Chiho Matsumoto

An Essential Role of Cytosolic Phospholipase A2α in Prostaglandin E2–mediated Bone Resorption Associated with Inflammation

Prostaglandin E (PGE)2 produced by osteoblasts acts as a potent stimulator of bone resorption. Inflammatory bone loss is accompanied by osteoclast formation induced by bone-resorbing cytokines, but the mechanism of PGE2 production and bone resorption in vivo is not fully understood. Using cytosolic phospholipase A2α (cPLA2α)-null mice, we examined the role of cPLA2α in PGE2 synthesis and bone resorption. In bone marrow cultures, interleukin (IL)-1 markedly stimulated PGE2 production and osteoclast formation in wild-type mice, but not in cPLA2α-null mice. Osteoblastic bone marrow stromal cells induced the expression of cyclooxygenase (COX)-2 and membrane-bound PGE2 synthase (mPGES) in response to IL-1 and lipopolysaccharide (LPS) to produce PGE2. Osteoblastic stromal cells collected from cPLA2α-null mice also induced the expression of COX-2 and mPGES by IL-1 and LPS, but could not produce PGE2 due to the lack of arachidonic acid release. LPS administration to wild-type mice reduced femoral bone mineral density by increased bone resorption. In cPLA2α-null mice, however, LPS-induced bone loss could not be observed at all. Here, we show that cPLA2α plays a key role in PGE production by osteoblasts and in osteoclastic bone resorption, and suggest a new approach to inflammatory bone disease by inhibiting cPLA2α.

Membrane-Bound Prostaglandin E Synthase-1-Mediated Prostaglandin E2 Production by Osteoblast Plays a Critical Role in Lipopolysaccharide-Induced Bone Loss Associated with Inflammation

PGE2 acts as a potent stimulator of bone resorption in several disorders including osteoarthritis and periodontitis. Three PGE synthases (PGES) were isolated for PGE2 production, but which PGES has the major role in inflammatory bone resorption is still unclear. In this study, we examined the role of PGE2 in LPS-induced bone resorption using membrane-bound PGES (mPGES)-1-deficient mice (mPges1−/−). In osteoblasts from wild-type mice, PGE2 production was greatly stimulated by LPS following the expression of cyclooxygenase 2 and mPGES-1 mRNA, whereas no PGE2 production was found in osteoblasts from mPges1−/−. LPS administration reduced the bone volume in wild-type femur that was associated with an increased number of osteoclasts. In mPges1−/−, however, LPS-induced bone loss was reduced. We next examined whether mPGES-1 deficiency could alter the alveolar bone loss in LPS-induced experimental periodontitis. LPS was injected into the lower gingiva and bone mineral density of alveolar bone was measured. LPS induced the loss of alveolar bone in wild-type, but not in mPges1−/− mice, suggesting an mPGES-1 deficiency resistant to LPS-induced periodontal bone resorption. To understand the pathway of LPS-induced PGE2 production in osteoblast, we used C3H/HeJ mice with mutated tlr4. Osteoblasts from C3H/HeJ mice did not respond to LPS, and PGE2 production was not altered at all. LPS-induced bone loss in the femur was also impaired in C3H/HeJ mice. Thus, LPS binds to TLR4 on osteoblasts that directly induce mPGES-1 expression for PGE2 synthesis, leading to subsequent bone resorption. Therefore, mPGES-1 may provide a new target for the treatment of inflammatory bone disease.

Toll-like receptor 2 heterodimers, TLR2/6 and TLR2/1 induce prostaglandin E production by osteoblasts, osteoclast formation and inflammatory periodontitis

TLR2 forms heterodimers with TLR1 and TLR6, and regulates host defense mechanisms against pathogens. We examined the role of TLR2 heterodimer signaling in osteoclast formation and inflammatory periodontitis. In co-cultures of mouse bone marrow cells and osteoblasts, a TLR2/6 ligand (diacylated lipopeptide designed from Gram-positive bacteria) markedly induced osteoclast formation. A TLR2/1 ligand (triacylated lipopeptide designed from Gram-negative bacteria) also induced osteoclast formation. The osteoclast formation induced by TLR2/6 and TLR2/1 ligands was completely suppressed by indomethacin. Osteoblasts expressed TLR1, 2, 4, and 6 mRNAs, and both TLR2/6 and TLR2/1 ligands induced the expression of COX-2, mPGES-1, and RANKL mRNA, as well as PGE production in osteoblasts. Both TLR2/6 and TLR2/1 ligands induced the resorption of mandibular alveolar bone in organ cultures, and elicited inflammatory periodontitis in vivo. Therefore, TLR2 heterodimer signaling may play a key role in PGE-mediated inflammatory bone loss in periodontal disease.

A novel carborane analog, BE360, with a carbon-containing polyhedral boron-cluster is a new selective estrogen receptor modulator for bone

Carboranes are a class of carbon-containing polyhedral boron-cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors. Estrogen deficiency results in marked bone loss due to increased osteoclastic bone resorption in females, but estrogen replacement therapy is not generally used for postmenopausal osteoporosis due to the risk of uterine cancer. We synthesized a novel carborane compound BE360 to clarify its anti-osteoporosis activity. BE360 showed a high binding affinity to estrogen receptors (ER), ERalpha and ERbeta. In ovariectomized (OVX) mice, femoral bone volume was markedly reduced and BE360 dose-dependently restored bone loss in OVX mice. However, BE360 did not exhibit any estrogenic activity in the uterus. BE360 also restored bone loss in orchidectomized mice without androgenic action in the sex organs. Therefore, BE360 is a novel selective estrogen receptor modulator (SERM) that may offer a new therapy option for osteoporosis.

Epigallocatechin gallate (EGCG) suppresses lipopolysaccharide-induced inflammatory bone resorption, and protects against alveolar bone loss in mice

Epigallocatechin gallate (EGCG), a major polyphenol in green tea, possesses antioxidant properties and regulates various cell functions. Here, we examined the function of EGCG in inflammatory bone resorption. In calvarial organ cultures, lipopolysaccharide (LPS)‐induced bone resorption was clearly suppressed by EGCG. In osteoblasts, EGCG suppressed the LPS‐induced expression of COX‐2 and mPGES‐1 mRNAs, as well as prostaglandin E2 production, and also suppressed RANKL expression, which is essential for osteoclast differentiation. LPS‐induced bone resorption of mandibular alveolar bones was attenuated by EGCGin vitro, and the loss of mouse alveolar bone mass was inhibited by the catechinin vivo.

Hyaluronan inhibits bone resorption by suppressing prostaglandin E synthesis in osteoblasts treated with interleukin-1.

Hyaluronan (HA), a large glycosaminoglycan, is a component of the extra-cellular matrix in various tissues. HA is essential for matrix assembly and fluid viscosity in cartilage, but the roles of HA in bone are unclear. Bone resorption associated with inflammation is closely related to prostaglandin E (PGE) synthesis by osteoblasts induced by cytokines such as interleukin-1 (IL-1). In mouse calvarial cultures, HA inhibited osteoclastic bone resorption and PGE production induced by IL-1. In mouse osteoblasts, HA suppressed IL-1-induced expression of cyclooxygenase(COX)-2 and membrane-bound PGE synthase (mPGES)-1 mRNAs, and PGE2 production. Matrix metalloproteinases (MMPs), including MMP-2 and MMP-13, were produced by osteoblasts in response to IL-1, and were clearly suppressed by HA. In osteoblasts, HA suppressed the NFkappaB-dependent transcription in a luciferase assay. Therefore, HA acts on osteoblasts to suppress the production of PGE2 and MMPs, and inhibits bone resorption, suggesting critical roles of HA in pathological bone loss with inflammation.

Indoxyl sulfate exacerbates low bone turnover induced by parathyroidectomy in young adult rats

Low-turnover bone disease is one of the bone abnormalities observed in patients with chronic kidney disease (CKD) and is recognized to be associated with low serum parathyroid hormone (PTH) level and skeletal resistance to PTH. Indoxyl sulfate (IS) is a representative uremic toxin that accumulates in the blood as renal dysfunction progresses in CKD patients. A recent in vitro study using an osteoblastic cell culture system suggests that IS has an important role in the pathogenesis of low bone turnover through induction of skeletal resistance to PTH. However, the effects of IS on the progression of low bone turnover have not been elucidated. In the present study, we produced rats with low bone turnover by performing parathyroidectomy (PTX) and fed these rats a diet containing indole, a precursor of IS, to elevate blood IS level from indole metabolism. Bone metabolism was evaluated by measuring histomorphometric parameters of secondary spongiosa of the femur. Histomorphometric analyses revealed significant decreases in both bone formation-related parameters and bone resorption-related parameters in PTX rats. In indole-treated PTX rats, further decreases in bone formation-related parameters were observed. In addition, serum alkaline phosphatase activity, a bone formation marker, and bone mineral density of the tibia tended to decrease in indole-treated PTX rats. These findings strongly suggest that IS exacerbates low bone turnover through inhibition of bone formation by mechanisms unrelated to skeletal resistance to PTH.

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth, Angiogenesis, and Metastasis

Background: Prostaglandin E2 (PGE2) is an inflammatory mediator produced in cancer. Results: B16 melanoma cells injected into mice metastasized to bone and soft tissues by activating PGE2-EP4 signaling in stromal cells, inducing osteoclast activation, angiogenesis, and cancer cell proliferation. Conclusion: Stromal cell PGE2 is a key mediator of melanoma tumorigenesis and metastasis. Significance: EP4 receptor blockade is a new potential therapy for tumor metastasis. The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1−/−) mice, we show that prostaglandin E2 (PGE2) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE2 production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE2 further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE2 production by the osteoblasts. PGE2, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE2 acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4−/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE2 effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE2 signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors.

BA321, a novel carborane analog that binds to androgen and estrogen receptors, acts as a new selective androgen receptor modulator of bone in male mice.

Carboranes are a class of carbon-containing polyhedral boron cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors such as estrogen receptor (ER) and androgen receptor (AR). We have synthesized BA321, a novel carborane compound, which binds to AR. We found here that it also binds to ERs, ERα and ERβ. In orchidectomized (ORX) mice, femoral bone mass was markedly reduced due to androgen deficiency and BA321 restored bone loss in the male, whilst the decreased weight of seminal vesicle in ORX mice was not recovered by administration of BA321. In female mice, BA321 acts as a pure estrogen agonist, and restored both the loss of bone mass and uterine atrophy due to estrogen deficiency in ovariectomized (OVX) mice. In bone tissues, the trabecular bone loss occurred in both ORX and OVX mice, and BA321 completely restored the trabecular bone loss in both sexes. Cortical bone loss occurred in ORX mice but not in OVX mice, and BA321 clearly restored cortical bone loss due to androgen deficiency in ORX mice. Therefore, BA321 is a novel selective androgen receptor modulator (SARM) that may offer a new therapy option for osteoporosis in the male.

The Protective Effects of β-Cryptoxanthin on Inflammatory Bone Resorption in a Mouse Experimental Model of Periodontitis

We examined the effects of β-cryptoxanthin, a typical carotenoid, on inflammatory periodontitis. β-Cryptoxanthin suppressed lipopolysaccharide (LPS)-induced osteoclast formation in co-cultures of bone marrow cells and osteoblasts. In a mouse model of periodontitis, it suppressed bone resorption in the mandibular alveolar bone in vitro and restored alveolar bone loss induced by LPS in vivo. β-Cryptoxanthin might protect against periodontal disease.