(Received for publication, June 19, 1995; and in revised form, December 29, 1995)
From the
Previously, our laboratory has shown that lactosylceramide
(LacCer) can serve as a mitogenic agent in the proliferation of aortic
smooth muscle cells ``a hallmark in the pathogenesis of
atherosclerosis'' (Chatterjee, S.(1991) Biochem. Biophys. Res.
Commun. 181, 554-561). Here we report a novel aspect of
LacCer-mediated signal transduction. We demonstrate that LacCer (10
µM) can stimulate the phosphorylation of mitogen-activated
protein (MAP) kinase p44 to phosphorylated p44
in aortic smooth muscle cells from rabbit or human origin.
Western immunoblot assays and direct measurement of activity in
immunoprecipitated MAP kinase revealed that within 5 min of incubation
of cells with LacCer there was a 3.5-fold increase in the activity of
p44
. This continued up to 10 min of incubation;
thereafter, the MAP kinase activity decreased in these cells.
Phosphoamino acid analysis revealed that the tyrosine and threonine
moieties of p44
was phosphorylated by LacCer. Incubation
of cells with ceramide and glucosylceramide did not significantly
stimulate p44
activity. Preincubation with tyrphostin
(20 µM; a potent and specific inhibitor of tyrosine
kinase) markedly inhibited the LacCer mediated stimulation in
p44
activity. Next we investigated the upstream and
downstream parameters in MAP kinase signaling pathways. We found that
lactosylceramide stimulated (7-fold) the loading of GTP on Ras.
Concomitantly, LacCer stimulated the phosphorylation of MAP kinase
kinases (MEK) and Raf within 2.5 min. Lactosylceramide specifically
induced c-fos mRNA expression (3-fold) in these cells as
compared to control. In summary, one of the biochemical mechanisms in
LacCer mediated induction in the proliferation of aortic smooth muscle
cells may involve Ras-GTP loading, activation of the kinase cascade
(MEK, Raf, p44
), and c-fos expression.
Glycosphingolipids are minor, yet integral, constituents of cell
membranes(1) . Glycosphingolipids and its metabolic products
have been recently shown to play critical roles as bioregulators of a
variety of processes such as cell
proliferation(2, 3) , cell mobility(4) , and
programmed cell death (apoptosis)(5) . Previously a
ganglioside, GM, (
)was shown to inhibit the
phosphorylation of epidermal growth factor. That may in part,
contribute to the inhibition of cell proliferation by this compound (3) . We found that lactosylceramide (LacCer) exhibited a time
and concentration-dependent proliferation of aortic smooth muscle
cells(2) . Since proliferation of smooth muscle cells is
considered a hallmark in the pathogenesis in atherosclerosis, we
measured the level of LacCer and other glycosphingolipids in human
subjects who had this disease. We found that the level of
glucosylceramide (GlcCer) and LacCer were markedly elevated in the
plaque and calcified plaque compared to unaffected aorta from patients
who died from atherosclerosis at The Johns Hopkins Hospital. (
)Moreover, LacCer derived from plaque was at least 2-fold
more potent in stimulating the proliferation of smooth muscle cells
compared to LacCer from unaffected aorta tissues. Although our findings
may suggest an important role for LacCer in cell proliferation, the
mitogenic signaling events predicted by this lipid is not clear.
The
best known mitogen activated protein (MAP) kinases are: p44 (extra cellular signal regulated kinase, ERK1), and p42
(ERK2). These are a group of serine/threonine protein kinases
that constitute an activation process triggered by a variety of growth
stimuli(6, 7, 8, 9, 10) .
Such protein kinases have been suggested to phosphorylate and activate
transcriptional factors such as
c-myc(11, 12, 13) ,
c-fos(14) , and
p62
(15, 16) , which regulates the
expression of genes essential for cell proliferation(17) . MAP
kinase kinase (MAPK-K) identified as a dual-specificity kinase, are
involved in the activation and phosphorylation of the tyrosine and
threonine residues in ERKs (MAP kinases) within the TEY motif in the
conserved domain VIII(18) . MAPK-K is a substrate for Rous
sarcoma associated factor (Raf-1), a serine-threonine
kinase(19) . The latter has been shown to integrate the
signaling of various receptor tyrosine kinases(9) : G-protein
coupled receptors(20, 21) , upstream serine/threonine
kinase, for example, protein kinase C, and other kinases(9) .
In this paper, we present evidence that LacCer specifically
activates the phosphorylation of p44 in cultured human
aortic smooth muscle cells. Upstream activators, Ras/Raf/MEK, are
involved in this signal kinase cascade. Concomitantly, LacCer
specifically stimulated c-fos proto-oncogene expression. Such
a combination of biochemical pathways may delineate signaling events
involved in LacCer mediated induction in aortic smooth muscle cell
proliferation.
Figure 1:
Effects of lactosylceramide,
glucosylceramide, and ceramide on MAP kinase activity in cultured human
aortic smooth muscle cells. Cells were incubated with various
concentrations (0-50 µM) of LacCer, ; GlcCer,
; and ceramide,
, for 5 min and lysed in lysis buffer.
Immunocomplex assay of the MAP kinase activity was measured as
described under ``Materials and Methods.'' Each point is the
mean ± S.D. of three individual
experiments.
Figure 2:
Effect of time of incubation with LacCer
on MAP kinases. Cells were incubated with 10 µM LacCer for
various time intervals (0-60 min) as indicated, harvested, and
lysed. Whole cell lysates (40 µg of protein/lane) were subjected to (A) Western immunoblot assays after separation on 12.5%
SDS-PAGE, transferred to nitrocellulose paper, and blotted with
anti-MAP kinase antibody and photographed. B, densitometric
scan of phosphorylated p44 (upper band). C, MAP kinase activity was assayed after immunoprecipitation
as described under ``Materials and
Methods.''
Figure 3:
Phosphoamino acid analysis of p44 kinase by thin layer chromatography. LacCer stimulated
P-labeled cells were lysed and immunoprecipitated with the
use of p44
antibody. The immunoprecipitates were
digested with 6 N HCl along with unlabeled phosphoserine,
phosphotyrosine, and phosphothreonine and separated by two-dimensional
TLC. The solvents in the first and second dimensions were:
butanol:pyridine:acetic acid:water (13:10:2:8, v/v) and
butanol:pyridine:acetic acid:water (15:10:3:12, v/v), respectively.
Nonradioactive phosphoamino acid standards were detected by ninhydrin
staining. P-Y, phosphotyrosine; P-T, phosphothreonine; and P-S,
phosphoserine.
Figure 4: Effect of protein tyrosine kinase inhibitor, tyrphostin, on MAP kinase activity. Cells were treated with 20 µM tyrphostin AG 126 for 30 min prior to the addition of 10-25 µM LacCer for 5 min. Next, the cells were harvested and lysed. The MAP kinase activity was assayed after immunoprecipitation of cell lysates as described in legend to Fig. 1. Tyr, tyrphostin.
Figure 5:
Effects of LacCer on p21activation in aortic smooth muscle cells.
P-Labeled
cells were stimulated with LacCer (10 µM) for different
times as indicated. The cell lysate was immunoprecipitated with
p21
antibody. The bound nucleotides (both GTP
and GDP) were eluted and separated on polyethyleneimine TLC plate using
0.75 M KH
PO
, pH 3.4, as solvent and
subjected to autoradiography (A) and densitometric scanning (B).
Figure 6: The electrophoretic mobility of Raf-1 in aortic smooth muscle cells treated with LacCer. Whole cell lysates were prepared as described under ``Materials and Methods'' and 40 µg of protein/lane were separated by 10% SDS-PAGE and transferred onto polyvinylidine difluoride membrane and detected with anti-Raf-1 antibody.
Figure 7: Gel shift assay of MEK (MAP kinase kinase) in aortic smooth muscle cells incubated with LacCer. Whole cell lysate protein (40 µg of protein/lane) was separated by 12.5% SDS-PAGE and (A) subjected to Western immunoblot assay employing MEK2 antibody (slower migrating upper bands represent the phosphorylated form of MEK2, MEK(P)). B, densitometric scan MEK(P) (upper band).
Figure 8:
Northern blot analysis of the expression
of proto-oncogene mRNAs in aortic smooth muscle cells stimulated by
LacCer. Aortic smooth muscle cells were incubated with 10 µM LacCer for 1 h and total cellular RNA was isolated. Twenty
micrograms of total RNA were analyzed by Northern blotting (A)
followed by densitometric scanning (B) for c-fos,
c-jun, c-myc, and GAPDH transcripts using respective P-labeled cDNA probes. Analysis of total cellular RNA for
each condition from three separate experiments provided similar
results. Lane 1, control; lane 2, 20 µM linoleic acid; lane 3, 10 µM LacCer.
Our studies generated several novel findings. First, we found
that LacCer specifically stimulated the phosphorylation of
p44, whereas GlcCer and Cer did not. Second, tyrphostin
(tyrosine kinase inhibitor) markedly abrogated LacCer mediated
induction in the phosphorylation of p44
. Third, LacCer
stimulated Ras-GTP loading and the phosphorylation of MEK2 and Raf.
Fourth, LacCer specifically stimulated the mRNA level of c-fos proto-oncogene in A-SMC.
To delineate the signal transduction
events in LacCer-mediated proliferation in human A-SMC, we examined its
effects on various parameters in the signal transduction cascade under
carefully controlled yet, varying conditions. The cells were grown to
confluence, washed, and incubated with Ham's F-10 medium without
serum for 2 h. Next, various agonists and antagonists were added. We
were concerned that contamination of any reagents or cell cultures with
bacteria or lipopolysaccharide may also effect the phosphorylation of
MAPK(31) . Accordingly, appropriate control experiments were
pursued to demonstrate that lipopolysaccharide (10 µg/ml) did not
alter the phosphorylation of either p44 or p42
in cultured human A-SMC (data not shown). Next, we analyzed the
effects of LacCer and other glycosphingolipids simultaneously employing
two methods, these were: 1) the measurement of MAPK activity of
immunoprecipitated MAPK employing myelin basic protein fragment
(APRTPGGRR) as substrate and [
P]ATP as the
phosphate donor; and 2) Western immunoblot assay followed by
densitometric scanning of individual gel bands. As shown in various
figures, both of these approaches yielded similar results confirming
the validity of our experimental techniques and the interpretation of
results.
Our studies clearly revealed that LacCer induced the
phosphorylation of tyrosine and threonine residues in
p44. This phenomenon was abrogated by an inhibitor of
tyrosine kinase, e.g. tyrphostin(32) . However,
staurosporine, an inhibitor of protein kinase C, failed to impair this
process (
)(data not shown). These findings reveal that
LacCer mediated p44
phosphorylation and cell
proliferation are independent of protein kinase C. This is in contrast
to a previous study in which GlcCer mediated proliferation in a
continuous renal cell line, Madin-Darby canine kidney, was shown to be
abrogated by inhibitors of protein kinase C(33) .
Recent
studies from several laboratories reveal that the induction of MAPKs
may also be due to inactivation of MAPK phosphatases(34) . In
particular, generation of free oxygen radicals and hydrogen peroxide
were shown to inhibit MAPK phosphatase and consequently increase the
level of p44 in neutrophils(35) . To rule out
this possibility, we pursued Northern blot assays in cells incubated
± LacCer and found that the mRNA level of MAPK phosphatase was
similar (data not shown). Thus, our findings indicate that LacCer
mediated induction of p44
is not due to the inhibition
of phosphatase activity.
The catabolic products of LacCer are
GlcCer, Cer, sphingosine, and stearic acid. In order to determine the
specificity of LacCer mediated stimulation of p44 phosphorylation, we also investigated the effects of GlcCer and
Cer that are highly enriched in atherosclerotic plaque intima and
media, but not in unaffected intima and media.
We found
that GlcCer and Cer did not alter p44
phosphorylation.
The effects of LacCer on upstream regulators, for example, Ras, Raf,
and MEK of MAPK, were investigated. The proto-oncogene p21 has been identified as a key molecular switch involved in
regulating cell activation triggered by various
mitogens(29, 36, 37) . In its resting state,
p21
is in a GDP-bound state. Upon in vivo activation p21
releases GDP and binds
GTP(38, 39) . Immunoprecipitation of p21
from stimulated and nonstimulated cells followed by nucleotide
(both GTP and GDP) elution revealed that a substantial increase in GTP
bound p21
occurred upon LacCer treatment. The maximal
effect was observed at 1 min after LacCer addition. Thereafter, GTP
bound p21
was decreased. This time dependent increase of
p21
GTP loading suggests that LacCer activates
p44
via the activation of p21
. We
speculate that the relatively short duration of LacCer induced
p21
GTP loading may be due to the interaction of the
latter with its effector or GTPase activating protein. An investigation
is required whether the LacCer mediated activation occurs by direct
interaction with p21
or indirectly through secondary
factors.
Raf-1, the product of the c-Raf 1 proto-oncogene is pivotal
in transmitting signals from Ras in the plasma membrane to cytosolic
nuclear compartments of the cells(40, 41) . The
stimulation of Raf-1 activity depends on the activation of the small
G-protein p21(42) . Active GTP-ras binds
to the NH
-terminal domain of Raf-1 and recruits Raf-1 to
the plasma membranes, which is sufficient to cause its
activation(40, 41, 43) . We investigated
whether Raf-1 is phosphorylated/activated after stimulation of
p21
(measured by p21
GTP loading) by
LacCer. Significant phosphorylation of Raf-1 was observed within
2.5-5 min of incubation of cells with LacCer. Thus the activation
of Raf-1 by LacCer was transient in A-SMC. Since Raf-1 activity is
known to participate in the G
/G
transition of
cells(42) , we may speculate that LacCer mediated Raf-1
activation may similarly help in the transition of A-SMC from the
G
phase to the G
phase of the cells. Next, we
measured the phosphorylation of MEK by LacCer to assess whether the
Raf-1, a serine-threonine kinase, transmits proliferative signals to
downstream effectors, e.g. p44
via the
activation of putative MAPK activator MEK. MEK2 phosphorylation was
stimulated within 2.5-5 min after the addition of 10 µM LacCer, and then it was dissipated at 10 min. Taken together,
these findings lead us to speculate that LacCer induced activation of
p44
may be mediated by a kinase cascade that includes
Ras, Raf, and MEK2 in a descending order.
Induced expression of
proto-oncogene c-fos, c-jun, and c-myc mRNA
is an early response to various growth
stimuli(44, 45, 46) . These proto-oncogenes
encode for nuclear binding transcriptional factors (44, 45) and play a crucial role in mitogen-induced
cell proliferation(47) . To determine whether these
proto-oncogenes are also involved in LacCer induced A-SMC
proliferation, subsequent to p44 activation, we measured
mRNA levels of these proto-oncogenes. Among the three proto-oncogenes
tested, only the c-fos mRNA level increased after 1 h of
LacCer incubation as compared to control. In contrast, cellular levels
of c-jun or c-myc mRNA in LacCer treated and control
cells were similar. Equal amounts of RNA loading was confirmed by GAPDH
analysis. This data suggested that LacCer specifically induced
c-fos proto-oncogene expression via activation of
p44
.
Why LacCer specifically induced the
phosphorylation of p44 and c-fos mRNA
expression is not clearly understood from our studies. Previous studies
with smooth muscle cells have revealed that fatty acids, e.g. linoleic acid and arachidonic acid, stimulate the phosphorylation
of both p42
and p44
(48) . In
addition, linoleic acid stimulated the mRNA levels of c-fos,
c-myc, and c-jun in rat vascular smooth muscle cells (49) and also in our studies with human A-SMC. Furthermore,
recent studies have shown that tumor necrosis factor-
specifically
stimulate MEK1, that in turn induce the activation of
p42
(50) . Our preliminary studies indicate that
LacCer did not stimulate MEK1, instead LacCer stimulated MEK2. Thus
there may be a clear dichotomy in regard to agonist specific induction
of MEK2 and activation/phosphorylation of p44
(ERK1).
Since other studies have shown that the ERK group of MAP kinases
phosphorylate Elk-1 and increases ternary complex
formation(15, 16) and activation of c-fos downstream in the signaling pathway, it is possible that LacCer,
like other growth factors, specifically mediates this process. This may
ultimately increase transcriptional activity and DNA synthesis,
subsequently cell proliferation. Further work in this area is warranted
to explain this phenomena to understand the pathophysiology of LacCer
mediated A-SMC proliferation in atherosclerosis.