From the Department of Cell Biology, Lerner Research
Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the
§ Sealy Center for Oncology and Hematology, University of
Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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Our previous studies have shown
that human native low density lipoprotein (LDL) can be oxidized by
activated human monocytes. In this process, both activation of protein
kinase C (PKC) and induction of superoxide anion (O It has been suggested that monocyte-mediated low density
lipoprotein (LDL)1 oxidation
may play an important role in atherogenesis and inflammatory tissue
injury (reviewed in Ref. 1). Previous studies in our laboratory have
shown that human monocytes develop the ability to oxidize native LDL
lipids only upon activation, thereby transforming LDL into an in
vitro cytotoxin (2, 3). The mechanisms involved in
monocyte-mediated LDL lipid oxidation are not fully understood. We
previously reported that superoxide anion (O To date at least 12 isoenzymes of PKC having differential activator
responsiveness, cellular distribution, and substrate specificity have
been reported in mammalian tissue. They are divided into three major
groups: conventional PKCs ( In previous studies, we found that a
Ca2+-dependent cPKC was involved in the process
of O Materials--
Cytochrome c and superoxide dismutase
(SOD) from bovine erythrocytes were purchased from Sigma. SOD was
dissolved in phosphate-buffered saline as a 100-fold stock solution and
stored at Isolation of Human Monocytes--
Human monocytes were isolated
from heparinized whole blood by sequential centrifugation over a
Ficoll-Paque density solution and adherence to serum-coated cell
culture flasks as described previously (2, 3). After washing away
nonadherent cells, the adherent cells were released with 5 mM EDTA and plated into multiwell dishes at 1.0-2.5 × 106 cells/ml. This cell population consisted of greater
than 95% monocytes (3). The isolated human monocytes were then
cultured overnight in Dulbecco's modified Eagle's medium with 10%
bovine serum before use in experiments. The monocytes were then washed twice with RPMI 1640 (Whittaker, Walkersville, MD) and incubated in
RPMI 1640 with or without LDL (0.5 mg cholesterol/ml) in the presence
or absence of ZOP (2 mg/ml).
Isolation of Human LDL--
LDL was prepared from human plasma
by sequential density ultracentrifugation as described previously (12).
During the preparation procedure, LDL was protected from exposure to
light and oxidation (6, 7). LDL was filter sterilized and stored in 0.5 mM EDTA. The concentration of LDL was adjusted to 10 mg
cholesterol/ml. Each batch of LDL was also assayed for endotoxin
contamination by the limulus amebocyte lysate assay (kit QCL-1000,
Whittaker Bioproducts Inc., Walkersville, MD). Final endotoxin
contamination was always <0.03 unit/mg LDL cholesterol. Immediately
before use, LDL was dialyzed at 4 °C against phosphate-buffered
saline without calcium or magnesium (Life Technologies, Inc.) in the
dark. 1 g/liter of Chelex was added to the dialysis buffer. In all
experiments, LDL was used at a final concentration of 0.5 mg
cholesterol/ml.
Measurement of LDL Lipid Oxidation--
Human monocytes (1 × 106 cells/ml) pretreated with ODN diluent or with ODN
(see "Treatment of cells with ODN") were incubated with LDL (0.5 mg
cholesterol/ml) in the presence or absence of activator (ZOP, 2 mg/ml)
in RPMI 1640 in 96-well flat bottomed culture plates (Costar,
Cambridge, MA) for 24 h. After incubation, cell-mediated LDL lipid
oxidation was determined by a modified thiobarbituric acid (TBA) assay
described by Schuh et al. (13). This assay detects
malondialdehyde (MDA) and MDA-like compounds reacting with TBA in
oxidized LDL. Briefly, 5 µl of 1 mM butylated hydroxytoluene, 5 µl of 10 mg/ml EDTA, and 50 µl of 25%
trichloroacetic acid were added to 100-µl samples, followed by 75 µl of 1% TBA. The samples were then incubated at 60 °C for 40 min. After incubation fluorescence at 515 nm excitation and 553 nm
emission was determined. Malondialdehyde bis (dimethyl acetal),
i.e. 1,1,3,3-tetramethoxypropane (Aldrich), was used as
the standard at concentrations of 0-10 nmol MDA/ml. Samples were
assayed in triplicate and expressed in terms of MDA equivalents (nmol
of MDA/ml).
PKC Activity Assay--
Human monocytes (2.5 × 106 cells/ml, a total of 10 × 106
cells/group) and ZOP (2 mg/ml) were incubated for 24 h. After
incubation, cell lysates were prepared using a buffer containing 20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 µg/ml leupeptin, and 25 µg/ml aprotinin. Following centrifugation at 1000 × g for 15 min, postnuclear supernatants were collected, and
protein concentrations of supernatants were determined by the Lowry
assay (7). PKC activity was determined in 20 µg of cell lysate
protein using a PKC assay kit (Life Technologies, Inc.). This assay is
based on measurement of the phosphorylation of acetylated myelin basic
peptide (amino acids 4-14) in the presence and absence of the PKC
pseudosubstrate inhibitor peptide, PKC (amino acids 19-36). The PKC
activity measurements were predetermined to be in the linear range of
the assay.
In studies of intracellular location of PKC activity, human monocytes
(2.5 × 106 cells/ml, a total of 10 × 106 cells/group) and ZOP (2 mg/ml) were incubated for
24 h. After incubation, cell lysates were prepared by sonication
in hypotonic buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgSO4, 0.5 mM EGTA, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and
20 µg/ml leupeptin) as described previously (14). Postnuclear lysates were centrifuged at 100,000 × g for 1 h at
4 °C, and supernatants were collected and are referred to as the
cytosol fractions. The pellets were resuspended in the same buffer and
incubated at 4 °C for 1 h. After centrifugation at 1000 × g for 10 min, supernatants were collected and are referred
to as the particulate/membrane fractions. The protein concentrations in
both fractions were determined by the Lowry assay using delipidated
albumin as the standard (15). PKC activity in both cytosol and
particulate/membrane fractions was assessed as described above, and
data are expressed as pmol phosphate incorporated per 106 monocytes.
Western Blotting Analysis--
Human monocytes (2.5 × 106 cells/ml, a total of 10 × 106
cells/group) were incubated in the presence or absence of ZOP and test reagents as indicated in figure legends. After incubation, cells were
harvested, and for total cell analysis the cells were resuspended in
100 µl of hypotonic lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgSO4, 0.5 mM EGTA, 0.1%
2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 100 µg/ml DNase, 100 µg/ml RNase, and 0.5%
Nonidet P-40), and the cells were vortexed for 15 s and then
subjected to centrifugation at 1000 × g for 10 min.
For analysis of proteins in the cytosolic and membrane/particulate
fractions, cell lysates were prepared by sonication in the above buffer
but in the absence of detergent. Fractions were obtained as described under "PKC Activity Assay." The samples were then prepared for 8%
SDS-PAGE (16). The proteins from the SDS-PAGE gels were transferred to
PVDF membranes (17). After blocking the nonspecific binding sites with
5% milk in Tris buffer (20 mM Tris-base, 1.5 M
NaCl, and 0.1% Nonidet P-40, pH 7.4) for 1 h at room temperature,
PKC isoenzymes were detected using rabbit anti-human PKC
isoenzyme-specific polyclonal antibodies (1:1000 dilution), followed by
incubation with goat anti-rabbit IgG (1:1000 dilution) conjugated with
horseradish peroxidase. The PVDF membrane was then developed by
enhanced chemiluminescence (Amersham Pharmacia Biotech).
The identity of the different PKC isoforms was confirmed by testing the
isoenzyme specificity of the antibodies using recombinant isoforms of
PKC and by confirming appropriate migration of cell-derived, immunoreactive bands on SDS-PAGE. Appropriate migration was determined by running molecular weight markers in adjacent lanes of the gel and
calculating the predicted molecular weight of immunoreactive bands.
Treatment of Cells with ODN--
The sequence for PKC Measurement of Superoxide Anion Production--
Superoxide
anion (O Statistical Analysis--
Data were collected and analyzed based
on triplicate or quadruplicate samples of each determination.
Experimental results are presented as the means ± S.E. of data
obtained from three experiments or the means ± S.D. of data
obtained from one of three experiments yielding similar results. The
significant difference between experimental groups and control groups
was determined by the unpaired, two-tailed Student's t
test. Statistical tests were performed with GraphPAD InStat software
(GraphPAD Software Inc., San Diego, CA). Data points were considered
significantly different if the p value was <0.05.
PKC activity in cytosol and particulate/membrane fractions was
investigated at various times after human monocyte activation. ZOP was
used as the monocyte activator. The data from a representative experiment are summarized in Fig. 1. PKC
activity was induced very quickly in ZOP-activated human monocytes.
Activity was first detected in the cytosolic fraction and then in the
particulate/membrane fraction. At 1 h post activation, the
predominant activity was found in the membrane-containing particulate
fraction. Although cytosolic PKC activity reached a maximum within
1 h of activation, activity dropped somewhat to a plateau level
that remained stable and substantially higher than basal levels
throughout the 24 h incubation. On the other hand, PKC activity in
the particulate/membrane fraction accounted for the majority of the
activity by 1 h after activation and maintained this higher level
of activity throughout the 24-h incubation. These data indicate that
upon activation monocyte PKC activity is substantially induced in both
fractions but to a greater degree in the particulate fraction.
2)
production are required. PKC is a family of isoenzymes, and the
functional roles of individual PKC isoenzymes are believed to differ
based on subcellular location and distinct responses to regulatory
signals. We have shown that the PKC isoenzyme that is required for both
monocyte O
2 production and oxidation of LDL is a member of the
conventional PKC group of PKC isoenzymes (Li, Q., and Cathcart, M. K. (1994) J. Biol. Chem. 269, 17508-17515). The
conventional PKC group includes PKC
, PKC
I, PKC
II, and PKC
.
With the exception of PKC
, each of these isoenzymes was detected in
human monocytes. In these studies, we investigated the requirement for
select PKC isoenzymes in the process of monocyte-mediated LDL lipid
oxidation. Our data indicate that PKC activity was rapidly induced upon
monocyte activation with the majority of the activity residing in the
membrane/particulate fraction. This enhanced PKC activity was sustained
for up to 24 h after activation. PKC
, PKC
I, and PKC
II
protein levels were induced upon monocyte activation, and PKC
and
PKC
II substantially shifted their location from the cytosol to the
particulate/membrane fraction. To distinguish between these isoenzymes
for regulating monocyte O
2 production and LDL oxidation,
PKC
or PKC
isoenzyme-specific antisense oligonucleotides were
used to selectively suppress isoenzyme expression. We found that
suppression of PKC
expression inhibited both monocyte-mediated
O
2 production and LDL lipid oxidation by activated human
monocytes. In contrast, inhibition of PKC
expression (including both
PKC
I and PKC
II) did not affect O
2 production or LDL
lipid oxidation. Further studies demonstrated that the respiratory
burst oxidase responsible for O
2 production remained
functionally intact in monocytes with depressed levels of PKC
because O
2 production could be restored by treating the monocytes with arachidonic acid. Taken together, our data reveal that
PKC
, and not PKC
I or PKC
II, is the predominant isoenzyme required for O
2 production and maximal oxidation of LDL by
activated human monocytes.
INTRODUCTION
Top
Abstract
Introduction
References
2) released upon activation of human monocytes was required for monocyte-mediated LDL
oxidation, because removal of O
2 by superoxide dismutase prevented LDL oxidation (2, 3). General antioxidants such as butylated
hydroxytoluene, vitamin E, and ascorbate also inhibited monocyte-mediated oxidation of LDL (2, 4, 5). We also found that
increases in intracellular Ca2+ levels, cytosolic
phospholipase A2 activity, and protein kinase C (PKC)
activity were required for monocyte-mediated LDL lipid oxidation
(6-8). We routinely measure monocyte-mediated LDL oxidation at 24 h after monocyte activation, a time when considerable oxidation products have accumulated and the oxidation begins to plateau. In our
earlier studies, PKC activity was shown to contribute to LDL lipid
oxidation both early and late during the 24-h incubation period
(7).
,
I,
II, and
), novel PKCs (
,
,
, and
), and atypical PKCs (
,
, µ, and
)
(reviewed in Ref. 9). Although only cPKCs are
Ca2+-dependent, all three groups of PKCs are
believed to participate in signal transduction (9, 10), and
accumulating evidence suggests that PKC isoenzymes likely play unique
roles and induce different functional changes within cells.
2 production and required for LDL lipid oxidation by
activated monocytes (7), we therefore initiated studies to identify
which cPKC isoenzyme(s) was involved in these processes. In the present
studies, PKC isoenzyme-specific antisense oligonucleotides (ODN) were
carefully designed and then used to suppress the expression of
individual isoenzymes. After establishing efficacy and selectivity of
antisense inhibition by Western analysis, the ODN were then tested for
their effects on monocyte-mediated O
2 production and LDL lipid
oxidation. Our data demonstrate that PKC
, but not PKC
I or
PKC
II, regulates monocyte O
2 production as well as
monocyte-mediated LDL lipid oxidation.
EXPERIMENTAL PROCEDURES
20 °C prior to use. Anti-human PKC isoenzyme antibodies
directed to synthetic isoenzyme peptides and recombinant PKC isoenzymes
were purchased from Oxford Biomedical Research, Inc. (Oxford, MI) and
Biomol (Plymouth Meeting, PA). Secondary antibodies were purchased from Kirkegaard & Perry (Gaithersburg, MD). Zymosan obtained from ICN Biochemicals (Cleveland, OH) was opsonized (11) and used at a
concentration of 2 mg/ml to activate human monocytes. Opsonized zymosan
(ZOP) was suspended in phosphate-buffered saline as a 20-fold stock
solution and stored at
70 °C prior to use. PVDF membranes were
from Micron Separations, Inc. (Westborough, MA).
isoenzyme-specific antisense ODN was 5'-CGC CGT GGA GTC GTT GCC CG-3',
and its control sense ODN sequence was 5'-CGG GCA ACG ACT CCA CGG
CG-3'. The sequence of PKC
isoenzyme-specific antisense ODN was
5'-AGC GCA CGG TGC TCT CCT CG-3', and its control sense ODN sequence
was 5'-CGA GGA GAG CAC CGT GCG CT-3'. The PKC
sequence was selected
from an area of conserved sequence between PKC
I and PKC
II. All of
the oligomers were phosphorothioate modified and HPLC-purified. They
were selected by our previously described criteria (7, 18). Briefly,
the sequences were selected from areas of the mRNA that are
relatively free of secondary structure as predicted by Mulfold 211. In
each case the translation start site was avoided as a target for
antisense recognition to avoid the recognition of concensus sites that
often characterize this region of the mRNA. The selected sequences
were also tested for lack of internal secondary structure and oligo
pairing using Mulfold 211 (18). Sequences were then screened for
uniqueness using Blast 211. For experiments using ODN treatment, human
monocytes (1.0-2.5 × 106 cells/ml) were cultured in
Dulbecco's modified Eagle's medium with 10% bovine serum albumin in
the presence or absence of sense or antisense oligomers for 20 h.
Levels of PKC
or PKC
isoenzyme expression were determined at
either 1 h or 24 h post-activation by ZOP to match the times
of the O
2 and LDL oxidation assays, respectively. For
activation the medium was changed to serum-free RPMI 1640, and for the
24 h activation experiments fresh ODN were added along with ZOP.
Monocytes were then assessed for PKC
or PKC
isoenzyme
expression, cell-mediated LDL lipid oxidation, and O
2
production as described.
2) production was measured by the cytochrome
c reduction assay as described previously (19). Briefly, human monocytes (1 × 106 cells/ml) and cytochrome
c (320 µM; Sigma) were incubated in the
presence or absence of 150 unit/ml SOD (from bovine erythrocytes; Sigma) in 96-well cell culture plates in RPMI 1640 without phenol red
(Whittaker, Walkersville, MD). ZOP (2 mg/ml) was then added for 1 h at 37 °C in a humidified incubator with 10% CO2.
After incubation, the absorbance was measured with a Thermo Max
Microplate Reader at 550 nm using Softmax software (PerSeptive
Biosystems, Framingham, MA). The following equation was used to
determine the nmol of O
2 produced: O
2 nmol/ml = [A550 (in the absence of SOD) × 158.73]
[A550 (in the presence of SOD) × 158.73].
RESULTS
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Fig. 1.
PKC activity is induced upon human monocyte
activation. Human monocytes (2.5 × 106 cells/ml,
a total of 10 × 106 cells/group) and ZOP (2 mg/ml)
were incubated together for different periods of time as indicated.
After incubation, cell lysates were prepared and fractionated, and PKC
activity assays were performed as described under "Experimental
Procedures." Closed circles represent PKC activity in
cytosol fractions. Open circles represent PKC activity in
particulate/membrane fractions. Data represent the averages ± data ranges of duplicate samples obtained in one of three experiments
giving very similar results.
The presence of various PKC isoenzyme proteins was then evaluated in
unactivated and activated human monocytes by Western blotting using PKC
isoenzyme-specific antibodies. As summarized in Fig.
2, PKC and PKC
I were detected in
unactivated monocytes. Although not evident in this blot low levels of
PKC
were sometimes observed in immunoblots of unactivated monocytes.
PKC
II was barely detectable in unactivated monocytes as well.
Interestingly, each of the PKC isoenzymes were induced upon activation,
and as expected, PKC
was not detected in human monocytes regardless
of the activation state. Because our previous studies indicated that
cPKC activity was required for monocyte-mediated LDL lipid oxidation
(7), in further studies we focused on the members of the cPKC group of
isoenzymes that are detectable in these cells, namely PKC
,
I, and
II.
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The cellular partitioning of PKC and PKC
isoenzymes was then
examined in unactivated and activated (24 h) human monocytes. In Fig.
3, a Western blot is shown where equal
amounts of protein are loaded in each lane. To adequately assess the
induction and intracellular location of the isoenzymes, the relative
density of each band was corrected for the total protein content in the fraction, and these data are given in Table
I. Of the three isoforms of PKC that we
examined, only PKC
I was predominantly located in the
particulate/membrane fraction in unactivated monocytes. PKC
,
PKC
I, and PKC
II all increased in the particulate/membrane fraction upon monocyte activation. When corrected for the total protein
content of the fractions, it is evident that although PKC
was
induced to a lesser extent than PKC
I or PKC
II, it showed the most
dramatic relocation to the particulate/membrane fraction upon
activation. It should be noted that the apparent induction level of the
PKC
II isoform may be inflated due to the barely detectable basal
level and calculation as fold induction.
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To further discriminate between PKC and PKC
isoenzymes as
regulators of O
2 production and monocyte-mediated LDL
oxidation, PKC isoenzyme-specific antisense ODN treatment was used. The
antisense ODN sequences for PKC
(recognizing only this isoenzyme)
and PKC
(recognizing both PKC
I and PKC
II) were selected as
described under "Experimental Procedures." In these experiments,
human monocytes were pretreated in the presence or absence of PKC
isoenzyme-specific antisense or sense ODN for 20 h. Monocytes were
then exposed to fresh ODN and ZOP for 24 h in RPMI 1640. After
activation, cell lysates were then prepared, and the expression of
PKC
, PKC
I, and PKC
II isoenzymes was examined by Western
blotting (Fig. 4A). Treatment
of human monocytes with either PKC
or PKC
antisense ODN markedly
inhibited their relevant specific PKC isoenzyme expression. In each of
these cases, the level of expression in antisense-treated monocytes was
reduced in an isoenzyme-selective manner as compared with either the
untreated or sense-treated monocytes. The lower expression of PKC
I
by activated monocytes depicted in lane 2 of Fig.
4A was not characteristic of other blots that showed
approximately equal levels of this isoform in unactivated and activated
monocytes. The PKC
antisense ODN treatment consistently inhibited
the expression of both PKC
I and PKC
II. The sense ODN control for
the PKC
antisense sometimes caused partial inhibition of PKC
expression, but the sense control ODN did not affect either LDL lipid
oxidation or O
2 production (see Fig. 6). These data, taken
together, provide further support for the independence of these
processes from PKC
activity.
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The PKC antisense ODN was also examined for potential nonspecific
inhibition of other isoforms of PKC as illustrated inFig. 4B. It is evident from these results that PKC
antisense
ODN treatment selectively inhibited the expression of PKC
and not
PKC
I, PKC
II, PKC
, PKC
, or PKC
and that treatment with
PKC
antisense ODN selectively inhibited the expression of both
PKC
isoforms without inhibiting the expression of PKC
.
To investigate the potential influence of PKC and PKC
on LDL
lipid oxidation, human monocyte-mediated LDL lipid oxidation was
examined after PKC isoenzyme-specific antisense ODN treatment. Human
monocytes were pretreated in the presence or absence of PKC
isoenzyme-specific antisense or sense ODN. After activation with ZOP
for 24 h in the presence of additional ODN, LDL lipid oxidation
was determined by the TBA assay. As shown in Fig.
5, substantial LDL lipid oxidation was
observed upon human monocyte activation. LDL lipid oxidation was
markedly inhibited in the presence of PKC
isoenzyme-specific
antisense ODN. In contrast, LDL lipid oxidation was unaffected by
treatment with PKC
sense ODN or PKC
isoenzyme-specific antisense
or sense ODN. These data suggest that PKC
is an essential enzyme for
activation-induced monocyte-mediated LDL lipid oxidation and that
PKC
I and PKC
II are not involved.
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In previous studies, we found that O2 production was required
for the process of monocyte-mediated LDL lipid oxidation, therefore, the effects of PKC isoenzyme-specific antisense ODN treatment on
O
2 production were also examined. Human monocytes were
preincubated with either PKC
or PKC
isoenzyme-specific antisense
or sense ODN for 20 h. After preincubation, monocytes were
activated with ZOP for 1 h during which time monocyte-mediated
O
2 production was quantified as described under
"Experimental Procedures" or lysates were prepared for analysis of
isoenzyme expression. As expected, O
2 production was induced
in ZOP-activated human monocytes (Fig.
6A). PKC
antisense ODN
treatment substantially inhibited O
2 production by activated
human monocytes, whereas other ODN were without effect. Data
presented in Fig. 6B indicate that antisense ODN
treatment caused isoenzyme inhibition under these conditions. The
levels of PKC isoenzymes in the sense ODN pretreated and 1 h
activated monocytes was similar to that in untreated, 1 h
activated monocytes (data not shown). Additionally, antisense ODN to
PKC
did not inhibit expression of PKC
isoforms and vice
versa (data not shown), thus confirming similar results
illustrated in Fig. 4.
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Evidence that treatment with PKC antisense ODN did not damage the
components of the respiratory burst oxidase (NADPH oxidase) complex
were derived from experiments in which superoxide anion production by
antisense pretreated, activated monocytes was restored by inclusion of
the anionic amphiphile, arachidonic acid. Arachidonic acid has been
shown to directly activate the NADPH oxidase and circumvent
requirements for PKC activity in guinea pig macrophages activated by
Fc
(20). The results of one of three experiments illustrating this
restoration of O
2 production are depicted in Fig.
7. Treatment with antisense ODN
significantly inhibited superoxide anion production (p < 0.05). This inhibition was ablated by the inclusion of arachidonic
acid resulting in enhanced superoxide anion production to levels that
were not significantly different from the amount produced by activated
monocytes or activated monocytes treated with sense ODN.
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DISCUSSION |
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Previously, our laboratory published studies indicating that the
activity of a member of the cPKC isoenzyme family was required for
human monocyte-mediated LDL lipid oxidation and for the production of
superoxide anion (7). The cPKC group of PKC isoenzymes consists of four
individual isoenzymes, including PKC, PKC
I, PKC
II, and PKC
.
Although PKC
is not present in human monocytes, the other three cPKC
isoenzymes remained as viable candidates for the requisite cPKC
activity required in the process of monocyte-mediated LDL oxidation and
O
2 production.
Activation of PKC is believed to involve the translocation of the enzyme from a cytosolic location in unstimulated cells to new subcellular sites present in the nucleus or particulate/membrane fractions of stimulated cells (reviewed in Ref. 21). Intracellular translocation is believed to be mediated by intracellular receptors for PKC termed RACKs because inhibition of the RACK binding site of a specific isoenzyme can block its translocation and relevant biologic response (22). Our studies presented in Fig. 1 reveal that PKC activity is substantially induced in both the cytosol and particulate fractions of activated human monocytes and that the predominant activity resides in the particulate fraction after 1 h of activation. These findings are similar to those reported by Kadri-Hassani et al. (23) in studies of human monocytes stimulated with PMA or diacylglycerol. We did not detect PKC activity in unactivated monocytes, a finding that appears to differ from several other studies indicating detectable levels of PKC activity in unactivated cells (23-27). A substantial difference between those studies and the ones presented here are the assay that was used to measure PKC activity. We used an assay that measured PKC activity based on measurement of the phosphorylation of acetylated myelin basic peptide in the presence and absence of the PKC pseudosubstrate inhibitor peptide that is believed to inhibit all isoforms of PKC. Only the pseudosubtrate-inhibitable activity was considered to be mediated by PKC. Studies that have detected activity in unactivated monocytes used assays measuring the total phosphorylation of a typical PKC substrate, such as histones I and III, that might be phosphorylated by other kinases. Indeed, we observed phosphorylation of the acetylated myelin basic peptide substrate in our studies that was not inhibited by the pseudosubstrate, thus implicating phosphorylation of this substrate by kinases that are not inhibited by the pseudosubstrate peptide. Despite these differences in base-line levels of PKC activity in unactivated monocytes, all of these studies agree in finding that activation by a variety of stimuli induces PKC activity and relocation of the majority of the PKC activity to an insoluble fraction.
The activator used in our studies is a yeast cell wall (zymosan) that
was boiled (1 h), extensively washed, and then opsonized with fresh
human serum. It is known that ZOP is a broad spectrum activator. We
have previously shown that ZOP induces human monocyte O2
production and LDL lipid oxidation by stimulating PKC and cPLA2
activity (7, 8). We have also found that other signal transduction
pathways, e.g. phospholipase C
, are activated by ZOP but
not required for superoxide anion production or ZOP-induced monocyte
oxidation of LDL (28). Using this broad activator, we observed
substantial induction of numerous PKC isoforms upon activation by ZOP
(Fig. 2). In unactivated cells, PKC
was located predominantly in the
cytosol and PKC
I in the particulate membrane fraction (Fig. 3), a
finding similar to other analyses of PKC isoform distribution in
monocytes (25, 27, 29). Both PKC
and PKC
II levels in the
particulate/membrane fraction increased as a result of monocyte
activation (Fig. 3). Similarly, Zheng et al. (25) have
reported the translocation of PKC
and PKC
in human monocytes with
OAG and PMA, but ZOP activation was not investigated. In our studies,
we detected expression of PKC
that was not detected in studies by
others (25, 27, 29). This difference may be due to the fact that we
used ECL development of our Westerns, a method that likely enhances the
sensitivity and detection of isoforms present at lower levels.
Although it has previously been reported that human monocyte-mediated
O2 production is correlated with PKC expression, PKC activity,
or PKC translocation, the identity of the relevant PKC isoenzyme was
not investigated (7, 23, 30-33). Furthermore, no studies have to date
identified the relevant isoenzyme of PKC that participates in
monocyte-mediated LDL oxidation, although our previous studies
implicated a member of the cPKC group of isoenzymes (7). To address
this issue, we designed and used PKC isoenzyme-specific antisense ODN
to selectively suppress the expression of different PKC isoforms. The
antisense approach has proven quite successful in this and other
studies by our laboratory (e.g. see Refs. 7, 8, and 34). Two
factors likely contributing to the effectiveness of this approach are
the use of monocytes as the target cells and the careful selection and
purity of the ODN. Monocytes rapidly equilibrate with their environment
through endocytotic mechanisms (35), thereby maximizing uptake of ODN. Monocytes are also resting, Go cells, and many of the
target proteins that we are studying are induced upon activation, thus
minimizing the need to ablate high constitutive levels of already
expressed protein. We have also found that phosphorothioate ODN cause
minimal nonspecific effects on monocytes at the dose ranges employed in these studies. Antisense ODN sequences were carefully selected to
target sequences of mRNA devoid of predicted secondary structure while avoiding the translation start site that may contain concensus sequences. Finally, the use of HPLC-purified antisense ODN avoids variable levels of contamination of the ODN preparation with incomplete synthesis products that will diminish the predicted concentration and
effectiveness of the full-length 20-mers and likely contribute to nonspecificity.
Evidence that the PKC antisense was working selectively to inhibit
PKC
expression is derived from the data indicating that the
expression of other PKC isoforms that we examined was not inhibited by
this treatment (Fig. 4) and by the finding that superoxide anion
production by PKC
antisense-treated monocytes can be restored by the
addition of arachidonic acid (Fig. 7), indicating that the NADPH
oxidase enzyme complex remains functional in cells that have been
treated with antisense ODN.
The important mechanistic finding of our studies is that PKC is
required for superoxide anion production (Fig. 6) and monocyte-mediated LDL lipid oxidation (Fig. 5) and that PKC
I and
II are not, even though all three of these isoforms of PKC are induced by monocyte activation. This is the first direct evidence that PKC
is required for human monocyte-mediated O
2 production and LDL lipid
oxidation. It is consistent with our previous observations that both
PKC activity and O
2 production are required for LDL lipid oxidation.
The relationship between PKC and O2 production has received
considerable attention. O
2 is formed during the respiratory burst by the NADPH oxidase of monocytes, neutrophils, and other phagocytic cells. NADPH oxidase consists of two membrane-bound subunits
(gp91 and p22phox) and several cytosolic subunits
(p47phox, p67phox, p40phox, and Rac2) (see
reviews in Refs. 29 and 35). In unactivated neutrophils the NADPH
oxidase is inactive, and its subunits are distributed between the
cytosol and the membrane (36-38). When the phagocytes are activated,
several of the components become phosphorylated and the cytosolic
subunits migrate to the membrane, where they associate with the
membrane-bound subunits to assemble the catalytically active oxidase
(see reviews in Refs. 30 and 39).
Several laboratories have presented evidence showing that a
PKC-dependent signaling pathway is involved in the
induction of NADPH oxidase and O2 production in neutrophils.
The NADPH oxidase component first shown to be phosphorylated upon
activation of neutrophils was p47phox (40). Multiple
phosphorylation sites have been identified on p47phox (41).
Among these sites, several were predicted to be phosphorylation sites
for PKC. PKC
, the predominant PKC isoenzyme in human neutrophils (42), has been shown to regulate phosphorylation of p47phox and
O
2 production by these cells (43). Recently,
PKC-dependent activation of p47phox was
reported in a cell-free system from neutrophils (32, 33) thus
substantiating earlier studies by Cox et al. (44); however, Waite et al. (45) also recently reported evidence to support the role for a novel, phosphatidic acid activated kinase in
p47phox phosphorylation. Another component of the oxidase
complex, p67phox, has recently been shown to be phosphorylated
upon PMA activation of neutrophils, suggesting PKC involvement as well
(46).
In human monocytes, inhibition of PKC activity suppresses O2
production (7, 23), and PMA, a potent activator of PKC, induces the
phosphorylation of p47phox (23). It has also been reported that
PMA- and ZOP-stimulated O
2 production is inhibited in
bacteria-infected human monocytes, and in these infected monocytes both
PKC
and PKC
were suppressed, suggesting that a
PKC-dependent pathway was involved (31). We report
herein that PKC
plays a major role in regulating O
2
production in activated human monocytes. Taken together, these data
suggest that PKC activity is required for cell-mediated O
2
production and that different PKC isoenzymes appear to be involved in
this process in different cells, i.e. PKC
is required in
neutrophils, whereas PKC
and not PKC
is required in monocytes.
Although our data indicate that PKC is involved in the
monocyte-mediated O
2 production, whether or not PKC
regulates or directly mediates phosphorylation of p47phox or
other components of the respiratory burst complex remains to be
determined. Currently, the relationship among phosphorylation of
p47phox, O
2 production, and PKC
is under study in
our laboratory. Recent studies suggest that more than one kinase may be
required to accomplish the phosphorylation of p47phox (32, 43,
47), thus PKC
may be only one of several kinases involved. We have
also found that PKC
is involved in regulating cPLA2, an enzyme that
generates free arachidonic acid, an anionic amphiphile that regulates
superoxide anion production by NADPH oxidase.2
In summary, our studies demonstrate that suppression of PKC
expression, but not PKC
I or PKC
II expression, results in
inhibition of O
2 production and LDL lipid oxidation by
activated human monocytes. These results suggest that PKC
is a
critical regulator of both of these inflammatory processes.
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FOOTNOTES |
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* These studies were supported by National Institute of Health Grants HL51068 (to M. K. C.) and CA56869 (to A. P. F.) and Grant AHA-NEO-315F from the Northeast Ohio Affiliate of the American Heart Association (to Q. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Scholar of the Leukemia Society of America.
To whom correspondence should be addressed: Dept. of Cell
Biology, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH
44195. Tel.: 216-444-5222; Fax: 216-444-9404; E-mail:
cathcam{at}cesmtp.ccf.org.
The abbreviations used are: LDL, low density lipoprotein; MDA, malondialdehyde (1,1,3,3-tetramethoxypropane); ODN, oligodeoxyribonucleotide(s); PKC, protein kinase C; cPKC, conventional PKC; SOD, superoxide dismutase; ZOP, opsonized zymosan; HPLC, high pressure liquid chromatography; PVDF, polyvinylidene difluoride; TBA, thiobarbituric acid; PAGE, polyacrylamide gel electrophoresis.
2 Q. Li and M. K. Cathcart, manuscript in preparation.
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REFERENCES |
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