prostaglandin e2 ( pge2 ) is mainly produced by osteoblasts during bone resorption associated with inflammation and acts as a potent stimulator of bone resorption .
inflammatory cytokines , such as interleukin ( il)-1 and tumor necrosis factor  ( tnf  ) which induces pge2 production by osteoblasts , and pge2 are able to induce the expression of receptor activator of nf-b ligand ( rankl ) on the surface of osteoblasts .
two types of cyclooxygenase ( cox ) , cox-1 and cox-2 , are expressed in osteoblasts , and the expression of cox-2 is markedly induced by inflammatory stimulants .
the blockage of pge2 synthesis by nonsteroidal anti - inflammatory drugs ( nsaids ) could suppress rankl - dependent osteoclastic bone resorption associated with inflammation [ 2 , 3 ] .
membrane - bound pge synthase-1 ( mpges-1 ) is also essential for pge2 synthesis in osteoblasts and pge - mediated osteoclast differentiation [ 2 , 3 ] . an mpges-1-null mouse demonstrated that pge2 production by osteoblasts is essential for inflammatory bone loss induced by lipopolysaccharide ( lps ) in vivo .
the transient receptor potential ( trp ) subfamily of ion channels contains six members ( trpv1trpv6 ) , most of which were thermal - sensitive cation channels .
trpv1 has been cloned from a cdna library of the dorsal root ganglion in sensory neurons , and the trpv1 signal is suggested to be a potential regulator of pain associated with thermal stimulation [ 5 , 6 ] .
capsaicin is a natural ligand for trpv1 that regulates nerve - related pain - sensitive signals , inflammation , and cancer growth .
mouse osteoblasts express trpv1 , and capsaicin suppresses il-1-induced osteoclast differentiation in the cocultures of mouse bone marrow cells and osteoblasts .
rossi et al .   reported that human pre - osteoclasts express trpv1 and its signal modulates rankl - mediated osteoclast differentiation .
recent studies suggesting some of the possible roles of trpv1 in bone metabolism are controversial , and the physiological and pharmacological effects of trpv1 signals are not known in bone tissues .
the present study examined the effects of capsaicin , a natural trpv1 ligand , on inflammatory bone resorption in vitro and in vivo .
these results showed that the trpv1 signal suppressed osteoclastic bone resorption associated with pge2 production in vitro and attenuated inflammatory bone loss induced by lps in vivo .
pge2 was obtained from sigma - aldrich co. llc ( st louis , mo ) .
newborn ( 2-day - old and 5-day - old ) and adult ( 6-week - old ) ddy mice were obtained from japan slc inc .
primary osteoblastic cells were isolated from newborn mouse calvariae after five routine sequential digestions with 0.1% collagenase ( wako pure chemical ) and 0.2% dispase ( godo shusei co. ltd , tomakomai , japan ) as previously described .
osteoblastic cells collected from fractions 24 were combined and cultured in -modified mem ( mem ) supplemented with 10% fetal calf serum ( fcs ) at 37c under 5% co2 in air .  
the cells adhering to the well surface were stained for tartrate - resistant acid phosphatase ( trap ) , and trap - positive multinucleated cells containing three or more nuclei per cell were counted as osteoclasts as previously described .
the concentrations of pge2 in the cultured medium were determined using an enzyme immunoassay , as previously described .
mouse calvariae were collected from 5-day - old mice and then were cultured for 24  h in bgjb medium containing 1  mg / ml bovine serum albumin ( bsa ) .
the calvaria was transferred into new medium , with or without lps after 24  h , and then was cultured for 5 days at 37c under 5% co2 in air .
the bone - resorbing activity was determined by measuring the concentration of calcium in the conditioned medium using a calcium kit ( calcium c test ; wako pure chemical ) as reported previously .
the bone - resorbing activity was expressed as an increase in medium calcium , which is consistent with the osteoclastic bone resorption as shown in the previous studies .  
primary mouse osteoblastic cells were cultured for 24  h in mem containing 1% fcs then treated with lps for 3  h. the total rna was extracted from mouse osteoblasts using the acid guanidium - phenol - chloroform method .
cdna was synthesized from 10  g of total rna by reverse transcriptase ( superscript ii preamplification system ; invitrogen life technologies co. , carlsbad , ca ) and amplified using pcr .
primers for the mouse cox-1 , cox-2 , mpges-1 , and glyceraldehydes-3-phosphate dehydrogenase ( gapdh ) genes were used in pcr as reported previously .
the pcr product was separated on a 1% agarose gel and stained with ethidium bromide .
six - week - old mice were i.p . injected with lps ( 10  mg / kg body weight ) on days 0 and 4 .
some mice were i.p . injected with capsaicin ( 1  mg / kg body weight ) .
the femurs were collected 8 days after the first injection of lps or phosphate - buffered saline ( pbs ) . for each group ,
was measured by dual x - ray absorptiometry ( model dcs-600r ; aloka , co. ltd , tokyo , japan ) as reported previously .
the bmd was calculated by dividing the bone mineral content of the measured area by the area .
lps markedly induced osteoclast differentiation in cocultures of mouse bone marrow cells and osteoblasts on day 7 , while 30  m of capsaicin completely suppressed the osteoclast formation induced by lps ( figure 1(b ) ) .
the conditioned coculture medium was examined to elisa to determine the effects of capsaicin on pge2 production induced by lps .
the level of pge2 in the conditioned medium treated with lps was higher than that of the control and was clearly suppressed by adding 30  m capsaicin in the coculture ( figure 1(c ) ) .
bone - resorbing activity was measured by the increase in calcium in the conditioned medium .
the addition of 30  m capsaicin significantly suppressed the bone - resorbing activity induced by lps ( figure 1(d ) ) .
these results indicate that capsaicin clearly suppressed the osteoclastic bone resorption associated with inflammation in vitro .
the expression of cox-2 and mpges-1 is essential for pge2 production by osteoblasts treated with bone - resorbing cytokines such as il-1 and lps , and the pge2 production is essential for inflammatory bone resorption .
an rt - pcr analysis showed the expression of cox-2 and mpges-1 mrnas to be markedly induced by lps in mouse primary osteoblasts at 3  h , and simultaneous addition of capsaicin clearly suppressed both the cox-2 and mpges-1 expression ( figure 2(a ) ) .
a low level cox-1 mrna expression was detected in the osteoblasts , and this expression was not influenced by lps or capsaicin .
capsaicin significantly suppressed lps - induced pge2 production in mouse primary osteoblast culture ( figure 2(b ) ) .
lps administration induces the severe loss of trabecular bone in distal femurs in mice , and mpges-1 knockout mice are resistant to the bone loss induced by lps .
injected with capsaicin with or without lps , and the femurs were collected on day 8 for measurement of femoral bmds .
distal femoral bmd was markedly suppressed by the treatment with lps , and simultaneous treatment with capsaicin significantly restored the lps - induced bone loss in mice ( figure 3 ) .
the present study showed that capsaicin , a trpv1 ligand , suppresses lps - induced osteoclastic bone resorption associated with inflammation by inhibiting pge2 production by osteoblasts .
we have reported that pge2 binds to the ep4 receptor , one of the pge receptor subtypes ep1-ep4 , and induces the rankl expression to stimulate bone resorption , using agonist and antagonist of eps and respective ep - knockout mice [ 2 , 11 ] .
pge2 produced by osteoblasts binds to ep4 in osteoblasts and induces rankl expression via ep4-mediated signals .
lps markedly induces the expression of cox-2 and mpges-1 mrnas in osteoblasts , and the pge2 production is essential for rankl - dependent osteoclast formation .
the transcriptional regulation is important to understand the biological significance in target tissues because mpges-1 is an inducible terminal enzyme associated with pge2 biosynthesis .
the mrna expression of mpges-1 is coupled with cox-2 , and the induction of cox-2 mrna proceeded mpges-1 after adding lps .
the mouse cox-2 gene promoter possesses functional regulatory elements for nf-b , nfil-6 , ap-1 , and c / ebp .
in contrast , the mouse mpges-1 gene promoter possesses ap-1 and c / ebp , but not nf-b .
capsaicin clearly suppressed the lps - induced expression of cox-2 and mpges-1 mrnas in osteoblasts in the present study .
further studies are needed to define the transcriptional regulation of cox-2 and mpges-1 by capsaicin in osteoblasts .
capsaicin , a typical ligand for trpv1 , is derived from chili peppers , which elicit a burning sensation through trpv1 [ 5 , 6 ] .
mouse primary osteoblasts and osteoblast cell line mc3t3-e1 express trpv1 , but expression of trpv1 mrna is not detected in bone marrow macrophages , and that capsaicin suppresses il-1-induced osteoclast differentiation in the cocultures of mouse bone marrow cells and osteoblasts .
in addition , both resiniferatoxin , a natural trpv1 agonist , and olvanil , a synthetic trpv1 agonist , suppress osteoclast formation induced by il-1 in the cocultures ( miyaura and inada ; unpublished data ) .
the present study found that capsaicin suppressed the pge2 production induced by lps in osteoblasts and suppressed lps - induced osteoclast formation ( figures 1 and 2 ) .
these results indicate that osteoblasts are main target cells for trpv1 ligand in bone tissues and regulate osteoclast differentiation associated with inflammation .
chen et al .   reported that capsaicin acts on the macrophage cell line raw264.7 to attenuate lps - induced cox-2 expression , but raw 264.7 cells do not express trpv1 . on the other hand , rossi et al .  
have shown that human mature osteoclasts express trpv1 isoforms and trpv1 agonist enhances the expression and the activity of trap and cathepsin k , two specific osteoclast biomarkers .
the addition of capsaicin to bone marrow macrophages cultures in the presence of soluble rankl and macrophage colony stimulating factor ( m - csf ) does not influence osteoclast differentiation from macrophages ( miyaura and inada ; unpublished data ) .
idris et al .   have shown that capsazepine , a trpv1 antagonist , acts on preosteoclasts and suppresses the differentiation into mature osteoclasts .
therefore , the roles of trpv1 signals still remain controversial in macrophages , preosteoclasts , and mature osteoclasts .
the current study showed that capsaicin significantly restored inflammatory bone loss in the femur induced by lps in mice ( figure 3 ) .
therefore , trpv1 ligands possess potential as clinical drugs targeting bone diseases associated with inflammatory bone resorption .
sancho et al .   reported that capsaicin has the potential to protect against inflammatory bowel diseases associated with severe inflammation , thus indicating the anti - inflammatory effects of trpv1 signal in intestinal diseases .
idris et al .   reported that the trpv1 antagonist capsazepine inhibits bone loss due to estrogen deficiency in ovariectomized ( ovx ) mice .
however , the mechanisms of bone loss in ovx animals differ from that in lps - treated animals accompanied with severe inflammation . in addition , clark et al .  
reported that trpv4 , a calcium permeable ion channel , possess chondroprotective role and deletion of trpv4 gene resulted in severe osteoarthritis using trpv4 knockout mice .
further studies using several animal models for bone and cartilage diseases are needed to define the possible roles of trpvs signals in bone .
 capsaicin , a transient receptor potential vanilloid type 1 ( trpv1 ) ligand , regulates nerve - related pain - sensitive signals , inflammation , and cancer growth . 
 capsaicin suppresses interleukin-1-induced osteoclast differentiation , but its roles in bone tissues and bone diseases are not known . 
 this study examined the effects of capsaicin on inflammatory bone resorption and prostaglandin e ( pge ) production induced by lipopolysaccharide ( lps ) in vitro and on bone mass in lps - treated mice in vivo . 
 capsaicin suppressed osteoclast formation , bone resorption , and pge production induced by lps in vitro . 
 capsaicin suppressed the expression of cyclooxygenase-2 ( cox-2 ) and membrane - bound pge synthase-1 ( mpges-1 ) mrnas and pge production induced by lps in osteoblasts . 
 capsaicin may suppress pge production by inhibiting the expression of cox-2 and mpges-1 in osteoblasts and lps - induced bone resorption by trpv1 signals because osteoblasts express trpv1 . 
 lps treatment markedly induced bone loss in the femur in mice , and capsaicin significantly restored the inflammatory bone loss induced by lps in mice . 
 trpv1 ligands like capsaicin may therefore be potentially useful as clinical drugs targeting bone diseases associated with inflammatory bone resorption . 