Hepatic processing of transforming growth
factor beta in the rat. Uptake, metabolism, and
biliary excretion.
R J Coffey Jr, … , H L Moses, N F LaRusso
J Clin Invest.
1987;
80(3)
:750-757.
https://doi.org/10.1172/JCI113130
.
Transforming growth factor beta (TGF beta), a recently discovered polypeptide, modulates
growth of normal and neoplastic cells. Since little is known concerning in vivo disposition of
TGF beta, we performed studies to examine the hepatic processing of biologically active
125I-TGF beta in the rat. After intravenous injection, 125I-TGF beta disappeared from the
plasma with an initial t1/2 of 2.2 min; partial hepatectomy delayed the plasma
disappearance of 125I-TGF beta by 80%. 60 min after intrafemoral injection, 63% of the
recovered label was present in liver and/or bile; by 90 min, most of the label removed by the
liver (83%) had been slowly excreted into bile. Nearly all the label in bile (96%) was soluble
in trichloracetic acid and not immunoprecipitable by specific antiserum. Colchicine and
vinblastine inhibited cumulative biliary excretion of label by 28 and 37%, respectively;
chloroquine and leupeptin each increased the amount of label in bile that was precipitable
by trichloracetic acid and that coeluted with authentic 125I-TGF beta on molecular sieve
chromatography. There was efficient first-pass hepatic extraction of 125I-TGF beta (36%) in
the isolated perfused rat liver, which was inhibited by unlabeled TGF beta (but not by
epidermal growth factor, EGF) and by lectins in a dose-dependent manner; prolonged
fasting also decreased clearance (26%). After fractionation of liver by differential or
isopycnic centrifugation, radiolabel codistributed with […]
Research Article
Find the latest version:
Hepatic
Processing of
Transforming
Growth
Factor
ft
in
the
Rat
Uptake,
Metabolism, and
Biliary
Excretion
R. J.Coffey, Jr., L. J. Kost, R. M.Lyons, H. L. Moses, and N. F. LaRusso
GastroenterologyResearch Unit, Department ofInternalMedicine, andtheDepartment ofCellBiology, MayoMedicalSchool, Clinic
andFoundation, Rochester,Minnesota 55905
Abstract
Introduction
Transforming growth
factor
beta(TGFjO),
arecently
discov-eredpolypeptide,
modulatesgrowth
of normal andneoplastic
cells. Since little is known
concerning
in vivodisposition
ofTGF,#,
weperformed studies
toexamine
thehepatic
process-ingof biologically active '25I-TGF()
in therat. After intrave-nousinjection,
'25I-TGF,&
disappeared
from theplasma
withaninitial
tI/2
of
2.2min;
partial hepatectomy
delayed
theplasma
disappearance
of1251I-TGFI
by 80%.
60min
after intrafemoral injection,63% of
the recovered label was present in liverand/
or
bile; by
90min,
most of the label removedby
the liver(83%)
had been slowly excreted
into
bile.Nearly
all the label inbile
(96%)
was soluble intrichloracetic
acid and notimmunopre-cipitable by
specific
antiserum.Colchicine
andvinblastine
in-hibited cumulativebiliary excretion
of labelby
28 and37%,
respectively; chloroquine and
leupeptin
eachincreased
the amount of label in bile that wasprecipitable
by
trichloraceticacid
andthat coeluted with authentic'25I-TGFB
onmolecular sieve chromatography. There wasefficient
first-pass
hepatic
extraction
of125I-TGFi (36%)
in theisolatedperfused
ratliver,
which
wasinhibited
byunlabeled
TGFft
(but
notbyepidermal
growth
factor, EGF)
and by lectins in adose-dependent
man-ner,prolonged fasting
also decreased clearance(26%). After
fractionation of liver by differential
orisopycnic centrifugation,
radiolabel codistributed with
marker enzymes forlysosomes.
The results
indicate
rapid,
extensive,
inhibitable,
andorgan-selective extraction of
TGFjO
by
theliver.
Afterextraction,
TGF,8
undergoesefficient transhepatic
transport,extensive in-tracellularmetabolism,
andslow but completebiliary
excretionof its metabolites. Liver fractionation
studies andpharmaco-logic manipulations
suggest that these processes areassociatedwith organelles
thatinclude microtubules
andlysosomes.
The data suggest that theliver is
amajor
targettissue
or site of metabolism forbiologically
activeTGFft.
Addressreprintrequests to Dr.LaRusso. Dr. Coffey's present address
isDepartments of Medicine and Cell Biology, Vanderbilt University
School of Medicine,Nashville, TN 37232. Dr. Moses' present address
isDepartment of Cell Biology, Vanderbilt University School of
Medi-cine, Nashville,TN37232.
Receivedfor publication 12 August 1986 and in revised form 10
March 1987.
Transforming growth
factor :(TGF(3)'
is 25 kD homodimer with two 112 amino acid chains linked by disulfide bonds (1).TGFO
causes reversiblemorphologic
transformation andgrowth
insoft agar ofmesenchymally
derivedmouseAKR-2B and rat NRK cells(2, 3).
AKR-2B cellsarestimulatedtodividein monolayer by
theaddition of TGF/3
butonly
afterapro-longed
prereplicative
phase
compared
toother
growth
factorssuch
asepidermal growth
factor(EGF)
(4). Also,
TGF(#
causesthe
early
induction of c-sis followedby
release ofplatelet
de-rived growth factor
(PDGF)-like
material and
theinduction of
PDGF-inducible
genes(e.g., c-fos
c-myc),
butwith
delayed
kinetics
relative
toinduction by
PDGF(5). Thus,
themito-genic effect of
TGF(#
on AKR-2B cells appearstobe indirectthrough
theinduction of c-sis
andsubsequent autocrine
stimu-lation by PDGF-like material.
In contrast toits growth
stimu-latory effects
onmesenchymal cells,
TGF(#
inhibits the
mono-layergrowth of
anumber
of normal
epithelial
cells,
including
rat hepatocytes
(6)
and humankeratinocytes
(7),
aswell asinhibiting the soft
agargrowth of
anumber
of carcinoma cell
lines (8).
Infact,
TGF#
is similar if
notidentical
tothegrowth
inhibitor isolated by Holley and co-workers from medium
conditioned by African
greenmonkey
kidney
cells(9).
TGF(P
appears to
be
animportant
regulator of cell
proliferation
for
which
manycell
types havespecific, high affinity,
membrane receptors(10).
TGFj3
is
ubiquitous; it
has been detectedin
normalliver,
lung,
kidney, submaxillary
gland,
brain,
placenta
andheart
tissue,
aswell
asin embryos (1 1,
12).
Platelets
havebeen
shown to
be
arich
sourceof
TGF(P
and
manylaboratories
purify TGF(1
from outdated
platelets (1).
Themolecule
is
re-leased
from cells in
alatent,
high
molecularweight form;
acti-vation in vitro
canbe
achieved by
extremesof pH
(13)
and by
plasmin,
acell-associated
protease (Lyons et al., unpublishedobservations).
Whether
this
high
molecularweight
form
repre-sents a precursor moleculeand/or
attachment to abinding
protein,
and whetherin vivo conversion
of
latent toactive
form
occurs intheplasma
orelsewhereis presently
unknown. The genefor
TGF1B
has been cloned byDerynck
and co-workersfrom
a humangenomic
library
andfrom
cDNAli-braries derived from
human termplacenta
and the humanfibrosarcoma
line HT 1080(14).
Itistranscribedinto
a2.5-kb1.Abbreviations usedin thispaper: EGF, epidermal growth factor; IPRL, isolated perfused rat liver; ML, mitochondrial lysosomal; PAGE, polyacrylamidegelelectrophoresis; PDGF, platelet-derived
growthfactor;TGF,6,transforming growth factor beta.
750 R. J. Coffey, Jr.,L.J.Kost,R. M.Lyons,H. L.Moses,and N. F.LaRusso
J.Clin.Invest.
© TheAmerican Societyfor Clinical Investigation, Inc.
0021-9738/87/09/0750/08 $2.00
mRNA present in a wide variety of normal andtransformed cells.
Of interest,
mRNA forTGF#
has not been detected in normal liver (14). Indeed, the liver may not synthesizeTGF3,
but may play a role in clearing this polypeptide from the plasma. As there is ample precedent for the hepatic extraction and biliary excretion of a number of circulating macromole-cules (15-17), including growth-active polypeptides such as EGF(18, 19), we examined the hepatic processing of
TGF,6.
Our results strongly suggest that the liver plays a major role inthe
clearance and metabolism of biologically activeTGFj3.
Methods
Peptides and reagents.
TGF6I
was purified from human platelets by the method of Assoianet al.(1) withtheaddition ofafinal purification stepconsisting ofreversephase C-18highperformance liquid chroma-tography(HPLC) witha 45% to60%acetonitrile gradient inwaterwith 0.1% trifluoroacetic acid (10). Thisplatelet-derivedTGFj has been showntobehomogeneousongelelectrophoresis under denaturing and reducing conditions (8).EGFwaspurified from adult malemousesubmaxillary glands by the methodof Savage and Cohen (20); gel electrophoresis under
dena-turing conditions revealedasingle polypeptide band.
Colchicine and leupeptinwerepurchased from Sigma Chemical Company (St.Louis, MO); vinblastinewasfrom EliLilly&Co.
(India-napolis, IN); chloroquine wasfromWinthrop Laboratories (New York); andconcanavalinA andwheat-germagglutininwerefrom
Cal-biochem Corp. (La Jolla,CA).
Preparation ofradiolabeled compounds. Purified TGF6 was ra-dioiodinated with '25I-labeled Bolton-Hunter reagent from Amersham Corp.(Arlington,IL)aspreviouslydescribed(10).Biological activity
was determined by demonstrating growth-stimulating activity for
AKR-2Bcells inasoft agar assay andshowingcompetitionina
ra-dioreceptor assay using AKR-2B (clone84A)cells(10).Specific activ-ity was determinedtobe - 80-100
sCi/Mg.
Purified EGFwasiodinated with Nal25l(Amersham Radiochemi-cals, Arlington Heights, IL) byamodification of the chloramine T
reaction(21). Biologicactivitywasdetermined inamitogenicassay with AKR-2B cells (21). The labeled EGFhadaspecific activityof 100-1 50
MCi//Ag.
Experimentalmodels.Allexperimentswereconducted with male Sprague-Dawleyratsweighingbetween 225 and 320 g. Forin vivo
experiments,weusedratsanesthetized with
intraperitoneal
pentobar-bital (40mg/kg)andpreparedwithbile fistulaeaspreviouslydescribed (22).In someoftheseexperiments,weperformedahepatectomy(70%)
or sham operationandtook serial blood samplesfrom the femoral artery after administration of the labeled
peptide (23).
For in vitroexperiments,weusedanisolatedratliver model
perfused
inanonre-circulatingfashion witha
nonhemoglobin
perfusate
aspreviously
de-scribedindetail(24).Insomeoftheseexperiments,
rats weresubjected
toprolongedfasting (72 h)beforeisolation of livers.
Toassess thedistribution of
'25I-TGF#
withinthe liver cell,weperformedthree types of tissue fractionation studies after
homogeniza-tionof liver with aPotter-Elveheimhomogenizer aspreviously
de-scribed(25).First,toprovideanestimate of theamountof label
asso-ciated withorganelles, we prepareda postnuclear supernatant
con-tainingboth organellesand cell sap(extract or E fraction) by differential centrifugation and subjectedit to ultracentrifugation to
separateahigh-speed pellet (75,000 gX40min)orparticulate fraction fromasupernatant devoid of organelles.In someof theseexperiments, we added adetergent, Triton X-100 (1% vol:vol), to theE fraction beforeultracentrifugation.In asecondsetof experiments,the E frac-tionwasfurther separated into four additional fractions by differential
centrifugationasdescribed (25). These fractionswereenriched in mi-tochondria(heavymitochondrialorMfraction), lysosomes (light
mi-tochondrialorLfraction), endoplasmic reticulum (microsomalorP
fraction),andcytosol(finalsupernatant or S fraction). In a third set of
experiments,weperformed isopycniccentrifugation of MLfractions
onlinearsucrosegradients(10to60%)asdescribed (25).
In separate experiments, we mixed lectins, unlabeled
TGFII
or EGF in theperfusate of isolated livers to provide a steady-stateenvi-ronment toaffectthehepaticextraction of the labeled peptide.
Inanotherseriesofexperimentsinratswithbile fistulae, we
in-jectedcolchicine (5gmol/kg)orvinblastine (10mg/kg)into the femo-ral vein 2 h before administration of labeled peptide. The dose of colchicinewassimilartothat used inassessing the effect of this micro-tubulebindingagentonthehepatictransport of IgA (26),while the dose ofvinblastinewassimilartothatusedin assessing the effect ofthis microtubulebindingagentonthebiliaryexcretion of lysosomal en-zymes(22). Inthoseexperimentsaimed at studying the effects of
lysosomotropicagentsonthemetabolism of
'25I-TGF#,,
weinjectedleupeptin(5 mg)orchloroquine(50 mg/kg)intravenouslyor
intraperi-toneally,respectively,intoratswith bile fistulae 1hbeforeinjectionof labeledpeptide.These dosesweresimilartothose usedby others
de-scribingtheinhibitoryeffect ofleupeptinonthehepatic metabolism of
asialoglycoproteins (27) and theinhibitoryeffect ofchloroquineon
hepatic metabolismof EGF(19).
Characterizationof radiolabel in bilewasperformedbythree
sepa-ratetechniques.TCAprecipitationwasperformedwith 2mlof 10% TCA after additionof 1% BSAto 100 Ml ofbile.Immunoprecipitation
was performed using apolyclonal antibody directed against native
TGFB, which wasraised in rabbits(Keski-Ojaetal., manuscriptin
preparation).Antibody-antigencomplexeswereprecipitatedwith 10% (wt/vol)StaphylococcusA(Pansorbin, Calbiochem)andthe
precipi-tated radioactivity analyzed by polyacrylamide gel electrophoresis (PAGE) using 0.1%
sodium
dodecyl sulfate (SDS) or in a gammacounter.Molecular sievechromatographywasperformedon a
Sepha-dexG-50column(1.0X 117cm)witharunningbuffer of 1.0Macetic
acid,0.05%TritonX-100,and 0.1% BSA. Thevoid and total column volumeswere determined with dextran blueand potassiumiodide, respectively.
Countingoftissue,blood, andbilesamplesfor1251 wasdoneby
gamma scintillation. Statisticalanalysesweredoneusingatwo-tailed
Student'st test.
Results
Hepatic
uptake
of
'25I-TGF,8.
The rate ofdisappearance
ofTGF,3
from
plasma
wasstudied
by
injecting
'25I-TGF(3
into
therat
femoral
vein, taking
serialsamples
ofblood,
anddeter-mining
thedisappearance
of label
from theplasma. Fig.
1 shows that theplasma
disappearance
ofradioactivity
wasrapid
after
systemic
administration
of'25I-TGFtB
(initial
t1/2
of
2.2±0.1
min)
andthat the
disappearance
wasprolonged
nearly
twofold
(P
<0.03)
after 70%hepatectomy (initial t1/2
of4.0±0.4
min).
Thesedeterminations
weremade aftera5-min-ute
equilibration
period following
hepatectomy.
The
uptake
of'25I-TGFf3
in variousorganswasalsodeter-mined.
Fig.
2shows that 43% of theinjected
radioactivity
wasfound
in liver and bile 60 min afterintravenousadministra-tion
of'25I-TGFfl;
this amount was twofoldgreater
than allother organs combined.The
hepatic
extraction aftersystemic
administration
of'25I-TGF#
and'25I-EGF
wasthencompared.
Fig.
3
again shows that themajority
of'25I-TGFO
radioactivity
(63%)
wasfound in liver andbile with lesser amounts inkid-ney
(20%)
and blood(15%);
incontrast toFig.
2,
the dataareexpressed
asthepercentage
ofrecoveredradioactivity.
Incom-parison
to'25I-TGFfl,
aftersystemic
administration of'25I-EGF,
alesser
percentageof
recoveredradioactivity (41
%)
wasfound
in liver andbile
and agreater percentage
in other'25I-2- 80
:, 60 0o
c
40
ID
-E 20
c~
0
100
11 H-- patectomy
II ---- Sham operated
-
-- --o -a<
10 15
Time, min
Figure 1.Plasmadisappearanceof
'25I-TGF#
inbile fistularatsafter shamoperationor70% hepatectomy. Results(mean±SEM)arefrom threeratsineach group.TGFB
radioactivity
and31% ofinjected '25I-EGF radioactivity
in all the
organs examined.Fig.
4 comparesthe
hepatic extraction (hepatic uptake
andbiliary
excretion)
of
radioactivity
after administration of
ra-diolabeled
TGF#
and
EGF in both theisolated
perfused
ratliver
(IPRL)model and in the
intact animal after either
intra-portal
orintrafemoral
delivery.
Results
fromthe
isolated
per-fused model demonstrate
agreater(P
<0.001)
first-pass
he-patic
clearance forradiolabeted
EGF than for TGFj(82.5±1.9%
vs.36±4.3%).
Theextraction
percentage of 1251TGFft
in the
IPRL was constant over the rangeof 6.4
to128
pM.
Invivo,
little
difference
exists in the
amountof'25I-TGF(#
extracted
by the liver after portal vein
versusfemoral
veininjection (52.5±1.5%
vs.49.6±0.5%).
In contrast, more(P
<0.001)
25I-EGF is extracted
by
the liver after
intraportal
injection compared
tointrafemoral
injection
(70±0.7%
vs.44.8±4.
1%).
Modulation of
hepatic
extraction.
Wedetermined
theeffect
of
fasting
onthe
uptake
process inorder
toprovide
initial
insight into
thefactors that regulate the hepatic extraction of
TGFft.
Wefasted
ratsfrom solid
food for
72h,
preparediso-lated
livers from
fasted
andnonfasted
controls (n= 5), and measuredfirst-pass extraction
of'25I-TGF#.
Asignificant
de-crease
(P
<0.03)
infirst-pass
clearance
occurredafter
pro-longed
fasting, from 35.8±2.4%
to26.4±2.8% of
theinjected
dose.
To assess
the
specificity of hepatic clearance of
125I-TGF#,
we
tested the
effect of unlabeled
TGF,#
andof
unlabeled EGF onthe
first-pass
extraction of
'25I-TGFj#
in
theIPRL.
As60
r-)
0
10
4-C._
._
0 0
40
I-20 F
n
Liver- Kidney- Blood Others*
bile urine
Figure 2.Organdistribution of radioactivity 60 minutes after intrafe-moralinjection of
125I-TGF#
in bilefistula rats. Results(mean±SEM) are from three rats and are expressed as percent
in-jectedradioactivity. *Spleen,adrenals,stomach, and duodenum.
so
0
._
Tc:
~0
0 0
._
ED
0 80
60
40
0
Liver- Kidney- Blood Others* bile urine
Figure 3. Organ distribution ofradioactivity60 minafter
intrafemo-raladministrationof'25I-TGF3 (o) and
'25I-EGF
(i) in bile fistularats.Results(mean±SEM)arefromthree ratsin eachgroupandare
expressedaspercentrecoveredradioactivity: *Spleen, adrenals,
stom-ach,and duodenum.
shown in Fig. 5, unlabeled
TGFP,
added to the perfusate in concentrations ranging from 1 to 20nM,
inhibited hepatic extraction of'25I-TGF,0
in adose-dependent
mannerup to 73.9% of control. In contrast, cold EGF, at a concentration of 20 nM(an amountcomparable
tothe concentration of coldTGFft
that achieved maximal inhibition in these experiments) had no effect onfirst-pass hepatic
extraction of'25I-TGFf.
Lectins with different
carbohydrate specificities
were then tested to determine their effects on hepatic extraction ofTGFP
(Fig. 5). Concanavalin
A,
which binds to mannose residues, yielded first-pass hepatic extractions of 34.9±3.3%, 25.3+2.7%,24.4+2.3%,
17.7±2.6%, and 13.4±2.3% of thein-jected
dose whenpremixed
into theperfusate
atconcentra-tions of 5, 50, 100, 250, and 500
,ug/ml,
respectively.
Wheat-germ agglutinin, which binds toN-acetylglucosamine
and si-alic acidresidues,
also decreasedhepatic
extraction.Concentrations
of0.5,
5, and 504g/ml
resulted
in extraction of31.7+4.7%,
27.3±1.2%,
and 19.4±2.6% oftheinjected dose,
respectively.
Biliary
excretionof25Ifrom
TGF#.
Weexamined
thebili-ary excretion of radiolabel in order to determine the fate of
In vitro
100 _
>. 80
Isolatoed
0
0o
60-0
1..40
-0
0)
20
Isolated
perfused
ratliver
In vivo Portal
vein injection
Bilefistularat
Femoral vein injection
Mean-±SEM;n=3 in each group
Figure 4.Hepatic extraction of radioactivity at 20 min after adminis-tration of
'25I-TGFft
(0)and'25I-EGF(i)
invitro (isolated perfusedratliver model) and in vivo (via portal and femoral vein bile fistula rats).Inthe invitroexperiments,results represent first-pass hepatic clearance. Results (mean±SEM)areforatleast threeratsin each group andareexpressed as percentageofinjected radioactivity.
752 R.J.
Coffey,
Jr.,L.J.Kost,R. M.Lyons,H. L. Moses, andN.F. LaRusso30
I
0
60 1
0 O Concanavalin A 0 A Wheat germagglutinin 40 0 TGFp
0EGF
o
f..,
0 1 10 100 1.000
Concentration of lectins,
ALg/
mlConcentration of unlabeled
TGFq,
nMFigure5.Effectsoflectins, unlabeledTGF#,andunlabeled EGF, on
thefirst-pass hepatic extractionof
'25I-TGF#
in theisolated perfused ratliver.Results are expressed as percent of control and are meandata(±SEM)for threerats eachwithconcanavalinA or wheat germ
agglutininand meandata for two rats each with unlabeled
TGF#
andunlabeled EGF.
1251I-TGF#
taken
up by theliver.
Fig. 6 shows thedistribution
of radioactivity in liver
andbile collected over three separatetime periods after
systemicadministration
of1251I-TGF,8.
Theoverall
hepatic extraction
(hepatic uptake and biliaryexcre-tion)
at20,
60, and 90min
was similar (49.9±0.2%,45.3±1.0%,
and48.1±2.0%,respectively).
However,increas-ing
amounts of radioactivity appeared in bile over time, com-prising 7.0±1.2% of hepatic extraction at 20min,
68.2±0.6%
at
60 min, 84.0±0.9%
at 90min.
The
kinetics of
appearance of radioactivity from'251I-TGF,6
in bile
wasexamined. Fig. 6 shows that after systemicadmin-istration of
1251-TGF,8,
theappearance of radioactivity in bileincreased rapidly,
peaked at 45min,
and gradually declined over90 min.
After 60 min, 20.1±6.4%
of theinjected
radioac-tivity
appearsin bile (Fig.
7). This issignificantly
greater (P<
0.05)
thanthe cumulative
amount of the percentage ofin-jected '25I-EGF,
which
appearsin bile
(5.1±0.3%) over the sametime
(Fig. 7).
Characterization
of
125lfrom
TGFj6
in
bile. To define the
nature
of
theradiolabel in bile after in
vivo administration ofKinetics ofappearance
F
I
0 20 40 60 80 Time,min
Distribution between liver and bile
100
-EM Biliary
80 _ excretion
r
Liver60 _ uptake
40
-
20-20 60 90 Time afterinjection,
min
n-3 in eachexperiment
Figure6.Kinetics ofbiliary excretion of radioactivity after
intrafemo-ralinjectionof '25I-TGF#8tobilefistularats.Results(mean±SEM)
arefor threerats.Cumulativehepatic uptakeandbiliaryexcretionof
radioactivity expressedaspercentageofinjected radioactivity for
three separate timeperiodsafterintrafemoralinjection of'25I-TGF(3
tobilefistula rats.Results(mean)arefrom threeratsateach time.
W~.
E v.
0
2D
X4- ZD
_
_
0-5D
20
10
0
o TGFp
o EGF
20 30
Time,min
40 50 60
n=3
Figure 7. Cumulative biliary excretion of radioactivity after intrafem-oralinjection of
T251-TGF#
or'25I-EGFin bile fistula rats. Results(mean±SEM)areforthree rats in each group.
'25I-TGF#,
weexamined bile containing 1251 by TCA
precipita-tion,
immunoprecipitation,
and molecular sieve
chromatogra-phy. When bile collected serially after
systemic administration
of
'25I-TGF#
wasexposed
toTCA,
amodest
amount wasTCA-precipitable
(27.7±0.8%
at10
min); this
percentagede-clined
rapidly (6.4±0.6%
at 25min)
andremained
<4% forthe entire 90-min collection
period.
In contrast, 94.9±0.7% ofradioactivity
wasTCA-precipitable
when
125I-TGF,3
wasadded
directly
tobile
("spiked" bile)
collected from
rats(n
=3)
to whom
'25I-TGF#
had not beenadministered.
Only
28.3±0.5% of
radioactivity
in
spiked
bile could
beimmuno-precipitated
using specific
polyclonal
antiserum
toTGF#,
pos-sibly due
tothe
specificity
and
affinity
ofTGF,
antibodies
and/or
thecomplex
natureof bile.
Analysis
ofimmunopreci-pitated material after
electrophoresis
on 7.5-15%polyacryl-amide
gradient
gels
containing
0.1% SDS
followedby
autora-diography demonstrated
asingle
band
thatmigrated
identi-cally to
'25I-TGFj3
(data
notshown).
In contrast, we wereunable
todemonstrate
anyimmunoprecipitable radioactivity
in
bile collected after
systemic
administration of
125I-TGFfi.
Finally, Fig. 8 shows results of
analysis
of
radioactivity
inbile
using molecular sieve
chromatography.
Bile that
had beenspiked
with
'25I-TGF,8
exhibited
asingle
peak
ofradioactivity
eluting
nearthe void volume
presumably representing
intact10
-° 8_ l
to
4I
0
0
0 20 40 60 80 100 120
Fraction number
Figure 8.Molecular sievechromatographyof
'25I-TGF3
inbileon Sephadex G50.Chromatographywasperformed
on ratbiletowhich'25I-TGFj
hadbeenaddeddirectly(-)
orthathad beencollected from bile fistulae for 30minaftertheintrafemoral administration of125I-TGFj#
toratspretreatedwith saline ----)orthelysosomotropic
agent,chloroquine
(-).
6
*
*01
-0.=
C o
._
4
2
TGF#.
Incontrast, aftersystemic
administration of'25I-TGF#,
molecular
sievechromatography demonstrated a single peakof radioactivity eluting just
before the total volume of thecolumn, representing
smaller molecularweight, degradation
products
of
'25I-TGF,3.
These
combined studies(TCA
precipi-tation, immunoprecipiprecipi-tation, and molecular sieve chromatog-raphy) suggest
that
after systemicinjection,
theTGF,3
that appears inbile
islargely degraded.
Effects ofpharmacologicagentson
biliary
excretionof'25I
from
TGF#.
Since
microtubulebinding
agents andlysosomo-tropic agents are known to affect the
hepatic
transport andbiliary
excretion of a number of macromolecules, their effect onthe
hepatic processing
of1251I-TGF(3
wasexamined. Table Isummarizes results of
experiments with administration ofmi-crotubule
binding
agents(vinblastine, colchicine)
andlysoso-motropic
agents(chloroquine, leupeptin)
torats with bilefis-tulae.
There wasnosignificant change
inhepatic
extraction ofradioactivity (hepatic uptake
and
biliary
excretion)
at60minafter administration of
'25I-TGFfl
with both
groups ofagents. However,both
groupsof
agentssignificantly
reduced the
ra-dioactivity
in bileoverthis same time interval with acorre-sponding increase in radioactivity
remaining
within the
liver
(Table
I).
Ofparticular
note,pretreatmentwith
chloroquine and,
to a lesser extent,leupeptin
increased the percentageof
radioactiv-ity in bile that
wasTCA-precipitable (Table I).
Although only0.4% of
radioactivity
in bile
wasimmunoprecipitable after
pretreatment
with
chloroquine, this immunoprecipitable
ma-terial
migrated
as asingle
band
identical
toauthentic
'25I-TGFfl
onSDS-PAGE followed
byautoradiography
(data notshown). Also,
chloroquine
pretreatmentof
ratsgiven
12511
TGF#l
systemically altered
theelution profile ofradioactivity
in
bile subjected
to molecularsieve
chromatography(Fig. 8); twopeaks,
onecoeluting with intact
'25I-TGF,3
near the voidvolume,
andthe
othereluting
near the total volume(represent-ing
smaller molecularweight degradation
products) became apparent.Intracellular
compartmentalization of
'25I-TGFO.
Weex-amined the distribution
of125I between
ahigh-speed
pellet and a supernatantof homogenized
ratliver
20min afterintrafem-oral
administration
of125I-TGFfl
toprovide initial insight
intothe
subcellular
localization of
'25I-TGF#
within the liver (TableII).
Intheseexperiments
the majority of1251
wasasso-ciated with
theparticulate fraction. After
exposureof
thepost-nuclear
supernatant to Triton X-100, approximatelytwo-thirds of
theradioactivity
previously associated with thepartic-ulate
fraction
wasreleased into
the supernatant.To
extend
ourinitial studies
thatsuggested
theassociationof
'25I-TGFf
withspecific organelles,
we nextdetermined thesubcellular
distribution pattern of'25I-TGFfi
among liverfrac-tions
enriched in variousorganelles
which werepreparedby
differential centrifugation.
As shown inFig. 9,
whichcom-pared
distribution
patterns of 125I and marker enzymes for variousorganelles, the distribution
ofl251
in ratliver 20minafter intrafemoral
injection
of'25I-TGF#
wasmostsimilartothe
distribution
ofN-acetyl-ft-glucosaminidase,
alysosomal
enzyme,
and
different from the
distribution patterns foren-zymes
associated
with otherorganelles. InFig. 10,
weplot
the distribution of1251
and marker enzymes for lysosomes andmitochondria after density equilibration
in a sucrosegradient
of
afraction enriched
inthese
twoorganelles (i.e.,
MLfrac-tion)
under
two circumstances:first,
125I-TGFft
wasinjected
into
rats20
minbefore the preparation of the
MLfraction,
which
wasthen placed
onthe
gradient; second, '25I-TGF(3
wasadded
directly
tothe
MLfraction
prior toplacing
thefraction
on
the
gradient.
When'25I-TGFj#
wasinjected
into the rat andextracted by
the liver beforepreparation
of theML
fraction,
the
radioactivity entered
thegradient
anddisplayed
adistribu-tion
patternsimilar
tothose for
N-acetyl-ft-glucosaminidase,
alysosomal
enzyme,and malate
dehydrogenase,
a markeren-zyme
for mitochondria (Fig. 10).
In contrast, whenlabeled
TGFft
wasadded
directly
tothe
MLfraction,
the
radioactivity
remained
attopof the
gradient (Fig. 10).
Discussion
We
havedemonstrated that
biologically active,
125I-TGFfl
is
taken
upby the liver (36%
first-pass hepatic
clearance) in
arelatively organ-specific
manner.This
efficient
clearance is
decreased
by
fasting
and
inhibited by lectins
andby
TGF(#
in
aconcentration-dependent
manner;it is
notaffected by EGF.
After
extraction,
radioactivity
is
excretedinto bile
slowlybut
efficiently, with
80%
of the extracted
radioactivity
presentin
bile after 90 min;
virtually
allof the
radioactivity
in
bile is
TCA-soluble,
notprecipitable with specific antiserum
toTGF#,
and
demonstrates
anelution
profile
bygel
filtration
different from that of authentic
'25_-TGF(3.
All of these
obser-vations
suggestextensive metabolism of
the peptide. The amountof radioactivity excreted into bile is reduced by
mi-crotubule
binding
and
lysosomotropic
agents, anobservation
compatible
withvesicular transport.
Themajority
of theradio-activity in the liver associates with
aparticulate fraction
and canbe
released by
exposure to adetergent,
anobservation
alsocompatible
with
sequestration
of label in vesicles.
Lysosomes TableI.Effect of
PharmacologicAgents ontheHepaticExtraction, Biliary Excretion, and Metabolism of'25ITGFt
Percent hepatic Percentradioactivity Percentradioactivity Percenttrichloroaceticacid-precipitable
extraction in bile in liver radioactivity in bile
Control 45.09 (±1.86) 30.84(±1.70) 14.25 (±0.36) 7.43(±1.68)
Vinblastine 41.14(±0.91) 19.41(±2.39)* 21.73 (±1.49)* 8.28(±0.95) Colchicine 43.82(±0.27) 22.19(±1.14)* 21.62(±1.40)* 7.39 (±0.66)
Chloroquine 39.41(±3.68) 19.01 (±0.93)* 20.40(±2.77) 15.36(±1.50)* Leupeptin 42.52 (±0.39) 15.45(±0.21)* 27.07 (±0.59)* 13.37 (±2.30)
tData
representmean±SEM
from at least three rats with bile fistulae in each group and are expressed as a percentage of the injected dose at 60 minafterinjection. Statistical analysis was performed by a two-tailed Student's t test.All
comparisons were made relative to thecorrespond-ingcontrol data and P values.0.02 are indicated by asterisks.
TableII. PercentageofRadiolabel Recovered
inSupernatants and Pellets ofHomogenized Rat Liver inAbsence andPresenceofDetergent
Distribution ofradioactivity Distribution ofradioactivity
in absence ofdetergent in presence ofdetergent
Supernatant 40.3 (±6.3) 79.5(±3.0)
Pellet 59.7 (±6.3) 20.5(±3.0)
'251-TGFflwas injectedinto the femoral vein of rats (n=3) and the
liverremoved 20minlater. The liver was homogenized and the amountof radioactivityin the supernatant and pellet after
ultracen-trifugation determined.In separate experiments (n=3), Triton
X-100wasadded to thehomogenatebefore centrifugation. Data are
mean±SEM.
appear toconstitute one subset of vesicles that sequester
1251.
TGFft,
as suggested by the findings that lysosomotropic agents inhibit the degradation of this molecule during its transhepatic transport and that radioactivity and marker enzymes for lyso-somescodistribute after
tissue fractionation and differential orisopycnic
centrifugation. Overall, we interpret these results asindicating
that the liver is the major organ involved in thehandling of
biologically activeTGF,6.
Because the hepatic processing of another growth active
polypeptide, EGF,
had been extensively studied(18,
19), wecn
2 0
6
2
0
MDH Microsomal
esterase
MLP S
LDH
u 0 20 40 60 80 100
Cumulative protein,%
Figure 9. Distributionpatternsof constituents after fractionation ofa
homogenate ofratliver by differentialcentrifugation 20minafter in-trafemoralinjection
'25I-TGF#.
Fractions enriched in nuclei (N), mi-tochondria (M),lysosomes(L), microsomes(P),andcytosol (S)areplotted from lefttoright in order of theaveragecoefficient of sedi-mentation oftheir subcellularcomponents.Each fraction is
repre-sentedseparatelyontheordinate by the relativespecific activityof
theconstituent (percentage of totalactivity recovered/percentageof totalproteinrecovered)andcumulativelyontheabscissa by its per-centageprotein. ,-NAG (N-acetyl-,B-glucosaminidase), a
lysosomal
enzyme; APDI
(alkaline
phosphodiesteraseI),aplasmamembraneenzyme; MDH(malate dehydrogenase),alargely mitochondrial
en-zyme.Esterase,whenmeasuredusingO-nitrophenylacetate,isa mi-crosomalenzyme; LDH(lactate dehydrogenase),acytosolicenzyme.
Recovery ofenzymesis calculated bysummingtheactivities in the five fractions foragivenenzyme anddividing thisnumberbythe totalactivityfor thesameenzymein the homogenate. Results
repre-sent meandatafrom threeexperiments;recoveryof constituents averaged 96% andranged from 87to102%ofstartingmaterial.
-^
a-NG
125I-TGFq
processed
by
liver---MDH I
. 1251-!
% 201L
v 60 r
1251-TGF
added to MLfraction
IL
40
[4
~~~~,j
20
-1.03 1.09 1.15 1.21 1.27
Density
Figure10. Distributionpatternsof constituents after isopycnic
cen-trifugation ofanMLfraction ofratliverthrough a linear sucrose
gradient (10-60%)20 min after intrafemoral administration of 125I-TGF#(top)orafter the addition of
'251-TGFj#
directly to afractionprepared fromratliver (bottom).The averagefrequency of the com-ponentsis calculatedfor each fractionaspreviously described (24)
a-NAG,
N-acetyl-0-glucosaminidase,
alysosomalenzyme;MDH,malate
dehydrogenase,
alargely
mitochondrialenzyme.compared the hepatic handling of
TGF#
to that of EGF. In vitro, first-pass hepatic clearance of EGF is greater than forTGF#.
Invivo, there
is asignificant
decrease in the hepatic extraction of EGF whenit is administeredsystemically rather than intotheportal vein; such a decrease does not occur with TGF#. These observations suggest that, while the liver has ahigh capacity
for extracting EGF, other organs, if presented with EGF first, will also avidly take up this polypeptide. In contrast, while the liver has a lower capacity for extractingTGFf3,
theuptake
is more liverspecific
inthat otherorgans
do notavidly
extract TGF#. Differences also exist in the biliaryexcretion
ofTGF,6
and EGF.Quantitatively,
the liver excretesTGFf
moreefficiently into
bile than EGF. Nearly four times asmuch
radioactivity
from labeledTGF#
is excreted into bile ascompared
with labeled EGF during a comparable timepe-riod. This difference
may also relate in part to the differentlabeling
procedures weemployedfor
EGF(i.e., chloramine
Treaction)
andTGF3(i.e.,
Bolton-Hunterreagent).
When1251
isincorporated
into a molecule by the former, but not by the latter technique, the 1251 appears to be sensitive to cellulariodinases (28); therefore,
'25I may diffuse out of the cell and notbe
excretedinto
bile.Fasting caused
asignificant
reduction in thehepatic
clear-ance of125I-TGFj3,
anobservationanalogous
tothedecreasedbinding
of'25I-EGF toliverplasma
membranes from fastedrats(29). This observation suggests that hepatic extraction of
TGFj#
is
regulatable
andcanrespond
tophysiologic
stimuli.Also, the concentration-dependent
inhibitory
effect of lectins onthehepatic
extraction
ofTGF,
is consistent with thepossi-bility
that aglycoprotein
is involved in theuptake
process. Along theselines, inhibition
of insulinbinding
toitsglycopro-tein
receptoronhepatocytes
has beenobserved with compara-bleconcentrations
of concanavalinA(30). Moreover,
resultsusing affinity labeling
techniques
suggest that the receptorforTGFB is
aglycoprotein (31)
andhepatocytes
inculturehavebeen demonstrated
to havespecific
receptors
forTGFB
(6).
Finally, unlabeled TGF,,
but not coldEGF,
inhibited thefirst-pass
extraction of'25I-TGF(#
by
the IPRL.Allthese data areconsistent with thepossibility
that thehepatic
extraction ofvia a glycoprotein receptor on thesinusoidalpole ofthe hepa-tocyte, areceptor that isdifferent from the
receptor
for EGF.Inthis context, our data also suggestthat the
transhepatic
transport of TGF occurs across the
hepatocyte
rather thanbetween cells,adistinction thatisimportantsince a paracellu-lar route for the biliary excretionof some
circulating
polypep-tides has been proposed (32).The slow kinetics of
biliary
ex-cretion and the extensive metabolism of theligand are both
consistent with a transcellularrouteof transport.In
addition,
preliminary data from our laboratory
using
light microscopic
autoradiography and isolated liver cells suggestthat
hepato-cytes but not
nonparenchymal
cells(e.g.,
Kupffer
cells andendothelial cells)cantakeup'25I-TGF,3.
Significant inhibition of biliary excretion of
TGF,3 by
mi-crotubule binding agents(vinblastine,
colchicine)
wasdemon-strated,
implying involvement of
microtubules
andvesicles
in the transportand
excretory processes. Theassociation
of125I1
TGF,#
with
aparticulate
fraction of
aliver homogenate after
high-speed centrifugation, and the release ofthe label from this fraction after exposure to a
detergent,
arealsocompatible
with sequestration of125I-TGF,3
inintracellularvesicles(33).
Lyso-somotropic agents
(chloroquine,
leupeptin)
inhibiteddegrada-tion of
'25I-TGF(B,
asevidencedby
the presence in bile of(a)
significantly
moreTCA-precipitable
radioactivity,
(b)
immun-oprecipitable
radioactivity,
and(c)
apeak of
radioactivity
co-eluting with authentic
'251I-TGFj3
onmolecular
sieve
chroma-tography
afteradministration of
chloroquine.
This
observa-tion suggests thatlysosomes
are onesubclass
of vesicles
involved in the hepatic processing of
TGF,#.
Additional
sup-portfor
therole of
hepatocyte lysosomes in the intracellular
metabolism and biliary secretion of TGFB
comesfrom
our studiesdemonstrating
codistribution of'25I-TGFB
with
marker
enzymesfor lysosomes after differential
orisopycnic
centrifugation.
Wehave
previously described the existence of
alysosome-to-bile hepatic
excretorypathway
that may be afinal
common
pathway
whereby the
hepatocyte disassembles
somemacromolecules
andreleases
into
bile
someend-products of
partial
orcomplete lysosomal hydrolysis (34-36). Based
onthe
data presented here, this pathway would
appear toconstitute
the most
likely
routefor
thetranshepatic
transport andbiliary
excretion of TGF,. Experimental approaches other
thanthe
biochemical
one takenin these experiments, such
aselectron
microscopic autoradiography,
would beuseful in further
de-fining the pathway of transhepatic
transport ofTGFfl.
The
physiological
significance of
ourobservations
remains tobe
determined.
Assuggested
earlier,
thedata are consistentwith the
liver being either
amajor
target organ and/or a site ofmetabolism of biologically active
TGF,3.
TGFP
has been shown toinhibit
EGF-stimulated
DNAsynthesis of
normal rathepatocytes
(6). It isprovocative
toattribute control of hepa-tocytegrowth
anddifferentiation
tothe modulating influencesof stimulatory
(e.g., EGF) and inhibitory (e.g.,TGFj#)
growthfactors
(37). Also, serum containsdetectable
amounts ofTGF,#
(80-200
ng/ml) that is derived from platelets (38). Cur-rentdata suggest that the TGFB released by platelets in vivo is in a latent form of higher molecular weight than biologicallyactive
TFG,3
(13).
While it is
knownthat this latent form ofTGFft
can beactivated
invitro by extremes of pH(13)
andplasmin (Lyons
etal., unpublishedobservations),
themecha-nism by which
latentTGF#
isactivated in vivo is unclear. Onepossibility
is that activation
occurs byproteolytic
cleavageei-ther in
theplasma
compartmentor atsites of
vascularinjury
after
releaseof latent
TGFB from platelets. Removal of the resulting biologically active form ofTGF,#
from plasma byefficient
hepatic clearance and subsequent lysosomaldegrada-tion
would then be importanttopreventunwantedbiologicaleffects
ofthe peptide
atdistant sites. The data presented here, coupled with information regarding low plasma levels ofTGFjO,
suggestthatthe liver may play a major role inregu-lating
levelsof
circulating, biologically activeTGFIO.
Acknowledgments
The authorswould like to thank Drs.Gregory Gores and Brent Myers forreviewing themanuscriptandMarilyn Breyer,VelWoyczikand RebeccaKoransky fortypingit.
This work was supported by grants AM-2403 1,CA-42749,
CA-42750, and CA-42572 from theNational Institutes of Health and by the MayoFoundation.
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