Prostaglandin E2 (PGE2) increases the number of rat bone marrow osteogenic stromal cells (BMSC) via binding the EP4 receptor, activating sphingosine kinase and inhibiting caspase activity.

Prostaglandin E(2) (PGE(2)) is bone-anabolic, i.e. stimulates bone formation and increases bone mass. In this study, we explored possible intracellular mechanisms of its increase of osteogenic cells in rat bone marrow. Adherent rat bone marrow cells were counted after 12-48 h or cultured for 21 days and mineralized nodules were counted. Also, apoptosis of marrow cells was measured after in vivo PGE(2) injection. PGE(2) (100 nM) increased 2-3 fold the number of adherent BMSC, an effect which was mediated via binding the EP(4) receptor since it was mimicked by forskolin and 11-deoxy-prostaglandin E(1) (PGE(1)) and was blocked by DDA and L-161982 (EP(4) antagonist). PGE(2) stimulated sphingosine kinase (SPK) activity since its effects were blocked by DMS (SPK inhibitor) and mimicked by SPP (SPK product). PGE(2) reduced the activity of caspase-3 and -8 in BMSC and their inhibitors increased BMSC number and nodule formation. In vivo, PGE(2) prevented the increase in the apoptosis of bone marrow cells caused by indomethacin. We propose that PGE(2) exerts an anti-apoptotic effect on BMSC, thereby increasing their number and subsequent osteoblastic differentiation. Such an effect could explain how PGE(2) stimulates bone formation in vivo.

Preservation of thoracic spine microarchitecture by alendronate: comparison of histology and microCT.

The effect of bisphosphonates on trabecular microarchitecture may contribute to the reduced risk of vertebral fracture with treatment independent of the bone volume. Trabecular structure was examined at the twelfth thoracic vertebra after 2 years of treatment of two groups of ovariectomized baboons on high and low doses of alendronate, compared with ovariectomized and non-ovariectomized controls. Standard 2D histological measurements showed that alendronate treatment of ovariectomized animals resulted in significantly higher total trabecular length and a lower marrow star volume in comparison with ovariectomized controls indicating preservation of connectivity. Similarly when the vertebrae were examined using a novel thick slice technique that combines 2D and 3D information, ovariectomy produced a significantly higher number of "real" trabecular termini in comparison with normal. When ovariectomized animals were treated with increasing doses of alendronate, fewer "real" termini were seen. MicroCT analysis (2D and 3D) correlated well with the histological measurements, although more variability and less discrimination between groups was seen, with no statistically significant differences with alendronate treatment. Reduced vertebral fracture risk with alendronate may be due to a combination of factors including the increased bone volume, reduced turnover and greater mineralization reported by others. Added to this is now suggested the preservation of several aspects of vertebral cancellous architecture, with microscopy the most sensitive method of analysis.

Osteoclast formation, survival and morphology are highly dependent on exogenous cholesterol/lipoproteins.

Osteoporosis is associated with both atherosclerosis and vascular calcification. No mechanism yet explains the parallel progression of these diseases. Here, we demonstrate that osteoclasts (OCL) depend on lipoproteins to modulate cellular cholesterol levels and that this controls OCL formation and survival. Removal of cholesterol in OCL via high-density lipoprotein or cyclodextrin treatment dose-dependently induced apoptosis, with actin disruption, nuclear condensation and caspase-3 activation. One mechanism linked to the induction of OCL apoptosis was the cell-type-specific failure to induce HMG-CoA reductase mRNA expression, suggesting an absence of feedback regulation of de novo cholesterol biosynthesis. Furthermore, cyclodextrin treatment substantially suppressed essential M-CSF and RANKL-induced survival signaling pathways via Akt, mTOR and S6K. Consistent with these findings, cholesterol delivery via low-density lipoprotein (LDL) significantly increased OCL viability. Interestingly, OCLs from the LDL receptor (LDLR)-/- mouse exhibited reduced size and lifespan in vitro. Remarkably, LDLR+/+ OCL in lipoprotein-deficient medium phenocopied LDLR-/- OCL, while fusion and spreading of LDLR-/- OCL was rescued when cholesterol was chemically delivered during differentiation. With hyperlipidemia being associated with disease of the vascular system and bone, these findings provide novel insights into the selective lipoprotein and cholesterol dependency of the bone resorbing cell. Cell Death and Differentiation (2004) 11, S108-S118. doi:10.1038/sj.cdd.4401399 Published online 12 March 2004

M-CSF, TNFalpha and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase.

Multinucleated bone-resorbing osteoclasts (Ocl) are cells of hematopoietic origin that play a major role in osteoporosis pathophysiology. Ocl survival and activity require M-CSF and RANK ligand (RANKL). M-CSF signals to Akt, while RANKL, like TNFalpha, activates NF-kappaB. We show here that although these are separate pathways in the Ocl, signaling of all three cytokines converges on mammalian target of rapamycin (mTOR) as part of their antiapoptotic action. Accordingly, rapamycin blocks M-CSF- and RANKL-dependent Ocl survival inducing apoptosis, and suppresses in vitro bone resorption proportional to the reduction in Ocl number. The cytokine signaling intermediates for mTOR/ribosomal protein S6 kinase (S6K) activation include phosphatidylinositol-3 kinase, Akt, Erks and geranylgeranylated proteins. Inhibitors of these intermediates suppress cytokine activation of S6K and induce Ocl apoptosis. mTOR regulates protein translation acting via S6K, 4E-BP1 and S6. We find that inhibition of translation by other mechanisms also induces Ocl apoptosis, demonstrating that Ocl survival is highly sensitive to continuous de novo protein synthesis. This study thus identifies mTOR/S6K as an essential signaling pathway engaged in the stimulation of cell survival in osteoclasts.

Inhibition of bone resorption by alendronate and risedronate does not require osteoclast apoptosis.

Bisphosphonate inhibition of bone resorption was proposed to be due to osteoclast apoptosis. We tested this hypothesis for both the N-containing bisphosphonates alendronate and risedronate, which inhibit farnesyldiphosphate synthase and thus protein isoprenylation, and for clodronate and etidronate, which are metabolized to adenosine triphosphate (ATP) analogs. We found, in dose-response studies, that alendronate and risedronate inhibit bone resorption (in pit assays) at doses tenfold lower than those reducing osteoclast number. At an N-bisphosphonate dose that inhibited resorption and induced apoptosis, the antiapoptotic caspase inhibitor, Z-VAD-FMK, maintained osteoclast (Oc) number but did not prevent inhibition of resorption. Furthermore, when cells were treated with either alendronate alone or in combination with Z-VAD-FMK for 24 or 48 h, subsequent addition of geranylgeraniol, which restores geranylgeranylation, returned bone resorption to control levels. On the other hand, Z-VAD-FMK did block etidronate and clodronate inhibition of resorption. Moreover, in cells treated with etidronate, but not alendronate or risedronate, Z-VAD-FMK also prevented actin disruption, an early sign of osteoclast inhibition by bisphosphonates. These observations indicate that, whereas induction of apoptosis plays a major role in etidronate and clodronate inhibition of resorption, alendronate and risedronate suppression of bone resorption is independent of their effects on apoptosis.

Alendronate increases degree and uniformity of mineralization in cancellous bone and decreases the porosity in cortical bone of osteoporotic women.

The strength of bone is correlated with bone mass but is also influenced significantly by other factors such as structural properties of the matrix (e.g., collagen mutations) and the mineral. Changes at all levels of this organization could contribute to fracture risk. We investigated the effects of alendronate (Aln) treatment on the density of mineralization and the ultrastructure of the mineral/collagen composite, size and habitus of mineral particles in iliac cancellous bone, as well as on the porosity of iliac cortical bone from postmenopausal osteoporotic women. Twenty-four transiliac bone biopsies from Phase III Aln (10 mg/day) trials (placebo and Aln after 2 and 3 years of treatment, n = 6 per group) were studied. The mineral structure was investigated by quantitative backscattered electron imaging (qBEI) and by scanning small-angle X-ray scattering (scanning-SAXS). qBEI histograms reflect the bone mineralization density distribution (BMDD), whereas SAXS patterns characterize the size and arrangement of the mineral particles in bone. We found that: (i) the relative calcium content of osteoporotic bone was significantly lower than that of data-base controls; (ii) mineralization was significantly higher and more uniform after Aln treatment; (iii) size and habitus of the mineral particles was not different between placebo and Aln-treated groups; and (iv) the porosity of cortical bone was reduced significantly by Aln treatment. We conclude that Aln treatment increases the degree and uniformity of bone matrix mineralization without affecting the size and habitus of the mineral crystals. It also decreases the porosity of the corticalis. Together these effects may contribute to the observed reduction in fractures.

Prostaglandin receptor EP(4) mediates the bone anabolic effects of PGE(2).

Prostaglandin (PG) E(2) is a potent inducer of cortical and trabecular bone formation in humans and animals. Although the bone anabolic action of PGE(2) is well documented, the cellular and molecular mechanisms that mediate this effect remain unclear. This study was undertaken to examine the effect of pharmacological inactivation of the prostanoid receptor EP(4), one of the PGE(2) receptors, on PGE(2)-induced bone formation in vivo. We first determined the ability of EP(4)A, an EP(4)-selective ligand, to act as an antagonist. PGE(2) increases intracellular cAMP and suppresses apoptosis in the RP-1 periosteal cell line. Both effects were reversed by EP(4)A, suggesting that EP(4)A acts as an EP(4) antagonist in the cells at concentrations consistent with its in vitro binding to EP(4). We then examined the effect of EP(4) on bone formation induced by PGE(2) in young rats. Five- to 6-week-old rats were treated with PGE(2) (6 mg/kg/day) in the presence or absence of EP(4)A (10 mg/kg/day) for 12 days. We found that treatment with EP(4)A suppresses the increase in trabecular bone volume induced by PGE(2). This effect is accompanied by a suppression of bone formation indices: serum osteocalcin, extent of labeled surface, and extent of trabecular number, suggesting that the reduction in bone volume is due most likely to decreased bone formation. The pharmacological evidence presented here provides strong support for the hypothesis that the bone anabolic effect of PGE(2) in rats is mediated by the EP(4) receptor.

Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase Cgamma in prefusion osteoclasts.

The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function. This study examines M-CSF- and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130(Cas), paxillin, and PLC-gamma. However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner. Involvement of PLC-gamma activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF-mediated cell spreading. Furthermore, in Src(-/-) pOCs M-CSF, together with filamentous actin, causes recruitment of beta(3) integrin and PLC-gamma to adhesion contacts and induces stable association of beta(3) integrin with PLC-gamma, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-gamma can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-gamma is a common downstream mediator for adhesion and growth factor signals. M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.

Expression of the prostaglandin E(2) (PGE(2)) receptor subtype EP(4) and its regulation by PGE(2) in osteoblastic cell lines and adult rat bone tissue.

Prostaglandins E (especially PGE(2)) stimulate bone formation and increase bone mass in several species including man. The mechanism for this effect, the target cells, and the receptors involved are not known. Specific cell-surface receptors for PGE(2) (EP(1-4)) have been cloned and characterized. EP(4) was reported to be the major receptor in embryonic and neonatal bone tissue in mice, especially in preosteoblasts; however, no data are available regarding its expression in adult bone. This study examines the expression of EP(4) in bone tissue of young adult rats, in which PGE(2) is markedly anabolic, and in various osteoblastic cell lines. Using northern blot analysis, we found that osteoblastic cell lines RCT-1, RCT-3, TRAB-11, and RP-1, primary osteoblastic cells harvested from fetal rat calvaria, as well as tibiae and calvariae of 5-week-old rats express 3.8 kb EP(4) messenger RNA (mRNA). Treatment of periosteal cells (RP-1) in vitro with 10(-6) mol/L PGE(2) increased the levels of both EP(4) mRNA and EP(4) protein, peaking at 1-2 h. Similarly, systemic administration of an anabolic dose of PGE(2) (3-6 mg/kg) to young adult rats upregulated the expression of EP(4) in the tibia and calvaria, also peaking at 1-2 h. Using in situ hybridization, we found increased expression of EP(4) in bone marrow cells of the tibial metaphysis in response to systemic PGE(2) treatment. The preosteoblastic nature of these EP(4)-expressing cells was suggested by the fact that dexamethasone-treated bone marrow stromal cells in culture express EP(4) mRNA, which is upregulated by PGE(2). Northern blot analysis failed to detect both basal and PGE(2)-induced EP(2) mRNA in the bone samples or cell lines tested. Taken together, these data implicate EP(4) as the major cyclic AMP-related PGE(2) receptor subtype expressed in bone tissue and osteoblastic cells and indicate that this receptor is upregulated by its ligand, PGE(2).