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Am J Physiol Heart Circ Physiol 284: H644-H653, 2003; doi:10.1152/ajpheart.00494.2001
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Vol. 284, Issue 2, H644-H653, February 2003

OxLDL stimulates cell proliferation through a general induction of cell cycle proteins

Marjorie E. Zettler1, Michele A. Prociuk1, J. Alejandro Austria1, Hamid Massaeli1, Guangming Zhong2, and Grant N. Pierce1

1 Cell Biology Laboratory, Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre, and the Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R2H 2A6; and 2 Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oxidized low-density lipoprotein (oxLDL) may be involved in atherosclerosis by stimulating proliferation of cells in the vessel wall. The purpose of this study was to identify the mechanism by which oxLDL induces proliferation. Quiescent human fibroblasts and rabbit smooth muscle cells were treated with 0, 10, or 50 µg/ml oxLDL for 24-48 h. This resulted in significant increases in total cell counts at both concentrations of oxLDL, at both time points, for both types of cells. Western blot analysis revealed that oxLDL-stimulated cell proliferation was associated with significant increases in the expression of proteins that regulate entry into and progression through the cell cycle [cell division cycle 2, cyclin-dependent kinase (cdk) 2, cdk 4, cyclin B1, cyclin D1, and PCNA]. Surprisingly, the expression of cell cycle inhibitors (p21 and p27) was stimulated by oxLDL as well, but this was to a lesser extent than the effects on cell cycle-activating proteins. OxLDL also induced nuclear localization of all cell cycle proteins examined. The similar effects of oxLDL on the translocation and expression of both cell cycle-activating and -inhibiting proteins may explain the controlled proliferative phenomenon observed in atherosclerosis as opposed to the more rapid proliferative event characteristic of cancer.

atherosclerosis; cyclins; cyclin-dependent kinases; low-density lipoproteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OXIDIZED LOW-DENSITY LIPOPROTEIN (oxLDL) plays a critical role in atherogenesis, in part by stimulating proliferation of cells within the vessel wall (41). In vitro, mildly oxidized LDL is capable of evoking a proliferative response in a variety of cell types, including smooth muscle cells (3, 11, 25, 46), macrophages (5, 20, 31, 42), fibroblasts (5), and endothelial cells (30). The mechanism whereby oxLDL induces proliferation is not clear.

Proliferation of any cell type is dependent on changes in the expression or activation of cell cycle regulatory proteins (21). Progression through the cell cycle is controlled by a series of cyclin-dependent kinases (cdk) that must bind to a cyclin to be active (35). Passage from one stage of the cell cycle to the next requires different cyclin/cdk complexes (16). For example, passage from the quiescent G0 state through G1, the first gap phase, requires both cyclin D1/cdk 4 and cyclin E/cdk 2 complexes. DNA synthesis in the S phase requires cyclin A/cdk 2 complexes as well as the DNA polymerase cofactor PCNA (39). Cyclin A/cdc 2 and cyclin B1/cdc 2 complexes are necessary to move through G2, the second gap phase, and the M (mitosis) phase, where the cell divides (24). The activity of these kinases is directed in part by inhibitors of cdk, such as p21 (50) [induced by p53 (49)] and p27 (40). Compelling evidence for the participation of cell cycle regulatory proteins in the pathogenesis of atherosclerosis and restenosis has come from studies involving the induction of cell cycle proteins in the balloon-injured vessels of animal models of restenosis and the ability of cell cycle inhibitors to prevent intimal thickening in these models (1, 7, 8, 12, 36-38, 44, 45, 51, 52).

Although the cell cycle represents the final common pathway of all mitogenic signaling cascades, there has been no evidence to date linking oxLDL to the induction of cell cycle proteins. Furthermore, the pattern of expression of cell cycle proteins and the upstream signaling pathway by which they are induced in the progression of vascular disease have not been elucidated. The purpose of the present study, therefore, was to determine whether oxLDL is capable of inducing proliferation in quiescent cells, to identify whether oxLDL is capable of altering the expression and distribution of specific cell cycle proteins, and finally to identify the signaling pathways involved in the mitogenic response.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell cultures. Confluent cultures of human neonatal fibroblasts and rabbit vascular smooth muscle cells [VSMC; isolated as described previously (34)] were trypsinized and seeded at 500,000 cells/100 × 20-mm dish. After 24 h in DMEM supplemented with 5% FBS, the cells were washed twice with PBS. The medium was replaced with serum-free DMEM supplemented with transferrin (5 µg/ml), selenium (1 nM), ascorbate (200 µM), and insulin (10 nM) for 6 days to induce growth arrest. Cells were then incubated with this medium and 10 or 50 µg cholesterol/ml LDL or oxLDL for various time points for up to 48 h. Cholesterol concentrations were assessed before oxidation, and these concentrations were used for both native LDL- and oxLDL-treated groups. Protein concentrations were unchanged throughout the course of the experiments. Cultures were maintained at 37°C in humidified 5% CO2, and the medium was replaced every 24 h. Freshly prepared oxLDL was also replaced on a daily basis. Control cells were maintained in an identical medium (in the absence of oxLDL) for the same period of time. For experiments involving inhibitors, cells were pretreated for 15 min with either 25 µg/ml polyinosinic acid (25), 20 µg/ml LY-294002 (32), 50 µg/ml 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (33), or 4 µg/ml PD-98059 (23) before exposure to oxLDL. These concentrations were maintained in the media for the duration of the experiments. To demonstrate that the concentrations of oxLDL utilized in these experiments were not toxic, LDH release into the culture medium was assayed as an indicator of cell damage according to the method of Bergmeyer (18). No significant differences were observed in levels of LDH release between oxLDL-treated cells and untreated controls over 24 or 48 h for either 10 or 50 µg/ml oxLDL (data not shown). Furthermore, we could detect no significant increases in the release of LDH after cells were incubated with any of the drugs, either alone or in combination with oxLDL.

Plasma lipoprotein isolation and oxidation. LDL (density 1.019-1.063 g/ml) was prepared by sequential ultracentrifugation as described previously (28, 33). The protein content of LDL was determined by Lowry's method (29), and cholesterol (free and esterified) was measured enzymatically as described. The absence of LDL oxidation during isolation or before its use in experiments was determined by an absence of malondialdehyde (MDA)-reactive products (15) and oxidized cholesterol (27). LDL was oxidized with a Fe-ADP free radical-generating system (19). In a typical experiment, 1 mg/ml LDL was incubated at 37°C for 3 h with freshly prepared 0.05 mM Fe and 0.5 mM ADP in sterile filtered 150 mM NaCl, pH 7.4. The extent of oxidation was determined by an MDA assay (15). The same concentrations of Fe and ADP added to control cells in the absence of LDL had no effect (data not shown).

Cell cycle analysis by flow cytometry. After exposure to 0, 10, or 50 µg/ml oxLDL for 2, 6, 12, 24, and 48 h, cells were trypsinized, fixed in ice-cold 100% ethanol, and treated with RNase A (500 U/ml in 1.12% sodium citrate) for 15 min at 37°C. DNA was stained with propidium iodide (5% solution in 1.12% sodium citrate) for 30 min at room temperature in the dark. Samples were analyzed on a Becton-Dickinson FacsCalibur flow cytometer. The percentage of cells in each phase of the cell cycle was estimated using CellQuest software.

Measurement of cell numbers. For quantification of the number of cells in culture after treatments, cells were trypsinized and counted in a hemacytometer. For each condition and time point, 18 fields were counted.

Immunocytochemistry. Cells were seeded onto glass coverslips and maintained as described above. After oxLDL treatment, cells were fixed in 50% acetone-50% methanol for 3 min. A blocking solution of wash buffer [10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20] plus 10% skim milk powder was used before antibody treatments. Cells were then immunostained with primary antibodies to cell division cycle (cdc) 2, cdk 2, cdk 4, cyclin A, cyclin B, cyclin D1, p21, p27, p53, Rb (Transduction Laboratories), cyclin E sc-481 (Santa Cruz Biotechnology), and PCNA (Sigma) according to the directions of the manufacturer. After incubation with the primary antibody for 1 h at room temperature, coverslips were rinsed repeatedly with wash buffer before incubation with a secondary antibody conjugated to FITC (Sigma) for an additional hour at room temperature in the dark. Coverslips were mounted on slides using Fluorsave reagent (Calbiochem). Fluorescence of cell cycle proteins was observed using a Bio-Rad MRC600UV confocal microscope and quantified using Molecular Dynamics Imagespace software (version 3.2.1).

Preparation of cell extracts and Western blot analysis. Cells treated as described above were washed twice with PBS and lysed with SDS lysis buffer [62.5 mM Tris · HCl (pH 7.6), 100 mM NaCl, 1% SDS, 1 mM PMSF, and 21 µM leupeptin]. Protein concentrations of each sample were determined using the modified Lowry assay (29). For each sample, 20 µg total protein was fractionated by SDS-PAGE in a gradient gel for 4 h at 550 mV, 80 mA (constant current). Gels were calibrated using prestained molecular weight markers (GIBCO-BRL). Transfer onto nitrocellulose membranes was performed using a Bio-Rad apparatus for 75 min at 50 V (constant voltage). After completion of the transfer, equal loading of the lanes was confirmed by staining with Ponceau S stain (Sigma) for 5 min. The membrane was then placed in blocking buffer for an hour at room temperature. Antibody treatments were performed according to the manufacturer's instructions. Membranes were washed five times in wash buffer, and antibody reactions were detected using horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) and enhanced chemiluminescent detection reagents (Pierce) according to the manufacturer's instructions. Densitometry was performed on a BioRad GS-670 Imaging Densitometer.

Assay of kinase activity. Immunoprecipitation of cdk 4 was carried out by adding 1 µg cdk 4 antibody (Transduction Laboratories) to 200 µg total cell lysate, 250 µl of 2 × immunoprecipitation buffer [2% Triton X-100, 300 mM NaCl, 20 mM Tris (pH 7.4), 2 mM EDTA, 2 mM EGTA (pH 8.0), 0.4 mM sodium orthovanadate, 0.4 mM PMSF, and 1% NP-40], and H2O to a final volume of 500 µl. The immunoprecipitation reaction was carried out overnight at 4°C with gentle rotation. The next day, 20 µl of 50% protein G agarose beads (Calbiochem) were added, and the sample was incubated at 4°C with gentle rotation for 30 min. The beads were collected by centrifugation (1 min at 7,000 rpm, 4°C), and the supernatant was removed. The bead pellet was washed with 1× immunoprecipitation buffer and centrifuged (4 min at 14,000 rpm, 4°C), and the supernatant was discarded. The washing step was repeated twice more using 1× immunoprecipitation buffer, and a final wash was done using kinase reaction buffer [40 mM HEPES (pH 7.4), 10 mM MgCl2, 1 mM DTT, and 2 mM EDTA (pH 8.0)]. After the last wash, the bead pellet was resuspended in 30 µl of kinase reaction buffer plus 0.2 µCi/µl [gamma -32P]ATP and the kinase substrate (0.01 µg/µl GST-pRb, Santa Cruz Biotechnology). The reaction was carried out for 30 min at 30°C and stopped by the addition of 4× SDS-PAGE loading buffer. Samples were then loaded onto a 10% gel and separated by SDS-PAGE. The gel was stained with Coomassie blue stain to confirm equal amounts of kinase substrate in each sample, destained, and then dried. Phosphorylated substrate was visualized by autoradiography and quantitated by densitometry.

D-Myo-inositol 1,4,5-trisphosphate assay. Cells treated as described were washed with PBS, scraped down, and homogenized. The D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] content of the homogenate was measured using a radioisotopic assay kit (Amersham) according to the manufacturer's instructions.

Data analysis. Data are reported as means ± SE. Results were analyzed by one-way ANOVA (control vs. oxLDL treatments) followed by a Dunnett's post hoc test. The statistics were computed with the SigmaStat program. A value of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Proliferation of fibroblasts after exposure to oxLDL. The ability of oxLDL to stimulate entry of cells into the cell cycle was first analyzed by flow cytometry. Cells maintained in starvation medium (no oxLDL) for 24 h remained at ~92-95% G0/G1 arrested. In contrast, cells treated with 10 or 50 µg/ml oxLDL had substantial decreases in the proportion of cells in G0/G1 over time. For example, after treatment with 10 µg/ml oxLDL, 91%, 93%, 82%, and 78% of cells were in G0/G1 at 2, 6, 12, and 24 h. After treatment with 50 µg/ml oxLDL, 89%, 91%, 74%, and 72% of the cells remained in G0/G1 at 2, 6, 12, and 24 h. Therefore, oxLDL released cells from growth arrest in a time- and dose-dependent manner.

The ability of oxLDL to stimulate proliferation was then assessed by total cell counts (Fig. 1). Treatment of cells with 0, 10, and 50 µg/ml oxLDL for 24 or 48 h resulted in significant increases in the numbers of both fibroblasts and smooth muscle cells. At least 995 cells were counted for each treatment and time point. Exposure to 10 µg/ml oxLDL resulted in increases of 39% at both 24 and 48 h in fibroblasts. The same concentration of oxLDL increased VSMC numbers by 25% and 27% at 24 and 48 h, respectively. Exposure of fibroblasts to 50 µg/ml oxLDL increased cell numbers by 59% at 24 h and 40% at 48 h, whereas VSMC numbers increased by 55% (24 h) and 33% (48 h) under the same conditions. Treatment of both types of cells with native LDL did not result in the same magnitude of change in cell numbers at either concentration or time point.


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  		Fig. 1.   Increase in total number of cells after exposure of quiescent fibroblasts (A and C) and vascular smooth muscle cells (VSMC; B and D) to oxidized low-density lipoprotein (oxLDL; A and B) and native LDL (C and D). Mean numbers of cells per field, as counted using a hemacytometer, are expressed as percentages of the control ± SE (*P < 0.05). Data represent at least 4 independent experiments. A minimum of 1,046 cells was counted per treatment. In some cases, the SE bars are too small to resolve.

For comparative purposes, we examined the effects of exposing quiescent fibroblasts to bFGF. Over 24 h of exposure, fibroblast cell counts increased 39.4% with 10 µg/ml oxLDL and 33.6% with 10 ng/ml bFGF. Over 48 h of exposure time, fibroblast cell counts increased 38.7% with 10 µg/ml oxLDL and 86% with 10 ng/ml bFGF. Thus oxLDL appears to possess a mitogenic activity similar to bFGF for 24-h exposure times but does not induce as sustained a proliferative effect over 48 h.

To evaluate the possible role of the scavenger receptor in the proliferative mechanism of oxLDL, the scavenger receptor blocker polyinosinic acid was used. At a concentration of 25 µg/ml, polyinosinic acid effectively inhibited the mitogenic action of oxLDL on serum-starved fibroblasts (Fig. 2). The phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 (at a concentration of 20 µg/ml) also prevented oxLDL-induced proliferation, as did the phospholipase C (PLC) inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) (used at a concentration of 50 µg/ml). The MEK 1/2 inhibitor PD-98059 (used at a concentration of 4 µg/ml), while effective in preventing growth in response to 10 µg/ml oxLDL, did not prevent growth in response to 50 µg/ml oxLDL. In each experiment, cells treated with the inhibitor in the absence of oxLDL showed no evidence of cell death compared with cells maintained in starvation medium (Fig. 2).


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  		Fig. 2.   Effect of inhibitors on cell numbers in fibroblasts exposed to oxLDL. Cell counts are expressed as means per field ± SE (*P < 0.05). Cells were pretreated with inhibitors alone for 15 min before exposure to oxLDL in combination with the inhibitors for 24 h. Data represent at least 4 independent experiments. A minimum of 112 cells was counted per treatment. NCDC, 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate.

Because the PLC signaling pathway appeared to be involved in the proliferative action of oxLDL, we speculated that the signaling molecule Ins(1,4,5)P3 might also play a role. Fibroblasts were treated with 0 or 50 µg/ml oxLDL in the presence or absence of 50 µg/ml NCDC for 24 h. Treatment of fibroblasts with 50 µg/ml oxLDL resulted in a significant increase in Ins(1,4,5)P3 levels (Fig. 3). This increase was prevented by NCDC treatment.


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  		Fig. 3.   D-Myo-inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] content in oxLDL-treated cells. Fibroblasts were treated with 0 or 50 µg/ml oxLDL in the presence or absence of 50 µg/ml NCDC for 24 h. Data represent 4 independent experiments. Ins(1,4,5)P3 contents are expressed as picomoles per milligram of protein ± SE (*P < 0.05 vs. all other treatments).

Cell cycle protein expression after exposure to oxLDL. Western blot analysis was used to determine whether oxLDL could induce changes in the total cellular levels of cell cycle proteins. Expression of the cell cycle proteins was examined in whole cell extracts of fibroblasts exposed to 10 and 50 µg/ml oxLDL for 24 and 48 h. Total cellular levels of PCNA were significantly increased at both concentrations and time points with respect to controls (Fig. 4A). Exposure to 10 µg/ml oxLDL resulted in an increase of 39% over control at 24 h and 34% over control at 48 h. Higher concentrations of oxLDL caused similar effects. Surprisingly, the expression of a cell cycle inhibitor, p27, was also induced by oxLDL treatment (Fig. 4B). Exposure of cells for 24 h to 10 and 50 µg/ml oxLDL resulted in increases in expression of 36% and 47% over control. Longer exposure times did not change expression. Not all cell cycle proteins were affected by oxLDL treatment. No significant changes in the expression of cdk4 were observed (Fig. 4C).


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  		Fig. 4.   Expression of PCNA (A), p27 (B), and cyclin-dependent kinase (cdk) 4 (C) in fibroblasts after exposure to oxLDL. Top: representative bands from Western blots of whole cell extracts from oxLDL-treated cells. Bottom: densitometric comparisons of expression in cells exposed to 0, 10, or 50 µg/ml oxLDL for 24 or 48 h expressed as percentages of control ± SE (*P < 0.05). Data represent at least 3 independent experiments for each protein examined.

The effects of oxLDL on both cell cycle activators and inhibitors in Fig. 4 prompted us to examine other representative proteins in greater depth. We examined cyclin D1 and p21 expression at earlier time points (6-48 h) after exposure of cells to oxLDL (Fig. 5). These targets were chosen because of their importance in regulating the cell cycle. Cyclin D1 is the first cyclin necessary for movement of the cells from a growth-arrested state into the cell cycle, and p21 is a potent inhibitor of cell proliferation throughout the entire cycle (17). Expression of cyclin D1 increased over control as early as 6 h after exposure to 10 µg/ml oxLDL. Maximal effects were observed at 24 h, followed by a sharp decline in expression at 48 h. Although the effects of oxLDL on p21 expression followed a similar pattern, the induction in expression was delayed and less pronounced. After exposure to a higher oxLDL concentration (50 µg/ml) for 24 h, total cellular levels of p21 were also significantly increased by 20% over control (data not shown).


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  		Fig. 5.   Expression of cyclin D1 and p21 in quiescent fibroblasts after exposure to oxLDL. Data represent at least 3 independent experiments for each protein and time point. Densitometric comparisons of expression in cells exposed to 50 µg/ml oxLDL for 6, 12, 24, and 48 h are expressed as percentages of control ± SE (*P < 0.05, significant difference from respective untreated values).

Total cellular levels of cdc 2, cdk 2, and cyclin B1 were difficult to detect in control cells (Fig. 6). However, by 24 and 48 h after exposure to oxLDL, the levels of these proteins clearly increased. Both 10 and 50 µg/ml oxLDL induced significant changes in the expression of all of these proteins. However, because of the low levels of expression in the control cells, it was not possible for us to quantitate this increase. The results depicted in Fig. 6 are representative of several experiments (n = 4).


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  		Fig. 6.   Increased expression of cell division cycle (cdc) 2, cdk 2, and cyclin B1 in fibroblasts after exposure to oxLDL. Bands from Western blots represent 3 independent experiments. Because control expression was minimal, quantification via densitometric comparisons could not be completed in a reliable fashion.

Cell cycle protein distribution after exposure to oxLDL. Cellular distribution of cell cycle proteins was then studied in cells exposed to 10 and 50 µg/ml oxLDL for 24 and 48 h. Translocation into the nucleus is a key step in the activation of cyclin/cdk complexes (17). Nuclear levels of PCNA were significantly higher than those of controls in fibroblasts treated with 50 µg/ml oxLDL for 24 h (38% over control; Fig. 7). Forty-eight hours of 10 and 50 µg/ml oxLDL treatment elevated nuclear levels of PCNA by 92% and 124% over control, respectively (P < 0.05).


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  		Fig. 7.   Increased nuclear fluorescence of PCNA in fibroblasts after 48 h of 50 µg/ml oxLDL treatment. A: cells treated with 0 µg/ml oxLDL stained with primary antibody to PCNA and secondary antibody to FITC. B: cells treated with 0 µg/ml oxLDL stained with secondary antibody to FITC (no primary antibody). C: cells treated with 50 µg/ml oxLDL stained with primary antibody to PCNA and secondary antibody to FITC. D: cells treated with 50 µg/ml oxLDL stained with secondary antibody to FITC (no primary antibody). Magnification, ×400. E: comparisons of nuclear fluorescence in cells exposed to 0, 10, or 50 µg/ml oxLDL for 24 or 48 h expressed as percentages of control ± SE (*P < 0.05); n = 3 for each condition and time point.

At the 24-h time point, nuclear levels of cyclin D1 were significantly increased at both 10 and 50 µg/ml oxLDL, by 49% and 45%, respectively, compared with control (Fig. 8). By 48 h, levels of nuclear cyclin D1 had risen by 119% (10 µg/ml oxLDL) and 221% (50 µg/ml oxLDL) versus control.


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  		Fig. 8.   Increased nuclear fluorescence of cyclin D1 in fibroblasts after 48 h of 50 µg/ml oxLDL treatment. A: cells treated with 0 µg/ml oxLDL stained with primary antibody to cyclin D1 and secondary antibody to FITC. B: cells treated with 0 µg/ml oxLDL stained with secondary antibody to FITC (no primary antibody). C: cells treated with 50 µg/ml oxLDL stained with primary antibody to cyclin D1 and secondary antibody to FITC. D: cells treated with 50 µg/ml oxLDL stained with secondary antibody to FITC (no primary antibody). Magnification, ×400. E: comparisons of nuclear fluorescence in cells exposed to 0, 10, or 50 µg/ml oxLDL for 24 or 48 h expressed as percentages of control ± SE (*P < 0.05); n = 4 for each condition and time point.

Similar comparisons were made for the cell cycle proteins cdc 2, cdk 2, cdk 4, cyclin A, cyclin B1, cyclin E, p21, p27, p53, and Rb (Table 1). After 24 h of exposure to 10 µg/ml oxLDL, significant increases in nuclear levels of cdc 2 and cdk 4 were noted. After 48 h of exposure to 10 µg/ml oxLDL, significant increases were observed in nuclear levels of all cell cycle proteins but p27. Twenty-four hours of exposure to 50 µg/ml oxLDL induced significant increases in nuclear levels of every cell cycle protein examined but cyclin B1. Exposure to 50 µg/ml oxLDL for 48 h resulted in significant increases in nuclear levels of all cell cycle proteins but cyclin B1 and cyclin E. Therefore, these data suggest that exposure of fibroblasts to oxLDL induces increases in the nuclear levels of cdc 2, cdk 2, cdk 4, cyclin A, cyclin B1, cyclin D1, cyclin E, p21, p27, p53, PCNA, and Rb.

                              
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  		Table 1.   Increased nuclear fluorescence of cell cycle proteins after exposure of quiescent fibroblasts to oxLDL

Kinase activation after exposure to oxLDL. Although translocation of cell cycle proteins into the nucleus suggests the activation of cyclin/cdk complexes, it is not direct proof of such activation. We examined cdk4 kinase activity after oxLDL exposure as a representative marker of kinase activation under our experimental conditions. Exposure of cells to 50 µg/ml oxLDL for 24 h resulted in a significant increase of 20% in cdk activity compared with control cells (Fig. 9).


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  		Fig. 9.   Increased kinase activity of cdk 4 in fibroblasts after exposure to oxLDL. Top: representative autoradiograph showing cdk 4 activity (with GST-pRb as a substrate) in whole cell extracts from fibroblasts treated with 0, 10, or 50 µg/ml oxLDL for 24 h. Bottom: densitometric comparisons of cdk 4 activity expressed as percentages of control ± SE (*P < 0.05); n = 4 for each condition and time point.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OxLDL induced a significant increase in the total number of cells in culture in the absence of any other cytokines or growth factors. Therefore, this study identifies oxLDL as a compound capable of inducing proliferation in the absence of any other mitogenic factors. The mitogenic action of oxLDL was similar to bFGF but did not maintain as large or as sustained a proliferative effect as bFGF. This mitogenic effect was specific to oxidized LDL (native LDL did not have the same magnitude of effect) and showed time and dose dependency. The effect was not dependent on cell type, because both fibroblasts and VSMC responded in a similar manner. We may safely conclude that oxLDL acts as an independent mitogen, as shown by others previously (9, 32).

The present investigation also identified several components of the cellular signaling pathway associated with the proliferative effects of oxLDL. We identified both cell surface and intracellular sites of action. The scavenger receptor blocker polyinosinic acid prevented oxLDL-induced increases in cell number. This suggests that oxLDL induces its proliferative action through an interaction with the scavenger receptor. If so, one would suspect that receptor stimulation would lead to activation of an intracellular signaling pathway. Our data would suggest that the PI3K pathway appears to be involved in the proliferative effects of oxLDL. This is consistent with results reported previously (32). The PLC pathway and the intracellular signaling molecule Ins(1,4,5)P3 also appear to be involved. The association of oxLDL, proliferation, and PLC has not been identified previously. However, lysophosphatidylcholine (LPC; a component with oxLDL) has been identified as an activator of PLC (4). The observation that PD-98059, a selective MEK1/2 inhibitor, was less effective in blocking proliferation in response to higher concentrations of oxLDL is somewhat surprising, given that numerous studies have shown activation of the MAPK pathway after exposure to oxLDL (10, 14, 22, 26). However, it is possible that the inhibition by PD-98059 of the MAPK pathway is incomplete, and the activation by higher concentrations of oxLDL simply overwhelms the inhibitory effect. Furthermore, activation of MEK1/2 does not necessarily imply its involvement in growth (48). Similarly, the inability of PD-98059 to block the mitogenic effect of oxLDL does not rule out the participation of other members of the MAPK family (22).

The most important and surprising observation in the present study is that oxLDL induced the simultaneous induction of both cell cycle activators and suppressors. In a state where cell proliferation is stimulated, one would have expected an increased expression of proteins responsible for the activation of the cell cycle and/or an inhibition of cell cycle inhibitory proteins. This is the case in other conditions of rapid cell proliferation like cancer or in development. Malignant cell growth is typically characterized by high levels of one or more cell cycle inducers and low levels (or a complete absence) of functional cell cycle inhibitors (43). However, this seemingly contradictory situation has previously been observed in other disease states, such as liver regeneration (2). It has been hypothesized that, by activating both inducers and inhibitors simultaneously, the cell effectively regulates its own growth. Induction of p21 serves to regulate the rate of progression through G1, whereas p27 modulates cdk 2 activity before and after the S phase (2). The cell cycle will proceed forward (presumably due to an imbalance of inducers over inhibitors), but high levels of inhibitors ensure that it may be shut down rapidly in response to changes in the cellular environment. This cooperation between cell cycle regulators is proposed to lead to a precisely controlled type of growth (2). This observation of a controlled proliferative response due to a generalized induction of all cell cycle proteins is consistent with the slower, nonmalignant cell growth typical of an atherosclerotic or restenotic plaque. The time dependency that we observed is consistent with this observation and further demonstrates that the expression of activators and inhibitors of the cell cycle is not exactly "simultaneous." The induction of a cell cycle activator like cyclin D1 occurred faster and to a greater degree than the induction of an inhibitory protein like p21. One may conclude, therefore, that p21 expression represents an adaptive response that may regulate the initial proliferative effects.

An increase in the expression of cell cycle proteins does not necessarily mean that functional changes exist. Translocation of cell cycle proteins into the nucleus is thought to activate cyclin/cdk complexes (17). Movement of cell cycle proteins into the nucleus would, therefore, represent strong indirect evidence in support of an activation of the cell cycle. In the present study, the increases in the total levels of these proteins, as determined by Western blot analysis, were generally accompanied by increases in the levels of cell cycle proteins in the nucleus. Nuclear localization of the cell cycle inducers cdc 2, cdk 2, cdk 4, cyclin A, cyclin B1, cyclin D1, cyclin E, and PCNA were all significantly increased with respect to controls after oxLDL treatment. Direct analysis of cdk4 activity confirmed the hypothesis that the kinase complexes were not only importing into the nucleus but were active and associated with the proliferative event. Consistent with the expression data, the cell cycle inhibitors p21, p27, p53, and Rb were all found in greater concentrations in the nucleus of the cell. These data are consistent with the hypothesis that oxLDL is inducing a proliferative event by increasing the expression and nuclear translocation of both inhibitors and activators of the cell cycle.

The mechanism responsible for the movement of cell cycle proteins into the nucleus of the cell by oxLDL is unclear from the results of the present study. Several possibilities exist based on previously published reports. It may occur through a PLC or PI3K pathway, as indicated above. However, no study has yet examined the potential for these pathways to regulate nuclear protein import. Alternatively, two other studies have demonstrated that nuclear protein import is sensitive to oxidative reactions and LPC (13, 47). LPC is a major byproduct of the oxidation of LDL and has proliferative action (6). It is possible that its entry into the cell may have altered nuclear translocation of cell cycle proteins. This awaits further experimentation.

In summary, oxLDL was capable of inducing proliferation in fibroblasts and smooth muscle cells in the absence of other mitogens. We may conclude that oxLDL is a potent independent mitogenic factor. Under some conditions, oxLDL can be cytotoxic. OxLDL was not cytotoxic under any of the conditions used in the present study. The mitogenic effect of oxLDL occurred through an interaction of oxLDL with scavenger receptors on the cell surface and an augmentation of intracellular signaling through the PI3K and PLC pathways. The stimulation was accompanied by a significant increase in the total cellular expression of cell cycle proteins as well as a redistribution of the cell cycle proteins into the nucleus of the cell. Our results provide the first demonstration that a known atherogenic lipoprotein, oxLDL, can induce changes in cell cycle protein distribution and expression characteristic of a controlled, adaptive response to a chronic pathological condition. These effects may play an important role during the early proliferative phases of atherosclerotic and restenotic vascular disease.


    ACKNOWLEDGEMENTS

M. Zettler received a studentship from the Deer Lodge Hospital Association Memorial Fund and a Doctoral Research Partnership Award from the Canadian Institutes of Health Research/Heart and Stroke Foundation of Canada. H. Massaeli received a Research Traineeship from the Heart and Stroke Foundation of Canada. G. N. Pierce is a Senior Scientist of the Canadian Institutes of Health Research.


    FOOTNOTES

Address for reprint requests and other correspondence: G. N. Pierce, Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, MB, Canada R2H 2A6 (E-mail: gpierce{at}sbrc.ca).

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.

10.1152/ajpheart.00494.2001

Received 6 June 2001; accepted in final form 15 September 2002.


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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 284(2):H644-H653
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