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Research Communications |
Institute of Biomedicine, Department of Anatomy and Medcity Research Laboratory, University of Turku, Turku, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: oxidative stress • skeletal muscle • overexpression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to its carbon dioxide hydratase activity, CA III has been demonstrated to have a carboxyl esterase activity and phosphatase activity, which suggests that it is a tyrosine phosphatase (9) . In a recent study, the role of CA III in carbohydrate utilization was also suggested (10) . CA III forms a disulfide link between glutathione and two of its five cysteine residues in a process termed S-glutathiolation (11) . Protein S-thiolation/dethiolation is an early cellular response to oxidative stress 12-14) . It has been demonstrated recently that glutathiolation of CA III, which occurs in vivo and is increased during aging, reversibly regulates its tyrosine phosphatase activity (15) . Glutathiolation of Cys-186 is required for the phosphatase activity of mammalian CA III, whereas glutathiolation of Cys-181 blocks it. This tyrosine phosphatase activity, which is dependent on cellular redox state, suggests that CA III may have a role in intracellular signaling, particularly in response to oxidative stress.
Aerobic metabolism leads to the production of reactive oxygen species (ROS), including singlet oxygen, superoxide, hydrogen peroxide (H2O2), nitric oxide (NO), and hydroxyl radical. At moderate concentrations, ROS serve as signal transduction messengers, but their excessive production causes oxidative damage. Such damage may afflict all types of biological molecules, including DNA, proteins, lipids, and carbohydrates. Cells have thus evolved an antioxidant defense system, consisting of antioxidant vitamins, glutathione, sulfhydryls, and antioxidant enzymes such as peroxidases, catalase, and superoxide dismutases (16) . CA III has two sulfhydryl groups; on this basis it has been suggested that it may have a role in scavenging oxygen radicals in skeletal muscle cells 17-19) since strenuous physical exercise has been shown to enhance generation of free radicals (20 , 21 ). Oxidative stress has been suggested to play a role as a common mediator of apoptosis or programmed cell death. In addition, a wide variety of antioxidants function as antiapoptotic agents in several different cell types (22) . CA III is expressed in high quantities in skeletal muscle, especially in type I muscle fibers, which have oxidative metabolism and thus produce a lot of ROS as a response to exercise.
We hypothesized that CA III may be an important protective mechanism against oxidative damage and ROS-induced cell death. To test this hypothesis, we created transfected cells expressing CA III at a high level and tested the basal intracellular ROS-levels and ability of the cells to resist oxidative stress generated by exposure to H2O2 in transfected and parental cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression plasmid and transfection
The plasmid vector used in the CA III transfections was pRC/CMV
(InVitrogen, San Diego, Calif.). This plasmid carries a neomycin
resistance gene and SV40 origin, which allows episomal replication in
COS7 cells. Rat CA III cDNA was a generous gift from Dr. Nicholas
Carter (Medical Genetics Unit, St. George's Hospital Medical School,
London). The pRC/CMV-CA III was constructed by cloning the polymerase
chain reaction (PCR) -amplified CA III cDNA (full coding sequence
~800 nt) with inserted Kozak sequence, at the Hind III-Not I site of
the expression vector. Cells were transfected by using a cationic
liposome reagent (Boehringer Mannheim, Mannheim, Germany). Transfected
COS7 cells were fixed for immunofluorescence staining or lysed for
immunoblotting 72 h after transfection. Transfected NIH/3T3 cells
were selected by 500 µg/ml geneticin (G418; Calbiochem-Novabiochem,
San Diego, Calif.), replacing the antibiotic containing medium every 3
days over 14 days. Isolated clones were maintained in 300 µg/ml
geneticin. Geneticin was omitted from the medium in experiments.
CA II expression construct was created by inserting the reverse transcription (RT) -PCR-cloned rat CA II cDNA into a pLTRpoly expression vector (24) that contains the Moloney murine leukemia virus LTR promoter. This construct was stably cotransfected with a SV2neo selection marker vector (25) into S115 cells by using Lipofectin method (GIBCO-BRL, Paisley. U.K.). Control clones were prepared similarly by using the pSV2neo vector alone. Transfected cells were selected with 750 µg/ml G418, after which the clones were maintained in complete medium containing 300 µg/ml G418.
Immunofluorescence staining
Cells were rinsed with phosphate-buffered saline (PBS), fixed
with 3% paraformaldehyde (15 min), permeabilized with 0.5% Triton
X-100 (4 min), blocked with PBS/3% bovine serum albumin/0,1% glycine
(30 min), and incubated for 1 h in monoclonal human CA III
antiserum (diluted 10 µg/ml with PBS/0.1% glycine). Texas
red-conjugated goat antimouse immunoglobulin G (IgG) (1:200) was used
as a secondary antibody. Samples were embedded in Moviol-488 (Hoechst,
Frankfurt, Germany) -2.5% diazabicyclo-octane (Sigma).
To determine the apoptosis index, paraformaldehyde-fixed cells were subjected to Hoechst 33258 staining (1 mg/ml stock diluted 1:800 in PBS) for 5 min. Samples were then maintained as above.
RNA isolation and RT-PCR
Total RNA was isolated from the cells using RNA STAT-60
isolation reagent (TEL-TEST, Friendswood, Tex.). Ethanol precipitated
RNAs were dissolved in diethyl pyrocarbonate-treated water.
First-strand cDNA was synthesized in a 20 µl reaction mixture
containing 15 µl total RNA, 0.5 mM dNTP (Pharmacia, Uppsala, Sweden),
0.5 µg random hexamer (Promega, Madison, Wis.), 2 µl of AMV buffer
(50 mM Tris-HCl, pH 8.3, 6 mM MgCl2, 40 mM KCl, 4.0 mM
DTT), and 20 units AMV reverse transcriptase (Finnzymes, Espoo,
Finland). The mixture was incubated at 42°C for 60 min.
CA III sense and antisense primer sequences were taken from exons 2 and 3 in order to avoid PCR products from possible genomic DNA contaminations. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers from exons 4 and 8 were used to assess the amount of RNA. PCR was carried out in 50 µl reaction mixture containing 5 µl PCR buffer (10 mM Tris-HCl, pH 8.8, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100), 0.2 mM dNTP, 0.8 µM of sense and antisense primers, 2 µl of RT cDNA, 1 unit DyNAzyme DNA polymerase (Finnzymes), and 38.5 µl sterile distilled H2O. PCR was conducted in a Peltier Thermal Cycler (MJ Research, Watertown, Mass.) for 27 cycles. After RT-PCR, samples were electrophoresed on 1% agarose gel containing 10 µg/ml ethidiumbromide, and intensity of the bands was measured by image analysis system (MCID/M2, Imaging Research Inc., Brock University, Ontario, Canada).
Isolation of genomic DNA and PCR
The plasmid copy number was estimated by PCR. Genomic DNA was
isolated from the cells using the method described by Sambrook et al.
(26)
. To detect incorporated CA III cDNA, sense and
antisense primers were chosen to amplify the whole CA III cDNA. For
endogenous gene, primers were chosen from exons 4 and 5 to amplify
intron 4. PCR was performed as described above, except that 1 µl of
genomic DNA was used as a template, the amount of sterile
H2O was 39.5 µl and the number of cycles was 30.
Amplicons were analyzed as RT-PCR products. CA III copy numbers for
transfected clones were estimated by using the ratio of the 800 nt CA
III cDNA signal and the 850 nt genomic signal (which corresponds to two
copies of the CA III gene in the genomic DNA).
Western blot analysis
For total cellular protein extraction, cells were washed with
PBS and lysed in lysis buffer [10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10
mM NaCl, 0.5% Triton X-100, 0.3% sodium dodecyl sulfate (SDS), 1 mM
PMSF, 5 µg/ml aprotinin]. Lysed cells were harvested by scraping
with a rubber policeman and sonicated on ice. Protein content of the
samples was measured using Bio-Rad protein assay (Bio-Rad Laboratories,
Hercules, Calif.). Cytoplasmic and nuclear protein extracts were
prepared essentially as described previously (27)
.
Briefly, cells were harvested from cell culture media by centrifugation
(at room temperature, 5 min at 3000 rpm, Beckman GH 3.7 rotor).
Pelleted cells were suspended in five volumes of PBS and collected by
centrifugation as above. Subsequent steps were performed at 4°C. The
cells were suspended in five volumes of buffer A and allowed to stand
for 10 min. The cells were collected by centrifugation as before,
suspended in two volumes of buffer A, and lysed by passing through a 20
gauge needle. The homogenate was centrifuged (10 min, 10,000 rpm in an
Eppendorf centrifuge) to pellet nuclei. The supernatant containing
cytoplasmic proteins was decanted and the pellet was suspended in
buffer A. Protein content of both fractions was measured as above.
Aliquots of the samples were electrophoresed on a 12% SDS polyacrylamide gel according to the method of Laemmli (28) . After electrophoresis, proteins were transferred onto nitrocellulose membranes in a Bio-Rad Mini Trans-Blot apparatus (Bio-Rad Laboratories). Membranes were stained by using polyclonal rabbit antiserum against human CA III, which has also been shown to cross-react with rat (29) in 1:200 dilution. Horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories) at a 1:3000 dilution was used as a secondary antibody.
Measurement of intracellular oxidized state
Measurements were made using a fluorescent dye,
2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), a
nonpolar compound that is converted into a nonfluorescent polar
derivative, DCFH, by cellular esterase after incorporation into cells.
DCFH is membrane impermeable and is oxidized to highly fluorescent DCF
in the presence of intracellular hydrogen peroxide and peroxidases
(30)
. For assays, cells were grown almost confluently on
glass coverslips. Medium was replaced with Hanks' solution containing
10 mM H2DCFDA, and the cells were incubated for 20 min at
37°C. After loading, the cells were rinsed briefly in the incubation
buffer containing 127 mM NaCl, 5 mM KCl, 2 mM MgCl2, 0.5 mM
NaH2PO4 2 mM CaCl2, 5 mM
NaHCO3, 10 mM glucose, 10 mM Hepes, and 0.1% bovine serum
albumin (pH 7.4). The measurements were performed with an image
analysis system (MCID/M2, Imaging Research Inc., Brock University,
Ontario, Canada) consisting of an Intel 403 E microcomputer linked to
an Image 1280 image processor (Matrox Dorral/Quebec, Canada). Cells
were kept at 37°C under a thermostated incubation hood (Nikon, Tokyo,
Japan). A Sony CCD 72E camera (Dage-MTI Inc. Michigan City, Mich.) and
a Videoscope KS-1381 signal amplifier (Washington) were used to collect
the data. Cells were excited 495 nm wavelengths, using a
computer-driven filter wheel (MAC 2000, Ludl Electronic Products Ltd,
New York) and 32 D neutral filter to avoid UV-induced radical
production. Emitted light was collected through a dichroid mirror and
an interference filter at 510 nm. Results are presented as intensity of
density levels (IOD). Measurement of intracellular oxidized state was
also done with CA II-transfected cells and their control cells in order
to evaluate the role of carbon dioxide hydratase activity to results.
Cell growth kinetics
Cell growth was determined using a nonradioactive cell
proliferation assay (Promega) or by measuring the absorbance at 260 nm
(A260), directly related to cell mass, as Mirault et al.
(31)
have described. When using the proliferation assay,
cells were seeded at a density of 1 x 104 cells/well
in 96-well dishes. After 2 h, H2O2 was
added and the cells were incubated overnight under normal growth
conditions before determining the number of living cells according to
manufacturer's instructions. When A260 was measured, cells
were seeded at 3 x 105 in 35 mm dishes, left to
attach, and treated for 1 h with H2O2.
After treatment, incubation medium was replaced by fresh medium and
cellular growth was recorded.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCR from the genomic DNA was used to detect integration of the plasmid-DNA into NIH/3T3 genome. PCR from the C1 and C3 clones yielded a 800 nt product, which was missing from the parental cell line (Fig. 2 A). By comparing the ratio of CA III cDNA signal and 850 nt genomic signal, we found that clone C1 had three and clone C3 two exogenous CA, III cDNA copies.
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CA III mRNA expression in cells was detected by RT-PCR (Fig. 2B ). The CA III-specific primers produced a 290 nt fragment in the samples of C1 and C3 none in control cells, which suggests that NIH/3T3 cells do not have endogenous CA III expression or it is below the limit of detection by this method.
In immunofluorescence staining with a polyclonal human CA III antiserum, CA III protein expression was detected in C1 and C3 clones; there was also some staining in parental cells (Fig. 3 A). C3 cells showed heterogeneity in CA III expression that was apparently caused by difficulties in isolating the clone. Western blot analysis agreed with results from PCR and immunofluorescence staining. CA III protein level was higher in C1 clone, with a higher copy number and CA III mRNA level than in C3 clone (Fig. 3B ). CA III concentration in the C1 clone was estimated to be at least 0.1% of the total cellular protein. The parental cell line did not seem to contain any CA III.
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Intracellular oxidized state and response to
H2O2 in CA III transfectants
The effect of CA III expression on the intracellular pool of ROS
was analyzed using a fluorescent probe, H2DCFDA. Increase
in fluorescence intensity reflected increase in cellular oxidized
state. The level of basal DCFH-fluorescence was about 60% lower in CA
III-expressing cells than in control cells (Fig 4A
). Addition of H2O2 to
incubation buffer led to a three-to fivefold increase in intracellular
oxidized state in control cells whereas no change in fluorescence
intensity was observed in transfectants (Fig. 4
C). The corresponding measurements were also done with CA
II-overexpressing cells to find out whether CA II also affects the
intracellular oxidized state. A similar rise in fluorescence intensity
was detected in these cells and control cells after addition of
H2O2 (Fig. 4D
). Because the final
concentration of H2O2 was only half of that
used in CA III experiments, the increase in IOD levels was smaller.
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Cell growth and sensitivity to H2O2-induced
apoptosis in CA III transfectants
Growth rate of the C1 clone and parental cells was
determined by a nonradioactive proliferation assay. As shown in
Fig. 5
, transfected cells grew significantly faster than control cells.
In a series of similar experiments, C1 clone usually had a higher
growth potential than C3 and also resisted oxidative stress more
effectively. When H2O2 was added at moderate
concentrations to the cell culture, parental cells were clearly more
sensitive to the treatment than CA III-expressing cells. In
proliferation assay, treatment with 25 µM oxidant caused a 50%
reduction in growth of the control cells when, in the transfected
clones C1 and C3, reduction was 20% and 28%, respectively (Fig. 5)
.
At higher concentrations of H2O2, growth
inhibition was about the same in control and transfected cells (data
not shown). In Fig. 6
the effect of a 1 h oxidative shock on cell growth kinetics
is shown. Cells were treated with 250 µM
H2O2, fresh medium was changed and cellular
growth was recorded on ensuing days. A slower growth rate of the
control cells was confirmed: their cell mass was only 52% of that of
CA III-expressing cells on the third day after plating.
H2O2 treatment resulted in an almost total
growth arrest in control cells, in which the growth rate was 20% of
that in untreated cells on the third day after oxidative shock. In
transfectants the cell growth was also remarkably retarded, but the
reduction was smaller than in parental cells (57%).
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The proportion of apoptotic cells after a 12 h H2O2 treatment was determined by Hoechst staining (Fig. 7 ). In control plates, 83% and 100% of the cells were apoptotic after treatment with H2O2 at a low and high concentrations, respectively. In C1 clone, the corresponding percentages were 13% and 31%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Measurement of intracellular oxidized state revealed a lowered intracellular steady-state level of ROS in transfected cells when compared to parental cells. CA III transfectants did not react to exogenously added H2O2, which resulted in increased oxidized state in control cells. This suggests continuous trapping of ROS in CA III-transfected cells. The cells overexpressing CA II did not seem to have this capacity, which suggests that this phenomenon is independent of carbon dioxide hydratase activity of CA III.
It has been reported that H2DCFDA detects oxidation caused by H2O2 and peroxidase, but not directly superoxide radical 33-35) . Additional experiments are required to clearly identify the radical species, which are eliminated, but H2O2 and its conversion product hydroxyl radical are possible candidates. The conversion of H2O2 to hydroxyl radical is catalyzed by Fe2+ in the Fenton reaction. Chelation of Fe3+ was reported to decrease the toxicity of H2O2 in cultured hepatocytes, an effect that was suppressed by addition of Fe2+ (36) . This suggests that a majority of cytotoxic effects of H2O2 could be mediated by the formation of hydroxyl radicals via Fenton-type reactions.
The putative ability of CA III transfectants to eliminate radicals was also assayed in cell proliferation measurements. After treatment with H2O2, growth reduction occurred and there were fewer apoptotic cells in CA III-transfected cells than in parental cells. There has been some controversy concerning ROS-induced apoptosis. However, several reports suggest that mild oxidative stress can initiate apoptosis rather than necrosis (37) . It was recently shown that ROS are able to reduce proliferation rate and induce apoptosis in human fibroblasts by increasing the levels of p53 and the cdk inhibitor WAF1/CIP1(38) . Myoblasts have also been shown to undergo apoptosis when exposed to reactive oxygen species and NO (39) . Furthermore, autocrine production of extracellular catalase prevented apoptosis in a human T cell line (40) , which indicates that H2O2 is a major factor in the induction of apoptosis. In our experiments, H2O2 induced apoptosis in NIH/3T3 cells, and this deleterious effect was clearly diminished in CA III-expressing cells.
Taken together, these results indicate that CA III could have a direct role in cellular response to oxidative damage. Tissues that have a high oxygen consumption rate such as liver, heart, brain, and skeletal muscle also possess high antioxidant activity (41) . CA III is abundant in skeletal muscle and some other tissues, which may make it physiologically a significant pool of reactive sulfhydryls that function as oxyradical scavengers. The concentration of CA III in these cells could reach the same order of magnitude as that of glutathione. However, myocardium and type IIa skeletal muscle fibers, for instance, do not express CA III although they have a high oxidative capacity. It is possible that other scavenger systems are more powerful in these particular cells. Chatterjee et al. (42) have described a protein (senescence marker protein-1) that is present in high amounts in adult rat liver and decreases during aging. This protein was later identified to be CA III (19) . A possible decrease in the concentration of CA III or of its radical-eliminating capacity during aging could have deleterious effects. A widely accepted hypothesis is that oxygen toxicity may be implicated in aging and in the etiology of a wide variety of pathophysiological conditions, e.g., muscular dystrophy and diabetes.
Experimental data indicate that the function of many growth
signal-transducing proteins depends on their redox state. Such proteins
include growth factor receptors, protein kinases, protein phosphatases,
and a number of important transcription factors including NF-
B and
AP-1 43-46)
. Since tyrosine phosphatase activity of CA
III is also dependent on the cellular redox state, it is possible that
some effects of the redox state on cellular metabolism could be
mediated by the phosphatase action of CA III. Unfortunately, it is not
yet known which are the physiological substrates of CA III phosphatase
activity in the cells. Furthermore, H2O2
produced endogenously suppressed DNA synthesis and addition of catalase
stimulated it in quiescent mouse osteoblastic cells (47)
.
Recently, a novel growth-promoting factor, which stimulated a variety
of cell types, was identified as catalase (48)
. Also, the
Cu/Zn-superoxide dismutase transfected cells, which have elevated
levels of H2O2, exhibited features of cellular
senescence (49)
. It is also possible that the lowered
level of intracellular oxyradicals in CA III-transfected cells in our
study had a positive effect on cell growth. However, it cannot be ruled
out that the faster growth rate of the transfected cells was a
secondary effect due to integration of the plasmid-DNA into such a
location in the genome that could affect cellular growth. Since the
increased growth rate was observed in two transfected clones, it is
unlikely that the integration as such explains the faster growth rate
of the CA III overexpressing cells.
In conclusion, we found that overexpression of CA III reduces steady-state levels of intracellular ROS, increases proliferation rate, and can protect cells against H2O2-induced apoptosis. These observations, together with the fact that CA III in humans as well as in other species is abundantly expressed in skeletal muscle, suggest that it may provide an important physiological mechanism of protecting muscle tissue against oxidative damage.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: CA, carbonic anhydrase; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; H2DCFDA;
2',7'-dichlorodihydrofluorescein diacetate;
H2O2, hydrogen peroxide; Ig, immunoglobulin;
IOD, intensity of density; NO, nitric oxide; PBS, phosphate-buffered
saline; ROS, reactive oxygen species; RT-PCR, reverse transcription
polymerase chain reaction; S115, Shionogi 115; SDS, sodium dodecyl
sulfate.
Received for publication September 4, 1998.
Revision received October 29, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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