Lysophosphatidylcholine activates mesangial cell PKC and MAP
kinase by PLC
-1 and tyrosine kinase-Ras pathways
Babu V.
Bassa1,2,
Daeyoung D.
Roh1,2,
Nosratola D.
Vaziri2,
Michael A.
Kirschenbaum
,1,2, and
Vaijinath
S.
Kamanna1,2
1 Nephrology Section,
Department of Veterans Affairs Medical Center, Long Beach 90822;
and 2 Division of Nephrology and
Hypertension, Department of Medicine, University of California,
Irvine, California 92697
 |
ABSTRACT |
Although lysophosphatidylcholine (LPC)-mediated cellular
responses are attributed to the activation of protein kinase C (PKC), relatively little is known about the upstream signaling mechanisms that
regulate the activation of PKC and downstream mitogen-activated protein
(MAP) kinase. LPC activated p42 MAP kinase and PKC in mesangial cells.
LPC-mediated MAP kinase activation was inhibited (but not completely)
by PKC inhibition, suggesting additional signaling events. LPC
stimulated protein tyrosine kinase (PTK) activity and induced Ras-GTP
binding. LPC-induced MAP kinase activity was blocked by the PTK
inhibitor genistein. Because LPC increased PTK activity, we examined
the involvement of phospholipase C
-1 (PLC
-1) as a key participant
in LPC-induced PKC activation. LPC stimulated the phosphorylation of
PLC
-1. PTK inhibitors suppressed LPC-induced PKC activity, whereas
the same had no effect on phorbol 12-myristate 13-acetate-mediated PKC
activity. Other lysophospholipids [e.g., lysophosphatidylinositol
and lysophosphatidic acid (LPA)] also induced MAP kinase
activity, and only LPA-induced MAP kinase activation was sensitive to
pertussis toxin. These results indicate that LPC-mediated PKC
activation may be regulated by PTK-dependent activation of PLC
-1,
and both PKC and PTK-Ras pathways are involved in LPC-mediated
downstream MAP kinase activation.
atherogenic lipoproteins; oxidatively modified
lysophosphatidylcholine; intracellular signaling; atherosclerosis; glomerulosclerosis; protein kinase C; mitogen-activated protein kinase; phospholipase C
-1
 |
INTRODUCTION |
A GROWING BODY OF EVIDENCE suggests that
lysophosphatidylcholine (LPC), a principal component of oxidatively
(ox) modified forms of low-density lipoproteins (LDL), can modulate
vascular cell activation processes, culminating in the gene expression of various cytokines, growth factors, and other proteins associated with vascular diseases (19, 20, 28, 43, 44). Similarly, we have shown
that the incubation of glomerular endothelial cells with LPC induced
intercellular adhesion molecule 1 (ICAM-1) expression and increased
monocyte adhesion to glomerular endothelial cells (9). LPC associated
with ox-LDL or exogenous LPC has been shown to induce selective
impairment of receptor-mediated endothelium-dependent arterial
relaxation (17). The in vivo generation of LPC involved in
pathobiological cellular events is not completely understood; however,
the hydrolysis of sn-2 fatty acid of phosphatidylcholine by plasma
lecithin-cholesterol acyltransferase can produce LPC (14). Furthermore,
phospholipase A2 associated with
apolipoprotein B of LDL has been implicated in the generation of LPC
during oxidative modification of LDL (31). After its formation, LPC, a
relatively polar molecule, gets associated with serum albumin or LDL
and is carried to various cellular sites to participate in cellular, metabolic, and pathophysiological pathways. Increased concentrations of
plasma LPC (including in LDL fractions) have been observed in
atherosclerosis and nephrosis in humans and experimental animals (14,
34). Higher circulating levels of LPC have also been reported during
ischemia in both animals and humans (1, 26).
Recently, we have reported that the stimulation of mesangial cells with
LDL, ox-LDL (with higher potency), and LPC (major active component of
ox-LDL) increased the activation of p42 mitogen-activated protein (MAP)
kinase (2). MAP kinases are a family of serine threonine kinases that
are proposed to converge diverse signal transduction events associated
with various cellular responses, including mitogenesis and
differentiation (33). Atherogenic lipoproteins (e.g., LDL, ox-LDL, or
its major components) can activate cellular protein kinase C (PKC; see
Refs. 18 and 29), and PKC in turn can activate MAP kinases through the
activation of Raf-1 and a MAP kinase kinase pathway (12, 40, 22). The activation of membrane or cytoplasmic protein tyrosine kinase (PTK)
pathways may regulate both PKC and downstream MAP kinase activation
through phospholipase C
-1 (PLC
-1) and Ras-mediated pathways (25,
27). However, the involvement of any of the above-noted multiple
signaling events in atherogenic lipoprotein- and LPC-mediated responses
are not known so far.
Based on the current understanding, the activation of PKC has been the
primary mechanism implicated in several actions of LPC (18, 29, 36).
However, the molecular basis for the activation of PKC, especially the
identification of the upstream signaling mechanism(s) of the
phosphoinositide turnover and the generation of diacylglycerol (DAG),
which is absolutely required for the activation of PKC, is poorly
understood so far. Furthermore, the involvement of LPC-induced PKC
and/or other signaling events in the regulation of MAP kinase is not
clearly understood. With the use of stably transformed murine
glomerular mesangial cells (SV40 transformed) and primary cultures of
rat glomerular mesangial cells (in selective MAP kinase experiments),
this study was designed to identify the upstream signaling mechanisms
of LPC-mediated PKC activation and the involvement of PKC and/or PTK-
and Ras-mediated pathways in downstream MAP kinase activation by
LPC. We report here that LPC, through the stimulation of
phosphorylation of PLC
-1 and activation of Ras, important members of
the mitogenic signaling cascade, regulates PKC activation and
downstream MAP kinase signaling. The activation of membrane or
cytoplasmic tyrosine kinases may be an early principal event in
LPC-mediated signaling processes.
 |
MATERIALS AND METHODS |
Materials. Antibodies for PKC (
,
, and
reacting), PKC-
, p42 MAP kinase, PLC
-1, Shc,
pp60src, and phosphotyrosines were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Ras
antibody (clone Y 13-259) was obtained from Calbiochem (San Diego,
CA). [32P]ATP was
obtained either from Amersham (Arlington Heights, IL) or from DuPont
New England Nuclear Research Products (Boston, MA). Pertussis toxin
(PTX), genistein, tyrphostin B46, and bisindolylmaleimide GF109203-X
(GFX) were obtained from Calbiochem (La Jolla CA). Protein A-Sepharose,
myelin basic protein (MBP), phorbol 12-myristate 13-acetate (PMA), LPC
(palmitoyl and oleolyl), lysophosphatidic acid (LPA),
lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI),
FBS, and other chemicals were purchased from Sigma Chemical (St. Louis, MO).
Cell culture and treatment. The murine
mesangial cells (stably transformed with SV40) were obtained from the
American Type Culture Collection (ATCC, Rockville, MD). The cells were
routinely grown in DMEM supplemented with 5% FBS. Primary cultures of
rat glomerular mesangial cells were isolated by a previously described procedure (30), and cells for MAP kinase assays were used within 4-10 passages. Cell cultures were prepared by seeding 5 × 105 cells/well in 30-mm six-well
cluster dishes. When cell monolayers were ~80% confluent, the medium
was replaced by serum-free DMEM. After 24 h, the medium was replaced by
fresh serum-free DMEM and LPC, or other agonists and protein kinase
inhibitors were added in appropriate quantities. LPC and other
lysophospholipid stock solutions were prepared in absolute ethanol
(10-20 mM). A working solution was prepared by diluting the stock
solution (in serum-free DMEM) and then adding aliquots of this solution
to the cultures at selected concentrations. A stock solution of PMA (10 mg/ml) was prepared in ethanol, and working solutions were prepared by diluting the stock appropriately in serum-free DMEM. GFX, herbimycin, and genistein were dissolved in DMSO. Appropriate quantities of DMSO
(5-10 µl) were added to the control dishes. After being
incubated for selected time intervals, cells were washed with 1 ml of
cold Hanks' balanced salt solution and were processed with an
appropriate cell lysis buffer for the determination of PKC, PLC
-1,
MAP kinase, tyrosine kinase, or Ras activities.
Assay of p42 MAP kinase. Mesangial
cells were stimulated with LPC and other agonists at various
concentrations for different time periods. After the stimulation, cells
were scraped in 0.5 ml of MAP kinase lysis buffer [50 mM HEPES,
pH 7.5, 100 mM NaCl, 2 mM EDTA, 1 mM vanadate, 40 mM paranitrophenyl
phosphate, 1 µM pepstatin, 1 µg/ml leupeptin, 2 µg/ml aprotinin,
and 1% Nonidet P-40 (NP-40)]. MAP kinase in cell lysate
(20-30 µg) was immunoprecipitated using goat polyclonal anti-p42
MAP kinase antibody (0.4 µg) and protein A-Sepharose (50%). After
washing, the kinase activity of the immune complex was assayed with MBP
as a substrate in a reaction mixture containing 7.5 mM HEPES, pH 7.5, 10 mM magnesium acetate, 50 µM ATP, and 4 µCi
[32P]ATP in a total
volume of 40 µl (35). The reaction was performed at 30°C for 20 min, and the reaction was terminated by adding 20 µl of 3×
Laemmli sample buffer. Samples were heated at 80°C for 5 min and
resolved by SDS-PAGE (10%). The gel was stained with Coomassie blue,
dried, and exposed to X-ray film for 1-4 h. The MBP bands were
then cut out, and the radioactivity was measured by liquid
scintillation spectrometry. MAP kinase activity was expressed as
picomoles phosphate incorporated in MBP per milligram cell lysate protein.
Assay of PKC. Cell cultures were
prepared as described above. After incubation with LPC at appropriate
concentrations and time intervals, the cells were washed and disrupted
in 20 mM Tris · HCl, 0.5 mM EGTA, 0.25 mM sucrose, 50 µM digitonin, and 50 µg/ml leupeptin at pH 7.5. After
centrifugation at 13,000 g, the
membrane fraction was solubilized in a buffer containing 1.0% Triton
X-100, 20 mM Tris · HCl, 2 mM EDTA, 0.5 mM EGTA, 2 mM
phenylmethylsulfonyl fluoride (PMSF), and 5 µg/ml leupeptin. The PKC
protein from this membrane solution was precipitated with anti-PKC
antibody that had been conjugated to protein A-Sepharose (30 µl of
50% protein A-Sepharose, 0.5 µg anti-PKC antibody) by mixing for 2 h
at 4°C. The immune complex was resuspended in 15 µl of micellar
mixture prepared as described earlier (11), and 25 µl of the assay
buffer (10 mM Tris · HCl, pH 7.5, 5 mM
MgCl2, 50 mM
CaCl2, 5 µM ATP, 0.6 mg/ml
histone III, and 2.5 µCi
[
-32P]ATP) were
then added. The kinase reaction was carried out at 30°C for 10 min,
and the reaction was terminated by the addition of 25 µl of Laemmli
sample buffer. After being heated at 80°C for 5 min, the samples
were resolved on SDS-PAGE (10% gel). The gels were dried and exposed
to X-ray films for autoradiography. The degree of histone
phosphorylation, as a measure of PKC activity, was determined by
densitometric scanning of autoradiograms.
PKC activation was also assessed by determining the membrane
association of PKC isoforms (e.g.,
/
,
, and
) by Western blot analysis in control and LPC-treated cells. Mesangial cells (serum
starved for 24 h) were stimulated with LPC (10 and 25 µM) for
5-15 min. Cells were scraped in PBS and sonicated for 15 s. The
membrane fraction was isolated by centrifugation at 100,000 g for 30 min. The membrane pellet was
solubilized in a lysis buffer (25 mM HEPES, pH 7.8, 10 mM EGTA, 10 µg/ml leupeptin, 1 mM PMSF, 50 mM NaF, 0.5 M NaCl, 0.2 mM
Na3VO4,
1.5% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS), samples
were resolved on SDS-PAGE (12% gel), and proteins were transferred to
the nitrocellulose membrane. The membranes were probed with antibodies
specific for PKC isoforms.
Ras activation analysis. Ras
activation analysis was done by the method of Downward et al. (4) and
modified as described below. Cells were cultured in six-well plates as
described earlier. After 24 h of serum starvation, cells were incubated
for 3 h in phosphate-free DMEM containing 300 µCi of
[32P]orthophosphate.
Cells were then stimulated with LPC (10 µM) for various time periods.
The cells were then washed with Tris-buffered saline and scraped in 0.5 ml of Ras lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM
MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 9 µg/ml PMSF, 1% Triton X-100, 1 mg/ml BSA, and
10 mM benzamidine). The cell lysate was first mixed with an equal volume of a solution of 1 M NaCl, 0.1% SDS, and 1% sodium
deoxycholate and was then treated with 100 µl of BSA-coated charcoal
(4). After a brief mixing and centrifugation, the supernatant was
immunoprecipitated with anti-Ras antibody (0.5 µg, conjugated to
protein A-Sepharose) by mixing for 40 min at 4°C. The beads were
washed four times with Ras lysis buffer, and the guanine nucleotides
were then eluted with potassium phosphate buffer (1 M, pH 3.4) by
heating at 80°C for 5 min. GTP and GDP were resolved by TLC on
polyethyleneimine cellulose plates and were detected by autoradiography.
Phosphorylation of PLC
-1 assay.
After 24 h of serum starvation, cells were stimulated with LPC
(10-25 µM) for 5-20 min. Cells were washed and lysed with a
buffer containing 10 mM Tris · HCl, pH 7.6, 5 mM
EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM
Na3VO4,
and 1% Triton X-100. After a brief centrifugation, the cell lysate was
immunoprecipitated with anti-PLC
-1 conjugated with protein
A-Sepharose. The immune complex was mixed with Laemmli sample buffer,
heated at 80°C for 5 min. The sample was resolved on SDS-PAGE
(12.5% gel), transferred to nitrocellulose membranes, and
immunoblotted with anti-phosphotyrosine, and phosphorylated PLC
-1
was detected by enhanced chemiluminescent (ECL) reagents.
Assay of membrane tyrosine kinase
activity. Tyrosine kinase activity in mesangial cell
membranes was measured by an in vitro phosphorylation technique as
described in instructions for a commercially available assay kit (Life
Technologies, GIBCO-BRL, Grand Island, NY). Mesangial cell membranes
were prepared by centrifugation at 21,000 rpm for 20 min. Membrane
preparations were incubated on ice with 10 µM LPC for 30 min.
Tyrosine kinase activity of the activated membranes was determined in
the presence or in the absence of genistein (100 µM) using a
synthetic peptide [12-amino acid sequence surrounding tyrosine
phosphorylation site in pp60src,
specific for epidermal growth factor (EGF) receptor]
as a substrate and
[
-32P]ATP. The
tyrosine kinase activity was expressed as counts per minute (cpm),
phosphate incorporation, per microgram of membrane protein.
For cellular protein tyrosine phosphorylation studies, quiescent
mesangial cells were treated with LPC (25 µM) for 5-10 min, and
the cells were lysed. The cell lysate was immunoprecipitated with
anti-phosphotyrosine antibodies. The immunoprecipitates were incubated
with [
-32P]ATP in a
kinase assay buffer (50 mM HEPES, pH 7.5, 0.1 mM EDTA, 0.01% NP-40,
and 75 mM NaCl) for 20 min at 30°C. The samples were resolved on
7.5% SDS-PAGE, and phosphorylated proteins were detected by
autoradiography. In additional studies, the phosphorylation of Shc and
the activity of pp60src were
measured. For Shc phosphorylation studies, LPC-treated cells were lysed
and immunoprecipitated with anti-Shc antibodies. The immunoprecipitates
were resolved on SDS-PAGE, transferred to the membranes, and
immunoblotted with anti-phosphotyrosine antibodies, and the
phosphorylation was detected by the ECL method. For Src activity
measurement, LPC-treated cells were lysed and immunoprecipitated with
anti-Src antibodies, and the Src activity was determined by using
enolase as a substrate (8).
Statistical analysis. Results are
presented as representative studies or by displaying mean values ± SE for three to four separate experiments, each assayed in duplicate or
triplicate. A Student's t-test was
used to compare the means, and a P
value of <0.05 was considered significant.
 |
RESULTS |
The incubation of mesangial cells with LPC (10-25 µM) for 15 min
to 3 h, as used in this study, did not alter the viability, cell
number, or cellular protein content as assessed by the trypan blue
exclusion criterion and by measuring cell number and protein content,
respectively. The cell numbers in control and LPC-treated cells were as
follows (1 × 106/dish,
average of 2 determinations): 15 min incubation, control = 1.98, 10 µM LPC = 2.30, 25 µM LPC = 2.25; 3 h incubation,
control = 1.76, 10 µM LPC = 1.63, 25 µM LPC = 1.61. The
viability of cells in control and LPC-treated cells for 15 min to 3 h
was similar (95-98% viable, as assessed by trypan blue exclusion method).
Activation of PKC by LPC and inhibition by tyrosine
kinase inhibitors. One of the aims of the present
studies is to understand the mechanism by which LPC activates PKC. For
the assay of PKC, membrane-bound PKC was purified by immnoprecipitation
with anti-PKC (reacting with
,
, and
isoforms), and the
kinase assay was performed using histone III as a substrate. We found
this method of assaying PKC to be more reproducible, as we encountered
high background radioactivity in the phosphocellulose disc method used for the separation of the substrate. The activity of PKC in the membrane fraction, obtained from LPC-treated cells, increased by
~2.5- to 3.5-fold during 5-10 min of stimulation (Fig.
1A). Preincubation of cells with the PTK inhibitor herbimycin completely suppressed the activation of PKC by LPC. The quantitative densitometric analysis (as arbitrary units, average from 2 experiments) of the autoradiograms for PKC activity showed the following data: control = 1.02; 10 µM LPC, 5 min = 2.39; 25 µM LPC, 5 min = 3.09; 10 µM LPC, 10 min = 3.32; 25 µM LPC, 10 min = 3.42. The corresponding quantitative data for LPC-induced PKC activity in the presence of
herbimycin was as follows: control = 1.02; 10 µM LPC + herbimycin, 5 min = 0.85; 25 µM LPC + herbimycin, 5 min = 1.01; 10 µM LPC + herbimycin, 10 min = 1.13; 25 µM LPC + herbimycin, 10 min = 1.47. PMA
markedly activated PKC activity, and this activation was not inhibited
by herbimycin (quantitative arbitrary units: control = 1.02; PMA = 6.4;
PMA + herbimycin = 6.2). Similarly, another tyrosine kinase inhibitor,
genistein (which is structurally different from herbimycin), also
suppressed the activation of PKC by LPC (quantitative data: control = 1.77; 10 µM LPC, 10 min = 2.95; 10 µM LPC + genistein, 10 min = 1.81; Fig. 1B). Additionally, pretreatment of cells with genistein did not significantly alter PMA-induced PKC activity (Fig. 1B).


View larger version (51K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of lysophosphatidylcholine (LPC) on protein
kinase C (PKC) activity: role of herbimycin and genistein (Gen) on the
activation of PKC by LPC. A: mesangial
cells were stimulated with LPC (10 or 25 µM) for 10 min. In
additional experiments, cells were preincubated with herbimycin
(5 µM) or vehicle (DMSO) for 1 h before stimulation of
cells with LPC (10 or 25 µM) or PMA (100 nM) for 10 min. Cells were
processed for the determination of PKC activity as described in
MATERIALS AND METHODS. C, control; Inc. time, incubation
time; PMA, phorbol 12-myristate 13-acetate.
B: mesangial cells (serum starved for
24) were incubated in the absence or presence of genistein (100 µM)
for 90 min. Cells were then treated with 10 or 25 µM LPC or PMA (100 nM) for 10 min, and cells were then processed for the determination of
PKC activity as described in MATERIALS AND METHODS.
|
|
In other studies, Western blot analysis of membrane association of PKC
isoforms indicated that LPC induced the membrane association of
/
and
isoforms of PKC (by 2- to 3-fold compared with controls; Fig.
2A), but
LPC had no effect on the membrane association of PKC-
(Fig.
2B). The PKC antibody used in Fig.
2A recognizes all three isoforms of
PKC (
,
, and
). Because the molecular masses for
and
PKC isoforms are very similar (~82 kDa), only a single band was noted
for both
and
isoforms, whereas the
isoform was detected as
a separate band of molecular mass of ~78 kDa.


View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of LPC on membrane association of PKC- /
and - isoforms (A) and PKC-
(B). Mesangial cells (serum starved
for 24 h) were stimulated with LPC (10 and 25 µM) for 5-15 min.
Cellular membrane fractions (100,000 g) were resolved on SDS-PAGE, and
proteins were transferred to the nitrocellulose membrane. Membranes
were probed with antibodies specific for either / / or isoforms of PKC. Membrane association of / , , or PKC
isoforms was detected by the enhanced chemiluminescent (ECL) method.
PKC antibody used in A recognizes all
3 isoforms of PKC ( , , and ; PKC-MC5, cat no. sc-80; Santa
Cruz Biotechnology). Because the molecular masses for and PKC
isoforms are very similar (~82 kDa), only single band was noted for
both and isoforms, whereas the isoform was detected as a
separate band of molecular mass ~78 kDa.
|
|
Activation of MAP kinase by LPC: Its partial
dependency on the activity of PKC. Activated PKC can
stimulate the activity of MAP kinases in the cell. In the present
studies, we examined the effect of LPC on the p42 MAP kinase isoform
because preliminary studies indicated that the p42 isoform is more
predominant in the cell type used. LPC activated MAP kinase in a time-
and dose-dependent manner (Fig. 3).
Significant activation of MAP kinase occurred as early as 5 min with
both 5 and 10 µM doses. With the use of 5 and 10 µM doses of LPC,
peak activities were observed at 15 min, and the activity returned to
basal levels between 30 min and 3 h. [Control values
were pooled as they did not vary significantly at different time points
(Fig. 3).] On the other hand, with a 25 µM dose, maximum
activation of MAP kinase (~3-fold) occurred at 10 min, and the
activation persisted for a longer period. At 3 h of stimulation with 25 µM of LPC, the MAP kinase activity was about twofold higher than that
of controls (Fig. 3). LPC did not affect MAP kinase protein content as
measured by Western blot analysis. The quantitative arbitrary data
(measured by densitometric scanning of Western blots) for MAP kinase
content were as follows: control = 65.8; 25 µM LPC, 10 min = 65.2; 25 µM LPC, 3 h = 62.2 (data are average of 2 experiments). In contrast,
phosphatidylcholine (parent molecule for the formation of LPC) did not
induce MAP kinase activity (data not shown). Previous studies reported
by this and other laboratories have shown similar morphological and functional characteristics between stably transformed murine mesangial cells and the primary cultures of mesangial cells (24, 30, 32). In the
present study, using primary cultures of rat glomerular cells, we have
found that LPC at 10 and 25 µM stimulated the MAP kinase activity by
125 and 160%, respectively.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of LPC on the activity of p42 mitogen-activated
protein kinase (MAPK): time and dose response. Mesangial cells (serum
starved for 24 h) at ~80% confluence in 30-mm dishes were incubated
with various concentrations of LPC for selected time intervals as
indicated. Cells were then washed with cold Hanks' balanced salt
solution and were extracted with MAPK assay buffer. The p42 MAPK
activity in the extracts was determined as described in MATERIALS
AND METHODS.
|
|
To test the PKC dependency of LPC-mediated MAP kinase activation, in
the first method, the cells were treated for 24 h under serum-free
conditions with 10 nM PMA to deplete the cellular PKC. PMA (10 nM)
stimulated MAP kinase activity by about fourfold in 15 min (Table
1). However, when the cells were
preincubated for 24 h with 10 nM PMA to deplete PKC, further addition
of PMA did not activate MAP kinase, confirming the degree of PKC
depletion (Table 1). On the other hand, depletion of cellular PKC did
not completely suppress the activation of MAP kinase by LPC. In
PKC-depleted cells, LPC at the doses of 10 and 25 µM induced MAP
kinase activity by 38 and 89%, respectively (Table 1). Under the same
experimental conditions, in control cells (without PKC depletion), LPC
at 10 and 25 µM doses activated MAP kinase by 135 and 138%,
respectively (Table 1). In the second method, the PKC inhibitor GFX was
used to inhibit PKC activity (37). Cells were incubated with GFX (5 µM) for 30 min before the addition of 10 or 25 µM LPC, and the
activity of MAP kinase was determined at 15 min of incubation. As shown
in Table 2, GFX suppressed MAP kinase
activity stimulated by LPC, at 10 and 25 µM, only by 31 and 63%,
respectively.
View this table:
[in this window]
[in a new window]
|
Table 1.
Effect of depletion of cellular PKC (by incubation with PMA, 24 h)
on the activation of p42 MAP kinase by LPC
|
|
Effect of PTX on the activation of MAP kinase by LPC
and other lysophospholipids. We also studied the effect
of PTX on the activation of MAP kinase by LPC and other
lysophospholipids to understand the role played by the
Gi proteins. PTX covalently modifies and inactivates Gi
proteins (7). In addition to LPC, LPI and LPA (but not LPE) activated
MAP kinase (Table 3). In these studies,
cells were incubated with PTX (500 ng/ml) for 6 h before stimulation
with LPC or other lysophospholipids (10 µM, 15 min). Thrombin, a
known agonist for G protein-mediated activation of MAP kinase, was also
included as a positive control. Preincubation of cells with PTX did not
affect the activation of MAP kinase by either LPC or LPI (Table 3). The
induction of MAP kinase activation by LPA and thrombin was
significantly suppressed by PTX (Table 3).
Effect of genistein on the activation of MAP kinase by
LPC: Involvement of PTK. Because
Gi protein-mediated pathways did
not appear to play a role in LPC stimulation of MAP kinase in mesangial cells and because PKC inhibition did not completely block LPC-induced MAP kinase, we next proceeded to examine if the inhibition of tyrosine
kinases would modulate the LPC-induced MAP kinase activity. Cells were
incubated with 50-100 µM genistein for 150 min before the
addition of LPC (10 or 25 µM), and the MAP kinase activity was
determined after 10 or 15 min of stimulation. Genistein nearly completely blocked LPC-induced MAP kinase activity, and genistein had
no significant effect on basal MAP kinase activity (Fig.
4). The MAP kinase activities (pmol/mg
protein), from three experiments, in LPC-treated cells (15 min) in the
absence or presence of genistein (100 µM) were as follows: control = 17.6 ± 2.5; 10 µM LPC = 37.8 ± 0.6; 10 µM LPC + genistein = 20.9 ± 6.1; 25 µM LPC = 47.8 ± 5.8; and 25 µM LPC + genistein = 21.8 ± 5.1. Similar inhibition of MAP kinase activity
by genistein was also observed in cells stimulated with LPC (10 and 25 µM) for 10 min (data not shown). Parallel experiments using 50 µM
of genistein also showed marked inhibition of LPC-induced MAP kinase
(data not shown). Incubation of cells with genistein at similar
concentrations and incubation time did not alter either the morphology
or viability of cells (data not shown). Similarly, tyrphostin B46
(50-100 µM, another specific PTK inhibitor) inhibited
LPC-induced mesangial cell MAP kinase activity (data not shown).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of genistein on the activation of p42 MAPK by
LPC in mesangial cells. Cells at nearly 80% confluence in 30-mm dishes
were serum starved for 24 h and were then preincubated in the absence
or presence of 100 µM genistein (stock dissolved in DMSO) for 3 h.
Cells were then stimulated with LPC (10 or 25 µM) for 10 or 15 min.
Extraction of the cells and assay of p42 MAPK activity were done as
described in MATERIALS AND METHODS. MBP, myelin basic
protein.
|
|
Effect of LPC on PTK activity. Because
LPC-mediated activation of MAP kinase was found to be dependent on PKC
and PTK, additional experiments were performed to examine the direct
effect of LPC on mesangial cell PTK activity. In these experiments,
plasma membranes (isolated from control cells) were incubated with 10 µM LPC on ice, and the membrane tyrosine kinase activity was
subsequently determined in these activated membranes. LPC at 10 µM
concentrations significantly increased membrane PTK activity by 20.3%
(PTK activity, as cpm/µg membrane protein, in control = 26,586 ± 590 and in 10 µM LPC = 32,183 ± 869, data are means ± SE of 3 separate determinations, statistically significant at
P < 0.05, control vs. LPC
treatment). Furthermore, addition of genistein completely inhibited the
stimulation of PTK activity by LPC (data not shown). Additionally, the
effect of LPC on the tyrosine phosphorylation of cellular proteins
(membrane/cytoplasmic) was assessed. The results indicated that LPC
induced a modest increase (40-60% compared with control) in the
phosphorylation of mainly two proteins in the molecular mass range of
~80 and 140 kDa (Fig. 5). Additional
studies indicated that LPC had no effect on the activation of Shc and
Src. The quantitative arbitrary data (as measured by densitometric
scanning of Western blots) for the activation of Shc were as follows:
control = 38.3; 25 µM LPC, 5 min = 40.2; 25 µM LPC, 30 min = 39.6 (data are average of 2 experiments). Similarly, the quantitative
arbitrary data for the activation of Src were as follows: control = 51.0; 25 µM LPC, 5 min = 49.9; 25 µM LPC, 30 min = 44.5 (data are
average of 2 experiments).

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of LPC on cellular protein tyrosine
phosphorylation profile. Quiescent mesangial cells were treated with
LPC (25 µM) for 5-10 min, and the cells were lysed. Cell lysate
was immunoprecipitated with anti-phosphotyrosine antibodies.
Immunoprecipitates were incubated with
[ -32P]ATP in a
kinase assay buffer for 20 min at 30°C. Samples were resolved on
7.5% SDS-PAGE, and phosphorylated proteins were detected by
autoradiography.
|
|
Effect of LPC on PLC-
1
phosphorylation. In the cell, DAG is one of the
principal activators of PKC. Phospholipases are the enzymes that
produce DAG by the hydrolysis of phospholipids during cell signaling.
Among various phospholipases, only PLC
-1 is known to be activated by
membrane tyrosine kinases and to be involved in the phosphoinositide
turnover and the generation of DAG. The observation that the activation
of PKC and MAP kinase by LPC was dependent on tyrosine kinases prompted
us to explore the possibility that the activation of PLC-
1 was
involved in LPC-mediated effects. Cells were incubated with 10 µM LPC
for 5-20 min and were processed for the assessment of PLC
-1
phosphorylation. As shown in Fig. 6, the
stimulation of mesangial cells with LPC markedly, but transiently (within 5-10 min), stimulated the phosphorylation of PLC
-1. The phosphorylation of PLC
-1 induced by LPC returned to basal levels in
20 min of stimulation (Fig. 6). The quantitative densitometric data for
the phosphorylation of PLC
-1 were as follows: control = 0.46; 10 µM LPC, 5 min = 1.52; 10 µM LPC, 10 min = 1.24; 10 µM LPC, 20 min = 0.20.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of LPC on phospholipase C -1 (PLC -1)
phosphorylation. Mesangial cells (serum starved for 24 h) were
stimulated with LPC (10 µM) for 5-20 min, and the cells were
lysed. An aliquot of cell lysate was immunoprecipitated with
anti-PLC -1 conjugated with protein A-Sepharose. Immune complex was
mixed with Laemmli sample buffer, heated at 80°C for 5 min. Sample
was resolved on SDS-PAGE (12.5% gel), transferred to nitrocellulose
membranes, and immunoblotted with anti-phosphotyrosine, and
phosphorylated PLC -1 was detected by ECL reagents.
|
|
Activation of Ras by LPC. In the
present studies, we also investigated the effect of LPC on Ras because
Ras is an important member of the tyrosine kinase signaling cascade,
and it lies outside the cross talk between the PKC and the growth
factor signaling cascade. Serum-starved cells were first incubated with
[32P]orthophosphate
for 3 h. LPC (10 µM) was then added, and the cells were processed
after 2, 5, 10, or 60 min of incubation. FBS (5%) was used as a
positive control. As shown in Fig. 7, LPC markedly increased GTP binding to Ras within 2 min, and the effect lasted for nearly 1 h. The ratios of GTP to GDP associated with Ras
were as follows: control = 0.032; FBS = 0.09; 10 µM LPC, 2 min = 0.124; 10 µM LPC, 5 min = 0.10; 10 µM LPC, 10 min = 0.08; 10 µM LPC, 1 h = 0.05.

View larger version (73K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of LPC on the activation of Ras. Mesangial
cells were grown in 30-mm dishes to 80% confluence. After 24 h of
serum starvation, cells were labeled with
[32P]orthophosphate
(50 µCi · 2 ml 1 · dish 1)
for 3 h. After this labeling period, cells were stimulated either with
FBS (5%) for 2 min or with 10 µM LPC for 2 min to 2 h. Cellular Ras
was then immunoprecipitated, and the guanine nucleotides associated
with Ras were analyzed by TLC as described in MATERIALS AND
METHODS.
|
|
 |
DISCUSSION |
Mesangial cells, specialized vascular pericytes within the glomerulus,
perform a fundamental role in the homeostasis of glomerular function
and are influenced by various circulating chemical modulators (3).
Recently, we have reported that the activation of mesangial cells with
circulating LDL and oxidatively modified LDL (with greater potency)
stimulated the expression of various cytokines and growth factors
involved in monocyte infiltration and proliferation of intrinsic
resident cells (10, 15, 16, 30). Because increased levels
of LPC is one of the major characteristics of ox-LDL, the atherogenic
potential of ox-LDL to modulate cellular responses has been attributed
to the presence of LPC (2). Presently, PKC activation is believed to be
the principal mediator in various cellular responses stimulated by LPC
(18, 29, 36). However, PKC activation alone may not explain all of the
diverse actions of LPC, because, for example, the stimulation of
endothelial cell ICAM-1 by LPC is insensitive to PKC inhibition (21).
In many cell types, active forms of PKC are capable of activating MAP kinases by a pathway that involves Raf (12, 22, 40). As reported by us
in our recent preliminary communication (2), LPC indeed markedly
activated MAP kinases in murine mesangial cells. LPC was also shown to
activate c-Jun NH2-terminal kinase (23). Our studies were originally aimed at identifying the principal early signaling events stimulated by LPC in mesangial cells. To understand the upstream intracellular signaling events stimulated by
LPC, we assessed the activities of either MAP kinase or both PKC and
MAP kinase under various experimental conditions. LPC activated both
PKC and MAP kinase. Interestingly, the activation of both PKC and MAP
kinase by LPC was inhibited by the tyrosine kinase inhibitors. The
effect of these inhibitors (herbimycin and genistein) on PKC is
noteworthy, because the activation of PKC is not mediated through its
tyrosine phosphorylated state, and it is less likely that these
inhibitors directly acted on PKC. It is possible that the upstream
membrane/cytoplasmic tyrosine kinases may be involved in LPC-induced
PKC activation. In this regard, LPC induced modestly the tyrosine
phosphorylation of cellular proteins of the molecular mass range of 80 and 140 kDa. Based on the molecular mass range, we reasoned that these
proteins may represent intracellular signaling molecules, including
PLC
-1, pp60src, Shc, etc.
However, we were unable to detect measurable activation of Shc or Src
by LPC.
There are two classical pathways by which various agonists stimulate
distinct forms of phospholipases to generate DAG, which is absolutely
required for the activation of PKC. The first one is dependent on
serpentine receptor- and Gi
protein-mediated activation of PLC-
(5). The inability of PTX to
inhibit LPC-induced MAP kinase in our experiments apparently excludes
this pathway as a participant in the activation of cells by LPC. Unlike
LPC, LPA-mediated MAP kinase activation was suppressed by PTX in
mesangial cells. LPA has been shown to stimulate MAP kinase through
PTX-sensitive G protein-coupled pathways in other cell types (13). The
second route is dependent on PTKs. For example, the activation of PKC by platelet-derived growth factor in many tissues involves
phosphorylation and activation of PLC
-1 (42). The induction of DAG
synthesis by EGF in A431 cells (38) or in Swiss 3T3 cells (41) and by LPI in thyroid cells (6) is also dependent on the phosphorylation and
stimulation of PLC
-1 by receptor tyrosine kinases. The generation of
DAG and the activation of PKC have been shown to be insensitive to PTX
in all of these instances. Based on these earlier reports and our
observations on the effects of PTK inhibitors on PKC activity, we
conducted additional experiments to find out if LPC activated membrane
tyrosine kinases and PLC
-1. LPC activated total membrane tyrosine
kinase activity as assayed using a synthetic peptide substrate.
Furthermore, LPC markedly, but transiently, activated PLC
-1, and the
time course of activation was consistent with the premise that PLC
-1
was involved in LPC-induced PKC and MAP kinase activation.
Our data indicate that both PKC-dependent and PKC-independent
mechanisms contributed to the activation of MAP kinase by LPC. First,
MAP kinase activity, especially in the early phases of activation,
showed clear dependency on the availability of PKC, but the depletion
or inhibition of PKC did not completely suppress the maximum activation
of MAP kinase at the later period of 15 min. We also found that LPC did
not activate PKC-
, suggesting that PMA-insensitive isoforms of PKC
are not involved in LPC-mediated MAP kinase activation. Second,
tyrosine kinase inhibitors nearly completely inhibited the activation
of MAP kinase by LPC. The concentrations of PTK inhibitors used in
these experiments were not known to inhibit the MAP
kinase-phosphorylating dual specific kinase, namely MAP kinase kinase
(40). Based on the conclusion that both PKC-dependent and
PKC-independent pathways contributed to the activation of MAP kinase,
we examined the effect of LPC on Ras. Ras is a G protein and is a key
member of the growth factor signaling cascade that is upstream of the
cross talk between PKC and the growth factor signaling cascades. LPC
markedly stimulated Ras-GTP binding. It is also interesting because, in
addition to activating the downstream members of the growth factor
signaling cascade, Ras is also known to directly modulate gene
expression (39).
As summarized in the proposed model (Fig.
8), our results suggest that the activation
of a membrane tyrosine kinase is the key early event in LPC-induced
cellular signaling responses. Activation of Ras and PLC
-1 by LPC and
the inhibition of the activation of PKC and MAP kinase by tyrosine
kinase inhibitors support this view. However, additional yet
unidentified signaling molecule(s) may be involved in LPC-mediated
cellular responses (Fig. 8). Because PKC, MAP kinases, and Ras are
involved in modulating nuclear events associated with gene expression,
our data may provide a more logical support for the earlier findings
that LPC stimulates diverse responses in various cell types.
Furthermore, because glomerular mesangial cells and vascular smooth
muscle cells share many morphological and functional characteristics,
the results presented in this study may shed light on the roles played
by LPC and LDLs in the pathobiology of renovascular and vascular
diseases, such as glomerulosclerosis and
atherosclerosis.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 8.
Proposed model for the involvement of tyrosine kinase
pathway in LPC-mediated activation of PKC and mitogen-activated protein
(MAP) kinase. Data suggest that the activation of membrane/cytoplasmic
tyrosine kinase(s) plays a key role in LPC-induced mitogenic signaling
responses. The specific upstream signaling events that modulate
LPC-mediated activation of PLC -1 and Ras, associated with PKC and
MAPK activation, are yet to be identified. DAG, diacylglycerol; MEK,
MAP kinase kinase.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. Rama Pai for technical assistance in tyrosine kinase assay.
 |
FOOTNOTES |
Deceased 21 June 1997.
This work was supported by a Merit Review from the Department of
Veterans Affairs.
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: V. S. Kamanna,
Nephrology Research Laboratories (151), Dept. of Veterans Affairs
Medical Center, 5901 East Seventh St., Long Beach, CA 90822 (E-mail:
kamanna.vaijinath_s.{at}long_beach.va.gov).
Received 7 August 1998; accepted in final form 7 May 1999.
 |
REFERENCES |
1.
Akita, H.,
M. H. Creer,
K. A. Yamada,
B. E. Sobel,
and
P. B. Corr.
Electrophysiologic effects of intracellular lysophosphoglycerides and their accumulation in cardiac lymph with myocardial ischemia in dogs.
J. Clin. Invest.
78:
271-280,
1986[Medline].
2.
Bassa, B. V.,
D. D. Roh,
M. A. Kirschenbaum,
and
V. S. Kamanna.
Aherogenic lipoproteins stimulate mesangial cell p42 mitogen-activated protein kinase.
J. Am. Soc. Nephrol.
9:
488-496,
1998[Abstract].
3.
Brenner, B. M.,
R. Zatz,
and
I. Ichikawa.
The renal circulation. In
In: The Kidney (3rd ed.). Philadelphia, PA: Saunders, 1986, p. 107.
4.
Downward, J.,
J. D. Graves,
P. H. Warne,
S. Rayter,
and
D. A. Cantrell.
Stimulation of p21ras upon T-cell activation.
Nature
346:
719-723,
1990[Medline].
5.
Exton, J. H.
Phosphoinositide phospholipases and G proteins in hormone action.
Annu. Rev. Physiol.
56:
349-69,
1994[Medline].
6.
Falasca, M.,
M. G. Silletta,
A. Carvelli,
A. L. D. Francesco,
V. Ramakrishna,
and
D. Corda.
Signalling pathways involved in the mitogenic actions of lysophosphatidylinositol.
Oncogene
10:
2113-2124,
1995[Medline].
7.
Gilman, A. G.
G proteins: transducers of receptor-mediated signals.
Annu. Rev. Biochem.
56:
615-649,
1987[Medline].
8.
Gould, K. L.,
and
T. Hunter.
Platelet-derived growth factor induces multiple phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity.
Mol. Cell. Biol.
8:
3345-3356,
1988[Medline].
9.
Ha, H.,
R. Pai,
V. S. Kamanna,
and
M. A. Kirschenbaum.
Oxidatively modified (ox)-LDL stimulates monocyte (M
) adhesion to glomerular endothelial cells (EC) mediated by ICAM-1 (Abstract).
J. Am. Soc. Nephrol.
6:
830A,
1995.
10.
Ha, H.,
D. D. Roh,
M. A. Kirschenbaum,
and
V. S. Kamanna.
Atherogenic lipoproteins enhance mesangial cell expression of platelet-derived growth factor: role of protein tyrosine kinase and cyclic AMP-dependent protein kinase A.
J. Lab. Clin. Med.
131:
456-465,
1998[Medline].
11.
Hannun, Y. A.,
C. R. Loomis,
and
R. M. Bell.
Activation of protein kinase C by triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine.
J. Biol. Chem.
260:
10039-10043,
1998[Abstract/Free Full Text].
12.
Howe, C. R.,
S. J. Leevers,
N. Gomez,
S. Nakielny,
P. Cohen,
and
C. J. Marshall.
Activation of the MAP kinase pathway by the protein kinase raf.
Cell
71:
335-342,
1992[Medline].
13.
Howe, L. R.,
and
C. J. Marshall.
Lysophosphatidic acid stimulates mitogen-activated protein kinase activation via a G-protein-coupled pathway requiring p21ras and P74raf-1.
J. Biol. Chem.
268:
20717-20720,
1993[Abstract/Free Full Text].
14.
Joles, J. A.,
N. Willekes-Koolschijn,
L. M. Scheek,
H. A. Koomans,
T. J. Rabelink,
and
A. V. Tol.
Lipoprotein phospholipid composition and LCAT activity in nephrotic and analbuminemic rats.
Kidney Int.
46:
97-104,
1994[Medline].
15.
Kamanna, V. S.,
B. V. Bassa,
and
M. A. Kirschenbaum.
Atherogenic lipoproteins and human disease: extending concepts beyond the heart to the kidney.
Curr. Opin. Nephrol. Hypertens.
6:
205-211,
1997[Medline].
16.
Kamanna, V. S.,
R. Pai,
D. D. Roh,
and
M. A. Kirschenbaum.
Oxidative modification of low-density lipoprotein enhances murine mesangial cell cytokines associated with monocyte migration, differentiation and proliferation.
Lab. Invest.
74:
1067-1079,
1996[Medline].
17.
Kugiyama, K.,
S. A. Kerns,
J. D. Morrisett,
R. Roberts,
and
P. D. Henry.
Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins.
Nature
344:
160-162,
1990[Medline].
18.
Kugiyama, K.,
M. Ohgushi,
S. Sugiyama,
T. Murohara,
K. Fukunaga,
E. Miyamoto,
and
H. Yasue.
Lysophosphatidylcholine inhibits surface receptor-mediated intracellular signals in endothelial cells by a pathway involving protein kinase C activation.
Circ. Res.
71:
1422-1428,
1992[Abstract].
19.
Kume, N.,
M. I. Cybulsky,
and
M. A. Gimbrone, Jr.
Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells.
J. Clin. Invest.
90:
1138-1144,
1992[Medline].
20.
Kume, N.,
and
M. A. Gimbrone, Jr.
Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human vascular endothelial cells.
J. Clin. Invest.
93:
907-911,
1994[Medline].
21.
Kume, N.,
H. Ochi,
E. Nishi,
M. A. Gimbrone, Jr.,
and
T. Kita.
Involvement of protein kinase C-independent mechanisms in endothelial ICAM-1 up-regulation by lysophosphatidylcholine.
Ann. NY Acad. Sci.
748:
54-542,
1995.
22.
Kyriakis, J. M.,
H. App,
X-F. Zhang,
P. Banerjee,
D. L. Brautigan,
U. R. Rapp,
and
J. Avruch.
Raf-1 activates MAP kinase kinase.
Nature
358:
417-421,
1992[Medline].
23.
Lang, X.,
S. Gibson,
C. Bast, Jr.,
and
G. B. Mills.
Lysophosphatidylcholine stimulates activator protein 1 and the c-Jun N-terminal kinase activity.
J. Biol. Chem.
272:
13683-13689,
1997[Abstract/Free Full Text].
24.
Mackay, K.,
L. J. Striker,
and
S. Elliot.
Glomerular epithelial, mesangial and endothelial cell lines from transgenic mice.
Kidney Int.
33:
677-684,
1988[Medline].
25.
Marshall, M. S.
Ras target proteins in eukaryotic cells.
FASEB J.
9:
1311-1318,
1995[Abstract/Free Full Text].
26.
McHowat, J.,
and
P. B. Corr.
Thrombin-induced release of lysophosphatidylcholine from endothelial cells.
J. Biol. Chem.
268:
15605-15610,
1993[Abstract/Free Full Text].
27.
Nakamura, S.,
and
Y. Nishizuka.
Lipid mediators of protein kinase C activation for the intracellular signaling network.
J. Biochem. (Tokyo)
115:
1029-1034,
1994[Abstract].
28.
Nakano, T.,
E. W. Raines,
J. A. Abraham,
M. Klagsbrun,
and
R. Ross.
Lysophosphatidylcholine upregulates the level of heparin-binding epidermal growth factor-like growth factor mRNA in human monocytes.
Proc. Natl. Acad. Sci. USA
91:
1069-1073,
1994[Abstract].
29.
Ohgushi, M.,
K. Kugiyama,
K. Fukunaga,
T. Murohara,
S. Sugiyama,
E. Miyamoto,
and
H. Yasue.
Protein kinase C inhibitors prevent impairment of endothelium-dependent relaxation by oxidatively modified LDL.
Arterioscler. Thromb.
13:
1525-1532,
1993[Abstract].
30.
Pai, R.,
M. A. Kirschenbaum,
and
V. S. Kamamma.
Low-density lipoprotein stimulates the expression of macrophage colony-stimulating factor in murine glomerular mesangial cells.
Kidney Int.
48:
1254-1262,
1995[Medline].
31.
Parthasarathy, S.,
and
J. Barnett.
Phospholipase A2 activity of low density lipoprotein: evidence for an intrinsic phospholipase A2 activity of apoprotein B-100.
Proc. Natl. Acad. Sci. USA
87:
9741-9745,
1990[Abstract].
32.
Satriano, J. A.,
M. Shuldiner,
K. Hora,
Y. Xing,
Z. Shan,
and
D. Schlondorff.
Oxygen radicals as second messengers for expression of the monocyte chemoattractant protein JE/ MCP-1 and the monocyte colony stimulating factgor, CSF-1, in response to tumor necrosis factor-
and immunoglobulin G.
J. Clin. Invest.
92:
1564-1571,
1993[Medline].
33.
Seger, R.,
and
E. G. Krebs.
The MAPK signaling cascade.
FASEB J.
9:
726-735,
1995[Abstract/Free Full Text].
34.
Stroes, E. S. G.,
J. A. Joles,
P. C. Chang,
H. A. Koomans,
and
T. J. Rabelink.
Impaired endothelial function in patients with nephrotic range proteinuria.
Kidney Int.
48:
544-550,
1995[Medline].
35.
Sturgill, T. W.,
L. Bryan Ray,
N. G. Anderson,
and
A. K. Erickson.
Purification of mitogen-activated protein kinase from epidermal growth factor-treated 3T3-L1 fibroblasts.
Methods Enzymol.
200:
342-351,
1991[Medline].
36.
Sugiyama, S.,
K. Kugiyama,
M. Ohgushi,
K. Fujimoto,
and
Y. Hirofumi.
Lysophosphatidylcholine in oxidized low-density lipoprotein increases endothelial susceptibility to polymorphonuclear leukocyte-induced endothelial dysfunction in porcine coronary arteries.
Circ. Res.
74:
565-575,
1994[Abstract].
37.
Toullec, D.,
P. Pianetti,
H. Coste,
P. Bellevergue,
T. Grand-Perret,
M. Ajakanes,
V. Baudet,
P. Boissin,
E. Boursier,
F. Loriolle,
L. Duhamel,
D. Charon,
and
J. Kirilovsky.
The bisindolylmaleimide GF 109203X is a potent, and selective inhibitor of protein kinase C.
J. Biol. Chem.
266:
15771-15781,
1991[Abstract/Free Full Text].
38.
Wagk, M. I.,
S. Nishibi,
P. G. Suh,
S. G. Rhee,
and
G. Carpenter.
Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalization and extracelluar calcium.
Proc. Natl. Acad. Sci. USA
86:
1568-1572,
1989[Abstract].
39.
White, M. A.,
C. Nicolette,
A. Minden,
A. Polverino,
L. V. Aelst,
M. Karin,
and
M. H. Wigler.
Multiple Ras functions can contribute to mammalian cell transformation.
Cell
80:
533-541,
1995[Medline].
40.
Winitz, S.,
M. Russel,
N. X. Qian,
A. Gardner,
L. Dwyer,
and
G. L. Johnson.
Involvement of Ras and Raf in the Gi-coupled acetylcholine muscarinic m2 receptor activation of mitogen-activated protein (MAP) kinase kinase and MAP kinase.
J. Biol. Chem.
268:
19196-19199,
1993[Abstract/Free Full Text].
41.
Yeo, E. J.,
and
J. H. Exton.
Stimulation of phospholipase D by epidermal growth factor requires protein kinase C activation in swiss 3T3 cells.
J. Biol. Chem.
270:
3980-3988,
1995[Abstract/Free Full Text].
42.
Yeo, E.,
A. Kazlauskas,
and
J. H. Exton.
Activation of phospholipase C-
is necessary for stimulation of phospholipase D by platelet-derived growth factor.
J. Biol. Chem.
269:
27823-27826,
1994[Abstract/Free Full Text].
43.
Zembowicz, A.,
S. L. Jones,
and
K. K. Wu.
Induction of cyclooxygenase-2 in human umbilical vein endothelial cells by lysophosphatidylcholine.
J. Clin. Invest.
96:
1688-1692,
1995[Medline].
44.
Zembowicz, A.,
J.-L. Tang,
and
K. K. Wu.
Transcriptional induction of endothelial nitric oxide synthase type III by lysophosphatidylcholine.
J. Biol. Chem.
270:
17006-17010,
1995[Abstract/Free Full Text].
Am J Physiol Renal Physiol 277(3):F328-F337
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society