bone mass is maintained by a homeostatic balance between the activities of osteoclasts , which resorb bone , and those of osteoblasts , which make bone . excessive osteoclast activity leads to bone loss in pathologic or inflammatory conditions , such as rheumatoid arthritis and postmenopausal osteoporosis . osteoclasts originate from hematopoietic stem cell monocyte / macrophage lineage that differentiate and fuse into multinucleated bone resorbing osteoclasts in the presence of tumor necrosis factor ( tnf ) family , receptor activator of nuclear factor - kappab ( rank ) ligand ( rankl ) and macrophage colony - stimulating factor ( m - csf).[2 - 4 ] m - csf is a secreted cytokine from osteoblasts / stromal cells and induces colony formation of monocyte / osteoclast precursor cells . rankl is expressed on the membrane of osteoblasts / stromal cells and binds to and activates rank on osteoclast precursor cells . upon binding to its receptor rank , rankl initiates the recruitment of tnf - associated factor 6 ( traf6 ) and immunoreceptor tyrosinebased activation motif ( itam)-containing adaptor proteins , dap12 and fcr. then , rankl activates the intracellular signaling pathways , including mitogen - activated protein kinase ( mapk ) and nuclear factor - kappab ( nf-b ) . ultimately , rankl leads to osteoclast differentiation by inducing transcription factors , including c - fos and nuclear factor of activated t cells cytoplasmic 1 ( nfatc1 ) . in particular , nfatc1 rescues osteoclast differentiation in cells lacking c - fos and can induce osteoclast differentiation in the presence of rankl . recently , many plant - derived natural products have been used in traditional medicine for the treatment of various diseases . we previously reported that several extracts derived from natural products , including papaya , deer antler , and drynariae rhizome , had inhibitory effects on osteoclast differentiation.[9 - 11 ] cornus officinalis , a species of dogwood , was used as a food plant . in particular , the fruit of cornus officinalis was widely used for its chemotherapeutic benefits . recently , cornuside , isolated from the fruit of cornus officinalis , suppressed inflammation by inhibiting tnf - alpha ( )-induced nf-b p65 translocation to the nucleus . cornuside has also been reported to inhibit lipopolysaccharide - induced inflammation through inhibition of nf-b activity . recently , an aqueous extract prepared from the fruit of a popular chinese herb cornus officinalis had strong anti - proliferative effect on estrogen receptor - positive breast cancer cells . taken together , it is possible that an extract obtained from cornus officinalis suppresses rankl - induced osteoclast differentiation by inhibiting nf-b activity . an extract of cornus officinalis was from plant extract bank ( yuseong , daejeon , korea ) . anti - actin antibody and tartrate - resistant acid phosphatase ( trap ) staining kit were from sigma - aldrich ( st . louis , mo , usa ) . the 2,3-bis ( 2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2 tetrazolium hydroxide ( xtt ) assay kit was from roche ( indianapolis , in , usa ) . antibodies against phosphor ( p)-38 , p38 , p - c - jun n - terminal kinase ( jnk ) , jnk , and i-b were from cell signaling technology ( beverly , ma , usa ) . bone marrow cells ( bmcs ) were freshly isolated from the long bones of 5-week - old male icr mice as described previously . briefly , bmcs were isolated from long bones by flushing with -minimum essential medium ( -mem ; gibco brl , grand island , ny , usa ) containing antibiotics ( sigma ) . red blood cells ( rbc ) were removed in rbc lysis buffer ( sigma ) . following centrifugation , cells were suspended in -mem supplemented with 10% fetal bovine serum ( fbs ; gibco brl , grand island , ny , usa ) and antibiotics , and then cultured for 3 d in the presence of m - csf . non - adherent cells were removed and adherent cells were used as bone marrow - derived macrophages ( bmms ) . bmms were seeded at 3.5  10 cells / well in 48-well plates and cultured for 4 d with m - csf and rankl in the presence or sbaence of cornus officinalis . bmms were seeded at 1  10 cells / well in 48-well plates in triplicate and cultured for 3 d with m - csf in the presence or absence of cornus officinalis . after 3 days , 50 l of xtt reagent was added to each well and further incubated for 6 h. the optical density at 450 nm was analyzed using an enzyme linked immunosorbent assay ( elisa ) reader . cells were lysed in lysis buffer containing 50 mm tris - cl , 150 mm nacl , 5 mm ethylenediaminetetraacetic acid ( edta ) , 1% triton x-100 , 1 mm sodium fluoride , 1 mm sodium vanadate , 1% deoxycholate , and protease inhibitors . equal amounts of proteins were boiled in sds sample buffer , resolved by 10% sds - polyacrylamide gel electrophoresis ( page ) , and transferred to a polyvinylidene fluoride ( pvdf ) membrane ( amersham biosciences , piscataway , nj , usa ) . the membrane was blocked and probed with primary antibodies , as indicated , washed extensively , and then incubated with secondary antibody conjugated to horseradish peroxidase . protein - antibody complexes were detected with enhanced chemiluminescence ( ecl ; amershan biosciences , buckinghamshire , uk ) . total rna was extracted from cultured cells using the trizol reagent ( invitrogen , carlsbad , ca , usa ) following the manufacturer 's instructions . one g of rna wasreverse - transcribed using oligo dt primers , deoxynucleotide triphosphate ( dntp ) , buffer , dithiothreitol , rnase inhibitor , and superscript ii rt ( invitrogen , carlsbad , ca , usa ) . cdna was amplified using the following primer sets : trap sense , 5'-acttccccagcccttactac-3 ' ; trap antisense , 5'-tcagcacatagcccacaccg-3 ' ; c - fos sense , 5'-ctggtgcagcccactctggtc-3 ' ; c - fos antisense , 5'-ctttcagcagattggcaatctc-3 ' ; nfatc1 sense , 5'-caacgccctgaccaccgatag-3 ' ; nfatc1 antisense , 5'-ggctgccttccgtctcatagt-3 ' ; osteoclast - associated receptor ( oscar ) sense , 5'-gaacaccagaggctatgactgttc-3 ' ; oscar antisense , 5'-ccgtggagctgaggaaaaggttg-3 ' ; gapdh sense , 5'-accacagtccatgccatcac-3 ' ; gapdh antisense , 5'-tccaccaccctgttgctgta-3 ' . pcr products were electrophoresed by using 1% agarose gels and visualized by staining with ethidium bromide . densitometric analysis was performed on the relative intensity of each band using the image pro - plus program , version 4.0 ( media cybernetics , carlsbad , ca , usa ) . statistical significance was analyzed by a student 's t test , and significant results ( p < 0.1 ) are marked by asterisks . an extract of cornus officinalis was from plant extract bank ( yuseong , daejeon , korea ) . anti - actin antibody and tartrate - resistant acid phosphatase ( trap ) staining kit were from sigma - aldrich ( st . louis , mo , usa ) . the 2,3-bis ( 2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2 tetrazolium hydroxide ( xtt ) assay kit was from roche ( indianapolis , in , usa ) . antibodies against phosphor ( p)-38 , p38 , p - c - jun n - terminal kinase ( jnk ) , jnk , and i-b were from cell signaling technology ( beverly , ma , usa ) . bone marrow cells ( bmcs ) were freshly isolated from the long bones of 5-week - old male icr mice as described previously . briefly , bmcs were isolated from long bones by flushing with -minimum essential medium ( -mem ; gibco brl , grand island , ny , usa ) containing antibiotics ( sigma ) . red blood cells ( rbc ) were removed in rbc lysis buffer ( sigma ) . following centrifugation , cells were suspended in -mem supplemented with 10% fetal bovine serum ( fbs ; gibco brl , grand island , ny , usa ) and antibiotics , and then cultured for 3 d in the presence of m - csf . non - adherent cells were removed and adherent cells were used as bone marrow - derived macrophages ( bmms ) . bmms were seeded at 3.5  10 cells / well in 48-well plates and cultured for 4 d with m - csf and rankl in the presence or sbaence of cornus officinalis . bmms were seeded at 1  10 cells / well in 48-well plates in triplicate and cultured for 3 d with m - csf in the presence or absence of cornus officinalis . after 3 days , 50 l of xtt reagent was added to each well and further incubated for 6 h. the optical density at 450 nm was analyzed using an enzyme linked immunosorbent assay ( elisa ) reader . cells were lysed in lysis buffer containing 50 mm tris - cl , 150 mm nacl , 5 mm ethylenediaminetetraacetic acid ( edta ) , 1% triton x-100 , 1 mm sodium fluoride , 1 mm sodium vanadate , 1% deoxycholate , and protease inhibitors . equal amounts of proteins were boiled in sds sample buffer , resolved by 10% sds - polyacrylamide gel electrophoresis ( page ) , and transferred to a polyvinylidene fluoride ( pvdf ) membrane ( amersham biosciences , piscataway , nj , usa ) . the membrane was blocked and probed with primary antibodies , as indicated , washed extensively , and then incubated with secondary antibody conjugated to horseradish peroxidase . protein - antibody complexes were detected with enhanced chemiluminescence ( ecl ; amershan biosciences , buckinghamshire , uk ) . total rna was extracted from cultured cells using the trizol reagent ( invitrogen , carlsbad , ca , usa ) following the manufacturer 's instructions . one g of rna wasreverse - transcribed using oligo dt primers , deoxynucleotide triphosphate ( dntp ) , buffer , dithiothreitol , rnase inhibitor , and superscript ii rt ( invitrogen , carlsbad , ca , usa ) . cdna was amplified using the following primer sets : trap sense , 5'-acttccccagcccttactac-3 ' ; trap antisense , 5'-tcagcacatagcccacaccg-3 ' ; c - fos sense , 5'-ctggtgcagcccactctggtc-3 ' ; c - fos antisense , 5'-ctttcagcagattggcaatctc-3 ' ; nfatc1 sense , 5'-caacgccctgaccaccgatag-3 ' ; nfatc1 antisense , 5'-ggctgccttccgtctcatagt-3 ' ; osteoclast - associated receptor ( oscar ) sense , 5'-gaacaccagaggctatgactgttc-3 ' ; oscar antisense , 5'-ccgtggagctgaggaaaaggttg-3 ' ; gapdh sense , 5'-accacagtccatgccatcac-3 ' ; gapdh antisense , 5'-tccaccaccctgttgctgta-3 ' . pcr products were electrophoresed by using 1% agarose gels and visualized by staining with ethidium bromide . densitometric analysis was performed on the relative intensity of each band using the image pro - plus program , version 4.0 ( media cybernetics , carlsbad , ca , usa ) . statistical significance was analyzed by a student 's t test , and significant results ( p < 0.1 ) are marked by asterisks . to examine the effect of cornus officinalis on osteoclast differentiation , bmms were cultured with m - csf and rankl in the presence or absence of cornus officinalis . osteoclasts were formed from bmm in the presence of m - csf , rankl , and dissolved in dimethyl sulfoxide ( dmso ) as vehicle . however , cornus officinalis greatly inhibits the differentiation of bmms , as evaluated by the number of trap - positive osteoclasts ( fig . 1a , 1b ) . to exclude the possibility that cornus officinalis can cause cytotoxicity on bmms during osteoclast differentiation , cornus officinalis can no cytotoxic effects at the same doses which considerably inhibited osteoclast differentiation ( fig . next , we examined the inhibitory effect of cornus officinalis on osteoclast differentiation by analyzing osteoclast - specific gene expression and transcription factors . bmms stimulated with m - csf and rankl showed high expression levels of trap and oscar mrna after 48 h. however , cornus officinalis inhibits the expression of trap and oscar mrna in bmms treated with m - csf and rankl . also , rt - pcr results revealed that c - fos and nfatc1 mrna levels were increased by m - csf and rankl , but both c - fos and nfatc1 expression was greatly inhibited by cornus officinalis ( fig . these results suggest that cornus officinalis may inhibit osteoclast differentiation through the inhibition of rankl - induced c - fos and nfatc1 expression . osteoclast differentiation is tightly regulated by the induction of various transcription factors . in particular , c - fos and nfatc1 play an essential role in osteoclast differentiation . to further confirm the above results , we examined the effects of cornus officinalis on c - fos and nfatc1 protein expression . bmms were stimulated with m - csf and rankl in the presence or absence of cornus officinalis . as shown in figure 3 , cornus officinalis suppresses the protein expression of c - fos and nfatc1 in bmms treated with m - csf and rankl . these results suggest that the inhibitory effects of cornus officinalis involved the inhibition of c - fos and nfatc1 induced by rankl . it is well known that rankl activates multiple intracellular signaling cascades that lead to osteoclast differentiation . to examine the molecular mechanism of the inhibitory effects of cornus officinalis on osteoclast differentiation , we examined the effects of cornus officinalis on the early signaling pathways in bmms treated with rankl . rankl - induced phosphorylation of p38 and jnk was increased at 5 min after rankl treatment , but both p38 and jnk phosphorylation was inhibited by cornus officinalis treatment . also , rankl induces degradation of i-b within 5 min , but cornus officinalis suppresses i-b degradation ( fig . these results suggest that the inhibition of early signaling pathways is , at least , involved in the cornus officinalis - induced inhibition of transcription factors and osteoclast differentiation . to examine the effect of cornus officinalis on osteoclast differentiation , bmms were cultured with m - csf and rankl in the presence or absence of cornus officinalis . osteoclasts were formed from bmm in the presence of m - csf , rankl , and dissolved in dimethyl sulfoxide ( dmso ) as vehicle . however , cornus officinalis greatly inhibits the differentiation of bmms , as evaluated by the number of trap - positive osteoclasts ( fig . 1a , 1b ) . to exclude the possibility that cornus officinalis can cause cytotoxicity on bmms during osteoclast differentiation , cornus officinalis can no cytotoxic effects at the same doses which considerably inhibited osteoclast differentiation ( fig . next , we examined the inhibitory effect of cornus officinalis on osteoclast differentiation by analyzing osteoclast - specific gene expression and transcription factors . bmms stimulated with m - csf and rankl showed high expression levels of trap and oscar mrna after 48 h. however , cornus officinalis inhibits the expression of trap and oscar mrna in bmms treated with m - csf and rankl . also , rt - pcr results revealed that c - fos and nfatc1 mrna levels were increased by m - csf and rankl , but both c - fos and nfatc1 expression was greatly inhibited by cornus officinalis ( fig . these results suggest that cornus officinalis may inhibit osteoclast differentiation through the inhibition of rankl - induced c - fos and nfatc1 expression . osteoclast differentiation is tightly regulated by the induction of various transcription factors . in particular , c - fos and nfatc1 play an essential role in osteoclast differentiation . to further confirm the above results , we examined the effects of cornus officinalis on c - fos and nfatc1 protein expression . bmms were stimulated with m - csf and rankl in the presence or absence of cornus officinalis . as shown in figure 3 , cornus officinalis suppresses the protein expression of c - fos and nfatc1 in bmms treated with m - csf and rankl . these results suggest that the inhibitory effects of cornus officinalis involved the inhibition of c - fos and nfatc1 induced by rankl . it is well known that rankl activates multiple intracellular signaling cascades that lead to osteoclast differentiation . to examine the molecular mechanism of the inhibitory effects of cornus officinalis on osteoclast differentiation , we examined the effects of cornus officinalis on the early signaling pathways in bmms treated with rankl . rankl - induced phosphorylation of p38 and jnk was increased at 5 min after rankl treatment , but both p38 and jnk phosphorylation was inhibited by cornus officinalis treatment . also , rankl induces degradation of i-b within 5 min , but cornus officinalis suppresses i-b degradation ( fig . these results suggest that the inhibition of early signaling pathways is , at least , involved in the cornus officinalis - induced inhibition of transcription factors and osteoclast differentiation . in human , the function of bones is to mechanically support the body , protect the soft vital organs such as brain and heart , serve as levers for muscle action , store and maintain mineral , and support hematopoiesis . thus , osteoporosis is one of the major health problems in our world and is especially troublesome for postmenopausal females who suffer from estrogen deficiency . it is well known that an imbalance between bone - resorbing osteoclasts and bone - forming osteoblasts is the main pathogenesis of bone diseases including osteoporosis.[2 - 4 ] osteoporosis , most common metabolic bone disorder , is one of the major age - related diseases and responsible for increasing bone fracture risk . upon binding to rankl , rank activates various transcription factors including c - fos and nfatc1 which thereby induces osteoclast - specific gene expression including trap and oscar.[5 - 7 ] in this study , cornus officinalis inhibits rankl - mediated osteoclast differentiation in a dose - dependent manner without cytotoxicity against bmms ( fig . 1 ) . also , we found that cornus officinalis suppresses the expression of trap , oscar , c - fos , and nfatc1 in bmms treated with rankl and m - csf . these results suggest that cornus officinalis inhibits osteoclast differentiation through inhibition of c - fos and nfatc1 induced by rankl . although the mechanism of rankl - induced c - fos expression in bmms remains unclear , they are likely to be related to nf-b and mapk , such as p38 and jnk.[17 - 19 ] both c - fos and nfatc1 are key transcription factors on osteoclast differentiation . c - fos is an upstream component relevant to nfatc1 during rankl - induced osteoclast differentiation . it has recently been reported that rankl can induce osteoclast differentiation from nf-b p50/p52 double knock - out ( dko ) osteoclast precursor cells when c - fos is overexpressed . also , several reports have indicated that p38 mapk regulates rankl - mediated c - fos expression in osteoclast precursors . this raises the possibility that cornus officinalis may regulate osteoclast differentiation through inhibition of c - fos expression via inhibition of nf-b activity . nf-b is usually accumulated in the cytoplasm through association with its inhibitory protein i-b . following cellular stimulation , i-b is phosphorylated , ubiquitinated , and degraded by the proteasome , which leads to the translocation of nf-b into the nucleus , where it acts as a transcription factor . in this study however , rankl - induced i-b degradation was greatly inhibited in the presence of cornus officinalis . also , cornus officinalis significantly inhibited the phosphorylation of p38 and jnk induced by rankl ( fig . these results demonstrate that cornus officinalis may inhibit osteoclast differentiation that may be involved in the inhibition of the signaling cascades nf-b / c - fos / nfatc1 . single compounds obtained from cornus officinalis is known to inhibit inflammation by regulating nf-b . in this study , we found that an extract derived from cornus officinalis inhibited rankl - induced osteoclast differentiation without cytotoxicity . the expression of c - fos and nfatc1 , the master transcription factors of osteoclast differentiation , was inhibited by cornus officinalis treatment . cornus officinalis inhibited p38 and jnk phosphorylation and i-b degradation in bmms treated with rankl .
objectivesosteoporosis is a disease of bones that is thought to result from an imbalance between bone resorption and bone formation . 
 although osteoporosis itself has no symptoms , osteoporosis caused by osteoclasts leads to an increased risk of fracture . 
 here we examined the effects of cornus officinalis on receptor activator of nuclear factor - kappab ligand ( rankl)-mediated osteoclast differentiation.methodswe evaluated the effects of cornus officinalis on rankl - induced osteoclast differentiation from bone marrow - derived macrophages ( bmms ) and performed a cytotoxicity assay , reverse transcriptase - polymerase chain reaction ( rt - pcr ) , and western blot analysis.resultscornus officinalis significantly inhibits rankl - mediated osteoclast differentiation in a dose - dependent manner , but without cytotoxicity against bmms . 
 the mrna expression of tartrate - resistant acid phosphatase ( trap ) , osteoclast - associated receptor ( oscar ) , c - fos , and nuclear factor of activated t cells cytoplasmic 1 ( nfatc1 ) in bmms treated with rankl was considerably inhibited by cornus officinalis treatment . 
 also , cornus officinalis inhibits the protein expression of c - fos and nfatc1 . 
 cornus officinalis greatly inhibits rankl - induced phosphorylation of p38 and c - jun n - terminal kinase ( jnk ) . 
 also , cornus officinalis significantly suppresses rankl - induced degradation of i-b.conclusionstaken together , our results suggest that cornus officinalis may be a useful the treatment of osteoporosis .