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Cardiovascular Research 2003 59(4):934-944; doi:10.1016/S0008-6363(03)00526-1
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

PKC/Raf/MEK/ERK signaling pathway modulates native-LDL-induced E2F-1 gene expression and endothelial cell proliferation

Gianfranco Pintusa,b,*, Bruna Tadolinib, Anna M Posadinoa, Bastiano Sannaa, Marcella Debiddaa, Ciriaco Carruc, Luca Deianac and Carlo Venturaa,b

aDepartment of Biomedical Sciences, Division of Biochemistry, Laboratory of Cardiovascular Research, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy
bDivision of Cell Biology, National Institute of Biostructures and Biosystems, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy
cInstitute of Clinical Biochemistry, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy

gpintus{at}uniss.it

gionfry_p{at}yahoo.it

* Corresponding author. Department of Biomedical Sciences, Division of Biochemistry, Laboratory of Cardiovascular Research, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy. Tel.: +39-079-228-121; fax: +39-079-228-120.

Received 10 March 2003; revised 29 June 2003; accepted 16 July 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background and objectives: The interactions of low-density lipoprotein (LDL) with the endothelium are thought to play a major role in the development of atherosclerosis. Due to this reason, the molecular sequelae of events resulting from native LDL (N-LDL) interaction with human endothelial cells (HECs) are largely under investigation. Methods and results: Here, we report that the exposure of serum-free HECs to different concentrations of N-LDL-cholesterol (LDL-chol) elicited a time- and dose-dependent induction of DNA synthesis. The exposure of serum-free HECs to N-LDL was able to elicit a time- and dose-dependent increase of protein kinase C (PKC) activity that, along with the activation of the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, leads to an increase in E2F-1 gene expression. In addition, the treatment of HECs with N-LDL was also able to induce both E2F-1 gene transcription and protein expression. These N-LDL-aroused responses were dramatically counteracted by PKC inhibition or down regulation. Similarly to what observed for Raf/MEK/ERK activation and E2F-1 gene expression, the inhibition of PKC as well as its down regulation, significantly lowered the DNA synthesis induced by N-LDL in serum-free HECs. Conclusions: These results suggest that the activation of PKC/Raf/MEK/ERK-mediated events controlling E2F-1 gene expression by N-LDL may represent an important mechanism in the regulation of HECs proliferation during normal and pathological processes.

KEYWORDS Endothelial function; Gene expression; Lipoproteins; Protein kinases; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
It is increasingly recognized that the development of atherogenesis is a complex and multifactorial pathology that depends on signal exchange between different cell types resident within the vessel wall. This interplay sets the molecular plight that is responsible for the progression and the clinical manifestations of vascular diseases. Low-density lipoprotein (LDL) interaction with endothelium seems to be a critical factor in vascular lesion development, as indicated by the observation that an increased plasma LDL-cholesterol is associated with enhanced atherogenic risk [1]. Consistent with the atherogenic role of LDL is the finding that coronary events and total mortality can both be reduced by pharmacological treatment of coronary-prone patients with lipid lowering agents [1–3]. In this context, a crucial role is sustained by native LDL (N-LDL), which has been shown to modulate critical functions of endothelial cells (ECs) involved in both atherogenesis development and progression. It has been reported that N-LDL binding to LDL receptor triggers a rise in intracellular calcium, promoting the appearance of vascular cell adhesion molecule-1, E-selectin [4] and intracellular adhesion molecule-1 increasing EC adhesiveness [5]. Atherogenic concentrations of N-LDL also enhance EC endocyctic activity [6], permeability [7] and the recruitment of mononuclear cells [8]. The finding that these in vitro responses closely resemble many of the early changes observed in vivo and ex vivo in human and animals during hypercholesterolemia [9–12] suggests that N-LDL may have a role during the pathological commitment toward atherogenic processes. Within this context, a central role is played by vascular remodeling [13]. This is an active process of structural alteration that involves changes in several cellular processes, including cell proliferation, migration and cell death, thus allowing the adaptation and repair of adult blood vessels. Inappropriate remodeling, including its absence, underlies the pathogenesis of major cardiovascular diseases, such as atherosclerosis and restenosis [13]. For this reason, understanding both the molecular mechanisms and the different factors involved in the regulation of this essential process is of extreme interest.

Although it has been shown that N-LDL can affect both proliferation and gene expression in ECs [14], the molecular patterning by which N-LDL activates ECs thus influencing vascular remodeling remains an area of inquiry. Protein kinase C (PKC) activation, as well as the activation of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, plays a pivotal role in transducing extracellular stimuli that modulate a number of cellular processes, including proliferation and angiogenesis [15,16]. In this context, it has recently been proposed that the levels of E2F-1, a heterodimeric transcription factor that controls transcription of several cell growth-related genes [17], are regulated at least in part by a signaling pathway that includes Ras. In fact, this Ras-induced increase in E2F-1 mRNA levels is dependent on both MEK and protein kinase B (PKB), is retinoblastoma (RB)-independent and seems to constitute a novel functional link between the Ras/MEK/ERK signaling pathway and E2F-1 [18].

In order to improve the knowledge of the molecular mechanisms by which N-LDL can affect EC functions, here we provide evidence that exposure of human endothelial cells (HECs) to N-LDL is able to induce PKC activation along with a PKC-dependent Raf/MEK/ERK phosphorylation. We also show that the modulation of this signaling pathway by N-LDL can account for both E2F-1 transcriptional gene expression and HEC proliferation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture and determination of DNA synthesis
HECs were isolated and cultured as previously described [19]. Cultured cells were identified as endothelial by their typical cobblestone appearance and production of von Willebrand factor as measured by enzyme-linked immunosorbent assay [20]. Cell viability was checked by the trypan blue-exclusion method and used within three passages at a density of 70,000 cells/ml. To determine DNA synthesis, HECs cultured in 24-well plates (Falcon) were serum-starved following a 24-h incubation in serum-free medium and then treated as described in the figure legends. During the last 24 h, to cells from each experimental group was added 1 µCi/ml [3H]thymidine (specific activity 5 Ci/mmol, Amersham) and DNA synthesis was determined as previously described by assessing [3H]thymidine incorporation [19].

2.2 LDL preparation and extraction
Blood was collected from fasted healthy volunteers by venopuncture into sampling vials containing EDTA. Human subjects gave informed consent and the investigation conformed with the principles outlined in the Declaration of Helsinki. Plasma was prepared by centrifugation at 2,000 g for 10 min at 4°C. LDL fraction was isolated by a very fast ultracentrifugation. Briefly, plasma (0.9 ml) was added to a centrifuge tube containing KBr (0.4451 g) adjusting the density of plasma to 1.3 g/ml. This solution was then overlaid with 2.1 ml of 150 mM NaCl and centrifuged for 2 h at 10°C at 440,000 g. The LDL orange colored band was recovered by suction, adjusted to a density greater than the LDL density (1,120 g/ml) by adding solid KBr and centrifuged for 2 h at 10°C at 440,000 g. The LDL fraction was dialysed and passed through a Sephadex column equilibrated with phosphate-buffered saline (PBS) to remove EDTA and other interfering compounds. The cholesterol content of the low-density lipoprotein, named native-LDL, was determined, adjusted to 1 mg/ml with PBS and immediately added to cell culture [21].

2.3 Determination of Raf/MEK/ERK activation and E2F-1 protein expression by immunoblotting
Immunoblotting analysis was performed as previously described [22]. Serum-starved HECs were treated as described in the figure legends. Cell lysates (10 µg/lane) were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with a 12% acrylamide separation gel and transferred to nitrocellulose membranes which were incubated with primary antibody. Proteins of interest were detected using specific antibodies against E2F-1, Raf, MEK and ERK. Whereas, Raf, MEK and ERK activation were assessed by using antibodies targeting their phosphorylated form (Cell Signaling). Antibody binding was detected using horseradish peroxidase-linked anti-IgG secondary antibody (Bio-Rad) and the ECL Plus system (Amersham).

2.4 PepTag assay for non-radioactive detection of PKC activity
Serum-starved HECs were treated as described in the figure legends. At the end of each experimental point, the medium was removed and cells were washed in ice-cold PBS and removed from the flask by gentle scraping in chilled lysis buffer [50 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES), pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium vanadate, 50 mM sodium fluoride, 20 mM β-glycerophosphate, 0.1 µM okadaic acid, 1 mM phenylmethylsulfonylfluoride, 20 µg/ml aprotinin, 50 µg/ml leupeptin, and 10 µM pepstatin]. After 15 min on ice, insoluble material was removed by sedimentation for 20 min at 100,000 g, and total PKC was recovered by immunoprecipitation with an anti-PKC antibody that recognizes an epitope located within the amino acid sequence 296–317, at the hinge region, close to the trypsin cleavage site of PKC (Sigma–Aldrich). Each cell extract (400 µg) was mixed with 10 µl of anti-PKC antibody for 1 h, and then 30 µl of 50% protein A–Sepharose in lysis buffer was added for an additional 1 h. The immune complex was recovered by sedimentation for 5 min in a microcentrifuge, washed three times with 0.5 ml PBS containing 1% Nonidet P-40 and 2 mM sodium vanadate and then dissolved in the PKC reaction mixture. Total PKC activity was assessed as previously described [23] by the aids of a PepTag PKC enzyme assay system (Promega).

2.5 Determination of E2F-1 gene expression by reverse transcription polymerase chain reaction (RT-PCR)
Serum-starved HECs were treated as described in the figure legends. At the indicated times, total RNA was extracted, reverse transcribed and amplified according to the procedure previously described [24]. As a check for genomic DNA contamination a no-RT control was added in RT experiments, while in PCR experiments the number of amplification cycles was determined experimentally for each primer pairs by establishing the point at which exponential accumulation plateaus [24]. The position of PCR fragments was evaluated by comparison with DNA molecular weight markers (Gibco). GAPDH mRNA was used for each sample as an internal control for mRNA integrity and equal loading. PCR conditions and specific primers directed against human sequences for E2F-1 and GAPDH were previously described [25].

2.6 Analysis of E2F-1 gene transcription by nuclear runoff assay
Serum-starved HECs were treated as described in the figure legends and processed for nuclear runoff assay at the indicated times. To prepare HEC nuclei, cells were washed with ice-cold PBS and lysed with 0.5% Nonidet P-40 solution (10 mM Tris–HCl, 10 mM NaCl, 3 mM MgCl2, and 0.5% NP-400, pH 7.4). Nuclei were isolated by centrifugation and resuspended in a 40% glycerol buffer (50 mM Tris–HCl, 40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA, pH 8.3). Nascent transcription in vitro as well as solution hybridization RNase protection assay used for esteeming radiolabeled nuclear RNA were performed as described in detail elsewhere [22,26]. A 396-base pair (bp) fragment amplified from the human genomic E2F-1 gene [25] was inserted into pCRII-TOPO (Invitrogen). Equal counts of radiolabeled RNA were hybridized in the presence of an unlabeled antisense E2F-1 RNA probe generated by transcription of the above mentioned plasmid linearized with BamHI. Radiolabeled nuclear RNA was also hybridized with unlabeled antisense GAPDH mRNA synthesized from a SacI-linearized pCRII-TOPO vector, containing a 788-bp fragment amplified from the human genomic GAPDH gene [25]. GAPDH mRNA was utilized as a constant mRNA for control.

2.7 Other cell treatments
PKC down-regulation was accomplished by exposing HECs to serum-free medium containing 0.1 µM phorbol 12-myristate 13-acetate (PMA) (Sigma) during the last 24 h of serum starvation. The PKC inhibitors chelerythrine and calphostin-C (Alexis) were added at the indicated concentrations during the last 15 min of serum starvation. Indeed, similar conditions have been previously reported to inhibit PKC in HECs with satisfactory results [27]. The MEK/ERK inhibitor PD98059 (Alexis), was added at a concentration of 10 µM during the last hour of serum starvation.

2.8 Statistical analysis
The statistical analysis of the data was performed by using the unpaired Student’s t-test, assuming a P<0.05 as the limit of significance. All values are given as means±S.E. of at least three independent experiments.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 N-LDL induces HEC proliferation
To test whether N-LDL may affect cell proliferation, we first examined the rate of DNA synthesis by following [3H]thymidine incorporation in HECs. Serum-starved cells were stimulated to leave the growth-arrested state by incubation in medium M199 in the absence or presence of 50 µg/ml N-LDL cholesterol (LDL-chol). As observed in Fig. 1A, the treatment of serum-starved HECs with N-LDL elicited a time-dependent induction of DNA synthesis, which was evident at 24 h, peaked at 48 h and declined at 72 h. Fig. 1B shows that the exposure for 48 h of HECs to the indicated concentrations of LDL-chol was able to induce a dose-dependent increase in DNA synthesis that reached the maximal amplitude at 50 µg/ml over a concentration range of 5–100 µg/ml.


Figure 1
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  		Fig. 1 N-LDL induces HEC proliferation. (A) Time–course of [3H]thymidine incorporation in serum-starved HECs exposed to 50 µg/ml LDL-chol. Serum-starved HECs were treated for the indicated times with medium M199, in the absence ({blacksquare}) or presence ({square}) of 50 µg/ml LDL-chol. At the indicated times, cells were processed for liquid scintillation counting as described under Methods. Data are expressed as % of the control (time 0). *, Significantly different from the control (P<0.01). (B) Dose–response effect of the indicated concentrations of LDL-chol on [3H]thymidine incorporation in serum-starved HECs. Serum-starved cells were treated for 48 h with medium M199 in the absence (UNTR) or presence of the indicated concentrations of LDL-chol. Data are expressed as % of control (UNTR). *, Significantly different from the control (P<0.01).

 
3.2 N-LDL induces PKC activity
We have previously reported that serum-induced HEC proliferation can occur both via PKC-dependent and independent pathways [19]. Therefore, we investigated whether N-LDL interaction with HECs may trigger PKC activation, which in turn could activate intracellular signaling pathways, ultimately ensuing in the transcriptional control of cell growth-related genes. By assessing PKC we found a dose- and time-dependent increase of enzyme activity in serum-starved HECs exposed to N-LDL. Time-related activation peaked at 5 min (Fig. 2A), while at the same time point, a dose-dependent effect was noticed eliciting a maximal response at 50 µg/ml of LDL-chol (Fig. 2B). Panels C–D of the same figure show that the PKC activity assessed after 5 min of HEC exposure to N-LDL was dose-dependently counteracted and ultimately abolished in the presence of 5 and 1 µM chelerythrine or calphostin C, respectively. A complete inhibition of N-LDL-generated PKC activity was also observed in HECs that had been PKC down-regulated before receiving N-LDL treatment (Fig. 2C). In the absence of N-LDL, the levels of PKC activity assessed in cells subjected to PKC inhibitors or PKC down-regulation did not differ significantly from the levels detected in control cells (Fig. 3C, D). The assay of lactate dehydrogenase release in culture medium, performed prior to and after the exposure of HECs with either PKC inhibitors or PMA down-regulation, revealed that these treatments did not exert significant toxic effect on given cells (not shown).


Figure 2
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  		Fig. 2 N-LDL induces PKC activity. (A) Serum-starved HECs were stimulated for the indicated times with medium M199 in the absence ({blacksquare}) or presence ({square}) of 50 µg/ml LDL-chol. PKC activities are expressed as % of control (time 0). *, Significantly different from the control (P<0.01). (B) Serum-starved cells were treated for 5 min with medium M199 in the absence ({blacksquare}) or presence ({square}) of the indicated concentrations of LDL-chol. PKC activities are expressed as % of control (0 µg/ml LDL-chol). *, Significantly different from the control (P<0.01). (C), (D) Serum-starved cells were pretreated for 15 min with the indicated concentrations of chelerythrine (CHE) and calphostin-C (CALF) or PKC-downregulated (DR+) and then stimulated for 5 min with medium M199 in the absence (LDL–) or presence of 50 µg/ml LDL-chol (LDL+). PKC activities are expressed as % of control (untreated). *, Significantly different from the control (P<0.01); §, significantly different from LDL+ (P<0.01).

 

Figure 3
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  		Fig. 3 N-LDL-induced Raf/MEK/ERK phosphorylation is mediated by PKC. Serum-starved HECs were PKC-down-regulated (DR) or pretreated for 15 min with 5 µM chelerythrine (CHE) and then stimulated for the indicated times with medium M199 in the absence (UNTR) or presence of 50 µg/ml LDL-chol (LDL). The left part of the figure shows representative autoradiograms corresponding to the immunoreactivity of phospho Raf (A), phospho MEK (B) and phospho ERK1/2 (C). The right part of the figure reports the quantitative analysis of phospho Raf (D), phospho MEK (E) and phospho ERK1/2 (F) immunodensity expressed as % of control (untreated). (individual densities from double bands were added up to generate one single value). {blacksquare}, Untreated; {square}, LDL; {image}, LDL+chelerythrine; blk12, LDL+downregulation. No consistent changes in the total protein levels of Raf, MEK and ERK1/2 were detected. *, Significantly different from untreated cells; §, significantly different from LDL.

 
3.3 N-LDL-induced PKC activation mediates Raf/MEK/ERK phosphorylation
In order to investigate the possibility that N-LDL-induced PKC activity could trigger cell growth-related intracellular pathways, we assessed the phosphorylation state of the Raf/MEK/ERK kinase with antibodies recognizing their activated phosphorylated form. Exposure of serum-starved HECs to 50 µg/ml LDL-chol elicited a time-dependent activation of Raf, MEK and ERK. Such an effect was evident after 5 min of treatment, reaching a maximum at 30 min then progressively declining during the following 30 min (Fig. 3A–C). The same panels also underline that PKC down regulation, as well as the treatment of cell culture with 5 µM chelerythrine, markedly, although not completely, counteracted the enzyme activation induced by N-LDL. The observations from immunoblotting experiments are further supported from the quantitative analysis of the phosphorylated forms corresponding to Raf, MEK and ERK1/2 immunodensity, shown in Fig. 3D–F.

3.4 N-LDL-induced E2F-1 gene expression is mediated by PKC/MEK/ERK-dependent pathway
Here we investigate whether the current N-LDL-induced PKC/Raf/MEK/ERK activation could correlate with an E2F-1 up regulation by assessing its gene expression in HECs exposed to N-LDL. Fig. 4A shows that the exposure of serum-starved HECs to N-LDL was able to increase the basal levels of E2F-1 in a time-dependent fashion with a maximum peak of expression at 3 h. The same figure (panel B) shows the dose-dependence of the E2F-1 gene expression on N-LDL concentration, which reached the maximal amplitude at 50 µg/ml of LDL-chol. The N-LDL-evoked E2F-1 gene expression, assessed after 3 h of cell treatment, was dramatically counteracted by chelerythrine-induced PKC inhibition or enzyme down regulation as well as by the block of the MEK/ERK signaling pathway (Fig. 5). The same figure also underlines that, at the same time points, the addition of PD98059 to PKC downregulated cells, as well as to HECs exposed to 5 µM chelerythrine failed to produce any additive inhibition in the residual effect of N-LDL-primed E2F-1 expression.


Figure 4
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  		Fig. 4 N-LDL induces E2F-1 gene expression. (A) Serum-starved HECs were stimulated for the indicated times with medium M199 containing 50 µg/ml LDL-chol. *, Significantly different from the control (time 0). (B) Serum-starved HECs were stimulated for 3 h with medium M199 in the presence of the indicated concentrations of LDL-chol. *, Significantly different from the control (0 µg/ml LDL-chol). The upper part of each panel shows representative ethidium bromide-stained gels of the reaction products obtained using 5 µl of the RT products after 30 cycles of PCR amplification for E2F-1 and GAPDH. The lower part of each panel reports the expression of E2F-1 mRNA levels detected by [32P]dCTP-PCR (30 cycles). Individual results were normalized to GAPDH mRNA detected in each sample and expressed as a ratio to GAPDH.

 

Figure 5
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  		Fig. 5 PKC/MEK/ERK-dependent pathway regulates N-LDL-induced E2F-1 gene expression: the levels of E2F-1 mRNA were detected by [32P]dCTP-PCR (30 cycles). Serum-starved cells were PKC-downregulated (DR+) or pretreated either for 15 min with 5 µM chelerythrine (CHE+) or for 1 h with 10 µM PD98059 (PD+) and then stimulated for 3 h with medium M199 in the absence (LDL–) or presence of 50 µg/ml LDL-chol (LDL+). In selected experiments, either downregulated or chelerythrine-treated cells were concomitantly exposed to 10 µM PD98059. The upper part of the panel shows representative ethidium bromide-stained gels of the reaction products obtained using 5 µl of the RT products after 30 cycles of PCR amplification for E2F-1 and GAPDH. The lower part reports the expression of E2F-1 mRNA levels detected by [32P]dCTP-PCR (30 cycles). Individual results were normalized to GAPDH mRNA detected in each sample and expressed as a ratio to GAPDH. *, Significantly different from the control (untreated); §, significantly different from (LDL+).

 
3.5 N-LDL-modulated PKC/MEK/ERK signaling pathway regulates E2F-1 gene transcription and protein expression
To establish whether the increase in E2F-1 mRNA expression elicited by N-LDL might have occurred at the transcriptional level, we assessed the rate of transcription of the E2F-1 gene by using an in vitro nuclear run-off transcription assay. We found that E2F-1 gene transcription was increased in nuclei isolated from cells that had been exposed for 3 h to 50 µg/ml LDL-chol (Fig. 6A). As performed in RT-PCR experiments, we examined whether PKC/MEK/ERK activation may be a signaling mechanism responsible for the transcriptional effect of N-LDL. PKC-downregulation, as well as the treatment with either 5 µM chelerythrine or 10 µM PD98059, markedly affected the E2F-1 gene transcription elicited by N-LDL (Fig. 6A). Furthermore, the addition of PD98059 to chelerythrine-treated or PKC down-regulated cells did not to produce any additive inhibition in the residual N-LDL-induced E2F-1 transcription (Fig. 6A). In addition, the transcriptional effect induced by N-LDL exhibited a pattern that overlaid that observed in RT-PCR experiments (Fig. 5) and, as confirmed by immunoblotting experiments, such an effect was paralleled by similar results concerning E2F-1 protein expression (Fig. 6B).


Figure 6
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  		Fig. 6 N-LDL induces E2F-1 gene transcription and protein expression. (A) Nuclear run-off transcription assay. Serum-starved cells were PKC-downregulated (DR+) or pretreated either for 15 min with 5 µM chelerythrine (CHE+) or for 1 h with 10 µM PD98059 (PD+) and then stimulated for 3 h with medium M199 in the absence (LDL–) or presence of 50 µg/ml LDL-chol (LDL+). In selected experiments, chelerythrine-treated cells were concomitantly exposed to 10 µM PD98059. Representative autoradiograms of the ribonuclease protection analysis of E2F-1 mRNA are shown in panel A. Autoradiographic exposure was for 2 days on Kodak X-Omat film with an intensifying screen. Row a, transcription of the E2F-1 gene. Row b, GAPDH mRNA. On the right are indicated the position of 400- or 800-base pair (bp) radiolabeled DNA markers, showing that the single protected fragments migrated with a molecular size comparable to E2F-1 (396 bp) or GAPDH (788 bp) mRNA. (B) Immunoblot analysis of E2F-1. Cells were treated as for nuclear run-off transcription; equal amounts of protein from each sample were subjected to SDS–PAGE and analyzed by immunoblotting. The upper and lower parts of panel B show, respectively, the E2F-1 immunoreactivity and the quantitative immunodensity expressed as % of control (untreated). *, Significantly different from the control (untreated); §, significantly different from (LDL+).

 
3.6 The inhibition of a PKC-dependent pathway down regulates HEC proliferation elicited by N-LDL
As currently reported, stimulation of HECs with N-LDL elicited a PKC inhibitable increase of Raf/MEK/ERK activation and E2F-1 gene expression. Exposure to N-LDL was also able to evoke a dose-dependent increase of HEC proliferation, which reached the maximal amplitude at 50 µg/ml N-LDL chol (Fig. 1). Fig. 7 shows that treatment in the presence of 5 µM chelerythrine or PKC downregulation dramatically counteracted the effect of N-LDL on DNA synthesis, suggesting the involvement of PKC-activated signaling in the modulation of N-LDL-induced HEC proliferation.


Figure 7
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  		Fig. 7 PKC-dependent pathway regulates HEC proliferation elicited by N-LDL. Serum-starved cells were PKC-downregulated (DR+) or pretreated for 15 min with 5 µM chelerythrine (CHE+) and then stimulated for 48 h with medium M199 containing the indicated concentrations of LDL-chol. Data are expressed as % of the control (untreated). *, Significantly different from control; §, significantly different from (LDL-chol).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
A hallmark of hyperlipidemia-induced atherosclerosis is altered gene expression that initiates cell proliferation and (de)differentiation in the intima of the arterial wall. In this context, a crucial role is played by angiogenesis, a phenomenon characterized by endothelial cell proliferation, migration and formation of new blood capillaries from preexisting vessels, whose last consequence is vascular remodeling [28,29]. The molecular signaling that mediates this process in vivo still remains to be identified. Mitogen-activated protein kinase (MAPK)/ERK are thought to play a pivotal role in transmitting signals required for cell proliferation in vitro. The present study was designed to determine whether N-LDL activates HECs by promoting Raf/ERK/MEK phosphorylation and E2F-1 gene expression, and whether N-LDL-induced PKC activation mediates the modulation of the Raf/ERK/MEK/E2F-1 pathway.

Cell exposure to N-LDL was able to evoke a time- and dose-dependent induction of PKC activity that was completely abolished either by PKC inhibitors or enzyme down regulation. It is broadly documented that PKC activation can modulate a great variety of cellular processes, either in a direct way or through the commitment of other intracellular pathways such as the MAPK/ERK pathway [30,31]. A number of extracellular stimuli have been reported to activate the MAPK/ERK pathway via Ras/Raf signaling [32]. This activation is mediated, at least in part, by G-protein coupled receptors (GPCRs), stimulation of membrane phospholipid hydrolysis, and activation of the diacylglycerol sensitive isoforms of PKC [32]. The finding that exposure of HECs to N-LDL resulted into a significant up-regulation of the Raf, MEK, ERK phosphorylation and the observation that this effect was reversed by PKC inhibition, may suggest the involvement of PKC-dependent signals in the activation of this intracellular pathway by N-LDL. Such a hypothesis is further inferred from the finding that PKC down-regulation prevented N-LDL-induced Raf/MEK/ERK activation. Although PKC inhibition as well as its down-regulation completely counteracted the N-LDL-elicited PKC activity, failure to achieve a complete blockade of phosphorylation of PKC-targeted enzymes under the same experimental conditions suggests the recruitment of additional PKC-independent pathway(s) in the activation of Raf, MEK and ERK1/2 by N-LDL.

PKC activation, and the activation of Ras/Raf/MEK/ERK signaling have been reported to be involved in the modulation of E2F-1 gene expression [33,18]. Here, we show that the exposure of serum-free HECs to N-LDL enhanced the expression of E2F-1 in a time- and dose-dependent fashion. PKC inhibition, as well as its down-regulation, significantly reduced the stimulatory effect of N-LDL on E2F-1 mRNA expression, suggesting the involvement of this enzyme in E2F-1 gene modulation by N-LDL. An involvement of MEK/ERK activation in such a response was further confirmed by the use of PD98059. This specific MEK inhibitor was also able to blunt the N-LDL-induced up-regulation of E2F-1 mRNA. Since no additive effect has been observed upon PD98059 treatment after PKC down-regulation or inhibition it may be envisaged that PKC and MAPK could represent sub-sequential steps in the same pathway responsible for the N-LDL-induced E2F-1 gene expression. The involvement of E2F-1 in growth regulation is well recognized [17]. In addition, an implication of E2F-1 in modulating both cell death and proliferation within the vessel wall has been recently proposed [34], suggesting the involvement of E2F-1 in vascular remodeling. The current finding that the effect of N-LDL was exerted at the transcriptional level and was paralleled by an increase in E2F-1 protein expression strongly suggests that N-LDL may have role in the growth regulatory responses controlled by this transcription factor. A link between N-LDL and HECs proliferation was also inferred from the analysis of [3H]thymidine incorporation. As reported, the induction of PKC activity by N-LDL proved to be responsible for the increase in the rate of HEC DNA synthesis, since the stimulatory effect of N-LDL on [3H]thymidine incorporation was significantly counteracted by PKC inhibition or its down regulation. However, also in this case the observation that these interventions failed to completely block the N-LDL-primed increase in E2F-1 gene expression and HEC proliferation further supports the guess that PKC-unrelated pathway(s) may intervene in N-LDL-mediated responses.

Although it has been extensively reported that different vascular cells may exhibit dissimilar responses to N-LDL, which seems to be dependent from both the N-LDL concentration and the duration of cell treatment, our in vitro results point out that N-LDL may be able to exert a mitogenic effect even at a concentration lower than that considered to be atherogenic, suggesting the involvement of N-LDL in the modulation of normal vascular growth and homeostasis. Nevertheless, it is now widely accepted that physiological angiogenesis results from a fine tuning of a wide range of molecular events that ultimately ensue in a balance among anti-angiogenic and pro-angiogenic stimuli controlling several features of the vascular remodeling, including cell proliferation, migration and death. Hence, a deregulation in such a critical homeostasis, may pave the way to molecular patterning contributing to the onset of circulatory disorders and progression to atherogenesis. The signaling targets activated by N-LDL are associated with the modulation of critical regulatory parameters in endothelial cells [35,36]. Activation of these mechanisms within a pathological environment may conceivably contribute to the atherogenic process, either by activating HECs and leading to a PKC-dependent increase in mononuclear cell binding [4,5] or by modulating the PMA-induced macrophage cholesterol accumulation in atherosclerotic plaques within the intima of the arterial wall [37].

The exact molecular sequelae of events underlying the observed effects of N-LDL on PKC/Raf/MEK/ERK activation, as well as E2F-1 gene expression and HEC proliferation, remain to be elucidated. In particular, assessing the involvement of selected PKC isozymes in N-LDL-mediated responses remain a scheming area of interest and is the subject for future investigations.

Time for primary review 30 days.


   Acknowledgements
 
This work was supported by grants from the "Regione Autonoma della Sardegna (Assessorato dell’Igiene, Sanità e Assistenza Sociale)", "Ministero dell’Istruzione, dell’Università e della Ricerca (Fondo Integrativo Speciale per la Ricerca—2001; Programmi di Ricerca Cofinanziati—2001)" and "Ministero della Sanità (Attività di Ricerca Finalizzata—2000)".


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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