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StimulationofFibroblastCellGrowth,MatrixProduction,andGranulationTissueFortnation
by
ConnectiveTissueGrowthFactor
KenFrazier,ShawnWilliams,DevashishKothapalli,HeleneKlapper,andGaryR.Grotendorst
DepartmentofCellBiologyandAnatomy,UniversityofMiamiSchoolofMedicine,Miami,Florida,U.S.A.
Connectivetissuegrowthfactor(CTGF)isa36-to38-kDapeptidethatisselectivelyinducedbytrans-fornUnggrowthfactor-fJ(TGF-fJ)infibroblasticcelltypes.WecomparedthebiologicactivitiesofCTGFwith
TGF-J3
onfibroblastsincultureandinanimalmodelsoffibroplasia.CTGFwasactiveasamitogeninmonolayerculturesofnormalratkidneyfibro-blasts.CTGFdidnotstimulateanchorage-indepen-dentgrowthofNRI{fibroblasts,however,orinhibitthegrowthofminklungepithelialcells,distinguish-ingCTGF'sgrowth-regulatoryactivitiesfromthoseofTGF-J3.InNRKfibroblasts,both
TGF-J3
andCTGFsignificantlyincreasedthetranscriptsencoding0:1typeIcollagen,
as
integrin,andfibronectin.Stimu-lationoftypeIcollagenandfibronectinproteinsynthesisby
TGF-J3
andCTGFwasconfirmedbypulselabelingofcellswith[
35
S]methionine.Subcu-taneousinjectionofTGF-J3andCTGFintoneonatalNIHSwissm.iceresultedinalargestimulationofgranulationtissueandfibrosisatthesiteofinjection.
Itt
siuc
hybridizationstudiesrevealedthatTGF-J3injectioninducedhighlevelsofCTGFmRNAinthedermalfibroblastsattheinjectionsite,demonstrat-ingthatTGF-{3caninducetheexpressionofCTGFinconnectivetissuecells
ill
vivo.
NoCTGFtranscriptsweredetectedintheepidermalcellsineithercontrolorTGF-{3-injectedskinorinfibroblastsincontrol(saline-injected)skin.Theseresultsdemonstratethat,likeTGF-I3,CTGFcaninduceconnectivetissuecellproliferationarrdextracellularrrratzixsynthesis.
Keywords:TGF-J3lcollagenlwomldrepairlfihrosis.
J
InvestDer-matol.
107:404-411,1996
T
ssueregenerationandrepairproceedinacascadefashionbeginningwithacoagulationand
inflamma-
tOI),phase,folJowedbygranulationtissueformationandfinallyextracellulnrmatrixdepositionandtermi-nationoftheresponse.Peptidegrowthfactorsplayacentralroleinthisprocess.
It
islikelythatfactorsreleasedbyplateletsand
inflammatory
cellsserve
as
initiatorsoftheregenera-tion/repaitresponse.Similarly,woundrepairdisorders,aswellasorgan-specificfibrosis,maybecausedbydysfunctionalcascades.Oneoftheprincipalregulatoryfactorsthatappearsto
function
asaninitiatorintheseprocessesis
rransforming
growthfactor-{3(TGF-f3)
(Arnento
andBeck,1991;
Rnghow,
1991;Wahl,1992;
Roberts
and
Sporn,
1993).TGF-,Bhasbeenshowntoactasapotent
stimulatory
signalforconnectivetissueformationduringwoundrepairandinfibroticconditions.ElevatedTGF-f3mRNAorproteinlevelshavebeendocumentedintissueduringnormalwoundrepair(Igarashi
etal,
1993;Levine
et
al,
1993),andfibroticdisordersoftheskin(Kulozik
et
ill,
1990;
Peltonen
etai,
1990;Smithand
Lek.oy,
1990)andinternalorgansandtissues(Nagy
et
ill,
1991;Kagami
etal,1993;
Bahadori
etill,
1995).TheincreasedfibrotictissuehasbeenattributedtoseveralactionsofTGF-,B,including:(i)increasedManuscriptreceivedFebruary23,1996;revisedApril29,1996;acceptedforpublicationMay21,1996..Reprintrequests
to:
Dr.Gary
R..
Grotendorst,DepartmentofCellBiologyandAnatomy,UniversityofMiamiSchoolofMedicine(R-124),
1600NW10th
Avenue,Miami,FL33136.Abbreviations:CTGF,connectivetissuegrowthfactor;rCTGF,recom-
binantconnectivetissuegrowthfactor.
fibroblastproliferation(Del.arcoandTodaro,1978;Assoi.an
e(
ill,
1984;Leofel
ill,
1986;SomaandGrorcndorst,1989;Ishikawa
dill,
1990;Battegay
ct
ill,
1990);(ii)elevatedsynthesisofextracellularmatrix
components
includingfibronectin,type
I
collagen,
inregrins,
laminin,
and
glycosarninoglycans
production
(Ignotz
andMas-sague,:1986;Roberts
ct
al,
1986;
Raghowdill,
1987;Varga
et.
III,
1987;Penttinen
ctai,
1988),and(iii)decreaseddegradationofextracellularmatrixduetodirectinhibitionofproteaseactivityandstimulationofthesynthesisofproteaseinhibitors(Laiho
etal,
1986;
Lnnd
etal,
1987;Kerr
ctill,
1990).Previousstudieshavedemon-
stnated
that
'.1
largeportionoftheTGF-{3inductionofmatrixproteinsynthesisisnotsharedbyothergrowthfactorssuchas
fibroblast
growth
factor
(FGF)orplateletderived
growth
factors(PDGF)(IgnotzandMassague,1986;Roberts
e/
ill,
1986;Penttinen
etai,
1988).
Connectivetissuegrowthfactor(CTGF)isacysteine-richmitogenicpeptidethatwas
originally
identifiedas
a
growthfactorsecretedbyvascularendothelialcellsinculture(Bradham
et
ill,
1991).CTGFisselectivelyinducedinfibroblastsafteractivationwithTGF-{3(SomaandGrotendorst,1989;Igarashi
e(ill,1993).
CTGFisamemberofafamilyofpeptidesthatincludeserum-inducedgeneproductsceflO(Simmons
etai,
1989),
cyro
l(O'Brien
et
al,
1990),flsp12/{3IGMl(Brunner
etai,
1991;
Ryscck
et
Ill,
1991)
and
11
chickentransforminggene,
nov(joliet
et
al,
1992).CTGFalsosharessignificantsequencehomologywitha
Drosopliiln
geneproduct,twistedgastrulation(twg)(Mason
etill,
1994),thatdeterminescellfatesduring
dorsal/ventral
patternformationintheembryo.PreviousstudieshavedemonstratedcoordinateexpressionofTGF-/31andCTGFingranulationtissuebedsduringwound0022-202X/96/S10.S0Copyright©1996byTheSocietyforInvestigativeDermatology,Inc.
404
 
VOL.107,NO.3SEPTEMBER
1996
repair(Igarashi
et
til,
1993)and
found
thatdermalfibroblasts
in
sc1er:odermalesions
overexpress
CTGF(Igarashi
etai,
1995).TheCTGFmRNA
isselectively
inducedin
fibroblastsby
TGF:"'{3,butnotby
other
growthfactors,
suchas
epidermalgrowthfactor(EGF),PDGF,orFGFs(acidicFGF,basicFGF)(Igarashi
etal,
1993).ProteinsynthesisisnotrequiredforTGF-J!stimulationofCTGFgene
expression
(Igarashi
etal,
1993;
Grotcridorst
etal,
1996),indicatingthattheCTGFgeneisdirectlyregulatedbyTGF-/3.Wehavenow
identifieda
novelTGF-{:lresponse
element
(T/31lE)intheCTGI'promoter
(Grotendorst
etal,
1996),whichbeginsto
define
themolecularbasisfortheselectiveregulationof
tins
genebyTGF-{3.TheTf3REsequencepresent
in
thehumanand
mouse
CTGFgenesisnot
present
inthepromotersofotherTGF-/3-regulafedgenethathasbeendescribedtodate.Becauseofthepleiotropic
actions
of
TGF-f3
on
cells
andtheselectiveiudu.ctionofCTGFby
TGF-f3
infibroblasts,we
wanted
tocomparethebiologicactivitiesofCTGFwithTGF-f3.Untilnow,thelimited
availability
ofnaturalCTGFmade
it
difficult
to
performstudiesonthebiologicactionsofthe
molecule.
WehaveproducedrecombinantCTGFtoaddressthis
question.
Omstudiesindicatethatrecomb-inantCTGFismitogenicfor
Nl~K
fibroblasts
inmonolayerculture.TGF-f3inthepresenceofEGFandserumiscapableof
stimulating
anchorage-independentgl'owt.hof
NIU(
fibroblasts(DeLarcoandTodaro,
1978;Assoian
ctal,
1994),butCTGF
does
notsubstituteforTGF-{3inthis
assay.
Also,thegrowth-inhibitoryactionsofTGF-f3onepithelialcells(Tucker
etal;
1984;Ogawaand
Seyedin,
1991)are
not
sharedbyCTGF.CTGFinduceselevatedexpressionand
synthesis
ofbothcollagenandfibronectill
in
fibroblastsincultureandinduces
fibroplasia
inthe
skin
ofnewbornmice.TheseresultsdemonstratethatCTGFstimulatesfibroblastproliferationandextracellular
matrix
synthesis
ina
manner
similar
toTGF-{3.CTGFdoesnotshareTGF-f3'sabilitytostimulateanchorage-independentgrowth
of
normalfibro-blastsoractasagrowthinhibitorforepithelialcells.MATERIALSANDMETHODSCellCulturesSf9cellswere
grown
inspinnerandmonolayercultureandmaintainedinTNM-FHmediaJGracc'sInsect
Culture
Media,(GIBCO,Gaithersburg,.MD)
with
3.33glactalbuminhydrolysateper
1111,
3.33gyeastolatcper
rnl,S'v"
fetalbovineserum,
5
'v"
Nuscrum
(CollaborativeBiomedical,Bedford,MA),10
JLg
gentamicinper
rnl,
and
O.
'1.%
Pluronic
F68
(spinnerculturesonly)].HighFivecells
(Invirrogcu,
SanDiego,CAlweregrowninshakerandmonolayercultureandmaintainedinExCel1405serum-freeinsect
cell
media
(JRH
Bioscience,Lenexa,KS).NR:I;C49FandMv1Lu
cells
wereobtainedfromAmericanTypeCultureCollection(Rockville,MD)andmaintainedin
Dulbeccos
modifiedEagle'smedium(DMEM)with5'){,fetalbovineserumand10JLggentamicinper
1111.
Sourcesof
Growrh
FactorsTGF-J3isolated
from
plateletswasagiftfrom
RichardAssoian
(UniversityofMiami).
Recombinant
platelet-derivedgrowthfactor
EE
wasobtained
fi:0111
Chiron
(Emeryville,CAl.
Purified
murineEGFwas
purchased
fromBiomedicalTechnologies,Inc.(Stough-ton,
Ma).Samples
wereassayedforendotoxinusing
an
endotoxinenzyme-
Iinkcd
immunosorbentassay
kit
(Sigma,St.Louis,MO).RecombinantCTGFwasproducedinthelaboratoryusingabncculoviralexpressionsystememploying
theplslucbacII
transfer
plasmid
(Invitrogen)containingtheCTGFopenrendingframe,
cotrausfcctcd
withlinearizedwild-typeAcMNPVintoSf')cellsaccordingto
prescribed
techniques(Ausubel
"I
nl,
1990).ForlargescaleproductionofrecombinantCTGF(rCTGF),HighFivecellsweregrowninshakerculturesto
a
density
0(3.5X
:J
0"ce:llsper
ml
andinfectedwithrCTGFbaculovirusatanMOl
=
7.TherecombinantCTGFwaspurifiedusingheparinSepharoseaffinitychromatography.Peak
frac-
rionscontainingrCTGFweredeterminedbyimmunoblottingandCOO.l11as-siestainingof
sodium
dodecylsulfate(SDS)-polyacrylamidcgelelectro-phoresisgels.GelElectrophoresisand
Imrrruuoblorrtng
Electrophoresiswas
per-
formed
using12'X,polyacrylamidegelscontainingSDSas,
described
by
Laernmli
~1970).
Immunobloccing
wasperformedbyelectroblotting
the
proteinsinthe
acrylarnide
geltoanitrocellulosemembraneasdescribedpreviously(SomaandGrotendorst,19-89).MitogenicandAnchorage-IndependentGrowthAssaysMitogenicassayswereperformedinmonolayerculturesusing48-wellplatesandNRKCTCFSTIMULATIONOFFlBROPLASIA
405
49Ffibroblasts
as
targetcellsasdescribedpreviously(SomaandGroten-
dorst,1989).Anchorage-independentgr-owthassayswereperformedessen-
tiallyasdescribed'byGuadagnoandAssoian(1991).GrowthInhibitionAssaysTheminklung
epithelialcellline,
MvlLu(ATTCNo.CCL64),wasusedastargerforgrowrh-inhibitoryassayswithTGF-/3andCTGF.AssayswereperformedasdescribedbyOgawaandScycdin(1991).ExtracellularMatrixProteinmRNA
Induction
AssaysNRKrat
fibroblasts'weregrowntoconAucnccinDMEMwith
50/0
feralbovineserum
andchenserumstarvedinDM.EMwith1%bovineserum
albumin
for24h.Growthfactorswere
added
tothecellcultures;totalcellularRNAwas
extracted
after24
.h,
andnorthernblotanalysiswas
performed
asdescribedpreviously(Igarashi
etal,
1993).To
ensure
thatequivalentamountsoftotalRNAwereaddedtoeachlaneon
a
gel,RNAwasquantitatedby
.A260/2S0
ratios.andequivalenttransferwasassured
by
comparingribosomal28Sand
18SRNAbandsin
each
laneafter
staining
with
ethidiurn
bromide.Asanadditionalcontrol,blotswere
reprobed
with
anactin
cDNAprobe.Double-strandedcDNAfragmentsused
for
probeswerelabeledwith
C
2
p]dCTPusing
arandom
primelabelingkit(Boehringer
Mannhcim,
Indianapolis,IN).TheCTG!'probewasderived
from
a·.1.1-kbhumaneDNAfragmentthatencompassedtheopenreading
frame
oftheCTGFtranscript.TheTGF-{31probewas
a
Lfl-kb
IVm:!
fragmentderived
6'0111
<J
2.0-kbhumanTGF-J31cDNApresent(G.LBell,H.H.
MedicalInsritutc,
UniversityofChicago).Thecd.-type1humancollagenprobewasderived
froma
J.5-khopenreadingframefragment
at
the
3'
end(ATCCNo.61323).Thet1'5
intcgrin
probewasproducedfromacDNAinsertcontain-ing
a
portionofthehumaneDNAcontainingtheopenreading
frame
(R.Assoian,UniversityofMiami).ThehumanfibronectinprobewasaO.9-kb
EwIU/Hi"dlII
fragmentderived
6'0111
a
2.2-kbeDNAclonecon-tainingthe
3'
regionoftheopenreading
frame
provided
by
F.Woessner(UniversityofMiami).Thehumanactin(controlRNA)probewaspur-chasedfromOncorCo.(Gaithersburg,MD).
Stfmularlon
ofMatrix
Protein
SynthesisCell
cultures
weregrownto.confluence,thenserum-starvedinDMEMandinsulin,transferrin,andselenium(ITS,CollaborutivcP...csearch,Bedford,MA)for18h.CultureswerethenplacedinDMEMcontaining100JLgbovineserumalbuminpcr
1111
andeither10
ug
TGF-{3per
ml,
20ngCTGFper
rnl,
ornothing(negativecontrol)andincubatedfor22h.Afterthistime,cultureswerepulselabeled'with50{LCi[
35
S]med1ionilleper
1111
inmethionine-freeDMEMfor2
h
inthepresenceoftheindicatedgrowth
factor.
Cellproteinswereextractedfromthecellpelletwith0.1
%
TritonXl00,0.5111M
phenylmethylsulfonylfluoride,
and
leupeptin
for10min.
Fibronecrin
was
immunoprecipitared
usingProteinA-SephaJ"oseusingprescribedmethods(Roberts
etal,
1988).Type
I
collagenwasanalyzedbyovernightpepsindigestion(5JLgper
ml
in
'I
Maceticacid)inthepresenceoflOJLgofcarrierbovinetypeIcollagen.Samplesusedforthe.
.i1l1.11TUnoprec::ipitation
andpepsindigestionwereadjustedsothatequivalentamountsoftotalincor-porated
C
5
S]meth,ioninewereusedineachsample.SDS-polyacrylamidegelelectrophoresiswasperformed,andsampleswere
autoradiographed.
SubcutaneousAdministrationofGrowthFactorstoMiceNeonatalN1J-lSwissmicewereinjectedinthenapeof
the
neckdailyfor3dwith20JLIoftotal
volume
ofeither
salinecontrol
(n
=
12);400or800ngofTGF-/3
(II
=
15);200or800
ng
ofPDGFBB
(n
=
12),800ngofEGF(n
=
8)or10,20,50,100,200,400,or800ngofrecombiuanrCTGF(n
=
38),basedonapreviousprotocol(Roberts
ct
nt,
1.986).Animalsweresacrificed24h
afterthelastinjection,andlargebiopsiesofthearea
containing
and
surrounding
the
injectionsitewereremoved.Sectionswerepreparedfor
histopathologicexaminationafterroutine
formalin.fixation,
processing,and
stainingwithMayer'shcmatoxvlinandeosin.
III
Siru
Hybridization
NIH
Swiss
neonatal
micewereinjected
as
described
aboveexceptthat.samples
ofdennis
and
subcutis
weretaken
6:01l1
thesiteofinjection
8
hafterthesecondinjection.Thesetissuesampleswereimmediatelyplacedin4.0%paraforrnaldehydefor1.5handthen
flash-
frozenandembedded.Sectionswere
cur
at
5
JLI11nd
placed
onTESPA-coatedslides(Oucor,Gaithersburg.MD.)
[II
situ
hybridizationforCTGFmRNAwasperformedusingpreviouslydescribedmethods(Pava
etal,
1990).Theslidesweredippedinphotographicemulsion(IlfordK-5.Polysciencc)andincubatedfor8dat4°C.Slideswerethendevelopedand
sectionscounterstainedinMayer'shematoxylinandeosin.
RESULTSProductionofRecomb
ill
antCTGFRecombinantCTGFwasproducedusingabaculoviralvectorsystemthat
contained
theopenreadingframeofthehumanCTGI'transcriptundertheregulation
 
406
FRAZIERETilL
u,
so
u,
C)
rn
o
I-
m
0
l-
o
l-
LL
LL
o
U.
en
o
o
W
o
~
I-
....
u,
>
C
o
U
rJ)
::::>
a.
2
fo..
....
II
....
97-
45-
30-
21-14-
CoornassieImrnunoblotanti-PDGF
R-250
Figure
1.
GelelectrophoreticanalysisofCTGF.rCTGFproducedinHighFivecellsbyabacculovirusexpressionsystemwaspurifiedbyaffinitychromatographyonheparinSepharose.Thepurityof
the
rCTGFisdemonstratedinlane2ofthe
fiji
pond
where
5
/LgofrCTGFarecomparedto
Bio-Rad
proteinstandards
(5
/Lg/ea).ComparisonofrCTGFwithCTGFsecretedbyhumanskinfibroblasts(HSF)afteractivationwithTGF-/3andhumanumbilicalveinendothelialcells(HUVE)andrPDGFBB
(rightpanel],
Allsamples
represent
approximately10
ng
ofCTGForPDGF.Peptidesweredetectedaftertransfertonitrocellulosebyananti-humanPDGFIgGandanalkalinephosphatase-conjugatedsecondantibodyasdescribedin
Materialsalld
Methods.
ofthebaculoviralpolyhedrongenepromoter.CTGFwaspurifiedbyaone-stepisolationprocedureusinghcparin-Sepharoseaffinitychromatographyasdescribedin
MaterialsandMethods.
ThepurityoftherCTGFwasdeterminedbySDS-PAGEandstainingwithCoornassieBlue
K-250.
AsshowninFig1,twopeptidesaredetectedthatco-migratewithbandsdetectedinaparallelimmu-noblotperformedonthesamematerial.Bothofthesepeptidesare
A
B
100
'70'70
75
-e-
)(
><
EE
Q.Q.
o
o
>,>,
.<:.<:
l-
I-
i
-.i:.
M
1020304050600025
[CTGF](ng/mll
THEJOURNALOFINVESTIGATIVEDERMATOLOGY
CTGF,basedonaminoacidcornposmonandpeptidesequenceanalysis.Thedoubletpatternobservedintherecombinantmaterialissimilartothatofthenaturalproductisolatedfromhumanskinfibroblastorendothelialcell-conditionedmedium(Fig1).TheCTGF
is
greaterthan
95'Yc,
purebasedonquantitativescansoftheCoornrnassic-stainedgels.ThebasisforthemultiplebandsappearstobeduetodifferencesinglycosylationasdetectedbydifferencesinreactivitywithconcanavalinA(GrotendorstGR,WilHams5,SegarurnP,unpublishedobservations).MitogenicActivityofCTGFWeinitiallytestedthebiologicactivityoftheCTGFwithNRKfibroblastsusingamitogenicassayperformedonmonolayercultures.CTGFstimulatedaconcentra-tion-dependentincreaseinDNAsynthesisinthemonolayercul-tures,withamaximalstimulationof6-foldovernon-treatedculturesatconcentrationsof
20-50
ngrCTGFper
1111
(Fig
2A).
ThisactivitywasenhancedbythepresenceofsmallamountsofEGF(Fig
2A),
althoughlessthanthatobservedwhenEGFwasaddedwithTGF-f3(Fig2C).BecauseCTGFexhibitedahigh
affinity
forheparin,andbecausepreviousstudieshavedemon-stratedthatheparin-bindinggrowthfactorssuchasacidicFGFcanexhibitmuchhighermitogenicactivityintbepresenceofheparin,weexaminedtheeffectsofheparin
011
CTGFactivity.Additionof
10
/.Lg
heparinpermltothemediumresultedinasignificantincreaseintheamountofmitogenicactivityexhibitedbyCTGF(3-foldovernon=beparin-treated)withnosignificantchangeintheconcentrationsofCTGFrequiredtogivehalf-maximalormaximalstimulationofDNAsynthesis(Fig
2B).
Lastly,wecomparedtheactivityofTGF-/3aloneorincombinationwitheitherEGForCTGF
in
theNIUZmonolayermitogenicassay(Fig2C).TGF-/3aloneexhibitedlittleactivityinonrDNAsynthesisassay.AlargesynergisticstimulationoccurredwithlowconcentrationsofEGF,ashasbeen,previouslyreported(DelarcoandTodaro,1978;Assoian
et
at,
1984).ThepresenceofCTGFstimulatedonlyaslightincreaseintheabilityofTGF-f3toinduceDNAsynthesisinthesecultures(the
arroul
inthefigureindicatesbaselineofCTGFactivitywithnoTGF-/3present).ThesedatasuggestthatatleastsomepartofthesynergisticactionofTGF-f3andEGFissharedbyCTGFandEGF,butthatthereisnosynergisticactivitybetweenTGF-f3andCTGFinthis'Issay.InabilityofCTGFtoStimulateAnchorage-IndependentGrowthorActasaGrowthInhibitorforEpithelialCellsTGF-f3wasoriginallyidentifiedduetoitsabilitytostimulatetheanchorage-independentgrowthofnormalfibroblasts(DelarcoandTodaro,1978).Whileothergrowthfactorsarerequiredforcelldivisionundertheseconditions
(Assoian
e/al,
1984),
110
othergrowthfactorhasbeenfoundtopossessthisuniqueactivity.TGF-/3hasalsobeenshowntoactasaspecificinhibitorofepithelialcells
--+
Heparin(10ug/ml)
C
'7
30
-a-
EGF0.2ng/ml
~
..
><
E
20
--
---CTGF20ng/ml
0..
o
-
,
"4
<:
I-
10
i
-0-
NoAdditions
01'"02
4
-0-
NoAdditions
5075100125
[CTGFJ(ng/mll[TGF-BJ(ng/ml)
Figure2.MitogenicactivityofCTGF.
(A)
MitogenicactivityofCTGFaloneandinthepresenceofEGFfor
NRK
fibroblasts.Cellswereassayedasdescribedin
MaterialsandMethods.
TheindicatedamountsofCTGFwereaddedtothemediaalone
(0)
orinthepresenceofEGF(0.2ngperml,.).
(B)
EffectofheparinonthemitogenicactivityofCTGFon
NRK
cells.CellsWereassayedasin
A.
TheindicatedamountsofCTGFwereaddedalone
(0)
orinthepresenceofheparin(10
peg
per
1111,.)
SynergismofTGF-B1andEGFbutnotCTGFtostimulateDNAsynthesisof
NRK
cellsinmonolayercultures,TheindicatedamountsofpureTGF-/3wereaddedalone
(0),
illthepresenceofCTGF(20ngperml,.)orEGF(0.2ngperml,
0).
ATOIII
pointstobaselineofCTGFstimulation..
Errorbars,
mean:':SD.

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