Continuously applied compressive pressure induces bone resorption by a mechanism involving prostaglandin E2 synthesis

In previous research, we devised a specific culture chamber to examine the effect of continuously applied compressive pressure (CCP) on bone formation and resorption. The chamber was infused with compressed mixed gases with different O2 and CO2 composition to maintain the pO2, pCO2, and pH in the culture medium under pressures of +0.5 atm (1.5 atm total) to +2.0 atm (3.0 atm total) at the same levels as those at the ordinary pressure (1 atm). Using the specific culture chamber, we demonstrated that CCP greatly suppressed the differentiation of mouse osteoblast-like MC3T3-E1 cells. The inhibition by CCP appeared to be mediated by prostaglandin E2 (PGE2). In the present study, we examined the effect of CCP on osteoclastic bone resorption. CCP treatment of mouse bone marrow culture markedly increased both the PGE2 production and the number of tartrate-resistant acid phosphatase (TRACP)-positive mononuclear cells (possibly precursors of multinucleated osteoclasts). An autoradiographic study using [125I]-salmon calcitonin showed clearly that those TRACP-positive cells had calcitonin receptors. The CCP effect was the greatest at +1.0 atm (2.0 atm total). Isobutylmethylxanthine potentiated the production of TRACP-positive cells induced by CCP. Adding indomethacin completely inhibited both the TRACP-positive cell formation and the PGE2 production induced by CCP. CCP also increased the release of 45Ca from prelabeled mouse calvaria during later stages (2-6 days) of the 6-day culture period. CCP markedly increased PGE2 but not interleukin 1 in the culture media of mouse calvaria. These results indicate that, besides inhibiting osteoblast differentiation, CCP stimulates bone resorption by generating new osteoclasts through a mechanism involving PGE2 production.     

Contribution of membrane-associated prostaglandin E2 synthase to bone resorption

This study initially confirmed that, among prostaglandins (PGs) produced in bone, only PGE(2) has the potency to stimulate osteoclastogenesis and bone resorption in the mouse coculture system of osteoblasts and bone marrow cells. For the PGE(2) biosynthesis two isoforms of the terminal and specific enzymes, membrane-associated PGE(2) synthase (mPGES) and cytosolic PGES (cPGES) have recently been identified. In cultured mouse primary osteoblasts, both mPGES and cyclooxygenase-2 were induced by the bone resorptive cytokines interleukin-1, tumor necrosis factor-alpha, and fibroblast growth factor-2. Induction of mPGES was also seen in the mouse long bone and bone marrow in vivo by intraperitoneal injection of lipopolysaccharide. In contrast, cPGES was expressed constitutively both in vitro and in vivo without being affected by these stimuli. An antisense oligonucleotide blocking mPGES expression inhibited not only PGE(2) production, but also osteoclastogenesis and bone resorption stimulated by the cytokines, which was reversed by addition of exogenous PGE(2). We therefore conclude that mPGES, which is induced by and mediates the effects of bone resorptive stimuli, may make a target molecule for the treatment of bone resorptive disorders.     

Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice

Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP-2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4-knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor.     

Adrenic acid inhibits prostaglandin synthesis

Adrenic acid inhibits oxygenation of arachidonic acid by homogenates of rabbit renal medulla and blunts the vascular effects of bolus injections of arachidonic acid in the rat. Adrenic acid may be a naturally occurring modulator of cyclo-oxygenase activity.     

Endosomal TLR3 signaling in stromal osteoblasts induces prostaglandin E2–mediated inflammatory periodontal bone resorption

Toll-like receptors (TLRs) are pattern recognition receptors that play a critical role in innate immune diseases. TLR3, which is localized in the endosomal compartments of hematopoietic immune cells, is able to recognize double-stranded RNA (dsRNA) derived from viruses and bacteria and thereby induce innate immune responses. Inflammatory periodontal bone resorption is caused by bacterial infections, which initially is regulated by innate immunity; however, the roles of TLR3 signaling in bone resorption are still not known. We examined the roles of TLR3 signaling in bone resorption using poly(I:C), a synthetic dsRNA analog. In cocultures of mouse bone marrow cells and stromal osteoblasts, poly(I:C) clearly induced osteoclast differentiation. In osteoblasts, poly(I:C) increased PGE₂ production and upregulated the mRNA expression of PGE₂-related genes, Ptgs2 and Ptges, as well as that of a gene related to osteoclast differentiation, Tnfsf11. In addition, we found that indomethacin (a COX-2 inhibitor) or an antagonist of the PGE₂ receptor EP4 attenuated the poly(I:C)-induced PGE₂ production and subsequent Tnfsf11 expression. Poly(I:C) also prolonged the survival of the mature osteoclasts associated with the increased mRNA expression of osteoclast marker genes, Nfatc1 and Ctsk. In ex vivo organ cultures of periodontal alveolar bone, poly(I:C) induced bone-resorbing activity in a dose-dependent manner, which was attenuated by the simultaneous administration of either indomethacin or an EP4 antagonist. These data suggest that TLR3 signaling in osteoblasts controls PGE₂ production and induces the subsequent differentiation and survival of mature osteoclasts. Endogenous TLR3 in stromal osteoblasts and osteoclasts synergistically induces inflammatory alveolar bone resorption in periodontitis.     

Effects of prostaglandin E2 on alveolar bone resorption during orthodontic tooth movement

Twenty Sprague-Dawley rats weighing 280-300 g were divided into two groups of ten animals each. They were treated by daily submucosal injections of 50 micrograms prostaglandin E2 (PGE2) per kilogram body weight into the region below the apex of the left first maxillary molar (experimental), or vehicle into the region below the apex of the right first molar (control), for a period of 5 days. The animals of the first group were sacrificed immediately following the treatment period, while those of the second group were sacrificed 5 days after the treatment period. Twenty-two hours prior to sacrifice, a piece of latex orthodontic elastic was secured to the adjacent area between the first and second maxillary molars of both sides of each rat by using two mosquito hemostats. The periodontal ligament (PDL) mesial to the mesiobuccal root of the first maxillary molar was assayed for changes in PDL cell factors. The results showed that immediately following the 5-day treatment period the left PDL had a significant decrease in the total number of fibroblasts and a significant increase in the total number of both osteoclasts and nuclei per osteoclast, while no significant changes in the osteoblasts when compared with those of the right control PDL. The left PDL of animals which were sacrificed 5 days after the treatment period revealed a significant decrease in the number of total fibroblasts and only a slight decrease in both numbers of total osteoclasts and total nuclei per osteoclast, but again no significant changes in osteoblasts when compared with those of the right control PDL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of bone formation by prostaglandin E2

We examined the effect of prostaglandin E2 (PGE2), in the presence or absence of cortisol, on bone formation in 21-day fetal rat calvaria maintained in organ culture for 24 to 96 h. [3H]Thymidine and [3H] proline incorporation were used to assess DNA and collagen synthesis, respectively. Changes in dry weight and DNA content were assessed after 96 h. PGE2 (10(-7) M) stimulated both DNA and collagen synthesis in calvaria. The effect on DNA synthesis was early (24 h), transient and limited to the periosteum. Collagen synthesis was stimulated at a later time (96 h), predominantly in the central bone. Cortisol (10(-7) M) inhibited DNA and collagen synthesis. The addition of PGE2 reversed the inhibitory effects of cortisol on DNA synthesis and content and increased collagen synthesis in central bone to levels above control untreated cultures. We conclude that PGE2 has stimulatory effects on bone formation and can reverse the inhibitory effects of cortisol. Hence the effects of cortisol may be mediated in part by their ability to reduce the endogenous production of prostaglandins.     

Ametantrone inhibits prostaglandin — mediated resorption in bone organ culture

Prostaglandins (PG) have been postulated to be involved in both tumor metastases to bone and in tumor-induced bone resorption. The anthracenedione antineoplastic agents ametantrone (HAQ) and mitoxantrone are potent antioxidants and inhibit hydroperoxide-dependent initiation and propagation reactions. Therefore, these compounds may inhibit PG production and could also inhibit tumor metastases and tumor-induced resorption. The ability of HAQ, a prototypic anthracenedione, to inhibit PG synthesis and PG-mediated bone resorption was investigated using neonatal mouse calvaria in organ culture. Epidermal growth factor (EGF) stimulates bone resorption in this tissue by inducing PG synthesis. Consequently, if HAQ inhibits EGF-stimulated PG synthesis, it should also inhibit EGF-stimulated bone resorption. HAQ, at 10 μM, completely abolished EGF-stimulated PG synthesis and calcium release. Moreover, HAQ (1.0–30 μM) inhibition of EGF-stimulated PGE₂ synthesis correlated with the inhibition of EGF-stimulated Ca release in a concentration-dependent manner. In contrast to EGF, parathyroid hormone stimulates resorption by a PG-independent pathway. HAQ at 10 μM had no effect on parathyroid hormone stimulated Ca release. These results suggest that HAQ inhibition of bone resorption appears to be primarily mediated by inhibition of PG biosynthesis.     

Stimulation of prostaglandin E2 and bone resorption by recombinant human interleukin 1 alpha in fetal mouse bones

Recombinant human interleukin 1 alpha (rhIL-1 alpha) stimulates prostaglandin E2 and bone resorption in cultured forearm bones of fetal mouse in a dose-dependent manner: the minimal rhIL-1 alpha to elicit a significant bone resorption was 1.6 ng/ml (89 pM). The half maximal concentrations to elicit bone resorption and thymocyte proliferation were 3.3 ng/ml (183 pM) and 0.31 ng/ml (17 pM), respectively. The bone resorbing activity induced by IL-1 was partially inhibited by indomethacin and hydrocortisone, and completely inhibited by anti-IL 1-antibody. There was a good correlation between PGE2 production and bone resorption induced by IL-1 alpha. These results suggest that rhIL-1 alpha stimulates bone resorption at approximately 10 times the concentrations necessary for thymocyte proliferation and that PGE2 produced in the bone is at least in part involved in osteoclastic bone resorption.     

Endogenous prostaglandin E2 synthesis inhibits growth of polyoma virus-transformed 3T3 fibroblasts.

Indomethacin lowered the cellular content of adenosine 3 ′: 5 ′-monophosphate (cAMP) and stimulated growth of polyoma virus-transformed 3T3 fibroblasts. Exogenous prostaglandin E₂, at concentrations produced in the absence of inhibitor, reversed the effects of indomethacin on cAMP levels and cell proliferation. Therefore, endogenously produced prostaglandin E₂ decreases cell growth and raises the levels of cAMP in these cells.     

Gallium inhibits bone resorption by a direct effect on osteoclasts

Gallium nitrate has been used clinically to treat cancer-related hypercalcemia. It has been suggested that gallium may reduce calcium release from bone by inhibiting bone resorption, but the mechanism(s) involved remain to be elucidated. Therefore, we have examined the effect of gallium on bone resorption in vitro using osteoclasts isolated from neonatal rat long bones cultured on slices of cortical bone. Gallium nitrate (0.01–100 μg/ml) produced a concentration-dependent inhibition of bone resorption. Morphological studies showed that even (100μg/ml) gallium nitrate induced no light microscopical change in osteoclast morphology and did not affect their survival on bone slices. Pretreatment of bone slices with gallium nitrate (100μg/ml for 18 h), followed by extensive washing also inhibited subsequent osteoclastic bone resorption. These results suggest that gallium can be adsorbed onto the calcified surface of bone and inhibit osteoclastic bone resorption.     

2-Carba cyclic phosphatidic acid inhibits lipopolysaccharide-induced prostaglandin E2 production in a human macrophage cell line

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator that contains a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. Using mouse models for multiple sclerosis (cuprizone-induced demyelination and experimental autoimmune encephalomyelitis) and traumatic brain injury, we revealed that cPA and its metabolically stabilized cPA derivative, 2-carba-cPA (2ccPA), have potential to protect against neuroinflammation. In this study, we investigated whether 2ccPA has anti-inflammatory effect on peripheral immune function or not using inflammation-induced macrophages-like cell line, THP-1 monocytes differentiated by phorbol 12-myristate 13-acetate (PMA). Lipopolysaccharide (LPS)-stimulated THP-1 cells were found to have higher expression of the mRNAs of several inflammation-related cytokines and of the enzyme cyclooxygenase-2 (Cox-2); however, when THP-1 cells were stimulated by LPS in the presence of 2ccPA, the increase in the expression of pro-inflammatory cytokine and Cox-2 mRNA was attenuated. 2ccPA treatment also decreased the amount of prostaglandin E2 (PGE2) produced by LPS-stimulated THP-1 cells and decreased expression of the mRNA of prostaglandin E receptor 2 (EP2, PTGER2), a PGE2 receptor that mediates inflammation. These results indicate that 2ccPA has anti-inflammatory properties.     

Human macrophage colony-stimulating factor inhibits bone resorption by osteoclasts disaggregated from rat bone

Colony stimulating factors (CSFs) regulate the survival, proliferation and differentiation of haemopoietic progenitor cells, as well as the functional activity of mature cells. Because the osteoclast is derived from haemopoietic tissue, and because osteoblastic cells produce CSFs, we tested the effects of several CSFs on bone resorption by osteoclasts disaggregated from neonatal rat long bone. We found that recombinant macrophage (M)-CSF was a potent inhibitor of bone resorption, causing significant inhibition at concentrations similar to those required to support the growth of macrophage colonies in agar. Unlike other inhibitors of osteoclastic resorption, M-CSF did not alter cytoplasmic motility in time-lapse recordings, suggesting that M-CSF may inhibit osteoclasts through a different transduction mechanism. None of the remaining cytokines tested (granulocyte-macrophage CSF, interleukin 3, interleukin 6, or interferon γ) influenced bone resorption. M-CSF production may be a mechanism by which osteoblastic cells, which produce M-CSF, may regulate osteoclastic function. Alternatively, inhibition of osteoclastic resorption by a CSF that is responsible for amplification of the macrophage compartment may reflect a close lineage relationship between mononuclear phagocytes, in which M-CSF induces a diversion of lineage resources away from osteoclastic function.     

Phytocomponent p-hydroxycinnamic acid stimulates bone formation and inhibits bone resorption in rat femoral tissues in vitro

The objective of this study was to examine alcohol-induced changes of bone in hormone-deficient males using the developed method. In the process of bone resorption, type I collagen crosslinking molecules, pyridinoline (PYD), are released into the circulation and cleared by the kidneys. ²H₂O as a tracer has been applied to measure the synthesis rates of slow-turnover proteins and successfully applied to bone collagen synthesis in our hormone deficiency rats. This study demonstrated for the first time, the early changes of the femur bone degradation in hormone-deficient male individuals, more influenced by alcohol through histopathological study, serum PYD assay, and ²H₂O labeling. We also observed that serum PYD was a sensitive pathological marker of bone degradation in castrated osteoporosis males and the unique features of ²H₂O labeling to measure the bone turnover collagen synthesis rates were excellent markers of bone degradation and aging.     

Gamma interferon inhibits basal and interleukin 1-induced prostaglandin production and bone resorption in neonatal mouse calvaria

Production of the osteolytic arachidonic acid metabolites, prostaglandin (PG) E2, PGI2 and PGF2 alpha, by neonatal mouse calvariae was quantitated by gas chromatography/mass spectrometry. Mouse recombinant interleukin 1 (rIL-1) raised medium levels of PGE2 and PGI2 (measured as 6-keto-PGF1 alpha) in the dose range tested (1.0-10.0 U/ml culture medium), while an effect on PGF2 was only observed at 10 U/ml. Bone resorption in response to rIL-1 reached a plateau at 3.0 U/ml. Mouse recombinant gamma-interferon (rIFN-gamma) between 100-500 U/ml suppressed basal PG synthesis and spontaneous resorption of cultured bone. In addition, IFN-gamma at 100 U/ml prevented stimulation of PG synthesis by 3.0 U/ml rIL-1 and thereby reduced the bone resorbing activity of the cytokine by at least 60%. 5 X 10(-7) M indomethacin was equally effective in suppression of PG synthesis and bone resorption. The present study provides evidence that IFN-gamma inhibits PG synthesis and consequently resorption of cultured bone.     

2-Methoxycinnamaldehyde inhibits tumor angiogenesis by suppressing Tie2 activation

Blood vessels are mainly composed of intraluminal endothelial cells (ECs) and mural cells adhering to the ECs on their basal side. Immature blood vessels lacking mural cells are leaky; thus, the process of mural cell adhesion to ECs is indispensable for stability of the vessels during physiological angiogenesis. However, in the tumor microenvironment, although some blood vessels are well-matured, the majority is immature. Because mural cell adhesion to ECs also has a marked anti-apoptotic effect, angiogenesis inhibitors that destroy immature blood vessels may not affect mature vessels showing more resistance to apoptosis. Activation of Tie2 receptor tyrosine kinase expressed in ECs mediates pro-angiogenic effects via the induction of EC migration but also facilitates vessel maturation via the promotion of cell adhesion between mural cells and ECs. Therefore, inhibition of Tie2 has the advantage of completely inhibiting angiogenesis. Here, we isolated a novel small molecule Tie2 kinase inhibitor, identified as 2-methoxycinnamaldehyde (2-MCA). We found that 2-MCA inhibits both sprouting angiogenesis and maturation of blood vessels, resulting in inhibition of tumor growth. Our results suggest a potent clinical benefit of disrupting these two using Tie2 inhibitors.     

Cellular confluence determines injury-induced prostaglandin E2 synthesis by human keratinocyte cultures

When keratinocyte cultures become confluent, their prostaglandin E2 synthesis is suppressed. To determine whether the injury response is characterized by increased prostaglandin E2 synthesis, an in vitro injury model was developed. When confluent keratinocyte cultures were focally lethally irradiated using ultraviolet light B, a dose-dependent increase in prostaglandin E2 synthesis was induced by the injury. After irradiation, confluent cultures' prostaglandin E2 synthesis increased for 2 days to 8-fold more than controls, then decreased to control values by day 6. Increased prostaglandin E2 synthesis was first detected 8 h after injury. Focal irradiation of non-confluent cultures (killing isolated colonies) caused no change in prostaglandin E2 synthesis, indicating that culture continuity must be disrupted before synthesis increases. In addition, partial irradiations of petri dishes demonstrated that enhanced metabolism was confined to cells adjacent to the injury site and was not mediated by a soluble factor. When confluent and injured cultures were incubated with [14C]arachidonic acid, and the products formed analyzed by thin layer chromatography, 10-fold more prostaglandin E2 microgram protein was seen in irradiated cultures relative to confluent controls. The products formed by each group were the same, and no consistent increases in metabolites other than prostaglandin E2 were observed. The increased synthesis of prostaglandin E2 by injured cultures was apparently due to an increase in cyclooxygenase activity as determined by kinetic experiments. These data indicate that the pattern of metabolism of arachidonic acid seen in non-confluent cultures is similar to that seen in injury, and that cell-cell contact modulates enhanced prostaglandin E2 synthesis.