Elizabeth Allan

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 >or=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 (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-delta (C/EBP delta) 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.

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals. A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.

Specific binding of leukemia-inhibitory factor (LIF) to osteoblasts, but not multinucleated osteoclasts, was demonstrated by receptor autoradiography by using cells isolated from newborn rat long bones. The clonal rat osteogenic sarcoma cells, UMR 106-06, which have several phenotypic properties of osteoblasts, expressed 300 LIF receptors per cell, with an apparent KD of 60 pM. Treatment of calvarial osteoblasts or UMR 106-01 cells with LIF resulted in a dose-dependent inhibition of plasminogen activator (PA) activity. Both calvarial osteoblasts and osteogenic sarcoma cells were shown by Western blotting and reverse fibrin autography to produce plasminogen activator inhibitor-1 (PAI-1), the production of which was increased by LIF treatment. Northern blot analysis revealed that LIF treatment resulted in a rapid (peak 1 hour), dose-dependent increase in mRNA for PAI-1. LIF treatment of the preosteoblast cell line, UMR 201, enhanced the alkaline phosphatase response of these cells to retinoic acid. Each of the osteoblast-like cell types (calvarial osteoblasts, UMR 106-06, and UMR 201) was shown to produce LIF by bioassay and, by using the polymerase chain reaction (PCR), was shown to express low levels of mRNA for LIF. These data establish that cells of the osteoblast lineage are targets for LIF action. The reported anabolic effects of this cytokine on bone formation in vivo could be related to inhibition of protease activity. LIF may be an important paracrine modulator in bone, or perhaps an autocrine one, based on the evidence for its production by osteoblasts and osteoblast-like cells.

Osteoblasts secrete transforming growth factor beta (TGF beta) as a biologically inert, latent complex that must be dissociated before the growth factor can exert its effects. We have examined the production and proteolytic activation of latent TGF beta (LTGF beta) by clonal UMR 106-01 rat osteosarcoma cells and neonatal mouse calvarial (MC) osteoblast-like cells in vitro. Synthetic bPTH-(1-34) increased the activity of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PA) in cell lysates (CL) of UMR 106-01 cells. The concentration of active TGF beta in serum-free CM from cultures treated with bPTH-(1-34) and plasminogen was significantly greater than in CM from untreated controls and cultures treated with either bPTH-(1-34) or plasminogen alone. This effect occurred at concentrations of PTH-(1-34) that increased PA activity and was prevented by aprotinin, an inhibitor of plasmin activity. Treatment with bPTH-(1-34) had no effect on the concentration of TGF beta in acid-activated samples of CM. Functional consequences of proteolytically activated TGF beta was examined in primary cultures of neonatal MC osteoblast-like cells. Human platelet TGF beta 1 caused a dose-dependent increase in the migration of these cells in an in vitro wound healing assay. Cell migration was also stimulated in cultures treated with bPTH-(1-34) and plasminogen together. This effect was blocked by an anti-TGF beta 1 antibody. The results of these studies demonstrate that (1) LTGF beta secreted by osteoblasts in vitro is activated under conditions where the plasmin activity in the cultures is increased, and (2) the TGF beta generated by plasmin-mediated proteolysis is biologically active. We suggest that the local concentration of TGF beta in bone may be controlled by the osteoblast-associated plasminogen activator/plasmin system. Furthermore, since several calciotropic factors influence osteoblast PA activity, this system may have an important role in mediating their anabolic and/or catabolic effects.