Renal Section, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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We have previously shown that lysophosphatidic acid (LPA), an abundant serum lipid that binds with high affinity to albumin, is a potent survival factor for mouse proximal tubular cells and peritoneal macrophages. We show here that BSA also has potent survival activity independent of bound lipids. Delipidated BSA (dBSA) protected cells from apoptosis induced by FCS withdrawal at concentrations as low as 1% of that in FCS. dBSA did not activate phosphatidylinositol 3-kinase, implying that its survival activity occurs via a mechanism distinct from that for most cytokines. On the basis of the following evidence, we propose that dBSA inhibits apoptosis by scavenging reactive oxygen species (ROS): 1) FCS withdrawal leads to ROS accumulation that is inhibitable by dBSA; 2) during protection from apoptosis, sulfhydryl and hydroxyl groups of dBSA are oxidized; and 3) chemical blockage of free sulfhydryl groups or preoxidation of dBSA with H2O2 removes its survival activity. Moreover, dBSA confers almost complete protection from cell death in a well-established model of oxidative injury (xanthine/xanthine oxidase). These results implicate albumin as a major serum survival factor. Inhibition of apoptosis by albumin occurs through at least two distinct mechanisms: carriage of LPA and scavenging of ROS.
apoptosis; reactive oxygen species; renal epithelial cells
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INTRODUCTION |
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ALBUMIN IS A 69-kDa plasma protein with a variety of physiological functions, including maintenance of plasma oncotic pressure, buffering of acid-base changes, and transport to and from tissues of multiple substances within the bloodstream (7, 40, 41). Molecules carried by albumin include free fatty acids, phospholipids such as lysophosphatidic acid (LPA), steroid-based hormones, prostaglandins, and heavy metals (25, 27, 40, 41). Albumin is unique among plasma proteins in having a free sulfhydryl group at Cys34 as well as being devoid of any carbohydrate side chains (22, 41). The redox state of the free sulfhydryl group at Cys34 has been shown to be important in regulating the ligand binding capacity of albumin (22).
Albumin is frequently used as a carrier protein in cell culture
systems. However, the potential role of albumin, or its multiple ligands, on the regulation of cell survival and apoptosis has not been
fully explored. Recently, we have shown that albumin-bound LPA is a
major serum noncytokine survival factor for primary cultures of murine
peritoneal macrophages (m) and renal tubular epithelial cells (25,
27). Because LPA was able to promote survival at concentrations as low
as 50 nM, it is clear that even trace amounts of BSA can have a
substantial effect on cell survival. During these studies, we observed
that 99.95% delipidated BSA alone, which is devoid of LPA, was itself
able to inhibit apoptosis of murine peritoneal m
subjected to serum
withdrawal (25).
We show here a novel role for albumin as a potent survival factor for
primary cultures of murine m and renal tubular epithelial cells.
This effect of albumin is independent of both bound lipids, such as LPA
or free fatty acids, and the oncotic properties of albumin. The
survival activity of albumin appears to depend on its ability to act as
an antioxidant, most likely through scavenging of reactive oxidative
species (ROS) by the free hydroxyl groups of albumin plus its free
sulfhydryl group at Cys34.
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METHODS |
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Reagents.
Neutral dextran-70 was obtained from Pharmacia (Uppsala, Sweden).
1-Glycoprotein (orosomucoid;
1GP) and
2-microglobulin (
2MG) were both generous gifts of Dr. Michael Shia (Boston Univ. School of Medicine, Boston, MA). All other reagents, unless otherwise stated, were obtained from Sigma Chemical (St. Louis, MO).
Primary culture of mouse peritoneal
m.
Peritoneal exudate cells were harvested by lavage from C57Bl/6 mice
(The Jackson Laboratory, Bar Harbor, ME) 3 days after ip injection of
1.5 ml of 4.05% thioglycollate broth (28). Cells were washed twice in
RPMI 1640 and plated in 24-well tissue culture plates at 2 × 105 cells/well in R.10 culture
medium (RPMI 1640 plus 10% FCS, with 2 mM
L-glutamine, 5 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin). After a 4-h incubation at
37°C, nonadherent cells were removed by washing with RPMI 1640. The
remaining adherent cells, >98% m
, as determined by morphological
examination and nonspecific esterase staining (28), were cultured in
R.10, R.0 medium (R.10 minus FCS), or R.0 plus various concentrations
of BSA or 99.95% delipidated fraction V BSA (dBSA).
Primary culture of mouse proximal tubular cells. Mouse proximal tubular (MPT) cells were cultured from collagenase-digested fragments of proximal tubules isolated from the cortices of kidneys of C57Bl/6 mice by a modification of previously described methods (30). Cortical tubules were plated in FCS-free, defined culture medium (1:1 mixture of DMEM and Ham's F-12, with 2 mM L-glutamine, 1 mM HEPES, 5 µg/ml transferrin, 5 µg/ml insulin, 50 nM hydrocortisone, 50 U/ml penicillin, and 50 µg/ml streptomycin), denoted as "full medium". Growth factor-free medium is defined as full medium minus insulin and hydrocortisone. MPT cells grew to confluence from tubules over 5-7 days and were studied within 2 wk of achieving confluence. Cell monolayers were previously shown to be of proximal tubular origin by a combination of morphological, biochemical, and transport characteristics (30). After confluence was achieved, cells were washed twice in Ham's F-12 and subsequently grown in full medium, growth factor-free medium, or growth factor-free medium plus various concentrations of BSA or dBSA.
Phase contrast and immunofluorescent microscopy. Nuclear morphology was assessed by staining with H33342 (Calbiochem, San Diego, CA), a supravital DNA dye with excitation and emission wavelengths of 348 and 479 nm, respectively. H33342 enters live cells and so stains the nuclei of viable as well as apoptotic and necrotic cells. The nuclei of apoptotic cells are readily distinguishable from those of viable and necrotic cells on the basis of chromatin condensation, nuclear fragmentation, and increased brightness of H33342 fluorescence (25, 27, 30, 31). Adherent cells and cells that had detached spontaneously from culture wells were washed separately in PBS before being stained with H33342 (1.0 µg/ml) for 10 min at 37°C. Wet preparations were made on glass slides and examined under phase contrast and epifluorescence microscopy for visualizing cell morphology and H33342 nuclear staining in the same cells.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide assay.
M and MPT cells were cultured for 72 h and 7-10 days,
respectively, in R.0 and growth factor-free medium alone or with FCS, high-dose insulin, and various concentrations of BSA or dBSA. The
number of viable cells remaining was determined with the use of a
modification of the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (37). This assay is based on the ability of
mitochondria from viable cells to cleave the tetrazolium rings of the
pale yellow MTT and form a dark blue formazan product. The number of
surviving cells is directly proportional to the level of the formazan
product created. After removal of the growth medium, 165 µl of MTT
dissolved in R.0 (m
) or growth factor-free medium (MPT cells) at 1 mg/ml were added to each well. After incubation at 37°C for 4 h,
the MTT formazan was dissolved by addition of 165 µl of 10% SDS in
0.01 N HCl. Aliquots from each well were read with the use of a
Dynatech microELISA plate reader with a test wavelength of 570 nm and a
reference wavelength of 650 nm.
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M survival assay for
N-acetylcysteine.
N-acetylcysteine alone, in the absence
of cells, promotes the conversion of MTT to its blue formazan product,
so that an MTT assay could not be used to assess the effect of
N-acetylcysteine on m
survival.
Instead, viability was determined by counting the number of cells that
remained adherent to the culture well and excluded trypan blue. Percent
increased viability above R.0 was then calculated identically as for
data obtained by MTT assay.
Thymidine incorporation.
M and MPT cells were cultured for 24 h in survival factor-free
medium alone or with FCS, high-dose insulin, and various concentrations of BSA or dBSA. A quantity of 2 µCi of
[3H]thymidine (2 Ci/mmol; NEN, Boston, MA) was added for the final 12 h. Cells were
washed three times with RPMI 1640, then incubated with 2.0 ml of
ice-cold 5% TCA for 1 h at 4°C. The TCA was removed, and cells
were washed once with fresh TCA. A quantity of 2.0 ml of ice-cold
ethanol containing 200 µM potassium acetate was added to each well
for 5 min, after which cells were incubated twice in 2.0 ml of a 3:1
mixture of ethanol and ether for 15 min per incubation. After air
drying, cells were solubilized in 1.0 ml of 0.1 N NaOH. We measured
[3H]thymidine counts
per minute by adding samples to scintillation fluid and counting with
the use of a model no. 1600TR Tri-Carb liquid scintillation analyzer
-counter (Packard Instrument, Meriden, CT).
Oncotic pressure. Oncotic pressure (in mmHg) was measured with the use of a Wescor oncometer (model no. 4400; Logan, UT).
ROS accumulation.
M were cultured in R.10, R.0, or R.0 plus dBSA (10 mg/ml). MPT cells
were cultured in full medium (high-dose insulin), growth factor-free
medium, or growth factor-free medium plus dBSA (5 mg/ml). Assays were
conducted at the onset of visible apoptosis: after 72 h for m
and
after 7-10 days for MPT cells. Cells were loaded with
dichlorodihydrofluorescein diacetate (DCF) (3, 12, 38) for 45 min,
after which point the dye was removed and the cells were washed and
incubated in PBS for 60 min at 37°C. After collection of
supernatants, cells were lysed in 1% Triton X-100 in PBS. DCF
fluorescence of supernatants and lysates was measured with a
Perkin-Elmer 650-105 fluorescence spectrophotometer (Norwalk, CT) with
the use of excitation and emission wavelengths of 488 and 530 nm,
respectively. Values were normalized for differences in cell number by
quantitation of cell protein in parallel identically treated wells with
the use of a microtiter bicinchoninic acid (BCA) protein assay (Pierce,
Rockford, IL), according to manufacturer's instructions.
Phosphatidylinositol 3-kinase assay.
Cell lysates were normalized for protein content and then incubated for
24 h with polyclonal anti-phosphatidylinositol 3-kinase (PI3K)
antibodies directed against the 85-kDa regulatory subunit of the
p85/p110 isoform (Upstate Biotechnology, Lake Placid, NY). Immune complexes were adsorbed onto protein A (p85/p110)-Sepharose; washed twice with PBS containing 1% Nonidet P-40 and 1 mM
Na3VO4; washed three times with 100 mM Tris, pH 7.4, containing 5 mM LiCl and 1 mM
Na3VO4;
and finally washed twice with 10 mM Tris, pH 7.4, containing 160 mM
NaCl, 5 mM EDTA, and 1 mM
Na3VO4.
PI3K assays were carried out for 10 min in a buffer containing 10 mM HEPES, pH 7.2, 1 mM EGTA, 20 mM
MgCl2, 100 µM ATP, 10 µg
phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL), and 20 µCi
[-32P]ATP (20,000 Ci/mmol; NEN). Reactions were run for 10 min and then stopped by
addition of 2 N HCl. Lipids were extracted with a 1:1 mixture of
CHCl3 and
CH3OH and then analyzed by TLC.
Preparation of BSA and dBSA.
BSA and dBSA were dissolved at a concentration of 20 mg/ml in growth
factor-free medium or R.0 for experiments involving MPT cells or m,
respectively. Before use, solutions were filtered with a 0.2-µm
filter. Protein content was determined with the use of a BCA protein
assay (Pierce).
Modifications of dBSA. Carboxyamidation of the free sulfhydryl groups of dBSA was performed by treating dBSA with equimolar iodoacetamide at pH 8.0 for 10 h in the dark at 37°C (22). Peroxidation of dBSA was performed by treating dBSA with a 700 µM solution of H2O2 for 2 h at room temperature (44). Modified dBSA was then dialyzed extensively against growth factor-free medium or R.0 to remove excess iodoacetamide or H2O2.
Determination of protein free sulfhydryl
content.
The reaction of 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with
free sulfhydryl groups was used to determine the free sulfhydryl content of dBSA, carboxyamidated dBSA, peroxidized dBSA, or dBSA that
had been exposed to cells in culture for 72 h (6). A quantity of 3 ml
of protein solution was mixed with 2 ml of PBS, pH 8.0, and 5 ml of
H2O. Then 20 µl of DTNB was
added to 3 ml of this mixture, and absorbance was measured at 412 nm
with the use of a Spectronic 1201 spectrophotometer (Milton Roy,
Rochester, NY). All values were zeroed by subtracting the absorbance at
412 nm for 20 µl of DTNB in 3 ml of either RPMI 1640 or growth
factor-free medium. Free sulfhydryl concentration (M) was then
calculated with the use of the following formula
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Determination of protein carbonyl
content.
The reaction of 2,4-dinitrophenylhydrazine (DNPH) with protein carbonyl
groups was used to determine the carbonyl content of freshly prepared
dBSA or dBSA that been exposed to cells in culture for 72 h (29). A
quantity of 500 µl of protein solution was precipitated in 10% TCA
at 4°C. After microcentrifugation at 11,000 g for 15 min, supernatants were
discarded and precipitates were resuspended in 500 µl of 10 mM DNPH
in 2 N HCl. Samples were incubated at room temperature for 60 min, with
vortexing every 15 min. After addition of 500 µl of 20% TCA, samples
were microcentrifuged at 11,000 g for
3 min and the supernatants were discarded. Precipitates were washed
three times in ethanol-ethyl acetate (1:1, vol/vol), then redissolved
at 37°C in 6 M guanidine in 20 mM potassium phosphate adjusted to
pH 2.3 with 0.05% trifluoracetic acid. After removal of any remaining
insoluble material by microcentrifugation, the absorbance at 374 nm was
measured with the use of a Spectronic 1201 spectrophotometer (Milton
Roy). All values were zeroed by subtracting the absorbance at 374 nm
for a control dBSA sample treated identically except that no DNPH was
added. Protein carbonyl concentration (M) was calculated by using the
same formula as for sulfhydryl content except that A is the absorbance
at 374 nm, the dilution factor D is 1, and the molar extinction
coefficient is 22,000 M
1 · cm
1.
Protein content was determined with the use of a BCA protein assay (Pierce).
Xanthine/xanthine oxidase model. Xanthine was prepared as a 287 mM stock solution in 1 N NaOH. This stock solution was further diluted to 0.8 mM in R.0 or growth factor-free medium, and the pH was adjusted to 7.4. Xanthine oxidase was reconstituted in H2O at 5 U/ml. Before use, all reagents were filtered through a 0.2-µm filter.
Measurement of H2O2. Concentrations of H2O2 were determined with the use of the PeroXOQuant quantitative peroxide assay (Pierce), according to manufacturer's instructions.
Statistics.
Quadruplicate wells for m and duplicate wells for MPT cells were
examined in each experiment, and the results were averaged. A minimum
of three experiments was performed for all data points. Data are
expressed as means ± SE of the averaged values obtained from each
experiment. Statistical significance was determined by a two-tailed
Student's t-test.
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RESULTS |
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dBSA acts as a survival factor for primary cultures
of murine peritoneal m and MPT cells.
We have previously shown that primary cultures of murine peritoneal
m
and MPT cells undergo apoptosis on withdrawal of serum or growth
factors (25, 27, 30). Loss of viability occurs asynchronously over
several days, leading to gradual loss of the monolayer. On the basis of
previous observations suggesting that BSA has survival activity for
m
independent of bound lipids (25), we assessed the ability of dBSA
(99.95% delipidated) to inhibit apoptosis of m
and MPT cells.
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dBSA is not a mitogen for m and MPT
cells.
The increased numbers of viable cells seen with addition of dBSA could
potentially be the result of two processes: inhibition of apoptosis
and/or stimulation of proliferation. We assessed the contribution of
dBSA-induced proliferation to increased m
and MPT cell viability by
measuring
[3H]thymidine
incorporation as an index of DNA synthesis. Cells were cultured for 24 h in growth factor-free medium alone or growth factor-free medium
supplemented with FCS, high-dose insulin, BSA, or dBSA.
[3H]thymidine was
added for the final 12 h.
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Survival activity of dBSA is not due to trace
contamination with lipopolysaccharide.
The remainder of our studies were directed toward elucidating the
lipid-independent mechanism by which BSA inhibits apoptosis. We first
sought to exclude the possibility that contamination with
lipopolysaccharide (LPS) might account for the survival activity of
BSA. Although delipidation of BSA should remove most contaminating LPS,
LPS is an extremely potent m survival factor, acting at concentrations as low as 100 pg/ml (34), so that even trace contamination with LPS could profoundly affect cell survival. We
therefore determined the effect of BSA on survival in the presence and
absence of polymyxin B, a cationic polypeptide antibiotic that
neutralizes the effects of LPS by binding avidly to its lipid A core
moiety (36). As shown in Fig. 4, the
addition of polymyxin B (10 µg/ml) had no effect on the survival of
m
and MPT cells cultured in the presence of BSA, dBSA, FCS, or
high-dose insulin. As a positive control, polymyxin inhibited
LPS-induced release of interleukin-1 by m
(not shown). These studies
rule out any contribution from LPS contamination.
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Survival activity of dBSA is independent of its
oncotic properties.
Recent data have established the importance of adhesion-mediated
changes in cell shape for the survival of endothelial cells (5).
Because albumin is the major protein responsible for maintaining the
colloid oncotic pressure of blood, we examined the possibility that the
survival activity of BSA may be due to its oncotic properties and
consequent changes in cell volume or shape. We compared the survival
activity of dBSA (5 mg/ml or 725 µM) to that of an equimolar concentration of neutral dextran-70. The colloid oncotic pressures of
these two solutions were measured independently and found to be 1.99 and 2.30 mmHg, respectively. Despite the slightly higher oncotic
pressure for neutral dextran-70, the viability of both m and MPT
cells in the presence of dBSA was significantly greater than that in
the presence of dextran-70 (Fig. 5). We
conclude that the survival activity of dBSA cannot be attributed to its oncotic properties.
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Inhibition of apoptosis by dBSA is independent of
PI3K activation.
Activation of PI3K plays a critical role in survival factor signaling
by a variety of cytokines. We have previously shown that LPA, a major
serum survival factor tightly bound to albumin, prevents apoptosis of
m and MPT cells subjected to survival factor withdrawal via
activation of PI3K (25, 27). We therefore determined whether dBSA might
also be activating PI3K. M
were FCS-starved for 6 h, then stimulated
with dBSA or BSA. Consistent with its known carriage of LPA,
nondelipidated BSA activated the p85/p110 isoform of PI3K within 2 min
of stimulation, with peak activity occurring at 5 min (Fig.
6). These kinetics are consistent with our
previously published data for LPA (25). In contrast, dBSA did not
activate PI3K above baseline levels. We have previously shown that
baseline PI3K activity is dependent on m
adhesion and likely
accounts for the ~25% survival seen after 72 h of FCS-free culture
(25). We conclude that dBSA does not activate PI3K and that inhibition
of apoptosis by dBSA occurs through a mechanism distinct from that for
most cytokines.
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dBSA inhibits the accumulation of ROS after
withdrawal of survival factors.
Unlike the majority of circulating plasma proteins, in which cysteine
residues form intermolecular or intramolecular disulfide bonds, albumin
possesses a free sulfhydryl group at
Cys34 (22, 41). By virtue of its
reduced state, the sulfhydryl group at
Cys34 may enable albumin to
function as a circulating antioxidant. This property of albumin may be
important to the regulation of apoptosis, since we have previously
shown that apoptosis of MPT cells induced by growth factor withdrawal
can be inhibited by a number of antioxidants and scavengers of ROS
(30). Similarly, N-acetylcysteine (10 mM), which uses a free sulfhydryl group to scavenge ROS, promoted the
survival of m cultured in R.0 (44 ± 17% survival,
P < 0.05).
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BSA is oxidized after withdrawal of survival
factors.
If dBSA inhibits apoptosis by scavenging of ROS, then dBSA should
itself undergo progressive oxidation during coculture with m and MPT
cells. Such oxidation would manifest as a decrease in free sulfhydryl
content via oxidation of Cys34 and
as an increase in carbonyl content via oxidation of free hydroxyl groups.
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Oxidation and/or chemical blockade of the free
sulfhydryl group in dBSA inhibits survival activity.
Because coincubation of dBSA with MPT cells in growth factor-free
medium led to a decrease in the free sulfhydryl content and an increase
in the carbonyl content of dBSA, we next determined whether chemical
blockade of the free sulfhydryl group and/or oxidation of dBSA would
attenuate its survival activity during survival factor withdrawal.
Modification of the free sulfhydryl group at
Cys34 was accomplished in one of
two ways. Oxidation by
H2O2
reduced the free sulfhydryl content of dBSA (72.5 µM) from 41 ± 1 to 28 ± 3 µM, whereas irreversible carboxyamidation with
iodoacetamide reduced the free sulfhydryl content to 17 ± 2 µM.
As shown in Fig. 9, modification of dBSA by
either or both of these two methods abolished most of the survival
activity of dBSA for m and MPT cells undergoing survival factor
withdrawal. We cannot be sure in these studies whether modification of
the free sulfhydryl group at Cys34
and/or oxidation of hydroxyl groups are the only chemical changes induced in dBSA by
H2O2
or iodoacetamide. Nonetheless, by showing that prior oxidation of dBSA
and/or blockade of free sulfhydryl and hydroxyl groups profoundly
diminish the survival activity of dBSA, these data further suggest that
dBSA functions as a scavenger of ROS.
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BSA is a potent antioxidant, protecting in a
well-established model of oxidant injury.
As a final test of our hypothesis, we determined whether BSA would
protect cells from ROS generated by the reaction between xanthine and
xanthine oxidase, a well-established model of oxidant injury (39, 44).
Xanthine oxidase catalyzes the oxidation of both xanthine and
hypoxanthine to yield a number of ROS, including O2,
H2O2,
and · OH (22). M
and MPT cells were incubated for 24 h in
the presence of a nonlimiting concentration of xanthine. As shown in
Fig. 10,
A and
B, the addition of xanthine oxidase
led to the dose-dependent death of both cell types. At the highest
concentration of xanthine oxidase (12.5 mU/ml), cell death occurred
predominantly by necrosis (as assessed by increased cell size, loss of
membrane integrity, and normal Hoechst nuclear staining), with only
~40% of cells still alive at 24 h. The addition of dBSA or BSA at 5 mg/ml led to nearly 100% protection from cell death at all
concentrations of xanthine oxidase (Fig. 10,
A and B). The limited cell death in the
presence of dBSA or BSA was predominantly apoptotic (as assessed by
decreased cell size, maintenance of membrane integrity, and nuclear
condensation). Conversion from necrosis to apoptosis further implies a
protective role for dBSA and is consistent with previous work showing
that the severity of cellular injury determines the mechanism of cell
death (31). Preoxidation of dBSA with
H2O2
abolished most of the protective effect of dBSA for MPT cells (Fig.
10C).
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Scavenging of ROS may be a general property of
proteins.
Proteins and amino acid acids vary in their susceptibility to oxidative
attack (see DISCUSSION). Although
albumin is unique among plasma proteins in having a free sulfhydryl
group, we hypothesized that the presence of free hydroxyl groups and
other sites capable of scavenging ROS should confer survival activity
on a variety of proteins. We therefore determined the survival activity
for m and MPT cells of three unrelated proteins, each having minimal free sulfhydryl content. These were ovalbumin,
1GP, and
2MG. The
free sulfhydryl content of 72.5 µM solutions of these proteins was
7.1, 5.4, and 6.7 µM, respectively, compared with a free sulfhydryl content of 40 µM for 72.5 µM dBSA. For m
, only
2MG showed
significant survival activity. In contrast, for MPT cells, all three
proteins showed significant survival activity, with
1GP and
2MG
actually possessing survival activity equal to that of dBSA. Given
probable differences in the ROS scavenged by these three proteins, our findings are consistent with at least two interpretations. MPT cells
and m
might differ in the type and quantity of ROS generated as a
result of survival factor withdrawal, or, alternatively, the signaling
pathways leading to induction of apoptosis in response to ROS might
differ between these two cells.
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DISCUSSION |
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Albumin is a major circulating plasma protein with a variety of
functions, including maintenance of oncotic pressure and transport of
multiple bioactive substances (7, 41, 42). Recently, we have shown that
albumin-bound LPA is a major serum noncytokine survival factor for
primary cultures of m and MPT cells (25, 27). In the course of these
studies, we found that dBSA (99.95% delipidated) alone, in the absence
of any other survival factors, was able to inhibit apoptosis of m
subjected to FCS withdrawal (25).
Our objective here was to examine the lipid-independent role of BSA as
a survival factor for m and MPT cells. We report several novel
findings. First, dBSA alone is a highly potent survival factor. A
significant effect on viability was seen at concentrations of dBSA as
low as 1% of those normally found in plasma. At concentrations equivalent to those of plasma, the survival activity of dBSA was
50%
of that achieved with FCS. Second, dBSA does not activate PI3K, which
implies that its survival activity is attributable to a mechanism
different from that for most cytokines. Third, the survival activity of
dBSA seems to be attributable, at least in part, to its antioxidant
properties through scavenging of ROS. Biochemical features of dBSA that
are important in this regard include free hydroxyl groups and a free
sulfhydryl group at Cys34, a
unique feature of albumin in comparison with other plasma proteins (22,
41). Fourth, the antioxidant effect of dBSA is not limited to apoptosis
induced by survival factor withdrawal. dBSA alone conferred almost
complete protection from cell death in a well-established model of
oxidative injury (xanthine/xanthine oxidase). Finally, protection
against apoptosis induced by survival factor withdrawal may be a
general feature of proteins, as
1GP and
2MG inhibited apoptosis
of MPT cells to a degree similar to dBSA. Protection, however, did not
extend to all proteins (see ovalbumin in Fig.
11), nor did protection in one cell type
generalize to protection in another cell type (compare data for m
and MPT cells).
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In establishing the role of dBSA as an antioxidant, we ruled out
several other mechanisms by which dBSA might promote cell survival.
First, dBSA was not a mitogen for m or MPT cells, indicating that
increased viability was the result of inhibition of apoptosis rather
than stimulation of proliferation. Second, survival was not the result
of trace contamination with LPS, as the addition of polymyxin B had no
effect on dBSA-mediated survival. Finally, in light of a recent report
indicating the importance of cell shape in promoting survival (5), we
showed that oncotic effects did not play a major role, as the survival
activity of neutral dextran-70 was significantly less than that of
dBSA, despite equal oncotic pressures.
Although the precise role of ROS in apoptosis remains undefined (2,
21), antioxidants have been shown to be protective in several models of
apoptosis induced by survival factor withdrawal, for example,
sympathetic neurons deprived of nerve growth factor (13) or FL5.12
cells (a murine pro-B lymphocyte cell line) deprived of interleukin-3
(15). ROS most likely promote apoptosis by signaling its initiation,
either by mediating cell damage or by acting as second messengers in
the complex signal-transduction pathways involved in switching on the
death program (2, 21). The role of ROS in the actual execution of
apoptosis is far more uncertain. In accordance with these ideas, we
have shown that apoptosis of MPT cells induced by survival factor
withdrawal can be inhibited by a number of antioxidants and ROS
scavengers (30). In this model, ROS appeared to act upstream of
caspases in the apoptotic pathway (30). Here, we show that antioxidants
also protect m, as
N-acetylcysteine, which like albumin
possesses a free sulfhydryl group, inhibited apoptosis of m
subjected to FCS withdrawal.
The mechanism by which albumin and other plasma proteins exert their
antioxidant effects is most likely multifactorial. Proteins such as
transferrin, ceruloplasm, and albumin bind the transition metals
Fe2+ and
Cu2+, thereby preventing the
generation of · OH via the Fenton reaction (15, 18, 32, 45).
In addition, albumin and other proteins can act as sacrificial sinks
for attack by ROS, either directly through oxidation of amino acid side
chains or indirectly through reaction with lipid species and radicals
arising from the peroxidation of cell membrane lipids (8, 9, 16). The
amino acids cysteine, histidine, methionine, tyrosine, and tryptophan
are particularly susceptible to direct oxidative attack, whereas lysine
is most susceptible to attack by malondialdehyde, one of the principal products of lipid peroxidation (8, 9, 16). Albumin is unique among
plasma proteins in possessing a free sulfhydryl group, making it a
particularly effective scavenger of ROS (22, 41). In contrast, most
sulfhydryl residues in other proteins form either inter- or
intramolecular disulfide bonds (22). The role of albumin as an ROS
scavenger has been confirmed in cell-free systems with the use of a
wide variety of oxidative species, including HOCl, H2O2,
· OH, carbon radicals, and peroxynitrite (8-11, 16, 17, 42-44, 46, 49-51). Finally, it is possible that the free
sulfhydryl group of albumin enables it to act not only as an
antioxidant but also as a reducing agent, affecting a wide variety of
cell proteins, the function of which is dependent on redox state,
including the
N-methyl-D-aspartic
acid receptor and the DNA binding proteins activator
protein-1 and nuclear factor-B (2, 21, 26, 35).
We explored the antioxidant role of dBSA by focusing on two potential
oxidative reactions, namely, loss of the free sulfhydryl group at
Cys34 and oxidation of hydroxyl
groups to carbonyls. As assessed by oxidation of the fluorogenic dye
DCF, dBSA inhibited the accumulation of ROS after withdrawal of
survival factors by >10× in MPT cells and >100× in
m. During protection of MPT cells and m
from apoptosis, dBSA
underwent progressive oxidation. Coincubation of dBSA with MPT cells
and m
subjected to 72 h of survival factor withdrawal led to an
~50% decrease in the sulfhydryl content of dBSA and an ~5×
increase in carbonyl content. Importantly, peroxidation of dBSA by
treatment with
H2O2
or chemical blockade of the free sulfhydryl group at
Cys34 by carboxyamidation almost
completely eliminated the survival activity of dBSA for both MPT cells
and m
. In addition, peroxidation of dBSA also inhibited its ability
to prevent the oxidative death of MPT cells and m
exposed to
xanthine and xanthine oxidase. These results strongly implicate free
sulfhydryl and hydroxyl groups in the survival activity of dBSA.
Nevertheless, it should be noted that our data do not rule out
protective effects from these groups independent of oxidative changes,
nor do they rule out the contribution of other potentially reactive
sites in dBSA.
It is interesting to note that albumin has been previously reported to have survival activity in a number of systems. Lornage et al. (33) found that BSA was a survival factor for spermatozoa, and Twigg et al. (48) extended these findings by showing that BSA protected spermatozoa from oxidant injury. Burleson et al. (1) found that perfusion of transplantable kidneys with albumin produced viability of the organ for up to 50 h without structural damage. Guilbert and Iscove (14) reported that BSA was necessary for the survival of bone marrow-derived hematopoietic cell colonies. In two studies, dBSA also prevented injury to hepatocytes subjected to ischemia-reperfusion damage (4, 47). Finally, Zoellner et al. (52) showed that BSA, human serum albumin, and recombinant human albumin all inhibited apoptosis of adherent, but not nonadherent, endothelial cells.
Our studies provide a framework for interpreting these protective
effects of albumin. Thus BSA may protect cells from apoptosis and/or
oxidant injury by several distinct mechanisms. First, as shown here,
dBSA is a potent antioxidant through scavenging of ROS. Second,
nondelipidated BSA contains high concentrations of bioactive
phospholipids. We have recently shown that LPA is a highly potent
survival factor for m and MPT cells, acting at concentrations as low
as 50 nM (25, 27). Finally, free unsaturated fatty acids, which also
bind with high affinity to albumin, are potent survival factors for
m
(but not MPT cells) via activation of PI3K and nuclear factor-
B
(24).
In conclusion, we have shown that BSA is a major survival factor in
FCS. BSA inhibits apoptosis of primary cultures of m and MPT cells
subjected to survival factor withdrawal by both lipid-dependent and
-independent mechanisms. The lipid-dependent mechanism depends on
activation of PI3K and relates to carriage of LPA and other bioactive
lipids. The lipid-independent mechanism is independent of PI3K and is
attributable, at least in part, to the ability of dBSA to act as a
potent antioxidant through scavenging of ROS.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Ronald McCaffrey for use of the microELISA plate reader.
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FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants AR/AI-42732 (to J. S. Levine), DK-375105, and HL-53031 (both to W. Lieberthal) and a Clinical Scientist Award from the National Kidney Foundation (to J. S. Levine).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. S. Levine, Renal Section, E428, Boston Medical Center, One Boston Medical Center Place, Boston, MA 02118 (E-mail: jlevine{at}bu.edu).
Received 4 March 1999; accepted in final form 15 June 1999.
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