Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900
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ABSTRACT |
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In a
companion paper (Vivekananda J, Smith D, and King RJ. Am J
Physiol Lung Cell Mol Physiol 281: L98-L107, 2001), we
demonstrated that tumor necrosis factor (TNF)- inhibited the
activity of CTP:phosphocholine cytidylyltransferase (CT), the
rate-limiting enzyme in the de novo synthesis of phosphatidylcholine
(PC), and that its actions were likely exerted through a metabolite of
sphingomyelin. In this paper, we explore the signaling pathway employed
by TNF-
using C2 ceramide as a cell-penetrating
sphingolipid representative of the metabolites induced by TNF-
. We
found that in H441 cells, as reported in other cell types, cytosolic
phospholipase A2 (cPLA2) is activated by
TNF-
. We also observed that the inhibiting action of C2
ceramide on CT requires protein kinase C-
, p38 mitogen-activated protein kinase, and cPLA2. The actions of C2
ceramide on CT activity can be duplicated by adding 2 µM lysoPC to
these cells. Furthermore, we found that the effects of C2
ceramide are dependent on 5-lipoxygenase but that cyclooxygenase II is
unimportant. We hypothesize that CT activity is inhibited by the lysoPC
generated as a consequence of the activation of cPLA2 by
protein kinase C-
and p38 mitogen-activated protein kinase. The
other product of the activation of cPLA2, arachidonic acid,
is a substrate for the synthesis of leukotrienes, which raise
intracellular Ca2+ levels and complete the activation of
cPLA2.
lung injury; pulmonary surfactant; phosphatidylcholine synthesis; leukotrienes; cytidine 5'-triphosphate; protein kinase C; mitogen-activated protein kinase; cytosolic phospholipase A2
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INTRODUCTION |
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THE METABOLISM OF
PULMONARY SURFACTANT is impaired during the development of
chronic lung diseases of the newborn (28) and adult
(37), presumably in response to unidentified materials generated during the course of these injuries. Among the several growth
factors and cytokines that are released is tumor necrosis factor
(TNF)-, an important cytokine that mediates a variety of cellular
functions in the inflammatory response (for example, see Refs.
30, 34). TNF-
has also been shown to
inhibit the synthesis of surfactant in pulmonary epithelial cells
(21, 36, 45, 55), and in the preceding companion paper
(53), our laboratory demonstrated that its effects
on phosphatidylcholine (PC) synthesis may be through the actions of
metabolic products of sphingomyelin hydrolysis acting on
CTP:phosphocholine cytidylyltransferase (CT; EC 2.7.7.15). In this
paper, we studied the signaling pathway(s) initiated by sphingomyelin
metabolites to effect this inhibition of CT. Using plasmid vector
constructs of dominant negative protein kinase C (PKC)-
,
hypothesized to be involved in the signaling pathway together with
relatively specific inhibitors of other purported intermediates, we
found that PKC-
, p38 mitogen-activated protein kinase (MAPK), and
cytosolic phospholipase A2 (cPLA2; type IV) are
all required for the ceramide-induced inhibition of CT activity.
Furthermore, we determined that an inhibitor of 5-lipoxygenase was also
able to nullify the actions of ceramide, whereas an inhibitor of
cyclooxygenase-2 (COX-2) had no effect. A hypothesis is presented,
based on these data as well as findings in the literature
(11), which suggests that the inhibition of CT is from the
generation of lysoPC through the action of activated cPLA2.
Arachidonic acid, the other product of the hydrolysis of sn-2-arachidonoyl PC, is a substrate for the synthesis of
leukotrienes, which may stimulate Ca2+ influx
(23, 46) and thereby further activate cPLA2
(33) or may directly stimulate the transcription of
TNF-
(48).
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EXPERIMENTAL PROCEDURES |
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Materials.
NCI-H441 (adult human adenocarcinoma) cells were obtained from the
American Type Culture Collection (ATCC, Manassas, VA). TransIT-LT1 transfection reagent was from PanVera (Madison,
WI). Polyclonal antibodies against the carboxy-terminal ends of PKC- and -
II isoforms were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA). Anti-p38 MAPK antibody was from New England Biolabs. The
myelin basic protein (MBP) 4-14 peptide fragment (Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu) was obtained from Sigma
(St. Louis, MO). Bromoenol lactone (BEL), arachidonyltrifluoromethyl ketone (AACOCF3), C2 ceramide
(N-acetyl-D-erythro-sphingosine), and
SB-203580 were from Calbiochem (La Jolla, CA).
[
-32P]ATP, [32P]orthophosphoric
acid,
L-
-1-palmitoyl-2-arachidonyl-[arachidonyl-1-14C]PC,
and [methyl-14C]phosphorylcholine were
obtained from NEN (Boston, MA). NS-398 and nordihydroguaiaretic acid
(NDGA) were from BIOMOL (Plymouth Meeting, PA).
Plasmid constructs.
Kinase-inactive PKC- (lysine in ATP-binding
site mutated to methionine) (7) cloned into pTB701/HA was
obtained from Dr. Robert V. Farese (University of South Florida College
of Medicine, Tampa, FL). Antisense (dominant negative) and wild-type
PKC-
cloned into pRSV (43) were obtained from Dr.
Robert I. Glazer (Georgetown University Medical Center, Washington, DC)
(2).
Cell culture. H441 cells (human lung adenocarcinoma cells) were obtained at the 50th passage from the ATCC. The cells were maintained in McCoy's 5A medium containing 5% fetal bovine serum (FBS) and 50 µg/ml of gentamicin sulfate at 37°C in a 5% CO2-95% air humidified atmosphere. Before all experiments, the medium was changed to McCoy's 5A medium without FBS and the cells were conditioned overnight. All experiments were conducted in medium without FBS.
Stable transfection with constructs of PKC-.
We used TransIT-LT1 transfection reagent (PanVera) following
the manufacturer's instructions. Briefly, H441 cells at 30-40% confluence were incubated with a complex of plasmid DNA and the LT1
transfection reagent (1 µg to 4 µl) and were cotransfected with
pCEV or pCDNA (neor) plasmids to allow selection with
Geneticin (G418, Life Technologies, Gaithersburg, MD). The cells were
washed twice with serum- and gentamicin-free McCoy's 5A medium, and
the transfection mix of DNA and transfection reagent prepared in the
same medium was overlaid on the cells. After 48 h of incubation,
the transfected cells were trypsinized and selected with 500 µg/ml of
Geneticin for a period of 3-4 wk. Heterogeneous populations of
transfected cells were then trypsinized and maintained in medium
containing G418 for >4 passages.
PKC activity. PKC activity in the cytosolic and membrane fractions was measured by the method of Chakravarthy et al. (13). Briefly, cells were grown to 60-70% confluence and then deprived of serum for 24 h. After treatment, the cells were washed twice with Ca2+- and Mg2+-free PBS and scraped into ice-cold lysis buffer containing 1 mM NaHCO3, pH 7.5, 5 mM MgCl2, 100 µM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml of leupeptin, 10 µg/ml of pepstatin, and 100 µM sodium orthovanadate. Cells were homogenized and vortexed for 1 min, and the homogenates were centrifuged at 45,000 rpm (TLA 45 rotor, Beckman TLX centrifuge) for 15 min at 4°C. Supernatant (cytosolic fraction) was collected, and the pellet (membrane fraction) was homogenized in 2× assay buffer (50 mM Tris · HCl, pH 7.5, 10 mM CaCl2, 200 µM sodium pyrophosphate, 2 mM sodium fluoride, and 200 µM PMSF). The activity assay was carried out on the same day.
The assay was standardized for all the fractions (cell lysate, cytosol, and membrane) with different amounts of protein and time and temperature of the incubation. PKC activity [counts per minute (cpm) per microgram of total protein] was linear for 2-30 µg of protein and the 10- to 30-min incubation period. The correlation coefficient was 0.97-0.99. The assay was carried out with 5-20 µg of protein in buffer containing 25 mM Tris · HCl, pH 7.5, 2.5 mM MgCl2, 0.5 µM CaCl2, 50 µM sodium pyrophosphate, 0.5 mM sodium fluoride, 50 µM PMSF, 20 µM ATP, 3 µg of the MBP4-14 peptide fragment substrate, 1 µCi of [32P]ATP, 0.015 µg of diolein, and 0.1 µg of phosphatidylserine. After 15 min of reaction at room temperature, 20 µl of the reaction mixture were spotted on phosphocellulose paper disks (Life Technologies). The disks were washed three times with 0.1% acetic acid and twice with deionized water and counted in 5 ml of scintillation fluid. Counts per minute were normalized with total protein in each fraction.PKC isoform-specific kinase activity.
Isoform-specific activity was determined by slight modifications of the
methods described by Denning et al. (17) and Bandyopadhyay et al. (8). The assay was standardized with different
amounts of protein from different cellular fractions, with different
amounts of antibody, and by varying the incubation time for
immunoprecipitation (4 and 16 h). The standard curves for PKC-
and PKC-
II activities were linear up to 500 µg of protein for all
the cellular fractions. Correlation coefficients for standard curves
for PKC activity (cpm/µg protein) varied from 0.89 to 0.995.
MAPK phosphorylation (activation).
Cells seeded in 60-mm tissue culture dishes were treated with TNF-
(10 ng/ml) for various time periods. The cells were washed twice with
PBS and lysed in 200 µl of Tris · HCl buffer (pH 7.4) containing 25 mM Tris, 1% Igepal, 150 mM NaCl, 50 mM NaF, 200 µM
sodium orthovanadate, and 1 mM PMSF. The lysates were probe-sonicated for 5 s and centrifuged at 14,000 rpm for 10 min to remove the cell debris. Lysate protein was separated on 10% SDS-polyacrylamide, and the gels were blotted with anti-phospho-specific p38 MAPK. Blotting
the membranes with anti-p38 MAPK antibodies confirmed equal protein
loading in the gels. The proteins of interest were detected with an
enhanced chemiluminescence kit. The MAPK antibodies were obtained from
New England BioLabs (Beverly, MA) and were used at 1:1,000 dilution in
Tris-buffered saline containing 5% BSA and 0.2% Tween 20. The
anti-rabbit IgG-horseradish peroxidase secondary antibody was from
Santa Cruz Biotechnology and was used at 1:5,000 dilution with blocking
buffer (10% milk in Tris-buffered saline-2% Tween 20).
CT activity. We used the method of Ansel and Chojnacki (3). Cells (0.2-0.3 × 106) were seeded on 60-mm petri dishes, grown to 40-50% confluence, and then treated with C2 ceramide (10 µM) as described in Ref. 53. For investigating the effect of inhibitors of kinases, the cells were pretreated for 30 min to 1 h with 1 and 2 µM SB-203580 [a p38 MAPK inhibitor (32)]; 5, 10, and 20 µM BEL [an inhibitor of Ca2+-insensitive PLA2 (iPLA2; type VI) (1, 6)]; 0.1, 1.0, and 10 µM AACOCF3 [an inhibitor of type IV cPLA2 (1)]; 0.1 and 1.0 µM NS-398 [an inhibitor of COX-2 (18)]; or 1 and 5 µM NDGA (an inhibitor of 5-lipoxygenase) (22). Cells were washed twice with normal saline, harvested, and homogenized in 200 µl of homogenization buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM dithiothreitol, 175 µg/ml of PMSF, 5 mM sodium fluoride, and 0.1 mM sodium orthovanadate). Homogenates were sonicated in a water bath sonicator for 20 min and centrifuged (14,000 rpm) for 5 min to isolate the membrane fraction. Membrane pellets were then homogenized in 200 µl of the homogenization buffer.
For the activity assay, 20 µg of membrane protein were mixed with assay buffer (400 mM Tris · HCl, pH 7.5, 30 mM magnesium acetate, 10 mM CTP, and 50,000 cpm of [14C]phosphorylcholine) and incubated at 37°C for 1 h. To stop the reaction, the tubes were immersed in a boiling water bath for 2 min. Radioactive CDPcholine was extracted on acid-washed charcoal, and the charcoal was washed three times with water before extraction with formic acid for scintillation counting.CT phosphorylation.
Cells were labeled with [32P]orthophosphate (100 µCi/60-mm dish) for 18 h in phosphate-free DMEM. The labeled
cells were stimulated with TNF- (100 ng/ml) or ceramide (10 µg/ml). After specified times (0, 1, 3, and 5 h), the cells were
washed with ice-cold PBS and lysed in Tris · HCl buffer (pH
7.4) containing 25 mM Tris, 1% Igepal, 150 mM NaCl, 50 mM NaF, 200 µM sodium orthovanadate, and 1 mM PMSF. One hundred micrograms of
lysate protein were mixed with 10 µl of a polyclonal antiserum
against human CT and incubated overnight at 5°C. The antiserum was a
mixture of antibodies developed to the peptide sequences
acetyl-AKVNARKRRKEAPGC-amide and acetyl-NEKKYHLQERVDKVC-amide (QCB,
Hopkinton, MA). The immune complex was captured on protein A agarose
beads, and the bound radioactivity was eluted in SDS-PAGE loading
buffer (75 µl). A portion of the elute was directly counted by
scintillation counting, and the rest was loaded on a 10%
polyacrylamide gel for further separation and autoradiography.
PLA2 activity.
We measured PLA2 activity in the cytosol of the cells by
the hydrolysis of a substrate of radioactive
sn-2-arachidonoyl PC. Cells were seeded in 60-mm plates and
allowed to attach for 4-6 h in 1.5 ml of 10% FBS-McCoy's 5A
medium. The cells were then washed twice with serum-free McCoy's
medium and equilibrated overnight (17-18 h) in 1.5 ml of
serum-free McCoy's medium. After treatment with 10 µM C2
ceramide, the cells were washed twice with ice-cold PBS and harvested
in 100 µl of a buffer containing 250 mM sucrose, 2 mM EDTA, 50 mM
HEPES, pH 7.5, 20 µg/ml of leupeptin, and 1 mg/ml of PMSF. The cells
were sonicated and centrifuged at 45,000 rpm for 30 min. The cytosolic
fraction was used for the assay of cPLA2. The substrate,
L--1-palmitoyl-2-arachidonyl-[arachidonyl-1-14C]PC
(NEN), was suspended in DMSO with vigorous vortexing. Five microliters
of the substrate were mixed with 10 µl of CaCl2 solution and 10 µl of BSA solution in a glass tube (final assay concentration of the substrate was 6 µM). The assay was initiated by adding 30-40 µg of the protein and incubating for 30-60 min at
37°C. The products of the reaction were separated by silicic acid
TLC, and the radioactive fatty acid was spot-scraped and counted.
Incorporation of [32P]orthophosphoric acid into PC.
Cells (0.2-0.3 X 106/well), either normal H441 or
cells transfected with PKC- constructs, were seeded on six-well
tissue culture plates (Corning, New York, NY). After overnight growth,
the cells were starved for 18-24 h in phosphate-free DMEM without
serum. The cells were then treated with 10 µM C2 ceramide
or DMSO (vehicle control) for varying times. At the end of the
treatments, the cells were further incubated for 5 h with 10 µCi
of [32P]orthophosphoric acid without removing the
C2 ceramide (a total of 6 h of ceramide exposure).
Cells were washed twice with PBS, harvested in 400 µl of deionized
water, and homogenized.
Statistical analyses. We used one-way ANOVA or t-test to test for significance. Significance was assumed for P < 0.05, with P < 0.1 noted.
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RESULTS |
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TNF- and C2 ceramide increase PKC-
activity in
H441 cells.
TNF-
(38, 44) and ceramide (24, 40) have
been shown to both activate and inhibit PKC depending on cell type and
PKC isoform. In bronchial epithelial cells (56), TNF-
is an activator. We tested whether this TNF-
-induced stimulation
holds in H441 cells when focusing on PKC-
. The results are
shown in Fig. 1. Cells treated with 10 ng/ml of TNF-
showed PKC-
activity that was elevated threefold.
Peak activity occurred within 30 min, the earliest time interval
analyzed. There was no change in the activity of PKC-
II (data not
shown). In one experiment, we also found that 10 µM C2
ceramide increased PKC-
activity by ~50% within 1 min, and this
remained elevated through 3 h.
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TNF- increases MAPK activity in H441 cells.
TNF-
and ceramide have been widely reported to activate MAPKs (see,
for example, Ref. 47), and this was reproduced in H441 cells as
shown in Fig. 2. TNF-
activates
(phosphorylates) p38 MAPK maximally within 15 min, consistent with a
report in other cell types (39). Similar results were
found in separate experiments with 10 µM C2 ceramide
(Fig. 2), with peak activation occurring after 5 min.
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C2 ceramide inhibits CT activity; the effect is
abolished in cells transfected with dominant negative clones of
PKC-.
We first established that PKC-
activity was altered in the cells
that we transfected with PKC-
constructs. The results are shown in Fig. 3. Activity of PKC-
in
the two dominant negative transfected clones was reduced to ~60% of
that in nontransfected control cells but was unchanged in the wild-type
PKC-
transfectants.
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Ceramide-induced inhibition of CT activity requires a functional
cPLA2 but not iPLA2.
cPLA2 has been reported to be activated in a number of cell
types in response to TNF- (19, 33, 41). We
confirmed this activity in H441 cells with C2
ceramide. cPLA2 was rapidly activated, and activity
remained elevated at ~150% of control value for at least 60 min
(Fig. 5). Consistent with this response,
we also found in one experiment that the phosphorylation of
immunoprecipitated cPLA2 was increased by 58% after cells
were exposed to 10 µM C2 ceramide for 15 min. This
increase in phosphorylation was maintained through 30 min but reversed
by 1 h. The results indicate that C2 ceramide exerts
an activation of cPLA2 similar to that reported for
TNF-
.
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The signaling pathway for ceramide-induced inhibition of CT
involves a leukotriene generated through 5-lipoxygenase.
We used NDGA (22), a compound that inhibits the
lipoxygenases, principally, 5-lipoxygenase. We used two concentrations
of NDGA, 1 and 5 µM, which we gave to the cells together with 10 µM
C2 ceramide. The results obtained with both concentrations were indistinguishable, and the data from the use of 1 µM
NDGA are shown in Fig. 8. NDGA completely
prevented the C2 ceramide inhibition of CT.
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Inhibition of CT does not involve an eicosanoid generated through COX-2. Two COXs have been described in the literature: a constitutively expressed enzyme, COX-1, and an induced enzyme, COX-2 (50). Most cytokine-induced actions involving COXs are through COX-2 (50). We used a relatively specific inhibitor for COX-2, NS-398 (18), and tested the effects of 10 µM C2 ceramide on the activity of CT in the presence and absence of three concentrations of this compound, 0.1, 1, and 10 µM. Neither 0.1 nor 1 µM NS-398 had an effect on either basal CT activity or the ability of 10 µM C2 ceramide to inhibit the activity of CT. At a concentration of 10 µM, NS-398 had no effect on the response to C2 ceramide, but it did reduce basal activity (data not shown).
LysoPC inhibits CT activity just as effectively as C2
ceramide.
One explanation for the data presented is that cPLA2 is
activated by TNF- or ceramide and that a metabolite of the reaction catalyzed by cPLA2 is responsible for the effects on CT
activity. We tested this hypothesis by treating cells with either 10 µM C2 ceramide or 2 µM 1-palmitoyl-2-lysoPC and
measuring the effects on CT activity after 1 h. The effects of
both compounds were identical as shown in Fig.
9. The results indicate that the lysoPC
generated through a ceramide-stimulated signaling pathway could be the
compound actually responsible for the ceramide-induced inhibition of
CT.
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CT is not phosphorylated in response to TNF-.
Changes in the phosphorylation of CT have been observed in connection
with certain physiological events such as mitosis (42a), but
their significance is not clear. There is no proven cause and effect
relationship between CT phosphorylation and its activity (42a).
Consistent with that conclusion, we did not observe a strong correlation between the phosphorylation of CT and the changes in its
activity induced by TNF-
. The data are presented in Fig. 10. CT phosphorylation was reduced to
~80% of control value 1-5 h after the cells were presented with
10 ng/ml of TNF-
, but only the effects at 5 h were
statistically significant and then only at P < 0.1. In
some experiments, we ran part of the immunoprecipitates in SDS-PAGE and
quantified by autoradiography the amount of
[32P]orthophosphate incorporated into the CT band. The
results confirmed data obtained by direct counting of the
immunoprecipitates. In one experiment, we found that 10 µM
C2 ceramide had almost identical effects to those of
TNF-
(data not shown).
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Neither PKC- nor p38 MAPK affect the inhibition by ceramide of
orthophosphate incorporation into PC despite their effects on CT
activity.
CT is the rate-limiting enzymatic activity in the Kennedy pathway for
PC synthesis, and changes in its activity should be reflected by
differences in synthetic rates, which is consistent with the data
presented in the companion paper (53). However, C2 ceramide may also inhibit other enzymes in the Kennedy
pathway that could likewise affect PC synthesis. To investigate this
possibility, we measured the rate of incorporation of
[32P]phosphate into cellular PC in cells
transfected with dominant negative constructs of PKC-
or in
nontransfected H441 cells that had been pretreated with 1 and
2 µM SB-203580. The results are shown in Fig.
11. [32P]phosphate
incorporation was inhibited by 10 µM C2 ceramide just as
effectively as in control H441 cells, even when PKC-
or p38 MAPK was
blocked, despite their apparent importance in the regulation of CT
activity. The results suggest that ceramide inhibits multiple enzymes
in the Kennedy pathway, only one of which is CT.
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DISCUSSION |
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The activity of CT is controlled by its association with a lipid environment. CT is a lipid-dependent enzyme, the activity of which is regulated by its interaction with cytoplasmic and nuclear membranes (15). Diacylglycerol (DAG) and certain other lipids are potent activators (5). CT shuttles between nuclear and cytoplasmic sites during the course of physiological events, and changes in CT enzymatic activity correlate with its intracellular localization (42a). The mechanisms by which CT is translocated between the cytoplasmic membranes (active) and nuclear membranes (inactive) are unknown, but they are likely to involve associated lipids. Jamil et al. (25) have presented intriguing evidence that activity (and translocation) may be related to the ratio of bilayer-forming to non-bilayer-forming associated lipids.
The signaling pathway for ceramide-induced inhibition of CT
involves cPLA2.
cPLA2 is an 85-kDa protein that is widely distributed,
including in lung tissue (19). cPLA2 is an
attractive candidate as a regulator of the TNF- or ceramide
inhibition of CT. An inhibitor of cPLA2, but not of
iPLA2, nullifies the ceramide-induced inhibition of CT
activity (this study). TNF-
stimulates cPLA2 activity, even when given in low concentrations (19).
cPLA2 has a relatively high specificity for arachidonic
acid (20:4) at the sn-2 position (32), and
arachidonic acid, in turn, may further activate sphingomyelinases (26). LysoPC given to H441 cells (data reported here) and
to BAC 1.2F5 cells, a macrophage-like cell line (11),
inhibits CT activity. LysoPC also promotes bilayer formation,
consistent with the hypothesis of Jamil et al. (25) for
the regulation of CT activity, but this may be coincidental with the
observed inhibition of CT rather than causative. LysoPC itself
stimulates Ca2+ influx, thereby potentially activating
cPLA2, reinforcing the elevation in lysoPC content
(20).
The function of PKC and p38 MAPK may be to activate cPLA2. cPLA2 is regulated by phosphorylation (12). Activity is dependent on two serine phosphorylation sites: Ser228 in the lipase sequence and Ser505 in a proline-dependent kinase sequence. Micromolar concentrations of Ca2+ are required, but the Ca2+ is not involved in the catalytic action but rather serves to anchor the enzyme to lipid membranes. Activated cPLA2 is principally found at the nuclear membrane.
One of the phosphorylation sites, Ser505, is a consensus sequence for proline-dependent kinases. PD-98059 and SB-20358, inhibitors of p42 and p38 MAPKs, respectively, inhibit phosphorylation and activity (12). PKC is in the signaling sequence (40). There are now numerous studies that show that the activation of MAPKs frequently proceeds through a PKC intermediary (for example, Ref. 29). The role of PKC in this activation is known for one of the classes of MAPK, p42/p44 (extracellular signal-regulated kinase). PKC activates Raf-1 by phosphorylation (directly or possibly through an intermediate) (29), which functions as a MAPK kinase kinase (i.e., two levels upstream) for p42/p44. The mechanisms by which PKC functions in the other MAPK pathways are unknown; presumably, PKC also activates upstream kinases. In addition, PKC may directly phosphorylate cPLA2 on several serine sites, but the effect of this phosphorylation is not fully known (12). PKCs are activated by TNF-Leukotrienes may be involved in the ceramide inhibition.
We have found that inhibiting the initial enzyme 5-lipoxygenase in the
principal pathway of leukotriene synthesis abolishes the ability of
C2 ceramide to inhibit CT. A similar observation has been
reported by Arias-Diaz and coworkers (4). The mechanisms by which leukotrienes interact in this system have not been determined, and we can only speculate as to its pathways. Leukotrienes themselves stimulate the transcription of cytokines (48), which could
amplify the overall effect. In addition, certain of the leukotrienes, LTB4 (46), LTD4 (23,
51), and LTE4 (51), increase
concentrations of intracellular Ca2+. There are two
mechanisms: activation of a phosphatidylinositol-specific PLC through
pertussis toxin-sensitive G proteins (23, 51) to hydrolyze
L--phosphatidylinositol 4,5-diphosphate to inositol 1,4,5-trisphosphate and release intracellular stores of
Ca2+ and the opening of receptor-operated Ca2+
channels through pertussis toxin-insensitive G proteins
(51). Elevated Ca2+ could then activate a
variety of calcium-dependent enzymes including cPLA2. This
would, of course, result in a renewed synthesis of the arachidonic acid
substrate, perpetuating the cycle. Although we consider it plausible
that leukotrienes could interact in the signaling process through their
effects on intracellular pools of calcium, the data in this study do
not directly measure these concentrations. Confirmation of this
purported effect will require further exploration in separate studies.
Ceramide must also inhibit other enzymes in the pathway(s) for PC
synthesis.
We have shown that when either PKC- or p38 MAPK is inhibited,
C2 ceramide does not inhibit CT activity. Despite this
reversal of CT inhibition, however, C2 ceramide inhibits PC
synthesis. We think that this indicates that C2 ceramide
acts simultaneously on several enzymatic activities, only one of which
is CT. We have not identified which of these enzymes are also affected
by ceramide, although our previous results suggest that the last enzyme
in the Kennedy pathway, CDPcholine:1,2-DAG cholinephosphotransferase, is not affected. An interesting candidate is phosphatidic acid phosphohydrolase (phosphatide phosphatase). Bleasdale and Johnston (9), in work that is nearly 20 years old, proposed that
the activity of phosphatidic acid phosphohydrolase is critical in maintaining the balance between PC, phosphatidylglycerol, and phosphatidylinositol. An inhibition of phosphatidic acid
phosphohydrolase should result (by their hypothesis) in a drop in PC, a
decrease in phosphatidylglycerol, and an increase in
phosphatidylinositol. This is exactly what happens in chronic lung
injury (for example, see Ref. 27).
A hypothesis for the effects of ceramide on CT activity.
We suggest that ceramide activates PKC and p38 MAPK and that these
contribute to the activation of cPLA2 by phosphorylation. Activated cPLA2 hydrolyzes sn-2-arachidonoyl PC
to lysoPC and arachidonic acid. LysoPC interacts with the cytoplasmic
membrane lipids associated with CT and induces the translocation of CT to the nucleus where it is in an inactive state. Arachidonic acid, through 5-lipoxygenase, generates leukotrienes that further activate cPLA2 through their elevation of intracellular
Ca2+ pools. In addition, these leukotrienes stimulate the
production of TNF-. In this manner, the pathway is self-sustaining
and reinforcing, consistent with the persistent effects of ceramide on
CT activity. A signaling scheme consistent with our data is
shown in Fig. 12. Although this scheme
is plausible, alternative relationships among the intermediaries are
also possible. For instance, our data do not distinguish whether the
activation of p38 MAPK in response to ceramide is sequential through
PKC-
or in parallel. Both PKC-
and p38 MAPK activate
cPLA2 (12). TNF-
has been shown to activate p38 MAPK in a variety of cell types (31), and more
recently, Mallampalli and coworkers (35) have shown that
it activates p42/p44 MAPK in type II cells. However, these studies do
not distinguish whether the activation also involves PKC-
.
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Multiple mechanisms may be involved in the regulation of CT.
Mallampalli and coworkers (36), in a paper that appeared
while this work was being completed, convincingly demonstrated that 500 ng/ml of TNF- reduced CT protein after 24 h of exposure and
that this phenomenon may involve proteasome-associated processing. However, there were modest or no changes at shorter times (4 and 12 h). This is in seeming contrast to our observations with 10 µM C2 ceramide in which changes in CT activity were
evident as early as 2 h. These differences could relate to
differences in cell types, to the fact that C2 ceramide
rapidly traverses cell membranes and thereby acts more rapidly, or from
some subtle and unrecognized procedural differences inherent in these
two studies. We propose an additional possibility, that signaling
through cPLA2 is a relatively rapid mechanism responsible
for the earliest changes in CT activity, whereas protein processing and
degradation require longer time intervals. This possibility seems to be
hinted at in the study by Mallampalli et al. (36) as
evident from comments in the discussion. An alternative explanation is
that proteasome-associated processing requires cPLA2, but a
thorough literature search uncovered no evidence for this.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert V. Farese (University of South Florida College of Medicine), Dr. Robert I. Glazer (Georgetown University Medical Center, Washington, DC), and Dr. Yoshitaka Ono (Faculty of Science, Kobe University, Kobe, Japan) for generous gifts of plasmid constructs of protein kinase C.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-52664.
Address for reprint requests and other correspondence: R. J. King, Dept. of Physiology, Univ. of Texas Health Science Ct., 7703 Floyd Curl Dr., San Antonio, TX 78229-3900 (E-mail: kingr{at}uthscsa.edu).
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.
Received 2 November 2000; accepted in final form 7 February 2001.
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