(Received for publication, December 16, 1994; and in revised form, January 25, 1995)
From the
Human peripheral blood monocytes responded to stimulation of
platelet-activating factor (PAF) with up-regulation of the transcript
for heparin-binding epidermal growth factor-like growth factor
(HB-EGF), a potent mitogen for vascular smooth muscle cells. This
function of PAF was observed at nanomolar concentrations of the ligand,
starting at 30 min after stimulation. The PAF-induced up-regulation of
HB-EGF mRNA was accompanied by an increase in B binding activity.
These functions of PAF appeared to be mediated through the cell surface
PAF receptors, as two PAF receptor antagonists, WEB 2086 and L-659,989,
blocked both the up-regulation of HB-EGF mRNA and
B binding
activity induced by PAF. The antagonists, however, had no effect on
phorbol ester-induced up-regulation of HB-EGF mRNA and
B binding
activity. Pretreatment of monocytes with pertussis toxin inhibited
these functions of PAF, whereas cholera toxin had no inhibitory effect.
Pyrrolidine dithiocarbamate, an inhibitor for NF-
B activation,
markedly reduced PAF-stimulated
B binding activity as well as
up-regulation of HB-EGF mRNA. These results suggest a potential role of
PAF in HB-EGF expression and provide evidence that this stimulation may
occur through increased
B binding activity.
Proliferation of smooth muscle cells (SMC) ()plays an
important role in the development of atherosclerosis into its advanced
stage(1, 2) . A number of growth factors and
cytokines, including platelet-derived growth factor, basic fibroblast
growth factor, transforming growth factor-
, and tumor necrosis
factor-
(TNF
), have been shown to stimulate SMC
proliferation(1) . These factors may be produced at the site of
lesion by endothelial cells as well as monocytes, which are attracted
to the subendothelial region by locally produced chemoattractants such
as monocyte chemotactic protein-1, colony-stimulating factor, and
transforming growth factor-
(1) . More recently, it was
shown that monocytic cells produce a protein that binds the epidermal
growth factor (EGF) receptor with 10-fold higher affinity than EGF
itself(3) . This molecule, termed heparin-binding EGF-like
growth factor (HB-EGF), is also produced by SMC and acts as a potent
mitogen for SMC by an autocrine
mechanism(4, 5, 6) . HB-EGF is a 22-kDa
single-chain polypeptide with an EGF-like domain and a heparin-binding
domain(3) . The cDNA as well as the gene for HB-EGF have been
cloned(3, 7) . The synthesis of HB-EGF is up-regulated
by a number of factors including lysophosphatidylcholine, phorbol
ester, angiotensin II, and
thrombin(6, 8, 9) . The mechanisms for
transcriptional activation of HB-EGF expression, however, are not fully
understood.
PAF, a biologically active phospholipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), is a substance released by many cells including monocyte/macrophages, endothelial cells, polymorphonuclear cells, and platelets (reviewed in (10) and (11) ). PAF exerts its effects through binding to specific receptors in target cells(12, 13) . Recent cloning of the PAF receptor indicates that it is a member of the G protein-coupled receptor superfamily(14, 15, 16, 17) . PAF has been suggested to play a role in atherogenesis and atherosclerosis, and clinical studies indicated higher levels of PAF in coronary artery samples from patients with severe atherosclerosis(18, 19) . A major part of the action of PAF during atherogenesis may be mediated by a juxtacrine intercellular signaling mechanism in which PAF synthesized by endothelial cells is attached to the surface of these cells, which could form a close interaction with leukocytes through P-selectin(20) . This interaction may be important for the subsequent monocyte/macrophage activation, secretion of proliferative agents, migration between the endothelium, and eventually accumulation of lipid within the cells.
In the current study, we investigated the
function of PAF in the regulation of expression of growth factors in
monocytes and attempted to establish the relationship between the
expression of HB-EGF and the activation of specific transcription
factors. Our results indicate that PAF is a strong inducer for HB-EGF
expression in monocytes, and this function is associated with an
enhanced B binding activity suggesting potential involvement of
NF-
B in PAF-induced gene regulation.
Figure 1:
PAF-induced expression of HB-EGF gene
in monocytes. Cells were stimulated under various conditions before RNA
extraction. Total RNA (20 µg) were subject to Northern blot
analysis with a P-labeled human HB-EGF probe. A,
autoradiograph of Northern blot with RNA from monocytes treated with
medium alone, PMA (100 nM), TNF
(40 ng/ml), and PAF (100
nM) for 60 min, as indicated. B, dose- and
time-dependent expression of HB-EGF mRNA. Total RNA was extracted from
cells stimulated with different doses of PAF for 60 min (lanes1-4) or after stimulation with 100 nM PAF
at the time points indicated (lanes5-8). C, effect of actinomycin D (ActD) on HB-EGF gene
transcription. The cells were stimulated with 100 nM PAF in
the absence (lane2) or presence (lane3) of 1 µg/ml actinomycin D for 60 min before
extraction of RNA. The bottompart of panelA is a photograph of the same gel before transfer to
membrane, with the relative molecular sizes marked by the 18 and 28 S
RNA species. Sample loading was similarly controlled for Northern blots
shown in other figure panels.
Figure 2: Effect of PAF antagonists, bacterial toxins, and protein kinase inhibitors on PAF-induced HB-EGF gene expression. Northern blot with total RNA (20 µg/lane) isolated after treatment of the cells as indicated below is shown. A, cells were pretreated for 30 min with L-659,989 (10 µM; lanes1 and 2) and WEB 2086 (10 µM; lanes3 and 4) before stimulation for 60 min with PAF (100 nM; lanes1 and 3) or PMA (100 nM; lanes2 and 4). B, cells were pretreated for 4 h with PTX (500 ng/ml) or CTX (5 µg/ml), followed by stimulation for 60 min with either PAF (100 nM) or PMA (100 nM). Lanes1-3, without toxin treatment. C, cells were pretreated for 40 min with herbimycin A (HA, 1 µM, lane3) or staurosporine (STSP, 200 nM, lane4), followed by PAF (100 nM) stimulation for 60 min. Lane1, no PAF or inhibitor treatment; lane2, PAF only.
PAF has been shown to bind a receptor that functionally
couples to G proteins(12, 13) , and recent cloning of
the PAF receptor confirmed that it possesses a typical structure for G
protein-coupled receptors (14, 15, 16, 17) . We examined
whether PAF-induced HB-EGF mRNA synthesis is subject to inhibition by
bacterial toxins that ADP-ribosylate the -subunits of certain G
proteins (Fig. 2B). Our results indicate that pertussis
toxin (PTX) significantly reduced PAF-induced HB-EGF message
up-regulation (lane4), while cholera toxin (CTX) has
no such inhibitory effect in monocytes (lane5).
Neither of the two toxins inhibited PMA-induced expression of the
HB-EGF gene (lanes5 and 7). In a
preliminary study of the potential mechanisms for PAF-stimulated gene
expression, we investigated the requirement for protein tyrosine kinase
(PTK) and protein kinase C (PKC) activity in PAF-induced HB-EGF
expression. Monocytes were treated with herbimycin A and staurosporine,
inhibitors for PTK and PKC, respectively. Herbimycin A treatment (1
µM, 40 min) completely inhibited PAF-induced HB-EGF gene
expression (Fig. 2C, lane3). Similar
results were obtained with staurosporine (lane4),
suggesting the functions of protein kinases in this process.
Figure 3:
PAF-induced B DNA binding activity in
monocytes. Shown are autoradiographs of EMSA results. A,
monocytes were stimulated with PAF (0.01-100 nM), PMA
(100 nM), TNF
(40 ng/ml), and lipopolysaccharide (LPS) (0.1 µg/ml) for 40 min or as indicated before
preparation of nuclear extracts. A
P-labeled 21-mer,
containing the consensus
B site (GGGACTTTCC), was used for EMSA. B, cells were pretreated for 30 min with L-659,989 (10
µM) and WEB 2086 (10 µM) before stimulation
with 100 nM PAF for 40 min.
We next examined
the effect of herbimycin A and staurosporine on PAF-induced B
binding activity. Both protein kinase inhibitors, at concentrations
that blocked HB-EGF gene expression, produced a marked decrease in the
B binding activity (not shown). Our data suggested the possible
involvement of PTK and PKC in PAF-stimulated
B binding activity in
monocytes.
Results presented above demonstrated parallel stimulation
of the HB-EGF gene expression and B binding activity upon PAF
stimulation. To determine whether HB-EGF gene expression could be the
result of PAF-induced
B binding activity, we examined the effect
of pyrrolidine dithiocarbamate (PDTC) on both the gene expression and
B binding activity in PAF-stimulated monocytes. PDTC is an
antioxidant that selectively blocks the dissociation of I
B from
the cytoplasmic NF-
B, thus preventing the activation and nuclear
translocation of NF-
B(27) . As shown in Fig. 4,
PDTC treatment of monocytes significantly inhibited the
B binding
activity induced by PAF (Fig. 4A). The same treatment
also nearly completely blocked PAF-induced expression of the HB-EGF
transcript. These results suggest that transcription factor(s) with
B binding activity participate in PAF-stimulated HB-EGF gene
transcription.
Figure 4:
Inhibition of PAF-induced B DNA
binding activity and HB-EGF gene expression by PDTC. A, PDTC
inhibits PAF-induced
B binding activity. Nuclear protein extracts
were prepared from monocytes preincubated for 40 min with PDTC at the
indicated concentrations, followed by PAF (100 nM) stimulation
for 60 min. The autoradiograph of EMSA results is shown. B,
PDTC inhibits PAF-induced HB-EGF gene expression. Cells pretreated with
PDTC, as indicated above, were stimulated with PAF (100 nM)
for 60 min. Total cellular RNA was extracted for Northern blot as
described in the legend for Fig. 1. The autoradiograph of the
Northern blot is shown.
HB-EGF was originally purified from conditioned
medium of cultured human mononuclear cells (28) and the
monocytic U-937 cell line(3) . It was subsequently found that
HB-EGF could be transcribed in vascular endothelial cells (29) and smooth muscle cells(6) ; both are associated
with atherogenesis. HB-EGF is a more potent mitogen than EGF for smooth
muscle cell proliferation(3) . The expression of HB-EGF in
these cells is highly regulated. It has been shown that factors that
participate in atherogenesis, such as TNF and thrombin, also
up-regulate the expression of HB-EGF. In spite of these findings, the
transcription factors involved in HB-EGF gene expression have not been
identified. Characterization of the gene for the human HB-EGF revealed
a 2-kilobase DNA fragment that contains promoter activity and a
potential AP-1 site (7) . The sequence of this AP-1 site,
however, contains a single residue mismatch (7) that was found
in a separate study to be defective in binding Fos and
Jun(30) . Although it was not clear from the above study
whether there are
B sites upstream of the HB-EGF gene, results
reported here suggest that transcription factors with
B binding
capability may contribute to the transcription of HB-EGF in monocytes.
NF-
B is a well characterized transcription factor whose activity
in non-B lymphocytes is regulated by agents including those associated
with inflammation and atherogenic lesions(31) . Thus, the
stimulating effect of several atherogenic factors on endothelial and
smooth muscle cells may be mediated in part through NF-
B
activation and up-regulation of growth factor synthesis.
PAF has
long been suggested to play a role in atherogenesis and atherosclerotic
lesions. Only recently, however, has evidence begun to accumulate
suggesting the function of PAF in gene regulation(32) . Our
results indicate that PAF at nanomolar concentrations is fully capable
of stimulating B binding activity. This action of PAF appears to
be mediated by the cell surface PAF receptor, which couples to a G
protein sensitive to pertussis toxin treatment. The signal transduction
mechanism for transcription factor activation by G protein-coupled
receptors remains to be explored. Our preliminary results indicate that
PAF-mediated
B binding activity involves kinase activation; thus
it is likely that multiple signal transduction pathways lead to the
phosphorylation and degradation of I
B. Although in physiological
conditions PAF released by cells is rapidly
degraded(33, 34) , prolonged exposure of leukocytes to
PAF could be achieved by the recently discovered juxtacrine
intercellular signaling mechanism in which newly synthesized PAF is
presented on the surface of endothelial cells that interact with
peripheral blood leukocytes as well as residential monocytes and
macrophages(35, 36) . Because of the subendothelial
localization of monocyte/macrophages in atherosclerotic lesions,
sustained high level synthesis of HB-EGF is possible through the
juxtacrine activation of the cells tethered by P-selectin and
stimulated by endothelial cell surface-associated PAF. We are currently
investigating this possibility as well as the transcription factors
involved in the process.
Note Added in Proof-Two B sites have
recently been identified in the mouse gene for HB-EGF (M. Klagsbrun,
personal communication). Weyrich et al. (37) found
that interaction of monocytes with P-selectin causes increased
expression of monocyte chemotactic protein-1, TNF
, and activation
of NF-
B in response to PAF stimulation.