©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Platelet-activating Factor Stimulates Transcription of the Heparin-binding Epidermal Growth Factor-like Growth Factor in Monocytes
CORRELATION WITH AN INCREASED kappaB BINDING ACTIVITY (*)

(Received for publication, December 16, 1994; and in revised form, January 25, 1995)

Zhixing Pan Vladimir V. Kravchenko Richard D. Ye (§)

From the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 kappaB 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 kappaB binding activity induced by PAF. The antagonists, however, had no effect on phorbol ester-induced up-regulation of HB-EGF mRNA and kappaB 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-kappaB activation, markedly reduced PAF-stimulated kappaB 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 kappaB binding activity.


INTRODUCTION

Proliferation of smooth muscle cells (SMC) (^1)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-beta, and tumor necrosis factor-alpha (TNFalpha), 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-beta(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 kappaB binding activity suggesting potential involvement of NF-kappaB in PAF-induced gene regulation.


MATERIALS AND METHODS

Preparation of Monocytes from Peripheral Blood

Heparinized human peripheral blood from health donors was fractionated on Percoll density gradients. The cells were then harvested and washed, and monocytes were isolated using gelatin/plasma-coated flasks. The purity of monocytes was greater than 95% as determined by staining with the anti-CD14 monoclonal antibody MY4. Cell viability was greater than 98% as measured by trypan blue exclusion. Monocytes were resuspended in RPMI 1640 medium (Irvine Scientific) with 10% (v/v) heat-inactivated fetal bovine serum. For isolation of total RNA and extraction of nuclear proteins, 5 times 10^6 cells were plated in 1 ml of the culture medium in sterile 1.5-ml polypropylene microcentrifuge tubes. The cells were incubated without serum for 12 h before stimulation with ligands.

RNA Extraction and Northern Blotting

Total RNA was isolated from monocytes by a modified guanidinium/acidic phenol method (21) . In some experiments the cells were treated with various ligands as well as the PAF antagonists WEB 2086 (kindly provided by Boehringer Ingelheim, Ridgefield, CT) or L-659,989 (a gift from Merck & Co.). About 20 µg of total RNA was subjected to electrophoresis in agarose gels containing 6% formaldehyde and transferred to the Hybond-N plus membranes (Amersham Corp.) by the diffusion method. A 616-base pair HindIII-XbaI fragment of the human HB-EGF cDNA was synthesized from U937 total RNA by reverse transcriptase polymerase chain reaction. The sequences of the primers are: 5-ATG AAG CTT CTG CCG TCG GTG-3 and 5-TGT CTA GAT GCC CAA CTT CAC TTT CTC-3. The polymerase chain reaction-amplified fragment was digested with the restriction enzymes HindIII and XbaI, and subcloned into the same sites in pBluescript SK(+) plasmid vector (Stratagene). For Northern hybridization, the fragment was labeled with [P]dATP to a specific activity of >5 times 10^8 cpm/µg of DNA by the random priming method(22) . Blots were prehybridized in 6 times SSC, 5 times Denhardt's solution, and 0.1% SDS at 65 °C for 2 h and further hybridized with the P-labeled probe (1 times 10^6 cpm/ml) for 16 h at the same temperature. Filters were washed with 2 times SSC and 0.1% SDS at 65 °C, followed by another wash with 0.1 times SSC and 0.1% SDS. The blots were exposed to Hyperfilm-MP (Amersham Corp.) with an intensifying screen. Relative hybridization was analyzed for quantitation of actual radioactivity by a PhosphorImaging System (Molecular Dynamics). The quantity of samples in each lane was standardized against relative staining of the 18 and 28 S RNA after gel electrophoresis.

Preparation of Nuclear Extracts

Nuclear extracts were prepared by a modified method of Dignam et al.(23) . Aliquots of monocytes (5 times 10^6 cells/sample) were stimulated with different agents as indicated in the text, after which the cells were washed 3 times with ice-cold phosphate-buffered saline, harvested, and resuspended in 0.4 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM dithiothreitol, 0.1 mM each of EDTA and EGTA, and 1 mM phenylmethylsulfonyl fluoride. After 10 min, 23 µl of 10% Nonidet P-40 was added and briefly mixed for 2 s. Nuclei were separated from cytosol by centrifugation at 13,000 times g for 10 s and were resuspended in 50 µl of buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride. After 30 min at 4 °C, lysates were separated by centrifugation (13,000 times g, 30 s) and the supernatants containing nuclear proteins were transferred to new vials. Samples were diluted to equal concentration in buffer B.

Electrophoretic Mobility Shift Assay (EMSA)

Electrophoretic mobility shift assays were performed essentially as described. (^2)Briefly, 2.5 µg of nuclear extracts were added to 12 µl of binding buffer (5 mM HEPES, pH 7.8, 5 mM MgCl(2), 50 mM KCl, 0.5 mM dithiothreitol, 0.4 mg/ml poly(dI-dC), 0.1 mg/ml sonicated salmon sperm DNA, and 10% glycerol) and incubated for 10 min at room temperature. Approximately 20-50 fmol of P-labeled oligonucleotide probe (30,000-50,000 cpm) was added, and the reaction mixture was incubated for 10 min at room temperature. The samples were analyzed on 5% acrylamide gel, made with buffers containing either 50 mM Tris borate, 1 mM EDTA (TBE) or 50 mM Tris, 380 mM glycine, 2 mM EDTA (TGE). After pre-electrophoresis for 2 h at 12 V/cm, electrophoresis was carried out at the same voltage for 2-2.5 h. The gel contents were transferred to Whatman DE-81 paper, dried, and exposed to either x-ray film or PhosphorImager screen.


RESULTS AND DISCUSSION

Stimulation of HB-EGF Gene Expression by PAF

Macrophage-like U-937 cells produce HB-EGF in response to treatment with PMA, which has also been shown to be an inducer of the HB-EGF mRNA in SMC(3, 4) . We investigated the effect of PAF, generated at the site of vascular injury during atherogenesis, on HB-EGF gene transcription in human monocytes. The expression of the HB-EGF message was examined by hybridization of a P-labeled human HB-EGF cDNA probe to total RNA isolate from human monocytes (Fig. 1A). A major species of 2.5 kilobases was found in cells stimulated with PMA (lane 2), TNFalpha (lane3), or PAF (lane4) but not in untreated monocytes (lane1). Although the base-line level of HB-EGF mRNA was very low, it increased rapidly after stimulation with PAF and was maintained for at least 4 h (Fig. 1B). PAF increased HB-EGF mRNA in a dose-dependent manner, with a maximal response achieved in approximately 10 nM PAF (Fig. 1B). Actinomycin D, an inhibitor for RNA synthesis, blocked PAF-stimulated increase of the HB-EGF mRNA (Fig. 1C). These results suggest that stimulation at the transcriptional level contributes to PAF-induced up-regulation of the HB-EGF message.


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), TNFalpha (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.



Effect of PAF Receptor Antagonists and a Bacterial Toxin on HB-EGF Gene Expression

To investigate whether PAF's effect on HB-EGF gene expression is mediated by the specific cell surface PAF receptor, we employed the PAF receptor antagonists, L-659,989 and WEB 2086(24, 25) . Monocytes were pretreated with L-659,989 (1 µM) or WEB 2086 (10 µM) prior to stimulation by PAF (100 nM). As shown in Fig. 2A, both antagonists markedly inhibited PAF-induced HB-EGF expression in monocytes (lanes1 and 3). In contrast, neither antagonist had an effect on PMA-induced expression of HB-EGF in the same cells (lanes2 and 4). Treatment of the cells with the antagonists alone did not induce HB-EGF gene expression (not shown).


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 alpha-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.

PAF-induced Gene Expression of HB-EGF Is Associated with a kappaB DNA Binding Activity

One of the mediators for immediate-early gene expression is the transcription factor NF-kappaB, which couples extracellular signals to gene-activating events. The prototypic NF-kappaB is a heterodimer consisting of p50 and p65 subunits and exists in the cytoplasm in an inactive form due to association with the specific inhibitor protein IkappaB. Phosphorylation of IkappaB by candidate kinases leads to the release and subsequent nuclear translocation of NF-kappaB, which then binds a decameric DNA sequence originally identified in the kappa light chain enhancer of immunoglobulin (26) . The potential involvement of PTK and PKC in HB-EGF gene expression prompted us to examine the possible induction of kappaB binding activity by PAF and its relationship to the expression of the HB-EGF gene in monocytes. Our results demonstrated that PAF stimulated a rapid rise in kappaB binding activity, observed within 20 min, peaked at 1 h, and returned to base line after 5 h (Fig. 3A, lanes10-14). PAF induced kappaB binding activity in a concentration-dependent manner proportional to the concentrations required for the expression of the HB-EGF gene (Fig. 3A, lanes 5-9). Maximal response was obtained at 10 nM PAF, and a higher concentration of PAF (100 nM) did not produce an additional increase in the kappaB binding activity. The PAF receptor antagonists L-659,989 and WEB 2086 both blocked kappaB binding activity stimulated by PAF (Fig. 3B, lanes3 and 4) but not that stimulated by PMA (Fig. 3B, lanes6 and 7). When the cells were treated with bacterial toxins as described above, PTX blocked PAF-induced kappaB binding activity but not that induced by PMA, whereas CTX had no inhibitory effect (data not shown). These results indicate that PAF could induce kappaB binding activity in monocytes through a receptor-mediated and G protein-coupled pathway.


Figure 3: PAF-induced kappaB DNA binding activity in monocytes. Shown are autoradiographs of EMSA results. A, monocytes were stimulated with PAF (0.01-100 nM), PMA (100 nM), TNFalpha (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 kappaB 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 kappaB binding activity. Both protein kinase inhibitors, at concentrations that blocked HB-EGF gene expression, produced a marked decrease in the kappaB binding activity (not shown). Our data suggested the possible involvement of PTK and PKC in PAF-stimulated kappaB binding activity in monocytes.

Results presented above demonstrated parallel stimulation of the HB-EGF gene expression and kappaB binding activity upon PAF stimulation. To determine whether HB-EGF gene expression could be the result of PAF-induced kappaB binding activity, we examined the effect of pyrrolidine dithiocarbamate (PDTC) on both the gene expression and kappaB binding activity in PAF-stimulated monocytes. PDTC is an antioxidant that selectively blocks the dissociation of IkappaB from the cytoplasmic NF-kappaB, thus preventing the activation and nuclear translocation of NF-kappaB(27) . As shown in Fig. 4, PDTC treatment of monocytes significantly inhibited the kappaB 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 kappaB binding activity participate in PAF-stimulated HB-EGF gene transcription.


Figure 4: Inhibition of PAF-induced kappaB DNA binding activity and HB-EGF gene expression by PDTC. A, PDTC inhibits PAF-induced kappaB 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 TNFalpha 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 kappaB sites upstream of the HB-EGF gene, results reported here suggest that transcription factors with kappaB binding capability may contribute to the transcription of HB-EGF in monocytes. NF-kappaB 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-kappaB 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 kappaB 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 kappaB binding activity involves kinase activation; thus it is likely that multiple signal transduction pathways lead to the phosphorylation and degradation of IkappaB. 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.


FOOTNOTES

*
This work was supported by American Heart Association Grant-in-aid 92-978 and by United States Public Health Service Grant GM46572. This is Publication 9054-IMM from The Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests and correspondence should be addressed: Dept. of Immunology, IMM-12, The Scripps Research Institute, La Jolla, CA 92037. Tel.: 619-554-8583; Fax: 619-554-8483.

(^1)
The abbreviations used are: SMC, smooth muscle cells; PAF, platelet-activating factor; HB-EGF, heparin-binding epidermal growth factor-like growth factor; NF-kappaB, nuclear factor kappaB; PMA, phorbol myristate acetate; PDTC, pyrrolidine dithiocarbamate; TNFalpha, tumor necrosis factor-alpha; PTK, protein tyrosine kinase; PKC, protein kinase C; EMSA, electrophoretic mobility shift assay; PTX, pertussis toxin; CTX, cholera toxin.

(^2)
V. V. Kravchenko, Z. Pan, J. Han, J.-M. Herbert, R. J. Ulevitch, and R. D. Ye, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Andrzej Ptasznik for assistance in blood cell preparation, Dr. Lili Feng for helpful discussion, and Drs. Eric R. Prossnitz and Darren D. Browning for critical reading of the manuscript.

Note Added in Proof-Two kappaB 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, TNFalpha, and activation of NF-kappaB in response to PAF stimulation.


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