leptin is a hormone that was initially found to be synthesized by white adipocytes and has a strong correlation with the amount of adipose tissue and with body mass index ( bmi ) .
leptin was first discovered in 1994 and found to act as a signal for the central nervous system to inhibit food intake and to stimulate energy expenditure .
more recent findings on the ubiquitous expression of leptin receptors in almost all tissues and on its cellular effects have revealed that leptin is also involved in the regulation of a variety of biological functions related to immune responses and inflammatory diseases , and to cardiovascular and respiratory pathophysiology [ 25 ] .
increased joint loading and altered mechanic loading axis has been proposed to explain the increased risk of oa in weight - bearing joints , including hip and knee joints .
surprisingly , there is also a positive association between obesity and oa in the hand , pointing to an obesity related metabolic factor that acts as a risk factor for oa .
recently , it has been shown that synovial fluid ( sf ) from oa patients contains leptin concentrations that are similar or higher than those measured in serum [ 79 ] .
furthermore , low soluble leptin receptor ( sob - r ) level in sf potentiates the biological activity of leptin in the joint as compared to that in serum .
articular cartilage has been reported to produce leptin [ 8 , 10 ] and express functional leptin receptor ob - r , and the expression of these two is further increased in advanced oa correlating with bmi of the patients . in vitro , leptin has been shown to potentiate interleukin-1 ( il-1 ) and interferon  ( ifn)-induced production of nitric oxide ( no ) in chondrocytes , which is a proinflammatory and destructive mediator in cartilage [ 12 , 13 ] .
leptin has been shown to decrease chondrocyte proliferation and to increase production of proinflammatory cytokine il-1 and destructive matrix metalloproteinases 9 and 13 ( mmp-9 and 13 ) in human chondrocytes [ 9 , 14 ] .
on the other hand , leptin has also been reported to increase proliferation and to enhance proteoglycan and collagen synthesis in human chondrocytes . in vivo ,
leptin injection into rat knee was reported to increase synthesis of insulin - like growth factor 1 ( igf - i ) and transforming growth factor  ( tgf ) both contributing to increased proteoglycan synthesis .
these effects are linked to increased cartilage matrix production , and also to osteophyte formation .
inducible nitric oxide synthase ( inos ) is expressed in oa cartilage , and there are markers of enhanced no production in oa joints [ 15 , 16 ] .
prostaglandins ( pgs ) , especially pge2 , mediate inflammation , tissue destruction , and pain in oa and in oa joints they are formed by cyclooxygenase ( cox ) enzymes ( particularly cox-2 ) and prostaglandin synthases .
interleukin-6 ( il-6 ) and interleukin-8 ( il-8 ) are produced by oa cartilage and have a proinflammatory and modulatory role in the pathogenesis of oa [ 10 , 18 ] .
the presence of bioactive leptin in oa joint and the effects of leptin on cartilage metabolism point to a pathophysiological role for leptin in oa .
the aim of the present study was to investigate the effects of leptin on mediators of cartilage metabolism by measuring its effects on the production of no , pge2 , il-6 , and il-8 in oa cartilage and by evaluating the signaling mechanisms involved in these effects by pharmacological means .
the study was approved by the ethics committee of tampere university hospital , and the patients gave their written approval .
the donor patients , age ranging from 53 to 87 years and body mass index ranging from 20 to 32 , were all diagnosed to have osteoarthritis .
cartilage samples were washed with phosphate buffered saline ( pbs ) and processed for the experiments within two hours after the operation .
full thickness pieces of articular cartilage from femoral condyles , tibial plateaus , and patellar surfaces showing macroscopical features of early oa were removed aseptically from subchondral bone with a scalpel and cut into small pieces ( about 2  2  2 mm ) .
cartilage cubes randomly selected from 3 different areas of the joint were incubated in one well of a 6-well plate in 3 ml of tissue culture medium ( dulbecco 's modified eagle 's medium ( dmem ) with glutamax - i containing 10% heat - inactivated fetal bovine serum , penicillin ( 100 units / ml ) , streptomysin ( 100 g / ml ) , and amphotericin b ( 250 ng / ml ) ; all obtained from invitrogen , carlsbad , calif , usa ) ) at 37c in humidified 5% carbon dioxide atmosphere . in the first two series of experiments , oa cartilage explants from 8 patients were incubated with leptin ( 0.1 g / ml or 10 g / ml ) alone or in combination with il-1 ( 10 pg / ml ) for 48 hours .
concentrations of no , pge2 , il-6 , and il-8 were determined in the culture medium . in the third series of experiments oa explants from 3 patients
were incubated with leptin ( 0.1 g / ml or 10 g / ml ) alone or in combination with il-1 ( 10 pg / ml ) for 48 hours .
cartilage samples were used to determine expression of inos and cox-2 protein . in the fourth series of experiments , signaling mechanisms involved in the leptin - induced no , pge2 , il-6 , and il-8 production were studied using pharmacological inhibitors .
oa explants from 7 patients were incubated for 48 hours with leptin ( 10 g / ml ) and following signaling pathway inhibitors : sp600125 10 m ( jnk inhibitor ) , sb220025 0.5 m ( p38 inhibitor ) , pd98059 10 m ( erk1/2 inhibitor ) , ag490 10 m ( jak2 inhibitor ) , whi - p154 10 m ( jak3 inhibitor ) , ro 31 - 8220 1 m ( pkc inhibitor ) , mg132 10 m ( nf-b inhibitor ) , and pdtc 10 m ( nf-b inhibitor ) .
concentrations of no , pge2 , il-6 , and il-8 were determined in the culture medium . in the fifth series of experiments , the effect of no on leptin - induced ( 10 g / ml ) il-6 , il-8 , and pge2 production
was studied by inhibiting leptin - induced endogenous no production with a selective inos inhibitor 1400 w ( 1 mm ) during a 48 hour incubation of oa explants from 8 patients .
the effect of no was further investigated by studying if exogenous no could reverse the effects of inos inhibitor 1400 w in leptin - treated cartilage .
this was made by adding no - donor snap ( 100 m ) together with 1400 w and leptin in the cartilage cultures .
concentrations of no , pge2 , il-6 , and il-8 were determined in the culture medium .
after the experiment the cartilage explants were weighed , and the results were expressed per mg of cartilage .
aliquots of the culture media were kept at 20c until assayed , and cartilage samples for western blotting were first snap frozen in liquid nitrogen and analysed as described below .
concentrations of nitrite , a stable product of no in aqueous solutions , were measured using the griess reaction .
the amount of pge2 released into the incubation medium was determined by radioimmunoassay , using reagents from the institute of isotopes ( budapest , hungary ) .
the concentrations of il-6 ( sanquin , pelipair , amsterdam , the netherlands ) and il-8 ( r&d systems , minneapolis , mn , usa ) in the culture medium were determined by elisa .
the results were expressed as pg of il-6/mg of cartilage , or pg of il-8/mg of cartilage .
after incubations , the cartilage specimen were snap frozen in liquid nitrogen , milled and lysed in extraction buffer ( 10 mm tris - hcl , 5 mm edta , 50 mm nacl , 1% triton - x-100 , 0.5 mm phenylmethylsulfonylfluoride ( pmsf ) , 1 mm sodiumorthovanadate , 20 g / ml leupeptin , 50 g / ml aprotin , 5 mm sodium fluoride , 2 mm sodium pyrophosphate , 10 m n - octyl - beta - d - glucopyranoside ) .
following incubation on ice for 15 minutes , samples were centrifuged and supernatants were mixed with sample buffer 1 : 4 ( 62.5 mm tris - hcl , ph 6.8 , 10% glycerol , 2% sds , 0.025% bromophenol blue , and 5%   -mercaptoethanol ) and stored at 20c until analyzed .
coomassie blue method was used to measure the protein content of the samples . after boiling , protein samples ( 20 g )
were separated with 8% sds - polyacrylamide electrophoresis gels and transferred to hybond enhanced chemiluminescence nitrocellulose membrane ( amersham biosciences uk limited , buckinghamshire , uk ) .
proteins were identified by western blotting using rabbit polyclonal antibody for human inos and goat polyclonal antibody for human cox-2 ( both obtained from santa cruz biotechnology , santa cruz , calif , usa ) .
actin was analysed as a loading control by using rabbit polyclonal antibody ( santa cruz biotechnology , santa cruz , calif , usa ) .
bound antibody was detected using supersignal west pico chemiluminescent substrate ( pierce , rockford , il , usa ) and fluorchem 8800 imaging system ( alpha innotech , san leandro , calif , usa ) .
results are expressed as mean       standard error of the mean ( sem ) .
recombinant human leptin and recombinant human il-1 beta were purchased from r&d systems ; manufacturer ensures low endotoxin level of the products ( < 1.0 eu per 1 g of the recombinant protein ) , and that amount in relation to the leptin concentrations used in the present study was tested to have no effect in our tissue culture conditions .
other reagents were obtained as follows : sp600125 , sb220025 , ag 490 and whi - p154 were from calbiochem ( merck , darmstadt , germany ) ; pd 98059 was from promega ( madison , wi , usa ) ; ro 31 - 8220 was from alexis corporation ( lausen , switzerland ) ; mg 132 was from tocris bioscience ( ellisville , mo , usa ) ; pdtc was from sigma chemical co ( st louis , mo , usa ) ; snap was from cayman chemical ( ann arbor , mi , usa ) .
1400 w was kindly given by dr richard g knowles , glaxo smithkline research & development , stevenage , uk .
leptin ( 0.1 g / ml and 10 g / ml ) enhanced no production in oa cartilage in a dose - dependent manner ( figure 1(a ) ) .
western blot analysis with human inos antibody showed that leptin ( 10 g / ml ) induced also inos expression in cultured cartilage tissue ( figure 1(b ) ) .
in addition , leptin ( 10 g / ml ) increased pge2 production and cox-2 expression , and il-6 and il-8 production in human oa cartilage during 48 hours incubation ( figures 1(c ) , 1(d ) , 1(e ) , and 1(f ) ) . a low concentration of proinflammatory cytokine il-1 ( 10 pg / ml ) had a slight stimulatory effect on no , pge2 , il-6 , and il-8 production and inos and cox-2 expression ( figure 2 ) .
leptin ( 10 g / ml ) enhanced no , pge2 , il-6 , and il-8 production , and inos and cox-2 expression in oa cartilage also in the presence of il-1 ( figure 2 ) .
the involvement of signaling pathways ( jnk , p38 , and erk1/2 map - kinases , jak2 and jak3 , pkc , and transcription factor nf-b ) in leptin - stimulated no , pge2 , il-6 , and il-8 production in oa cartilage was studied by pharmacological means .
inhibitors of transcription factor nf-b , mg 132 ( 10 m ) and pdtc ( 100 m ) , and jnk inhibitor sp600125 ( 10 m ) significantly inhibited leptin - induced no , pge2 , il-6 , and il-8 production ( figure 3 ) .
in addition to the effect of jnk inhibitor , inhibitors of other map - kinases , that is , sb220025
( p38 inhibitor ; 0.5 m ) and pd 98059 ( erk1/2 inhibitor ; 10 m ) inhibited leptin - induced pge2 production , but had no effect on no , il-6 , or il-8 production .
jak2 inhibitor ag 490 ( 10 m ) had no effect on leptin - induced no , pge2 , il-6 , or il-8 production , whereas jak3 inhibitor whi - p154 ( 10 m ) inhibited leptin - induced no synthesis , but not pge2 , il-6 , or il-8 production .
leptin - induced no , il-6 , and il-8 production was inhibitable with protein kinase c inhibitor ro 31 - 8220 ( 1 m ) , while it had no effect on pge2 production . a selective inos inhibitor 1400 w ( 1 mm ) inhibited leptin - induced no production almost completely indicating that it was synthesized through inos pathway ( figure 4(a ) ) .
interestingly , inhibition of no production with 1400 w reduced also the production of il-6 , il-8 , and pge2 ( figures 4(b)4(d ) ) .
the effect of 1400 w was reversed when no - donor snap ( 100 m ) was added into the culture .
those results suggest that the increasing effect of leptin on il-6 , il-8 , and pge2 production in human oa cartilage is dependent on no .
the course of the destructive process is determined by the balance between anabolic and catabolic mediators and their regulators in the joint , and the local distribution of these mediators in the cartilage .
leptin is an obesity related mediator , which has been suggested to take part in the regulation of anabolic and catabolic processes within the osteoarthritic joint and to play a role in the pathogenesis of oa . in the present study
, we found that leptin induced the production of no , pge2 , il-6 , and il-8 in human osteoarthritic cartilage and that leptin - induced pge2 , il-6 , and il-8 production was dependent on no .
no mediates many of the destructive effects of il-1 in inflamed joints [ 15 , 16 ] .
no has been reported to increase the production of matrix metalloproteinases ( mmps ) and to activate them [ 10 , 22 , 23 ] , to inhibit proteoglycan [ 2426 ] and collagen   synthesis and to induce chondrocyte cell death [ 28 , 29 ] .
no is also involved in the progress of inflammation by reducing the production of anti - inflammatory / anabolic factors tgf- , endogenous il-1 receptor antagonist ( il-1ra ) , and il-10 in chondrocytes [ 10 , 31 , 32 ] , and by contributing to the resistance against anabolic effects of igf-1 .
no has also been shown to sustain activation of nf-b providing a prolonged transcription of nf-b dependent genes . in support , pelletier et al .
reported reduced destruction of the articular cartilage by using inos - inhibitor l - nil in instability - induced oa in dogs .
in further studies with this model , l - nil was shown to reduce the levels of matrix metalloproteinase-1 and -3 ( mmp-1 and -3 ) , to inhibit chondrocyte apoptosis   and to reduce the interleukin-1 converting enzyme ( ice ) levels .
van den berg et al . studied the development of experimental osteoarthritis induced with intra - articular collagenase injection in inos knock - out mice . in this model ,
no has several catabolic and antianabolic actions in cartilage and thus it is identified as a possible target of treatment in osteoarthritis .
leptin has been shown to induce or to potentiate together with interferon  ( ifn ) , with tumor necrosis factor  ( tnf ) or with ifn and il-1 , no production in murine j774a.1 macrophages , rat adipocytes , c6 glioma cell - line , and rat vascular smooth muscle cells ( vsmcs ) [ 3942 ] . in human chondrocytes , leptin was reported to enhance interleukin-1 ( il-1 ) and interferon  ( ifn)-induced production of no [ 12 , 13 ] . in the present study
, we showed that leptin alone is sufficient to induce the inos expression and no production in human oa cartilage , and an enhancing effect was seen also in the presence of low concentrations of il-1 ( figures 1(a ) , 1(b ) , and 2(a ) , 2(b ) ) .
prostaglandins ( especially pge2 ) are produced in high amounts in oa cartilage and are modulators of inflammation , tissue destruction , and inflammatory pain .
prostaglandins are formed from arachidonic acid by the prostaglandin synthesizing cyclooxygenase ( cox ) enzymes and prostaglandin synthases .
cox-2 is highly expressed in oa cartilage and is induced by various cytokines that are involved in destructive processes in oa cartilage , for example , il-1 and tnf [ 4345 ] .
current drug therapy of oa is based on nonsteroidal anti - inflammatory drugs ( nsaids ) .
nsaids inhibit cox enzymes and prostanoid production , and they are used to relieve oa pain [ 46 , 47 ] . to our knowledge
, we show here for the first time that leptin increases cox-2 expression and pge2 production in human oa cartilage in the absence and in the presence of il-1 ( figures 1(c ) , 1(d ) and 2(c ) , 2(d ) ) .
previously , leptin has been shown to induce pge2 production in oe33 barret 's esophageal adenocarcinoma ( eac ) cell - line and in murine j774a.1 macrophages [ 39 , 48 ] .
oa cartilage produces il-6 and il-8 , and quantitative rt - pcr studies have shown elevated il-6 and il-8 mrna levels in oa cartilage as compared to normal cartilage [ 10 , 50 ] .
cytokines il-1 and tnf which induce destructive effects in cartilage both induce il-6 and il-8 production in human articular chondrocytes .
il-6 and il-8 may promote inflammation and cartilage destruction induced by il-1 or tnf and have a modulatory role in the pathogenesis of oa [ 18 , 5254 ] . in the present study
, we show for the first time that leptin enhances il-6 and il-8 production in human oa cartilage ( figures 1(e ) , 1(f )   and 2(e ) , 2(f ) ) .
leptin has previously been shown to induce il-6 production in human dendritic cells , human monocytes , human endometrial stromal cells ( esc ) , and in epithelial cells [ 5557 ] , and to stimulate il-8 production in human endometrial stromal cells ( escs ) and in epithelial cells .
leptin signaling through leptin receptors ( ob - r ) is thought to be mediated through janus kinase / signal transducer and activator of transcription ( jak / stat ) pathway and in addition to this , also mitogen - activated protein kinases ( mapks ) , protein kinase c ( pkc ) , phosphatidylinositol 3-kinase ( pi3k ) , and nuclear factor b ( nf-b ) pathways have been reported to mediate some effects of leptin , depending on the cell type [ 3 , 58 ] . in chondrocytes ,
otero et al . showed by using pharmacological inhibitors that induction of no production with a combination of leptin and il-1 or ifn was dependent on jak2 , pi3k , erk1/2 , and p38 [ 12 , 13 , 59 ] , but
these studies did not show signaling pathways involved in the responses induced by leptin alone .
we studied the effects of inhibitors of jak ( jak2 and jak3 ) , mapk ( erk1/2 , p38 , jnk ) , pkc and nf-b pathways on leptin - induced no , pge2 , il-6 , and il-8 production in oa cartilage .
there seems to be some differences in the signaling pathways important for production of the four different leptin - induced inflammatory molecules in oa cartilage .
leptin - induced no production was suppressed by inhibitors of jnk , jak3 , pkc , and nf-b , while leptin - induced pge2   production was reduced by inhibitors of jnk , p38 , and erk1/2 mapkinases and transcription factor nf-b .
leptin - induced il-6 and il-8 production was reduced by inhibitors of jnk , pkc , and nf-b ( figure 3 ) . in the present study , inhibition of leptin - induced endogenous no production in oa cartilage with a selective inos inhibitor
1400 w also suppressed the effects of leptin on pge2 , il-6 , and il-8 production .
the effect was reversed with exogenously added no ( no - donor snap ) ( figure 4 ) .
those results suggest that leptin induces pge2 , il-6 , and il-8 production in oa cartilage by an no - dependent manner .
the mediator role of no in leptin - induced metabolic changes in human oa cartilage has not been reported previously .
it is , however , supported by the previous findings on the involvement of no in the effects of leptin in some other organ systems , that is , in the secretion of luteinizing hormone - releasing hormone from the pituitary gland , in the control of blood pressure , and in the gastroprotection upon vagal activity [ 6062 ] . in conclusion ,
oa cartilage was shown to respond to leptin by producing increased amounts of no , pge2 , il-6 , il-8 , and all those effects can be considered harmful in cartilage metabolism .
those effects of leptin seem to be dependent on activation of transcription factor nf-b and the mapk pathway c - jun nh2-terminal kinase ( jnk ) in human oa cartilage .
in addition , jak3 signaling seems to be involved in leptin - induced no production , p38 , and erk1/2 mapk pathways in leptin - induced pge2 production , and pkc pathway in leptin - induced no , il-6 , and il-8 production .
inhibition of no production reduced the effects of leptin on pge2 , il-6 , and il-8 production pointing to a mediator role of no in these leptin - induced changes in cartilage metabolism and to a possible beneficial effect of inos inhibitors on oa cartilage .
these findings support the idea of leptin as a factor in the pathogenesis of osteoarthritis , and as a possible link between obesity and osteoarthritis .
 obesity is an important risk factor for osteoarthritis ( oa ) in weight - bearing joints , but also in hand joints , pointing to an obesity - related metabolic factor that influences on the pathogenesis of oa . 
 leptin is an adipokine regulating energy balance , and it has recently been related also to arthritis and inflammation as a proinflammatory factor . in the present paper , the effects of leptin on human oa cartilage were studied . 
 leptin alone or in combination with il-1 enhanced the expression of inos and cox-2 , and production of no , pge2 , il-6 , and il-8 . 
 the results suggest that the effects of leptin are mediated through activation of transcription factor nuclear factor b ( nf-b ) and mitogen - activated protein kinase ( mapk ) pathway c - jun nh2-terminal kinase ( jnk ) . 
 interestingly , inhibition of leptin - induced no production with a selective inos inhibitor 1400 w inhibited also the production of il-6 , il-8 , and pge2 , and this was reversed by exogenously added no - donor snap , suggesting that the effects of leptin on il-6 , il-8 , and pge2 production are dependent on no . 
 these findings support the idea of leptin as a factor enhancing the production of proinflammatory factors in oa cartilage and as an agent contributing to the obesity - associated increased risk for osteoarthritis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion