From the Departments of Biochemistry and Internal
Medicine and Touchstone Center for Diabetes Research, University of
Texas Southwestern Medical Center, Dallas, Texas 75235 and
¶ BetaGene, Inc., Dallas, Texas 75235
Received for publication, September 12, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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Destruction of pancreatic islet Type 1 diabetes is caused by autoimmune destruction of pancreatic
islet Unfortunately, enthusiasm for this important finding is tempered by the
fact that the number of human pancreata available for islet
transplantation in the United States is on the order of several
thousand per year (6, 7), which does not approach the number of
patients that could benefit from this new form of therapy. To deal with
this problem, we and others have been attempting to develop a
replenishable source of cells that could serve as islet surrogates for
cell-based insulin replacement in diabetes (9-13). One working concept
is that immortalized cell lines engineered for robust
glucose-stimulated insulin secretion can be transplanted in the context
of a cell-impermeant macroencapsulation device, preventing contact of
cellular elements of the immune system with the transplanted cells
(11). However, because such devices are envisioned to allow rapid exit
of insulin, as well as highly efficient equilibration of nutrients,
oxygen, and waste products, it is anticipated that soluble mediators of
immunological damage such as cytokines or reactive oxygen species will
readily gain access to the cells within the device. Thus, development
of methods to protect transplanted cells against diffusable,
noncellular mediators of immunological destruction may be important for
the eventual success of cell-based insulin replacement strategies.
Recently, we described a method for selection of cell lines with
resistance to the cytotoxic effects of exposure to a mixture of IL-1 The current study was undertaken to define the factor or factors
responsible for resistance to IFN- Cells and Reagents--
The insulinoma cell line INS-1 (15) and
clonal derivatives thereof (14, 16) were cultured in RPMI 1640 medium
containing 10% fetal calf serum, 10 mM Hepes, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µM Cloning and Overexpression of Rat
STAT-1 Measurement of Cytokine-mediated Cytotoxicity--
Cells were
inoculated at 50,000 cells/well into 48-well tissue culture plates
(Corning Glass, Corning, NY). After 24 h, cells were treated with
1.0 × 109 plaque-forming units (pfu) of
AdCMV-STAT-1 Measurement of Nitrite Formation--
NO production was
estimated by measurement of nitrite levels, as described previously
(14, 21), and data were normalized to protein content per well,
measured by the Bradford assay (Bio-Rad).
Insulin Secretion Experiments--
Cells were cultured in
24-well plates. Assays were initiated by removal of culture medium,
washing of cells with 1 ml of PBS (Life Technologies) and incubation in
1 ml of secretion assay buffer containing 3 mM glucose for
1 h, followed by incubation in secretion assay buffer containing 3 or 15 mM glucose for 2 h at 37 °C. Secretion assay
buffer contains 114 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.16 mM
MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3, 20 mM HEPES, and 0.25%
fatty acid-free bovine albumin (Sigma), pH 7.4. After centrifugation of
media samples at 3000 rpm for 6 min to remove floating cells, the
supernatants were subjected to insulin radioimmunoassay (DPC
Coat-A-Count). The cells remaining in the wells were then washed once
with PBS and lysed with 60 µl of PBS containing 0.5% Triton X-100
for measurement of total protein (Bio-Rad).
Immunoblot Analysis--
Cultured cells were washed once with
PBS and lysed with buffer containing 1% Triton X-100, 50 mM Hepes, 150 mM NaCl, 0.2 mg/ml phenylmethylsulfonyl fluoride, 100 mM NaF, 2 mM
sodium vanadate, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 5 µg/ml
leupeptin, pH 7.2. Lysates were vortexed, kept on ice for 20 min, and
centrifuged at 14,000 rpm in a refrigerated microcentrifuge.
Supernatant fractions were collected, and protein concentrations were
determined. Samples containing 25 or 50 µg of protein were suspended
in 5× sample buffer (300 mM Tris, 10% SDS, 0.5%
bromphenol blue, 50% glycerol, 300 mM RNA Isolation and Blot Hybridization Analysis--
Total RNA was
isolated by extraction with the TRIzol reagent (Life Technologies)
according to the manufacturer's protocol. 10 µg of total RNA was
resolved on a 1.5% formaldehyde/agarose gel, and samples were
transferred to nylon membrane and hybridized in Rapid-Hyb buffer
(Amersham Life Sciences) with 32P-labeled STAT-1 Statistical Methods--
Statistical analysis of the data was
performed using the two-tailed Student's t test, assuming
unequal variances.
Expression of STAT-1
The IFN- STAT-1 IL-1
The differential effects of IL-1 Adenovirus-mediated Expression of STAT-1
Based on these results, the cytokine-sensitive cell line 834/40 was
treated with 1 × 109 pfu/ml of AdCMV-STAT-1 Adenovirus-mediated Overexpression of STAT-1 Adenovirus-mediated Overexpression of STAT-1 In a previous study, we described a method for selecting
insulinoma cell lines with resistance to the cytotoxic effects of IFN- In the current study, these unresolved issues have been investigated.
By tracing the known signal transduction pathway for IFN- Our data also provide fresh insight into cross-talk between separate
cytokine signaling pathways in islet Another interesting example of cross-talk is the apparent interaction
of IFN- IFN- Within this framework, the site at which STAT-1 Our results suggest that expression of STAT-1 Nevertheless, further work will be required before we can conclude that
STAT-1-cells in type
1 diabetes appears to result from direct contact with infiltrating
T-cells and macrophages and exposure to inflammatory cytokines such as interferon (IFN)-
, interleukin (IL)-1
, and tumor necrosis factor TNF-
that such cells produce. We recently reported on a
method for selection of insulinoma cells that are resistant to the
cytotoxic effects of inflammatory cytokines
(INS-1res), involving their growth in progressively
increasing concentrations of IL-1
plus IFN-
, and selection of
surviving cells. In the current study, we have investigated the
molecular mechanism of cytokine resistance in INS-1res
cells. By focusing on the known components of the IFN-
receptor
signaling pathway, we have discovered that expression levels of signal
transducer and activator of transcription (STAT)-1
are closely
correlated with the cytokine-resistant and -sensitive phenotypes. That
STAT-1
is directly involved in development of cytokine resistance is
demonstrated by an increase of viability from 10 ± 2% in control
cells to 50 ± 6% in cells with adenovirus-mediated overexpression of STAT-1
(p < 0.001) after culture
of both cell groups in the presence of 100 units/ml IFN-
plus 10 ng/ml IL-1
for 48 h. The resistance to IL-1
plus IFN-
in
STAT-1
-expressing cells is due in part to interference with
IL-1
-mediated stimulation of inducible nitric-oxide synthase
expression and nitric oxide production. Furthermore, overexpression of
STAT-1
does not impair robust glucose-stimulated insulin secretion
in the INS-1-derived cell line 832/13. We conclude that expression of
STAT-1
may be a means of protecting insulin-producing cell lines
from cytokine damage, which, in conjunction with appropriate
cell-impermeant macroencapsulation devices, may allow such cells to be
used for insulin replacement in type 1 diabetes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells. Destruction of
-cells appears to result from direct
contact with infiltrating T-cells and macrophages and exposure to
inflammatory cytokines such as
IFN-
,1 IL-1
, and
TNF-
that such cells produce (1-5). Insulin replacement by
injection, the current treatment, fails to replicate the precise control of fuel homeostasis afforded by normal regulation of insulin secretion in response to glucose and other physiological cues. Islet
transplantation has therefore been investigated as an alternative to
insulin injection therapy for more than 3 decades (6, 7). Success in
this area had been very limited until a recent trial in Edmonton,
Alberta, Canada, in which a combination of mild immunosuppressive agents were used in conjunction with freshly isolated islet tissue to
achieve insulin independence in seven successive patients studied for
up to 14.9 months post-transplant (8).
and IFN-
, known mediators of
-cell destruction in type 1 diabetes
(14). This involved growth of INS-1 insulinoma cells in progressively
elevated concentrations of the two cytokines over an 8-week period and
selection of surviving cells. The resultant cell line, termed
INS-1res, was 80% viable after 5 days of exposure to the
combination of 10 ng/ml IL-1
and 100 units/ml IFN-
, while less
than 1% of parental INS-1 cells survived when treated in the same
fashion. Interestingly, resistance to IL-1
alone persisted for up to
6 months after removal of cytokines, while resistance to IFN-
or
IL-1
plus IFN-
was partially lost upon removal of cytokines and
restored upon their return to the culture medium (14).
or IFN-
plus IL-1
in selected insulinoma cell lines. By focusing on the known components of
the IFN-
receptor (IFN-R) signaling pathway, we have discovered that
expression levels of signal transducer and activator of transcription (STAT)-1
are closely correlated with the cytokine-resistant and -sensitive phenotypes. Furthermore, we demonstrate that
adenovirus-mediated overexpression of STAT-1
in cytokine-sensitive
INS-1 cell lines confers resistance to the combination of IL-1
plus
IFN-
. Our findings suggest that expression of STAT-1
in
insulin-secreting cell lines may be a means for enhancing their
survival in a transplant setting.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 100 units/ml
penicillin, and 100 µg/ml streptomycin (Sigma) at 37 °C and 5%
CO2. Recombinant rat IL-1
was obtained from Endogen
(Cambridge, MA). Recombinant rat
-IFN was obtained from Life
Technologies, Inc. Anti-STAT-1 and anti-phospho-STAT-1 antibodies were
purchased from New England Biolabs, Inc (Beverly, MA), and anti-iNOS
antibody was purchased from Transduction Laboratories (Lexington, KY).
--
Cytokine-resistant 833/117 cells (14) were kept in
medium containing IL-1
plus IFN-
for 24 weeks prior to RNA
isolation using TRIzol (Life Technologies). cDNA was
reverse-transcribed by the Advantage reverse
transcriptase-polymerase chain reaction kit
(CLONTECH Inc., Palo Alto, CA). Primers were
derived from the published mouse STAT-1 cDNA sequence
(GenBankTM accession number U06924) and included
BamHI restriction enzyme recognition sequences:
5'-GGATCCAGGATGTCACAGTGGTTCG (sense), encompassing nucleotides
4 to 16, and 5'-GGATCCTCGCCAGAGAGAAATTCGTG (antisense), encompassing nucleotides 2256-2275. Polymerase chain reaction products
were cloned into pCRII from the TA cloning kit (Invitrogen, Carlsbad,
CA). The amplified and subcloned rat STAT-1
cDNA was sequenced
with a DNA Sequencer 377 (PE Biosystems, Foster City, CA), and the
sequence was submitted to GenBankTM (accession number
AF205604). A 2.2-kilobase BamHI fragment of the STAT-1
cDNA containing the protein coding sequence was cloned into the
adenovirus vector pACCMV.pLpA (17), and a recombinant virus
(AdCMV-STAT-1
) was prepared by previously described methods (18).
Control experiments were conducted with a recombinant virus containing
the bacterial
-galactosidase gene (AdCMV-
GAL), prepared and used
as described previously (19, 20).
or AdCMV-
-galactosidase recombinant viruses. After
12 h of viral treatment, culture medium was changed, and cells
were incubated in the absence of virus for an additional 24 h
prior to the addition of cytokines. After 48 h of cytokine
treatment, media samples were collected for nitrite assay and replaced
with 250 µl/well of medium with 75 µg/ml
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) for 1.5 h at 37 °C and 5% CO2. The
resulting formazan crystals were solubilized in 250 µl of 0.04 N HCl in isopropyl alcohol. The optical density of the
solubilized formazan was read at 575 and 650 nm using a SpectraMax 340 (Molecular Devices, Inc., Sunnyvale, CA) plate reader. The reduction in
optical density caused by cytokine treatment was used as a measurement
of cell viability, normalized to cells incubated in control medium,
which were considered 100% viable.
-mercaptoethanol,
500 mM dithiothreitol, pH 6.8), heated for 5 min in boiling
water, and electrophoresed using 10% precast Tris-glycine gels
(Bio-Rad). Protein was transferred to polyvinylidene difluoride
membranes (Millipore Corp., Bedford, MA) and blocked with 5% dry milk
in TBST (10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 8.0). Blots were incubated with anti-STAT-1,
anti-phospho-STAT-1, or anti-iNOS antibodies according to protocols
supplied by the manufacturers. Protein bands were visualized with
horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham
Pharmacia Biotech) and enhanced chemiluminescence (Amersham Pharmacia
Biotech). Immunoblots were scanned with a UMAX UC840 scanner, and band
intensities were measured by NIH Image 1.59.
cDNA probe, prepared with the Redi-priming labeling kit (Amersham
Life Sciences). After hybridization and washing, nylon membranes were
exposed to film to create autoradiographs.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Correlates with Cytokine
Resistance--
We have previously isolated cytokine-resistant
insulinoma cell lines (INS-1res) by culture of the cells in
the presence of increasing concentrations of IL-1
plus IFN-
for a
period of 8 weeks (14). INS-1res cells exhibited impaired
translocation of the NF-
B transcription factor in response to
IL-1
, which may explain their attendant block of NO production and
iNOS expression. However, iNOS expression and NO production did not
account for resistance to IFN-
or IFN-
plus IL-1
, as evidenced
by the fact that
NG-monomethyl-L-arginine, an
iNOS inhibitor, only partially blocked killing of INS-1 cells induced
by IL-1
plus IFN-
(14). To gain understanding of the mechanism by
which the selection process confers resistance to IFN-
or IFN-
plus IL-1
, we have analyzed known components of the IFN-
signal
transduction system in cytokine-resistant and -sensitive cell lines.
receptor transduces signals via its associated Jak kinase
activity, which in turn binds to and phosphorylates STAT-1 transcription factors (22-24). We therefore treated clonal cell lines
derived from INS-1res cells (lines 833/15 and 833/117) or parental INS-1 cells (line 834/40) acutely with IFN-
or IL-1
+ IFN-
for a period of 20 min and then assayed phosphorylation and
expression of STAT-1
by immunoblot analysis. Stimulation of
STAT-1
phosphorylation in response to acute cytokine treatment was
similar in all groups of cells, as shown in a representative experiment
in Fig. 1. However, there was a clear
correlation between susceptibility to cytokine damage and STAT-1
protein levels. Thus, 833/15 and 833/117 cells that are grown in the
continual presence of cytokines are IFN-
- and IFN-
plus
IL-1
-resistant (14), and contain 6.9 and 7.0 times as much STAT-1
protein, respectively, as the same cell lines that had been grown in
the absence of cytokines for 4 weeks or more (rendering them sensitive to IFN-
- and IL-1 plus IFN-
-mediated killing). Essentially
identical results were obtained in a second independent experiment
(data not shown). STAT-1
levels were also low in 834/40 cells, which had not been through the selection procedure and are thus highly susceptible to cytokine damage. These data suggest that STAT-1
expression may directly contribute to cytokine resistance induced by
the selection protocol.
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Fig. 1.
STAT-1 expression
and phosphorylation in INS-1-derived cell lines. INS-1-derived
(833/40) and INS-1res-derived (833/15, 833/117) clonal cell
lines were grown in the presence or absence of IL-1
plus IFN-
(100 units/ml and 10 ng/ml, respectively) for a period of 48 h
(culture condition) in 12-well plates. Cells were then shifted to
serum-free medium without cytokines overnight, treated with or without
the indicated concentrations of cytokines (stimulants) for 20 min,
washed with PBS, and lysed. 50 µg of total protein was loaded in each
lane. Activated STAT-1 was detected by using anti-phospho-STAT-1
(Tyr701) antibody. Total STAT-1
was measured with an
anti-STAT-1 antibody. The results show that resistance to IFN-
and
IFN-
plus IL-1
-mediated cytotoxicity (achieved in the
INSres-derived lines only when cultured in cytokines) is
correlated with a large increase in expression of STAT-1
. The data
shown are representative of two independent experiments.
Expression in Resistant Cells Decreases upon Withdrawal
of Cytokines from the Culture Medium--
To learn more about the
regulation of STAT-1
expression by cytokines in the culture medium,
we measured STAT-1
protein levels as a function of time after
cytokine withdrawal. As shown in Fig. 2,
culture of the INS-1res-derived cell lines 833/15 and
833/117 in the continual presence of cytokines resulted in very high
STAT-1
protein levels. 2 days after cytokine removal, STAT-1
expression was unchanged in 833/15 cells and only mildly decreased in
833/117 cells. However, by 6 days after cytokine removal, STAT-1
protein levels were decreased by 48 and 67% in the two lines,
respectively, relative to starting levels. The time course and
magnitude of the decrease in STAT-1
expression was confirmed in a
second independent experiment identical in design to the representative
experiment shown in Fig. 2 (data not shown). Thus, constant IFN-
treatment is required to maintain the high STAT-1
protein levels
associated with cytokine resistance in INS-1res cells.
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Fig. 2.
Time course of decrease in
STAT-1 protein levels in response to cytokine
removal. INS-1res clones (833/15 and 833/117) were
cultured in the presence of IL-1
(10 ng/ml) plus IFN-
(100 units/ml) for 48 h. Cells were then washed with PBS (day 0) and
cultured in medium with or without cytokines as indicated. At 2 and 6 days after the start of this culture period, cells were harvested for
immunoblot analysis, using 25 µg of total protein per lane and an
anti-STAT-1 antibody. These data show that STAT-1
expression is
decreased when cytokines are withdrawn from resistant cell lines,
consistent with the return of IFN-
- and IFN-
plus
IL-1
-mediated cytotoxicity, providing support for a protective role
of elevated STAT-1
expression. Data shown are representative of two
independent experiments.
Inhibits IFN
-induced STAT-1
Expression in
Cytokine-sensitive INS-1 Cells--
We initially assumed that
STAT-1
protein levels were being regulated mainly by IFN-
in the
tissue culture medium, as has been reported in other systems (25, 26).
Indeed, as shown in Fig. 3, treatment
with IFN-
alone stimulates STAT-1
expression to a similar degree
in cytokine-resistant and sensitive clones. However, the coaddition of
IL-1
and IFN-
to a cytokine-sensitive cell line (line 834/40) for
12 or 24 h resulted in accumulation of only 8.1 and 20% as much
STAT-1
protein as observed with IFN-
alone, while in
cytokine-resistant cells (line 833/117), no inhibitory effect of
costimulation with IL-1
was observed. This result was confirmed in
three other independent experiments, similar in design to the
representative experiment shown in Fig. 3. Thus, in unselected cells,
IL-1
inhibits and delays IFN-
-induced expression of STAT-1
. In
contrast, selected cells lose IL-1
-mediated suppression of IFN-
-mediated up-regulation of STAT-1
expression. The implication is that cytokine resistance in selected INS-1 cells may be a sequential process, in which development of IL-1
resistance alleviates
inhibition of IFN-
-mediated up-regulation of STAT-1
, allowing
STAT-1
to accumulate to levels that protect against the cytotoxic
effects of IFN-
alone or the combination of IFN-
plus
IL-1
.
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Fig. 3.
IL-1 impairs
IFN-
-mediated induction of
STAT-1
expression in cytokine-sensitive but
not -resistant INS-1-derived cell lines. INS-1-derived (834/40)
and INS-1res-derived (833/117) cells were cultured in
normal medium lacking cytokines for a period of 6 months. Cells were
then plated in 12-well plates and treated with IFN-
(100 units/ml)
or IFN-
(100 units/ml) plus IL-1
(10 ng/ml). At the indicated
time, cells were washed with PBS and lysed in 200 µl of lysis buffer.
25 µg of total protein was separated by 10% SDS-PAGE and subjected
to immunoblot analysis with rabbit anti-STAT-1 antibody. In the
cytokine-sensitive line, induction of STAT-1
expression by cytokines
is suppressed by normal IL-1
signaling, rendering these cells
vulnerable to IFN-
-mediated cytotoxicity. This IL-1
-mediated
suppression of IFN-
signaling is lost in the IL-1
-resistant cell
line. Data shown are representative of four independent
experiments.
on cytokine-sensitive and
-resistant cell lines were also apparent at the level of STAT-1
mRNA. In both a sensitive (834/40) and resistant (833/117) clone, IFN-
administration caused an increase in STAT-1
mRNA within 4 h of treatment. However, STAT-1
mRNA levels were much
lower in 834/40 cells than in 833/117 cells 24 h after treatment
with the combination of IL-1
plus IFN-
, and IL-1
clearly
attenuated the IFN-
-mediated increase in mRNA levels in the
former but not the latter cells (data not shown).
in Cytokine-sensitive
Cells Confers Protection against Cytokine Damage--
To investigate
whether STAT-1
expression in cytokine-sensitive INS-1 cell lines can
confer protection against cytokine damage, it was first necessary to
clone the rat STAT-1
cDNA. This was achieved by reverse
transcriptase-polymerase chain reaction-mediated preparation of cDNA
from the INS-1 cell line 833/117 after treatment with IFN-
for
48 h to induce STAT-1
expression. The sequence of the rat
STAT-1
cDNA was determined and deposited in
GenBankTM (accession number AF205604), and a fragment
containing the entire protein coding region was used to prepare a
recombinant adenovirus (AdCMV-STAT-1
). Treatment of INS-1 cells with
a range of AdCMV-STAT-1
titers from 2.5 × 108 to
1 × 109 pfu/ml resulted in
titer-dependent increases in STAT-1
protein overexpression, as evaluated by immunoblot analysis (Fig.
4A).
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Fig. 4.
Adenovirus-mediated expression of
STAT-1 in a cytokine-sensitive INS-1-derived
cell line confers broad resistance to cytokine-induced
cytotoxicity. A, INS-1 cells were treated with the
indicated concentrations of AdCMV-STAT-1
for 12 h and then
incubated in fresh medium for 24 h prior to cell harvesting and
immunoblot analysis with an anti-STAT-1
antibody. Control cells were
incubated for the same time period but without virus treatment.
B, the cytokine-sensitive cell line 834/40 was treated with
1 × 109 pfu/ml of AdCMV-STAT-1
, the same amount of
AdCMV-
GAL virus, or no virus. These cell groups were cultured in
normal medium for 24 h and then treated with 10 ng/ml IL-1
, 100 units/ml IFN-
or both cytokines for 48 h. Cell viability was
then determined with the MTT assay, as described under "Materials and
Methods." Data represent the mean ± S.E. for four independent
experiments. The symbols refer to comparisons of viability
of STAT-1
-overexpressing cells with other groups as follows. @,
p < 0.001, versus untreated control
and p < 0.03 versus
AdCMV-
GAL-treated control; #, p = 0.05 versus untreated control and p < 0.007 versus AdCMV-
GAL-treated control; *, p < 0.002 versus either control group.
or, as
a control, the same amount of AdCMV-
GAL virus. These cell groups, as
well as a group of cells not treated with adenovirus, were cultured in
normal medium for 24 h and then treated with 10 ng/ml IL-1
, 100 units/ml IFN-
or the combination of IL-1
plus IFN-
. After
48 h of cytokine treatment, viability was measured by the MTT
assay. As shown in Fig. 4B, adenovirus-mediated
overexpression of STAT-1
in 834/40 cells increased the viabilities
of the IL-1
-, IFN-
-, and IL-1
plus IFN-
-treated groups from
41.7 ± 2.0, 56.5 ± 3.2, and 19.0 ± 0.9%,
respectively (control group with no viral treatment), or 60.9 ± 7.8, 44.0 ± 4.9, and 10.1 ± 2.4% (control group treated with AdCMV-
GAL) to 81.1 ± 3.8, 72.9 ± 7.9, and 50.1 ± 5.6% (AdCMV-STAT-1
-treated group), respectively. These data show
that STAT-1
overexpression is sufficient to provide substantial,
albeit incomplete, protection of insulinoma cells from cytokine-induced
cytotoxicity. Our results also suggest that the induction of STAT-1
observed in response to IFN-
treatment of insulinoma cells
represents a defense mechanism for protection against cytokine attack.
Finally, the data support the notion that the synergistic effects of
IL-1
plus IFN-
in promoting islet
-cell destruction may be
related to the capacity of IL-1
to interfere with the normal
IFN-
-mediated induction of STAT-1
expression in
cytokine-sensitive cells.
Reduces NO
Production and iNOS Expression--
IL-1
is known to stimulate NO
production in islet
-cells via induction of iNOS, and NO production
has been implicated in IL-1
-mediated
-cell destruction (2-5,
21). In previous studies, we have shown that the iNOS inhibitor,
NG-monomethyl-L-arginine can
block cytotoxicity mediated by IL-1
and that INS-1res
cells do not produce NO in response to acute treatment with IFN-
,
IL-1
, or IL-1
plus IFN-
(14, 21). To test whether STAT-1
overexpression affects NO production and iNOS expression in
cytokine-sensitive 834/40 cells, cells were treated as described in the
legend to Fig. 5 with AdCMV-STAT-1
, AdCMV-
GAL, or no virus and were then exposed to 10 ng/ml IL-1
, 100 units/ml IFN-
, or the combination of IL-1
+ IFN-
for
48 h. As shown in Fig. 5, after 48 h of treatment with
IL-1
or Il-1
plus IFN-
, nitrite production (a measure of NO
accumulation) was significantly reduced in STAT-1
-overexpressing
834/40 cells compared with either control group. The impairment in NO
production in STAT-1
-expressing cells was already apparent within
9 h of cytokine addition, proving that expression of the
transcription factor effectively blocks the acute response to cytokine
treatment (data not shown). Consistent with these findings,
AdCMV-STAT-1
-treated cells exhibited a 70% reduction in
IL-1
-induced accumulation of iNOS protein relative to untreated or
AdCMV-
GAL-treated control cells and a 50% reduction following
treatment with IL-1
plus IFN-
, based on densitometric scanning of
the two representative immunoblots shown in Fig.
6. Taken together, these data suggest that protection against cytokine-mediated cytotoxicity in
STAT-1
-overexpressing cells is in part attributable to reduced NO
production and iNOS expression. It is interesting to note that the
impairment of cytokine-induced NO production in STAT-1
overexpressing cells is partial, in contrast to our findings in cells
taken through the cytokine selection process, in which NO production
was completely blocked (14). These results imply that factors other
than STAT-1
expression, possibly including impairment of
IL-1
-induced NF-
B translocation (14), contribute to the more
complete blockade of NO production noted in the INS-1res
cell lines.
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Fig. 5.
Adenovirus-mediated expression of
STAT-1 in a cytokine-sensitive INS-1-derived
cell line partially blocks cytokine-induced NO production. The
cytokine-sensitive cell line 834/40 was treated exactly as described in
the legend to Fig. 4, and medium was collected for assay of nitrite
levels. Data represent the mean ± S.E. for five independent
experiments. An asterisk indicates that
AdCMV-STAT-1
-treated cells produced less nitrite than either control
group, with p < 0.01.
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Fig. 6.
Adenovirus-mediated expression of
STAT-1 in a cytokine-sensitive INS-1-derived
cell line partially blocks cytokine-induced iNOS expression. The
cytokine-sensitive cell line 834/40 was treated exactly as described in
the legend to Fig. 4. Following 48 h of culture in the presence or
absence of the indicated cytokines, cells were harvested for immunoblot
analysis with an anti-iNOS antibody. Data from two independent
experiments are shown.
Does Not Impair
Robust Glucose-stimulated Insulin Secretion in INS-1-derived Cell
Lines--
If STAT-1
expression is to serve as a viable strategy
for protecting insulin-producing cells against cytokine-mediated
damage, it is important to demonstrate that expression of this gene
does not impair glucose-stimulated insulin secretion. We have recently demonstrated that the rat insulinoma cell line INS-1 (15) is composed
of a mixture of glucose-responsive and -unresponsive cells (14, 16). We
therefore tested the effect of STAT-1
expression in a robustly
glucose-responsive INS-1-derived cell line, 832/13, which was obtained
from parental, unselected INS-1 cells by stable transfection with a
plasmid containing the neomycin resistance gene and harvesting of
independent colonies (16). As shown in Fig.
7, incubation of 832/13 cells with 15 mM glucose stimulated insulin secretion by 12-14-fold
relative to cells incubated at 3 mM glucose, regardless of
whether they had first been treated with AdCMV-STAT-1
or
AdCMV-
GAL or left untreated. We conclude that STAT-1
overexpression has no effect on insulin secretion in a robustly
glucose-responsive INS-1-derived cell line.
View larger version (34K):
[in a new window]
Fig. 7.
Overexpression of STAT-1
does not affect glucose-stimulated insulin secretion. The
INS-1-derived cell line 832/13 (16) was left untreated or was treated
with 1 × 109 pfu/ml of AdCMV-STAT-1
or
AdCMV-
GAL adenoviruses. 36 h after viral treatment, cells were
treated with 3 mM glucose or 15 mM glucose for
2 h, and media were collected for insulin radioimmunoassay. Data
are normalized to total cell protein and represent the mean ± S.E. for three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
plus IL-1
(INS-1res), involving culture of the
cells in increasing concentrations of the cytokines over an 8-week
period (14). A potential mechanistic explanation for the development of
resistance to IL-1
was provided by our finding that activation of
NF-
B translocation by the cytokine was impaired in
INS-1res cells relative to cells that had not undergone
selection. NF-
B is a known activator of iNOS expression, possibly
explaining the blockade of IL-1
-induced NO production that was also
observed in INS-1res cells (14). However, incubation of
INS-1 cells with NG-monomethyl-L-arginine, an
inhibitor of iNOS, only partially prevented the cytotoxic effects of
the combined addition of IL-1
plus IFN-
and had no effect on
IFN-
-mediated cytotoxicity. Furthermore, levels of IFN-R mRNA
were not reduced in INS-1res cells relative to parental
INS-1 cells. Thus, the mechanism by which INS-1res cells
gained resistance to IFN-
and the combination of IFN-
plus
IL-1
was not resolved.
-mediated
signaling in mammalian cells, we have discovered that there is a tight
correlation between the level of expression of the transcription factor
STAT-1
and resistance to cytokine damage. Thus, cells that have
undergone the selection process and have been grown in the continual
presence of IL-1
plus IFN-
are resistant to the combined effects
of the two cytokines (14). These cells have very high levels of
STAT-1
expression. Withdrawal of the cytokines from the culture
medium for periods of as little as 6 days and as long as 32 weeks
results in a loss of resistance to IFN-
and a partial loss of
resistance to IL-1
plus IFN-
but full retention of resistance to
IL-1
(14). These changes are correlated with a sharp drop in
STAT-1
protein levels, such that expression becomes equal to that in
cells that have not been through the selection procedure (see Figs. 1
and 2). Adenovirus-mediated expression of STAT-1
in naive INS-1
cells confers partial resistance to IL-1
, to IFN-
, and, most
prominently, to the combined cytotoxic effects of IL-1
plus IFN-
.
We conclude that the induction of STAT-1
expression that occurs
during selection by growth in the presence of cytokines is directly
linked to the resistant phenotype of INS-1res cells.
-cells, as exemplified by prior
studies in which IFN-
was shown to prime rat islets for
IL-1
-induced iNOS expression (27). An important finding of the
current work is that IL-1
impairs IFN-
-induced expression of
STAT-1
in unselected INS-1 cells. This means that in cells that have
been subjected to the selection procedure, the ability of IL-1
to
suppress induction of STAT-1
expression by IFN-
is partially
blocked. Since we have shown that increased expression of STAT-1
is
sufficient to induce resistance to IFN-
- and IL-1
plus
IFN-
-mediated cytotoxicity, it follows that ablation of IL-1
-mediated suppression of STAT-1
expression is an important event in the development of resistance to these cytokines.
and IL-1
signaling pathways in control of NO production
in INS-1 cells. In previous studies, we have shown that incubation of
nonresistant INS-1 cells with IL-1
alone induces a large increase in
iNOS expression and NO production, while IFN-
has no effect on these
variables (14). However, overexpression of STAT-1
, a transcription
factor that clearly participates in IFN-R-mediated signaling, is able
to partially block NO production and iNOS expression in response to
IL-1
(Figs. 6 and 7). STAT-1
is not known to participate in
IL-1
-mediated signaling. However, the promoter region of the iNOS
gene does contain several copies of the consensus sequence that
mediates IFN-
regulation of gene expression through STAT-1
binding (GAS sites) (5). Thus, possible mechanisms by which STAT-1
overexpression interferes with iNOS expression and NO production could
include a change in transcription factor binding to the iNOS promoter
or interference of STAT-1
with more proximal steps in the
IL-1R1/NF-
B/iNOS signaling pathway. Further experimentation will be
required to resolve these possibilities.
exerts its biological effects by binding to its cell surface
receptor, which is a heterodimer composed of
and
subunits (22-24). Binding of IFN-
causes receptor dimerization and
association of soluble Jak kinases with distal regions of the
-chain
of the IFN-R receptor. Jak kinase then phosphorylates a nearby tyrosine in the receptor
-chain, allowing it to bind to STAT-1
via its C-terminal Src homology 2 domain. Juxtaposition of STAT-1
and Jak
kinase results in phosphorylation of receptor-associated STAT-1
, rendering it competent for homo- or heterodimerization (e.g.
with other members of the STAT family of proteins), nuclear
translocation, DNA binding, and activation of transcription.
overexpression
causes resistance to IFN-
- and IFN-
plus IL-1
-mediated cytotoxicity remains to be elucidated. It has been appreciated for some
time that levels of STAT-1
mRNA and protein are increased in
response to IFN-
treatment of a variety of different cell types (25,
26), but the significance of this increase has remained obscure.
Particularly puzzling is the finding that the effects of IFN-
on
expression of its target genes wanes within 2-4 h of application of
the cytokine, while STAT-1
expression continues to increase in this
time frame and then remains at high levels for as long as IFN-
is
applied (22, 25, 26). The results presented here suggest that increases
in expression of STAT-1
serve as a mechanism for blunting untoward
effects of IFN-
on cellular processes, possibly by direct impairment
of IFN-
signal transduction or by cross-talk with other cytokine signaling pathways (e.g. that for IL-1
). Interestingly,
accumulation of STAT-1
protein in response to long term culture of
cells in cytokines does not cause a proportional increase in
phosphorylated STAT-1
(see Fig. 1), suggesting that the
overexpressed protein is not efficiently phosphorylated, dimerized, or
translocated to the nucleus. Possibilities for future investigation
include interference of the overexpressed, unphosphorylated STAT-1
with normal homodimer formation or nuclear translocation or
interference with other cellular processes via "promiscuous" Src
homology 2 domain-mediated complex formation.
in cell lines designed
for insulin replacement in type 1 diabetes may be a means of protecting
them against cytokine-induced damage occurring in response to
transplantation. An apparent advantage of this approach is that it
confers partial protection against a mixture of two cytokines
prominently associated with inflammation, IFN-
and IL-1
. This is
in contrast to the more specific protection conferred by other genetic
engineering strategies, such as expression of Mn-superoxide dismutase,
which provides protection against IL-1
and IL-1
plus TNF-
, but
not against IFN-
(21), or expression of dominant-negative FADD
domain proteins, which partially block TNF-
signaling (28, 29).
Expression of anti-apopototic members of the bcl gene
family have been reported to confer partial protection against cytokine
mixtures (30, 31), but at high levels of expression, these proteins may
also impair glucose metabolism and glucose-stimulated insulin secretion
(32). In contrast, adenovirus-mediated expression of STAT-1
in the
strongly responsive INS-1-derived cell line 832/13 (16) did not impair
the 12-14-fold increase of insulin secretion induced by raising the
glucose concentration from 3 to 15 mM (Fig. 7).
expression is a practical or broadly applicable strategy for
cell transplantation. One concern is that STAT proteins are involved in
a wide variety of receptor pathways, including those of the gp130,
growth hormone, and receptor tyrosine kinase families (22). Thus, long
term expression of STAT-1
may alter signaling through pathways other
than that mediated by IFN-
and IFN-R, affecting the viability,
growth, or function of cells in as yet undefined ways. Furthermore, the
protection conferred by STAT-1
expression in naive INS-1-derived
lines is incomplete, in contrast to the complete protection afforded by
the selection procedure (14), suggesting that IL-1
resistance
occurring through impairment of NF-
B signaling must be present as a
complement to STAT-1
overexpression for complete protection to be
gained. Studies involving stable transfection of INS-1res
cell lines (which exhibit long term IL-1
resistance (14)) with
STAT-1
and evaluation of the growth and function of these cells in
macroencapsulation devices will be required to fully evaluate the
potential of this new approach.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Roger Unger for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by NIDDK, National Institutes of Health (NIH) Juvenile Diabetes Foundation International Grant DK-55188 and a grant from the Cabe Foundation (to C. B. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by NIH Grant T32 GM08203.
To whom correspondence should be addressed: Touchstone Center
for Diabetes Research, Room Y8.212, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: 214-648-2930; Fax: 214-648-9191; E-mail:
newgard@utsw.swmed.edu.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M008330200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; IFN-R, IFN receptor; STAT, signal transducer and activator of transcription; pfu, plaque-forming units; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; iNOS, inducible nitric-oxide synthase.
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