Cellular distribution of transforming growth factor beta 1 and procollagen types I, III, and IV transcripts in carbon tetrachloride induced rat liver fibrosis

Cellular distribution of transforming growth

factor-beta 1 and procollagen types I, III, and IV

transcripts in carbon tetrachloride-induced rat

liver fibrosis.

H Nakatsukasa, … , E Marsden, S S Thorgeirsson

J Clin Invest.

1990;

85(6)

:1833-1843.

https://doi.org/10.1172/JCI114643

.

The cellular distribution and temporal expression of transcripts from transforming growth

factor-beta 1 (TGF-beta 1) and procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) genes

were studied in carbon tetrachloride (CCl4)-induced rat liver fibrosis by using in situ

hybridization technique. During the fibrotic process, TGF-beta 1 and procollagen genes

were similarly and predominantly expressed in Desmin-positive perisinusoidal cells (e.g.,

fat-storing cells and myofibroblasts) and fibroblasts and their expression continued to be

higher than those observed in control rats. These transcripts were also observed in

inflammatory cells mainly granulocytes and macrophage-like cells at the early stages of

liver fibrosis. The production of extracellular matrix along small blood vessels and fibrous

septa coincided with the expression of these genes. Expression of TGF-beta 1 and

procollagen genes were not detected in hepatocytes throughout the experiment. No

significant differences in cellular distribution or time course of gene expression among

procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) were observed. Desmin-positive

perisinusoidal cells and fibroblasts appeared to play the principal role in synthesis of

collagens in CCl4-induced hepatic fibrosis. The simultaneous expression of TGF-beta 1

and procollagen genes in mesenchymal cells, including Desmin-positive perisinusoidal

cells, during hepatic fibrosis suggests the possibility that TGF-beta 1 may have an important

role in the production of fibrosis.

Research Article

Cellular Distribution of Transforming Growth

Factor-fil

and

Procollagen

Types

1,

Ill,

and IV Transcripts

in

Carbon

Tetrachloride-induced

Rat

Liver

Fibrosis

HarushigeNakatsukasa, PeterNagy, RitvaP. Evarts, Chu-Chieh Hsia,Elizabeth Marsden, and Snorri S.Thorgeirsson

Laboratory ofExperimental Carcinogenesis, NationalCancer Institute, NationalInstitutesofHealth, Bethesda, Maryland 20892

Abstract

The cellular distribution and temporal expression of tran-scripts from transforming growth

_{factor-,l (TGF-,f1)}

and procollagen al(I), al(III), and al(IV) genes were studied in carbontetrachloride

(CC14)-induced

rat liverfibrosis by using in situ hybridization technique. During the fibrotic process,

TGF-ft1

and procollagen genes were similarly and predomi-nantly expressed inDesmin-positive perisinusoidal cells (e.g., fat-storing cells and myofibroblasts) and fibroblasts and their expression continued to be higher than those observed in con-trol rats.Thesetranscripts were also observed in inflammatory cells mainly granulocytes and macrophage-like cells at the early stages

of

liver

fibrosis.

Theproduction of extracellular matrix along small blood vessels and fibrous septa coincided with the expression of these genes. Expression of

_TGF-,81

and procollagen genes were not detected in hepatocytes throughout the

experiment.

No

significant

differences in cellular

distribu-tion

or time course of gene expressions among procollagen

al(I),

al(III),

and

al(IV)

were observed. Desmin-positive

perisinusoidal

cells and

fibroblasts

appeared to play the

prin-cipal

role in

synthesis of

collagens in

CC14-induced

hepatic

fibrosis.

Thesimultaneous expression of

TGF-ftl

and procol-lagen genes in mesenchymal cells, including Desmin-positive

perisinusoidal

cells,

during hepatic fibrosis

suggests the

possi-bility

that

TGF-,B1

may havean

important

role in the

produc-tion

of fibrosis.

(J. Clin. Invest. 1990.85:1833-1843.)

TGF-f

1

-

collagens

*

liver fibrosis

*

fat-storing

cell * in

situ

hybridization

Introduction

Hepatic fibrosis is frequently

observed

after

severe

liver

dam-age,and

is

alsoseen

in

connection

with

chronic liver diseases

(1). Fibrogenesis is

one

of

the most

important

process

in

the

development of liver cirrhosis.

The

extracellular matrix of

the

liver

is mainly composed

of

collagens, noncollagenous

glyco-proteins,

and

proteoglycans

(2). The collagens are the major

glycoproteins

in the extracellular

matrix,

and are greatly

in-creased

in

the

cirrhotic liver (2). Chronic stimulation of

rele-vant

cells

toproduce

extracellular matrix

components

is

there-fore

a

critical factor in the cirrhotic

process.

Furthermore, the

extent

of fibrogenesis

correlates well with

hepatic injury (3).

Recurrent

hepatic necrosis, inflammation,

and

regenerative

A preliminary report of this work appeared in 1988. (Hepatology.

8:1303).

AddressreprintrequeststoDr.Thorgeirsson, National Cancer

In-stitute, Building37, Room 3C28, Bethesda,MD20892.

Receivedfor publication8 June 1989 andinrevisedform5 Febru-ary 1990.

The Journalof ClinicalInvestigation,Inc.

Volume85,June1990,1833-1843

process of the liver or some

combination of

these are also

strongly related

to the

development of liver

cirrhosis.

How-ever,

the

factor(s) which initiate(s)

the

gradual

progression of

cirrhosis is/are

unknown.

Recently,

transforming

growth

factor-3 (TGF-,B)'

hasbeen

found

toregulate the

production and degradation of certain

extracellular

matrix

components

(4, 5).

The

TGF-,B family

has

multifunctional action

on

fibroblasts

and other cell types

(6).

TGF-,B1

increases

the

expression of

collagen and

fibronectin

as well as

increases

the

incorporation of

these componentsinto the

matrix

in

cultured

and

transformed cells (4).

In

addition,

TGF-j31

inhibits

collagenase

expression,

and also enhances the

expression

and

secretion

of tissue inhibitor of

metalloprotein-ases together

with certain

growth

factors

(5).

Therefore,

TGF-(31

may,under

certain conditions, disturb the balance of

synthesis

and

degradation of collagens

and cause

accumula-tion of extracellular matrix.

Since

many

cell

types

in the

liver,

such

as

perisinusoidal

cells

(e.g.,

fat-storing

cells,

myofibroblasts,

etc.) (7-9),

hepato-cytes

(10-12),

and

endothelial

cells

(13, 14), produce

some

components of extracellular

matrix,

the

identity

of the

specific

cell

type(s)

that

produce collagens

in

hepatic

fibrosis still

re-mains

controversial. Increased

procollagen

mRNA

levels

were

found in

therat

liver

treated with CC14 (15)

or

with

dimethyl-nitrosamine

(16)

as

well

asin

fibroblasts stimulated by

afactor

that

is

isolated from thioacetamide-induced

fibrotic ratliver (17). However, the cellular distribution andextent ofcollagen gene

expression

and

its time

course in

liver fibrosis

are

un-known.

The use

of histochemical

and

immunohistochemical

methods

provides

a

powerful

technique for

identifying

the components

of the extracellular

matrix.

However,

duetoboth the

multicellular

nature

of

the

liver

and the fact that both

parenchymal

and

nonparenchymal

liver cellsare

thought

tobe

capable of

producing

extracellular

matrix,

this

technique

can-not

reliably

identify

the cell

type(s)

that

is

predominant

in

synthesizing

the components

of

the

extracellular matrix

during

fibr~ogenesis.

The in

situ

hybridization

method isatpresent the

most accurate

procedure for

evaluating

the cellular

distribu-tion of

gene

expression

invivo. This method does not, how-ever,

address the role of

posttranslational

regulation

or

further

processing of

proteins

suchas

intracellular

degradation.

Nev-ertheless,

the in

situ

hybridization procedure provides

an

un-ambiguous identification of

the cell

type(s)

expressing

the genes of interests.

Theaimof the present

study

was todefine both

temporal

and cellular

distribution of

the

expression

of

TGF-#

1 and type

I,

III,

and

IV

collagen

genes

during

the

progression

ofrat

he-patic

fibrosis

by

using

in situ

hybridization technique.

The

1.Abbreviations usedinthis paper:GAPDH,

glyceraldehyde-3-phos-phatedehydrogenase;GST-P,glutathione

transferase-placental form;

TGF-01,

transforming growth

factor-#I.

.r: ...^

r ~~~~~~.. ... :a '

.

.

...

...

Figure

1.

(A)

Centrilobular

necrosisafter 1 wk

of

CC14treatment(H&E, X400).Proliferated mesenchymalcells andinfiltrated

inflamma-tory cells arepresent. Granulocytesand

phagocytegiant cells are alsoobserved.(B) Earlycirrhosisat 4 wk(Masson's trichrome staining,

X100).

(C)

Immunohistochemistry

(X400)withDesmin antibodyat 4 wkofCC14 treatment. Fibrousseptacomposedof

Des-min-positive perisinusoidalcells(arrow)and

fibroblasts(openarrow).(D) Immunohisto-chemistry

(X400)

withVimentin antibodyat 4 wk

of

CC14treatment.Most of thestromal cellsare Vimentinpositivewhichsuggests

fi-broblasts.(E)Advanced cirrhosisat 12 wk

Figure I (Continued)

main focus

wascentered on answering two questions: which

cell

type

is

the

predominant

producer of collagens during

he-patic fibrosis

and what, if any, is the role of TGF-,3 in the

fibrotic process.

Methods

Treatmentof animals. 67 Fischer male rats weighing - 200 gwere

used.Throughouttheexperimental periodall rats were fed standard

PurinaChow pellets (NIH Supply)containing0.05% phenobarbital (Sigma Chemical Co.,St.Louis, MO) starting1 wkbeforethe

adminis-tration of 0.33 ml/kg bodywtofCCI4(Fluka AG)once a week by gavage. Ratsfed thesamephenobarbital-containingpelletandreceived thesamevolume of saline instead ofCC14 wereusedascontrol. Rats were killedfollowingalightanesthesiawith ether by decapitation and

exsanguinated from carotid arteries1 wkafterthe lastadministration ofCC14at1,2,4,6, 8,10, and12wk. Inadditiontotheaboveanimals, six normalratswithoutanytreatment were also used.

Thepresentexperimentswereundertakenaccordingtothecriteria oftheNationalInstitutes of Health for thecare anduseof laboratory animalsinresearch.

Histological and immunohistochemical methods. Immediately

after its removal, smallpieces oftheliver, whichwere notperfused,

werefrozenat -50'C forinsitu hybridization, andtheresiduewas instantlyfrozen in liquid nitrogen and keptat-70'C.Frozensections further fixed withaceton wereusedfordesmin

immunohistochemis-try.Liversections fixed with Bouin's fixativewereused for vimentin immunohistochemistry and for hematoxylin and eosin. Masson's trichromewasused for demonstration of connective tissue.

Vectastain ABC Elite kit(VectorLaboratories, St. Louis, MO)was usedfor the identification of Desmin-positive perisinusoidalcells(e.g., fat-storing cells and myofibroblasts)(18)with Desmin antibody (Dako, Copenhagen, Denmark) and mesenchymalcellswithantibodies raised against Vimentin(Boehringer-Mannheim,Indianapolis, IN).

Probes. The followingprobes were used in this study.A 600-bp fragment ofthecomplementary deoxynucleic acid (cDNA) obtained fromthefragment ofpalR2fromrat procollagenal(I)(19),andan

1,800-bp fragment frommouseprocollagena

1(III)

(20) were generous

gifts of Dr. B. Peterkofsky (National Cancer Institute, NIH) and were subcloned into the pGEM3z (Promega, Madison, WI). A 830-bp frag-ment of mouse procollagenaI(IV) cDNA (21) subcloned into pGEM2 was a kind gift of Dr. Y. Yamada (National Institute of Dental Re-search, NIH). A600-bp fragment from human

TGF-,#1

cDNA sub-clonedinto pGEM4 was kindly provided by Dr. G.I. Bell (22). Single stranded RNA probesof

TGF-#

I and procollagens were labeled with 3p or35Sfor Northern blot and in situ hybridization analyses, respec-tively, following the run off of the plasmid with the RNA polymerases. ThecDNAfor glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was kindly provided by Dr. C. Rories (National Cancer Insti-tute),wasusedasinternal standard for the Northern blot analyses (23).

35S-labeledRNAprobeswerehydrolyzed to yield - 0.3 kb fragments

(24) before in situ hybridization. The specific activity of35S-labeled

RNAprobes was 1.9X 109

dpm/gg.

Northern blot analyses. Total RNA was isolated from the liver tissue by extraction inguanidine isothiocyanate and centrifugation in cesium chloride (25). Poly(A+)RNAwasselected byoligo

(dT)-cellu-lose chromatography(26). 10 ug of poly(A+)RNA from each sample was separated in 1% agarose gel containing 2.2 M formaldehyde (27), andtransferredontonitrocellulose. Blots were hybridized with

32P-la-beledRNAprobesat60°C overnight. After high stringency washing (0.1% SDS and 0.1 XSSC: 1.5 mM sodium citrate, 15mM NaCl, pH 7.0,at70°C, for15min, three times)theblotswereexposed to Kodak

XARfilm. Thenitrocellulose filters were boiled for 10 min to strip off theradioactive probes, thenrehybridized with32P-labeledcDNAprobe for GAPDH with the similarmanner as RNAprobes at42°C and washed inhigh stringency condition for showing the internal standard. In situ hybridization. In situ hybridization was performed with frozen liver sections with slight modification of our previous report (28).Briefly, paraformaldehyde fixed frozen liver sections were treated with 5mMMgCl2 in PBS and 0.2 M Tris (pH 7.4) containing0.1 M glycine for 10 min. each. Then the liver sections were postfixed with

4%paraformaldehyde in PBS for 20 min. After rinsing with PBS, tissue sectionsweredehydrated in ethanol. The liver sectionswere prehy-bridized for 2 hat48°C for procollagens andat37°C for

TGF-,3l.

Hybridization wasperformedfor 2-3 h atthe sametemperatureas

prehybridization using35S-labeled antisenseRNAprobes. This short

hybridization time brought sufficientspecific hybridization and avoidedhigh nonspecifichybridization.Tissueswerewashedwith0.1 XSSCcontaining 50% formamideat55°C for 1 h, andweretreated with ribonuclease A (50 ,g/ml; Boehringer-Mannheim, FRG) and with ribonuclease Tl (250 U/ml, Boehringer-Mannheim) for I h at

37°C. After washingtwice with 0.1 XSSC for 1 h the slides were

dehydrated in ethanol and coated with Kodak NTB-2 emulsion.The

exposure timewas2wkat4°C.Theslidesweredevelopedin Kodak D-19 developer and fixed in Kodak fixer and counterstained with

hematoxylinandeosin.Toruleout

false-positive

and

-negative

insitu

hybridization,

35S-labeled

sense RNAprobesfor

TGF-f31

and

procol-lagensaswellasirrelevantantisenseprobessuchasforratalbumin, alpha-fetoproteinandglutathione

transferase-placental

form(GST-P)

were also examined in thesame seriesof liver slides and underthe

identical conditions.

Results

Histology

of

the liver. Centrilobular necrosis of the liver with

infiltrated

inflammatory

cells

and

proliferated mesenchymal

cells was

found

following

a

single

administration of

CC14

(Fig.

1

A).

Slight

accumulation

of

fiber in the necrotic area,as

dem-onstrated

by

Masson's

trichrome,

was

already

observed

at

this

time.

By

week two, the presence

of

connective tissue

became

prominent

atthe

necrotic central

areasas

well

asin the

portal

tracts. Necrosis of the liver

significantly

decreased

after this

time

point.

The

Desmin-positive

perisinusoidal

cells

and

fibro-blastswere the

predominant

cell types in the fibrotic tissue.

After

2

wk, the connective tissue

was

increased

and

newly

produced

thin

fibers

were

found

by

Masson's

trichrome

along

some

sinusoids

or

small

blood

vessels

(including

capillarized

sinusoids).

By

week

4,

accumulated connective

tissues formed

bridging

fibrosis between central and

portal

area

dividing

the

hepatic

tissue into

pseudolobules

of different sizes

(Fig.

1

B),

with

Desmin-positive perisinusoidal

cells and

fibroblasts

pro-liferating along

the

connective tissue

(Fig.

1,

C and

D).

A

prominent

liver cirrhosis

waspresentat

week

8.

By

weeks 10

and

12,

a

gradual

increase in

the

extracellular matrix

was

ob-served

along

the

borders

of the

pseudolobules

(Fig.

1

E).

Livers

from animals fed

phenobarbital pellets

andwere not

treated

with

CC14

showed

occasional

single

cell

necrosis

with

macro-phage

infiltration

and a

slight

increase in connective tissue

around the

portal

tracts

(data

not

shown).

Northern blot

analyses.

The

expression

of

TGF-fl1

and

procollagen

a

_1(I),

a

_1(111),

and

al(IV)

was

analyzed by

North-ernblot

analyses.

Results

from

TGF-(

1

and

procollagen

a 1(I) anda

_1(III)

are

shown in

Fig.

2.

In

the

normal

liver,

procolla-gena

l(I)

wasvery

weak,

whereas

the

transcripts

for

TGF-#

I

and

procollagen

a

1(III)

and

a

1(IV) (not

shown)

were

detected.

The mRNA

levels

of

_TGF-#3I

and

procollagens

in the

fibrotic

livers

were

higher

than

those

observed in control animals.

However,

the

expression

of

these genes

varied from time

to

time. Similar variations of

the

procollagen

gene

expressions

during

the

fibrotic

process

have

also been

observed

by

other

investigators (15).

In

the livers

of

phenobarbital-treated

rats,

a

slight

increase in

the

expression

of

TGF-f31

(not

shown)

and

procollagen

genes was

observed,

however,

the

expression

of

these

genes

remained

at low level and somewhat varied

throughout

the

experiment

as seen

in

Fig.

2 B.

GAPDH

ex-pression

was

_unchanged

_throughout

the

_present

experiments

(Fig.

2

A).

Densitometric

analysis

of

TGF-BIl

and

a

_1(III)

(Fig.

2

A)

is documented in

Table I

after

the

standardization with

the

_expression

of GAPDH.

In situ

hybridization.

The

distribution of

TGF-,B1

and

pro-collagen

al(I),

al(III),

and

_{al(IV) transcripts}

in normal liver

was

localized in mesothelial cells in the

hepatic capsule,

around

the

large

blood vessels

and

_periductal

cells in

the

portal

tracts,as

well

as

around

the

central

_hepatic

veins.

Some

sinu-soidal-lining

cells also

_{weakly expressed}

these genes. No

ex-pression

of

TGF-,

I and

_procollagen

geneswas

observed

in the

hepatocytes.

The

cellular distribution and time

course

of

TGF-(31,

pro-collagen

a

_1(I),

a

1(III),

and a

I(IV) transcripts during

the

fi-brotic

process

is shown

in

_Fig.

3.

Both

the distribution and

time

course

of

gene

expression

of

TGF-#

1 and

procollagen

a

_l(I),

a

_1(III),

anda

_I(IV)

_were

similar.

_By

week

1,

thesegenes were

expressed

in the

necrotic centrilobular

areas

mainly

com-posed

of

_{Desmin-positive perisinusoidal}

cells,

fibroblasts,

and

inflammatory

cells,

whichare

_{mainly granulocytes}

and

macro-phagelike

cells

(Fig.

4

_A).

Various

cells

expressing

thesegenes

are

present

as a

network

surrounding

the

small

groupsor

single

necrotic

hepatocytes.

These geneswerenot

expressed

in

hepa-tocytes. In

addition,

the

_expression

of

_TGF-#

Iand

_procollagen

genes was

increased around the

_portal

tract at

this time.

_By

week

2,

the

area

occupied

by

silver

_grains

increased in size. The

tracks

of silver

grains

present

in both

central

and

_portal

areas,

formed thin

bridges

before

the

accumulation of

Masson's

trichrome

positive

connective tissue.

The

connective tissue

was

mainly

localized in

the

central

and

_portal

areas at

this

Weeks of

treatment

N

1|1

2 14

1

6

1

8

110 112

A

TG

_F-f1l

AN&

t

*

-2

5 kb

a,

11)^*

5.3

kb

-

1.4 kb

Weeks

of treatment

F~~

1 2 4

6

N

a b

Ia

b Ia

b a b

§

#e&

=~~~~5.7

kb

-4.7kb

Table I. Relative Densitometric Analysis of TGF-j31

and a) (III)mRNALevels

Weeks N 1 2 4 6 8 10 12

TGF-Pl/GAPDH

1 2.1 1.5 2.7 1.4 5.0 2.2 2.7

al(III)/GAPDH 1 8.5 8.2 3.5 1.4 4.6 11.7 3.6

Density ofTGF-#landa1(III)mRNAlevels (Fig. 2 A) were ana-lyzed by using a computerized densitometry. Each densitywas

stan-dardized by relevant GAPDH level.Data wereexpressed relative density while standardized density of normal liver (N)was set as 1.

Transcripts for

TGF-#l

and procollagen genes were not

detected

in hepatocytes throughout the experiment. The

ex-pression of

TGF-ft

1 and the procollagen genes in some

sinusoi-dal-lining

cells was

found in both

normal and CC14-treated animals.However, no increase in the expression of these genes in

sinusoidal-lining

cells in hepatic lobules was observed

dur-ing

the

progression of fibrosis.

In order to rule out

false-positive and -negative

in these

experiments, the

sense probes

for TGF-(

1, procollagenaI(I), a

1(111), and al(IV)

aswell as the

antisense

RNAprobes for rat

albumin, alpha-fetoprotein,

and

glutathione

transferase-pla-cental

form (GST-P)

wereused

for

in

situ

hybridization

in the same

series of liver slides

and

under identical conditions.

The sense RNA

probes

slightly labeled nonspecifically

but not

sig-nificantly

over

the background

(Fig.

5).

The

mRNA

distribu-tions

for

rat

albumin (Fig. 6 A), alpha-fetoprotein (Fig.

6 B) and

GST-P (Fig.

6

C) probes

were

specific for

the relevant mRNA

and

entirely different from

those

of

TGF-#lI

and

pro-collagens.

GAPDH

a

*

*

*

*

-1.4 kb

Figure2.Northern blotanalysesofTGF-#lI and procollagena1(I)

andaI(III) during CCl4-inducedfibrosis.10_,gofpoly(A+)RNAwas

appliedineach lane.Poly(A+)RNAswerehybridizedwith

32P-la-beled RNAprobesonthenitrocellulose filter.(A)Theexpressionof

TGF-#lI and procollagena1(III)genesincreasefollowing CC14-treat-mentascomparedtothatof normalliver(N).Theexpression of GAPDH,whichwasexamined with cDNAprobe,isunchanged throughouttheexperiments. (B)Procollagena1(I)geneexpressionin the liverofCC14-treated(a)and untreated(b) (fed pelletwith

pheno-barbital butnottreated withCC14)animals. Note thesignificant dif-ference oftranscriptlevelsbetweenCC14-treatedanduntreated ani-mals. Thesamefindingwasalsoobservedduring 8-12wk(datanot

shown).Aslightincrease in theexpressionofprocollagen al(I)gene

is observed inphenobarbital-treatedgroups(b)ascomparedto

nor-mal(N).

grainswerefoundalong the borders of the pseudolobules com-posed of mesenchymal cells and increased extracellular matrix (Fig. 3). The cell types expressing TGF-j3l and procollagen

genesalong the sinusoids and small blood vessels, where the fibersaccumulated,weremainly Desmin-positive

perisinusoi-dalcellsandfibroblastsasshown in Fig. 4, B and C. Atlater timepoints, the expression of thesegeneslocalized primarily in the fibrotic tissues and increased with the progression of

fibrosis(notshown).

Inthepresentstudy,wehave shown that the cellular

distribu-tion of

_TGF-P

I and procollagen al(I), a

1(III),

and al(IV)

geneexpressionswassimilarthroughout the hepatic

fibrogen-esis. In the normalliver, the transcripts of

TGF-l3I,

and

pro-collagena1(I),a1(111), anda1(IV)genes wereobserved in me-sothelialcellsinthehepatic capsule, around portal veins, peri-ductal cells, and central hepatic veins as well as in

sinusoidal-lining cells. At the earlystages ofthe fibrotic

pro-cess,mesenchymal cells principally Desmin-positive

perisinu-soidalcells, fibroblastsandinflammatorycellsexpressed

pro-collagen genes together with the

TGF-ft

l gene. In the later stages offibrosis,the expression ofthesegeneswith the

pro-duction ofcollagensandpossiblyother extracellular matrices

waspredominantly localized againinthesamemesenchymal cell types. Theexpressions ofTGF-,Bl and

procollagen

a

I(I),

a

I(111),

and al(IV) genescontinued tobe higher than those

observed in control ratsduring the fibrotic process. Neither

TGF-3 northeprocollagengeneexpressionswereobserved in

hepatocytes.

It has been found that certaintypesofcollagensare synthe-sizedby fat-storingcellsby demonstratingthe

immunoreactiv-ity oftypes IandIIIcollagenintheroughendoplasmic

reticu-JumandGolgi's apparatus followingcarbon tetrachloride

ad-ministration in rats(9). The presentstudy providesevidence

thatstrongexpressionof themostcommonprocollagengenes

is found inthe mesenchymal cell compartmentespeciallyin

TransformingGrowthFactor-f31 andProcollagen Transcripts 1837

._01

(APDH

B

F-U

lat

ktw

#S

8@

@t~;Qowo;

I

*

~~fii...'e

-1...

>,>@X

s+

' !, *666 e8D

/

.- .4; *

:

4'

-4

5-i4~~~~~

ex

+ j | ~,%now,

t.~@zmfX4

Aid~~~;A.

{i71

AT:An

W eS

,.-' +

:w~8t'iA

mV,

.

L4

~

.*.*9zf

~

- i

At**.

Wr'lk'-

o

'2

#w

A

4,,

ill

. 4-4~~~~~~~~~~~~~~~~~~~4

9 9

.4

0. 0.

IS

i2

Figure4.(A) ThedistributionofTGF-#1 transcripts in the

inflam-matory area at 1 wk(X400).Silver grainsare seeninperisinusoidal cells, fibroblasticcells(arrow) and in inflammatory cells

(arrow-head).Hepatocytes (open arrow)arenegative. See Fig. 1Afor

refer-ence.(B)Thedistribution ofprocollagenaI(IV)transcriptsatthe front edge ofthesilver graintracts at 4 wk(X400).Silver grainsare

found mainly inthecells inthefibrousseptaandalongthe

sinusoi-dal space.(C) Silver grains of

TGF-#31

transcriptsby week 4 are also seenalong thesmall blood vesselsasindicated byalumen(arrow) (X400).

Desmin-positive perisinusoidal cells and fibroblasts simulta-neously with an abundant production of extracellular matrix. These results indicate that the mesenchymal cell compartment plays

the

principal

role in the

CCL1-induced hepatic

fibrogen-esis. Incontrast, the expression of

_TGF-#

I and procollagen genes does not

increase in the perisinusoidal cells locating

in thehepatic lobules, in which no necrosis or accumulated

con-nective tissues is

observed, during the experiment. The in-volvement

of

other

sinusoidal-lining

cells, such asendothelial

cells,

Kupffer

cells

and

pit cells in

the

fibrotic

process

is

not clear at

this

point.

We and others have shown that hepatocytes can produce

collagens

and other extracellular

matrix

components

in

pri-mary culture (10, 1 1). Also, it has been suggested, based on

data

obtained

by

immunohistochemical

methods, that hepa-tocytes can produce types

I,

III, and IV collagens in

human

fibrotic

livers,

and

basement

membrane components in human chronic

active

liver diseases (29, 30). Martinez (31) reported that type I collagen as well as fibronectin were found

in

the rough

endoplasmic

reticulum of hepatocytes. Further-more,

Chojkier

et al. reported that hepatocytes produce the

majority

of

newly

synthesized

hepatic

collagen

in

CCL4-in-duced rat

liver fibrosis ( 12).

Our data

fail

todemonstrate any

significant increase

overbackground in mRNA transcripts

for

TGF-f1

and al(I),

a1(III),

and al(IV) procollagen genes in hepatocytes

from

either

normal

liver

or

during

CCL4-induced

hepatic fibrosis. Pierce

etal.

(15)

have shown that no signifi-cant

increase in the

DNA content

of the liver

takes

place

dur-ing

CC14-induced

hepatic fibrosis

strongly

suggesting

an

in-crease per cell in

procollagen synthesis.

The data presented here,

showing

a

selective increase in

the steady-state level of the

transcripts for the procollagen

genes

in

the

mesenchymal

cell compartment, are in agreement

with this

conclusion.

Mi-lani

et

al.

(32, 33) recently

reported that

by

using in situ

hy-bridization

types

I, III,

and IV

procollagen

gene

expressions

were observed

predominantly in

nonparenchymal cells and concluded that

hepatocytes

do not appear to be

significantly

involved in

procollagen production in CCL-induced liver

fi-brosis. Our dataarein

good

agreement

with this. The

_disparity

between

our

data

and

those

of Martinez and

Chojkier

et al.

is

atpresent unclear.

Nevertheless,

the

contribution of

hepato-cytestothe

increase in collagen

during

CCl4-induced

hepatic

fibrosis is

at best

questionable.

The

distribution

of

type

I, III,

and IV

collagens is

different,

since

type I

and

III

collagens

are the

main

components

of

interstitial collagens,

whereas type IV

collagen is

one

of

the

main

components

of basement membrane

(2, 31).

However,

the cellular

distribution

and the

time

course

of

the

expression

of procollagen

a

1(I),

a

1(III),

anda

I(IV)

geneswerevery simi-lar

throughout

the

experiment.

Procollagen

genes aswell as

TGF-#

Igene are all

similarly

and

simultaneously expressed

in

the

necrotic

areain the

early

stages. In

the

later stages,

they

are

expressed

along

the

small

blood vessels and the

fibrous

septa. The

expression of

these genes

is associated with

the

increase

in the number

of

Desmin-positive perisinusoidal

cells and

fibro-TransformingGrowth Factor-NJ andProcollagen Transcripts 1839

Figure3.Thecellulardistributionandthetimecourseof

TGF-#

1,procollagenal(I),a1(III),andal(IV) transcriptsat 1, 2, and 4 wkofCC14

treatment(X100).

TGF-#

1 andprocollagengenes are allsimilarlyexpressedduring fibroticprocess. At 1 wk, silver grainsarefound inthe

cen-trilobularareawherenecrosis, inflammation,andregenerationareobserved.At 2wk, thetracksof silver grains inboth central andportalarea

4

or

0*%,

u.A

ikw~

4

e10

*1..

44s

.7.

*0

A,

*%v.

# An.. ^

~~~~~~~~~~~~~~~~~~~~~~~~~~~~A

-wSF' M! ...4*~~J:

I

:#<

-

...,

t . r; ' >

*~~4

*^

o*r\*Ps^

ye

Figure5. Insituhybridization with antisense(a) and sense (s) RNA probes for

TGF-#

I (A),procollagenaI(I)(B), aI(III)(C),and al(IV)(D).

Silvergrainsfrom antisense probesareobserved inthefibroussepta and along thesinusoids, whereasthedensityof grains from the sense

probes isnotsignificantly differentfromnonspecific background level

_(CC14

8wk,X400).

blasts

along

the

connective tissue,

and

coincides

with the

pro-duction of

extracellular

matrix.

Although the cellular

distribu-tion

and

time

course

of procollagen

a

1(111)

expression were

similar

to

other

procollagens

and TGF-1

1,

the number

of

silver

grains

per cell

of

procollagen

a

1(III)

was

smaller

than othersasshown in

Fig.

3.The reasonfor

this difference

isnot

clear, however,one

could

speculate

that the

transcription

level

differ-Figure 5(Continued)

ent

from

the

other

procollagen

genes as suggested by Pierce et al. (15).

TGF-/#

has been

shown

to beboth

chemotactic

for

fibro-blasts and also able to greatly enhance

synthesis of

the

extra-cellular matrix

components

by these

cells

(6,

34).

TGF-,3

stim-ulates

the

proliferation

of fibroblasts and small vessels

(35),

as well asincreases the collagen

synthesis of

human dermal

fibro-I ₉

S I, ",& 1*I v-~~~~~~~~~~~_'k e

a9'I5

w* -.. ~tSt4j 0.

,* 9. 9**'. 0

L44

AM

~- as46

blasts (36). The increased mRNA level and production of both collagens and

fibronectin

by

_TGF-f3

are

found

in fibroblasts

obtained from

mouse, rat and human (36-38).

TGF-3

also causes

marked

increase

in

proline

and

leucine incorporation

into

collagens (35). In addition,

_TGF-f#

inhibits theexpression

of

tissue

collagenase, which

is

a

specific

enzyme

for

degrada-tion of mammalian

interstitial collagens, and enhances the

, ~., , w 4i

Figure6. Insituhybridization with irrelevant antisenseRNAprobes in fibrotic liver

(CCL4

8wk, X200). Transcripts ofratalbumin(A) are

observedonly in hepatocytes butnotin stromal cells.Alpha-feto

pro-tein (B) isnotsignificantly expressed in fibrotic liver. GST-P (C)

transcriptsareobservedonly in bile duct cells (not shown) butnotin hepatocytesorstromal cells.

production oftissue inhibitor of metalloproteases, which in-hibits the collagenase activity (5). Thus

_TGF-ft

iscapable of stimulating fibrogenesis by the fibroblast. Furthermore, it was recently suggested

in

anabstract that

_TGF-f3

may play arole in the hepatic fibrogenesis through its effect on fat-storing cell collagen

synthesis, because

of

the

observation

that cultured fat-storing cells contained

significant

amount of mRNA

of

both

_TGF-#31

and type I procollagen and these mRNA levels wereincreasedby TGF-(3 treatment (39). In the present study,

TGF-#

I gene

is

strongly expressed not only in the Desmin-positiveperisinusoidal cells but also in fibroblasts and inflam-matory cells together

with

the expression of procollagen genes in the same cell types

throughout

theprogression of

fibrosis.

It is therefore possible that

_TGF-#

I may have enhanced both

proliferation and collagen synthesis in

themesenchymal cells

by

autocrine and

paracrine mechanisms resulting in excess

accumulation of connective tissue throughout

the

fibrotic

pro-cess in the liver.

Acknowledgments

Authors thankA.Olsonfor densitometricanalyses. F. Williams and N.

Higginsarealso acknowledgedforsecretarialexpertise.

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