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Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94304, USA
1Correspondence: Department of Pediatrics, Stanford University School of Medicine, 750 Welch Rd #315, Palo Alto, CA 94304, USA. E-mail: dennery{at}leland.stanford.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: heme • hyperoxia • doxycycline • carbon monoxide • bilirubin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The
reason for the induction of HO-1 could be the protective effect against
oxidative injury since HO metabolic activity leads to the formation of
bile pigments, which have been shown to behave as an antioxidants
(2)
. Another possible cytoprotective effect of HO-1
induction may be related to the decrease in heme iron and hemoproteins
and the increased expression of iron responsive genes such as ferritin
and aconitase (3)
. The role of other metabolites of HO,
namely, carbon monoxide (CO), have not been as well understood in the
context of antioxidant defense, although recent reports indicate
increased CO in inflammatory lung diseases such as asthma
(4)
. Several studies have demonstrated that low
overexpression of HO-1 (less than a fivefold increase in HO activity)
is associated with protection against oxidative stress. For example, a
threefold overexpression of HO-1 was associated with protection against
heme-mediated damage (5)
, and fibroblasts with endogenous
1.8-fold overexpression of HO-1 protein were protected against oxygen
toxicity (6)
. Furthermore, suppressing HO activity using
antisense transfection (7
, 8)
or inhibitors (7
, 9)
led to worsened oxidative stress. Nonetheless, in some
clinically relevant circumstances (10
, 11)
and
experimental models (12
, 13)
, the resultant level of HO
activity is well beyond the modest elevation achieved with transfection
systems and it is not clear whether HO is beneficial at these higher
levels. Exaggerated HO activity may not be beneficial because in
addition to the formation of bile pigments and CO, iron is released
from the degradation of heme via HO. Iron is well known to mediate
reactions leading to the formation of hydroxyl radicals
(14)
This could result in increased susceptibility to
oxidative stress, most likely at the higher range of HO-1 expression.
We have shown that despite improved resistance to oxygen toxicity with
a 1.8-fold increase in activity, a mere doubling of the level of HO-1
expression in fibroblasts resulted in a loss of cytoprotection
(7)
. When HO activity was elevated eight- to ninefold in a
model of endotoxic shock, significant hypotension was observed
(11)
. It would therefore be crucial to understand what
mediates protection in the HO reaction and to identify circumstances
under which HO-1 induction is protective so as to allow for judicious
use of HO in therapeutic interventions. To systematically test whether
overexpression of HO-1 is beneficial, we established HO-1 transfected
cells with low (2–5-fold), moderate (10- to 14-fold), and high (15- to
18-fold) HO activity as compared to untransfected controls. After
careful accounting of all HO reaction products, a diametrically
opposed, concentration-dependent effect of HO-1 in susceptibility to
oxygen toxicity was observed due to reactive iron accumulation. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.3 µM per
flask.
Experimental design
Establishment and maintenance of variable HO-1 cell line
To establish variable HO-1 overexpression, the Tet-Off Gene
Expression System (CLONTECH Laboratories, Palo Alto, Calif.) was used.
As recommended, 1 x 105 HA-1 cells were
grown in 6-well dishes to 60% confluence in a 95% room air, 5%
CO2 incubator. The cells were then incubated
overnight with 2 µg of the pTet-Off plasmid complexed with 6 µl of
LipofectAMINE Reagent (Life Technologies, Inc., Gaithersburg, Md.).
Because the pTet-Off plasmid contains a neomycin resistance site,
transformed colonies were isolated after 3 wk by growing in the
presence of G418 (400 µg/ml, Life Technologies). Transient
transfection assays were performed with the luciferase containing
plasmid (pTRE-Luc) in order to identify a cell line that had a high
level of luciferase induction in the absence of Dox with low background
expression in the presence of Dox. This antibiotic is a derivative of
tetracycline and is as useful in regulating gene expression with the
Tet-Off system. Once identified, this cell line was transfected with 2
µg each of pBI-L containing the full-length rat HO-1 cDNA inserted at
the HindIII and EcoRV restriction sites and
pTK-Hyg to allow for selection of transformed colonies by growth in
hygromycin (Hyg) in a liposome complex. The cells were grown in G418
(400 µg/ml) and Hyg (200 µg/ml; Life Technologies). After 3 wk,
transformed colonies were screened using luciferase activity in order
to isolate a cell line with high induction of HO-1 in the absence of
Dox and low background in the presence of Dox. The cell line was
maintained in EMEM with 10% fetal calf serum supplemented with G418
(200 µg/ml) and Hyg (100 µg/ml). Regulation of variable HO-1
expression was accomplished by incubation in media containing 0 to 1
ng/ml of Dox.
Incubation in Dox and hyperoxia exposure
Four days before hyperoxic exposure, transfected cells were
plated at 2 x 105 cells per 75
cm2 flask and varying concentrations of Dox were
added (1, 0.1, 0.05, 0.01, 0.005, 0.001, and 0 ng/ml) to the
maintenance media. The HO-1-expressing cells and HA-1 controls were
then exposed to hyperoxia (95% O2/5%
CO2) at 37°C in a humidified chamber and
harvested for assays after 24 h of exposure.
Incubation with SnMP and DTPA
To verify that the results were due solely to the overexpression
of HO-1, high HO-1-expressing cells were incubated with an inhibitor of
HO activity, tin mesoporphyrin (SnMP, 10 µM; Porphyrin Products,
Logan, Utah). Cell were also incubated with an iron chelator,
diethylenetriaminepentaacetic acid (DTPA, 25 µM/ml), to determine
whether increased reactive iron associated with HO activity was
involved in mediating increased cell damage. After incubation with
either SnMP or DTPA for 4 h, cell cultures were exposed to
hyperoxia. After 24 h of hyperoxia, cells were harvested for
assays.
Verification of gene expression in cultured cells
Since the pBI-L/HO-1 transfected cells expressed the luciferase
gene, this was used as a reporter system to assess the level of gene
expression with doxycycline. Luciferase activity was monitored in the
cells incubated with varying concentrations of doxycycline by
incubating culture dishes with 20 µl of luciferin (30 mg/ml) for 20
min and then visualizing the photon emission from each dish on an
enhanced charge couple device (CCD) camera as described previously
(15)
.
Total HO activity
Assays were conducted in subdued lighting. Twenty
microliter aliquots of cell sonicates were reacted with 20 µl hemin
(150 µM; Sigma) and 20 µl NADPH (4.5 mM; Sigma) in a septum sealed
vial at 37°C. Blanks consisted of cell sonicates reacted with hemin
only. Vials were purged with CO-free air and allowed to incubate for 15
min. The reaction was stopped with dry ice (-78°C), and CO
generation in the vial gas head space was analyzed by gas
chromatography (16)
. HO activity was derived by
subtracting the blank value from the sample value and expressing this
quantity as nmol CO/mg protein (16)
.
Cell protein content
Sonicates were analyzed for protein content by the method of
Bradford (17)
and read at absorbance 595 nm.
Antibodies
Polyclonal rabbit anti-rat HO-1 antibodies were raised against a
30 kDa soluble HO-1 protein expressed in Escherichia coli
from rat liver cDNA (18)
(gift of Angela Wilks, University
of California San Francisco, Calif.) by Berkeley Antibodies (Berkeley,
Calif.), as described previously (6)
. Rabbit polyclonal
immunoglobulin G (IgG) anti-p53 corresponding to the full-length p53 of
human origin as well as mouse anti-PCNA IgG and goat polyclonal IgG
anti-FGF-2 corresponding to amino acids 40–63 of the amino-terminal
domain of the FGF-2 precursor (all antibodies from Santa Cruz
Biotechnology, Santa Cruz, Calif.) were used. This latter antibody is
specific for basic fibroblast growth factor (bFGF) and does not cross
react with acidic FGF.
Western analysis For HO-1, p53, PCNA, and bFGF
This technique was used to determine the level of HO-1
expression achieved in the transfected cells and to assess the effect
of HO-1 overexpression on markers of cellular proliferation. Aliquots
(20 µg protein) of cell sonicates or nuclear proteins (for PCNA
detection) were used. Nuclear proteins were derived from cell sonicates
collected in a buffer containing 0.5 M sucrose, 10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.9),
1.5 mM MgCl2, 10 mM KCl, 10% glycerol, 1 mM
ethylenediaminetetraacetic acid disodium dihydrate (EDTA), and 1 mM
phenylmethyl sulfonyl fluoride (PMSF). The sonicates were centrifuged
at 4000 x g for 20 min at 4°C and the pellet was
resuspended in a high-salt buffer containing 20 mM HEPES (pH 7.9), 25%
glycerol, 0.5M KCl, 1.5 mM MgCl2, 0.4 mM EDTA,
and 0.5 mM PMSF. After incubation on ice for 10 min, the supernatant
was centrifuged at 14,000 x g for 15 min at 4°C. The
supernatant was aliquoted and stored at -80°C. Prior to assays,
nuclear protein contents were determined as described above (see `Cell
protein content').
The samples were electrophoresed on a 12% polyacrylamide gel according
to the methods of Laemmli (19)
. Proteins from cell
sonicates or from nuclear extracts were transferred for 24 h to
PVDF membrane (Bio-Rad, Hercules, Calif.) with a Bio-Rad transblot
apparatus according to the method of Towbin et al. (20)
.
Blots were washed briefly in 1x Tris-buffered saline (TBS: 200 mM
Tris, 1.5 M NaCl), then incubated overnight at 25°C with rabbit
anti-rat HO-1 immunoglobulin G diluted 1:800 in blocking solution [1%
non-fat milk + 0.5% bovine serum albumin in 0.05% Tween 20-TBS
(T-TBS)]. Blots were washed in T-TBS and incubated for 2 h at
37°C with a 1:5000 dilution of horseradish peroxidase- (HRP)
conjugated goat anti-rabbit IgG (Caltag Laboratories, So. San
Francisco, Calif.). Antigen–antibody complexes were visualized with
the HRP chemiluminescence system according to the manufacturer's
instructions (Bio-Rad). Quantification was performed using Molecular
Analyst image analysis software (Bio-Rad).
Immunohistochemical detection of HO-1 protein
This method was used to further verify the level of HO-1
overexpression achieved. HO-1 transfected cells, grown to >80%
confluence on glass slides for 4 days in Dox (1, 0.5, 0.001, and 0
ng/ml), were washed in phosphate-buffered saline (PBS), then fixed in
ice-cold 100% acetone. The cells were permeabilized in 0.3% saponin
in PBS and blocked in a PBS solution containing 5% milk, 1% bovine
serum albumin, and 0.03% saponin. The slides were incubated with a
1:25 dilution of rabbit anti-rat HO-1 IgG overnight in a humidified
chamber. After incubation, the slides were washed twice in PBS with
0.03% saponin and 1% milk, subsequently incubated with a 1:50
dilution of fluorescein isothiocyanate- (FITC) conjugated goat
anti-rabbit IgG (Southern Biotechnologies Inc, Birmingham, Ala.) for
2 h at 37°C, and washed twice in PBS. The slides were then
mounted in phenylene diamine and viewed with an Axioskop fluorescent
microscope (Zeiss, Germany) fitted with a 100W Mercury HBO100W/2
(Zeiss) lamp at excitation 493 nm and photographed with a Nikon camera.
Determination of cell viability
Trypan blue exclusion was used to determine cell viability.
Cells were released with 0.05% trypsin-EDTA and 20 µl aliquots were
mixed with 20 µl of 0.5% trypan blue. The number of dead (stained)
cells was expressed as a ratio of the total (stained and unstained)
cells counted.
Determination of lactate dehydrogenase (LDH)
Release of LDH into the medium serves as a marker of altered
cellular membrane integrity (21)
. Therefore, we assessed
whether overexpression of HO-1 altered membrane function in hyperoxia.
Media from HO-1 transfected and sham transfected control samples (100
µl) was mixed with 200 µg of NADH in 0.1 M
KPO4 buffer and allowed to incubate for 10 min in
a multiwell plate. Sodium pyruvate (2.3 µmol) was then added and
samples were read at 340 nm at 2 s intervals for 2 min. LDH
concentration was calculated automatically from the slope of the
absorbance curve. Accuracy was determined with a standard LDH enzyme
solution (Enzyme control 2-E, Sigma).
Determination of protein oxidation
To evaluate changes in oxidative injury with HO-1 transfection
and hyperoxic exposure, protein oxidation was measured by detecting
oxidatively generated carbonyl groups using the OxyBlot Kit (Oncor,
Gaithersburg, Md.). The antigen-antibody signal was visualized by
chemiluminescence using the HRP chemiluminescence system according to
the manufacturer's instructions (Bio-Rad).
Detection of lipid peroxidation
To further corroborate cellular oxidative injury, formation of
thiobarbituric acid-reactive substances (TBA-RS) was measured as an
estimate of membrane lipid peroxidation using the method of Wright et
al. (22)
. The concentration of TBA-RS was calculated from
the molar extinction coefficient 1.55 x
10-3 M-1
cm-1.
Total glutathione content of cells
A change in cellular glutathione content can be used as a marker
of oxidative injury since the synthesis of this tripeptide can be
increased in oxidative stress (23)
. Total glutathione was
measured using the Glutathione Assay Kit (Cayman Chemical, Ann Arbor,
Mich.). The samples were deproteinated per the manufacturer's
protocol. The samples were read at 405 nm using a microtiter plate at 5
min intervals for 30 min. Total glutathione was determined by
comparison with standards and expressed per milligram of protein.
Heme content
To detect alterations in the substrate of the HO reaction with
transfection, cellular heme content was determined by quantitation of
the hemochrome in a reaction with pyridine in alkali. Cells were washed
twice with cold 0.15 M KCl, scraped, and centrifuged. The samples were
then resuspended in 0.9% NaCl, mixed with a solution of 25% (v:v)
pyridine in 0.075 M NaOH and scanned at absorbance 350–600 nm. The
hemochromes have strong Soret bands in the range of 405–440 nm. The
absorbance peak corresponding to the Sorret band (414 nm) in the
samples was quantitated using a published molar extinction coefficient
(24)
. Values were expressed as µM heme/mg protein.
Iron content
Since iron is released from the HO reaction and can serve in
oxidative reactions, cellular reactive iron was detected using the
bleomycin assay. Iron contamination was obviated by using Chelex-100
Resin (Bio-Rad) in all buffers. Fifty microliter aliquots of cell
lysates were incubated with a solution, pH 7.4, containing 500 µl DNA
[(1 mg/ml; Sigma), 50 µl bleomycin(1.5 U/ml, Sigma), 100 µl
MgCl2 (50 mM; Mallinkrodt Baker, Inc., Paris,
Ky.), and 100 µl L-ascorbic acid (75 mM; Sigma) in a 37°C shaking
water bath for 1 h. The reaction was stopped with 100 µl EDTA
(0.1 M; ICN Biochemical, Cleveland, Ohio). The samples were reacted
with 500 µl 2-thiobarbituric acid (1% w/v, in 50 mM NaOH; Sigma) and
500 µl hydrochloric acid (25%; Mallinkrodt Baker) in 80°C for 20
min. The absorbance at 532 nm was measured and the iron content is
expressed as µM/mg protein (25)
.
Determination of cellular bilirubin content
Bile pigments such as bilirubin are formed during the HO
reaction. These may alter oxidative injury due to their antioxidant
properties. The effect of HO-1 overexpression on cellular bilirubin
content was evaluated to document any dose-dependent increase in
bilirubin. The moist pellet from 10 to 15 million cells (~20 µl)
was mixed with 80–100 µl of 0.1 M di-octamylamine acetate in
methanol, sonicated, and centrifuged. Twenty microliters of the
supernatant were chromatographed by reversed phase high-performance
liquid chromatography (HPLC) as described previously (26)
.
Guanosine 3',5'-cyclic monophosphate (cGMP) content
Since CO is released from the HO reaction and this volatile gas
has been implicated in the activation of guanylate cyclase
(27)
, cGMP was measured in the HO-1 transfected cells
using the Format A cyclic GMP enzyme immunoassay kit (BIOMOL Research
Laboratories, Inc., Plymouth Meeting, Pa.). The samples were read at
405 nm and the cGMP content was determined by comparison to standards
per the manufacturer's instructions.
Statistical analysis
For comparison between treatment groups, the Null hypothesis
that there was no difference between treatment means was tested by a
single-factor analysis of variance for multiple groups or unpaired
t test for two groups (Statview 4.02; Abacus Concepts,
Berkeley, Calif.). Statistical significance (P<0.05)
between groups was determined by means of the Fisher method of multiple
comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A).
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HO expression in Tet-Off transfected cells
In transfected cells, HO activity was modulated 2.4 ± 0.1 to
17.6 ± 0.5-fold by incubation with 0–1 ng/ml of Dox (Table 1
). We also verified that Dox did not have any effect on HO-1 expression
in the absence of the Tet-Off promoter (data not shown).
|
Similarly, HO-1 protein was increased 2.8 ± 0.3 to 10.1 ±
0.3-fold with the varying Dox concentration (Table 1)
.
Immunohistochemical localization of HO-1 revealed intensity of HO-1
protein signal inversely related to the Dox concentration (Fig. 1B
). Based on the consistent levels of HO activity and HO-1
protein observed in repeated experiments (n=6 in each
group), HO-1 overexpression was classified as low, moderate, or high
based on the degree of HO activity and HO-1 protein achieved.
Therefore, for clarification, low was defined as 2–4 to 4.3-fold,
moderate was a 10- to 13.5-fold, and high was a 14.9–17.6-fold
increase in HO activity.
Cellular injury in HO-1-overexpressing cells
When cells with 2.8- and 10-fold increase in HO activity were
exposed to hyperoxia for 48 h, cell viability was significantly
increased compared to controls (Table 1)
. However, at other low or
moderate levels of HO-1 expression, no differences were observed. In
contrast, at high levels of HO-1 expression, cell viability was
significantly lowered consistent with cytotoxicity at high levels of
HO-1 overexpression despite protection at low levels (Table 1)
.
As another indicator of oxidative injury, LDH release was also
determined. Cellular LDH release was similar to that of control at the
low and moderate range of HO-1 expression. However, at the high level
of HO-1 expression, LDH release was significantly increased
(>2.5-fold) compared to controls (Table 1)
. This further documents
increased cytotoxicity when HO-1 expression is highest.
Effect of HO-1 overexpression on cell proliferation
The protective effect of HO-1 have been ascribed in some instances
to alterations in cellular proliferation (28)
. To assess
the effect of HO-1 expression on cell proliferation, we determined the
level of immunoreactive PCNA in cell nuclei. This protein was elevated
when HO-1 was expressed in the low range, but decreased at intermediate
levels of HO-1 expression. At high levels of HO-1 expression, increased
proliferation was again observed in the viable cells (Fig. 2
). With the increase in PCNA at high HO-1 expression, immunoreactive
bFGF, a fibroblast growth factor and marker of cellular proliferation
and transformation (29
30
31)
. was also detected (Fig. 2)
whereas low and moderate HO-1 expression was not associated with an
increased bFGF compared to controls, suggesting that the increased
proliferation denoted at the highest levels of HO-1 expression was more
likely abnormal. Increases in p53 were detected at high levels of HO-1
expression, further (Fig. 2)
suggesting that perturbations in the cell
cycle are noted when HO is maximally expressed in the HA-1 cells.
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Oxidative injury with HO-1 overexpression
As for more specific determinants of oxidative injury, the
formation of protein carbonyls was also evaluated. These are formed
during oxidative modification of proteins and have been correlated with
oxidative damage (32
, 33)
. There was a visible decrease in
protein carbonyl content at low levels of HO-1 expression, whereas
moderate levels of HO-1 overexpression did not demonstrate differences
compared to controls. At high levels of HO-1 overexpression, protein
carbonyl content was visibly increased compared to controls (Fig. 3
).
|
Another parameter of oxidative injury is the formation of lipid
peroxide byproducts such as TBA-RS. As with the other markers of
injury, these were significantly lower in cultured cells with low HO-1
expression, no different in cells with moderate HO-1 expression, and
significantly increased in the cells with high HO-1 expression compared
to controls (Table 1)
.
Total glutathione content was also evaluated since glutathione is an
important antioxidant molecule. At the low or moderate levels of HO-1
expression, no differences in glutathione content could be detected
when compared to controls. However, at the high levels of HO-1
expression, there was a 2.5-fold increase in total glutathione content
compared to controls (Table 1)
.
Determination of HO reaction products in HO-1-overexpressing cells
Since CO from HO metabolic activity can modulate guanylate cyclase
and thereby alter cGMP (27)
, we evaluated cGMP levels in
the HO-1-overexpressing cells to determine whether this by-product of
the HO-1 reaction could to lead to alterations in cellular toxicity in
hyperoxia. No significant differences in cGMP were observed at any
levels of HO-1 overexpression.
Bilirubin is another important byproduct of HO that may modulate
oxidative damage (34)
. This was determined in the high and
low HO-1-expressing cells. Neither bilirubin nor bilirubin glucuronides
could be detected in extracts of cells by a sensitive HPLC assay.
To evaluate whether the changes in oxidative injury were correlated to
the levels of heme or reactive iron within the cells, we evaluated
these parameters. Heme content was significantly lower at the low
levels of HO-1 expression, but returned to control levels and did not
vary significantly at the moderate or high levels of HO-1 expression
(Fig. 4
). As for reactive iron content, this was highest, as expected, at the
high levels of HO-1 expression compared to controls (Fig. 5
).
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Since induction of HO and consequent iron release is associated with
increased expression of ferritin protein (35)
, we
determined immunoreactive ferritin protein content of cells variably
expressing HO-1. Ferritin protein was consistently detected in the low
and moderate HO-1-expressing cells but was visibly increased in the
high HO-1-overexpressing cells (Fig. 6
), corroborating the iron-responsive nature of ferritin gene regulation
(36)
and increased iron release with HO-1 expression.
Nonetheless, despite increased ferritin protein with high HO-1
expression, no protection against oxidative injury was found, but
significant oxygen toxicity could be detected.
|
To further document that the differences in oxidative stress observed
were related to reactive iron content, cells with high levels of HO-1
expression were treated with SnMP, an inhibitor of heme oxygenase, or
DTPA, an iron chelator. Total HO activity was reduced by greater than
50% with 10 µM SnMP resulting in a level of HO activity equal in the
low to moderate range of HO-1 expression (specifically, a seven- to
eightfold increase in HO activity) (Table 2
). Treatment with DTPA however significantly increased HO activity in
the high HO-1-overexpressing cells (Table 2)
. Nonetheless, iron levels
were significantly lower in the DTPA-treated HO-1 high expressors than
in the untreated samples, resulting in iron levels similar to those of
untreated low and moderate HO-1-overexpressing cells (Table 2)
.
Treatment with SnMP resulted in a significant but less dramatic
lowering of reactive iron in the high HO-1-overexpressing cells than in
untreated high HO-1-expressing samples. The resulting iron levels were
comparable to that of the moderate HO-1-expressing cells (Table 2)
.
Whether achieved with SnMP or DTPA, the lowering of reactive iron
content was associated with a significant decrease in injury parameters
when compared to the untreated high HO-1-expressing cells, as
demonstrated by lowered LDH release (Table 2)
and lowered protein
oxidation (Fig. 3
, inset). It is important to note that treatment with
DTPA or SnMP did not increase the level of ferritin protein expression
in the HO-1-overexpressing cells, thereby precluding an increased
sequestration of redox active iron mediated by ferritin with the use of
these agents (Fig. 6)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 37)
. To corroborate this, some reports have suggested
a possible duality of effects of HO-1 overexpression in oxidative
stress (7
, 38
, 39)
. Since it is feasible that
catalytically active (reactive) iron would accumulate when HO-1
expression is highest, we used a variable overexpression model, where
HO activity varied between 3- and 17-fold and HO-1 protein content was
modified 3- to 10-fold. This level of expression was consistent from
experiment to experiment.
With hyperoxic exposure, protein oxidation, lipid peroxidation, and
glutathione content were significantly increased in the cells
expressing greater than 15-fold HO activity, demonstrating a strong
association between HO-1 expression and exacerbation of oxidative
injury per several reliable parameters. An increase in lung protein
carbonyl content was detected in premature infants exposed to high
oxygen, which correlated with the incidence of chronic lung disease
(40
, 41)
. A significant increase in total glutathione is
frequently observed early in the process of oxidative injury
(42)
.
Despite the cytotoxic effect of high HO-1 expression, there was also a
consistent cytoprotective effect of HO-1 at low levels of expression.
This finding was consistent with many other reports similarly
documenting a protective effect of HO-1 when HO activity achieved was
in the range of the low overexpressing cells of this current study
(5
, 7
, 9
, 28
, 43)
, but the deleterious effects of high
expression were unmasked with the present study.
Decreased cell proliferation has been noted with HO-1 transfection, and
this effect was tied to the protective nature of the HO-1 enzyme
(28)
. In the present study, moderate levels of HO-1
expression were associated with decreased cell proliferation, but this
was reversed at the high levels of HO-1 expression. Furthermore, the
increased cellular proliferation at the highest levels of HO-1
expression was associated with possible cellular transformation.
Increased expression of bFGF was noted in the high HO-1-expressing
cells. This growth factor is specific for fibroblasts, and increased
expression has been noted in several malignancies and with exposure to
carcinogens (29
30
31)
. In the high HO-1 expressors, there
was also increased expression of p53, a well-characterized oncogene,
further suggesting abnormal cell growth and differentiation (44
, 45)
. Taken together, these data suggest that high HO-1
expression perturbs the cell cycle. In clinical settings, abnormal
fibroblast proliferation is observed with oxidant stress and may lead
to abnormal lung architecture or malignancy (46
47
48)
.
We documented a lowered heme content in the low HO-1-overexpressing
cells. This corroborates other observations of a 40% reduction of heme
with a threefold increase in HO activity mediated by ultraviolet A
radiation (49)
and a lowered cellular heme iron with a
nitric oxide donor that increased HO activity fivefold
(3)
. Nonetheless, no reports have evaluated the effect of
high HO-1 expression on cellular heme content. A paradoxical increase
in cellular heme content was observed with moderate and high HO-1
expression compared to low HO-1-overexpressing cells. It is not clear
whether this finding is explained by increased iron release from the HO
reaction at the higher levels of HO-1 expression and up-regulation of
the heme synthetic pathway through iron response elements
(50)
. This remains to be systematically investigated.
To verify whether the increased oxidative injury was related to changes
in the levels of metabolic products of heme oxygenase, we looked at
bilirubin, reactive iron content, and cGMP levels. No significant
change in cGMP levels was detected with modification of HO expression.
Several studies have demonstrated activation of guanylyl cyclase
through NO and CO (51
52
53
54)
. Nonetheless, some evidence
suggests that NO is a better modulator of guanylyl cyclase than is CO
(55
, 56)
. Our studies would corroborate these
observations. We also did not see significant alterations in cellular
bilirubin content in the low or high expressors, implying that the
generation of bilirubin via the HO reaction is not sufficiently
different to explain the cytoprotective effect of low HO-1 expression
or that other factors may obviate the protective effects of bilirubin
at the high levels of HO-1 expression (57)
, at least in
this model.
Iron detected with the bleomycin assay is thought to be associated with
increased generation of oxygen radicals. In fact, many studies have
shown that reactive iron is associated with increased oxidative stress
in human disease (58
59
60)
. Cells with the highest levels
of HO-1 expression had the highest reactive iron content. At the high
levels of HO-1 expression, ferritin protein was up-regulated in the
presence of iron, but this did not result in increased cytoprotection.
In fact, worsened oxygen toxicity was observed, suggesting that
ferritin iron could participate in oxidative reactions when released
from ferritin in oxidative stress (61
, 62)
. Nonetheless,
it is not clear whether this ferritin was iron loaded and therefore not
useful in iron sequestration.
To further understand whether there was an association between the higher reactive iron content seen at the higher levels of HO-1 expression and the increased oxidative injury, iron levels were modified either by treatment by SnMP or with an iron chelator. As predicted, SnMP reduced heme oxygenase activity from 15-fold to 4- or 5-fold compared to controls. At this range of HO-1 expression, the cultured cells were relatively protected against oxidative injury, as demonstrated by decreases in LDH release and decreases in protein oxidation in hyperoxia compared to untreated high HO-1-expressing controls.
Treatment with DTPA resulted in increased HO activity compared to
controls, perhaps suggesting that reactive iron may serve to inhibit HO
activity as is the case with other enzymes (63)
. Despite
the increase in measured HO activity, reactive iron content was reduced
to control values with DTPA and there was a significant reduction in
oxidative injury, as evidenced by decreased LDH release and protein
oxidation. Since both SnMP and DTPA resulted in decreased reactive iron
content, the data strongly suggest that released reactive iron from the
HO reaction dictates the toxicity of HO-1 at high levels of expression.
In this study, the balance of heme and reactive iron determined the antioxidant effect of HO-1. At low HO-1 expression, low cellular heme and low iron may allow for decreased oxidative injury and up-regulation of important enzymes, whereas excessive accumulation of reactive iron at high HO-1 expression would result in increased oxidative stress, cytotoxicity, and abnormal cellular proliferation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 25, 1999. Revised for publication April 15, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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