Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and HuH7 Cells

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.


Introduction
Anti-atherosclerotic properties of HDL and apolipoprotein A-I (apoA-I) include their ability to promote reverse cholesterol transport (RCT), the removal of excess cholesterol from peripheral tissues to the liver for bile secretion [1][2][3]. HDL receptors and ABC transporters are key molecules in cholesterol efflux from macrophages, with ABCA1 facilitating transfer of cholesterol onto apoA-I, while ABCG1 and SR-BI augment export of cholesterol onto HDL. In addition, ABCA1 in the liver is required for cholesterol export during HDL biogenesis, while hepatic SR-BI has a prominent role for the selective uptake of cholesteryl esters from HDL [1][2][3]. The molecular mechanisms of cholesterol transfer via ABC transporters and SR-BI have been studied extensively, however the signaling events that trigger mobilization of cellular cholesterol pools, or alternatively, alter expression and activity of cholesterol transporters are not fully understood. An increasing number of studies suggest that cell surface binding and internalization of HDL and apo-AI activate signaling proteins such as protein kinase A and C (PKA, PKC), Rac/Rho GTPases, Janus Kinase 2 (JAK2), calmodulin and MAPK to modulate the ability of cells to export cholesterol [4][5][6].
Hence, differential regulation of ABCA1 and SR-BI protein levels in peripheral and hepatic cells by Mek/Erk kinases seem to involve multiple nuclear receptors. It is yet unknown if Mek1/2 inhibition can reduce ABCA1 protein levels in hepatic cells in a PPARa-dependent manner and if the Ras/MAPK pathway modulates protein levels of ABCG1. Here we demonstrate that Erk1/2 inhibition reduced ABCA1 protein stability in CHO and HuH7 hepatocarcinoma cells treated with PPARa agonists, while overexpression of constitutively active H-Ras, K-Ras or Mek1 increased ABCA1 expression. In addition, blocking Erk1/2 activity reduced ABCG1 protein levels in the presence and absence of LXR agonists in ABCG1 overexpressing CHO and HEK293 cells. This correlated with Erk1/2 inhibition strongly decreasing HDL-inducible cholesterol efflux in ABCG1 overexpressing CHO cells. Similar to Erk1/2 inhibition promoting SR-BI degradation in these cells [28], blocking MAPK activity reduced ABCA1 and ABCG1 protein stability without affecting ABC transporter mRNA expression. In contrast, Mek1/2 inhibitor PD98059 increased ABCA1 and ABCG1 protein levels in THP1 macrophages. In summary, depending on the cell-type and their repertoire of nuclear receptors, inhibition of the Ras/MAPK pathway could have opposite effects on ABCA1 and ABCG1 expression.

Real Time RT-PCR
Total RNA from HEK293 cells was extracted using the Trizol and RNeasy system (Macherey-Nagel, Germany) according to manufacturer's instructions. 1 mg of RNA was reverse transcribed with the High Capacity cDNA Archive Kit (Applied Biosystems) and Real Time RT-PCR was performed as described previously [33]. Assay-on-Demand primer sets to amplify cDNA fragments encoding ABCA1, ABCG1 and TATA Box Binding Protein (TBP) sequences were from Applied Biosystems. Relative ABCA1 and ABCG1 expression was calculated by normalization to the housekeeper mRNA (TBP) as described [34].

Cholesterol Efflux Assays
For the determination of cholesterol efflux, 2-5610 5 cells (in triplicate) were labeled overnight with [ 3 H]-Cholesterol (2610 6 cpm/ml) as described [28]. Non-internalized radioactivity was removed by extensive washing with PBS. Then cells were incubated in Ham's F12/0.1% BSA650 mg/ml HDL 3 (total protein) or 30 mg/ml apoA-I for 6 h, respectively. The media were harvested, cells were lysed in 0.1 N NaOH and the total cellular protein was determined [35]. The radioactivity in the media and cell lysate was measured by scintillation counting [28]. The ratio of released/(released and cell-associated) radioactivity6100 was calculated and is given in (%).

Subcellular Fractionations
For the isolation of plasma membrane-enriched fractions, lysates from 1610 7 CHO-ABCG1 cells were separated on Percoll gradients as described [26,28]. 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 (PNS) was layered on top of 10 ml of 30% Percoll and centrifuged at 84.000 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 ABCG1 and Ras.
ABCA1 is important for hepatic HDL biogenesis, and we next investigated if similar mechanisms exist in hepatic HuH7 cells ( Fig. 2A). Indeed, Mek1/2 inhibition in these cells reduced ABCA1 levels in the absence of Wy-14643 by 35 To substantiate the involvement of PPARa and Mek1/2 in ABCA1 expression, HuH7 cells were transfected with siRNA to knockdown endogenous PPARa (Fig. 2B). As shown above, Wy-14643 treatment of HuH7 cells transfected with control siRNA induced ABCA1 protein levels ( . This could be due to incomplete PPARa knockdown/inhibition or indicate that Mek1/2 inhibition in HuH7 hepatocytes modulates ABCA1 expression probably not exclusively via PPARa, but also via other nuclear factors, possibly PPARc and LXRb, as shown for HepG2 cells [24]. In contrast to the results obtained from CHO and HuH7 cells (see above), Mek1/2 inhibition increases ABCA1 expression in RAW and mouse peritoneal macrophages in a dose-dependent manner [23]. Mek1/2 activation in macrophages can occur through various signaling pathways. HDL-induced activation of Mek/Erk kinases has been demonstrated in various cell types [25][26][27][28][40][41][42] and Mek/Erk phosphorylation upon HDL incubation in TPA-differentiated THP1 monocytes indicates that this signaling cascade also exists in human macrophages (Fig. 2C,  compare lane 1 and 2). HDL-induced Mek/Erk phosphorylation in THP1 macrophages is effectively inhibited by PD98059 (compare lane 2 and 3). To determine if Mek/Erk inhibition could also elevate ABCA1 expression in a human macrophage model system, THP1 monocytes were differentiated with TPA for 24 h before treatment 610 mM PD98059 (Fig. 2D). Mek1/2 inhibition increased ABCA1 protein expression approximately 2-fold in these cells (*, p = 0.032; compare lane 1 and 2), which is comparable to data using similar PD98059 concentrations in RAW macrophages [23]. Consistent with our previous data [28], PD98059-incubated THP1 macrophages also showed a small, but not significant increase of SR-BI expression compared to controls (Fig. 2D). It remains to be determined if HDL-induced Erk1/2 activation contributes to modulate cholesterol efflux via controlling ABCA1 levels in macrophages in vivo, however findings from the hepatic HuH7 cells and THP1 macrophage model support opposite and cell-specific roles for Mek/Erk kinases in the regulation of ABCA1 expression.

Blocking Mek1/2 Activity Reduces ABCG1-mediated Cholesterol Efflux in CHO Cells
Erk1/2 inhibition has been shown to affect both ABCA1 expression and activity [22,23], so we next aimed to identify if reduced ABCG1 levels in PD98059-treated CHO-ABCG1 cells would correlate with diminished ABCG1 transporter activity. We first determined ABCG1 cell surface expression. Western blot analysis of Ras-containing plasma membrane fractions isolated from Percoll gradients identified decreased ABCG1 levels in CHO-ABCG1 cells treated with PD98059 (*, p = 0.028; Fig. 4A). Next we examined the ability of PD98059 to inhibit HDLinducible Ras/MAPK activation and cholesterol efflux in ABCG1 overexpressing CHO cells. As shown for CHOwt and CHO cells overexpressing SR-BI [28], HDL strongly activates Mek1/2 and Erk1/2 kinases (P-Mek1/2, P-Erk1/2) in CHO-ABCG1 cells (Fig. 4B, compare lane 1 and 2). HDL-induced Mek1/2 and Erk1/2 phosphorylation (compare lane 2 and 3) in these cells was inhibited with PD98059, while phosphorylation of other Ras effectors, such as Akt, was not altered (not shown). We then analyzed HDL-inducible cholesterol efflux in CHO-ABCG1 cells 6 PD98059 (Fig. 4C). CHO-ABCG1 cells were labeled for 24 hours with [ 3 H]-Cholesterol, pre-incubated for 4 h610 mM PD98059, followed by an incubation with 50 mg/ml HDL or 30 mg/ml apoA-I for 6 h. Cells and media were assayed for radioactivity and efflux was determined as the percentage of total cholesterol in the culture. As shown previously [28], PD98059 alone did not affect [ 3 H]-cholesterol internalization, cell viability or basal cholesterol efflux, nor did apoA-I activate the Ras/MAPK pathway in CHOwt cells [26] (not shown). Furthermore, treatment with PD98059 alone, and consistent with previous studies in CHO cells [43], incubation with apoA-I (30 mg/ml) 6 PD98059 did not alter basal cholesterol efflux activities. In contrast, HDL effectively stimulated cholesterol efflux in CHO-ABCG1 cells (**, p = 0.0017; 5.660.7-fold). Yet, efflux of [ 3 H]cholesterol-loaded CHO-ABCG1 cells onto HDL in the presence of PD98059 was reduced by 53611% (*, p = 0.012). Hence, the Ras/MAPK activity modulates ABCG1 expression and activity in CHO-ABCG1 cells.
Primary hepatocytes and hepatic HuH7 cells express very little ABCG1, making it difficult to quantify ABCG1 protein degradation in these cells by western blotting. Therefore, to examine ABCG1 turnover, ABCG1 overexpressing CHO-ABCG1 cells were treated with cycloheximide 6 Mek1/2 inhibitor PD98059 and ABCG1 levels were monitored for 0-8 h (Fig. 5C). After 4 and 8 h, ABCG1 protein levels in cycloheximide-treated CHO-ABCG1 cells were reduced by 35.167.3 and 33.5613.3%, respectively (compare lane 1-4, white columns for quantification).
In the presence of PD98059, ABCG1 degradation was more pronounced after 8 h (54.465.0%, black columns).
In line with Mek1/2 inhibition increasing ABCA1 and ABCG1 protein levels in THP1 cells (Fig. 2D, 3C), addition of Mek1/2 inhibitors to cycloheximide-treated THP1 cells slightly increased ABCA1 and ABCG1 protein stability by 1.4-2.0 -fold after 4-8 h, respectively (Fig. 5D, compare lanes 1-3 with 4-6). These findings possibly suggest opposite effects of Erk1/2 inhibition on degradation pathways that regulate ABCA1 and ABCG1 protein levels in HuH7 and CHO cells (Fig. 5B-C) compared to THP1 cells. Erk1/ 2 inhibition protecting ABCA1 mRNA degradation in macro-phages, as shown by others [23], could further add to increase ABC transporter expression in PD98059-incubated THP1 cells. Taken together, Mek/Erk kinase inhibition is a common module to reduce not only SR-BI [28], but also ABCA1 and ABCG1 protein stability in HuH7 and CHO cells.

Discussion
In this study we demonstrate that Ras/MAPK inhibition reduced protein levels of ABCA1 and ABCG1 in CHO, HEK293 and HuH7 cells, possibly via nuclear receptor (PPARa, LXR) -inducible degradation pathways. In these cells, Ras/MAPK signaling modulates ABCA1 and ABCG1 cell surface expression and activity. In contrast, blocking Mek1/2 activity increased ABCA1 and ABCG1 expression in THP1 macrophages. Thus, cell-specific regulation of ABCA1 and ABCG1 protein stability and cell surface expression via the Ras/MAPK signaling cascade could contribute to fine-tune reverse cholesterol transport.
We previously demonstrated that enhanced Ras/MAPK signaling in CHO and HEK293 cells did not alter PPARa expression or localization, but increased Ser21-PPARa phosphorylation [28]. Erk1/2 phosphorylation events targeting PPARs are well documented [29,30] and both PPARa and PPARc contain MAPK phosphorylation sites that modulate their transcriptional activity. Erk1/2-mediated transactivation of PPARa occurs at serine residues S12 and S21 and PD98059 treatment blocks PPARa activity [29,30]. In contrast, Erk2-mediated phosphory-lation of S82 inhibits PPARc1 transcriptional activity [29,30]. These findings were predominantly derived from studies related to insulin signaling, but we showed that PD98059 interferes with HDL-induced S21-PPARa phosphorylation [28], indicating similar events during HDL-mediated signal transduction and RCT. Also, HDL-induced and MAPK-mediated phosphorylation of PPARc in RAW macrophages is associated with reduced expression of PPARc-responsive genes [53]. Most relevant to RCT, Erk1/2 inhibition correlated with decreased PPARc phosphorylation in vivo, which was associated with increased PPARc activity, elevated ABCA1 expression and reduced size of atherosclerotic lesions [56].
It remains to be determined how Mek1/2 inhibition alters ABCG1 protein expression in the presence of LXR agonists. In macrophages, Erk1/2 inhibition synergizes with LXR activation to induce ABCA1 expression [23]. Alternatively, in HepG2 cells it was proposed that Mek1/2 inhibition might interfere with direct interaction of LXRb with ABCA1 [24]. Similar mechanisms might exist in the CHO and HEK293 models analyzed in this study. However, although LXRa becomes phosphorylated at a MAPK consensus site at S198, the physiological relevance of this phosphorylation is still unclear [31].
We previously showed that Mek1/2 inhibition promotes proteasomal degradation of SR-BI [28]. Interestingly, ubiquitin- dependent ABCA1 and ABCG1 degradation has also been observed [10,11]. This degradation pathway is commonly initiated by serine/threonine phosphorylation or dephosphorylation events to recruit ubiquitin ligases, followed by ubiquitination of the target protein. Depending on the cell type, the same phosphorylation/ dephosphorylation event can activate or inhibit the ubiquitinproteasome machinery [57], hence providing opportunities for Mek/Erk kinases to prevent or enhance ABCA1, ABCG1 and SR-BI protein turnover in a cell-specific manner. Along these lines, comparison of gene arrays from livers of fibrate-treated wildtype and PPARa ko-mice and monkeys identified ,30 upregulated PPARa target genes encoding for proteasome subunits and ubiquitin-activating/conjugating enzymes [58][59][60]. Also, the E2conjugating enzyme Ubc9 and Sumo E3 ligase PIASc promote recruitment of co-repressor NCoR to PPARa in HuH7 cells [61]. Hence, nuclear receptor-dependent pathways seem to affect multiple proteins involved in the machinery that controls the protein turnover of ABC transporters.
The diverse action of phosphorylation events and nuclear receptors on the multifactorial proteasome machinery adds to the complexity of the physiological relevance of signal transduction targeting ABC transporters and HDL receptors in RCT. Since the key findings of our study were derived from various cell culture models, future in vivo studies using mouse models will have to substantiate a role for the Ras/MAPK pathway in modulating protein stability of ABC transporters, HDL metabolism and RCT. We speculate that HDL-induced activation of Ras/MAPK could be an opportunity to fine-tune the activity and contribution of Figure 5. Mek1/2 inhibition reduces ABCA1 and ABCG1 protein stability. (A) 1 mg of RNA extracted from HEK293 cells treated 6 LXR agonist (1 mM T0901317) and Mek1/2 inhibitor (10 mM U0126) was reverse transcribed and Real Time RT-PCR to amplify ABCA1, ABCG1, and TATA Box Binding Protein (TBP) cDNA fragments was performed as described [28]. Relative expression from 2 independent experiments with duplicate samples is given and was calculated by normalization to the housekeeper mRNA (TBP). PPARa, PPARc and LXR in peripheral and hepatic ABC transporters and SR-BI expression. Identifying the physiological stimuli that enable Mek/Erk kinases to differentially regulate the protein stability of ABC transporters and SR-BI may provide insights into the contribution of growth factors, insulin and hormones for hepatic ABCA1 and SR-BI activity and deliver clues to explain how Ras/MAPK overactivation contributes to atherosclerotic lesion development [62][63][64].