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(Circulation Research. 1995;77:530-535.)
© 1995 American Heart Association, Inc.

Articles

Elevated Levels of cAMP Inhibit Protein Kinase C– Independent Mechanisms of Endothelial Platelet-Derived Growth Factor–B Chain and Intercellular Adhesion Molecule-1 Gene Induction by Lysophosphatidylcholine

Hiroshi Ochi, Noriaki Kume, Eiichiro Nishi, Toru Kita

From the Department of Geriatric Medicine, Faculty of Medicine, Kyoto University, Japan.

Correspondence to Noriaki Kume, MD, PhD, Department of Geriatric Medicine, Faculty of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan.

	Abstract

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Abstract Lysophosphatidylcholine (lyso-PC), a polar phospholipid product increased in atherogenic lipoproteins and atherosclerotic lesions, has been shown to differentially induce functional intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 and mRNA for platelet-derived growth factor (PDGF)–A and –B chains and heparin-binding epidermal growth factor–like growth factor in various cultured endothelial cells. In this study, we have demonstrated increased expression of cell- and matrix-associated forms of PDGF–B chain (PDGF-B) protein elicited by lyso-PC and further characterized potential signal transduction mechanisms responsible for lyso-PC–induced gene expression, focusing on PDGF-B and ICAM-1 genes in cultured human umbilical vein endothelial cell models. Cycloheximide almost completely inhibited PDGF-B but not ICAM-1 mRNA induction elicited by lyso-PC, suggesting that dependence on de novo protein synthesis for PDGF-B is different from that for ICAM-1. Prolonged exposure to phorbol myristate acetate (PMA), which depletes protein kinase C (PKC), or staurosporine, a PKC inhibitor, did not block lyso-PC–induced increases in PDGF-B or ICAM-1 mRNA. Forskolin and dibutyryl cAMP, which elevate intracellular cAMP levels, blocked both PDGF-B and ICAM-1 upregulation elicited by lyso-PC; however, these cAMP-elevating agents did not suppress ICAM-1 upregulation by PMA. Taken together, PDGF-B and ICAM-1 gene induction by lyso-PC may involve different signaling mechanisms; however, both appear to be independent of PMA-regulatable PKC activation but are suppressed by increased levels of intracellular cAMP.

Key Words: protein kinase C • platelet-derived growth factor–B chain • intercellular adhesion molecule-1 • cAMP • lysophosphatidylcholine

	Introduction

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Vascular endothelium plays a pivotal role in the pathogenesis of vascular diseases, including atherosclerosis and inflammation, expressing endothelial-leukocyte adhesion molecules, growth factors, and cytokines in response to various pathophysiological stimuli.^{1 2 3} Lyso-PC, which is a prominent phospholipid component of atherogenic lipoproteins⁴ and is also generated in inflammatory lesions by extracellular phospholipase A₂ activities,⁵ has been shown to differentially upregulate VCAM-1 and ICAM-1 expression in various cultured endothelial cells, showing its potential role in mononuclear leukocyte recruitment into these lesions.⁶ Lyso-PC has been demonstrated to induce gene expression of potent smooth muscle growth factors such as PDGF–A and –B chains and HB-EGF in cultured human endothelial cells, thus suggesting its potential role in smooth muscle proliferation.⁷ Lyso-PC has been shown to act on human monocytes as well to induce HB-EGF gene expression.⁸ These effects of lyso-PC on endothelial genes appear to be specific and distinct from those elicited by cytokines, such as IL-1 and TNF-, since neither E-selectin nor IL-8 was upregulated in the same cells stimulated with lyso-PC. Nuclear runoff assays and the evaluation of mRNA half-lives have indicated that lyso-PC stimulates transcription of these genes⁷ ; however, signal transduction mechanisms responsible for the gene induction of adhesion molecules and growth factors by this lyso-PC stimulus have not been fully elucidated. Furthermore, the effects of lyso-PC on growth factor protein expression have not been determined in cultured endothelial cells.

In the present study, therefore, we have measured PDGF-B protein levels in lyso-PC–treated cultured HUVECs and explored the potential signal transduction mechanisms involved in both PDGF-B and ICAM-1 upregulation elicited by lyso-PC, focusing on roles of PKC and cAMP.

	Materials and Methods

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Cells
Cultured HUVECs were isolated by collagenase digestion and grown in medium 199 with 20% (vol/vol) heat-inactivated FBS (Irvine Scientific), supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, 25 U/mL heparin (porcine intestinal mucosa, Sigma Chemical Co), and 30 mg/mL ECGS (Sigma) as previously described.⁹ All experiments were carried out within 3 days after HUVECs reached confluence. Cells (passage numbers between 2 and 4) were incubated with or without test stimuli in medium 199 supplemented with 5% (vol/vol) FBS, without heparin or ECGS.

Reagents
Lyso-PC (palmitoyl, C16:0) was purchased from Avanti Polar Lipids. Cycloheximide, PMA, forskolin, and Bt₂cAMP were obtained from Sigma, and staurosporine was from Calbiochem. All other reagents were of reagent grade.

Northern Blot Analysis
Total cellular RNA, isolated from HUVECs by the acid guanidinium–phenol–chloroform method,¹⁰ was electrophoresed through 1% agarose gels containing formaldehyde, transferred onto 0.45-µm nylon membranes (Zeta-Probe, Bio-Rad), and fixed by UV cross-linking. Northern membranes were hybridized with human ICAM-1 and PDGF-B cDNA probes, which were labeled with [-³²P]dCTP (DuPont NEN) by using random hexanucleotide primers (Pharmacia) at 65°C for 18 hours in a mixture containing 1 mmol/L EDTA, 0.25 mol/L Na₂HPO₄ (pH 7.2), and 7% SDS. The filters were subsequently washed at 65°C twice with 1 mmol/L EDTA, 40 mmol/L Na₂HPO₄ (pH 7.2), and 5% SDS and once with 1 mmol/L EDTA, 40 mmol/L Na₂HPO₄ (pH 7.2), and 1% SDS and exposed to x-ray films. A 1.3-kb Xho I fragment of human ICAM-1, kindly provided by Dr Brian Seed (Massachusetts General Hospital, Boston), was used to detect ICAM-1 mRNA.^{6 7 11} A 2.1-kb BamHI fragment of human PDGF-B cDNA,¹² obtained from American Type Culture Collection, and a Pst I–EcoRI fragment of human PDGF-B,¹³ kindly provided by Dr Tucker Collins (Brigham and Women's Hospital, Boston, Mass) were used as hybridization probes. Northern analyses using these PDGF-B cDNAs gave similar results. Some blots were rehybridized with radiolabeled human ß-actin cDNA to control the amounts of RNA loaded. Densitometric scanning was performed to quantify the amounts of mRNA; an Image Master laser densitometer (Pharmacia) was used. Relative amounts of mRNA for PDGF-B and ICAM-1 were normalized to ß-actin mRNA levels.

Western Blot Analysis
Laemmli sample buffer (2% SDS, 10% glycerol, 60 mmol/L Tris [pH 6.8], and 0.001% bromophenol blue) was directly poured into HUVEC culture plates, and the cell lysates were passed through 25-gauge needles 10 times. After heating at 98°C for 10 minutes, samples were subjected to SDS–polyacrylamide (10% to 20% gradient) gel electrophoresis in nonreducing conditions and transferred onto nitrocellulose filters (Hybond ECL filters, Amersham Corp) by electroblotting. After preincubation with TBS (50 mmol/L Tris-Cl [pH 8.0], 2 mmol/L CaCl₂, 100 mmol/L NaCl, and 5% [wt/vol] nonfat dry milk) for 3 hours at room temperature, filters were incubated with a rabbit polyclonal antibody directed to PDGF-BB homodimer (Genzyme Corp) diluted in TBS at room temperature for 2 hours, followed by washing twice with TBS without nonfat dry milk. Filters were then incubated with the horseradish peroxidase–conjugated anti-rabbit IgG antibody (Amersham) diluted in TBS for 2 hours at room temperature, washed twice in TBS without nonfat dry milk, and visualized by use of a chemiluminescence reagent (ECL kit, Amersham Corp).

Statistical Analysis
Statistical analyses were carried out by paired Student's t test. Percent changes in mRNA levels compared with respective controls are expressed as mean±SD.

	Results

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Lyso-PC Induces Cell- and Matrix-Associated Forms of PDGF-B Protein
Lyso-PC has been shown to increase the amounts of mRNA for PDGF-B.⁷ To examine the effects of lyso-PC on PDGF-B protein levels, we performed Western blot analyses using cell lysate from lyso-PC–treated HUVECs. As shown in Fig 1, multiple bands with approximate molecular masses between 38 and 43 kD, which appear to be compatible with previous reports,^{14 15 16} were detected by treatments with lyso-PC for 8 hours and continuously increased for at least 20 hours. Similar bands of 38 to 45 kD were detectable in HUVECs treated with PMA for 8 hours. These bands disappeared when excess amounts of recombinant human PDGF-BB homodimer were included in the incubation buffer containing anti-human PDGF-B antibody (data not shown). Two additional bands with approximate molecular masses of 85 and 55 kD, respectively, appeared to be nonspecific, since these bands were similarly detectable when anti-human PDGF-B was replaced at a concentration equivalent to that of nonimmune rabbit serum (data not shown).

View larger version (83K): [in this window] [in a new window]   Figure 1. Time course of PDGF-B protein expression induced by lyso-PC. Confluent monolayers of HUVECs were treated with or without test reagents (100 nmol/L PMA or 70 µmol/L lyso-PC in medium 199 with 5% FBS) for the indicated times. Cell lysate was subjected to Western blot analyses as described in "Materials and Methods." Recombinant human (rh) PDGF-BB (5 ng per lane, Genzyme Corp) dissolved in Laemmli buffer was also included as a reference. Molecular mass standards (in kilodaltons) are indicated on the left. One of three similar results is shown. * denotes bands for PDGF-B.

PDGF-B mRNA Upregulation by Lyso-PC Depends on De Novo Protein Synthesis, but ICAM-1 Does Not
In previous studies, lyso-PC has been shown to upregulate mRNA for both ICAM-1 and PDGF-B as early as 4 hours of treatment. To determine whether de novo protein synthesis is required in this process, we treated HUVECs with or without lyso-PC (or PMA as a control) in the presence or absence of cycloheximide for 4 hours, and Northern blot analyses were performed. Cycloheximide completely blocked lyso-PC–induced, as well as PMA-induced, increases in PDGF mRNA levels, although cycloheximide alone modestly increased the amount of PDGF-B mRNA (Fig 2A). In contrast, cycloheximide dramatically increased ICAM-1 mRNA levels in both untreated and lyso-PC–treated or PMA-treated cells (Fig 2B). These results clearly indicate that dependence of de novo protein synthesis in lyso-PC–induced, as well as PMA-induced, gene upregulation is different between PDGF-B and ICAM-1.

View larger version (96K): [in this window] [in a new window]   Figure 2. Dependence of PDGF-B and ICAM-1 mRNA induction by lyso-PC or PMA on de novo protein synthesis. Confluent monolayers of HUVECs were treated for 4 hours with media alone, lyso-PC (60 µmol/L), or PMA (100 nmol/L) in the presence (+) or absence (-) of cycloheximide (10 µg/mL). Total cellular RNA was isolated, and Northern blot analyses were performed as described in "Materials and Methods" to detect PDGF-B mRNA (A) and ICAM-1 mRNA (B). Each lane contained 10 µg total RNA. One of two similar results is shown.

Induction of mRNA for Both PDGF-B and ICAM-1 Is Independent of PMA-Regulatable PKC Activation
To investigate the potential role of PKC in PDGF-B and ICAM-1 mRNA induction by lyso-PC, we followed two strategies: depletion of PKC activities by prolonged exposure to PMA and inhibition of PKC by a pharmacological reagent. As shown in Fig 3, pretreatment with PMA for 24 hours significantly blocked PDGF-B mRNA induction elicited by the subsequent stimulation with PMA (63±18% reduction); however, this PMA pretreatment did not inhibit lyso-PC–induced increases in PDGF-B mRNA. Effects of prolonged exposure to PMA on ICAM-1 mRNA upregulation is similar to those observed in PDGF-B; pretreatment with PMA did not prohibit lyso-PC–induced increases in ICAM-1 mRNA levels but completely blocked the effect of the subsequent stimulation with PMA (>99% reduction). We also examined the effects of staurosporine, an inhibitor of PKC. Staurosporine (10 nmol/L) significantly reduced PMA-induced increases in PDGF-B mRNA levels (44±12% reduction); however, this agent did not inhibit but rather increased those elicited by lyso-PC (Fig 4). These results indicate that signal transduction mechanisms responsible for PDGF-B and ICAM-1 mRNA upregulation elicited by lyso-PC in HUVECs appear to be dissociated from PMA-regulatable PKC activation. We further tested the effects of higher concentrations of staurosporine; however, >50 nmol/L concentrations of this reagent with 60 µmol/L lyso-PC caused morphologically apparent cytotoxicity.

View larger version (41K): [in this window] [in a new window]   Figure 3. Effect of prolonged exposure to PMA on PDGF-B and ICAM-1 mRNA levels. Confluent monolayers of HUVECs were pretreated with (+) or without (-) PMA (500 nmol/L in 5% FBS/medium 199) for 24 hours and subsequently incubated with lyso-PC (60 µmol/L) or PMA (100 nmol/L) for 4 hours. Northern blot analyses were carried out to measure PDGF-B (A) and ICAM-1 (B) mRNA levels. Each lane contained 10 µg total RNA. Relative amounts of mRNA were calculated as described in "Materials and Methods" (C). Mean values from four separate experiments are shown. **P<.001 vs respective controls without PMA pretreatment (solid bars).

View larger version (42K): [in this window] [in a new window]   Figure 4. Effects of staurosporine on PDGF-B mRNA levels induced by lyso-PC or PMA. Confluent monolayers of HUVECs were treated with media alone, lyso-PC (100 µmol/L), or PMA (100 nmol/L) in the presence (+) or absence (-) of staurosporine (10 nmol/L) for 4 hours. Total cellular RNA was isolated from each sample, and Northern blot analyses were performed as described. Each lane contained 10 µg total RNA. A, One of four separate experiments is shown. B, Relative amounts of PDGF-B mRNA were calculated as described in "Materials and Methods." Mean values from four separate experiments are shown. **P<.001 vs respective controls without staurosporine (solid bars).

Elevated Levels of cAMP Inhibit PDGF-B and ICAM-1 mRNA Induction Elicited by Lyso-PC
To explore the potential role of cAMP in lyso-PC–induced PDGF-B and ICAM-1 gene expression, we examined the effects of forskolin and Bt₂cAMP, both of which can increase intracellular cAMP levels. We treated HUVECs with or without lyso-PC or PMA in the presence or absence of forskolin and measured mRNA levels for PDGF-B and ICAM-1 by Northern analyses. As shown in Fig 5, forskolin significantly reduced lyso-PC–induced, as well as PMA-induced, increases in PDGF-B mRNA (59±19% and 68±19% reduction, respectively). In contrast, this agent inhibited only lyso-PC–induced increases in ICAM-1 mRNA (67±22% reduction) and did not significantly block those induced by PMA. Bt₂cAMP exhibited effects similar to those observed with forskolin. Upregulated expression of PDGF-B mRNA elicited by lyso-PC, as well as PMA, was significantly prohibited by Bt₂cAMP (54% and 30% reduction, respectively); however, this agent blocked only lyso-PC–induced ICAM-1 mRNA upregulation (46% reduction) but did not show any significant inhibitory effect on PMA-induced ICAM-1 mRNA expression (Fig 6). To examine whether elevated levels of cAMP decrease PDGF-B protein levels, we performed Western blot analyses. Treatment with optimal doses of lyso-PC or PMA for 20 hours significantly increased the levels of cell- and matrix-associated forms of PDGF-BB, which were effectively suppressed by forskolin (Fig 7).

View larger version (41K): [in this window] [in a new window]   Figure 5. Effects of forskolin on PDGF-B and ICAM-1 mRNA levels induced by lyso-PC or PMA. Confluent monolayers of HUVECs were treated with media alone, lyso-PC (100 µmol/L), or PMA (100 nmol/L) in the presence (+) or absence (-) of forskolin (25 µmol/L) for 4 hours. Total cellular RNA (12 µg per lane) was subjected to Northern blot analyses. Representative results for PDGF-B (A) and ICAM-1 (B) mRNA are shown. PDGF-B and ICAM-1 mRNA levels were semiquantified as described in "Materials and Methods" (C). Mean values from three (for ICAM-1) and five (for PDGF-B) separate experiments are shown. *P<.05 and **P<.001 vs respective controls without forskolin (solid bars).

View larger version (44K): [in this window] [in a new window]   Figure 6. Effects of Bt2cAMP on PDGF-B and ICAM-1 mRNA induction by lyso-PC or PMA. Confluent monolayers of HUVECs were treated for 4 hours with lyso-PC (60 µmol/L) or PMA (200 nmol/L) in the presence (+) or absence (-) of Bt2cAMP (1 mmol/L). Northern blot analyses were performed as described in Fig 5. One of two similar results is shown (A). Densitometric analyses were performed as described in "Materials and Methods" (B). Solid bars indicate control (without Bt2cAMP). Representative data from two separate experiments are indicated.

View larger version (57K): [in this window] [in a new window]   Figure 7. Effects of forskolin on PDGF-B protein expression induced by lyso-PC or PMA. HUVECs were treated for 20 hours with or without test reagents (200 nmol/L PMA and 60 or 70 µmol/L lyso-PC) in the presence or absence of forskolin (50 µmol/L). Cell lysate was subjected to Western blot analyses as described in Fig 1. One of two similar results is shown. * denotes bands for PDGF-B.

We also have examined the effects of combinations of reagents used in the present study on cell viability. We did not detect any evidence of cytotoxicity by either microscopic observation or measuring lactate dehydrogenase released into culture media (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endothelial expression of smooth muscle growth factors and endothelial-leukocyte adhesion molecules appears to play an important role in atherogenesis and inflammatory disease processes.^{1 2 3} Lyso-PC, a phospholipid stimulus relevant to atherosclerosis and inflammation, has been shown to selectively upregulate gene expression of adhesion molecules and growth factors, including PDGF-B and ICAM-1. In the present study, we have shown, for the first time, that cell- and matrix-associated forms of PDGF-B protein are increased by lyso-PC treatment in HUVECs. We have also explored the potential signal transduction mechanisms responsible for the PDGF-B and ICAM-1 upregulation by lyso-PC in HUVEC models.

Inhibition of nascent protein synthesis by cycloheximide provided contrasting results between PDGF-B and ICAM-1 gene regulation. PDGF-B mRNA induction by lyso-PC, as well as that elicited by PMA, requires de novo protein synthesis, but ICAM-1 does not. In fact, cycloheximide enhanced the accumulation of ICAM-1 mRNA in both lyso-PC–treated and untreated HUVECs; this finding appears to be similar to the previous finding with PMA-treated and TNF-–treated HUVECs.¹⁷ These results clearly indicate that signal transduction mechanisms responsible for lyso-PC–induced gene expression might be different between PDGF-B and ICAM-1.

PKC activation has been reported to be involved in lyso-PC–induced inhibition of endothelium-dependent vasorelaxation ex vivo, and transient activation of PKC has been detected in lyso-PC–stimulated endothelial cells.¹⁸ Furthermore, a recent report has shown that lyso-PC treatment resulted in increased expression of ICAM-1 protein in isolated porcine coronary arteries, which was efficiently blocked by PKC inhibitors.¹⁹ Therefore, we have explored the potential role of PKC in lyso-PC–induced endothelial gene upregulation of PDGF-B and ICAM-1 in our HUVEC models, in which selective patterns of gene regulation by lyso-PC have been well characterized.⁷ Data presented here have clearly demonstrated that upregulated expression of PDGF-B and ICAM-1 by lyso-PC in our HUVEC models appears to depend on mechanisms other than PMA-regulatable PKC activation. Previous studies by others, using PMA and staurosporine, have indicated that ICAM-1 upregulation by TNF- also is dissociated from PKC activation,^{20 21} thus supporting the existence of PKC-independent mechanisms of ICAM-1 gene induction.

Transient increases in cytosolic calcium levels and turnover of phosphoinositides have been detected in lyso-PC–treated cultured endothelial cells and are reported to be involved in the inhibitory actions of lyso-PC on endothelium-dependent vasorelaxation.²² Our preliminary experiments, however, have revealed that the calcium ionophore ionomycin did not induce PDGF-B or ICAM-1 mRNA (data not shown), suggesting that calcium mobilization alone is not sufficient to induce endothelial genes such as PDGF-B and ICAM-1.

The cAMP–protein kinase A pathway is another possible signal transduction cascade that can transmit biological signals. Forskolin or Bt₂cAMP, which elevates intracellular cAMP levels, alone did not upregulate PDGF-B or ICAM-1 expression but rather suppressed lyso-PC–induced increases in both PDGF-B and ICAM-1 mRNA levels. These effects of cAMP-elevating agents do not appear to result from general inhibitory action, since forskolin or Bt₂cAMP inhibited lyso-PC–induced ICAM-1 mRNA upregulation but did not affect that induced by PMA. Although previous studies by others have demonstrated that elevated levels of intracellular cAMP can also antagonize the effects of thrombin or TGF-ß on PDGF-B mRNA levels²³ but do not suppress ICAM-1 expression induced by TNF-,²⁴ our results appear to be the first to demonstrate that elevated levels of cAMP can counteract the effect of lyso-PC on both PDGF-B and ICAM-1 expression.

Previous studies with Northern analyses using actinomycin D and nuclear runoff assays have revealed that lyso-PC does not appear to act on PDGF-B and ICAM-1 genes by stabilizing mRNA but rather by stimulating gene transcription.⁷ Consensus sequences for binding of known transcription factors, including activating protein 1 (AP-1), have been detected in 5' promoter regions of PDGF-B^{25 26 27} and the ICAM-1^{28 29} gene. Studies are in progress in our laboratory to identify which promoter elements and transcription factors are involved in lyso-PC–induced endothelial gene upregulation.

In summary, we have demonstrated that PDGF-B protein expression is upregulated by lyso-PC and have partially characterized potential signal transduction mechanisms responsible for endothelial gene induction elicited by lyso-PC; PDGF-B and ICAM-1 gene induction by lyso-PC appears to depend on signal transduction mechanisms other than PMA-regulatable PKC, which can be suppressed by elevated levels of cAMP. Dependence of de novo protein synthesis for PDGF-B is different from that for ICAM-1, suggesting that lyso-PC might stimulate multiple and diverse signaling pathways. Further studies related to signal transduction pathways and transcriptional regulatory mechanisms involved in this lipid stimulus relevant to atherogenesis and inflammation may provide new insights into endothelial activation in these pathophysiological settings and might provide potential therapeutic targets in preventing vascular diseases.

	Selected Abbreviations and Acronyms

 

Bt2cAMP = dibutyryl cAMP ECGS = endothelial cell growth supplement FBS = fetal bovine serum HB-EGF = heparin-binding epidermal growth factor–like growth factor HUVEC = human umbilical vein endothelial cell ICAM-1 = intercellular adhesion molecule-1 IL = interleukin lyso-PC = lysophosphatidylcholine PDGF = platelet-derived growth factor PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate TBS = Tris-buffered saline TNF- = tumor necrosis factor- VCAM-1 = vascular cell adhesion molecule-1

	Acknowledgments

 
This study was supported by a research grant from the Ministry of Education, Science, and Culture of Japan (No. 06671022) and a grant-in-aid from the Japan Research Foundation for Clinical Pharmacology, Tokyo, Japan. We thank the doctors and nursing staff in the obstetrics ward of Kyoto University Hospital for their help in obtaining human umbilical cords. We also gratefully acknowledge Dr Tucker Collins (Brigham and Women's Hospital, Boston, Mass) for his generous gift of PDGF-B cDNA and helpful discussions.

Received October 24, 1994; accepted April 19, 1995.

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