MEK-ERK Inhibition Corrects the Defect in VLDL Assembly in HepG2 Cells

【摘要】  Objective- Hepatic VLDL assembly is defective in HepG2 cells, resulting in the secretion of immature triglyceride-poor LDL-sized apoB particles. We investigated the mechanisms underlying defective VLDL assembly in HepG2 and have obtained evidence implicating the MEK-ERK pathway.

Methods and Results- HepG2 cells exhibited considerably higher levels of the ERK1/2 mass and activity compared with primary hepatocytes. Inhibition of ERK1/2 using the MEK1/MEK2 inhibitor, U0126 (but not the inactive analogue) led to a significant increase in apoB secretion. In the presence of oleic acid, ERK1/2 inhibition caused a major shift in the lipoprotein distribution with a majority of particles secreted as VLDL, an effect independent of insulin. In contrast, overexpression of constitutively active MEK1 decreased apoB and large VLDL secretion. MEK1/2 inhibition significantly increased both cellular and microsomal TG mass, and mRNA levels for DGAT-1 and DGAT-2. In contrast to ERK, modulation of the PI3-K pathway or inhibition of the p38 MAP kinase, had no effect on lipoprotein density profile. Modulation of the MEK-ERK pathway in primary hamster hepatocytes led to changes in apoB secretion and altered the density profile of apoB-containing lipoproteins.

Conclusion- Inhibition of the overactive ras-MEK-ERK pathway in HepG2 cells can correct the defect in VLDL assembly leading to the secretion of large, VLDL-sized particles, similar to primary hepatocytes, implicating the MEK-ERK cascade in VLDL assembly in the HepG2 model. Modulation of this pathway in primary hepatocytes also regulates apoB secretion and appears to alter the formation of VLDL-1 sized particles.

Inhibition of the overactive ras-MEK-ERK pathway in HepG2 cells was found to correct the defect in VLDL assembly (that normally secrete immature triglyceride-poor LDL-sized apoB particles) leading to the secretion of large, VLDL-sized particles, similar to primary hepatocytes. Our data implicate the MEK-ERK pathway in regulating the VLDL assembly process in HepG2 cells.

【关键词】  apolipoprotein B MEKERK HepG VLDL

Introduction

Apolipoprotein B100 (apoB) is the structural component of triglyceride (TG)-rich lipoproteins such as very low density lipoproteins (VLDL). 1 There is evidence that much of the newly-synthesized apoB is subject to both co- and posttranslational degradation before it can be secreted. 1-3 The key determinant of apoB folding and susceptibility to intracellular degradation is lipid availability. Nascent apoB, as it translocates across the ER membrane, is prone to rapid proteasomal degradation in the absence of sufficient neutral lipids to form a primordial lipoprotein particle. 4 This step has been found to be catalyzed by the microsomal triglyceride transfer protein (MTP). 5-7 It is also the first step in VLDL maturation. After apoB enters the ER lumen, it appears to remain susceptible to degradation by ER proteases. 8,9 As primordial lipoproteins move from the ER to the Golgi, these appear to fuse with lipid droplets and mature into VLDL-sized particles. 10 This is the second step in VLDL maturation. The enzyme/protein factors responsible for the second step have not been identified. However, several factors have been suggested to be involved in VLDL assembly, including diacylglycerol acyltransferase (DGAT), Arf-1, and phospholipase D (PLD). 11

DGATs are microsomal enzymes that catalyze the last committed step in TG synthesis. 12-14 Furthermore, recent studies have shown that the overexpression of DGAT results in increased apoB-containing lipoprotein secretion. 15,16 Conversely, inhibition of DGAT activity by niacin decreases TG level and secretion. 17 There are two DGAT proteins, DGAT1 and DGAT2, which have distinct tissue distribution. DGAT1 is expressed most highly in the small intestine whereas DGAT2 has high expression in the liver and adipose tissue. 12 Furthermore, DGAT activities can be separated into overt and latent activities. 18,19 It has been suggested that the overt activity of DGAT is responsible for cytosolic TG storage, whereas the latent activity contributes to the TG pool accessible to VLDL. 18 Arf-1 is a small GTPase that has been shown to activate PLD, which produces phosphatidic acid from phosphatidylcholine. 20 Phosphatidic acid is a precursor for TG. It has been shown that part of the VLDL-TG comes from phospholipids. 21 Inhibition of Arf-1 by low concentration of Brefeldin A has also been shown to inhibit VLDL assembly. 20 In addition, overexpression of an inhibitory Arf-1 mutant in rat hepatoma McArdle RH-7777 was found to decrease VLDL secretion, 20 suggesting the involvement of Arf-1 in VLDL maturation.

A key metabolic regulator of apoB assembly and secretion is insulin. Insulin inhibits apoB secretion as well as TG secretion, possibly by limiting the assembly of VLDL and reducing the intracellular association of apoB with lipid. 22,23 The insulin effect is not a consequence of decreased lipid availability as insulin simultaneously stimulates triglyceride synthesis. The direct inhibitory effect of insulin on apoB appears to be exerted by both a reduction in apoB mRNA translation and by favoring the turnover of freshly synthesized apoB. 24 Several reports have demonstrated that insulin requires phosphatidylinositol-3-kinase (PI3-K) to decrease apoB-VLDL secretion as PI3-K inhibitors, wortmannin and LY294002, reverse the insulin effect on apoB in cultured hepatocytes. 22,25 Furthermore, it has been suggested that PI3-K inhibits apoB secretion at the VLDL maturation stage as LY294002 restores the suppression of VLDL-apoB by insulin while having no effect on small dense immature VLDL-apoB particles. 22 It is thought that insulin may prevent the recruitment of lipid droplets onto apoB, thus promoting intracellular apoB degradation and suppressing VLDL-apoB secretion. 11 Despite the effect of PI3-K on apoB secretion, its downstream effector protein kinase B (PKB)/Akt does not appear to participate in apoB regulation. Overexpression of dominant active Akt1 does not inhibit apoB secretion in McArdle-RH7777 cells, 26 indicating that apoB secretion may be regulated by other signaling pathways that are parallel or upstream of the Akt cascade. Insulin may also regulate the assembly and secretion of apoB particles via modulation of MTP gene expression through the MEK-ERK pathway. 27,28 The larger catalytic subunit of MTP is essential for lipid transfer activity and apoB-containing lipoprotein secretion. ApoB secretion is decreased in a dose-dependant manner on treatment with specific MTP inhibitors, 29 and apoB secretion in non-hepatic and non-intestinal cells requires coexpression of apoB with MTP. 6

In the present study, we have exploited the defective lipoprotein assembly process in HepG2 cells, a popular model used in apoB studies, to identify the signaling mechanisms involving in regulating the apoB-VLDL assembly process. HepG2 is a human hepatoblastoma cell line that secretes the majority of apoB with low density lipoprotein (LDL) density. 30,31 Although the addition of oleic acid enhances apoB secretion from HepG2 cells, most of the apoB-containing lipoproteins remain in the LDL density range. 30,31 It has been suggested that in HepG2 cells, the defect in apoB-VLDL secretion is attributable to low mobilization of cytosolic TG storage as TG synthesis is active in this cell line. 31,32 Apparently, most of the newly synthesized TG is stored in the cytosol, with very little of it transferred to the microsomal TG storage. 32 Because microsomal TG is the lipid pool accessible to apoB, the lack of TG transfer to the microsomal pool results in the defect of VLDL maturation in HepG2 cells. 32 In the present report, we provide experimental evidence that defective VLDL assembly in HepG2 cells may be linked to hyperactivity of the MEK-ERK pathway, suggesting a critical role for this signaling cascade in regulating the intracellular assembly of apoB-containing lipoproteins.

Materials and Methods

Please see the supplemental materials, available online at http://atvb.ahajournals.org, for more detailed methods.

Cell Culture

HepG2 cells were maintained in MEM supplemented with 5% FBS and 1 x antibiotic-antimycotic at 37°C, 5%CO 2. Cells were passaged every 3 to 4 days. Freshly isolated Golden Syrian hamster hepatocytes and McArdle-RH7777 cells were maintained in DMEM supplemented with 20% FBS and 1 x antibiotic-antimycotic at 37°C, 5%CO 2.

Inhibitor Treatment

U0126, U0124, SB203580, and SB202474 were dissolved in DMSO to make up 20 mmol/L stock solutions. U0124 is a compound structurally similar to U0126, but it does not inhibit MEK1/2 activity. Similarly, SB202474 is the structural analogue of SB203580 and does not inhibit p38 activity. PD98059 was dissolved in DMSO to make a 50 mmol/L stock solution. Cells used for inhibitor dose study and for lipoprotein density profile were incubated in cysteine- and methionine-free media supplemented with 15% FBS and 360 µmol/L oleic acid for 1 hour, followed by a 3.5-hour pulse with 50 µCi/mL [ 35 S]-labeling mix.

Transient Transfection

HepG2 cells were seeded in 6-well plates (1 x 10 6 cells per well). Active MEK1 construct was transfected using FuGENE6, following the protocol provided by Roche Diagnostics. After 2-day transfection, cells were radiolabeled, lysed, immunoprecipitated, and subjected to SDS-PAGE.

Adenoviral Infection

HepG2 cells were seeded in 100 mm plates (6 x 10 6 cells per plate) and infected with 10 multiplicity of infection at room temperature for 10 minutes. The cells were then washed with PBS and incubated in DMEM containing 10% FBS for 2 days.

Metabolic Labeling, Immunoprecipitation, and Fluorography

HepG2 cells were treated with U0126, U0124, PD98059, SB203580, SB202474, transfected with active MEK1, or adenoviral infected with wild-type (wt) or dominant negative (C/S) PTEN as described above. Primary hamster hepatocytes were treated U0126, U0124, or adenoviral infected with active MEK1 as described above. Cells were pulsed with 50 µCi/mL [ 35 S] for 3.5 hours as described in the figure legends. Media was collected for immunoprecipitation or for NaBr gradient ultracentrifugation.

Ultracentrifugation and Fractionation

Salt density gradient was performed using a modified version of a previously described method (see detailed methods). Briefly, media was collected and adjusted to 1.1 g/mL with NaBr, which was overlaid with 1.06, 1.02, and 1.006 g/mL gradients and ultracentrifuged at 35 000 rpm for 18 hours using Beckman Optima LE-80K Ultracentrifuge. Samples were then fractionated, immunoprecipitated for apoB, and subjected to SDS-PAGE.

Real-Time Polymerase Chain Reaction Analysis of mRNA Levels

Total RNA was extracted using QIAGEN RNeasy mini kit. cDNA was synthesized using Applied Biosystems TaqMan Reverse Transcription Reagents. Real time polymerase chain reaction (PCR) was performed using the specific sets of primers to measure the mRNA levels of apoB, MTP, DGAT1, and DGAT2.

Results

Basal ERK Mass and Activity in HepG2 Cells Compared With That in Primary Hamster Hepatocytes and McArdle-RH7777, a Rat Hepatoma Cell Line

There is evidence from the literature suggesting that HepG2 cells may have a constitutively activated ERK pathway, which (we postulate) may contribute to the defective apoB-lipoprotein phenotype observed in this cell line. The relative expression and activity of ERK1/2 in HepG2 cells were assessed in comparison to 2 other hepatocyte systems: primary hamster hepatocytes and McArdle-RH7777 cells. All 3 cell types were lysed and then immunoblotted for phospho-ERK and ERK1/2 mass ( Figure 1 A). Equal amount of total protein was loaded in triplicate lanes from each of the 3 cell lines. HepG2 had the highest total ERK phosphorylation and protein mass compared with the other 2 hepatocytes. McArdle-RH7777 cells had considerably lower ERK activity and mass compared with HepG2 but had higher levels when compared with primary hamster hepatocytes which showed very little basal activity or mass.

Figure 1. Basal ERK1/2 phosphorylation and mass in HepG2 cells and the effect of ERK1/2 inhibition on apoB secretion. A, Immunblot analysis of phospho-ERK and ERK mass. B, Immunoblot analysis for both phosph-ERK1/2 and total ERK mass in the presence of the MEK1/2 inhibitior, U0126. C, Cells were metabolically labeled with [ 35 S]-labeling mix and the media was immunoprecipitated for apoB. n=3; * P <0.05 compared with U0124 treatment; P <0.05 compared with untreated control.

MEK1/2 Inhibition by U0126 Increases ApoB100 Secretion From HepG2 Cells in a Dose-Dependent Manner

To examine whether MEK1/2 inhibition would affect apoB production, HepG2 cells were treated overnight with 0, 0.1, 1, 5, or 10 µmol/L of U0124 or U0126, followed by a 3.5-hour pulse in the presence of oleic acid. The media was immunoprecipitated for apoB. Although U0126 decreased phospho-ERK level dose-dependently, U0124, a negative control, did not affect phospho-ERK significantly at doses lower than 10 µmol/L ( Figure 1 B), confirming U0126 specificity.

Cellular apoB was not affected significantly by MEK1/2 inhibition (data not shown). However, apoB secretion appeared to increase with U0126 ( Figure 1 C). The highest increase in apoB secretion was observed at 1 µmol/L of U0126 (55%, P =0.003), and the increase plateaued after 1 µmol/L ( Figure 1 C). Total radiolabeled protein counts, which estimate total protein synthetic activity, decreased 15% at 10 µmol/L U0126. Therefore, to avoid cytotoxicity, 1 µmol/L was chosen as the U0126 concentration to be used for later experiments.

MEK1/2 Inhibition Increases ApoB Secretion Under Basal Conditions, and Enhances the Secretion of VLDL-Sized ApoB-Containing Lipoprotein Particles in the Presence of Oleic Acid

The effect of the MEK1/2 inhibitor, U0126, on lipoprotein density was investigated by incubating HepG2 cells with 1 µmol/L of U0124 or U0126 overnight, followed by a 3.5-hour pulse in the presence or absence of oleic acid. The media was collected and subjected to gradient ultracentrifugation and fractionation. ApoB was immunoprecipitated from each fraction. At basal state, both U0124- and U0126-treated cells secreted apoB in mostly LDL-sized particles ( Figure 2 A). However, MEK1/2 inhibition by U0126 appeared to increase apoB secretion ( Figure 2 A). In the presence of oleic acid, apoB-containing lipoproteins significantly shifted to lower density fractions following U0126 treatment ( Figure 2 ). The shift in apoB-containing particle density induced by U0126 made the density profile more similar to that of lipoproteins secreted by primary hepatocytes ( Figure 2 A). U0124, the inactive analogue (negative control), was found to have no effect on apoB secretion or density profile (data not shown), suggesting the specificity of the effects observed with U0126.

Figure 2. MEK1/2 inhibition increases the assembly and secretion of VLDL-sized apoB-containing lipoprotein particles. A, HepG2 cells were treated with U0124 or U0126 (1 µmol/L) and metabolically labeled with [ 35 S]-labeling mix in the presence or absence of 360 µmol/L oleic acid. The media was collected and apoB-lipoprotein profile was determined by NaBr gradient ultracentrifugation. B and C, Similar experiments as above were conducted but following treatment with either DMSO or PD98059 (5 µmol/L). The graph is a representative of 5 experiments.

To determine whether the shift in apoB-containing lipoprotein density caused by U0126 was truly attributable to MEK-ERK inhibition, a structurally different inhibitor PD98059 was used. HepG2 cells were treated with PD98059 in the same fashion as U0126. There was a dose-dependent decrease in phospho-ERK level as PD98059 concentration increased ( Figure 2 B). Similar to U0126, PD98059 treatment at 5 µmol/L also induced an increase in VLDL-sized particles, though the shift was not as dramatic and there was still a prominent peak around fraction 5 ( Figure 2 C).

MEK1/2 Inhibition Significantly Increases Cellular and Microsomal TG Level

To elucidate the shift in apoB-containing lipoprotein density in the presence of U0126 and oleic acid, TG level was measured in cells treated overnight with U0124 and U0126. Compared with U0124, the TG level of U0126-treated cells increased by 37% ( P =0.00007; Figure 3 A). Moreover, microsomal TG level of U0126-treated cells was also 34% higher than U0124-treated cells ( P =0.042; Figure 3 B). The results demonstrate an overall increase in TG availability for VLDL assembly after ERK inhibition.

Figure 3. Effect of MEK-1 inhibition on cellular and microsomal TG as well as apoB, MTP, DGAT1, and DGAT2 mRNA levels. A and B, HepG2 cells were treated with U0124 or U0126 (1 µmol/L) in the presence of 360 µmol/L oleic acid. Cellular and microsomal TG levels were determined. n=3; * P <0.05 compared with U0124-treated samples. C, mRNA levels were also measured using real-time PCR. Values are mean±SD for 2 experiments performed in triplicate. * P <0.05 compared with U0124-treated samples.

MEK1/2 Inhibition Does Not Significantly Affect ApoB and MTP mRNA Levels, but Increases DGAT1 and DGAT2 mRNA Levels

To determine the underlying mechanisms for the shift in apoB-containing lipoprotein density profile and the increase in TG level, mRNA levels for apoB, MTP, DGAT1, and DGAT2 were measured using real-time PCR. Total RNA was extracted from U0124- and U0126-treated cells, and cDNA was made using appropriate primers, on which real-time PCR was performed. While MEK1/2 inhibition did not affect apoB mRNA significantly ( Figure 3 C), MTP mRNA increased slightly but insignificantly in the presence of U0126. In contrast, U0126 caused a significant 2-fold increase in DGAT1 and a 4-fold increase in DGAT2 mRNA level ( P =0.03 and P =0.04, respectively; Figure 3 C). The increase in DGAT1 and DGAT2 mRNA levels may underlie the observed increase in cellular and microsomal TG.

The density profile of apoB100-containing lipoproteins in HepG2 cells was found to be insensitive to (1) insulin treatment, (2) modulation of PI-3 kinase via PTEN overexpression, (3) p38 inhibition, and (4) MEK-1 overexpression. These control data are included in the online supplemental materials.

Effect of Modulating the MEK-ERK Pathway on the Density Profile of ApoB100-Containing Lipoproteins in Primary Hamster Hepatocytes

To determine whether the ERK pathway regulates VLDL assembly in other cell types, primary hamster hepatocytes were isolated and treated with U0126 overnight or infected with constitutively active MEK1 adenovirus. U0126 inhibited ERK phosphorylation dose-dependently and MEK1 overexpression increased ERK phosphorylation ( Figure 4 A). MEK1/2 inhibition appeared to increase and MEK1 overexpression appeared to decrease the total number of apoB particles secreted by primary hepatocytes ( Figure 4 B). Interestingly, MEK-1/2 inhibition by U0126 appeared to increase the amount of labeled apoB in the largest VLDL fraction (fraction 1, referred to as VLDL-1; Figure 4 B, top panel) while MEK-1 overexpression caused a considerable decrease in the same fraction (VLDL-1; Figure 4 B, bottom panel). TG and cholesterol mass were also measured in active MEK1-infected primary hepatocytes. Active MEK1-overexpressing cells induced significant decreases in total cellular cholesterol (100±19.9% versus 73.0±29.5% in ß-gal and MEK1 overexpressing cells, respectively; expressed as percent of ß-gal control, P <0.05) and microsomal cholesterol (100±25.2% and 69.2±34.5% compared with microsomal cholesterol in ß-gal and MEK1 overexpressing cells, respectively; expressed as percent of ß-gal control, P <0.05). Active MEK-1 overexpression also reduced total cellular and microsomal TG (by 12% and 11%, respectively, compared with ß-gal transfected control cells), although these changes in TG did not reach statistical significance (total cellular TG: 100±16.3% and 87.7±17.2%. in ß-gal and MEK-1 overexpressing cells, respectively; expressed as percent of ß-gal control, P =0.13; microsomal TG: 100±17.9 and 88.7±22.1 in ß-gal and MEK-1 overexpressing cells, respectively; expressed as percent of ß-gal control, P =0.25).

Figure 4. Modulating the MEK-ERK pathway in primary hamster hepatocytes alters the density profile of apoB-containing lipoproteins. Cells treated with U0124, U0126 (10 µmol/L), or adenoviral-infected with active MEK1 were immunoblotted for p-ERK, ERK2, and MEK1 (A). B, ApoB100-containing lipoprotein profile of U0126-treated or MEK1-infected cells. Primary hepatocytes were infected for 1 day with either ß-gal or active MEK1 adenovirus. Infected cells were radiolabeled and the media was fractionated by NaBr gradient ultracentrifugation. The graphs are representatives of 3 experiments for each condition.

We also measured MTP mRNA levels (as in Figure 3 ) in primary hepatocytes transfected with ß-gal or MEK-1 and found no significant change in MTP mRNA induced by MEK-1 overexpression (there was a nonsignificant 32% decrease in MTP mRNA but this did not reach significance because of animal to animal variability in message abundance [100±66.9% and 67.4±20.4% in ß-gal and MEK-1 overexpressing cells, respectively; expressed as percent of ß-gal control, P =0.65, n=4]). Finally, we attempted to measure DGAT-1/-2 mRNA in hamster hepatocytes, but the PCR probes designed based on human DGAT sequences did not appear to detect hamster DGAT mRNA transcripts.

Discussion

HepG2 cells have been used extensively for understanding apoB metabolism. Unlike primary hepatocytes, which secrete VLDL particles, HepG2 cells secrete little VLDL, and have a lipoprotein profile that is rich in low-density lipoprotein (LDL)-sized particles. 30-32 Supplementing the incubation media with oleic acid increases VLDL-apoB secretion slightly in HepG2 cells. 31 However, this increase is still incomparable to the amount of VLDL secreted by primary hepatocytes. 33,34 Because TG synthesis in HepG2 is active, the defect in VLDL-secretion appears to be attributable to inefficient TG mobilization. 32 In the present study, we have demonstrated that inhibiting MEK1/2 by U0126 increases apoB secretion significantly, consistent with the recent findings by Allister et al. 35 Moreover, we showed that in the presence of oleic acid, MEK1/2 inhibition enhances VLDL-apoB secretion to a degree that it resembles the lipoprotein profile secreted by primary hepatocytes. These observations are novel as HepG2 have never been reported previously to secrete apoB-containing lipoproteins with a density profile comparable to that of primary hepatocytes.

The use of PD98059 further confirmed that the shift in lipoprotein profile observed following U0126 treatment was attributable to MEK-ERK inhibition, and not an effect specific to one chemical inhibitor. PD98059 is structurally different from U0126 and preferentially inhibits MEK1 whereas U0126 inhibits both MEK1 and MEK2. 36,37

It appears that both cellular and microsomal TG levels increased in the presence of the MEK1/2 inhibitor which may underlie the observed increase in VLDL-apoB secretion. Consistent with the TG results, there was a significant increase in both DGAT1 and DGAT2 mRNA levels when MEK1/2 is inhibited. Similarly, 3T3-L1 cells treated with arachidonic acid exhibit both MEK-ERK activation and DGAT activity inhibition. 38 Inferring from the increased cellular and microsomal TG levels, it is reasonable to postulate that the overall increase in DGAT expressions translated into enhanced DGAT activities, which in term promoted TG production as well as the turnover rate between the cytosolic and microsomal TG pools, providing more substrate for apoB lipidation and resulting in a shift in apoB-containing lipoprotein density profile. However, a recent report has demonstrated that short-term overexpression of DGAT1 or DGAT2 is sufficient to increase cytosolic TG pool but insufficient to change VLDL production, 39 suggesting that the change in lipoprotein profile after MEK inhibition may not be necessarily linked to changes observed in DGAT1 and DGAT2. We also did not observe a significant change in cellular or microsomal cholesterol mass of HepG2 cells in the presence of the MEK1/2 inhibitor (data not shown).

Interestingly, MEK1 overexpression in primary hamster hepatocytes also induced changes in intracellular lipids, with significant decreases in cellular and microsomal cholesterol and an insignificant trend toward lower cellular and microsomal TG. The significance of the changes observed in cellular and microsomal cholesterol mass is currently unclear. It is normally the triglyceride content that determines the size of VLDL particles, although a role for cholesterol in modulating particle size cannot be ruled out. Experiments in HepG2 cells did not show changes in cellular or microsomal cholesterol. Although the changes in TG induced by MEK-1 overexpression in primary hepatocytes was small (11% to 12%), this correlates with a relatively small (but significant) effect on VLDL-apoB particle formation. Primary hepatocytes likely have a highly efficient VLDL assembly process and their secretory pools of lipoprotein lipids may be less sensitive to short-term MEK-1 overexpression. However, chronic perturbations in MEK-ERK cascade (as observed in insulin resistant states) may potentially exert more profound effects on hepatic VLDL assembly in the liver. Limited viability of primary hepatoyctes in culture did not allow us to perform chronic/long-term overexpression of MEK1 in hepatocytes (cells could only be transfected for 24 hours; longer transfections are not possible in primary cell cultures because of loss of viability and loss of differentiated functions).

Consistent with Au et al, 28 MTP mRNA also increased in the present study, though the change was insignificant. The increase in MTP mRNA may also have contributed to the shift in apoB-containing lipoprotein density profile as MTP is essential in stabilizing nascent apoB by loading neutral core lipids onto the protein. 5-7 MTP inhibition has been shown to decrease TG incorporation into VLDL in McArdle-RH7777 cells, 40,41 indicating the importance of MTP in VLDL-apoB secretion. However, the insignificant changes observed in MTP mRNA in HepG2 cells (as well as in primary hamster hepatocytes overexpressing MEK1) suggest that changes in MTP cannot account for the alteration in the density profile of apoB-lipoproteins induced by modulation of MEK-ERK pathway.

Interestingly, there was also a noticeable reduction in apoB secretion and the number of large VLDL particles after MEK1 overexpression. However, a total shift to highly-dense HDL-sized particles was not observed. These observations appear to suggest that overexpression of MEK1 only affected the second step in VLDL assembly and did not alter the initial lipidation of particles with densities in the range of HDL-LDL. It is also important to note that HepG2 cells already have high N-ras activity, and thus high ERK activity. 42,43 Further enhancement of ERK phosphorylation via MEK overexpression may not significantly enhance the basal inhibitory effect of this high ERK activity on apoB-containing lipoprotein assembly.

To determine whether our hypothesis that MEK-ERK regulates VLDL assembly is universal, we modulated the pathway in primary hamster hepatocytes using U0126 and active MEK1 adenovirus. U0126 dose-dependently decreased ERK phosphorylation and active MEK1 increased ERK phosphorylation. Modulation of ERK in primary hepatocytes also led to changes in level of the largest VLDL sized apoB particles (referred to as VLDL-1). In addition, MEK1/2 inhibition increased and MEK1 overexpression decreased overall apoB secretion. Thus, MEK-ERK appears to modulate the rate of apoB secretion and alter the normal VLDL assembly process in primary hamster hepatocytes.

HepG2, like many cancer cell lines, has an overactive ras protein. 42 This is attributable to a missense mutation in the N-ras gene, making it dominant active. 42 Because N-ras is upstream of the MEK-ERK pathway, this mutation leads to an overactive MEK-ERK cascade. 43 This may be the underlying cause of the inability of HepG2 to secrete VLDL as this defect could be corrected by inhibiting the MEK-ERK pathway. Interestingly, when basal ERK phosphorylation and mass in HepG2 cells were compared with other hepatocytes (primary hamster hepatocytes and McArdle-RH7777, a rat hepatoma cell line), HepG2 cells showed the highest total phospho-ERK level. A protein-protein BLAST showed ERK protein sequences across species to be highly identical (data not shown).

Taken together, the results suggest that the defective VLDL-apoB assembly in HepG2 cells can be fully restored by blocking the activity of the MEK-ERK pathway. Evidence that the MEK-ERK pathway also regulates apoB production in primary hepatocytes supports a potential role for ERK as a direct physiological regulator of the VLDL-apoB assembly process.

Acknowledgments

Sources of Funding

This study was supported by an operating grant (T-5323) from the Heart and Stroke Foundation of Ontario to K.A. J.T. was supported by a postgraduate scholarship from NSERC.

Disclosures

None.

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作者单位：Julie Tsai; Wei Qiu; Rita Kohen-Avramoglu; Khosrow AdeliFrom the Division of Clinical Biochemistry, Hospital for Sick Children, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.