Open Access
A ailable online h p://a h i is- esea ch.com/con en /7/3/R581
R581
Vol 7 No 3
Resea ch a icle
Signalling pa hway in ol ed in ni ic oxide syn hase ype II
ac i a ion in chond ocy es: syne gis ic e ec o lep in wi h
in e leukin-1
Miguel O e o1, Rocío Lago1, F ancisca Lago2, Juan Jesús Gomez Reino3,4 and O es e Gualillo1
1NEIRID (Neu oEndoc ine In e ac ions in Rheuma ology and In lamma o y Diseases) Labo a o y, San iago Uni e si y Clinical Hospi al, Resea ch
Labo a o y 4, San iago de Compos ela, Spain
2Labo a o y o Molecula and Cellula Ca diology, San iago Uni e si y Clinical Hospi al, Resea ch Labo a o y 1, San iago de Compos ela, Spain
3Rheuma ology Di ision, San iago Uni e si y Clinical Hospi al, San iago de Compos ela, Spain
4Depa men o Medicine, School o Medicine, Uni e si y o San iago de Compos ela, San iago de Compos ela, Spain
Co esponding au ho : O es e Gualillo, gualill[email p o ec ed]
Recei ed: 11 Aug 2004 Re isions eques ed: 16 Sep 2004 Re isions ecei ed: 14 Jan 2005 Accep ed: 3 Feb 2005 Published: 4 Ma 2005
A h i is Resea ch & The apy 2005, 7:R581-R591 (DOI 10.1186/a 1708)
This a icle is online a : h p://a h i is- esea ch.com/con en /7/3/R581
© 2005 O e o e al.; licensee BioMed Cen al L d.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/
2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Abs ac
The objec i e o he p esen s udy was o in es iga e he e ec
o lep in, alone o in combina ion wi h IL-1, on ni ic oxide
syn hase (NOS) ype II ac i i y in i o in human p ima y
chond ocy es, in he mouse chond ogenic ATDC5 cell line, and
in ma u e and hype ophic ATDC5 di e en ia ed chond ocy es.
Fo comple eness, we also in es iga ed he signalling pa hway
o he pu a i e syne gism be ween lep in and IL-1. Fo his
pu pose, ni ic oxide p oduc ion was e alua ed using he G iess
colo ime ic eac ion in cul u e medium o cells s imula ed o e
48 hou s wi h lep in (800 nmol/l) and IL-1 (0.025 ng/ml), alone
o combined. Speci ic pha macological inhibi o s o NOS ype II
(aminoguanidine [1 mmol/l]), janus kinase (JAK)2 ( y phos in
AG490 and Tkip), phospha idylinosi ol 3-kinase (PI3K;
wo mannin [1, 2.5, 5 and 10 µmol/l] and LY294002 [1, 2.5, 5
and 10 µmol/l]), mi ogen-ac i a ed p o ein kinase kinase
(MEK)1 (PD098059 [1, 5, 10, 20 and 30 µmol/l]) and p38
kinase (SB203580 [1, 5, 10, 20 and 30 µmol/l]) we e added 1
hou be o e s imula ion. Ni ic oxide syn hase ype II mRNA
exp ession in ATDC5 chond ocy es was in es iga ed by eal-
ime PCR and NOS II p o ein exp ession was analyzed by
wes e n blo . Ou esul s indica e ha s imula ion o
chond ocy es wi h IL-1 esul s in dose-dependen ni ic oxide
p oduc ion. In con as , lep in alone was unable o induce ni ic
oxide p oduc ion o exp ession o NOS ype II mRNA o i s
p o ein. Howe e , co-s imula ion wi h lep in and IL-1 esul ed in
a ne inc ease in ni ic oxide concen a ion o e IL-1 challenge
ha was elimina ed by p e ea men wi h he NOS II speci ic
inhibi o aminoguanidine. P e ea men wi h y phos in AG490
and Tkip (a SOCS-1 mime ic pep ide ha inhibi s JAK2)
blocked ni ic oxide p oduc ion induced by lep in/IL-1. Finally,
wo mannin, LY294002, PD098059 and SB203580
signi ican ly dec eased ni ic oxide p oduc ion. These indings
we e con i med in ma u e and hype ophic ATDC5
chond ocy es, and in human p ima y chond ocy es. This s udy
indica es ha lep in plays a p oin lamma o y ole, in syne gy wi h
IL-1, by inducing NOS ype II h ough a signalling pa hway ha
in ol es JAK2, PI3K, MEK-1 and p38 kinase.
In oduc ion
Chond ocy es a e he p edominan cells in ma u e ca ilage
ha syn hesize and main ain he in eg i y o ca ilage-speci ic
ex acellula ma ix. In heuma oid a h i is and os eoa h i is
he pheno ype o chond ocy es changes, and apop osis and
ex acellula ma ix deg ada ion occu [1-3]. These se e e pe -
u ba ions in ca ilage homeos asis may be media ed in pa by
ni ic oxide (NO). This gaseous media o is induced by se e al
p oin lamma o y cy okines, including IL-1.
Lep in, he OB gene p oduc , is a 16 kDa ho mone ha is syn-
hesized by adipocy es. Lep in egula es ood in ake and
ERK = ex acellula signal- egula ed kinase; GAPDH = glyce aldehyde-3-phospha e dehyd ogenase; IFN = in e e on; IL = in e leukin; JAK = janus
kinase; MAPK = mi ogen-ac i a ed p o ein kinase; MEK = mi ogen-ac i a ed p o ein kinase kinase; MMP = ma ix me allop o einase; NF-κB = nuclea
ac o -κB; NO = ni ic oxide; NOS = ni ic oxide syn hase; PBS = phospha e-bu e ed saline; PI3K = phospha idylinosi ol 3-kinase; RT-PCR = e e se
ansc ip ion polyme ase chain eac ion; SOCS = supp esso o cy okine signalling.
A h i is Resea ch & The apy Vol 7 No 3 O e o e al.
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ene gy expendi u e, bu i also modula es neu oend oc ine
unc ion [4]. I is in ol ed in immune modula ion in ha i in lu-
ences he inna e immune esponse by p omo ing ac i a ion o
monocy e/mac ophages, chemo axis and ac i a ion o neu-
ophils, and ac i a ion o na u al kille cells [5]. Fu he mo e,
lep in in luences adap i e immuni y by inc easing he exp es-
sion o adhesion molecules by CD4+ T cells, and p omo ing
p oli e a ion and sec e ion o IL-2 by naï e CD4+ T cells [5-7].
Lep in has also been ound o in luence bone g ow h [8] and
in lamma ion [9].
High lep in le els a e associa ed wi h obesi y, which is a isk
ac o o os eoa h i is [10-12]. In e es ingly, in pa ien s wi h
os eoa h i is lep in is p esen in syno ial luid and is exp essed
by a icula chond ocy es [13], and no mal human chond o-
cy es exp ess he unc ional Ob-Rb lep in ecep o iso o m
[14]. I is unlikely ha lep in alone ac s on ca ilage o igge
an in lamma o y esponse; a he , i may associa e wi h o he
p oin lamma o y cy okines o ampli y in lamma ion and
enhance damage o ca ilage. We ecen ly demons a ed a
syne gis ic e ec o lep in wi h IFN-γ on ni ic oxide syn hase
(NOS) ype II ac i i y in cul u ed chond ocy es ha was medi-
a ed by he janus kinase (JAK)2 [15]. In he p esen s udy we
in es iga ed whe he lep in syne gizes wi h IL-1, an abundan
media o o in lamma ion and ca ilage des uc ion [16,17], o
ac i a e NOS ype II in chond ocy es. To gain u he insigh s
in o he mechanism o ac ion o his pu a i e syne gism, we
also analyzed he ole played by se e al in acellula kinases by
using speci ic pha macological inhibi o s.
Ma e ials and me hods
Reagen s
Foe al bo ine se um, issue cul u e media, media supple-
men s, mouse and human ecombinan lep in, mouse ecom-
binan IL-1, y phos in AG490, wo mannin, LY294002,
PD098059 and SB203580 we e pu chased om Sigma (S
Louis, MO, USA) unless o he wise speci ied. RT-PCR ea-
gen s we e pu chased om In i ogen (Ca lsbad, CA, USA)
and S a agene (La Jolla, CA, USA). Tkip (WLVFFVIFYFFR), a
supp esso o cy okine signalling (SOCS)-1 mime ic pep ide
ha inhibi s JAK2 au ophospho yla ion, was gene ously p o-
ided by D Howa d M Johnson (Ins i u e o Food and Ag icul-
u al Science, Depa men o Mic obiology and Cell Science,
Uni e si y o Flo ida, Gaines ille, FL, USA).
Cell cul u e
The clonal chond ogenic cell line ATDC5 was chosen o
hese s udies because i has been shown o be a use ul in i o
model o examining he mul is ep di e en ia ion o chond o-
cy es. Undi e en ia ed ATDC5 cells p oli e a e apidly un il
hey each con luence, a which poin hey unde go g ow h
a es . When ea ed wi h insulin, ans e in and sodium
seleni e, con luen ATDC5 cells e-en e a p oli e a i e phase
and o m ca ilaginous ma ix nodules (ma u e chond ocy es).
As di e en ia ion p og esses, hese cells unde go a la e di e -
en ia ion phase, becoming hype ophic, calci ying chond o-
cy es ha syn hesize ype X collagen and os eopon in – a
ma ke o e minal chond ocy e di e en ia ion [18]. ATDC5
cells we e a kind gi om D Agamemnon E G igo iadis
(Depa men o C anio acial De elopmen , King's College,
London Guy's Hospi al, London, UK). Unless o he wise spec-
i ied, cells we e cul u ed in Dulbecco's modi ied Eagle's
medium/Hams' F12 medium supplemen ed wi h 5% oe al
bo ine se um, 10 µg/ml human ans e in, 3 × 10-8 mol/l
sodium seleni e and an ibio ics (50 U/ml penicillin and 50 µg/
ml s ep omycin).
In some expe imen s, conduc ed o demons a e ha lep in/IL-
1 syne gism does no appea o depend on he di e en ia ion
s a e o he chond ocy es, chond ogenic ATDC5 cells we e
di e en ia ed in o ma u e and hype ophic chond ocy es, as
desc ibed by Thomas and cowo ke s [19]. B ie ly, cells we e
pla ed a an ini ial densi y o 2 × 104 cells/well in 24-well
pla es. Cells we e cul u ed in he abo e-men ioned medium
supplemen ed wi h 10 µg/ml o human ecombinan insulin
(No o No disk A/S, Bags ae d, Denma k). Cul u e was con in-
ued o a u he 15 o 21 days, wi h eplacemen o medium
e e y o he day. As expec ed, ATDC5 cul u es ea ed wi h
insulin unde wen p og essi e di e en ia ion om 0 o 21 days
as compa ed wi h un ea ed cul u es. This di e en ia ion was
quali a i ely cha ac e ized by inc eased o ma ion o ca ilage
nodules and enhanced s aining wi h alcian blue dye, which is
indica i e o ca ilage p o eoglycan accumula ion.
In o he expe imen s (da a no shown), he di e en ia ion om
days 0 o 21 was u he e idenced by sequen ial inc eases in
ype II collagen, agg ecan and ype X collagen mRNAs. The
ea ly and ma u e chond ocy e ma ke ype II collagen was
exp essed in undi e en ia ed ATDC5 cells; he le el began o
inc ease a day 3, peaked a days 7–10 and g adually declined
a e day 15. The exp ession p o ile o agg ecan mimicked ha
o ype II collagen bu wi h a sligh delay o a couple o days.
The decline in exp ession o bo h chond ocy e ma ke s coin-
cided wi h he onse o la e-s age chond ocy e di e en ia ion.
The exp ession o he hype ophic chond ocy e ma ke ype X
collagen began a days 12 and 13. The exp ession pa e ns o
hese ea ly and la e chond ocy e ma ke s we e consis en wi h
p e ious indings in ATDC5 cells ega ding in i o chond o-
cy e di e en ia ion. We do no illus a e indings ega ding he
di e en ia ion o ATDC5 cells because hey a e ex ensi ely
epo ed in li e a u e [19].
Ca ilage ha es and human chond ocy e isola ion
Human no mal a icula ca ilage samples we e ob ained om
knee join s o pa ien s unde going leg ampu a ions om abo e
he knee because o pe iphe al ascula disease. (Pe mission
om he local e hical commi ee was g an ed.) None o he
pa ien s had a clinical his o y o a h i is o any o he pa hology
a ec ing he ca ilage, and he specimens appea ed no mal on
mo phological examina ion (no change in colou and no
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ib illa ion). Fo chond ocy e isola ion, asep ically dissec ed
ca ilage was subjec ed o sequen ial diges ion wi h p onase
(ca alogue numbe 165921; Roche Molecula Biochemicals,
Indianapolis, IN, USA) and collagenase P (ca alogue numbe
1213873; Roche Molecula Biochemicals) a a inal concen-
a ion o 1 mg/ml in Dulbecco's modi ied Eagle's medium/F12
plus 10% oe al cal se um and s e ilized by il a ion, in
acco dance wi h he manu ac u e 's ins uc ions. In ou hands,
his p ocedu e was supe io o enzyma ic isola ion wi h colla-
genase alone in e ms o chond ocy e yields and capaci y o
a achmen . Ca ilage specimens we e inely diced in phos-
pha e-bu e ed saline (PBS), and a e emo ing PBS diced
issue was incuba ed o 30 min wi h p onase in a shaking
wa e ba h a 37°C. P onase was subsequen ly emo ed om
he diges ion lask and he ca ilage pieces we e washed wi h
PBS. A e emo al o PBS, diges ion was con inued wi h
addi ion o collagenase P; his was done o e 6–8 hou s in a
shaking wa e ba h a 37°C. The esul ing cell suspension was
il e ed h ough a 40 µm nylon cell s aine (BD Biosciences
Eu ope, E embodegem, Belgium) in o de o emo e deb is.
Cells we e cen i uged and washed wice wi h PBS, coun ed
and pla ed in 24-well issue cul u e pla es o chond ocy e cul-
u e. Cells we e se ially passaged o ob ain a su icien numbe
o cells and used be ween he i s and second passages.
Cell ea men s and ni i e assay
ATDC5 cells and human p ima y chond ocy es, wi h a iabili y
g ea e han 95% as e alua ed using he ypan blue exclusion
me hod, we e cul u ed (as desc ibed abo e) in 24-well pla es.
A e 12 hou s o s a a ion in se um- ee medium, cells we e
s imula ed o 48 hou s wi h lep in (800 nmol/l), alone o in
combina ion wi h IL-1 (0.025 ng/ml). We wished o de e mine
whe he inc eased NO p oduc ion was due o NOS ype II
ac i a ion and o he in ol emen o JAK2, phospha idylinosi ol
3-kinase (PI3K), mi ogen-ac i a ed p o ein kinase kinase
(MEK)1 and p38 kinase. Fo his pu pose, he ollowing spe-
ci ic pha macological inhibi o s we e added 1 hou be o e
cy okine s imula ion: aminoguanidine (1 mmol/l) o NOS ype
II; y phos in AG490 (5 and 10 µmol/l) and Tkip (20 and 50
µmol/l) o JAK2; wo mannin (1, 2.5, 5 and 10 µmol/l) and
LY294002 (1, 2.5, 5 and 10 µmol/l) o PI3K; PD098059 (1,
5, 10, 20 and 30 µmol/l) o MEK-1; and SB203580 (1, 5, 10,
20 and 30 µmol/l) o p38 kinase. Cy okines and pha maco-
logical inhibi o doses we e selec ed on he basis o p io
dose– esponse expe imen s (da a no shown) o p e iously
published li e a u e [15].
Ni i e accumula ion was measu ed in cul u e medium using
he G iess eac ion. B ie ly, 100 µl cell cul u e medium was
mixed wi h 100 µl G iess eagen (equal olumes o 1%
[weigh / ol] sul anilamide in 5% [ ol/ ol] phospho ic acid and
0.1% [weigh / ol] naph yle hylenediamine-HCl), incuba ed a
oom empe a u e o 10 min, and hen he abso bance a 550
nm was measu ed using a mic opla e eade (Ti e ek-Mul i-
scan, Labsys em, Helsinki, Finland). F esh cul u e medium was
used as blank in all o he expe imen s. The amoun o ni i e in
he samples (in mic omola uni s) was calcula ed om a
sodium ni i e s anda d cu e eshly p epa ed in cul u e
medium.
RNA isola ion and eal- ime RT-PCR
ATDC5 chond ogenic cells we e seeded in P6 well pla es o
each 85–90% con luence. A e 8 hou s o s a a ion in
se um- ee medium, cells we e ea ed wi h lep in alone o in
combina ion wi h IL-1. In o de o es he in ol emen o JAK2,
PI3K, MEK-1 and p38 kinase on NOS ype II mRNA exp es-
sion, speci ic inhibi o s ( y phos in AG490 10 µmol/l, wo -
mannin and LY294002 10 µmol/l, PD098059 30 µmol/l and
SB203580 30 µmol/l) we e added 1 hou be o e cy okine
s imula ion. A e 48 hou s o ea men , RNA was isola ed
om cell cul u e using he T izol-LS®TM me hod (Gibco-BRL,
Li e Technologies, G and Island, NY USA), in acco dance wi h
he manu ac u e 's ins uc ions. B ie ly, 5 × 105 cells we e
lysed in 1000 µl T izol-LS® eagen , and eco e y o o al RNA
a e isop opanol p ecipi a ion was measu ed using a spec o-
pho ome e (Beckman DU62, Ame sham Biosciences, Chal-
on S . Giles, UK) a 260 nm.
Analysis o ni ic oxide syn hase ype II gene exp ession
using eal- ime RT-PCR
Real- ime RT-PCR analyses we e pe o med in a luo escen
empe a u e cycle (MX3000P Real Time PCR Sys em; S a -
agene), in acco dance wi h he manu ac u e 's ins uc ions.
To al RNA 1 µg was used o each RT eac ion. cDNAs we e
syn hesized using 200 uni s o Moloney mu ine leukaemia
e e se ansc ip ase (Gibco-BRL) and 6 µl dNTPs mix (10
mmol/l o each dNTP), 6 µl o i s s and bu e (250 mmol/l
T is-HCl [pH 8.3], 375 mmol/l KCl, 15 mmol/l MgCl2; Gibco-
BRL), 1.5 µl o 50 mmol/l MgCl2, 0.17 µl andom hexame
solu ion (3 µg/µl; Gibco-BRL) and 0.25 µl o RNAse Ou TM
( ecombinan ibonuclease inhibi o 40 µg/µl; Gibco-BRL), in
a o al olume o 30 µl. Reac ion mix u es we e incuba ed a
37°C o 50 min and a 42°C o 15 min. The RT eac ion was
e mina ed by hea ing a 95°C o 5 min and subsequen ly
quick chilled on ice. The 50 µl ampli ica ion mix u e (B illian
SYBR G een QPC Mas e Mix; S a agene) con ained 2 µl o
RT eac ion p oduc s plus 0.75 µl (30 nmol/l) dilu ed e e -
ence dye, 150 nmol/l o each p ime and nuclease- ee, PCR
g ade wa e o adjus he inal olume o 50 µl.
A e a i s enzyme ac i a ion s ep (95°C o 10 min), eac-
ions we e cycled 33 imes using he ollowing pa ame e s o
NOS ype II de ec ion: dena u a ion a 95°C o 40 s, anneal-
ing a 60°C o 1 min and ex ension a 72°C o 1 min. Mouse
glyce aldehyde-3-phospha e dehyd ogenase (GAPDH) cDNA
(5'-TCCATGACAACTTTGGCATCGTGG-3' o ups eam
p ime and 5'-GTTGCTGTTGAAGTCACAGGAGAC-3' o
downs eam p ime ; Genebank M32599) was ampli ied unde
he same condi ions and was used as a no malize gene. The
amoun o PCR p oduc s o med in each cycle was e alua ed
A h i is Resea ch & The apy Vol 7 No 3 O e o e al.
R584
on he basis o SYBR G een I luo escence. A inal ex ension
a 72°C o e 10 min was ollowed by mel ing cu e p o iles as
ollows: 95°C o 1 min, amping down o 45°C a a a e o
0.2°C/s, and hea ing slowly (0.5°C/cycle) o 95°C o a o al
o 81 cycles (30 s/cycle). Fluo escence was measu ed con in-
uously o con i m ampli ica ion o speci ic ansc ip s (da a no
shown).
The oligonucleo ide p ime s speci ic o mouse NOS ype II
we e as ollows: ups eam p ime 5'-CTCACTGGGACAG-
CACAGAA-3' and downs eam p ime 5'-TGGT-
CAAACTCTTGGGGTTC-3' ( om Genbank U43428).
Cycle- o-cycle luo escence emission eadings we e moni-
o ed and quan i ied using he second de i a i e maximum
me hod om he MX3000P Real Time so wa e package
(S a agene). This me hod de e mines he c ossing poin s o
indi idual samples using an algo i hm ha iden i ies he i s
u ning poin o he luo escence cu e. This u ning poin co -
esponds o he i s maximum o he second de i a i e cu e
and co ela es in e sely wi h he log o he ini ial empla e con-
cen a ion. NOS ype II mRNA le els we e no malized wi h
espec o mouse GAPDH le el in each sample.
Ni ic oxide syn hase ype II wes e n blo analysis
ATDC-5 chond ogenic cells we e seeded in P100 pla es un il
hey eached 85–90% con luence. A e o e nigh s a a ion
in se um- ee medium, cells we e s imula ed o 24 hou s wi h
lep in (800 nmol/l), alone o in combina ion wi h IL-1 (0.025
ng/ml). In o de o demons a e he in ol emen o JAK2, PI3K,
MEK-1 and p38 kinase, he ollowing speci ic pha macological
inhibi o s we e added 1 hou be o e cy okine s imula ion: y -
phos in AG490 (5 and 10 µmol/l) and Tkip (20 and 50 µmol/
l) o JAK2; LY294002 (1, 5 and 10 µmol/l) o PI3K;
PD098059 (1, 10 and 30 µmol/l) o MEK-1; and SB203580
(1, 10 and 30 µmol/l) o p38 kinase. A e s imula ion, cells
we e apidly washed wi h ice cold PBS and sc aped in lysis
bu e : 10 mmol/l T is-HCl (pH 7.5), 5 mmol/l EDTA, 150
mmol/l NaCl, 30 mmol/l sodium py ophospha e, 50 mmol/l
sodium luo ide, 1 mmol/l sodium o ho anada e (Na3VO4),
10% glyce ol, 0.5% T i on X-100, 1 mmol/l phenylme hylsul-
onil luo ide, ap o inin, leupep in and peps a in A (10 mg/ml).
Lysed cells we e cen i uged a 13000 g o 15 min. Lysa es
om con ol o s imula ed cells we e collec ed and sepa a ed
by SDS-PAGE on a 10% polyac ylamide gel. P o eins we e
subsequen ly ans e ed o a poly inylidene di luo ide ans e
memb ane (Hybond TM-P; Ame sham In e na ional, Li le
Chal on , UK) using a ans e semid y blo cell (BioRad Labo-
a o ies, He cules, CA, USA). Blo s we e incuba ed wi h he
app op ia e an ibody (mouse an i-NOS II an ibody; pu chased
om Ups a e Bio ech, Lake Placid, NY, USA). Immunoblo s
we e isualized using ECLPlus de ec ion Ki (Ame sham-Pha -
macia Bio ech, Ba celona, Spain) using ho se adish pe oxi-
dase labelled seconda y an ibody. To con i m equal load in
each sample, a e s ipping in glycine bu e a pH 3, mem-
b anes we e eblo ed wi h an i-ac in an ibody (San a C uz
Bio echnology Inc., San a C uz, CA, USA). The images o
au o adiog ams we e cap u ed and analyzed using a Typhoon
9410 digi al a iable mode image (Ame sham Bio ech, Li le
Chal on , UK).
Da a analysis
Da a a e exp essed as mean ± s anda d e o o he mean o
a leas h ee independen expe imen s, each wi h a leas
h ee o mo e independen obse a ions. S a is ical analysis
was pe o med using analysis o a iance ollowed by he S u-
den –Newman–Keuls o Bon e oni mul iple compa ison es
wi h he Ins a compu e ized package (G aphPad So wa e
Inc., San Diego, CA, USA). i < 0.05 was conside ed s a is i-
cally signi ican .
Resul s
Lep in syne gis ic e ec o e IL-1 induced ni i e
p oduc ion in chond ocy es
A lep in concen a ion o 800 nmol/l was ound o be op imal
o co-s imula o y expe imen s. This concen a ion was
selec ed based on a b aod se o p e ious dose– esponse
expe imen s (da a no shown). Because NOS ype II s imula-
ion wi h IL-1 a 0.05 ng/ml was maximal, a dose o 0.025 ng/
ml was selec ed in o de o a oid masking lep in syne gism. As
shown in Fig. 1, ATDC5 cells and human p ima y chond o-
cy es did no accumula e ni i es when s imula ed wi h lep in
alone; howe e , lep in was able o inc ease signi ican ly ni i e
accumula ion induced by IL-1 when cells we e co-s imula ed
wi h bo h cy okines (Fig 1a,c). This esul was con i med in
e ms o p o ein exp ession. Indeed, a clea -cu inc ease in le -
els o NOS ype II p o ein was obse ed when cells we e co-
s imula ed wi h lep in and IL-1 (Fig. 1b).
To con i m whe he NO o ma ion was p oduced ia NOS ype
II, ATDC5 cells and human chond ocy es we e incuba ed o
48 hou s wi h bo h cy okines in he p esence o he NOS ype
II inhibi o aminoguanidine (1 mmol/l), added 1 hou be o e
cy okine adminis a ion. Aminoguanidine comple ely inhibi ed
ni i e accumula ion in he cul u e supe na an o human p i-
ma y chond ocy es (Fig. 1c) and ATDC5 cells (Fig. 1d).
Janus kinase-2 inhibi ion blocks lep in/IL-1 induced
ni ic oxide p oduc ion and ni ic oxide syn hase ype II
p o ein exp ession
We also in es iga ed he ole played by JAK2 in ni i e p oduc-
ion e oked by co-s imula ion wi h lep in and IL-1 by using y -
phos in AG490. This JAK2 inhibi o , added 1 hou be o e
cy okine co-s imula ion, comple ely blocked ni i e p oduc ion
(Fig. 2a). This esul was con i med in e ms o p o ein exp es-
sion, because cell p e ea men wi h y phos in AG490 signi -
ican ly dec eased NOS II p o ein exp ession in lep in/IL-1 co-
s imula ed cells (Fig. 2d). In iguingly, y phos in AG490 was
also able o inhibi ni i e accumula ion induced by IL-1 alone,
sugges ing ha lep in syne gizes wi h undamen al pa hways
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in IL-1 esponses. To gain u he insigh s in o he in ol emen
o JAK2, Tkip (a 12-me SOCS-1 mime ic pep ide ha binds
o he au ophospho yla ion si e o JAK2) was added o ATDC5
cells 1 hou be o e hey we e s imula ed wi h lep in o IL-1, o
bo h cy okines. Tkip a 50 µmol/l was able o blun comple ely
lep in/IL-1 induced ni i e accumula ion and NOS II p o ein
exp ession (Fig. 2b,e). A lipophilic i ele an pep ide, MuIFN-
γ95–125 (AKFEVNNPQVQRQAFNELIRVVHQLLPESSL), was
used as con ol. In iguingly, Tkip was also able o inhibi , in a
dose– esponse manne , ni i e accumula ion and NOS II p o-
ein exp ession in ATDC5 cells s imula ed wi h IL-1 alone (Fig.
2c,e).
E ec o he speci ic signalling pa hways inhibi o s
LY294002, PD098059 and SB203580 on lep in/IL-1 co-
s imula ion
In o de o de ine he signalling pa hway in ol ed in he syne -
gis ic induc ion o NOS ype II media ed by co-s imula ion wi h
lep in and IL-1 in cul u ed ATDC5 cells, we e alua ed he
e ec s o speci ic pha macological inhibi o s on o he kinases,
speci ically PI3K, MEK-1 and p38 kinase.
We i s in es iga ed he e ec o a speci ic inhibi o o PI3K,
namely LY294002 (1, 2.5, 5 and 10 µmol/l) on lep in/IL-1
induced NO p oduc ion. The addi ion o LY294002 1 hou
be o e cy okine co-s imula ion esul ed in signi ican and dose-
dependen dec eases in NO p oduc ion and NOS ype II p o-
ein exp ession (Fig. 3a,a1).
In o de o es whe he MEK-1 ( he mi ogen-ac i a ed p o ein
kinase [MAPK] kinase in ol ed in ex acellula signal- egula ed
kinase [ERK]-1 and ERK-2 phospho yla ion/ac i a ion) pa ici-
pa es in NOS ype II induc ion ia lep in/IL-1 co-s imula ion,
we used he speci ic MEK-1 inhibi o PD98059. When his
inhibi o was added 1 hou be o e cy okine co-s imula ion, sig-
ni ican dose-dependen dec eases in NO p oduc ion and
NOS II p o ein exp ession we e obse ed (Fig. 3b,b1).
Figu e 1
Lep in syne gizes wi h IL-1 in inducing ni ic oxide syn hase (NOS) ype IILep in syne gizes wi h IL-1 in inducing ni ic oxide syn hase (NOS) ype II. Syne gis ic e ec o lep in (OB) on ni i e (NO2-) accumula ion and NOS
ype II p o ein exp ession induced by IL-1. S imula ions we e conduc ed in se um- ee condi ions (a,b) in ATDC5 chond ogenic cells and (c) in
human p ima y chond ocy es. NO2- accumula ion is selec i ely inhibi ed by aminoguanidine (AG) bo h in (d) ATDC5 cells and in (panel c) human p i-
ma y chond ocy es. Values a e exp essed as mean ± s anda d e o o he mean. WB, wes e n blo .

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Finally, because i has been shown ha p38 kinase is in ol ed
in apop o ic p ocesses induced by NO in chond ocy es, we
es ed whe he his MAPK is also in ol ed in NOS ype II syn-
e gis ic ac i a ion s imula ed by lep in/IL-1. Fo his pu pose,
we used he speci ic p38 kinase inhibi o SB203580. Addi ion
o his inhibi o 1 hou be o e lep in/IL-1 co-s imula ion caused
signi ican and dose-dependen dec eases in NO p oduc ion
and NOS II p o ein exp ession (Fig. 3c,c1 [lowe panel]).
Lep in syne gism does no depend on chond ocy e
di e en ia ion s a e
In o de o de e mine whe he lep in/IL-1 syne gism and i s sig-
nalling pa hway depend on he di e en ia ion s a e o chond o-
cy es, we conduc ed simila expe imen s in ma u e and
hype ophic chond ocy es. We di e en ia ed ATDC5 cells
(see Ma e ials and me hods, abo e) in o ma u e and hype -
ophic chond ocy es, and es ed co-s imula ion and ea -
men s wi h all speci ic inhibi o s. Ni i e accumula ion,
e alua ed in 15-day (ma u e) and in 21-day (hype ophic) di -
e en ia ed ATDC5 cells a 24 and 48 hou s a e ea men ,
was simila o ha obse ed in he ATDC5 chond ogenic
undi e en ia ed cell line (Fig. 4a–d). No e ha in o de o e al-
ua e he in ol emen o PI3K, in some expe imen s we also
used wo mannin a 10 µmol/l (a classical bu no e y speci ic
PI3K inhibi o ), yielding esul s simila o hose ob ained wi h
LY294002.
Finally, a simila pa e n was obse ed in human cul u ed p i-
ma y chond ocy es. In hese cells, lep in induced a s ong
inc ease in ni i e accumula ion o e ha induced by IL-1, and
Figu e 2
Janus kinase (JAK)2 inhibi ion blocks lep in/IL-1-induced ni ic oxide (NO) p oduc ion and ni ic oxide syn hase (NOS) ype II p o ein exp essionJanus kinase (JAK)2 inhibi ion blocks lep in/IL-1-induced ni ic oxide (NO) p oduc ion and ni ic oxide syn hase (NOS) ype II p o ein exp ession.
E ec o y phos in AG490 and Tkip on NO p oduc ion and NOS II p o ein exp ession. The e ec o y phos in AG490 was e alua ed in e ms o (a)
ni i e accumula ion in ATDC5 cells s imula ed wi h lep in and IL-1, and in e ms o (d) NOS II p o ein exp ession. The e ec o Tkip was e alua ed by
ni i e accumula ion in (b) lep in/IL-1 ATDC5 co-s imula ed cells and in (c) IL-1 s imula ed cells (panel c). (e) E ec o Tkip on NOS ype II p o ein
exp ession in lep in/IL-1 co-s imula ed cells.
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he syne gis ic esponse was signi ican ly inhibi ed by
y phos in AG490, wo mannin, LY294002, PD98059 and
SB203580 (Fig. 5).
E ec o lep in/IL-1 co-s imula ion on ni ic oxide
syn hase ype II RNA exp ession
We inally s udied NOS II mRNA exp ession in o de o de e -
mine whe he NO inc ease/inhibi ion was due o modula ion o
NOS ype II mRNA exp ession. As shown in Fig. 6, NOS ype
II mRNA, e alua ed using eal- ime PCR, was s ongly
exp essed when cells we e co-s imula ed wi h lep in plus IL-1,
and his exp ession was signi ican ly educed by y phos in
AG490, wo mannin, LY294002, PD098059 and SB203580.
Discussion
In he p esen s udy we in es iga ed he e ec o lep in on NO
p oduc ion s imula ed by IL-1. We ound ha lep in had a syn-
e gis ic e ec in he ATDC5 mu ine chond ogenic cell line, in
di e en ia ed ma u e and hype ophic ATDC5 chond ocy es,
and in human p ima y chond ocy es.
Lep in has been classi ied as a cy okine-like ho mone,
because o i s s uc u e and he homology o i s ecep o s wi h
membe s o he class I cy okine ecep o supe amily. A p oin-
lamma o y ole o lep in has p e iously been p oposed. Se -
e al da a show ha lep in le els a e inc eased by
p oin lamma o y cy okine adminis a ion and in animal models
o acu e in lamma ion [9]. In addi ion, lep in egula es no only
humo al bu also cellula immune esponses in an igen-
induced a h i is models [20]. Ne e heless, he e a e only ew
Figu e 3
In ol emen o phospha idylinosi ol 3-kinase (PI3K), mi ogen-ac i a ed p o ein kinase kinase (MEK)-1 and p38-kinase in lep in/IL-1-induced ni ic oxide syn hase (NOS)In ol emen o phospha idylinosi ol 3-kinase (PI3K), mi ogen-ac i a ed p o ein kinase kinase (MEK)-1 and p38-kinase in lep in/IL-1-induced ni ic
oxide syn hase (NOS). Dose-dependen e ec o (a,a1) LY294002, (b,b1) PD098059 and (c,c1) SB203580 on ni i e (NO2-) p oduc ion and NOS
ype II p o ein exp ession in s imula ed and uns imula ed ATDC5 cells. S imula ions we e conduc ed in se um- ee condi ions. Each inhibi o was
added 1 hou be o e cy okine co-s imula ion. Values a e exp essed as mean ± s anda d e o o he mean. OB, lep in; WB, wes e n blo .
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epo s o a di ec ac ion o lep in a he cellula le el in ca i-
lage [14,15].
NO con ols a a ie y o ca ilage unc ions, including loss o
chond ocy e pheno ype, chond ocy e apop osis, and ex acel-
lula ma ix deg ada ion [2,3]. NOS ype II is mainly exp essed
by immune cells in esponse o a wide ange o p oin lamma-
o y cy okines [21,22]. In i o, human a icula ca ilage is able
o p oduce la ge amoun s o NO [23], which can be enhanced
by p oin lamma o y cy okines. In addi ion, NO p oduc ion can
be signi ican ly inc eased by he p esence o lep in, as shown
in ou p e ious wo k [15] and in he p esen s udy.
He e, we show ha he IL-1 induced p oduc ion o NO by
ATDC5 mu ine chond ocy es and by human chond ocy es is
signi ican ly enhanced by lep in. I is no ewo hy ha , apa
om blood, se e al sou ces o lep in and IL-1 ha e been iden-
i ied in o a ound he join s in pa hological condi ions. IL-1 is
p oduced by in lamed syno ium and pe ia icula a pad [24].
In e es ingly, mul ipo en s omal cells om he in apa ella a
p oduce lep in [25]. In addi ion, os eoa h i ic human chond o-
cy es p oduce lep in, and lep in adminis a ion in a s induces
o e -exp ession o his ho mone by a icula chond ocy es
[13]. Thus, in pa ien s wi h in lamma o y syno i is o os eoa -
h i is, he e is a unique mic oen i onmen in he ca ilage cha -
ac e ized by ele a ed le els o bo h lep in and IL-1, due no
only o local p oduc ion bu also o sys emic inc ease
[10,13,26]. I is concei able ha in his scena io lep in plays a
signi ican p oin lamma o y ole, as sugges ed by he indings
p esen ed he e. O u he in e es is ou p e ious epo [15]
o he co-s imula o y e ec o lep in and IFN-γ a he chond o-
cy e le el.
Figu e 4
Lep in syne gism does no depend upon chond ocy e di e a ion s a eLep in syne gism does no depend upon chond ocy e di e a ion s a e. E ec o di e en inhibi o s on ni i e (NO2-) accumula ion in 15-day di e en-
ia ed ATDC5 cells s imula ed o no wi h lep in, alone o in combina ion wi h IL-1, du ing (a) 24 and (b) 48 hou s. The e ec o inhibi o s was also
e alua ed in 21-day di e en ia ed ATDC5 cells, a e (c) 24 o (d) 48 hou s o s imula ion wi h lep in and IL-1 (alone o in combina ion). Values a e
exp essed as mean ± s anda d e o o he mean. OB, lep in.
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We p e iously es ablished ha he ea ly e en in lep in/IFN-γ
syne gis ic NOS ype II ac i a ion was he in ol emen o JAK2
[15]; he p esen esul s con i m ha JAK2 ac i a ion is also an
ea ly s ep in lep in/IL-1 induced NOS ype II co-s imula ion.
The ac ha y phos in AG490 blocks he lep in/IL-1
esponse implies ha lep in syne gizes wi h c i ical pa hways
in IL-1 esponse. I was su p ising ha y phos in AG490 also
blocked he esponse o IL-1 alone, because JAK2 is no
known o be equi ed o IL-1 ecep o ansduc ion, and so
one would expec he e ec o y phos in AG490 o be pa ial.
Howe e , ou esul s a e in ag eemen wi h hose epo ed by
o he in es iga o s [27,28].
We also used Tkip in ou expe imen s; Tkip is a 12-me
SOCS-1 mime ic lipophilic pep ide (WLVFFVIFYFFR) ha
inhibi s JAK2 au ophospho yla ion [29]. In e es ingly, he
beha iou o his pep ide was simila o ha o y phos in
AG490 in e ms o NOS II inhibi ion. I is concei able ha his
pep ide, because o i s SOCS-1 mime ic p ope ies, could
inhibi IL-1/Toll-like ecep o unc ion in chond ocy es. SOCS-
1 is a nega i e egula o o lipopolysaccha ide-induced mac o-
phage ac i a ion [30,31] and has been shown o bind o IL-1
ecep o associa ed kinase [32]. This dis up s he cascade
ha leads o nuclea ac o -κB (NF-κB) signalling and causes
NOS inhibi ion. O no e, i has been demons a ed ha y -
phos in AG490 inhibi s IL-1 induced NF-κB ac i a ion in con-
cen a ions ha also inhibi NOS II mRNA and p o ein
syn hesis. These indings sugges ha JAK2 is equi ed o
NF-κB ac i a ion, which in u n media es IL-1 induced NOS II
exp ession in chond ocy es [28].
To gain u he insigh s in o he mechanism by which lep in,
oge he wi h IL-1, p omo es NO p oduc ion, we e alua ed he
oles played by downs eam signalling cascades using spe-
ci ic pha macological inhibi o s. Fi s , we analyzed he in ol e-
men o PI3K. The ole played by his kinase in he ac i a ion o
NOS ype II is qui e con o e sial and emains he subjec o
deba e. A numbe o s udies suppo he iew ha PI3K ac i -
i y down- egula es NOS ype II, bu he e a e se e al ca ea s
Figu e 5
Lep in ac s syne gis ically wi h IL-1 in human p ima y chond ocy esLep in ac s syne gis ically wi h IL-1 in human p ima y chond ocy es.
Ni i e (NO2-) accumula ion in lep in (OB)/IL-1 co-s imula ed human p i-
ma y chond ocy es. S imula ions we e conduc ed in se um- ee condi-
ions in he p esence o absence o y phos in AG490, wo mannin,
LY294002, PD98059 and SB203580 inhibi o s. Values a e exp essed
as mean ± s anda d e o o he mean.
Figu e 6
E ec o lep in/IL-1 co-s imula ion on ni ic oxide syn hase (NOS) ype II mRNA exp essionE ec o lep in/IL-1 co-s imula ion on ni ic oxide syn hase (NOS) ype II
mRNA exp ession. Real- ime RT-PCR analysis o he exp ession o he
inducible NOS ype II mRNA in lep in (OB)/IL-1 co-s imula ed ATDC5
cells. S imula ions (24 hou s) we e conduc ed in se um- ee condi ions.
Speci ic inhibi o s we e added 1 hou be o e cy okine co-s imula ion.
Values a e exp essed as mean ± s anda d e o o he mean.