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Am J Physiol Heart Circ Physiol 292: H2773-H2781, 2007. First published February 23, 2007; doi:10.1152/ajpheart.01018.2006
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Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation

Weilin Zhou, Basil O. Ibe, and J. Usha Raj

Division of Neonatology, Harbor-University of California, Los Angeles (UCLA) Medical Center, Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, California

Submitted 17 September 2006 ; accepted in final form 20 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH2 terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC.

fetal pulmonary circulation; pulmonary vein

PLATELET-ACTIVATING FACTOR (PAF), a naturally occurring phospholipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), is involved in a variety of physiological and pathological events in many cell types and tissues (40), including vasoconstriction (7, 14) and regulation of cell proliferation (29, 46). We have previously reported that in the near-term fetal lamb, circulating levels of endogenous PAF are very high and that the levels fall dramatically immediately after birth (14). PAF receptor binding and PAF synthesis decrease, whereas PAF catabolism increases, in an immediate newborn period (19–21). Blocking the effects of endogenous PAF with the infusion of a specific PAF receptor antagonist into fetal lambs in vivo dramatically decreased pulmonary vascular resistance, suggesting that PAF contributes significantly to the maintenance of high vasomotor tone in the pulmonary circulation in utero (14). In the fetus, both pulmonary arteries and veins contribute equally to the high pulmonary vascular resistance in utero, and PAF is a potent vasoconstrictor of both arteries and veins in the fetal lung (7, 14, 35, 42). In fetal pulmonary venous (PV) smooth muscle cells (PV-SMCs), PAF synthesis is more than twofold greater, and PAF catabolism by PAF acetylhydrolase is lower than in arterial SMCs (15, 18), indicating that PAF may have a more important role in fetal pulmonary veins than in arteries. Since PAF has been shown to have mitogenic effects in systemic arterial SMCs (8, 10, 34), we wanted to investigate whether PAF can induce proliferation in pulmonary vascular SMC.

It is well established that stimulation of many G protein-coupled receptors (GPCRs) can activate signaling in receptor tyrosine kinases by a process termed transactivation (22). For example, angiotensin II, bradykinin, endothelin, phenylephrine, etc., stimulate their specific GPCRs to transactivate epidermal growth factor receptor (EGFR) (47), leading to induction of different signal transduction pathways, including mitogen-activated protein (MAP) kinase signaling cascades and thus initiate cellular processes such as proliferation, differentiation, development, stress responses, and apoptosis. Since PAF receptor is a member of GPCRs, we hypothesized that PAF may act as a mitogen in SMC by activation of PAF receptor and subsequent transactivation of a specific receptor tyrosine kinase. Our results indicate that PAF has a mitogenic effect in ovine fetal pulmonary vascular SMCs and that transactivation of EGFR is involved in this process.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. PAF (C-16), epidermal growth factor (EGF), AG-1478 (selective inhibitors of EGFR kinase), and GM-6001 (inhibitors of pan matrix metalloproteinases, MMP) were obtained from Biomol (Plymouth Meeting, PA). CRM-197 [inhibitor of heparin binding-EGF, heparin-binding (HB)-EGF release] was obtained from Sigma (St. Louis, MO), and rabbit polyclonal antibodies against EGFR and phospho-c-Jun NH2-terminal kinase (JNK) were from Santa Cruz Biotech (Santa Cruz, CA). Rabbit polyclonal antibodies against phospho-EGFR (Tyr1068), p44/42 MAP kinase (ERK1/2), phospho-p44/42 MAP kinase, and phospho-p38 MAP kinase were purchased from Cell Signaling (Beverly, MA).

Cell culture. PV-SMCs used in this study were cultured from near-term ovine fetal fourth through sixth generation intrapulmonary veins and were prepared as previously reported (19). Briefly, loosely adhering adventitia of the isolated veins was removed by forceps dissection. Vessels were predigested for 30–45 min at 37°C in six-well plates with 2–3 ml of 2 mg/ml collagenase I solution and then washed with sterile phosphate-buffered saline (PBS) solution to remove the collagenase. Any remaining adventitia and inner layers were removed by gentle mechanical manipulation under a magnifying glass. The vessels were rinsed three times with PBS and then placed in 3 ml of Dulbecco Modified Eagle's medium (DMEM) containing 2 mg/ml collagenase II solution. This was incubated for <2 h with periodic agitation to aid in the digestion of the SMCs from the vessels. Digestion was quenched by the addition of 10 ml of DMEM supplemented with 10% fetal bovine serum (FBS) and then filtered through a cell strainer with a mesh size of 100 µm (BD Bioscience) to remove tissue debris. The filtrate containing the cells was centrifuged at 1,000 rpm for 10 min. Cell pellet was resuspended and seeded in cell culture plate at the concentration of 4,000 cell/cm2. Cells were confirmed to be SMCs by their typical "hill and valley" morphology and by {alpha}-smooth muscle actin immunofluorescent staining. Contamination with endothelial cells was ruled out by negative immunofluorescent staining with an anti-von Willebrand factor VIII antibody. The cells were grown at 37°C in DMEM with 10% FBS and were used between passage 4 and 6.

DNA constructs and transfections. Transfection was performed using Lipofectamin (Invitrogen). Briefly, cells were seeded at a density of ~6.3 x 104/cm2 in DMEM with 10% FBS and allowed to attach overnight. Transfection was performed 1 day after seeding by using 1 µg of DNA and 2 µl of Lipofectamin per 105 cells following the manufacturer's protocol. Selection was started 48 h after transfection using 500–1,000 µg/ml G418 (Invitrogen). A plasmid containing dominant-negative EGFR cDNA pcDNA-HERCD533 was provided by Dr. Sylvain Meloche, Institut de Recherches Cliniques de Montréal, Montreal, Canada. The EGFR mutant lacking 533 COOH-terminal amino acids was subcloned into pcDNA3 and then transfected to PV-SMCs (44). A construct with human HB-EGF tagged with alkaline phosphatase (AP) HB-EGF-AP cDNA in pRC/CMV vector, a gift from Dr. Shigeki Higashiyama, Ehime University School of Medicine, Shitsukawa, Japan (43), was also transfected to PV-SMCs.

Determination of DNA synthesis employing [3H]thymidine incorporation. To assess DNA synthesis in cells exposed to PAF with or without inhibitors, [3H]thymidine incorporation method was determined as previously described (17). Briefly, cells were seeded on 24-well plates, allowed to grow in standard culture conditions to subconfluence, and starved with DMEM with 0.5% FBS overnight. Cells were treated in the presence of different inhibitors or just the inhibitor solvent for 30 min, followed by addition of PAF or PAF vehicle (ethanol) for 24 h. The concentration of PAF used in all experiments was 10 nM because this was the optimal concentration determined from the concentration-response experiments. The inhibitors used were AG-1478 (250 nM dissolved in DMSO), GM-6001 (10 µM in DMSO), and CRM-197 (3 µg/ml in distilled H2O). During the last 6 h of the incubation period, the cells were labeled with [3H]thymidine (0.5 µCi/ml; Perkin-Elmer, Wellesley, MA) and then harvested 6 h afterward. The incubations were terminated by aspirating the medium and washing the monolayers three times with chilled PBS, two times with chilled 5% trichloroacetic acid (5 min). The monolayers were lysed with 0.5 N NaOH, and the aliquots were then used for liquid scintillation counting by a Beckman scintillation spectrometer. The relative [3H]thymidine incorporation was expressed as a percentage of cells treated with vehicle. Each data point represents the mean ± SE of three independent experiments, and each same condition was performed in at least six different wells.

Determination of cell number. Cell numbers were determined by cell-counting kit-8 (Dojindo Chemicals, Gaithersburg, MD), which is based on a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay, the WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] assay. WST-8 is reduced by dehydrogenases in cells to give a yellow-colored product (formazan), which is soluble in the tissue culture medium, and the amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. Cells were plated in 96-well plates at 2.5 x 103 cells per well, cultured in the growth medium, and starved overnight in medium with 0.5% FBS. After starvation, the cells were treated in the presence of different inhibitors or just the inhibitor solvent for 30 min, followed by addition of PAF or PAF vehicle (ethanol) for 72 h at 37°C in a 5% CO2 incubator. The concentrations of PAF and inhibitors were the same as in [3H]thymidine incorporation experiments. To compensate for degradation of drugs during the 72-h culture period, medium with inhibitors or PAF was changed everyday. At the indicated time points, 10 µl of WST-8 were added to each well, and after 2-h incubation under 95% N2-5% CO2 at 37°C, the solubilized formazan product was quantified spectrophotometrically by using a microplate reader under the absorbance at 450 nm. For each experimental condition, eight wells were used for each experimental and control condition. This experiment was then repeated three times and means ± SE were reported. To ensure the optical density at 450 nm was proportionate to the cell number in each well, a calibration curve was prepared by using the data obtained from the wells that contained known numbers of viable cells.

Preparation of cell extracts and Western blot. Cells were plated in 100-mm Petri dishes and rendered quiescent at confluence by incubation in fresh DMEM with 0.5% FBS for 24 h. Growth-arrested PV-SMCs were incubated with PAF or vehicle (ethanol) at 37°C for the indicated times. When inhibitors were used, they were applied 30 min before the addition of PAF. The concentrations of PAF and inhibitors were the same as in [3H]thymidine incorporation experiments. After incubation, the cells were then rapidly washed with ice-cold PBS and lysed with ice-cold cell lysis buffer (Cell Signaling). Protease inhibitors including phenylmethylsulfonyl fluoride (1 mM) and protease inhibitor cocktail tablets (added as instructed in the protocol, Roche) were freshly added to the cell lysis buffer. Phosphatase inhibitor sodium fluoride (1 mM) was also added, but sodium orthovanadate was omitted because it is already included in the cell lysis buffer from Cell Signaling. Concentration of protein was determined with the BCA reagents (Pierce, Rockford, IL). Samples were subjected to SDS-PAGE and transferred to membranes. Membranes were probed with primary antibodies at 4°C overnight. Antibody complexes were detected on immunoblots by chemiluminescence using SuperSignal West Pico substrate solution (Pierce) according to the manufacturer's instructions. Densitometric analysis of bands was performed using Un-Scan-it software (Silk Scientific). The phosphorylation ratio was normalized with total ERK expression and then expressed as fold increase compared with vehicle. Each bar represents the mean ± SE of at least three independent experiments.

Quantitative measurement of EGFR ligand shedding. Transfectants expressing AP-tagged HB-EGF were seeded in 96-well plates at 2 x 104 cells/well and incubated for 24–48 h under subconfluent conditions. After treatment, conditioned media from different time points were collected and assayed simultaneously using a commercially available secreted AP chemiluminescent assay kit (Roche), using purified secreted human AP as standard, to ensure the readings were within the linear range. AP activity of each unknown sample is expressed as the amount of AP (according to the calibration curve) normalized with the total protein in each well. Each data point represents the mean ± SE of three different experiments performed in triplicate.

Gelatin zymography. Gelatinolytic activity of proteins in conditioned medium of PV-SMCs was assayed as previously described (12). Cells were pretreated with or without inhibitors, including a specific PAF receptor inhibitor WEB-2170 (10 µM) (11) or pan MMP inhibitor GM-6001 (10 µM) 30 min, and then treated with 10 nM PAF for indicated times. After treatment, conditioned medium was collected and concentrated by Speed Vac (Savant). Gelatin zymography was performed on a 10% polyacrylamide gel containing 0.1% gelatin (Invitrogen) under nonreducing conditions. Samples were subjected to electrophoresis in 4°C at 125 V until the dye front reached the end of the gels. The gels were then washed with 2.5% Triton X-100 in room temperature for 1 h and incubated in development buffer with 50 mM Tris, 5 mM CaCl2, 0.5 mM ZnCl2, pH 7.0, for 24–48 h at 37°C. Gels were stained with Coomassie blue and destained in 40% methanol-10% acetic acid. Nonstaining regions of the gel corresponding to gelatinase activity were quantified by Un-Scan-it software and expressed as fold increase compared with vehicle. Each bar represents the mean ± SE of three independent experiments.

Statistical analysis. Significant differences between two means were determined by an unpaired Student's t-test. Differences among the means of more than two groups were determined by using analysis of variance and a Dunnett's multiple comparison tests. Statistical significance was accepted at P < 0.05 or <0.01.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PAF induces cell proliferation and phosphorylation of MAP kinases in a dose-dependent manner. We measured PAF effects on cell proliferation by using two methods. Data from the [3H]thymidine incorporation method showed that 24-h treatment of PAF in PV-SMCs induced statistically significant increases of DNA synthesis at the concentration of 1, 10, and 100 nM (~40%, 50%, and 70% increase, respectively, Fig. 1A). With the use of a modified MTT dye reduction assay, the cell number of PV-SMCs after 72-h treatment of PAF at the concentrations of 1, 10, and 100 nM also increased significantly by ~40%, 55%, and 70% increase, respectively (Fig. 1B). The cell proliferation data by both methods suggested that PAF induced PV-SMC cell proliferation in a dose-dependent manner. In all subsequent experiments, 10 nM of PAF was used because this level of PAF was measured in the circulation of fetal lambs in vivo (14).


Figure 1
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  		Fig. 1. Platelet-activating factor (PAF) induces smooth muscle cell-pulmonary venous (PV-SMC) proliferation in a dose-dependent manner. A: for [3H]thymidine incorporation studies, PV-SMCs were treated at different concentration of PAF or vehicle for 24 h. *Significant difference vs. vehicle (0 M PAF treatment) (P < 0.01). B: for determination of cell number by WST-8 assay, PV-SMCs were treated with different concentration of PAF or vehicle for 72 h. *Significant difference vs. vehicle (P < 0.01).

 
Activation of MAP kinase, including ERK, JNK, and p38 MAP kinase, plays an important role in cell proliferation (24). Treatment of PV-SMCs with 10 nM PAF caused a rapid increase in phosphorylation of ERK1/2 (2, 5, and 10 min) and p38 (2 and 5 min) MAP kinases (Fig. 2A). Phosphorylation of ERK1/2 and p38 MAP kinases decreased after 30 min of PAF treatment. There was no statistical difference between phosphorylation at 0- and 30-min treatment points. PAF treatment has no significant effect on phosphorylation of JNK MAP kinase. Treatment of PAF at different concentrations for 2 min in PV-SMVs also induced dose-dependent phosphorylation of ERK1/2 and p38 MAP kinases (Fig. 2B). These results provide evidence that in PV-SMCs, PAF can induce rapid activation of ERK1/2 and p38 MAP kinases, which may play an important role in PAF-induced cell proliferation.


Figure 2
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  		Fig. 2. PAF induces phosphorylation of mitogen-activated protein (MAP) kinases in a dose-dependent manner. A: representative Western blots (top) and bar graphs (bottom) from PV-SMCs treated with 10 nM PAF in different time points. *Significant difference vs. vehicle (0 min treatment) (P < 0.05). B: representative Western blots and bar graphs from PV-SMCs treated with PAF at different concentrations for 2 min. *Significant difference vs. vehicle (P < 0.05).

 
PAF-induced cell proliferation and MAP kinase phosphorylation are dependent on the transactivation of EGFR. Pretreatment of PV-SMCs with 250 nM AG-1478, a selective EGF tyrosine kinase inhibitor, abolished PAF-induced PV-SMCs proliferation (Fig. 3, A and B) and inhibited PAF-induced MAP kinase phosphorylation after 2 and 5 min (Fig. 4A). After exposure of PV-SMCs to 10 nM PAF, there was a significant increase in EGFR phosphorylation within 5 min (Fig. 4B), demonstrating an effect of PAF on activation of EGFR.


Figure 3
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  		Fig. 3. PAF-induced PV-SMC proliferation is blocked by inhibitors of epidermal growth factor receptor (EGFR), matrix metallaproteinase (MMP) or heparin-binding EGF (HB-EGF). A: for [3H]thymidine incorporation studies, PV-SMCs were pretreated with inhibitors or vehicle for 30 mins and then treated with 10 nM PAF or vehicle for 24 h. *Significant difference vs. vehicle (P < 0.01). #Significant difference vs. PAF only (P < 0.01). B: for determination of cell number, PV-SMCs were pretreated with inhibitors or vehicle for 30 min and then treated with 10 nM PAF or vehicle for 72 h. *Significant difference vs. vehicle (P < 0.01). #Significant difference vs. PAF only (P < 0.01).

 

Figure 4
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  		Fig. 4. PAF transactivates EGFR in PV-SMCs. A: representative Western blots (top) and bar graphs (bottom) from PV-SMCs pretreated with 250 nM AG-1478 or DMSO for 30 min followed by treatment with 10 nM PAF or PAF vehicle for indicated times. *Significant difference vs. vehicle (0 min treatment) (P < 0.05). #Significant difference vs. PAF+AG-1478 (P < 0.05). B: representative Western blots (top) and bar graph (bottom) from PV-SMCs with 10 nM PAF treatment for 5 min. *Significant difference vs. vehicle (0 min treatment) (P < 0.05). C: PAF induces less cell proliferation in a dominant-negative EGFR mutant-transfected PV-SMCs. The pcDNA3-HERCD533 (dominant-negative EGFR mutant) and pcDNA3 (vector only) transfected cells were treated with 10 nM PAF, 3 ng/ml EGF, or vehicle for 24 h. *Significant difference vs. control (P < 0.05). #Significant difference vs. in pcDNA3-HERCD533 transfected cells (P < 0.05).

 
Expression of a dominant-negative EGFR mutant, pcDNA3-HERCD533, in PV-SMCs resulted in less mitogenic effects from EGF stimulation (3 ng/ml) compared with expression of pcDNA3 vector alone, which confirmed that EGFR activity was partially blocked in pcDNA-HERCD533-transfected cells. In these cells expressing a dominant negative EGFR mutant, there was marked attenuation of PAF-induced cell proliferation (Fig. 4C), which also supports the observation that EGFR is involved in PAF-induced mitogenic effect in PV-SMCs. All the above results demonstrate that PAF-induced activation of MAP kinase and PV-SMC proliferation are at least in part mediated by EGFR transactivation.

PAF-induced MAP kinase phosphorylation is dependent on HB-EGF. EGFR ligand family includes EGF, transforming growth factor (TGF)-{alpha}, amphiregulin, HB-EGF, beta-cellulin, epiregulin, and epigen (33). There is evidence that EGFR transactivation occurs either by direct induction of EGFR tyrosine kinase activity or by release (shedding) of EGFR ligands such as HB-EGF (32). To determine whether HB-EGF is involved in PAF-induced EGFR transactivation, the effect of CRM-197 on PAF-induced MAP kinase phosphorylation was examined. CRM-197 is a nontoxic mutant of diphtheria toxin that binds and internalizes pro-HB-EGF and thus inhibits HB-EGF release from cells (22, 32). A 30-min pretreatment of PV-SMCs with 3 µg/ml CRM-197 inhibited PAF-induced MAP kinase phosphorylation (Fig. 5A) and also abolished PAF-induced cell proliferation (Fig. 3, A and B).


Figure 5
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  		Fig. 5. HB-EGF is released quickly after PAF treatment. A: representative Western blot (top) and bar graphs (bottom) from PV-SMCs pretreated with 3 µg/ml CRM-197 or vehicle for 30 min followed by treatment with 10 nM PAF or PAF vehicle for indicated times. *Significant difference vs. vehicle (0 min treatment) (P < 0.05). #Significant difference vs. PAF+CRM-197 (P < 0.05). B: PAF induced HB-EGF release in HB-EGF-AP transfected PV-SMCs. AP-tagged HB-EGF transfectants were treated with 10 nM PAF or ethanol for the indicated time and culture media were collected and assayed. *Significant difference vs. vehicle (basal) (P < 0.05).

 
Once released from pro-HB-EGF shedding, HB-EGF binds to heparan sulfate proteoglycans (HSPG), which restricts diffusion, provides a matrix depot, and increases the affinity of HB-EGF for EGFR (33). The binding of HB-EGF to HSPG is important for EGFR activation by HB-EGF, but it can also interfere with the measurement of pro-HB-EGF shedding in cell culture medium (32). To further test our hypothesis, we used an alternative way to measure HB-EGF release by measuring AP activity in AP-tagged HB-EGF-transfected cells. Our results showed that addition of PAF increased AP activity of the transfectants within 1 min, indicating that PAF quickly induces HB-EGF release (Fig. 5B). These experiments provide substantial evidence in support of the concept that stimulation of PV-SMC proliferation by PAF is mediated by cleavage of membrane-bound pro-HB-EGF and that the released HB-EGF transactivates EGFR.

MMP activation is required for PAF-induced HB-EGF release. To determine the mechanism by which HB-EGF release occurs, we examined the effects of GM-6001, a pan MMP inhibitor, on PAF-induced MAP kinase phosphorylation and cell proliferation by pretreating PV-SMCs with 10 µM GM-6001 for 30 min. GM-6001 significantly inhibited ERK1/2 and p38 MAP kinase activation induced by PAF (Fig. 6A) and abolished PAF-induced cell proliferation (Fig. 3, A and B). These results support our hypothesis that the mechanism of PAF-induced MAP kinase phosphorylation and cell proliferation involves MMP activation with subsequent HB-EGF release and EGFR transactivation.


Figure 6
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  		Fig. 6. PAF activates MMP in PV-SMCs. A: representative Western blot (top) and bar graphs (bottom) from PV-SMCs pretreated with 10 µM GM-6001 or DMSO for 30 min followed by treatment with 10 nM PAF or PAF vehicle for indicated times. *Significant difference vs. vehicle (0 min treatment) (P < 0.05). #Significant difference vs. PAF+GM-6001 (P < 0.05). B: representative gelatin zymography gel (top) and bar graphs (bottom) show that PAF activated MMP-2 and MMP-9 within 1 min. *Significant difference vs. vehicle (0 min treatment) (P < 0.05). C: representative gelatin zymograph gels show that GM-6001 (top) and WEB-2170 (bottom) inhibited PAF-induced MMP activation.

 
To identify the particular metalloproteinase(s) responsible for pro-HB-EGF cleavage in PV-SMCs, MMP activity in conditioned media of cultured PV-SMCs was analyzed by gelatin zymography. The culture media from PV-SMCs treated for 1 min with PAF contained higher proteolytic activity at 62 kDa (~30% increase) and 82 kDa (~9-fold increase), corresponding to MMP-2 and MMP-9, respectively, compared with the media from vehicle-treated cells (Fig. 6B). MMP-9 activity was also higher within 10 min after PAF treatment. However, under the same experimental conditions, the proteolytic activity at other apparent molecular weights was not significantly changed. These effects could be inhibited by pretreatment of PV-SMCs with the MMP inhibitor GM-6001 and the PAF receptor antagonist WEB-2170 (Fig. 6C). These results suggest that 10 nM PAF can induce MMP-2 and MMP-9 activation in PV-SMCs within 1 min.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of our study demonstrate that PAF induces fetal PV-SMC proliferation, and that this effect is mediated through transactivation of EGFR. This is the first demonstration of PAF as a stimulator of pulmonary vascular smooth muscle cell proliferation. Several studies have shown that arterial and venous SMC may have heterogeneous characteristics and may respond differently to various growth factors (23, 25). Saphenous vein SMC are more dedifferentiated and more proliferative than aortic SMC in response to platelet-derived growth factor or FBS (27, 48). Since the availability of PAF in that PV-SMC is much higher than that in pulmonary arterial SMC (15, 18), it is reasonable to speculate that the effect of PAF on pulmonary arterial SMC might be different from that in veins. In future studies, the effect of PAF on pulmonary arterial SMC proliferation will be studied.

Our studies also showed that PAF-induced ERK1/2 and p38 MAP kinase phosphorylation is dependent on EGFR transactivation. Our results parallel those of the effects of angiotensin II in rat thoracic aorta smooth muscle cells (6) but are different from the study with PAF in keratinocytes, which showed that only ERK1/2 activation, but not p38 MAP kinase, was dependent on EGFR transactivation (29). This discrepancy may be related to the different cell types studied.

Transactivation is considered to be one of various types of cross-talk occurring among different receptors. Activation of many GPCRs can transactivate receptor tyrosine kinases, such as EGFR, by several mechanisms. EGFR is endogenously expressed in numerous cell types and is an important factor in the control of many fundamental cellular processes (39). EGFR-binding members comprise EGF, TGF-{alpha}, HB-EGF, amphiregulin, epiregulin, beta-cellulin, and epigen (33). All family members are synthesized as membrane-anchored precursors that can be processed by specific metalloproteases to release soluble bioactive factors from the cell surface. Recently, many reports have shown that HB-EGF shedding plays a central role in GPCR-mediated EGFR transactivation. Our results also suggest that PAF-induced EGFR transactivation in PV-SMCs is mediated through HB-EGF release. HB-EGF is synthesized as a proligand (pro-HB-EGF) that is anchored to the plasma membrane and released upon proteolytic cleavage (32). HB-EGF is a potent mitogen of fibroblasts, keratinocytes, and SMCs. In SMC, HB-EGF and platelet-derived growth factor-B have similar mitogenic potency, whereas EGF is much less potent (33).

There is evidence that MMP and/or a disintegrin and metalloproteinases (ADAMs) are implicated in HB-EGF shedding. Several MMPs (MMP-3 and MMP-7) and ADAMs (ADAM-10, ADAM-12, and ADAM-17) have been identified as mediating EGFR ligand (including HB-EGF) shedding and/or EGFR transactivation in response to distinct GPCR agonists (9, 26, 28, 37, 38, 50). The results of the present studies demonstrate that PAF is able to increase MMP-2 and MMP-9 activity, which may play a role in HB-EGF shedding. Our data are different from the report by Lemjabbar and Basbaum (26) in a human epithelial cell line HM3. They found that a Gram-positive bacterial wall component lipoteichoic acid activates PAF receptor, which results in activation of ADAM-10, cleavage of pro-HB-EGF, activation of the EGFR and finally stimulation of mucin production (26). Therefore, which particular MMP or ADAM is responsible for pro-HB-EGF cleavage is dependent on the type of GPCR agonist as well as the type of cells or tissues studied.

Rapid transactivation of EGFR by different GPCRs in different cell types has been reported. In the colonic epithelial cell line T84 cell, EGFR reaches the maximal phosphorylation within 0.5 min after addition of vasoactive intestinal polypeptide (1). Phenylephrine induces rapid (≤5 min) phosphorylation of EGFR and ERK1/2 in the media of rat aorta maintained in organ culture (49), whereas increased EGFR phosphorylation is observed after 3 min treatment of various GPCRs, including angiotensin II, endothelin-1, and lysophosphatidic acid in rat fibroblasts, human renal carcinoma cell line ACHN cells, and TccSup bladder cancer cells (26, 38). Our data show that PAF induces MMP activation and HB-EGF release in PV-SMCs within 1 min. PAF also induces phosphorylation of EGFR, ERK1/2, and p38 MAP kinase within 5 min.

Little is known about PAF-induced transactivation of receptor tyrosine kinase in different cell types. There is only one study in keratinocytes that shows that PAF-induced ERK activation and increased cell proliferation were blocked by the selective EGFR tyrosine kinase inhibitor AG-1478 (29). In conflict with this finding, in HEK-293 cells, although of epithelial origin, PAF-stimulated ERK1/2 phosphorylation was not dependent on EGFR transactivation. AG-1478, even at the highest concentration of 250 nM, which is almost 100 times of the IC50 value, did not decrease PAF-induced ERK1/2 activation. However, at a concentration of 25 nM, AG-1478 significantly blocked EGFR- or beta2-adrenergic receptor-mediated ERK1/2 activation (4). These results suggest that PAF receptor-mediated transactivation of EGFR and subsequent MAP kinase activation may depend on cell types.

PAF has also been implicated in the pathogenesis of hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in animal models (2, 28, 31). Most studies have focused on remodeling of pulmonary arteries; however, there is increasing evidence of vascular remodeling in pulmonary veins in humans and in experimental models of pulmonary hypertension due to hypoxia and other causes (3, 5, 13, 30, 36, 41, 45). We have shown that under hypoxic conditions, similar to that in utero, fetal pulmonary vascular SMC demonstrate greater PAF receptor binding and cell signaling than in normoxia and that PAF receptor binding was greater in PV-SMCs than in fetal pulmonary arterial SMC (16). Our present results provide one mechanism for the remodeling of pulmonary veins in hypoxia-induced pulmonary hypertension.

In conclusion, this study is the first to demonstrate that PAF-stimulated cell proliferation in pulmonary venous SMC is dependent on transactivation of EGFR. PAF receptor activation results in activation of MMP-2 and MMP-9, leading to the release of HB-EGF and the resultant activation of ERK1/2, p38 MAP kinase, and cell proliferation. Our results provide a mechanism for PAF-induced pulmonary venous smooth muscle cell proliferation in the fetus. We speculate that in the fetus, PAF may not only serve as a physiological vasoconstrictor in the pulmonary circulation but may also play a role in pulmonary vascular smooth muscle cell proliferation and vessel growth.


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 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-077819 (to J. U. Raj).


   ACKNOWLEDGMENTS
 
The authors thank Drs. Y. Gao, S. Negash, and J. Liu for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. Zhou, Div. of Neonatology, Harbor-UCLA Medical Center, Los Angeles Biomedical Institute, 1124 West Carson St., Torrance, CA 90502 (e-mail: wzhou{at}labiomed.org)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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