TNF-
modulates expression of the tissue transglutaminase
gene in liver cells
Gerald S.
Kuncio1,
Mariya
Tsyganskaya1,
Jianling
Zhu1,
Shu-Ling
Liu1,
Laszlo
Nagy2,
Vilmos
Thomazy2,
Peter J. A.
Davies3, and
Mark A.
Zern1
1 Department of Medicine and
the Department of Pathology, Anatomy, and Cell Biology, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107;
2 Department of Integrative
Biology, Physiology, and Pharmacology, University of Texas Medical
School, Houston 77096; and
3 Department of Pharmacology,
University of Texas-Houston Medical School, Houston, Texas 77225
 |
ABSTRACT |
One of several
postulated roles for tissue transglutaminase (tTG) is the stabilization
and assembly of extracellular matrix via peptide cross-linking. We
previously determined that tTG activity increased in an animal model of
hepatic fibrogenesis and in human liver disease. To further study the
role of tTG in liver disease, we initiated investigations into the
effect of a proinflammatory mediator, tumor necrosis factor (TNF)-
,
on tTG activity in cultured liver cells. Treatment of human Hep G2
cells with 1 ng/ml TNF-
increased
[14C]putrescine
cross-linking to cellular proteins. An increase in tTG mRNA content was
observed 1 h after addition of TNF-
, and levels of tTG mRNA remained
elevated after 24 h. Hep G2 cells, transiently transfected with a
luciferase reporter containing 1.67 kb of the human tTG promoter,
showed an increase in reporter activity after addition of TNF-
. Gel
shift experiments using nuclear extracts from TNF-
-treated cells and
oligonucleotides containing the tTG nuclear factor (NF)-
B motif
revealed increased binding, concordant with mRNA data. Transient
transfections with a truncated reporter construct lacking the tTG
NF-
B sequence showed an attenuated response to TNF-
treatment.
Similar responses were seen in stably transfected HeLa cells. Primary
hepatocytes isolated from a trangenic mouse line containing the mouse
tTG promoter driving the
-galactosidase reporter, show similar
time-dependent increases in promoter activity when treated with
TNF-
. Furthermore, Hep G2 cells are incapable of upmodulating tTG
promoter reporter activity in the presence of TNF-
when those cells
overexpress a transdominant, negative mutant NF-
B subunit. Because
TNF-
expression is upregulated in hepatic inflammation, the data
suggest TNF-
-mediated increases in tTG expression may play an
important role in the process of hepatic fibrogenesis.
transcriptional regulation; nuclear factor-
B; hepatocytes
 |
INTRODUCTION |
THE CALCIUM-DEPENDENT specific cross-linking of
-amines and
-glutamyl residues is accomplished by a family of
enzymes termed transglutaminases (1, 9, 10). These enzymes participate in a variety of cellular and tissue functions and have been implicated in the formation of stabilizing cross-links among extracellular protein
components. For example, a distinct keratinocyte transglutaminase is
upregulated during epidermis formation and catalyzes significant cross-linking among keratin and several additional extracellular proteins (12). Furthermore, the establishment of fibrin clots is
dependent on activation of factor XIIIa, another transglutaminase (15).
An additional family member, the ubiquitous tissue transglutaminase (tTG), can be localized to the cell membrane and detected in a secreted
form, and tTG appears to be present intracellularly, partitioning
between membrane-bound and soluble compartments (1). The physiological
function of this intracellular form remains largely unknown. However,
it has been known for some time that tTG also has
guanosinetriphosphatase activity as well, and recent studies identify
tTG as the GTP-binding, Gh
subunit, which couples the
1
-adrenergic receptor to a
unique form of phospholipase C (19). Such data implicate tTG in a
variety of signal transduction events in addition to cross-linking
activity.
Several groups have described a role for tTG in cross-linking
fibronectin, osteonectin, osteopontin, laminin, nidogen, and other
extracellular matrix components (1, 9). Analogous to its noted
participation in wound healing, it is tempting to speculate that
extracellular tTG may participate in other tissue remodeling processes
subsequent to cell injury and death, such as fibrogenesis. Indeed, we
recently documented a dramatic rise in tTG activity during the
induction of liver fibrosis in rats by
CCl4 damage and in human patients
with acute liver disease (17).
Liver fibrosis represents a common terminal stage of disease
precipitated by a variety of etiologies leading to sustained cellular
injury. The initiation of fibrogenesis appears to involve several
events mediated by proinflammatory and cytotoxic cytokines, such as
tumor necrosis factor (TNF)-
, interleukin (IL)-1
, and later IL-6,
and transforming growth factor (TGF)-
isoforms (5, 7). It would
therefore be reasonable to suggest that tTG could participate in the
deposition of excess extracellular matrix seen in fibrotic diseases.
Because our earlier studies also revealed an increase in nuclear factor
(NF)-
B binding to tTG promoter sequences accompanying liver fibrosis
and TNF-
is known to be a major activator of NF-
B binding, a
potential role for TNF-
is strongly suggested in tTG expression. The
following in vitro experiments support a significant role for TNF-
modulation of tTG gene activity.
 |
MATERIALS AND METHODS |
Cell culture and manipulation.
Cultures of human hepatoblastoma cells (Hep G2) or cervical carcinoma
cells (HeLa) were incubated at 37°C in 5%
CO2 in modified Eagle's medium
supplemented with 100 U /ml penicillin, 100 U/ml streptomycin, and 10%
fetal calf serum (Life Technologies, Gaithersburg, MD). Cells were
inoculated into 10 ml of medium in 100-mm plastic petri dishes
(Corning, NY) or into 3-ml medium per well in six-well culture plates
and incubated until 70-80% confluency was achieved. We noted that
addition of large amounts of serum-containing medium stimulated an
increase in transglutaminase activity. As a result, no more than 20 ml
of fresh medium with or without 1 ng/ml (440 U) human TNF-
(Genzyme, Boston, MA) per milliliter of culture medium were added for a
period of 1-24 h before cell harvest. This protocol placed cells
at the final stages of exponential growth at the time of assay.
For some experiments we used cells from lineage 27, a mouse line
transgenic for the
-galactosidase reporter driven by 4 kb of the
murine tTG promoter (L. Nagy, V. A. Thomazy, M. M. Saydak, J. P. Stein, and P. J. A. Davies, unpublished data). Primary mouse hepatocytes, grown in six-well cultures containing William's E medium
supplemented with 10% fetal calf serum, were isolated as previously
described (17). TNF-
treatment was as previously described.
Assay for transglutaminase activity.
Cells were inoculated into six-well plates and grown as previously
described. TNF-
at 1 ng/ml was added to the medium at 1, 2, 6, or 24 h before terminating the experiment. One hour before cell harvest,
[14C]putrescine (100 µCi) was added to each culture. Cells were washed once with ice-cold
Hanks' buffered salts, scraped into centrifuge tubes, and pelleted by
low-speed centrifugation. The cell pellet was washed once more with
Hanks' salts and transferred to a 1.5-ml microfuge tube. After
low-speed centrifugation the cell pellet was precipitated with 5%
trichloroacetic acid (TCA) and placed on ice for 15 min. The insoluble
protein pellet was washed two times with ice-cold 5% TCA and recovered
by high-speed centrifugation. After solubilization of the pellet in 1 N
NaOH, radioactivity was determined by liquid scintillation counting.
Samples were normalized to protein by bicinchoninic acid (BCA; Ref. 24)
determination of an aliquot of each solublized pellet.
Plasmid transfection and stable line construction.
Equimolar amounts corresponding to 3 µg of the pHTGP2 or pHTGP2-mut3
(16) constructs were used in all calcium-phosphate transfections (20).
The plasmid DNA was mixed thoroughly in 220 µl of sterile
H2O in a 15-ml conical tubes.
Sterile 2 M CaCl2 (30 µl) was
added slowly to the surface of the DNA solution followed by the slow
addition of 250 µl of sterile 2× Hanks' buffered salt solution
(in mM: 280 NaCl, 10 KCl, 1.5 Na2HPO4,
12 glucose, 50 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid; pH 7.05) to the bottom of the tube. The solution was
then mixed gently and a DNA-calcium-phosphate precipitate was allowed
to form at room temperature. The precipitate was mixed and gently added
to a 10-ml 100-mm petri dish cell culture. The cultures were
subsequently incubated overnight for 16-20 h. The medium was
removed, and the cells were recovered by trypsinization and low-speed
centrifugation. The cells were then replated into wells of a six-well
plate, and incubation was continued for 24 h to allow the
cells to recover. Treatment with TNF-
began at this time. Control
experiments previously demonstrated that reporter expression continues
to be elevated during this period. Untreated, paired time controls were
comparable, indicating that expression of the transiently transfected
reporter was equivalent over the course of the experiment.
A stable Hep G2 cell line was produced by cotransfecting 10 µg of the
mutant p50 expression construct, cytomegalovirus (CMV)-
SP, a kind
gift of A. Israel (14), with 1 µg of a plasmid containing the
neomycin-coding region driven by the SV40 promoter and enhancer (25). After 24 h the cells were trypsinized, and
neomycin-resistant cells were selected by replating in medium
containing 700 µg/ml of the neomycin analog G-418. Incubation
continued for 3 wk with frequent replacement of selection medium.
Several clones were selected and expanded, and frozen stocks were
prepared. Experiments were performed on a clonal stock that had
previously tested as having no detectable nuclear extract binding to
NF-
B DNA motifs after 24 h of treatment with TNF-
.
Lines of HeLa cells stably transfected with either the full-length tTG
promoter pHTGP2 or the NF-
B-deficient promoter pHTGP2-mut3 were
generated as above.
Luciferase and
-galactosidase expression.
After incubation the cultures were washed once in phosphate-buffered
saline. Cells were lysed by the addition of 200 µl of cell lysis
buffer (Promega, Madison, WI) followed by gentle shaking. The lysate
was transferred to a 1.5-ml tube and clarified with a 2-min
centrifugation at 16,000 rpm. The pellets were discarded, and the
supernatants were either assayed immediately or stored at
70°C. The protein concentration of the lysate was determined by the BCA method of Smith et al. (24).
Luciferase activity was assayed, with modifications, according to deWet
et al. (4). Reactions were performed in 300-µl volumes containing
25-200 µl of cell lysate and reaction buffer at a final
concentration of 0.125 M tris(hydroxymethyl)aminomethane (pH 7.2)
and (in mM) 1 dithiothreitol, 1 ATP, 0.5 acetyl CoA, and 5 MgCl2. Light emissions were
measured directly and integrated over a 30-s period at room temperature
after the injection of 100 µl of freshly prepared
D-luciferin (Sigma
Chemical, St. Louis, MO) into a luminometer (Lumat LB9501, Berthold
Analytical, Nashua, NH). Blank reactions were determined with
equivalent volumes of lysis buffer substituted for cell lysates.
The data were expressed as relative activity calculated as the
luciferase activity per milligram protein of the test construct divided
by the luciferase activity per milligram protein of the untreated
samples.
-Galactosidase was measured on cell lysates as per the
manufacturer's recommendations (Promega, Madison, WI) and normalized to untreated control cell lysates.
mRNA analysis and electrophoretic mobility studies.
Northern analysis for tTG transcripts was performed as previously
described (17) on RNA extracted from Hep G2 cultures grown in T-25
flasks with 10 ml medium. Cultures were incubated with no or 1 ng/ml
TNF-
for 1, 2, 6, or 24 h before extraction.
Gel-mobility shift analyses were performed using nuclear extracts from
untreated or 1-, 6-, or 24-h TNF-
-treated Hep G2 cultures grown in
T-25 flasks as previously described (17).
32P-labeled, double-stranded
oligonucleotides of 30 bp, containing the NF-
B motif plus flanking
sequences at nucleotide positions
1,338 to
1,350 of the
tTG promoter, were used for nuclear extract binding reactions. Control
reactions were performed using cold, excess oligonucleotides as
competitors. The p50-p50 dimer-shifted bands and p50-p65-shifted bands
were identified by supershifting with p50 or p65 antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA).
 |
RESULTS |
Using the active incorporation of the externally applied amine donor
[14C]putrescine into
Hep G2 cellular proteins as an assay for transglutaminase, we noted a
time-dependent increase in incorporation after treatment with low
levels of TNF-
(Fig. 1). It can be seen
that activity is elevated after only 1 h of treatment of TNF-
and
that maximal activity is seen at 6 h. Activity is still increased after
a 24-h incubation with the cytokine. The increase in transglutaminase activity is not correlated to Hep G2 cell death because previous studies indicated that at the levels of TNF-
used in these
experiments there is no cell necrosis (21).

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Fig. 1.
Assay of transglutaminase activity in Hep G2 cells treated with tumor
necrosis factor (TNF)- . Hep G2 cells were incubated with no or 1 ng/ml TNF- for 1, 2, 6, or 24 h. One hour before harvest, 100 µCi
of[14C]putrescine were
added to each culture. Total radioactivity into protein was determined
by trichloroacetic acid extraction and liquid scintillation counting.
Protein was determined by bicinchoninic acid reaction. Results are
expressed in disintegrations per minute per milligram protein in
treated cultures vs. disintegrations per minute per milligram protein
in untreated cultures.
|
|
Because of complex regulation, the cross-linking activity of tTG can be
modulated without prior synthesis of protein (3, 23). Therefore, enzyme
levels cannot be used as a direct assay of mRNA levels or
transcriptional activity. We directly examined changes in the levels of
mRNA specific for tTG via Northern blot analysis after treatment with
TNF-
. The results depicted in Fig. 2
show good correlation with the previously determined enzyme levels.
Again, mRNA levels (normalized to glyceraldehyde-3-phosphate dehydrogenase levels) increase at 1 h to a maximum at 6 h, remaining high after 24 h of incubation with TNF-
.

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Fig. 2.
Tissue transglutaminase (tTG) mRNA levels in Hep G2 cells treated with
TNF- . Total RNA was extracted from Hep G2 cells treated for various
durations with TNF- , followed by gel electrophoresis and blotting.
Blots were probed with a mouse tTG cDNA or rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA and exposed to a
Phosphorimager screen. Data are presented as the density ratio of tTG
to GAPDH in treated cells normalized to untreated culture ratios.
|
|
Using subconfluent cultures of Hep G2 cells for transfection, a
luciferase construct containing 1.67 kb of the human tTG promoter (pHTGP2, nucleotide position +72 to
1,665 ), and an assay of luciferase activity as a reporter for tTG promoter response, we observed (Fig. 3) that treatment with
TNF-
stimulated the expression of luciferase activity. The time
course and magnitude of this response were almost identical to that
observed with the mRNA levels noted in Fig. 2. Because of the
transfection experimental protocol used with these experiments,
expression level data are normalized to untreated culture controls,
thus avoiding the need for transfection efficiency measurements. In
contrast, deletion of 1.1 kb of upstream sequences in the tTG promoter
(pHTGP2-mut3, nucleotide position +72 to
561) results in a
highly attenuated response to TNF-
(Fig. 3). The truncated 0.56-kb
construct, pHTGP2-mut3, was previously shown to have no decrease in
constitutive activity under normal conditions compared with the full
length 1.67-kb promoter construct pHTGP2 (16). The deleted region was
noted to contain several other transcription regulatory binding motifs, including a glucocorticoid response element at
1,399 and an IL-6 response element at
1,190, in addition to the putative NF-
B binding motif at
1,338. Therefore, sequences within the deleted upstream segment must be involved in the appropriate upregulation in
response to TNF-
.

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Fig. 3.
tTG promoter activity in Hep G2 cells treated with TNF- . Hep G2
cells were calcium phosphate transfected with either a 1.67- kb human
tTG promoter-luciferase construct (wt, ) or a 0.56-kb human tTG
promoter-luciferase construct lacking a nuclear factor (NF)- B
binding motif (mut-3, ). Transfected cells were replated and treated
for varying durations with TNF- . Luciferase and protein assays were
performed on cell lysates. Data are presented as the ratio of treated
light units per milligram protein to untreated light units per
milligram protein. Each point represents triplicate samples with SD
error bars.
|
|
To determine whether the effects we observed in the hepatoma-derived
cell line also occurred in other cells, we undertook studies with HeLa
cells. HeLa cells stably transfected with the 1.67-kb tTG reporter
construct also showed stimulation of activity after incubation for 24 h
with TNF-
(Fig. 4). Moreover, HeLa cells
stably transfected with the truncated, 0.56-kb tTG reporter construct
showed an attenuated response to TNF-
, as had occurred in the Hep G2
system. In addition, freshly isolated primary hepatocytes from a
transgenic mouse line, stably transfected with a 4-kb tTG promoter-
-galactosidase reporter construct, showed stimulation of
reporter activity after incubation for up to 24 h with TNF-
(Fig.
5).

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Fig. 4.
tTG promoter activity in HeLa cells treated with TNF- . Stippled
bars, control; solid bars, treated with TNF- . Stable HeLa cell
transfectants were made with either a full length 1.67-kb tTG promoter
construct (wt) or a truncated 0.56 kb-tTG promoter construct (mut-3) as
described in MATERIALS AND METHODS.
Each stable cell line was then treated for 24 h with TNF- .
Luciferase and protein assays were performed on cell lysates. Data are
presented as ratio of treated light units per milligram protein to
untreated light units per milligram protein. Each point represents
triplicate samples with SD error bars.
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Fig. 5.
tTG promoter activity in primary mouse hepatocytes treated with
TNF- . A stable transgenic line containing a 4-kb tTG
promoter- -galactosidase reporter construct was used to prepare
primary hepatocytes. Hepatocytes from the transgenic line were then
treated for 1, 5, 6, or 24 h with TNF- . -Galactosidase assays
were performed on cell lysates. Data are presented as the ratio of
treated enzyme activity to untreated enzyme activity. Each point
represents 3-8 samples with SD error bars.
|
|
With use of a segment of the tTG promoter containing the NF-
B
binding motif and nuclear extracts from untreated, control Hep G2 cells
or Hep G2 cells treated for varying times with TNF-
, gel-mobility
shift analyses were performed. A demonstrable increase in binding of
treated culture nuclear extracts to the NF-
B binding motif could be
seen compared with untreated, control extracts (Fig.
6). The rise in binding activity was seen
at 1 h of cytokine incubation, and like the protein, mRNA, and reporter
levels, increased at 6 h followed by a slight decrease (but still
elevated level) after 24 h of incubation. Control experiments with cold
excess oligonucleotides and with p50 and p65 antibodies confirm the
specificity of the binding reaction and identification of the p50
subunit as one of the bound NF-
B species (data not shown). These
findings therefore strongly point to a role of NF-
B binding to the
tTG promoter and upregulating gene activity in response to TNF-
treatment. To test this hypothesis further, we stably transfected Hep
G2 cells with a plasmid expressing a nonfunctional, transdominant mutant of the p50 NF-
B subunit. Control experiments show little binding of nuclear extracts from these cells to consensus NF-
B sequences even after incubation for 24 h with TNF-
(data not shown).
In contrast to cells lacking this mutant, transient transfection of
these cells with a full length, 1.67-kb promoter construct shows no
increase in activity with TNF-
treatment (Fig.
7).

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Fig. 6.
Electrophoretic mobility analysis of Hep G2 nuclear extracts and the
tTG promoter NF- B sequence motif. Nuclear extracts were prepared
from Hep G2 cells treated for varying periods with TNF- .
Gel-mobility shift analyses were performed after incubation of nuclear
extracts with a 32P-labeled,
double-stranded oligonucleotide containing the NF- B binding motif of
the human tTG promoter. Lane 1,
untreated cells; lane 2, cells treated
for 1 h with TNF- ; lane 3, cells
treated for 6 h; lane 4, cells treated
for 24 h. This assay is representative of 3 separate experiments.
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Fig. 7.
Promoter analysis of Hep G2 cells containing mutated NF- B subunit
p50. Hep G2 cells were stably transfected with a constitutively
expressing mutant p50 construct. No NF- B DNA binding could be
detected with nuclear extracts prepared from these cells. The stable
cell line was calcium phosphate transfected with a 1.67-kb human tTG
promoter-luciferase construct. After transient transfection, cells were
replated and treated for varying durations with TNF- . Luciferase and
protein assays were performed on cell lysates. Data are presented as
the ratio of treated light units per milligram protein to untreated
light units per milligram protein. Each point represents triplicate
samples with SD error bars.
|
|
 |
DISCUSSION |
We have been interested in the mechanisms by which fibrogenesis
proceeds in the liver after injury. Extensive fibrosis is a common
pathway in many progressive liver diseases, and inflammatory events
typically produce hepatocyte damage. Several cytokines are known to be
secreted by resident liver cells, as well as invading immune monocytes,
T cells, or B cells. Among these cytokines are TNF-
, IL-1
, the
TGF-
isoforms, and IL-6, whose effects on hepatic tissue during the
injury process are well studied (5, 7). Consequently, this cascade of
inflammatory mediators and their paracrine, juxtacrine, or autocrine
effects, when sustained, often leads to excess extracellular matrix
deposition and the architectural disruption of hepatic tissue and
function.
We have become intrigued by the expression of the enzyme tTG, whose
role in cross-linking extracellular matrix proteins is well documented
(1). Speculative roles of such cross-linking in wound healing (27) and
liver damage (11, 22) have been put forth. In addition, tTG activity in
hepatoma cell lines is stimulated by cytokines such as TGF-
(8) and
IL-6 (26). These observations suggest a possible involvement of
transglutaminase activity in the generation of stabilizing,
cross-linked extracellular matrix molecules during tissue assembly and
maintenance. In particular, increased activity could be regulated by
known matrix-promoting mediators of injury and inflammation. Because
TNF-
is known to be expressed in hepatic injury, regeneration, and
fibrosis and because it enhances NF-
B binding to a variety of genes
(5, 6), it seemed reasonable to investigate the role of this cytokine in affecting changes in the activity of the tTG gene via binding of
NF-
B.
This hypothetical role is supported by our previous studies, in which
liver fibrosis was induced by prolonged exposure to CCl4 (17). Those data show a rapid
rise in transglutaminase activity in all hepatic cell types. The rise
in enzyme activity is correlated with an increase in tTG mRNA activity
as well. When tTG mRNA levels were measured in normal human versus
fulminant liver disease, a large increase was seen in the diseased
livers. After a computer search of potential transcriptional motifs in the tTG promoter, a putative NF-
B site ~1.3 kb upstream from the
start site was identified. Binding of nuclear extracts to this region
increases in parallel to tTG mRNA activity during the course of induced
chronic liver disease.
To investigate the regulation of tTG expression in more detail we
turned to an in vitro model of hepatocyte response, the Hep G2
hepatoblastoma cell line. We show by analysis of enzymatic activity,
mRNA levels, and promoter activation that TNF-
produces a modest,
but significant, stimulation in all cases. Furthermore, 1) specific nuclear factor binding
activity to a tTG NF-
B promoter motif is enhanced by TNF-
treatment, 2) deletion of this motif in the tTG promoter regions leads to a loss of this TNF-
response, and 3) expression of a promoter
reporter is diminished in cells with an attenuated NF-
B binding
capacity. The data therefore implicate TNF-
in upmodulating the
activity of the tTG promoter. This response appears to be generalizable
because data obtained with HeLa cells corroborate the results observed
with Hep G2 cells. Similarly, the tTG promoter in primary murine
hepatocytes appears to respond to TNF-
treatment in an identical
fashion. Moreover, glial transglutaminases are elevated in the presence
of TNF-
(18).
Our results indicate a sustained increase in NF-
B binding over the
course of incubation with TNF-
. This phenomenon may be explained by
the subsequent activation of both TNF-
and IL-6 genes in cells by
NF-
B. This could result in a sustained autocrine stimulation of the
tTG promoter at both the NF-
B promoter motif and its neighboring
IL-6 response element. The deleted region in the truncated reporter
construct contains several transcription regulatory binding motifs,
including a glucocorticoid response element at
1,399 and the
IL-6 response element at
1,190, in addition to the putative
NF-
B binding motif at
1,338. It is therefore possible that
these elements could additionally modulate the level of tTG gene
expression during exposure of cells to TNF-
. In other experiments
(data not shown) we have also demonstrated elevation of tTG promoter
activity by either TGF-
or IL-6 after incubation for 24 h.
Therefore, our data may reflect a bipartite mode of regulation with a
TNF-
role in early events, followed by prolonged increases involving
other factors. We conclude that hepatocytes are susceptible to rapid
stimulation of the tTG gene by TNF-
, a cytokine that is upregulated
during the fibrogenic process. Chronic production of TNF-
and other
inflammatory mediators could stimulate the synthesis and release of
this enzyme, which in turn could play a significant role in the
initiation, formation, and sustained levels of the disruptive
extracellular matrix seen in many fibrotic diseases. Control of
transglutaminase activity therefore may result in important and
innovative therapies for this significant disease.
 |
ACKNOWLEDGEMENTS |
This study was supported in part by National Institutes of Health
Grants AA-06386 and DK-41875 to M. A. Zern.
 |
FOOTNOTES |
Address for reprint requests: M. A. Zern, Dept. of Medicine and Dept.
of Pathology, Anatomy, and Cell Biology, Thomas Jefferson Univ., 901 College Bldg., 1025 Walnut St., Philadelphia, PA 19107.
Received 14 April 1997; accepted in final form 22 October 1997.
 |
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