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Platelet-activating factor (PAF) induces activation of matrix metalloproteinase 2 activity and vascular endothelial cell invasion and migration ¹

T. WILLIAM AXELRAD^*,, DAYANAND D. DEO^*,, PAULO OTTINO, JENNEFER VAN KIRK^*,, NICOLAS G. BAZAN^*,, HAYDEE E.P BAZAN^*, and JAY D. HUNT^*,,2

^* Department of Biochemistry and Molecular Biology,
Stanley S. Scott Cancer Center and
Department of Ophthalmology, Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA

²Correspondence: LSU Health Sciences Center, Stanley S. Scott Cancer Center, 533 Bolivar St., CSB-4-18, New Orleans, LA 70112, USA. E-mail: jhunt{at}lsuhsc.edu

SPECIFIC AIMS

The aims of this study were to determine whether platelet-activating factor (PAF) stimulates migration and invasion of vascular endothelial cells, induces transcription of genes encoding matrix metalloproteinases, and if PAF stimulation leads to activation of MMP2 through increases in proMMP2 protein levels or as a result of increased MT1-MMP activity. A model was developed in which TIMP-2 binds to a membrane-bound, active MT1-MMP that serves as a receptor to direct proMMP2 to the extracellular matrix, where a second active MT1-MMP molecule can cleave the prodomain from proMMP2.

PRINCIPAL FINDINGS

1. Modulation of HUVEC growth involves PAF as a second message but not as a primary message
To elucidate the involvement of PAF in human umbilical vein endothelial cell (HUVEC) proliferation, HUVEC were grown in rich medium containing 2% FBS, bFGF, VEGF, EGF, and IGF-1 and treated with the PAF receptor antagonist LAU-8080. Significant inhibition of proliferation was observed in cells treated with PAF receptor antagonist LAU-8080 at concentrations of 1–10 µM. To determine whether PAF alone would increase proliferation, quiescent HUVEC were treated with either cPAF or vehicle. Because growth medium containing serum has high levels of PAF acetylhydrolase, cPAF C-16 was used. HUVEC were also treated with excess bFGF as a positive control for proliferation. Treatment of HUVEC with bFGF results in a significant increase in proliferation over control. Treatment with 1, 10, or 100 nM cPAF had no effect on proliferation whereas concentrations of 1000 nM caused cell lysis, possibly through a detergent effect. These data confirm earlier findings that although PAF does not stimulate proliferation alone, it is a second messenger involved in signaling cascades leading to cellular proliferation.

2. PAF induces invasion of HUVEC
To determine whether cPAF induces invasion of HUVEC through a matrix-coated membrane, cells were exposed to 0, 10, or 100 nM cPAF. At 10 and 100 nM cPAF, a significant increase in invasion occurred, with 10 nM cPAF producing a 60% and 100 nM an 80% increase in invasion (Fig. 1 A). Pretreatment with PAF receptor antagonists CV-3988, BN-52021 or LAU-8080 inhibited cPAF-induced invasion (Fig. 1B ). To determine whether PAF signaling is involved in the migration of vascular endothelial cells toward tumor cells, prostatic carcinoma PC-3 cells were allowed to condition basal endothelial growth medium for 48 h, then the medium was used in a Transwell assay with or without prior coating with Matrigel^TM. HUVEC layered onto Transwells without Matrigel^TM demonstrated a significant increase in migration toward PC-3-conditioned medium, which was inhibited by all PAF receptor antagonists (Fig. 1C ) and concentration dependent (Fig. 1D ). Similarly, HUVEC layered onto Transwells with Matrigel^TM demonstrated an increase in invasion by PC-3-conditioned medium that was significantly inhibited by the inclusion of each of the PAF receptor antagonists (Fig. 1E ) and concentration dependent (Fig. 1F ).

View larger version (33K): [in this window] [in a new window]   Figure 1. PAF increases invasion of HUVEC. A) HUVEC treated with 10 or 100 nM cPAF demonstrated increased invasion through Matrigel-coated membranes. B) HUVEC treated with 100 nM cPAF and 10 µM CV-3988 showed a significant reduction in invasion vs. cells treated with 100 nM cPAF alone. C) Basal medium conditioned by PC-3 cells for 48 h induces migration of HUVEC through uncoated membranes, which is inhibited by each of the PAF receptor antagonists. D) HUVEC were treated with rich endothelial growth medium (EGM-2) and exposed to increasing concentrations of BN-52021 or LAU-8080 (0.01, 0.1, 1, 10, or 100 µM) or CV-3988 (0.01, 0.1, 1, 3, or 10 µM). E) PC-3-conditioned basal medium induces invasion through Matrigel-coated membranes, which is also inhibited by each of the PAF receptor antagonists. F) HUVEC were induced to invade through Matrigel-coated membranes with EGM-2, which showed a concentration-dependent decrease in invasion when treated with PAF receptor antagonists (same concentrations as in panel D). Data values are expressed as mean ± SE (*P<0.05 using Student’s t test).

3. PAF induces TIMP-2 and MT1-MMP mRNA expression
Given that cPAF did not induce proliferation but did induce migration and invasion of HUVEC, we hypothesized that exposure of HUVEC to cPAF would result in activation of MMPs. To determine the effect of cPAF treatment on protease production in HUVEC, RT-PCR and quantitative real-time-PCR were performed. Quiescent HUVEC were treated with 100 nM cPAF or vehicle alone and mRNA was isolated at 2, 4, 5, 6, 7, 8, 9, 10, 11, and 12 h. mRNA was subjected to reverse transcription, followed by real-time PCR using the intercalating dye SYBR Green 1. cPAF induces a sixfold increase in TIMP-2 mRNA levels at 6 h and a nearly fourfold increase in MT1-MMP mRNA levels at 9 h. No changes in MMP1, MMP2, uPA, or TIMP-1 mRNA levels were detected up to 12 h after exposure to cPAF. Pretreatment with the PAF receptor antagonist LAU-8080 in cPAF-stimulated HUVEC significantly reduced TIMP-2 and MT1-MMP transcription.

4. PAF increases the ratio of active/inactive MT1-MMP in HUVEC
To determine whether MT1-MMP mRNA levels resulted in an increase in protein production, immunoblot analysis was performed. HUVEC pretreated with PAF receptor antagonists for 30 min were treated with cPAF and total cell extracts were collected at 0, 16, 24, and 36 h. Immunoblot analysis revealed a significant increase in proMT1-MMP in cPAF-treated cultures at 24 h vs. untreated control cultures. This increase was inhibited by CV-3988, LAU-8080, and BN-52021. PAF also induced the activation of MT1-MMP at 24 h, increasing the ratio of active MT1-MMP to latent proMT1-MMP. By 36 h, active MT1-MMP levels were similar to those of control. Cells treated with LAU-8080 remained inhibited at 36 h.

5. PAF induces activation of MMP2
The increases in active MT1-MMP levels observed in HUVEC after treatment with PAF should result in increased downstream proteolytic activation of latent proMMP2 (72 kDa) to active MMP2 (62 kDa). HUVEC were treated with 100 nM cPAF; media were collected at 0, 4, 8, 12, 16, 20, 24, and 36 h and analyzed by Western analysis or zymography. Results show increased activation of MMP2 by cPAF as early as 8 h and continuing through 36 h; LAU-8080 inhibits this cPAF-induced activation (Fig. 2 A). The fold induction of active MMP2 after exposure of HUVEC to cPAF is shown in Fig. 2B . To determine whether the increased activity of MMP2 observed by zymography (Fig. 2A ) resulted from increased conversion of proMMP2 to MMP2 or increased expression of MMP2 protein, immunoblot analysis was performed. As seen in Fig. 2C , stimulation of quiescent HUVEC with cPAF did not result in increased proMMP2 protein levels, suggesting that increases in MMP2 activity result from proteolytic activation of latent proMMP2.

View larger version (32K): [in this window] [in a new window]   Figure 2. PAF induces activation of MMP2. A) Media samples from HUVEC treated with cPAF or with vehicle alone for 36 h were subjected to zymography. cPAF-induced conversion of proMMP2 to MMP2 is observed at 36 h. Pretreatment of HUVEC with 10 µM LAU-8080 demonstrates that PAF receptor antagonists attenuate cPAF-induced activation of MMP2. B) Densitometry of zymography data demonstrate a fourfold induction of active MMP2 after exposure to cPAF for 36 h. C) Immunoblot analysis reveals that MMP2 protein levels are not increased by stimulation of HUVEC with cPAF as detected by sera specific for the prodomain (anti-proMMP2). STND = standard. Each blot was stripped and reprobed with anti-tubulin antibody.

CONCLUSIONS

Many angiogenic growth factors, including bFGF and VEGF, stimulate endothelial cell invasion through transmembrane protein kinase receptor dimerization and phosphorylation, leading to activation of phospholipase C (PLC) and Ras, both of which ultimately result in activation of cPLA₂ (Fig. 3 ). This in turn leads to the production of bioactive lipids such as PAF. We recently demonstrated that binding of PAF to its G-protein-coupled seven transmembrane receptor in HUVEC induces a signaling cascade involving Src and JAK-2, leading to phosphorylation of the transcription factor STAT-3. Others have demonstrated that stimulation of cells by PAF leads to activation of the transcription factor NF-B, shown to be involved in MT1-MMP induction in certain cancer cell lines, and induction of TNF-, IL-1, bFGF, and VEGF in HUVEC. Therefore, it is plausible that activation of endothelial cells by PAF leads to transcriptional activation of MMPs responsible for invasion of vascular endothelial cells. Here, we demonstrated that PAF is involved in endothelial cell invasion, possibly through the induction of specific proteases and activation of MMP2, but that PAF alone does not induce proliferation of vascular endothelial cells.

View larger version (47K): [in this window] [in a new window]   Figure 3. Schematic representation of MMP2 activation after PAF stimulation. [1] PAF is formed after stimulation of a receptor tyrosine kinase, which binds to and activates its G-protein-coupled seven-transmembrane receptor, leading to transcriptional activation of [2] proMT1-MMP and TIMP-2. [3] proMT1-MMP is processed by the proenzyme convertase furin, leading to cleavage of the prodomain. [4] Active MT1-MMP is bound to the extracellular surface of the membrane. [5] TIMP-2 is transported from the cell and binds to constitutively expressed latent proMMP2, which is [6] bound to the extracellular matrix (ECM). [7] Membrane bound MT1-MMP serves as a receptor for TIMP-2, localizing the TIMP-2/proMMP2 complex to the membrane, where a second active MT1-MMP cleaves the prodomain from proMMP2, releasing active MMP2 [8].

We show that antagonism of the PAF receptor inhibits proliferation of HUVEC in rich growth medium containing bFGF, VEGF, EGF, and insulin-like growth factor-1 (IGF-1), but that PAF alone does not induce proliferation in the absence of protein growth factors. This confirms our previously published findings that antagonism of the PAF receptor in HUVEC attenuates proliferation in the presence of bFGF. This indicates that in HUVEC, PAF is a necessary second messenger in bFGF-induced proliferation but is not sufficient to induce proliferation in the absence of the primary messenger bFGF, perhaps because stimulation of HUVEC with bFGF may induce a host of transcription factors that PAF alone cannot. Conversely, we demonstrate that PAF signaling is directly involved in vascular endothelial cell invasion and migration by basal medium conditioned by a human prostatic carcinoma cell line or by direct supplementation of culture medium with cPAF, indicating that the PAF signaling cascade alone is sufficient to induce migration and invasion. Because invasion requires the degradation of extracellular matrix components, our novel discovery that PAF activates MMP2 through the transcriptional activation of MT1-MMP and TIMP-2 further supports PAF as a key proangiogenic factor.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0479fje;

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