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INTRODUCTION |
Mesangial cells line the blood vessels of the renal
glomerulus, provide structural support, and regulate glomerular
ultrafiltration (1, 2). Importantly mesangial cells undergo a change in phenotype during glomerular inflammation in which they become proliferative and matrix-secreting myofibroblasts before they are
eliminated by apoptosis (2-4). Apoptosis has been identified as the
mechanism responsible for the deletion of excess myofibroblasts on
completion of the inflammatory response in skin (5), liver (6), and
renal glomerulus (7). In the glomeruli of patients experiencing acute
glomerular inflammation, referred to as glomerular nephritis
(GN),1 apoptotic bodies were
detected as a compensatory response, instigated to counterbalance
mesangial hypercellularity thereby permitting normal structure and
function to return (8). Mesangial/myofibroblast apoptosis was also
identified in the rat in vivo model of experimental GN,
anti-Thy1.1 nephritis (7, 9, 10). Proliferative forms of GN are
characterized by a dysregulation of mesangial cell apoptosis allowing a
chronic secretion of proinflammatory stimuli and prostaglandins (PG),
which leads to an excess deposition of extracellular matrix proteins,
post-inflammatory scarring, and renal failure (7, 10). Proliferative GN
remains a leading cause of end-stage renal failure (11). Consequently,
to avoid progression of glomerular inflammatory disease it is important
to define the mechanisms underlying the inhibition of mesangial cell apoptosis.
Cyclooxygenase-2 (COX-2) is an inducible form of cyclooxygenase
involved in chronic inflammation (12, 13). Several studies have
highlighted an up-regulation of COX-2 expression in proliferative GN
(14, 15). However, the precise role of COX-2 has not been investigated.
It is plausible that COX-2 is responsible for the progression of
proliferative GN by an anti-apoptotic mechanism. A growing body of
evidence that COX-2 has an anti-apoptotic role in the pathogenesis of
epithelial cell carcinomas, in particular colorectal cancer, supports
this hypothesis (16). An overexpression of COX-2 conferred a survival
advantage in rat intestinal epithelial cells by inhibiting
apoptosis (17). COX-2 selective inhibitors induced apoptotic cell
death in HCA-7, HT-29 (18), and CaCo-2 colon cancer cell lines, which
constitutively expressed COX-2 (19).
Within the inflamed glomerulus, TNF
is produced locally by mesangial
and epithelial cells as well as by infiltrating monocytes/macrophages (20). TNF
alone may be a key component for the resolution phase of
glomerular inflammation and may enhance death receptor
initiated-apoptosis of mesangial cells by an autocrine and/or paracrine
mechanism. TNF
can also stimulate the release of other
proinflammatory cytokines including IL-1
(21, 22). Endothelins,
particularly ET-1, are mitogenic to mesangial cells in vivo
and may act in concert with other vasoconstrictor peptides or cytokines
to promote glomerular inflammation (23-25). Several studies utilizing
rat primary cultures of renal mesangial cells (RMC) have demonstrated
enhanced COX-2 protein levels induced by either ET-1 (26, 27) or
IL-1
(28, 29). TNF
alone had little effect on COX-2 but the
combination of TNF
plus IL-1
dramatically increased COX-2
expression (28). The contribution of TNF
may depend on its mediation
of either one of two conflicting pathways; cell survival via activation of NF-
B (30) or caspase-mediated apoptosis (30, 31). In previous
studies of RMC apoptosis, sensitivity to TNF
could only be achieved
in the presence of cycloheximide or actinomycin D, to prevent synthesis
of survival factors (32) or by a specific inhibition of NF-
B (33,
34).
The overall objective of this study was to determine whether COX-2
expression inhibits apoptosis in RMC. Because mesangial cells express
TNF
receptors and represent a potential route of apoptosis induction
in the resolution of proliferative GN, we evaluated the extent of this
apoptosis, and its inhibition by COX-2. We first established a suitable
cell culture model in which TNF
-mediated apoptosis was measured in
multiple apoptosis assays. We then used two different methods to induce
COX-2 expression, i.e. induction by ET-1, or IL-1
, and
forced expression using adenoviral-mediated gene transfer. We also
investigated the effects of PGE2 and PGI2 on
TNF
-mediated apoptosis, after establishing that those PGs were the
major PG metabolites generated by COX-2 overexpression. Using these
multiple approaches combined with several apoptosis assays, an
anti-apoptotic effect of COX-2 on TNF
-mediated apoptosis was
conclusively demonstrated in renal mesangial cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant IL-1
was obtained from R&D Systems
(Minneapolis, MN). Recombinant ET-1 was obtained from Calbiochem (La
Jolla, CA). Annexin V and the caspase-3 apoptosis assay detection kits were purchased from Oncogene Research Products (Boston, MA) and BD
Pharmingen, respectively. NS398 was obtained from Cayman Chemical (Ann
Arbor, MI). RPMI 1640 medium and fetal bovine serum were from
Invitrogen. Polyclonal goat and polyclonal rabbit anti-COX-2 (N-20) and bcl-2 (N-19) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated donkey anti-goat and goat anti-rabbit immunoglobulins (IgGs) were from Bio-Rad. Protein was
determined by a BCA assay from Pierce (Rockford, IL). All other
reagents were obtained from Sigma. Flow cytometry experiments were
carried out using a BD Biosciences FACS calibur (Mountain View,
CA). In all experiments 10,000 live cells, based on light scatter
properties, were gated and analyzed using Cell Quest Software (BD Biosciences).
Cell Culture--
Primary glomerular mesangial cells from male
Sprague-Dawley rats were isolated and characterized as previously
described (35, 36). RMC were maintained in RPMI 1640 medium
containing 17% heat-inactivated fetal bovine serum, 5 µg/ml each of
insulin and transferrin, and 5 ng/ml sodium selenite, 100 µg/ml
penicillin, 100 µg/ml streptomycin at 37 °C in a humidified
incubator (5% CO2, 95% air). All experiments were
performed with cells cultured in 60-mm dishes and used at 8-20
passages. After the cells were grown to ~60% confluence they were
starved for 24 h in basal RPMI 1640 culture medium before the experiments.
Adenovirus-mediated COX-2 Gene Transfer--
The recombinant
adenoviral vectors (Ad) AdCOX-2 and Ad wild-type (AdWT) expressing the
COX-2 and empty adenovirus vector, respectively, were constructed from
the replication-deficient adenovirus type 5 (Ad5) as previously
described (37). RMC were incubated with AdCOX-2 or AdWT (at a
multiplicity of infection (m.o.i.) of 200) for 1 h at 37 °C
with periodic shaking, followed by addition of complete medium. At
24 h after infection, cells were lysed for Western blot analysis.
Efficiency of gene transfer in RMC was determined by adenoviral
infection of green fluorescent protein at an m.o.i. of 200 and
visualized by fluorescence microscopy at 24 h.
Western Blot Analysis--
Cells were washed in ice-cold
phosphate-buffered saline (PBS) and then harvested in lysis buffer as
previously described (35). Cleared total cell lysates (20-40 µg)
were resolved by criterion SDS-PAGE (Bio-Rad) and transferred to
nitrocellulose membranes (Micron Separation Inc.). Equal protein
loading was confirmed by staining the membranes with Ponceau S, which
stains all the proteins on the membrane; an intensely stained band of a
distinct molecular weight sufficiently different from the protein under investigation was selected from each membrane, to show that the amount
of protein in each lane was identical. Membranes were probed with
either COX-2 (1:1000 dilution) or bcl-2 (1:300 dilution) antibodies
overnight at 4 °C. Primary antibodies were detected with goat
anti-rabbit IgG-horseradish peroxidase conjugates for bcl-2
identification or anti-goat IgG-horseradish peroxidase antibodies for
COX-2 detection (1:2000 dilution), followed by extensive washing of the
membranes. The membranes were visualized by enhanced chemiluminescence (Amersham Biosciences).
Analysis of PG Production--
RMC were infected with AdCOX-2 or
AdWT as described above, but in this instance the conditioned medium
was replaced with new basal medium devoid of any previously formed PGs.
After the 24-h infection period, the supernatants were collected and
the cell monolayers were washed twice with a HEPES-buffered medium (pH 7.4) and were incubated in this buffer containing the calcium ionophore
A23187 (10 µm) for 15 min at 37 °C (38). The total cell
lysates were run on a solid phase extraction device for analysis of
PGE2, PGI2 (detected as
6-keto-PGF1
, a stable product of PGI2),
PGF2a, PGJ2, and PGD2 prior to
measurement by liquid chromatography mass spectrometry (39). Internal
standards, 1.0 ng each of
d4-PGE2,
d4-PGF2
,
d4-6-keto-PGF1
,
d4-PGD2, or
d4-PGJ2, were added to the samples
followed by ethanol, to give a final concentration of 15% in the
samples. Glacial acetic acid (10 µl/ml) was then added. The samples
were sonicated and vortexed three times before centrifugation at
300 × g, at room temperature for 5 min. The
supernatants were loaded onto the solid phase extraction columns
(C18Bond Elut SPE columns) that had been preconditioned with 5 ml of
ethanol and 15 ml of water. The columns were washed with 29 ml of water
and allowed to run dry. Then the PGs were eluted from the column with 6 ml of ethyl acetate. A vacuum was applied to a completely dry column.
The top layer of ethyl acetate was removed from the water layer at the
bottom of the reaction tubes. The compounds of interest were in the
ethyl acetate layer. The water layer was extracted twice with 1 ml of
ethyl acetate. The resulting two ethyl acetate portions were combined
for each sample and dried under a stream of nitrogen gas until
completely dry. The sample was then redissolved in 20 µl of
acetonitrile and transferred to an insert in the sample vial.
The derivatized extracts were subject to liquid chromatography-mass
spectrometry using LC-ESI-MS (Agilent 1100 LC/MSD, SL model) (39).
Concentrations of the different PGs were evaluated by comparing their
ratios of peak areas to the standard curves. Results were expressed as
picograms per milligram of protein per dish.
Morphological Analysis of Apoptosis--
RMC infected with AdWT
or AdCOX-2 (m.o.i. of 200) for 24 h were incubated with TNF
(100 ng/ml) or etoposide (100 µM). After treatment, cells
were morphologically assessed for apoptosis by acridine orange (AO)
staining using inverted fluorescence microscopy. The cell monolayers
were washed in PBS and incubated with AO in PBS (10 µg/ml) for
2
min. Typically 3 fields were randomly selected from each 60-mm dish so
that at least 80 RMC were counted. All cells produced a green
fluorescence in response to a high affinity binding of AO to DNA.
Apoptotic green cells were scored by their distinct morphology of
cellular shrinkage and chromatin condensation. Attached apoptotic cells
were scored as a percentage of the total number of cells counted for
each dish.
Cell Cycle Analysis--
RMCs (5 × 105 cells)
infected with either AdWT or AdCOX-2 were incubated with TNF
(100 ng/ml) or etoposide (100 µM). At the end of the
incubation time the supernatants were collected. The remaining cell
monolayer was washed in Hanks' buffered saline solution without
Ca2+ and Mg2+, and then incubated in a solution
of trypsin-EDTA at 37 °C. The reaction was terminated by addition of
basal media. The trypsinized cells were then added to the previously
collected culture supernatants. This cell suspension was centrifuged at
179 × g, 4 °C, for 5 min, and the resulting pellet
was resuspended in PBS. The cell suspension was centrifuged again at
179 × g, 4 °C, and the pellet was resuspended in
70% ice-cold ethanol. Cells fixed in ethanol were stored at
20 °C
for up to 3 days. The cells were precipitated from the ethanol by
high-speed centrifugation at 358 × g, 4 °C, for 15 min. The pellet was resuspended in PBS and centrifuged again at 358 × g, 4 °C, for 15 min. The final pellet was
resuspended in 1 ml of RNase A (prepared in PBS without
Mg2+ or Ca2+ and heat treated to inactivate
DNase) to give a final concentration of 250 µg/ml. The mixture was
incubated at 37 °C for 30 min. At the end of the incubation
propidium iodide (PI) (1 mg/ml) was added to give a final concentration
of 50 µg/ml. The suspension was incubated at room temperature for 10 min in the dark. The cells were then analyzed by flow cytometry.
Annexin-V FITC and Propidium Iodide Double Staining--
RMCs
(5 × 105 cells) infected with either AdWT or AdCOX-2
were treated with TNF
(100 ng/ml) or etoposide (100 µM) and stained with annexin-V (AV) labeled to FITC in
combination with PI, for FACS analysis of apoptosis. FITC-AV/PI
staining was optimized for attached cells according to the instructions
outlined by the manufacturers of the AV assay kit (Oncogene Research Products).
Caspase-3 Assay--
FITC conjugated to a monoclonal rabbit
antibody raised against the active fragment of caspase-3 was also used
to determine apoptosis in AdWT or AdCOX-2 cells treated with either
TNF
(100 ng/ml) or etoposide (100 µM), following cell
permeabilization and fixation according to the instructions outlined by
the manufacturer.
FITC-AV/PI and active-caspase-3-FITC staining was also carried out on
cells stimulated to express COX-2 by the addition of ET-1 (100 nM) or IL-1
(2 ng/ml) and on cells treated with either PGE2 (500 nM) or PGI2 (500 nM) to investigate the effects of these various treatments
on TNF
- (100 ng/ml) or etoposide- (100 µM) mediated
apoptosis. For PG addition, conditioned media was removed and replaced
with fresh basal media to ensure removal of any existing PGs. PGs were
readministered after 6 and 12 h of co-incubation with the
apoptotic inducers to replenish depleted PGs. Effect of NS398 (25 µM) on ET-1 protection of apoptosis was also assessed by
these assays. Control cells received Me2SO at the same
concentration and time of incubation as PGE2 (500 nM) and NS398 (25 µM).
 |
RESULTS |
Characterization of COX-2 Overexpression by Adenovirus-mediated
Infection--
Previous work from this laboratory demonstrated a
transient overexpression of COX-2 in SV40-transformed human mesangial
cells using an adenovirus-mediated transfer of COX-2 cDNA (37). In this study the AdCOX-2 construct was used to express COX-2 in rat
primary RMC. The transgenic AdGFP construct showed
100%
transfection efficiency in RMC at 24 h postadenoviral infection,
as visualized by fluorescence microscopy (Fig.
1B), compared with uninfected control cells (Fig. 1A). The ability of the virus to infect
the cell is governed by specific cell receptors, and this infection process is not specific to the gene insert. Therefore AdGFP is a
suitable control that can be used for assessing the level of expression
by adenovirus-mediated gene delivery (40). Western blot analysis
confirmed COX-2 protein expression, which was enhanced by a
dose-dependent increase in AdCOX-2 after 24 h of
infection (Fig. 1C). Certain regions of the kidney contain a
higher constitutive expression of COX-2 than most tissues, however,
mesangial cells are normally devoid of COX-2 (41). Uninfected
RMC and RMC infected with the AdWT construct did not reveal
any detectable COX-2 (Fig. 1C). In addition, we checked for
anti-apoptotic bcl-2 protein in our system because an overexpression of
COX-2 in colon cancer cells was accompanied by bcl-2 protein expression
(18, 19). However, we could not detect bcl-2 in RMC transfected with
AdWT or AdCOX-2 (Fig. 1D).

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Fig. 1.
Characterization of RMC overexpressing COX-2
by adenovirus-mediated infection. Subconfluent
serum-starved cells untransfected (A) or infected
(B) with the Ad cDNA construct for green fluorescent
protein at a multiplicity of infection (m.o.i.) of 200 for 24 h.
Expression of COX-2 (C) 24 h after infecting with
increasing m.o.i. of AdCOX-2, and bcl-2 expression after infection with
AdCOX-2 at a m.o.i. of 200 (D) was determined by Western
blot analysis with anti-COX-2 or anti-bcl-2 antibody, respectively.
Cell lysates from AdCOX-2 cells were standardized for protein, and
extracts from LNcaP prostate cancer cells were used as positive (+)
controls. AdWT construct was used as a transfection control. AdWT cells
did not show endogenous COX-2 or bcl-2 proteins (D). Ponceau
S staining of the membrane-bound protein confirmed equal protein
loading. ECL exposure and fluorescence micrographs are
representative of three independent experiments.
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Inhibition of TNF
-mediated Apoptosis in RMC by an Overexpression
of COX-2 as Determined by FITC-AV/PI Double
Staining--
Our first task was to develop an apoptosis model in RMC
cultures. To conclusively demonstrate the elicitation of apoptotic cell
death by a cytokine/receptor-mediated route, the effect of TNF
was
compared with a cytotoxic insult using the DNA topoisomerase II
inhibitor etoposide, which readily induces classic apoptotic changes
such as phosphatidylserine (PS) externalization in different cell
types. We found that TNF
at a dose of 100 ng/ml induced a
time-dependent increase in apoptotic cells, which became
significant by 24 h, and reached maximum effect by 40 h
incubation, by which time, however, higher levels of necrotic cells
were also appearing (results not shown). Lower doses of
TNF
were ineffective at inducing cell death (results not shown).
Because the 24-h incubation time frame imposed the earliest,
significant induction of apoptosis by TNF
, we used it to compare an
inhibition of apoptosis by COX-2 overexpression or following its
up-regulation.
PS externalization is a characteristic hallmark of apoptotic cells,
serving as a signal for their phagocytic recognition and removal
in vivo (42). PS externalization was detected by FITC-AV binding in combination with PI to distinguish between viable (V), early
membrane intact apoptotic (EA), and necrotic (N) cells. Because there
is no phagocytic disposal mechanism in vitro apoptotic cells
accumulate and continue to undergo degradation and membrane lysis. It
was crucial, therefore, to include PI in the reaction, and highlight PS
exposure on the surface of EA cells, and distinguish them from N cell
populations, which may or may not have transited the process of
apoptosis (43). By this analysis it was possible to both qualitatively
determine viable, V (AV(
)/PI(
)), EA (AV(+)/PI(
)), and N (AV(
and +)/PI(+)) cell fractions (Fig. 2) and
simultaneously quantify this transition (Table
I). AdWT cells incubated with either
TNF
(100 ng/ml) or etoposide (100 µM) produced
distinct fractions of EA cells (28 and 30%, respectively) (Table I and Fig. 2). However, some EA and N cell fractions were induced in control
AdWT-infected cells implicating adenovirus-mediated cell death as a
result of the infection procedure. Nevertheless the profile and extent
of apoptosis induction by TNF
or etoposide in AdWT-infected cells
was similar to uninfected cells treated with these inducers in which
cell death progressed from V to EA to N cell populations. For cells
overexpressing COX-2, the EA cell fraction induced in the AdWT cells by
TNF
was significantly suppressed (Fig. 2). The annexin V assay
highlighted a reduction in the maximum fraction of EA cells (from 28 to
6%) in AdCOX-2 cells treated with TNF
compared with AdWT-treated
cells (Table I and Fig. 2). A substantial fraction of AdCOX-2 cells
were N (~18%) in response to TNF
(Table I and Fig. 2). The
transition in cell death for AdCOX-2 cells exposed to TNF
was
therefore from V directly to N. AdCOX-2 cells treated with etoposide,
on the other hand, gave rise to a distinct EA cell fraction (~27%) (Table I and Fig. 2). It was noted, that AdCOX-2-infected cells displayed higher levels of N cell fractions in control cultures suggesting that the procedure of adenovirus infection was also inducing
necrotic cell death as shown for AdWT-infected control cells.

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Fig. 2.
Illustration of the transition in cell
viability by FITC-AV/PI staining following COX-2 overexpression.
Cells infected with AdWT or AdCOX-2 were incubated with TNF (100 ng/ml) or etoposide (100 µM) for 24 h and
subsequently stained with FITC-AV/PI to demonstrate fractions of V, EA,
and N cell populations. Cells that received no adenovirus are indicated
as uninfected. Dot plot is representative of five independent
experiments.
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Morphological Analysis of Apoptosis Induction and Its
Inhibition by an Overexpression of COX-2--
Chromatin condensation
is an early event of apoptosis that requires a supply of ATP (44) and
may be independent of internucleosomal DNA fragmentation (45). It was
measured by AO staining of AdWT and AdCOX-2 cells treated with either
TNF
(100 ng/ml) or etoposide (100 µM) for 24 h.
Nuclei of control AdWT cells were composed of diffuse chromatin as
depicted by a homogenous pattern of AO staining (Fig.
3, A and G). In
response to TNF
substantial chromatin margination and condensation
became apparent, compared with AdWT control cultures (from ~8 to
18%) (Fig. 3, B and G). Etoposide also increased
the proportion of AdWT cells with apoptotic nuclear phenotype (from
~8 to 28%) (Fig. 3, C and G). AdCOX-2 cells
treated with TNF
were prevented from undergoing chromatin
condensation (Fig. 3E), and appeared morphologically
identical to AdCOX-2 control cells (Fig. 3D). TNF
-induced
apoptosis was significantly reduced (from ~18 to 6%) in cells
overexpressing COX-2 compared with AdWT cells (Fig. 3G). By
contrast etoposide-treated AdCOX-2 cells were not protected
from apoptosis (Fig. 3F) and high levels of apoptotic cells were still observed (Fig. 3G).

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Fig. 3.
Apoptotic morphology as detected by
fluorescence microscopy (×40). AdWT-infected (A-C) or
AdCOX-2-infected (D-F) cells were stained with
AO and subsequently quantified for nuclear apoptotic morphology
(G). Cells were treated with either TNF (B and
E) or etoposide (C and F) for 24 h. Control cultures (A and D) were cultured in
complete medium for 24 h. Apoptotic cells were identified by
"dots" of chromatin condensation, which became pronounced in the
shrunken apoptotic cells (indicated by arrows). Fluorescence
micrograph is representative of four independent experiments. Data
points (G) show the mean ± S.E. of the mean of
triplicate readings in a representative experiment; significant
difference form AdWT control cells (#, p < 0.01), and
from AdWT-treated cells (*, p < 0.001).
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Inhibition of Caspase-3 Activity by an Overexpression of
COX-2--
Caspase-3 activation is a specific biochemical event in
apoptosis, responsible for cleaving cellular substrates that lead to
characteristic apoptotic morphology (46). It was measured by FACS
analysis of FITC conjugated to a monoclonal antibody raised against the
active fragment of capase-3. A marked increase in the fraction of
active caspase-3-FITC positive AdWT cells treated with either TNF
(100 ng/ml) (from 7 to 22%) or etoposide (100 µM) (from
7 to 35%) was observed by 24 h (Fig.
4). Caspase-3 inactive cells were the
predominant cell population (>90%) in control AdWT and uninfected
cells (Fig. 4). For cells overexpressing COX-2 the induction of the
active caspase-3 FITC positive population remained high in response to
etoposide (25%) (Fig. 4). Conversely, AdCOX-2 cells treated with
TNF
were prevented from the induction of a separate population of
cells, highlighting the increase in active caspase-3-FITC fluorescence.
Instead the majority of AdCOX-2 cells displayed inactive caspase-3
(93%) with TNF
(Fig. 4). Because the level of caspase-3 activation
in control AdWT or control AdCOX-2-infected cells was at a minimum
identical to uninfected control cells (Fig. 4), cell death was probably
not occurring specifically as a consequence of the adenovirus-mediated
infection procedure, as detected by this method.

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Fig. 4.
Effect of COX-2 overexpression on caspase-3
activation. AdWT- or AdCOX-2-infected cells were induced to
apoptosis with either TNF (100 ng/ml) or etoposide (100 µM) for 24 h and assayed for caspase-3 activation by
flow cytometry. Histograms of cell count versus active
caspase-3 labeled to FITC highlight the M1 and M2
regions, which represent cells with inactive and active caspase-3,
respectively. Histograms are representative of three independent
experiments.
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Suppression of the Sub-G1 Population by an
Overexpression of COX-2--
Further evidence for an apparent
inhibition of TNF
(100 ng/ml)-induced apoptosis by COX-2
overexpression was obtained by cell cycle analysis and the measurement
of a sub-G1 region. FACS analysis of nuclear DNA showed a
profound sub-G1 peak, produced by a leakage of DNA
fragments from apoptotic cells following their fixation, in response to
etoposide treatment in AdWT cells. A smaller but well defined
sub-G1 region was also produced by TNF
in AdWT cells
(Fig. 5). The sub-G1 region
was considerably smaller in AdCOX-2 cells compared with AdWT cells
following treatment with TNF
but was still visible in the presence
of etoposide (Fig. 5).

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Fig. 5.
Cell cycle analysis. Cells infected with
AdWT or AdCOX-2 were analyzed for nuclear DNA content by PI staining,
as described under "Experimental Procedures," after treatment with
TNF (100 ng/ml) or etoposide (100 µM) for 24 h.
Histograms are typical of three independent experiments.
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Profile of Prostaglandin Synthesis following Overexpression of
COX-2--
To assess the impact of an overexpression of COX-2 on
prostaglandin production in RMC, AdWT and AdCOX-2 cells were stimulated with the calcium ionophore A23187 (1 mM, 15 min at
37 °C) to release cellular arachidonic acid from membrane
phospholipids, as described elsewhere (38). The PGs were then extracted
and separated by liquid chromatography-mass spectrometry (39). By this
analysis it was possible to determine the conversion of arachidonic acid to a variety of COX-2-mediated PG metabolites. AdWT cells contained low levels of PGE2 and a substantial amount of
PGI2. When the cells were overexpressed with COX-2 there
was a selective enhancement of PG production. AdCOX-2 cells showed a
2-fold increase in PGE2 and PGI2 (detected as
6-keto-PGF1
). There was no evidence of
PGF2
, PGJ2, PGD2, and
thromboxane A2 production by AdWT or AdCOX-2 cells. Because
COX-2 was the major active isoform because of adenovirus transfection,
PGE2 and PGI2 were deemed to be the principal
PG metabolites induced by COX-2 in RMC.
Anti-apoptotic Effect of PGE2 and
PGI2--
Having established that PGE2 and
PGI2 were generated by an overexpression of COX-2, we
attempted to mimic COX-2 activity by exogenous addition of either
PGE2 or PGI2. PGE2 (500 nM) and PGI2 (500 nM) were each
co-incubated with either TNF
(100 ng/ml) or etoposide (100 µM) in uninfected RMC. Their effect on TNF
or etoposide-mediated apoptosis was monitored by FACS analysis using the
previously established assays in this study. The fraction of active
caspase-3-FITC cells was reduced from 16 to 1% by PGE2 (Fig. 6) and from 16 to 3% by exogenous
PGI2 (Fig. 6). Furthermore, the EA cell fraction was
significantly reduced in TNF
-treated cells, co-incubated with either
PGE2 or PGI2 (Table
II). In each instance the fraction of EA
cells was reduced from ~18.5 to 3.6%. The extent of apoptosis
inhibition by PGE2 and PGI2 was comparable with
the anti-apoptotic effect of an overexpression of COX-2, suggesting
that the cytoprotective effect of COX-2 on TNF
apoptosis was
mediated by PGE2 and PGI2. Moreover, the
inhibitory effect of PGE2 and PGI2 could not be
elicited in the presence of etoposide as shown by a significant
induction of EA cells (~20%) and cells with active caspase-3
(>25%) (Table II and Fig. 6, respectively). PGE2 or
PGI2 alone had no effect on apoptosis, as shown by the low
levels of caspase-3 activation (<5%, see Fig. 6) and EA cell induction (<10%, see Table II).

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Fig. 6.
Effect of PGE2 and
PGI2 on TNF -mediated
apoptosis. Cells were incubated for 24 h in fresh basal media
in the absence (control) or presence of PGE2 or
PGI2, or each PG was co-incubated with either TNF (100 ng/ml) or etoposide (100 µM). PGs were reapplied 6 and
12 h during incubation to offset their metabolic degradation as
described under "Experimental Procedures." Cells were analyzed for
active caspase-3 FITC staining. Control cultures were also composed of
an equivalent concentration of Me2SO used to dissolve
PGE2 (500 nM). Histograms of cell count
versus caspase-3 staining are representative of three
independent experiments.
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ET-1 Inhibition of TNF
-mediated Apoptosis--
Having
determined the anti-apoptotic effect of an overexpression of COX-2 and
the cytoprotection afforded by the PGs generated by this mechanism we
then wanted to examine the influence of other mediators that are
responsible for proliferative GN. ET-1 may be important in the
progression of proliferative GN (23, 24). Earlier investigations
demonstrated a rapid induction of COX-2 gene expression in response to
ET-1 in RMC (25, 26). Here COX-2 expression was enhanced in RMC by ET-1
(100 nM) alone and more so by a combination of ET-1 (100 nM) with TNF
(100 ng/ml) (Fig.
7A). In contrast, COX-2 was
barely detected in cells incubated with TNF
alone (Fig.
7A).

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Fig. 7.
Effect of ET-1 on
TNF -mediated apoptosis and COX-2
expression. Cells incubated with ET-1, TNF (100 ng/ml), or both
for 24 h were harvested and the whole cell lysates were analyzed
by immunoblotting with anti-COX-2 antibody. Ponceau S staining of the
membrane-bound protein confirmed equal protein loading (A).
Cells treated with ET-1 (100 nM), NS398 (25 µM), TNF (100 ng/ml), or etoposide (100 µM) alone or in several combinations as indicated were
stained with FITC-caspase-3 antibody and analyzed by flow cytometry
(B). Control cultures were also composed of an equivalent
concentration of Me2SO used to dissolve NS 398 (25 µM). Histograms and Western blot results are
representative of at least three independent experiments.
|
|
This synergistic effect of ET-1 and TNF
on COX-2 expression was then
tested on the inhibition of TNF
-mediated apoptosis. ET-1 in
combination with TNF
reduced the EA cell fraction induced by TNF
alone (from 19 to 8%) (Table III). A
fraction of TNF
-treated RMC with caspase-3 activation was also
reduced (from 21 to 8%) in TNF
cells pre-exposed to ET-1 (Fig.
7B). ET-1 did not prevent apoptosis induced by etoposide as
evidenced by the substantial number of cells with active caspase-3
(26%) (Fig. 7B). NS398 (25 µM), a COX-2
selective inhibitor, was administered in combination with ET-1 for
24 h prior to incubation with TNF
for an additional 24 h.
In this scenario the protective effect of ET-1 over TNF
-induced apoptosis was eliminated, presumably because of an inhibition of
COX-2 enzymatic activity by NS398 (Fig. 7B). NS398 alone
induced neither significant fractions of EA (<10%, Table III) cells
nor cells with active caspase-3 (
10%, Fig. 7B) as
predicted by the lack of up-regulation of endogenous COX-2 in RMC, and
the reported specificity of NS398 for this isoform. ET-1 alone failed
to induce EA cell fractions or caspase-3 active populations (Table III
and Fig. 7B).
Inhibition of TNF
-mediated Apoptosis by IL-1
--
IL-1
in
combination with TNF
was reported to elicit an additive induction of
COX-2 in RMC, with IL-1
being the more potent inducer of COX-2 (28).
With this observation in mind, RMC were treated with a combination of
cytokines, and the effects on apoptotic cell death were evaluated. RMC
stimulated with IL-1
(2 ng/ml) displayed increased endogenous COX-2
protein expression compared with TNF
(100 ng/ml)-treated cells (Fig.
8A). The combination of TNF
and IL-1
further enhanced COX-2 protein levels (Fig. 8A)
and mitigated apoptosis resulting from TNF
alone, because of their
synergism over COX-2 expression. As a result EA cell fractions and
cells with active caspase-3 were reduced to almost that of control
levels from 19 to 6% (Table IV) and 19 to 9% (Fig. 8B), respectively, in cells co-incubated with
TNF
and IL-1
. This response further supports our hypothesis that
COX-2 has an anti-apoptotic role during cytokine-mediated proliferative
GN.

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Fig. 8.
Effect of IL-1 on
TNF -mediated apoptosis and COX-2
expression. Cells stimulated with IL-1 or TNF or both
cytokines were harvested and whole cell lysates were assessed for COX-2
protein by Western blot analysis. Ponceau S staining of the
membrane-bound protein confirmed equal protein loading (A).
FITC-capase-3 staining of cells incubated in the absence (control) or
presence of IL-1 (2 ng/ml) and/or TNF (100 ng/ml) was carried out
after 24 h incubation (B). Histograms and Western blot
results are representative of at least three independent
experiments.
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|
 |
DISCUSSION |
Mesangial cells are myofibroblasts essential for maintaining
immunological functions of renal glomeruli (1-4, 7). The mechanisms
responsible for the impairment of myofibroblast/mesangial cell
apoptosis, in severe proliferative GN remain poorly characterized. To
address this issue our primary objective was to develop a suitable cell
culture model of apoptosis in RMC. We were able to induce ~20 to 25%
apoptotic cells in subconfluent RMC, in response to TNF
, highlighted
by a number of apoptotic indices, without prior cellular or molecular
manipulations as described in other published investigations (32-34).
Our findings are in agreement with one other study whereby
TNF
-mediated apoptosis was also restricted to subconfluent rat
mesangial cells, at a level of ~25% by 24 h incubation, in the
absence of any manipulations (47).
After establishing this primary cell culture model we were able to
investigate the relationship between COX-2 expression and TNF
-mediated apoptosis in RMC. We used recombinant
adenovirus-mediated gene transfer, which is an indispensable tool for
driving gene expression in primary cell types, and by this mechanistic
approach we were able to show that cells overexpressing COX-2 were
resistant to apoptosis induced by TNF
. PS exposure was reduced by
~50% in AdCOX-2 cells, as were nuclear apoptotic events, such as
chromatin condensation and DNA fragmentation. Protection from
TNF
-mediated apoptosis by an overexpression of COX-2 was because
of the suppression of caspase-3 activation. On the other hand, AdCOX-2
cells could not be rescued from etoposide-mediated apoptosis,
suggesting that COX-2 anti-apoptotic activity was confined to the
pathways mediated by TNF
. Although there is definitive evidence of
COX-2 suppression of apoptosis in cancerous or transformed cells
(16-19) this study is one of few to demonstrate the cytoprotection of
COX-2 overexpression in primary cell types such as RMC.
We were convinced of an anti-apoptotic activity of COX-2 expression
because we had utilized more than a single viability assay. There are
several points of controversy for relying exclusively on the
interpretation and specificity of a single assay. For instance the AV
assay using FITC-AV/PI staining is a sensitive FC method for detecting
PS exposure, which is an early and transient event in apoptosis that
may be difficult to distinguish from the necrotic cell fraction.
Studies utilizing the AV assay without including PI in the analysis run
the risk of overestimating the level of apoptosis and incorrectly
diagnosing necrosis as apoptotic cell death (43, 48). Consequently, the
level of EA cell induction by TNF
at 24 h was a significant
parameter that we had consistently highlighted, and inhibited by an
overexpression of COX-2. By contrast, the caspase-3 assay can
exclusively detect the level of apoptotic cell death because it
measures a specific event of apoptosis not present in necrosis. As a
result few caspase-3-positive cells were identified in AdWT and AdCOX-2
control cells, and as expected, the absolute quantity of apoptosis
measured by the two methods was different. Nevertheless, all of our
assays were consistent in showing the same ultimate conclusion: COX-2
inhibits TNF
-mediated apoptosis in RMC.
TNF
was investigated in this study because it plays an important
role in the physiology of RMC. Elimination of mesangial/myofibroblast cells by immune surveillance may depend on TNF
-initiated apoptosis. Consequently, many studies have emphasized the importance of TNF
in
the resolution of proliferative GN (32-34) and yet few have demonstrated the caspase-mediated pathway of TNF
, without
implementing a prior deletion of the NF-
B survival pathway (32-34).
We hypothesized that the observed cytotoxic effect of TNF
was a
result of the absence of COX-2 expression in our cells. TNF
alone,
at all the doses tested (25, 50, and 100 ng/ml), did not induce COX-2.
However, a stimulatory effect on COX-2 expression by TNF
became
synergistic with the proinflammatory cytokine IL-1
and the
vasoconstrictor peptide ET-1. This observation is consistent with
previous work showing that IL-1
potently induced COX-2 in RMC (28,
29), and in a host of other cell types relevant to the inflammatory process, e.g. in human gingival fibroblasts (49) and
osteoblasts (50). Furthermore, TNF
plus IL-1
was shown to have an
additive effect on COX-2 expression in RMC (29), and a synergistic
effect in human gingival fibroblasts (49) with IL-1
as the more
potent inducer of COX-2 in each instance (29, 49). Similarly, ET-1 by
itself was shown to rapidly induce COX-2 in RMC (26, 27), whereas its
anti-apoptotic effect was demonstrated in serum-deprived rat
fibroblasts (51) and endothelial cells (52).
We found that the respective combinations of TNF
with ET-1 or
IL-1
ameliorated TNF
-mediated apoptosis by >50%, as quantified by a reduction in PS exposure and caspase-3 activation. Because we also
show that the cytoprotection induced by ET-1 was reversed by NS398,
highlighting specific inhibition of COX-2 anti-apoptotic catalytic
activity, we suggest that renal inflammation may be propagated by at
least two pathways of COX-2 induction. TNF
may act as a bimodal
ligand, at least in mesangial cells, by promoting cell survival in a
synergistic action with other mediators, and cell death simultaneously,
which may be circumvented by up-regulated COX-2 expression. Hence our
results implicate a novel role for ET-1 and IL-1
as potent survival
factors for renal mesangial cells against TNF
-mediated apoptosis.
Another line of investigation is to analyze both the levels and types
of PGs generated by COX-2 metabolism, which can change significantly
during an inflammatory reaction. Several groups correlated a single
measurement of PGE2 production with COX-2 expression in RMC
(28, 29, 53, 54). Here, we sought to determine the profile of PG
release from endogenous arachidonic acid derived from an overexpression
of COX-2. By this analysis AdCOX-2 cells demonstrated a preferential
synthesis of PGI2 and PGE2 with very little if
any production of thromboxane A2, PGF2
, PGJ2, and PGD2. Accordingly, both
PGE2 and PGI2 inhibited the apoptotic
parameters elicited by TNF
, suppressing caspase-3 activation and PS
exposure, by ~80%. Therefore COX-2 may prevent TNF
apoptosis in
RMC, at least in part, by generating anti-apoptotic products PGE2 and PGI2. Generally, PGE2
production has been correlated with the inhibition of apoptosis as
shown in cancerous or transformed cells such as human colon cancer
cells (19) and cholangiocarcinoma cells (55). The results from the
present study support a growing recognition of both PGI2
and PGE2 participation in the progression of various
inflammatory conditions and cancer progression (38, 49).
Many studies correlate an overexpression of COX-2 and the
prevention of apoptosis with an enhanced expression of bcl-2
(16, 18, 19). In this investigation bcl-2 was not evident in
AdCOX-2 cells implying that an overexpression of COX-2 does not
regulate bcl-2 activity in RMC, and bcl-2
induction is dependent on the cell type and extent of expression or
configuration of the proto-oncogene. Recent work from this laboratory
identified an up-regulation of anti-apoptotic dynein light chain in
PC12 cells (37), and P-glycoprotein expression in RMC, in response to
an overexpression of COX-2 (40). In the former study dynein light chain
selectively prevented nitric-oxide synthase activity and caspase-3
activation, in response to a trophic nerve growth factor
withdrawal model of apoptosis, and the latter report correlated COX-2
activity with increased activity of P-glycoprotein. Studies are
underway to determine whether these mechanisms can be applicable to a
COX-2 suppression of TNF
-mediated apoptosis in RMC, enabling further
novel observations of COX-2 anti-apoptotic activity in primary cell types.
In summary, the presented data suggest that COX-2 overexpression or
induction may prevent apoptosis in renal mesangial cells. The
observations implicate COX-2 expression and catalytic activity in
proliferative GN by inhibiting TNF
-dependent apoptosis
perhaps via the generation of PGE2 or PGI2.
These results could be useful in elucidating the molecular mechanisms
underlying the regulation of COX-2 and may open up specific strategies
for the treatment of renal inflammatory diseases that specifically
target COX-2 or the downstream components of COX-2 rather than TNF
or its receptor.