mRNA was detected after 21 days in osteoblast differentiating medium. Alkaline phosphatase activity and mRNA in the three mineralizing clones were comparable with the parent clones. Osteocalcin mRNA and protein levels in the non‐mineralizing clone were low and non‐detectable, respectively, while both were elevated in the parent cells and mineralizing subclones after 21 days in differentiating medium. PTH receptor mRNA and activity increased in the four subclones and parent cells with differentiation. mRNA for two other osteoblast phenotypic markers, osteopontin and bone sialoprotein, were similarly expressed in the parent cells and subclones while mRNAs for the transcription factors, Runx2 and osterix, were detectable in both parent and subclone cells. Runx2 was unchanged with differentiation while osterix was increased. Interestingly, PPARγ mRNA expression did not correlate with cell line potential to differentiate into adipocytes. Indian hedgehog mRNA and its receptor (patched) mRNA levels both increased with differentiation while mRNA levels of the Wnt pathway components β‐catenin and dickkopf also increased with differentiation. Although we have focussed on characterizing these clones from the osteoblast perspective it is clear that they may be useful for studying both osteoblast and adipocyte differentiation as well as their transdifferentiation. J. Cell. Biochem. 90: 158–169, 2003.

Communication Between EphrinB2 and EphB4 Within the Osteoblast Lineage

Members of the ephrin and Eph family are local mediators of cell function through largely contact-dependent processes in development and in maturity. Production of ephrinB2 mRNA and protein are increased by PTH and PTHrP in osteoblasts. Both a synthetic peptide antagonist of ephrinB2/EphB4 receptor interaction and recombinant soluble extracellular domain of EphB4 (sEphB4), which is an antagonist of both forward and reverse EphB4 signaling, were able to inhibit mineralization and the expression of several osteoblast genes involved late in osteoblast differentiation. The findings are consistent with ephrinB2/EphB4 signaling within the osteoblast lineage having a paracrine role in osteoblast differentiation, in addition to the proposed role of osteoclast-derived ephrinB2 in coupling of bone formation to resorption. This local regulation might contribute to control of osteoblast differentiation and bone formation at remodeling sites, and perhaps also in modeling.

Zinc Finger Protein 467 Is a Novel Regulator of Osteoblast and Adipocyte Commitment

Osteoblasts and adipocytes are derived from common mesenchymal progenitor cells. The bone loss of osteoporosis is associated with altered progenitor differentiation from an osteoblastic to an adipocytic lineage. cDNA microarrays and quantitative real-time PCR (Q-PCR) were carried out in a differentiating mouse stromal osteoblastic cell line, Kusa 4b10, to identify gene targets of factors that stimulate osteoblast differentiation including parathyroid hormone (PTH) and gp130-binding cytokines, oncostatin M (OSM) and cardiotrophin-1 (CT-1). Zinc finger protein 467 (Zfp467) was rapidly down-regulated by PTH, OSM, and CT-1. Retroviral overexpression and RNA interference for Zfp467 in mouse stromal cells showed that this factor stimulated adipocyte formation and inhibited osteoblast commitment compared with controls. Regulation of adipocyte markers, including peroxisome proliferator-activated receptor (PPAR) γ, C/EBPα, adiponectin, and resistin, and late osteoblast/osteocyte markers (osteocalcin and sclerostin) by Zfp467 was confirmed by Q-PCR. Intra-tibial injection of calvarial cells transduced with retroviral Zfp467 doubled the number of marrow adipocytes in C57Bl/6 mice compared with vector control-transduced cells, providing in vivo confirmation of a pro-adipogenic role of Zfp467. Furthermore, Zfp467 transactivated a PPAR-response element reporter construct and recruited a histone deacetylase complex. Thus Zfp467 is a novel co-factor that promotes adipocyte differentiation and suppresses osteoblast differentiation. This has relevance to therapeutic interventions in osteoporosis, including PTH-based therapies currently available, and may be of relevance for the use of adipose-derived stem cells for tissue engineering.

Plasminogen Activator System in Osteoclasts

To determine which genes of the plasminogen activator (PA) system were expressed in osteoclasts, RNA extracted from microisolated mouse osteoclasts was used as template for reverse transcribed polymerase chain reaction (RT‐PCR) with gene‐specific primer pairs. Using this approach, the expression of RNAs for tissue‐type plasminogen activator, urokinase‐type plasminogen activator, plasminogen activator inhibitor‐1, plasminogen activator inhibitor‐2, protease nexin, and urokinase receptor isoform 1 (uPAR1) were detected in mouse osteoclasts. The expression of uPAR RNA in osteoclasts was confirmed by in situ hybridization with a uPAR1 probe. RNA encoding the uPAR isoform 2 was not detected in mouse osteoclasts, but a novel unspliced uPAR RNA variant was detected in these cells. The novel uPAR variant and uPAR1 RNA were also detected in mouse calvarial osteoblasts, kidney, muscle, and the mouse macrophage cell line J774A.1 by RT‐PCR. The presence of RNAs for most of the components of the PA system in osteoclasts suggests that it may have a functional role in this cell type.

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.

The Chemokine Cxcl1 Is a Novel Target Gene of Parathyroid Hormone (PTH)/PTH-Related Protein in Committed Osteoblasts

The PTH receptor (PTHR1) is expressed on osteoblasts and responds to PTH or PTHrP in an endocrine or autocrine/paracrine manner, respectively. A microarray study carried out on PTHR1-positive osteoblasts (Kusa 4b10 cells) identified the cysteine-X-cysteine (CXC) family chemokine ligand 1 (Cxcl1) as a novel immediate PTH/PTHrP-responsive gene. Cxcl1 is a potent neutrophil chemoattractant with recognized roles in angiogenesis and inflammation, but a role in bone biology has not been described. Cxcl1 mRNA levels were up-regulated 1 h after either PTH or PTHrP treatment of differentiated Kusa 4b10 osteoblasts (15-fold) and mouse calvarial osteoblasts (160-fold) and in rat metaphyseal bone (5-fold) 1 h after a single sc injection of PTH. Furthermore, PTH treatment stimulated a 10-fold increase in secreted Cxcl1 protein by both Kusa 4b10 cells and calvarial osteoblasts. Immunohistochemistry and PCR demonstrated that CXCR2, the receptor for Cxcl1, is highly expressed in osteoclast precursors (hemopoietic cells) but is predominantly undetectable in the osteoblast lineage, suggesting that osteoblast-derived Cxcl1 may act as a chemoattractant for osteoclast precursors. Confirming this hypothesis, recombinant Cxcl1 dose-dependently stimulated migration of osteoclast precursors in cell culture studies, as did conditioned media from Kusa 4b10 cells treated with PTH. These data indicate that local action through the PTHR1 could stimulate cells of the osteoblast lineage to release a chemokine capable of attracting osteoclast precursors to the bone environment.