Ras/Mitogen-activated Protein Kinase (MAPK) Signaling Modulates Protein Stability and Cell Surface Expression of Scavenger Receptor SR-BI*

The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPARα-inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPARα Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPARα-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPARα-dependent degradation of SR-BI in hepatocytes.


The mitogen-activated protein kinase (MAPK) Erk1/2 has been implicated to modulate the activity of nuclear receptors, including peroxisome proliferator activator receptors (PPARs) and liver X receptor, to alter the ability of cells to export cholesterol. Here, we investigated if the Ras-Raf-Mek-Erk1/2 signaling cascade could affect reverse cholesterol transport via modulation of scavenger receptor class BI (SR-BI) levels. We demonstrate that in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells, Mek1/2 inhibition reduces PPAR␣inducible SR-BI protein expression and activity, as judged by reduced efflux onto high density lipoprotein (HDL). Ectopic expression of constitutively active H-Ras and Mek1 increases SR-BI protein levels, which correlates with elevated PPAR␣ Ser-21 phosphorylation and increased cholesterol efflux. In contrast, SR-BI levels are insensitive to Mek1/2 inhibitors in PPAR␣-depleted cells. Most strikingly, Mek1/2 inhibition promotes SR-BI degradation in SR-BI-overexpressing CHO cells and human HuH7 hepatocytes, which is associated with reduced uptake of radiolabeled and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyane-labeled HDL. Loss of Mek1/2 kinase activity reduces SR-BI expression in the presence of bafilomycin, an inhibitor of lysosomal degradation, indicating down-regulation of SR-BI via proteasomal pathways. In conclusion, Mek1/2 inhibition enhances the PPAR␣-dependent degradation of SR-BI in hepatocytes.
Anti-atherosclerotic properties of HDL and the major HDL apolipoprotein, apoA-I, are believed to include their ability to induce signaling events that promote cholesterol export from peripheral cells to the liver for disposal. However, the signal transduction pathways that contribute to stimulate reverse cholesterol transport in macrophages and hepatocytes are not fully understood (1)(2)(3). HDL binding to receptors such as SR-BI 6 activates various cellular processes, including endothelial nitric-oxide synthase activation in endothelial cells (1)(2)(3) and proliferation in smooth muscle cells (4) but also cell surface localization of SR-BI in hepatocytes (5). Downstream targets of HDL include Src family kinases, phospholipase C and D, Ras, phosphatidylinositol 3-kinase (PI3K), Akt, the mitogen-activated protein kinase (MAPK) pathway (Mek1/2 and Erk1/2), and Rac/Rho GTPases (1)(2)(3)(4)(5)(6)(7)(8). Other kinases implicated in HDL-or apoAI-inducible cholesterol transport include protein kinase C (PKC), protein kinase A (PKA), c-Jun N-terminal kinase (JNK), and p38 MAPK (9 -14).
Alternatively, signaling pathways can modulate the activity of nuclear receptors, including PPAR and LXR, to alter the ability of cells to transport cholesterol (15)(16)(17)(18). Indeed, post-translational phosphorylation via various kinases, including Erk1/2, can alter PPAR␣ and PPAR␣ co-activator activity in a liganddependent and -independent manner (17)(18)(19)(20)(21)(22)(23)(24)(25). Recent findings link Erk1/2 kinases with nuclear receptors and lipid export via ABCA1. First, enhanced Erk1/2 signaling increases ABCA1 expression and ABCA1-mediated phospholipid efflux via upregulation of PPAR␣ levels in lung epithelial cells (26). Second, inhibition of Erk1/2 and activation of LXR synergistically induce macrophage ABCA1 expression and cholesterol efflux (27). Third, Mek1/2 inhibition is involved in the regulation of PPAR␥and LXR␤-dependent ABCA1 protein degradation in HepG2 cells (28). These findings indicate that Erk1/2 kinases might exert opposite effects on nuclear receptor-inducible lipid transport depending on the cell type analyzed. SR-BI is another PPAR␣ target gene that is up-regulated by PPAR␣ agonists in human and mouse macrophages to promote HDL-inducible cholesterol efflux (29). Although the role of SR-BI for cholesterol efflux in peripheral cells is not fully understood (2,30,31), SR-BI is also highly expressed in liver to regulate hepatic uptake of HDL cholesteryl esters for excretion into bile (2,(32)(33)(34)(35). Surprisingly, in hepatocytes PPAR␣ activation induces SR-BI protein degradation and reduces SR-BI cell surface expression (36,37). The mechanisms that down-regulate hepatic SR-BI have yet to be fully elucidated. Recently, the PDZ domain containing protein PDZK1 was identified to interact and stabilize SR-BI cell surface expression in mouse hepatocytes (38 -40). Loss of hepatic SR-BI in PDZK1 KO mice suggest that PDZK1 is essential for maintaining hepatic SR-BI levels (40). However, PPAR␣-inducible SR-BI degradation in mouse liver appears independent of PDZK1 (37).
In this study, we show that MAPK inhibition down-regulates SR-BI in CHO and HEK293 cells treated with PPAR␣ agonists, whereas enhanced Ras/MAPK activity increases PPAR␣ Ser-21 phosphorylation and SR-BI expression levels, respectively. This correlates with MAPK inhibition reducing SR-BI activity, as judged by reduced cholesterol efflux onto HDL. Most relevant for the function of SR-BI in the liver, Ras/MAPK inhibition reduces SR-BI protein stability in HuH7 hepatocytes, which correlates with reduced uptake of [ 3 H]cholesteryl ester and DiIlabeled HDL. Mek1/2 inhibition most likely promotes proteasomal degradation of SR-BI, whereas levels of the SR-BI adaptor PDZK1 remain unaffected. Thus, Erk kinases appear to target PPAR␣-dependent degradation pathways to regulate hepatic SR-BI protein stability. The role of the Ras/MAPK pathway to alter SR-BI protein levels and fine-tune hepatic cholesterol homeostasis is discussed.
Real Time RT-PCR-Total RNA from HEK293 cells was extracted using the TRIzol and RNeasy system (Macherey-Nagel, Germany) according to the manufacturer's instructions. 1 g of RNA was reverse-transcribed using the High Capacity cDNA archive kit (Applied Biosystems) as per the manufacturer's instructions. Real time RT-PCR was performed as described previously (42). Assay-on-Demand primer sets to amplify cDNA fragments encoding human SR-BI and TATA box-binding protein sequences were from Applied Biosystems. Relative SR-BI expression was calculated by normalization to the housekeeper mRNA (TATA box-binding protein) as described previously (43).
Cholesterol Efflux and Uptake Assays-For the determination of HDL 3 -induced cholesterol efflux, 2-5 ϫ 10 5 cells (in triplicate) were labeled overnight with [ 3 H]cholesterol (2 ϫ 10 6 cpm/ml) as described previously (44). Noninternalized radioactivity was removed by extensive washing with PBS. Cells were incubated in Ham's F-12, 0.1% BSA Ϯ 50 g/ml HDL 3 for 4 -8 h, respectively. The media were harvested; cells were lysed in 0.1 N NaOH, and the total cellular protein was determined (45). The radioactivity in the media and cell lysate was determined by scintillation counting (44,46). The ratio of released and cellassociated radioactivity was determined and is given in %.
For DiI-HDL uptake assays, HDL 3 was labeled with DiI according to the manufacturer's instructions (Molecular Probes). CHO-SRBI and HuH7 cells were preincubated Ϯ bafilomycin, lactacystin, and PD98059 as above and washed, and 10 g/ml DiI-HDL was added for 5-15 min at 37°C, respectively.
Subcellular Fractionations-For the isolation of plasma membrane-enriched fractions, lysates from 1 ϫ 10 7 cells were separated on Percoll gradients as described previously (7). Cells were washed twice in 0.25 M sucrose, 1 mM EDTA, 20 mM Tris-HCl, pH 7.8, plus protease inhibitors, collected, and centrifuged. The postnuclear supernatant was layered on top of 10 ml of 30% Percoll and centrifuged at 84,000 ϫ g for 30 min in a Beckman 70.1 TI rotor. The plasma membrane fraction in the middle of the gradient was isolated (1 ml), concentrated, and analyzed for the amount of SR-BI and Ras.
For the isolation of nuclear fractions (50), cells were harvested in 10 mM Tris-HCl, pH 7.5, 2 mM EDTA and incubated on ice for 10 min. An equal volume of 0.5 M sucrose, 0.1 M KCl, 10 mM MgCl 2 , 2 mM CaCl 2 , 2 mM EDTA in 10 mM Tris-HCl, pH 7.5, was added. Lysates were passed 10 times through a 25-gauge needle and centrifuged (700 ϫ g, 10 min, 4°C). Pellet and supernatant were collected as nuclear and cytoplasmic fractions, separated by SDS-PAGE, and transferred to Immobilon-P.
For the isolation of late endosomes, 4 -6 ϫ 10 7 CHO-SRBI cells were incubated overnight Ϯ bafilomycin and PD98059 and homogenized by 10 passages through a 22-gauge needle. Endosomes were separated on sucrose gradients as described previously (44). In brief, the cell homogenate was centrifuged, and the postnuclear supernatant was brought to a final 42% sucrose (w/v) concentration. Then 35% sucrose, 25% sucrose, and homogenization buffer were poured stepwise on top of the postnuclear supernatant. The gradient was centrifuged for 90 min at 35,000 rpm, 4°C in a Beckman SW40 rotor. After centrifugation, fractions representing late endosomes at the interphase of 25% sucrose and homogenization buffer were collected and analyzed by Western blotting.
Cell Growth-Cell proliferation in CHOwt and CHO-SRBI cells was determined using colorimetric proliferation assays (Promega; G5421 and G4000) according to the manufacturer's instructions. Cells (0.5 ϫ 10 5 , in triplicate) were plated in 96-well plates Ϯ 50 g/ml HDL, 10 M PD98059, or both and incubated with tetrazolium compound (MTS in G5421 or MTT in G4000) for 0 -6 h. MTS and MTT are reduced by cells into formazan products, which was determined by absorbance at 490 or 570 nm without any further processing, respectively.

Inhibition of MAPK Reduces SR-BI Expression-Recent stud-
ies implicate cell type-specific and differential effects of MAPK signaling on ABCA1 transporter expression and activity (26 -28). To address the potential involvement of the Ras/MAPK pathway in SR-BI expression, we first investigated if inhibition of Erk1/2 signaling would alter the expression of endogenous SR-BI in the presence or absence of the PPAR␣ agonist Wy-14643. Therefore, we initially analyzed CHO wild-type (CHOwt) cells, which have been proven valuable for Ras/ MAPK signaling studies (6 -8, 32, 51) and express low but detectable levels of SR-BI and PPAR␣ as judged by Western blot analysis using antibodies against human SR-BI and PPAR␣, respectively (Figs. 1, A and B, and 2A). Similar to results obtained from other cell types (29), Wy-14643 induced SR-BI expression 4.1 Ϯ 1.7-fold (Fig. 1A, compare lane 1 and 3) (**, p Ͻ 0.01; see quantification in Fig. 1A). Upon addition of the commonly used Mek1/2 inhibitor PD98059 (52), which reduced basal P-Erk1/2 levels by 46 Ϯ 5% in this set of experiments, the expression of SR-BI Ϯ Wy-14643 decreased by 57.5 Ϯ 9% (*, p Ͻ 0.05) and 73.1 Ϯ 19.5% (*, p Ͻ 0.05), respectively) (Fig. 1A, compare lanes 1 and 2 and 3 and 4). We next determined the effect of PD98059 on SR-BI protein levels in CHO cells incubated with the PPAR␣ antagonist MK886 (Fig.  1B). Incubation with MK886 reduced SR-BI levels by ϳ75% (Fig. 1B, compare lane 1 and 3), indicating that MK886 inhibits the involvement of PPAR␣ to establish basal SR-BI levels. To enhance detection of the low endogenous SR-BI levels in MK886-treated cells (Fig. 1B, lanes 3 and 4), overnight exposures of Western blots were analyzed. Similar to Fig. 1A, SR-BI expression was reduced by ϳ30% with PD98059 ( Fig. 1B, lanes  1 and 2). However, residual SR-BI expression in MK886-treated cells was not reduced any further by PD98059, supporting that Erk1/2 inhibition decreases SR-BI levels via modulating PPAR␣ activity (Fig. 1A, compare lanes 3 and 4).
To verify our findings in human cells, we examined the involvement of the Ras/MAPK pathway on the regulation of SR-BI expression in the human embryonic kidney cell line HEK293, a common model to study cholesterol transport and nuclear receptor activity (1, 14 -18). Cells were incubated Ϯ PPAR␣ agonist Fenofibrate (FF) or Wy-14643 in the presence or absence of the Mek1/2 inhibitor PD98059 (Fig. 1C, for representative Western blot see supplemental Fig. 1A). Consistent with data from CHOwt cells, FF and Wy-14643 increased SR-BI levels 1.7-2.0-fold (Fig. 1C), whereas Mek1/2 inhibition reduced SR-BI levels in FF-treated HEK293 cells by 45 Ϯ 7% and almost completely repressed the stimulatory effect of Wy-14643 on SR-BI expression (Fig. 1C). To further substantiate these findings, HEK293 cells were transfected with PPAR␣ siRNA to knock down the expression of endogenous protein (Fig. 1D). Similar to Fig. 1C, in HEK293 cells transfected with control siRNA, PD98059 reduced SR-BI protein levels by ϳ38 Ϯ 10% (compare lane 1 and 2). RNAi-mediated downregulation of PPAR␣ by 66 Ϯ 9% correlated with 63 Ϯ 6% reduced SR-BI levels (Fig. 1D, compare lane 1 and 3). Most strikingly, PPAR␣-depleted HEK293 cells did not exhibit further down-regulation of SR-BI upon Mek1/2 inhibition (54 Ϯ 8% compared with control), supporting a model of MAPK signaling modulating SR-BI expression via regulating PPAR␣ activity (Fig. 1D, compare lane 3 and 4).
Ras/MAPK Signaling Increases PPAR␣ Phosphorylation-Erk1/2 could increase PPAR␣ activity through transcriptional up-regulation (26), by promoting its nuclear translocation as shown for PPAR␥ (53), or through the phosphorylation of serine residues at positions 12 and 21 within the N-terminal region of PPAR␣ (17,19). To address these different modes of action, we first examined expression and cellular distribution of PPAR␣ upon overexpression of constitutively active H-Ras (HRasG12V). Results from Fig. 1D (lanes 1 and 2) already revealed that Mek1/2 inhibition did not down-regulate endogenous PPAR␣ expression in HEK293 cells. Conversely, as shown in Fig. 2A (compare lanes 1-3) and supplemental Fig. 2A  (lanes 1 and 2), HRasG12V overexpression did not increase endogenous PPAR␣ levels. Also, HRasG12V did not alter levels of ectopically expressed PPAR␣ (supplemental Fig. 2A). In addition, HRasG12V did not change the predominant nuclear localization of PPAR␣ as indicated by the constant nuclear/ cytoplasmic ratio of ectopically expressed PPAR␣ Ϯ HRasG12V (supplemental Fig. 2B). However, phosphorylation of Ser-21 in the N-terminal region of PPAR␣ was increased 4.0 -4.7fold upon overexpression of HRasG12V, KRasG12V, or Mek215-DD (**, p Ͻ 0.01; Fig. 3A) in CHO cells, respectively. Similarly, ectopically expressed active Mek1 increased Ser-21 phosphorylation of PPAR␣ ϳ4.0 -10-fold in HEK293 cells treated with or without Wy-14643 (Fig. 3B). Hence, phosphorylation events targeting the N-terminal Ser-21 residue, and possibly Ser-12 of PPAR␣, might alter PPAR␣ activity, which could contribute to the stimulatory effect of Ras/MAPK signaling on SR-BI expression.
Inhibition of Mek1/2 Reduces SR-BI Activity-We next reasoned that reduced SR-BI expression upon Mek1/2 inhibition should result in reduced SR-BI activity. Indeed, cell surface expression of SR-BI, as judged by Western blot analysis of Rascontaining plasma membrane fractions isolated from Percoll gradients, was robustly decreased in CHOwt cells treated with PD98059 (Fig. 4A). Together with the data presented above (Figs. 1-3), we hypothesized that Ras/MAPK inhibition could reduce SR-BI activity, as judged by HDL-inducible cholesterol efflux. As shown previously (6 -8), HDL 3 is a potent activator of the MAPK pathway in CHOwt cells. PD98059 strongly reduced HDL-induced Mek1/2 and Erk1/2 activity (Fig. 4B, compare  lanes 2 and 3). Other effectors downstream of Ras, such as PI3K/Akt, which could possibly control cholesterol transporter activity and HDL-inducible efflux (1-5), were not significantly affected by PD98059 (Fig. 4B).
Next CHOwt cells were labeled for 24 h with [ 3 H]cholesterol, preincubated for 3-4 h Ϯ PD98059, followed by an incubation with 50 g/ml HDL 3 (Fig. 4C). Cells and media were assayed for radioactivity after 4 h, and efflux was determined as the percentage of total cholesterol in the culture. Similar to previous experiments (44), HDL stimulated cholesterol efflux 2.0 -2.5fold in CHOwt compared with controls (Fig. 4C). However, incubation of HDL in the presence of PD98059 reduced efflux by ϳ15-20% (*, p Ͻ 0.05). Treatment with PD98059 alone did not affect basal cholesterol efflux levels. Comparable results were obtained when determining cholesterol efflux in CHOwt in the presence of U0126, another Mek1/2-specific inhibitor (data not shown) (54). To further validate that inhibition of Erk1/2 signaling reduces SR-BI activity, we transiently transfected CHOwt cells with dominant-negative Erk1 (DN-Erk1), which is known to inhibit Erk1/2 signaling (55). Similar to the results obtained with Mek1/2 inhibitors, overexpression of DN-Erk1 inhibited HDL-inducible cholesterol efflux by 43.7 Ϯ 4.3% (Fig. 4C). Reduced cholesterol efflux onto HDL upon addition of MAPK inhibitors was not due to unequal internalization of [ 3 H]cholesterol, which was comparable in cells incubated with and without HDL and treated Ϯ PD98059 (4.1 Ϯ 0.3 ϫ 10 8 cpm/mg cell protein) or transfected Ϯ DN-Erk1 (4.4 Ϯ 0.6 ϫ 10 8 cpm/mg cell protein), respectively. Furthermore, colorimetric (MTT) proliferation assays verified that 10 M PD98059 (and U0126; data not shown) did not significantly reduce cell growth in CHOwt within the time frame of the experimental setting (Fig. 4D).
CHO cells stably expressing SR-BI (CHO-SRBI) are very suitable for HDL-dependent cholesterol transport studies and are characterized by increased HDL surface binding (32). We therefore examined if MAPK signaling affects efflux pathways in CHO-SRBI. Consistent with our previous findings, HDLincubated CHO-SRBI displayed a strong activation of H-Ras (4.0 Ϯ 1.0) and Mek1/2 (Fig. 5, A and B). PD98059 efficiently reduced Mek1/2 phosphorylation and, as shown for CHOwt (Fig. 4B), HDL-induced activation of other Ras effectors such as Akt was not significantly affected by PD98059 in CHO-SRBI cells (Fig. 5B). Importantly, PD98059 reduced HDL-induced cholesterol efflux in CHO-SRBI by 28 -32% (*, p Ͻ 0.05) (Fig. 5C). As shown for CHOwt, cell growth of CHO-SRBI Ϯ 10 M PD98059 was not altered within the time frame of this efflux experiment, as determined by proliferation assays (data not shown).
HRasG12V or Mek215-DD. Similar to results presented above, HRasG12V stimulated HDL-inducible cholesterol efflux 3-4fold compared with the negative control (supplemental Fig.  3C). PD98059 reduced HDL-inducible efflux in HRasG12Vtransfected cells by 25-30%, supporting a model of Ras stimulating efflux via Mek1/2 and Erk1/2 signaling. Interestingly, 10 M PD98059 was insufficient to inhibit efflux in cells with highly elevated levels of the active Mek1 mutant.

Mek1/2 Inhibition Reduces SR-BI Protein Stability-Endoge-
nous SR-BI expression in CHOwt is driven by its homologous promoter, whereas ectopically expressed SR-BI in CHO-SRBI (33) is under the control of the heterologous CMV promoter. Hence, it appears unlikely that Ras/MAPK inhibition reduces SR-BI mRNA, possibly in a PPAR␣-dependent manner, in both cell lines. To identify if Ras/MAPK signaling modifies SR-BI mRNA or protein levels (29, 36, 37), we first measured SR-BI mRNA by real time PCR (Fig. 6A). However, treatment of HEK293 Ϯ Wy-14643 and U0126 did not significantly alter SR-BI mRNA levels.
In contrast, addition of PD98059 accelerated SR-BI protein degradation, and after 2 h, reduction of SR-BI protein levels by 57.4 Ϯ 2.5% was evident (Fig. 6B, lanes 5-8). Thus inhibition of MAPK signaling appears to reduce protein stability of SR-BI.

Mek1/2 Inhibition Enhances PPAR␣-inducible SR-BI Degradation in
Hepatocytes-SR-BI protein stability was then analyzed in other cell types. Like CHO cells, HEK293 exhibited enhanced SR-BI degradation in the presence of cycloheximide and PD98059 (data not shown). In contrast, Mek1/2 inhibitors did not significantly alter SR-BI protein stability in endothelial (bovine aortic endothelial cells) and monocytic (THP1) cell lines (Fig. 7A). These findings suggest that Erk inhibition reduces SR-BI protein stability in a cell-specific manner that does not include peripheral cells. Because fibrates induce SR-BI degradation in liver (36,37), we next analyzed SR-BI protein stability in human HuH7 hepatocytes. As shown for primary hepatocytes from fibrate-fed mice (36,37), Wy-14643 strongly down-regulated SR-BI protein levels in HuH7 cells (Fig. 7B,  compare lanes 1 and 3). Furthermore, PD98059 reduced SR-BI protein expression in the absence and presence of PPAR␣ agonists (Fig. 7B, compare lanes 1 and 2 and 3 and 4). Similar to the mRNA expression data obtained from HEK293 cells (Fig. 6A), SR-BI mRNA levels in mouse hepatocytes remained unchanged upon Mek1/2 inhibition (data not shown). Enhanced SR-BI degradation FIGURE 6. Mek1/2 inhibition reduces SR-BI protein stability. A, 1 g of RNA extracted from HEK293 cells treated Ϯ PPAR␣ agonists (20 M Wy-14643) and Mek1/2 inhibitor (10 M U0126) was reverse-transcribed, and real time RT-PCR to amplify SR-BI and TATA box-binding protein (TBP) cDNA fragments was performed as described previously (42). Relative expression from two independent experiments with duplicate samples is given and was calculated by normalization to the housekeeper mRNA (TBP) (43). B and C, CHOwt (B) and CHO-SRBI (C) were preincubated with 20 M Wy-14643 overnight, followed by the addition of 20 ng/ml cycloheximide (CHX) Ϯ 10 M PD98059 (PD) for 0 -8 h as indicated. Western blot analysis of SR-BI and ␤-actin in each lysate of a representative experiment is shown. The mean values Ϯ S.D. of SR-BI expression levels (n ϭ 2) are given.
in the presence of PD98059 and cycloheximide further suggest that Mek1/2 inhibition down-regulates SR-BI protein levels in HuH7 hepatocytes (Fig. 7C, compare lanes 4 and 8).
Results described above (Fig. 8B) implicated the accumulation of SR-BI in late endosomes of bafilomycin-treated HuH7 cells. Therefore, we compared SR-BI localization in the CHO-SRBI Ϯ bafilomycin and Mek1/2 inhibitor (Fig. 9A). As expected, large amounts of SR-BI are localized at the plasma membrane in control cells (see arrows in Fig. 9A, panel a). Consistent with SR-BI down-regulation in HuH7 cells by Western blot analysis (Fig. 8B), PD98059 strongly reduced SR-BI staining intensity and led to a punctate staining pattern throughout the cytoplasm (Fig. 9A, panel b). As hypothesized, bafilomycin induced an accumulation of SR-BI in perinuclear, possibly late endosomal vesicles (Fig. 9A, panel c). In agreement with the expression analysis (Fig. 8B), PD98059 reduced bafilomycininducible SR-BI accumulation (Fig. 9A, panel d) in perinuclear vesicles. To verify accumulation of SR-BI in late endosomes in the presence of bafilomycin, we performed co-localization studies of SR-BI with the late endosomal marker LBPA (44). Indeed, significant co-localization of SR-BI and LBPA in bafilomycin-treated cells was observed (Fig. 9B, see panel h and arrows in panel i) supporting a model of SR-BI being targeted to (pre)-lysosomes/late endosomes upon inhibition of lysosomal degradation. As control, lactacystin did not induce perinuclear accumulation of SR-BI (data not shown). In contrast, SR-BI staining is found in cellular sites not resembling late endosomes upon Mek1/2 inhibition (Fig. 9B, panels d-f). In addition, PD98059 treatment of cells incubated with bafilomycin reduced co-localization of SR-BI and LBPA (Fig. 9B, panels j-l). Western blot analysis of membrane fractions enriched with late endosomes from CHO-SRBI cells support these findings (Fig. 9,  panel c). SR-BI was not detectable in late endosomal fractions from untreated CHO-SRBI cells Ϯ PD98059 (data not shown) but accumulated in Rab7-positive late endosomes with bafilomycin. Incubation of bafilomycin together with PD98059 reduced late endosomal SR-BI levels, further indicating that Mek1/2 inhibition promotes degradation pathways that do not involve targeting of SR-BI to lysosomes.

DISCUSSION
In this study we demonstrate that members of the Ras/ MAPK signaling cascade, including H-Ras, K-Ras, Mek1/2, and Erk1/2, regulate the protein levels of SR-BI via PPAR␣-induci- C, late endosomes from 4 -6 ϫ 10 7 CHO-SRBI cells treated overnight with 20 ng/ml bafilomycin Ϯ 10 M PD98059 were isolated as described previously (44) and analyzed for SR-BI by Western blotting. The purity of isolated fractions was assessed by immunoblotting with a marker for late endosomes (Rab 7). ble degradation pathways in hepatocytes and fibroblasts. Modulating Ras/MAPK signaling correlates with altered SR-BI activity, as judged by cholesterol export onto HDL in fibroblasts and cholesteryl ester uptake in HuH7 hepatocytes. Thus, Ras isoforms and Mek1/2 kinases upstream of Erk1/2 are novel modulators of reverse cholesterol transport via regulating SR-BI protein stability and cell surface expression.
Earlier studies aiming to link Mek and Erk kinases with cholesterol transport showed that cholesterol loading and apoA-Imediated cholesterol efflux did not alter MAPK activity (41), However, NIH/3T3 cells transformed with constitutively active HRas (HRasG12V) exported cholesterol much more rapidly to HDL compared with controls (56). This was mainly attributed to Ras-mediated down-regulation of caveolin-1, a cholesterolbinding protein that inhibits HDL-induced cholesterol efflux in some cell types, including NIH3T3 (56,57). However, transient HRasG12V overexpression or Mek1/2 inhibition did not significantly alter caveolin expression in our cell model systems (see Figs. 2A and 5B). Results presented in this study suggest that Ras/MAPK signaling modulates SR-BI expression and activity via PPAR␣, a major regulator of cholesterol homeostasis (16,58). Yet the role of PPAR␣ in SR-BI expression is complex and can involve transcriptional and post-transcriptional mechanisms, depending on the cell type analyzed (2,29,36,37). PPAR␣ agonists enhance SR-BI mRNA expression in macrophages but down-regulate SR-BI protein in hepatocytes. Fibrates have therefore been proposed to possibly regulate SR-BI protein synthesis, trafficking, degradation, and interaction with SR-BI adaptors, such as PDZK1 (36,37). Results presented here suggest that Mek1/2 inhibitors enhance PPAR␣inducible proteasomal degradation pathways to down-regulate SR-BI in fibroblasts and hepatocytes, which is most relevant for the involvement of SR-BI in reverse cholesterol transport.
Our findings suggest that PPAR␣-inducible SR-BI degradation pathways identified in mouse liver (36,37) may also exist in human hepatocytes. Because fibrates down-regulate SR-BI, but also PDZK1, which is essential for maintaining hepatic SR-BI levels (2,36,37,40), it was originally speculated that decreased hepatic SR-BI levels upon PPAR␣ activation might be secondary to decreased PDZK1. Also, an atherogenic diet induces post-translational down-regulation of both SR-BI and PDZK1 in mouse liver (68). However, PPAR␣ agonists reduced hepatic SR-BI in PDZK1-deficient mice (37), pointing at PPAR␣ targeting SR-BI protein stability independent of PDZK1. Similar pathways may exist in humans, as PDZK1 levels remained unaffected in HuH7 hepatocytes treated with PPAR␣ agonists and Mek1/2 inhibitors.
Little is yet known about the degradation pathways that promote PPAR␣-inducible SR-BI down-regulation (37). This study provides insights into the mechanism driving SR-BI protein turnover. Inhibitors of proteasomal (lactacystin) as well as lysosomal (bafilomycin) degradation increased SR-BI protein levels in HuH7 hepatocytes, indicating that SR-BI can be targeted to the proteasome and lysosome for degradation. Importantly, Mek1/2 inhibition enhanced SR-BI degradation in the presence of bafilomycin, but not lactacystin, suggesting that MAPK are possibly involved in proteasomal degradation pathways. Subcellular fractionation and fluorescence microscopy demonstrate that SR-BI accumulates in (pre)-lysosomes/late endosomes upon inhibition of lysosomal degradation. PD98059 not only reduces SR-BI cell surface expression but also co-localization of SR-BI with the late endosomal marker LBPA in the presence of bafilomycin. Hence, SR-BI down-regulation might occur through various degradation pathways, with the Ras/ MAPK pathway regulating the targeting of SR-BI from the cell surface to the proteasome.
Identifying the molecular mechanism that enables Mek1/2 kinases to decrease SR-BI protein stability may provide new clues on the physiological and pathophysiological regulation of hepatic SR-BI. Mouse models have identified SR-BI as a key molecule facilitating hepatic uptake of HDL cholesterol and secretion into bile. This study indicates that PPAR␣-inducible SR-BI degradation pathways are triggered by HDL or other ligands that activate the Ras/MAPK pathway. As hepatic SR-BI expression positively correlates with biliary cholesterol secretion (2,(33)(34)(35)(36)(37)(38)(39)(40), one can speculate that HDL-induced activation of Mek1/2 kinases, followed by PPAR␣ phosphorylation and subsequent SR-BI degradation, is a feedback loop to fine-tune cholesterol homeostasis in the liver.
It is important to note that Erk2 can also phosphorylate PPAR␥, which is considered to decrease transcriptional activity of PPAR␥ (17-19, 69, 70). In fact, HDL-induced and MAPKmediated phosphorylation of PPAR␥ inhibited expression of PPAR␥-responsive genes in RAW macrophages (53). Hence Ras/MAPK-mediated phosphorylation events could be an important switch to determine the contribution of PPAR␣ and PPAR␥ in peripheral and hepatic cholesterol metabolism. Depending on the PPAR␣ and PPAR␥ levels in a given cell, one can envisage that enhanced Ras/MAPK signaling could have opposite effects on HDL receptor and ATP-binding cassette transporter expression.
The diverse action of Erk1/2 kinases on PPAR␣ and PPAR␥ activity in peripheral and hepatic cells reflects the complexity of signaling cascades and the still poorly understood physiological relevance of PPAR phosphorylation. To date, the following three kinase families have been implicated in PPAR phosphorylation: MAPK (Erk1/2, p38, and JNK), PKA, and PKC (17)(18)(19). All of these kinases are involved in interconnected signaling cascades and provide multiple controls at the level of the receptor, ligand, cell type, cofactors, and gene promoter. Several studies suggest that Ras/MAPK overactivation contributes to atherosclerotic lesion development (71). Ras activation because of oxidative stress and H-Ras minisatellite instability in atherosclerotic plaque indicated that increased Ras activity may be involved in atherosclerosis (71). Ras inhibition using farnesyltransferase inhibitors attenuated atherosclerotic lesion formation and reduced oxidative stress in apoE-deficient mice (71,72) and intimal thickening in the rat carotid injury model (73). Statins, which interfere with Ras prenylation and activity, reduce angiogenesis and plaque progression (74). Although those studies favor Ras/MAPK inhibition to be beneficial in the prevention of atherosclerotic lesion formation, results presented here implicate that Mek1/2 inhibition promotes PPAR␣-inducible SR-BI degradation in hepatocytes. Future studies to clarify how Ras/MAPK signaling affects hepatic SR-BI protein stability will add to a better understanding of HDL metabolism and reverse cholesterol transport in vivo.