Vol.
171,
No.
September
2, 1990 14,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Pages
647-654
TRANSFORMING GROWTH-FACTOR-8 (TGF-l3) INHIBITS ALBUMIN SYNTHESIS IN NORMAL HUMAN HEPAtOCYTES AND IN HEPATOMA HepGP CELLS’ Nathalie BUSSO, * Christophe CHESNE, + ’ # Francois DELERS , * Fabrice MOREL, + and Andre GUILLOUZO + * Laboratoires GLAXO, Centre de Recherches, 25 avenue du Quebec , 91951 Les Ulis Cedex, France + INSERM U49, Hopital de Pontchaillou , 35033 Rennes Cedex,
France
# BIOPREDIC , Hopital de Pontchaillou , 35033 Rennes Cedex, France * Laboratoire des Proteines de la Reaction lnflammatoire, 45 rue des Saints Peres, 75270 Paris Cedex 06, France Received
July
23,
1990
We explored the effect of transforming growth factor I3 (TGF-O), a cytokine that appears to play a central role in inflammatory events, on albumin expression by normal adult human hepatocytes and hepatoma cells. Addition of TGF-6 to primary human hepatocyte cultures resulted in a dramatic decrease in albumin accumulation and synthesis. This effect was dose-dependent, took place after a 48h incubation period and was maintained over 96h. TGF-B-decreased albumin protein levels were associated with reduced albumin mRNA content. Actin mRNA levels were concomittantly increased. Comparable qualitative effects of TGF-8 were observed on human hepatoma HepG2 cells. 0 1990Academic P7xSS. Inc. Tissue injury and inflammatory
processes
induce a host response commonly
referred to as the acute phase reaction (1). Adaptation of liver cells to the acute phase reaction is characterized derived
plasma
consequently
by changes in the biosynthetic
proteins
the plasmatic
(acute-phase concentration
whereas that of some others is reduced albumin, the most abundant hepatocytes. inflammatory
proteins).
pattern of a subset of liver-
The hepatic
production,
and
of some of these proteins, is increased, (for review see 2). Among the latter ones is
serum protein, the synthesis of which is restricted to
Indeed, hypoalbuminemia
has been frequently
found in patients with
diseases (3). Moreover inflammatory mediators secreted by mononuclear
‘This work was partly supported Recherche Medicale (INSERM).
by the lnstitut National de la Sante Et de la
The abbreviations used are : BSA, Bovine serum albumin; SDSSodium Dodecyl Sulfate; SDS-PAGE, Polyacrylamide gel electrophoresis in the presence of SDS; IL-l-8, Interleukin-16; IL-6, Interleukin-6; MEM, Minimum Essential Medium; PBS,Phosphate-Buffered Saline; TNF-a, Tumor Necrosis Factor-o!.
647
0006-291XE’O $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 171, No. 2, 1990 phagocytes
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
such as IL-l-O, TNF-a and IL-6, modulate acute phase protein expression,
and notably decrease albumin synthesis in a variety of in vivo and in vitro models (48). TGF-8, another multifunctional
molecule secreted
appears to play a central role in inflammatory
by mononuclear
phagocytes,
events (for review see 9). Thus far,
however, there is no direct evidence for TGF-8 as an acute phase mediator in human hepatocytes.
This prompted
us to investigate the effect of TGF-8 on the production
albumin by normal adult human hepatocytes
as well as by the human
of
hepatoma
HepG2 cell line.
EXPERIMENTAL PROCEDURES MATERIALS: Cell culture reagents were from Gibco (Cergy-Pontoise, France). TGF-8 type 1 purified from human platelets was from R & D (Minneapolis, MN). Recombinant human TNF-a! (specific activity 2.10’ u/mg) was from Genzyme (Boston, MA). Recombinant human IL-l-8 (specific activity 2.2 10’ u/mg) and a 21 bases oligonucleotide probe for human 8 actin were obtained from Glaxo Institute for Molecular Biology (Geneva, Switzerland). The human albumin cDNA probe was generously provided by A. Dugaiczyk (10). Rabbit anti-human albumin serum was from Behring (Rue&Malmaison, France) and protein A-Sepharose from Sigma (La Verpilliere, France). Amplify, [“Cl methylated protein standards, L-[?S] methionine (800 Ci/mmol), deoxycytidine 5’[a-=P] triphosphate (> 3000 Ci/mmol) and adenosine 5’[ “P] triphosphate (> 5000 Ci/mmol) were from Amersham (Les Ulis, France). All other chemicals were of the best commercial grade available. CELL CULTURES: Human hepatic tissue fragments were obtained from organ donors (3 male subjects, 22, 23 and 27 years old). Sampling was made in accordance with French legal considerations. Adult human hepatocytes were isolated by the two-step collagenase perfusion method as previously described (11). Hepatocytes were seeded at a density of 1.5 x lo5 cells per well in 24 well plates in 75 % MEM and 25 % medium 199 supplemented with 0.1 mg/ml BSA, 10 kg/ml bovine insulin and 10 % fetal calf serum (12). For RNA extraction cells were seeded at a density of 10’ cells per T75cm2 flask. After attachment the cells were maintained in serum-free medium complemented with 7 x 10”M hydrocortisone hemisuccinate. Primary cultures consisted of more than 95 % hepatocytes. All tested agents were added 24h or 48h after cell plating in serumfree medium with hydrocortisone and 0.1 mg/ml BSA. Neither morphological alterations nor lactate dehydrogenase leakage were observed during the incubation period in the presence of the agents. The human hepatoma cell line HepG2 was provided by the American Type Culture Collection (Rockville, MD). Cells were grown in MEM (Eagle) with non essential amino acids, sodium pyruvate and 10 % fetal calf serum. All tested agents were added at confluency in serum-free medium supplemented with 0.1 mg/ml BSA. ALBUMIN IMMUNOELECTROPHORESIS: This was performed method of Laurel1 (13) as previously described (14).
by a modification
ofthe
BIOSYNTHETIC LABELING AND IMMUNOPRECIPITATION: Cell monolayers in 24 wells were incubated with or without various TGF-8 concentrations for 48h. Medium was removed and monolayers were washed twice with methionine-deficient medium. Hepatocytes were labeled with 12.5 pCi/well of[J5S]-methionine for 4h in serum-free medium containing 5 % of normal concentration of methionine in the presence or absence of TGF-8. The medium was harvested, the monolayers washed twice with cold PBS and the cells were lysed in 0.1 M Tris-HCI pH 8.1, 0.4 % Triton X-100. The
Vol.
171, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
effect of TGF-8 on total protein synthesis was determined by measuring the incorporation of [?S]-methionine into trichloroacetic acid precipitable material. After normalization of the loaded media volumes with respect to the corresponding rate of protein synthesis media samples were electrophoresed in the presence of SDS in 10 % polyacrylamide gels under reducing conditions as described by Laemmli (15). Gels were treated with Amplify, dried and fluorographed. For immunoprecipitation of albumin, samples of labeled culture media, obtained as described above, were incubated overnight at 4°C with anti-human albumin or irrelevant rabbit antisera diluted l/5 with PBS. Immune complexes were pelleted with excess protein A-sepharose. After centrifugation the immune pellets were washed, eluted by boiling in sample buffer and electrophoresed under reducing conditions as below. [‘“Cl-methylated molecular mass markers (200, 92.5, 69, 46, 30, 21.5, 14.3 Kd) were included on all gels. RNA ANALYSIS: Total cellular RNA was isolated from monolayers of hepatocytes by the guanidinium isothiocyanate/cesium chloride method (16). For Northern-blot hybridizations, fractionated RNAs were transferred onto nylon membranes. The amount of RNA per lane was controlled by staining the bound RNA on filters with 0.05 % methylene blue and destaining in 70 % ethanol. The RNAs were hybridized to the nick-translated [3’P] labeled albumin probe. After this first hybridization, the probe was eluted by boiling the membrane 5 min in 20 mM Tris-HCI pH 8.1 and the filter rehybridized with the 5’end-[32P]labeled actin probe. Filters were exposed to Kodak XAR-5 films at -80°C between intensifying screens. Relative mRNA levels were determined by densitometric analysis of the autoradiograms. RESULTS When primary adult human hepatocytes were incubated for 48 h in the presence of 2 rig/ml of TGF-8, a striking inhibition (> 95 %) of albumin accumulation medium inhibition
was measured starting
(Tablei).
at 1 rig/ml
This effect was dose-dependent
of TGF-8 (data not shown). TABLE
into culture
with maximal
Concomitantly
with a
1
INHIBITION OF ALBUMIN ACCU ULATION IN PRIMARY CULTURES OF ADULT HUMAN HEPATOCMES BY TGF:: COMPARISON WITH THE EFFECT OF TWO OTHER CYTOKINES AGENTS
ALBUMIN
CONCENTRATION
(% of control cultures) 223
TGF-I3 2 rig/ml IL-l-0
100 u/ml
29 2 22
TNF-a
100 rig/ml
34 f 20
Hepatocytes were plated into 24 well plates at 1.5 x 10” cells/well. Duplicate or triplicate monolayers were treated after a recovery period of 24-48h with 0.5 ml of serum-free medium and the indicated factors. Following 24h stimulation the medium was replaced by 0.5 ml of fresh medium supplemented with the same agents. After an additional 24h, the medium was collected and aliquots were tested by immunoelectrophoresis for albumin concentration. Results are expressed as percent of the albumin concentration measured in untreated cultures (mean 2 SD). 649
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BIOCHEMICAL
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RESEARCH
COMMUNICATIONS
decreased albumin amount, we measured in samples from TGF-8 treated hepatocytes increased plasminogen
activator inhibitor type 1 (PAI-1) and urokinase
(manuscript in preparation),
(U-PA) levels
thus ruling out a non-specific action of TGF-8 on albumin
resulting from a general toxicity; similar results on PAI- and U-PA modulation
by TGF-
8 have been previously reported in other cell types (17, 18). The effect of TGF-8 on albumin supraoptimal
was compared
doses of IL-l-6 and TNF-a. These two cytokines
levels by 70 % (Tablel) 7). Taken together, compared
concentration
confirming
earlier observations
to the effects of decreased
on other cellular models (5
these results indicate that TGF-8 has a more potent
to IL-l-6 and TNF-a. At the concentrations
effect of TGF-8 on albumin accumulation,
as those of IL-l-6 and TNF-a, were barely after 48 h and
over a 96 h period of incubation (data not shown). The modulation
of albumin levels by TGF-8 in the human hepatoma comparable
effect
indicated in table I, the inhibitory
detectable after the first 24 h incubation but became more pronounced were maintained
albumin
to that observed
in albumin concentration
in primary
human
cell line HepG2 was qualitatively hepatocytes
(maximal
inhibition
of 50 % reached at 48 h with 10 rig/ml of TGF-13, data not
shown). To determine if the observed inhibitory effect of TGF-8 on albumin accumulation in the medium
occurred
at the biosynthetic
level, hepatocytes
were incubated
in
medium containing varying amounts of TGF-8 for 48 h, and biosynthetically labeled for 4 h in the presence of the same amounts of TGF-6. TGF-6, at an optimal dose, inhibited albumin synthesis by 5-fold (as measured by densitometric 1A). This effect was dose-dependent protein synthesis
(Figure 1B). Although
up to 50 % at the highest concentration,
synthesis was specific since TGF-8 in the same conditions immunologically uncharacterized
identified
as
PAI-
(manuscript
analysis of figure
TGF-8 decreased
increased a 47 Kd protein
in preparation)
and
another
27 Kd protein. In addition, TGF-8 increased actin biosynthesis
extracts (not shown). Analysis of the biosynthetic
total
the effect on albumin
in cell
labeling of secreted proteins by the
hepatoma
HepG2 cell line gave a similar pattern (Figure 1C). First, TGF-I3 decreased
albumin
synthesis,
higher concentrations
although
the
dose-response
in this cell line compared
effect
was
shifted
to primary hepatocyte
to
cultures.
Secondly, as in primary cultures, TGF-8 increased the synthesis of actin (not shown) and of PAI-1. Nevertheless TGF-8 did not affect total protein synthesis nor did it induce the 27 Kd protein
in HepG2 cells in contrast
with its effects in primary
human
hepatocytes. TO
further assess the effect of TGF-8 on the expression of the albumin gene, we
extracted total cellular RNAs and subjected them to Northern-blot
hybridization
2). Albumin mRNA level was significantly reduced in both primary hepatocyte 650
(Figure cultures
Vol. 171, No. 2, 1990
Normal
--ab
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS
hepatocytes
HepG2
cells
cd +
TGF-beta
1 rig/ml 10 -
1
+TGF
.l
50
.Ol
-
beta nglml
10
+TGF
1
5
s
.l
beta rig/ml
FIGURE 1 . EFFECT OF TGF-0 ON ALBUMIN SYNTHESIS Hepatocyte monolayers were incubated in medium without (-) or with (+) TGFR atthe~ ’ indicated doses for 4Bh and then radiolabeled for 4h in serum-free medium containing [“S] methionine (50 clCi/ml) in the presence or absence of TGF-0. Samples of culture media from primary hepatocytes (figure IB) or HepGP cell cultures (figure 1C) were analysed directly by electrophoresis under reducing conditions. Alternatively, the labeled culture media samples from primary hepatocyte cultures were immunoprecipitated with anti albumin (tracks b and d) or with irrelevant (tracks a and c) rabbit antisera. Immune pellets were eluted and electrophoresed under reducing conditions (figure 1A). Molecular weight markers were electrophoresed in parallel on all gels. Gels were treated with amplify, dried and fluorographed.
and
HepG2
densitometric
cells (74% and
as measured
by
analysis). As a control we analysed in parallel RNAs from IL-i-0
and
TNF-a treated cells. As expected
35% of decrease these inflammatory
respectively
decreased
albumin
mRNA steady state level by 70% and 60% respectively in primary hepatocyte
mediators
cultures.
The effect of TGF-6 on albumin mRNA was specific in both primary hepatocyte
cultures
and HepG2 cells since - 1) the amounts of 28s and 18s mRNA were the same in the different RNA samples analysed as quantified by methylene blue staining of ribosomal RNAs (not shown) - 2) actin mRNA content in TGF-I3 treated cells was significantly increased
(260 % in normal hepatocytes,
210% in HepG2 cells). Modulation
of actin
mRNA level is consistent with the TGF-I3 induced increase in biosynthetically
labeled
actin reported above. Moreover, this result is in agreement
with the findings of Czaja
et al. (19) who measured a slight increase of actin mRNA after TGF-f3 treatment hepatocytes,
and with the 2-fold induction
different cell types (20). 651
of O-actin mRNA content
of rat
by TGF-B in
Vol.
171, No. 2, 1990
BIOCHEMICAL
Normal
albumin
AND BIOPHYSICAL
hepatocytes
HepG2
RESEARCH COMMUNICATIONS
cells
-
-albumin
-
actin -
C IL-1 TNF
TGF
C
IL-1
TNF
actin
TGF
FIGURE 2 . EFFECT OF TGF-B ON ALBUMIN mRNA CONTENT Hepatocyte monolayers were incubated for 4Bh (normal hepatocytes) or for 24h (HepGP cells) in serum-free medium containing 0.1 mg/ml BSA (C = control) or in serum-free medium supplemented with 100 u/ml IL-I-B (IL-l), with 100 rig/ml TNFQ (TNF) or with 2 rig/ml (normal hepatocytes) or 10 rig/ml (HepG2 cells) TGF-R (TGF). Northern-blot hybridization analysis was performed using 12 pg of total cellular RNA per track. The filter was hybridized first to an albumin -P-labeled cDNA probe, then to a B-actin =P-labeled oligonucleotidic probe.
DISCUSSION The accumulation
results
reported
here
show
that
TGF-l3
(> 95 % inhibition) of the negative acute-phase
normal human hepatocyte same conditions,
and human hepatoma
dramatically
decreases
protein albumin in both
HepG2 cell line culture media. In the
albumin mRNA levels are decreased
by 2-4 fold. Thus, it appears
doubtful that these limited observed changes in albumin mRNA levels are sufficient to account for the marked effect of TGF-t3 on albumin. Several other factors may account for such a discrepancy
: translation efficiency of albumin mRNA may vary ; alternatively
albumin accumulation
may be influenced by clearance mechanisms.
Several other hepatocytes
proteins
are also modulated
by TGF-I3 in primary
and HepG2 cells ; amongst these is plasminogen
1 (PAL1 manuscript
in preparation),
a positive acute phase protein (21). In hepatoma
cells, it has been recently reported apolipoprotein
human
activator inhibitor type-
that in addition
to albumin,TGF-l3
decreases
A-l, another negatively regulated protein during acute phase reaction
(22). The fact that TGF-0 is able to act on negative (albumin and apolipoprotein
A-l)
as well as on positive (PAI-1) acute phase proteins, suggests that this cytokine may be an important
mediator of the acute phase response.
In this regard it would be of
interest to determine the effect of TGF-l3 on other acute phase proteins in normal adult human hepatocytes. Comparison
of the biosynthetic
protein pattern of primary human hepatocytes
and of HepG2 cells in the presence of TGF-8 revealed that essentially the same 652
Vol.
171, No. 2, 1990
spectrum
BIOCHEMICAL
of proteins was affected;
AND BIOPHYSICAL
nevertheless
RESEARCH COMMUNICATIONS
HepG2 cells appeared
to be less
sensitive to TGF-8 but still remain a suitable in vitro system to study TGF-8 effects. TGF-8 seems to have an important role in hepatic pathophysiology. reported to be a potent inhibitor of hepatocyte
proliferation in vitro (23-25). Moreover
hepatic TGF-8 mRNA levels increased after partial hepatectomy
in the nonparenchymal
liver cells (25) ; thus TGF-8 may function in vivo as the effector paracrine
mechanism
regeneration. production
to
prevent
uncontrolled
In view of these observations, during liver regeneration.
It has been
hepatocyte
of an inhibitory
growth
during
liver
it would be interesting to examine albumin
In two different in vivo models of hepatic fibrosis
it has been reported that TGF-8 gene expression in the liver was associated
with the
increase in collagen synthesis (19). In these models the authors have also observed that albumin mRNA content declined with the progression TGF-8 synthesis increased).
of fibrosis (i.e. as hepatic
However, these authors have not found a direct effect
of TGF-8 on albumin mRNA content of rat primary hepatocytes
after a 24h incubation
in the presence of the cytokine. This lack of effect may be due to a species difference or to the delayed action of TGF-8 on albumin ; indeed we have shown that the effect of TGF-I3 was barely detectable
after 24h but was drastic after 48h. In this context, it
seems likely that the decreased
level of albumin in hepatic fibrosis could result from
a autocrineiparacrine
modulation
by TGF-8.
ACKNOWLEDGMENT We thank E. Nicodeme for her expert technical assistance. REFERENCES 1. 2. 3.
4. 5. 6. 7. 8. 9. 10.
KOJ A. (1985). in The acute phase response to injury and infection (Koj A. and Gordon A.H. eds), vol 10, pp 139-232, Elsevier - Amsterdam - New York. BAUMANN H. (1989). In Vitro Cell. Dev. Biol., 25, 115-126. VAN TONGEREN J.H.M., CLUYSENAAR O.J.J., LAMERS C.B.H., DE MULDER P.H.M. and YAP S.H. (1978). in Clinical Aspects of Albumin (Yap S.H., Majoor C.L.H. and Van Tongeren J.H.M. eds), pp 117-133, Martinus Nijhoff Publishing Co., The Hague- Netherlands. RAMADORI G., SIPE J.D., DINARELLO C.A., MIZEL S.B. and COLTEN H.R. (1985). J. Exp. Med., 162, 930-942. PERLMUTTER D.H., DINARELLO C.A., PUNSAL P.I., COLTEN H.R. (1986). J. Clin. invest., 78, 1349-1354. MOSHAGE H.J., JANSSEN J.A.M., FRANSSEN J.H., HAFKENSCHEID J.C.M. and YAP S.H. (1987). J. Clin. Invest.,79, 1635-1641. BAUMANN H., ONORATO V., GAULDIE J. and JAHREIS G.P. (1987). J. Biol. Chem., 262, 9756-9768. CASTELL J.V., GOMEZ-LECHON M.J., DAVID M., HIRANO T., KISHIMOTO T. and HEINRICH P.C. (1988). FEBS Lett., 232, 347-350. WAHL S.M., MCCARTNEY-FRANCIS N. and MERGENHAGEN S.(1989).lmmunol. Today, 10, 258-261. DUGAICZYK A., LAW S.W. and DENNISON O.E. (1982). Proc. Natl. Acad. Sci., 79, 71-75. 653
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1990
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GUGUEN-GUILLOUZO C., CAMPION J.P., BRISSOT P., GlAlSE D., LAUNOIS B., BOUREL M. and GUILLOUZO A.(1982). Cell BioLlnt. Rep., 6, 625628. CLEMENT B., GUGUEN-GUILLOUZO C., CAMPION J.P., GLAISE D., BOUREL M. and GUILLOUZO A. (1984). Hepatology, 4, 373-380. LAURELL C.B.(1967). In Protides in Biological Fluids (Peeters H. ed), Vol 14, pp 499502, Pergamon press, Oxford. GUILLOUZO A., DELERS F., CLEMENT B., BERNARD N. and ENGLER R.(1984). Biochem. Biophys. Res. Commun., 120, 311-317. LAEMMLI U.K. (1970). Nature, 227, 690-695. GLISIN V., CRKVENJAKOV R. and BYUS C. (1974). Biochemistry, 13, 26332637. LAIHO M., SAKSELA 0. and KESKI-OJA J. (1986). Exp. Cell Res., 164, 399407. HELSETH E., DALEN A., UNSGAARD G., SKANDSEN T., GRONDAHL- HANSEN J. and LUND L.R. (1988). APMIS, 96, 845-849. CZAJA M.J., WEINER F.R., FLANDERS K.C., GIAMBRONE M.A., WIND R., BIEMPICA L. and ZERN M.A. (1989). J. Cell Biol., 108, 2477-2482. KESKI-OJA J., RAGHOW R., SAWDEY M., LOSKUTOFF D.J., POSTLETHWAITE A.E., KANG A.H. and MOSES H.L. (1987). J. Biol. Chem., 263, 3111-3115. JUHAN-VAGUE I., AILLAUD M.F., DE COCK F., PHILIP-JOET C. ARNAUD C., SERRADIMIGNI A. and COLLEN D. (1985). in Progress in fibrinolysis (Davidson J.F., Donati M.B. and Coccheri 8, eds), Vol 7, p 146 - Edinburgh, Churchill Livingstone. MORRONEG., CORTESE R. andSORRENTlNOV.(1989). EMBO J.,8,37673771 NAKAMURA T., TOMITA Y., HIRAI R., YAMAOKA K., KAJI K. and ICHIHARA A. (1985). Biophys. Res. Commun., 133,1042-1050. CARR B.I., HAYASHI I., BRANUM E.L. and MOSES H.l.(1986). Cancer Res., 46, 2330-2334. BRAUN L., MEAD J.E., PANZICA M., MIKUMO R., BELL G.I. and FAUST0 N (1988). Proc. Natl. Acad. Sci., 85, 1539-1543.
654
171,
No.
September
2, 1990 14,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Pages
647-654
TRANSFORMING GROWTH-FACTOR-8 (TGF-l3) INHIBITS ALBUMIN SYNTHESIS IN NORMAL HUMAN HEPAtOCYTES AND IN HEPATOMA HepGP CELLS’ Nathalie BUSSO, * Christophe CHESNE, + ’ # Francois DELERS , * Fabrice MOREL, + and Andre GUILLOUZO + * Laboratoires GLAXO, Centre de Recherches, 25 avenue du Quebec , 91951 Les Ulis Cedex, France + INSERM U49, Hopital de Pontchaillou , 35033 Rennes Cedex,
France
# BIOPREDIC , Hopital de Pontchaillou , 35033 Rennes Cedex, France * Laboratoire des Proteines de la Reaction lnflammatoire, 45 rue des Saints Peres, 75270 Paris Cedex 06, France Received
July
23,
1990
We explored the effect of transforming growth factor I3 (TGF-O), a cytokine that appears to play a central role in inflammatory events, on albumin expression by normal adult human hepatocytes and hepatoma cells. Addition of TGF-6 to primary human hepatocyte cultures resulted in a dramatic decrease in albumin accumulation and synthesis. This effect was dose-dependent, took place after a 48h incubation period and was maintained over 96h. TGF-B-decreased albumin protein levels were associated with reduced albumin mRNA content. Actin mRNA levels were concomittantly increased. Comparable qualitative effects of TGF-8 were observed on human hepatoma HepG2 cells. 0 1990Academic P7xSS. Inc. Tissue injury and inflammatory
processes
induce a host response commonly
referred to as the acute phase reaction (1). Adaptation of liver cells to the acute phase reaction is characterized derived
plasma
consequently
by changes in the biosynthetic
proteins
the plasmatic
(acute-phase concentration
whereas that of some others is reduced albumin, the most abundant hepatocytes. inflammatory
proteins).
pattern of a subset of liver-
The hepatic
production,
and
of some of these proteins, is increased, (for review see 2). Among the latter ones is
serum protein, the synthesis of which is restricted to
Indeed, hypoalbuminemia
has been frequently
found in patients with
diseases (3). Moreover inflammatory mediators secreted by mononuclear
‘This work was partly supported Recherche Medicale (INSERM).
by the lnstitut National de la Sante Et de la
The abbreviations used are : BSA, Bovine serum albumin; SDSSodium Dodecyl Sulfate; SDS-PAGE, Polyacrylamide gel electrophoresis in the presence of SDS; IL-l-8, Interleukin-16; IL-6, Interleukin-6; MEM, Minimum Essential Medium; PBS,Phosphate-Buffered Saline; TNF-a, Tumor Necrosis Factor-o!.
647
0006-291XE’O $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 171, No. 2, 1990 phagocytes
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
such as IL-l-O, TNF-a and IL-6, modulate acute phase protein expression,
and notably decrease albumin synthesis in a variety of in vivo and in vitro models (48). TGF-8, another multifunctional
molecule secreted
appears to play a central role in inflammatory
by mononuclear
phagocytes,
events (for review see 9). Thus far,
however, there is no direct evidence for TGF-8 as an acute phase mediator in human hepatocytes.
This prompted
us to investigate the effect of TGF-8 on the production
albumin by normal adult human hepatocytes
as well as by the human
of
hepatoma
HepG2 cell line.
EXPERIMENTAL PROCEDURES MATERIALS: Cell culture reagents were from Gibco (Cergy-Pontoise, France). TGF-8 type 1 purified from human platelets was from R & D (Minneapolis, MN). Recombinant human TNF-a! (specific activity 2.10’ u/mg) was from Genzyme (Boston, MA). Recombinant human IL-l-8 (specific activity 2.2 10’ u/mg) and a 21 bases oligonucleotide probe for human 8 actin were obtained from Glaxo Institute for Molecular Biology (Geneva, Switzerland). The human albumin cDNA probe was generously provided by A. Dugaiczyk (10). Rabbit anti-human albumin serum was from Behring (Rue&Malmaison, France) and protein A-Sepharose from Sigma (La Verpilliere, France). Amplify, [“Cl methylated protein standards, L-[?S] methionine (800 Ci/mmol), deoxycytidine 5’[a-=P] triphosphate (> 3000 Ci/mmol) and adenosine 5’[ “P] triphosphate (> 5000 Ci/mmol) were from Amersham (Les Ulis, France). All other chemicals were of the best commercial grade available. CELL CULTURES: Human hepatic tissue fragments were obtained from organ donors (3 male subjects, 22, 23 and 27 years old). Sampling was made in accordance with French legal considerations. Adult human hepatocytes were isolated by the two-step collagenase perfusion method as previously described (11). Hepatocytes were seeded at a density of 1.5 x lo5 cells per well in 24 well plates in 75 % MEM and 25 % medium 199 supplemented with 0.1 mg/ml BSA, 10 kg/ml bovine insulin and 10 % fetal calf serum (12). For RNA extraction cells were seeded at a density of 10’ cells per T75cm2 flask. After attachment the cells were maintained in serum-free medium complemented with 7 x 10”M hydrocortisone hemisuccinate. Primary cultures consisted of more than 95 % hepatocytes. All tested agents were added 24h or 48h after cell plating in serumfree medium with hydrocortisone and 0.1 mg/ml BSA. Neither morphological alterations nor lactate dehydrogenase leakage were observed during the incubation period in the presence of the agents. The human hepatoma cell line HepG2 was provided by the American Type Culture Collection (Rockville, MD). Cells were grown in MEM (Eagle) with non essential amino acids, sodium pyruvate and 10 % fetal calf serum. All tested agents were added at confluency in serum-free medium supplemented with 0.1 mg/ml BSA. ALBUMIN IMMUNOELECTROPHORESIS: This was performed method of Laurel1 (13) as previously described (14).
by a modification
ofthe
BIOSYNTHETIC LABELING AND IMMUNOPRECIPITATION: Cell monolayers in 24 wells were incubated with or without various TGF-8 concentrations for 48h. Medium was removed and monolayers were washed twice with methionine-deficient medium. Hepatocytes were labeled with 12.5 pCi/well of[J5S]-methionine for 4h in serum-free medium containing 5 % of normal concentration of methionine in the presence or absence of TGF-8. The medium was harvested, the monolayers washed twice with cold PBS and the cells were lysed in 0.1 M Tris-HCI pH 8.1, 0.4 % Triton X-100. The
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AND BIOPHYSICAL
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effect of TGF-8 on total protein synthesis was determined by measuring the incorporation of [?S]-methionine into trichloroacetic acid precipitable material. After normalization of the loaded media volumes with respect to the corresponding rate of protein synthesis media samples were electrophoresed in the presence of SDS in 10 % polyacrylamide gels under reducing conditions as described by Laemmli (15). Gels were treated with Amplify, dried and fluorographed. For immunoprecipitation of albumin, samples of labeled culture media, obtained as described above, were incubated overnight at 4°C with anti-human albumin or irrelevant rabbit antisera diluted l/5 with PBS. Immune complexes were pelleted with excess protein A-sepharose. After centrifugation the immune pellets were washed, eluted by boiling in sample buffer and electrophoresed under reducing conditions as below. [‘“Cl-methylated molecular mass markers (200, 92.5, 69, 46, 30, 21.5, 14.3 Kd) were included on all gels. RNA ANALYSIS: Total cellular RNA was isolated from monolayers of hepatocytes by the guanidinium isothiocyanate/cesium chloride method (16). For Northern-blot hybridizations, fractionated RNAs were transferred onto nylon membranes. The amount of RNA per lane was controlled by staining the bound RNA on filters with 0.05 % methylene blue and destaining in 70 % ethanol. The RNAs were hybridized to the nick-translated [3’P] labeled albumin probe. After this first hybridization, the probe was eluted by boiling the membrane 5 min in 20 mM Tris-HCI pH 8.1 and the filter rehybridized with the 5’end-[32P]labeled actin probe. Filters were exposed to Kodak XAR-5 films at -80°C between intensifying screens. Relative mRNA levels were determined by densitometric analysis of the autoradiograms. RESULTS When primary adult human hepatocytes were incubated for 48 h in the presence of 2 rig/ml of TGF-8, a striking inhibition (> 95 %) of albumin accumulation medium inhibition
was measured starting
(Tablei).
at 1 rig/ml
This effect was dose-dependent
of TGF-8 (data not shown). TABLE
into culture
with maximal
Concomitantly
with a
1
INHIBITION OF ALBUMIN ACCU ULATION IN PRIMARY CULTURES OF ADULT HUMAN HEPATOCMES BY TGF:: COMPARISON WITH THE EFFECT OF TWO OTHER CYTOKINES AGENTS
ALBUMIN
CONCENTRATION
(% of control cultures) 223
TGF-I3 2 rig/ml IL-l-0
100 u/ml
29 2 22
TNF-a
100 rig/ml
34 f 20
Hepatocytes were plated into 24 well plates at 1.5 x 10” cells/well. Duplicate or triplicate monolayers were treated after a recovery period of 24-48h with 0.5 ml of serum-free medium and the indicated factors. Following 24h stimulation the medium was replaced by 0.5 ml of fresh medium supplemented with the same agents. After an additional 24h, the medium was collected and aliquots were tested by immunoelectrophoresis for albumin concentration. Results are expressed as percent of the albumin concentration measured in untreated cultures (mean 2 SD). 649
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decreased albumin amount, we measured in samples from TGF-8 treated hepatocytes increased plasminogen
activator inhibitor type 1 (PAI-1) and urokinase
(manuscript in preparation),
(U-PA) levels
thus ruling out a non-specific action of TGF-8 on albumin
resulting from a general toxicity; similar results on PAI- and U-PA modulation
by TGF-
8 have been previously reported in other cell types (17, 18). The effect of TGF-8 on albumin supraoptimal
was compared
doses of IL-l-6 and TNF-a. These two cytokines
levels by 70 % (Tablel) 7). Taken together, compared
concentration
confirming
earlier observations
to the effects of decreased
on other cellular models (5
these results indicate that TGF-8 has a more potent
to IL-l-6 and TNF-a. At the concentrations
effect of TGF-8 on albumin accumulation,
as those of IL-l-6 and TNF-a, were barely after 48 h and
over a 96 h period of incubation (data not shown). The modulation
of albumin levels by TGF-8 in the human hepatoma comparable
effect
indicated in table I, the inhibitory
detectable after the first 24 h incubation but became more pronounced were maintained
albumin
to that observed
in albumin concentration
in primary
human
cell line HepG2 was qualitatively hepatocytes
(maximal
inhibition
of 50 % reached at 48 h with 10 rig/ml of TGF-13, data not
shown). To determine if the observed inhibitory effect of TGF-8 on albumin accumulation in the medium
occurred
at the biosynthetic
level, hepatocytes
were incubated
in
medium containing varying amounts of TGF-8 for 48 h, and biosynthetically labeled for 4 h in the presence of the same amounts of TGF-6. TGF-6, at an optimal dose, inhibited albumin synthesis by 5-fold (as measured by densitometric 1A). This effect was dose-dependent protein synthesis
(Figure 1B). Although
up to 50 % at the highest concentration,
synthesis was specific since TGF-8 in the same conditions immunologically uncharacterized
identified
as
PAI-
(manuscript
analysis of figure
TGF-8 decreased
increased a 47 Kd protein
in preparation)
and
another
27 Kd protein. In addition, TGF-8 increased actin biosynthesis
extracts (not shown). Analysis of the biosynthetic
total
the effect on albumin
in cell
labeling of secreted proteins by the
hepatoma
HepG2 cell line gave a similar pattern (Figure 1C). First, TGF-I3 decreased
albumin
synthesis,
higher concentrations
although
the
dose-response
in this cell line compared
effect
was
shifted
to primary hepatocyte
to
cultures.
Secondly, as in primary cultures, TGF-8 increased the synthesis of actin (not shown) and of PAI-1. Nevertheless TGF-8 did not affect total protein synthesis nor did it induce the 27 Kd protein
in HepG2 cells in contrast
with its effects in primary
human
hepatocytes. TO
further assess the effect of TGF-8 on the expression of the albumin gene, we
extracted total cellular RNAs and subjected them to Northern-blot
hybridization
2). Albumin mRNA level was significantly reduced in both primary hepatocyte 650
(Figure cultures
Vol. 171, No. 2, 1990
Normal
--ab
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS
hepatocytes
HepG2
cells
cd +
TGF-beta
1 rig/ml 10 -
1
+TGF
.l
50
.Ol
-
beta nglml
10
+TGF
1
5
s
.l
beta rig/ml
FIGURE 1 . EFFECT OF TGF-0 ON ALBUMIN SYNTHESIS Hepatocyte monolayers were incubated in medium without (-) or with (+) TGFR atthe~ ’ indicated doses for 4Bh and then radiolabeled for 4h in serum-free medium containing [“S] methionine (50 clCi/ml) in the presence or absence of TGF-0. Samples of culture media from primary hepatocytes (figure IB) or HepGP cell cultures (figure 1C) were analysed directly by electrophoresis under reducing conditions. Alternatively, the labeled culture media samples from primary hepatocyte cultures were immunoprecipitated with anti albumin (tracks b and d) or with irrelevant (tracks a and c) rabbit antisera. Immune pellets were eluted and electrophoresed under reducing conditions (figure 1A). Molecular weight markers were electrophoresed in parallel on all gels. Gels were treated with amplify, dried and fluorographed.
and
HepG2
densitometric
cells (74% and
as measured
by
analysis). As a control we analysed in parallel RNAs from IL-i-0
and
TNF-a treated cells. As expected
35% of decrease these inflammatory
respectively
decreased
albumin
mRNA steady state level by 70% and 60% respectively in primary hepatocyte
mediators
cultures.
The effect of TGF-6 on albumin mRNA was specific in both primary hepatocyte
cultures
and HepG2 cells since - 1) the amounts of 28s and 18s mRNA were the same in the different RNA samples analysed as quantified by methylene blue staining of ribosomal RNAs (not shown) - 2) actin mRNA content in TGF-I3 treated cells was significantly increased
(260 % in normal hepatocytes,
210% in HepG2 cells). Modulation
of actin
mRNA level is consistent with the TGF-I3 induced increase in biosynthetically
labeled
actin reported above. Moreover, this result is in agreement
with the findings of Czaja
et al. (19) who measured a slight increase of actin mRNA after TGF-f3 treatment hepatocytes,
and with the 2-fold induction
different cell types (20). 651
of O-actin mRNA content
of rat
by TGF-B in
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Normal
albumin
AND BIOPHYSICAL
hepatocytes
HepG2
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cells
-
-albumin
-
actin -
C IL-1 TNF
TGF
C
IL-1
TNF
actin
TGF
FIGURE 2 . EFFECT OF TGF-B ON ALBUMIN mRNA CONTENT Hepatocyte monolayers were incubated for 4Bh (normal hepatocytes) or for 24h (HepGP cells) in serum-free medium containing 0.1 mg/ml BSA (C = control) or in serum-free medium supplemented with 100 u/ml IL-I-B (IL-l), with 100 rig/ml TNFQ (TNF) or with 2 rig/ml (normal hepatocytes) or 10 rig/ml (HepG2 cells) TGF-R (TGF). Northern-blot hybridization analysis was performed using 12 pg of total cellular RNA per track. The filter was hybridized first to an albumin -P-labeled cDNA probe, then to a B-actin =P-labeled oligonucleotidic probe.
DISCUSSION The accumulation
results
reported
here
show
that
TGF-l3
(> 95 % inhibition) of the negative acute-phase
normal human hepatocyte same conditions,
and human hepatoma
dramatically
decreases
protein albumin in both
HepG2 cell line culture media. In the
albumin mRNA levels are decreased
by 2-4 fold. Thus, it appears
doubtful that these limited observed changes in albumin mRNA levels are sufficient to account for the marked effect of TGF-t3 on albumin. Several other factors may account for such a discrepancy
: translation efficiency of albumin mRNA may vary ; alternatively
albumin accumulation
may be influenced by clearance mechanisms.
Several other hepatocytes
proteins
are also modulated
by TGF-I3 in primary
and HepG2 cells ; amongst these is plasminogen
1 (PAL1 manuscript
in preparation),
a positive acute phase protein (21). In hepatoma
cells, it has been recently reported apolipoprotein
human
activator inhibitor type-
that in addition
to albumin,TGF-l3
decreases
A-l, another negatively regulated protein during acute phase reaction
(22). The fact that TGF-0 is able to act on negative (albumin and apolipoprotein
A-l)
as well as on positive (PAI-1) acute phase proteins, suggests that this cytokine may be an important
mediator of the acute phase response.
In this regard it would be of
interest to determine the effect of TGF-l3 on other acute phase proteins in normal adult human hepatocytes. Comparison
of the biosynthetic
protein pattern of primary human hepatocytes
and of HepG2 cells in the presence of TGF-8 revealed that essentially the same 652
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spectrum
BIOCHEMICAL
of proteins was affected;
AND BIOPHYSICAL
nevertheless
RESEARCH COMMUNICATIONS
HepG2 cells appeared
to be less
sensitive to TGF-8 but still remain a suitable in vitro system to study TGF-8 effects. TGF-8 seems to have an important role in hepatic pathophysiology. reported to be a potent inhibitor of hepatocyte
proliferation in vitro (23-25). Moreover
hepatic TGF-8 mRNA levels increased after partial hepatectomy
in the nonparenchymal
liver cells (25) ; thus TGF-8 may function in vivo as the effector paracrine
mechanism
regeneration. production
to
prevent
uncontrolled
In view of these observations, during liver regeneration.
It has been
hepatocyte
of an inhibitory
growth
during
liver
it would be interesting to examine albumin
In two different in vivo models of hepatic fibrosis
it has been reported that TGF-8 gene expression in the liver was associated
with the
increase in collagen synthesis (19). In these models the authors have also observed that albumin mRNA content declined with the progression TGF-8 synthesis increased).
of fibrosis (i.e. as hepatic
However, these authors have not found a direct effect
of TGF-8 on albumin mRNA content of rat primary hepatocytes
after a 24h incubation
in the presence of the cytokine. This lack of effect may be due to a species difference or to the delayed action of TGF-8 on albumin ; indeed we have shown that the effect of TGF-I3 was barely detectable
after 24h but was drastic after 48h. In this context, it
seems likely that the decreased
level of albumin in hepatic fibrosis could result from
a autocrineiparacrine
modulation
by TGF-8.
ACKNOWLEDGMENT We thank E. Nicodeme for her expert technical assistance. REFERENCES 1. 2. 3.
4. 5. 6. 7. 8. 9. 10.
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