Differential Roles of Extracellular Signal-regulated Kinase-1/2 and p38MAPK in Interleukin-1beta - and Tumor Necrosis Factor-alpha -induced Low Density Lipoprotein Receptor Expression in HepG2 Cells*

Amit KumarDagger , Ashley Middleton, Timothy C. Chambers, and Kamal D. Mehta§

From the Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The inflammatory cytokines interleukin-1beta (IL-1beta ) and tumor necrosis factor-alpha (TNF), elevated in inflammatory, malignant, and infectious diseases, induce low density lipoprotein (LDL) receptor transcription in HepG2 cells, and such an induction can account for hypocholesterolemia associated with these states. However, the signaling mechanisms of cytokine-mediated LDL receptor induction are largely unexplored. In the present studies, we examined the potential involvement of different mitogen-activated protein kinase (MAPK) pathways. Northern analysis demonstrated that IL-1beta or TNF significantly increased LDL receptor transcript in HepG2 cells, whereas expression of another tightly regulated sterol-responsive squalene synthase gene was unaffected. IL-1beta treatment resulted in transient activation of three MAPK cascades, namely p46/54JNK, p38MAPK, and ERK-1/2, with maximal activation of 20-, 25-, and 3-fold, respectively, occurring 15-30 min after cytokine addition. PD98059, a specific inhibitor of MAPK kinase activity, inhibited IL-1beta -induced LDL receptor expression. In contrast, SB202190, a specific inhibitor of p38MAPK, enhanced IL-1beta -induced LDL receptor expression, with a concomitant increase in ERK-1/2 activity. Similarly, TNF induced LDL receptor expression also required ERK-1/2 activation. Finally, sterols repressed IL-1beta induced receptor expression, without affecting ERK-1/2 activation. These results show that IL-1beta - or TNF-induced LDL receptor expression requires ERK-1/2 activation, that the p38MAPK pathway negatively regulates LDL receptor expression, and that sterols inhibit induction at a point downstream of ERK-1/2 in HepG2 cells.

    INTRODUCTION
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Cytokines interleukin-1 (IL-1)1 and tumor necrosis factor-alpha (TNF) are potent immunoregulatory and proinflammatory cytokines secreted by a variety of cells in response to infection, activated lymphocyte products, microbial toxins, and other stimuli (1, 2). These cytokines induce a variety of biochemical and functional responses in hepatic cells (3-6). IL-1, TNF, and other cytokines have been demonstrated to act as the principal mediators that influence liver metabolism and induction of a variety of hepatic genes during liver cell infection and injury. Elevated plasma levels of cytokines, including IL-1beta and TNF found in various inflammatory, infectious, and malignant diseases, are often associated with hypocholesterolemia (7-11). Systemic infusion of cytokines has been shown to lower serum cholesterol levels in animals and humans (12-14). The concentrations of IL-1beta and TNF similar to those found following infection induce low density lipoprotein (LDL) receptor expression in human hepatoma (HepG2) cells, and this effect is not part of its mitogenic response, as IL-1beta did not increase DNA synthesis (15-18). Interestingly, a 1.56-kilobase pair region of the 5'-flanking region of the human LDL receptor promoter has been shown to confer IL-1beta -dependent induction to an heterologous gene, suggesting that increased receptor expression results from activation of LDL receptor transcription and is not due to an alteration in LDL receptor mRNA stability (18). These studies have also established that, unlike TNF, IL-1beta -induced LDL receptor transcription does not require protein synthesis and, therefore, likely depends on activation of a preexisting component(s). The initial step in their action is the association of agonist with its cell surface receptors, followed by intracellular protein phosphorylation/dephosphorylation (19-21). The downstream effectors linking receptor activation with the different cellular responses are still largely to be defined, although several signaling pathways have been proposed (20, 21). The signal transduction mechanisms by which these cytokines stimulates LDL receptor expression are poorly characterized.

Many extracellular signals elicit specific biological responses through activation of MAPK cascades (22-26). The three major subfamilies of MAPKs in higher eukaryotes include ERK-1/2, 46/54JNK, and p38MAPK, all of which are activated by phosphorylation of a tyrosine and a threonine residue catalyzed by a dual specificity MAPK kinase. ERKs are most strongly activated by mitogenic signals such as growth factors or 12-O-tetradecanoylphorbol-13-acetate (TPA), whereas p46/54JNK, and p38MAPK are activated by stressful stimuli such as the inflammatory cytokines, IL-1beta , and TNF, thermal shock, and osmotic shock (23, 27). Activation of MAPKs leads to distinct cellular responses mediated by phosphorylation of specific target substrates (25). Recently, we have shown involvement of ERK-1/2 signaling cascade in TPA-induced LDL receptor expression in HepG2 cells (28).

In this study, we have investigated the early in vivo signaling events triggered by IL-1beta that elicit LDL receptor activation in HepG2 cells. We show that, although IL-1beta at physiological concentrations strongly activates the stress-activated p46/54JNK and p38MAPK in these cells, induction of LDL receptor expression actually depends on the mild induction of the ERK-1/2 signaling cascade by this cytokine. We also demonstrate that the stress-activated p38MAPK cascade negatively regulates LDL receptor expression via ERK-1/2. These results provide new insight into the mechanisms of IL-1beta action and suggest that the MAPKs may be the critical components that control LDL receptor expression.

    EXPERIMENTAL PROCEDURES
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Materials-- IL-1beta was purchased from R & D Systems Inc. (Minneapolis, MN). TNF was obtained from Collaborative Biomedical (Bedford, MA). The mitogen/extracellular-regulated protein kinase kinase-1 and -2 (MEK-1/2) inhibitor PD98059 and p38MAPK inhibitor SB202190 were purchased from Calbiochem. PD98059 from Research Biochemicals Inc. (Natick, MA) was also used in some experiments. Phospho-specific antibodies to the activated forms of ERK-1/2 (Thr-202/Tyr-204), p46/54JNK (Thr-183/Tyr-188), p38MAPK (Thr-180/Tyr-182), and MEK-1/2 (Ser-217/221) were from New England Biolabs (Beverly, MA). Antibodies to ERK-1/2 and MAPK phosphatase-1 (MKP-1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). TRIzol and all tissue culture supplies were from Life Technologies Inc. A Zeta probe blotting membrane and the protein assay reagent were purchased from Bio-Rad. [alpha -32P]dCTP (3000 Ci/mmol) was obtained from Dupont (Boston, MA), and the enhanced chemiluminescence (ECL) detection kit was obtained from Amersham International. Cholesterol, 25-hydroxycholesterol, and other chemicals were obtained from Sigma.

Cell Culture-- HepG2 cells were maintained as monolayer cultures in a humidified 5% CO2 atmosphere at 37 °C in Eagle's minimum essential medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (Life Technologies Inc.), 2 mM L-glutamine, 20 units/ml penicillin, and 20 µg/ml streptomycin sulfate.

Immunoblot Analysis-- Proteins were fractionated by SDS-PAGE with an 10% acrylamide separation gel. Proteins were transferred to nitrocellulose in 25 mM Tris-HCl, 192 mM glycine, and 10% methanol at 4 °C for 12-16 h at a constant current of 50 mA or for 2 h at 300 mA with similar results. Nitrocellulose membranes were processed as described previously (28, 29). Briefly, membranes were incubated in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.2% v/v Tween 20 (Tris/NaCl/Tween 20) with 5% w/v non-fat dried milk for 1 h, washed in Tris/NaCl/Tween 20 (3 × 5 min), and incubated for 1 h with primary antibody in Tris/NaCl/Tween 20 containing 1% milk at room temperature for non-phospho-antibodies and overnight at 4 °C for phospho-specific antibodies. The following dilutions were used for individual antibodies against different proteins: ERK-1/2 (1:1600), phospho-ERK-1/2 (1:1000), phospho-p46/54JNK (1:1000), phospho-p38MAPK (1:1600), phospho-MEK-1/2 (1:1000), and MKP-1 (1:1500). After further washing in Tris/NaCl/Tween 20, membranes were incubated for 1 h with horseradish peroxidase-linked anti-IgG secondary antibody (Bio-Rad, diluted 1:5000), and immunoreactive proteins were detected by ECL as described by the supplier. Quantitative analyses of protein levels were performed by scanning of the autoradiograms and are representative of three or more independent experiments.

Northern Analysis-- HepG2 cells were grown and treated as described in the figure legends. Total RNA was isolated using TRIzol, and Northern blotting was done essentially as described earlier (28, 30). Briefly, 20 µg of total cellular RNA were fractionated on 1% formaldehyde-agarose gel and transferred to a Zeta probe membrane by capillary blotting. RNA blots were hybridized with LDL receptor and squalene synthase-specific single-stranded M13 probes labeled with [alpha -32P]dCTP. Hybridized filters were washed and exposed to Kodak x-ray film. The relative intensities of specific bands were determined densitometrically within the linear range of film on a model 300A laser densitometer (Molecular Dyanamics, Sunnyvale, CA) and Image Quant software. LDL receptor mRNA was normalized to squalene synthase (included in this study) or to beta -actin (data not shown) mRNA level, and data for each points were plotted as the percentage of LDL receptor mRNA as compared with controls.

Transfection Studies-- Human LDL receptor promoter-reporter constructs were transfected into HepG2 cells using LipofectAMINE (31), and the effect of PD98059 on IL-1beta -induced expression of the luciferase gene was determined.

Measurement of ERK-1/2 Activity-- Cells were washed in ice-cold phosphate-buffered saline and removed from the flask by gentle scraping into 0.25 ml of lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium vanadate, 50 mM sodium fluoride, 20 mM beta -glycerophosphate, 0.1 µM okadaic acid, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 50 µg/ml leupeptin, and 10 µM pepstatin). After 15 min on ice, insoluble material was removed by sedimentation for 20 min at 100,000 × g, and ERK-1/2 activity was determined by an immune complex kinase assay with myelin basic protein as the substrate as described previously (32). Briefly, each cell extract (400 µg) was mixed with 10 µl of anti-ERK-1/2 antibody for 1 h, and then 30 µl of 50% protein A-Sepharose in lysis buffer was added for an additional 1 h. The immune complex was recovered by sedimentation for 5 min in a microcentrifuge, washed three times with 0.5 ml of phosphate-buffered saline containing 1% Nonidet P-40 and 2 mM sodium vanadate and once with ERKs reaction buffer (25 mM Hepes, pH 7.5, 10 mM MgCl2, 20 mM beta -glycerophosphate, 0.5 mM sodium vanadate, 0.5 mM EDTA, 10 mM dithiothreitol, 10 µg/ml leupeptin, 6 µM pepstatin). The immunoprecipitate was resuspended in 30 µl of reaction buffer containing 6 µg of myelin basic protein and 5 µl of 0.3 mM [gamma -32P]ATP (30,000 cpm/pmol). After incubation for 20 min at 30 °C, the reaction was terminated by 8 µl of 5× SDS sample buffer and heating to 95 °C for 5 min. Samples were analyzed by SDS-PAGE (15% acrylamide), and gels were stained with Coomassie Blue, dried, and subjected to autoradiography.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Kinetics of IL-1beta -induced LDL Receptor Expression-- Northern blot analysis of HepG2 cells that had been incubated in the absence and presence of 5 ng/ml IL-1beta for various periods of time showed that LDL receptor induction reached a peak at 4 h after IL-1beta stimulation and remained elevated thereafter (Fig. 1). Furthermore, we tested whether IL-1beta would similarly stimulate another tightly regulated gene involved in cholesterol homeostasis, squalene synthase. Our results show that, in contrast to LDL receptor gene, IL-1beta did not affect expression of the squalene synthase gene (Fig. 1).


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Fig. 1.   Time course for the induction of LDL receptor mRNA by IL-1beta in HepG2 cells. 1 × 106 cells were plated on day 0. On day 2, cells were refed with fresh medium. On day 4, it was changed to medium containing 0.5% serum, and 5 ng/ml IL-1beta was added to the cells. Total cellular RNA was extracted after the indicated times and hybridized with 32P-labeled LDL receptor and squalene synthase cDNA probes. Autoradiographs were quantified densitometrically. LDL receptor mRNA levels were normalized to squalene synthase mRNA levels. In the bottom panel, results are expressed as the fold stimulation by IL-1beta as compared with uninduced cells.

The ERK-1/2 Cascade Positively Regulates LDL Receptor Expression in HepG2 Cells-- The activation of MAPKs requires specific phosphorylation of the threonine and tyrosine residues within the TXY motif (33, 34). We first evaluated the temporal characteristics of MAPKs activation in response to 5 ng/ml IL-1beta stimulation in HepG2 cells by using antibodies specific for phosphorylated (activated) forms of ERK-1/2, p46/54JNK, and p38MAPK. These antibodies do not recognize inactive, unphosphorylated enzymes. As shown in Fig. 2A, stimulation by IL-1beta preferentially activated p46/54MAPK (20-fold) and p38MAPK (25-fold) as compared with ERK-1/2 (3-fold). The increased activation of p46/54JNK and p38MAPK were detected within 5 min of exposure to IL-1beta , reached a peak in 15-30 min, and returned to the basal level at 90 min. Consistent with the immunoblot analyses, an increase in ERK-1/2 activity after 30 min of IL-1beta treatment, which declined to the basal level at 90 min, was observed in immune complex kinase assays using myelin basic protein as a substrate (Fig. 2B). As a positive control for ERK-1/2 activation in the kinase assay, extracts from cells treated with 100 nM TPA for 15 min were included because we have observed earlier TPA-dependent ERK-1/2 activation in HepG2 cells (28). The kinetics and fold activation of ERK-1/2 by immune complex kinase assay were similar to those obtained on immunoblotting with phospho-specific antibody. Therefore, we have used the phospho-specific immunoblot as an index of kinase activity in our subsequent experiments. Taken together, these results establish that the different MAPKs are rapidly and transiently activated by IL-1beta in HepG2 cells.


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Fig. 2.   Kinetics of activation of ERK-1/2, p46/54JNK, and p38MAPK in HepG2 cells treated with IL-1beta . A, 2 × 105 cells were grown and treated as described in Fig. 1. After the indicated times, cells were washed with 1× PBS at 37 °C and lysed in 1× SDS sample buffer. Equal amounts of whole cell lysates were separated by SDS-PAGE in 10% gels. Phosphorylation levels of MAPKs were analyzed by Western blotting using phospho-specific antibodies, following electrotransfer of total proteins onto nitrocellulose. B, cells were treated with IL-1beta as above. ERK-1/2 was immunoprecipitated with specific antibody, and immune complex kinase assays were done using myelin basic protein as a substrate. After SDS-PAGE, gel was dried and exposed to autoradiography film. p46/54, p46/54JNK; p38, p38MAPK.

The activation of ERK-1/2 is catalyzed by the dual specificity kinase, MEK-1/2 (35, 36). To phosphorylate and activate ERK-1/2, MEK-1/2 must itself be serine-phosphorylated (37, 38). This portion of the ERK-1/2 activation cascade was analyzed by immunoblotting total lysates with an antibody specific for serine-phosphorylated (activated) MEK. As shown in Fig. 3, IL-1beta induced MEK-1/2 phosphorylation with kinetics similar to that of ERK-1/2 activation.


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Fig. 3.   Effects of IL-1beta on phosphorylation of MEK-1/2 and MKP-1 expression in HepG2 cells. Top panel, time course for the induction of MEK-1/2 by IL-1beta in HepG2 cells. The samples used in Fig. 2A were analyzed with phospho-specific anti-MEK antibody. Bottom panel, IL-1beta treatment does not affect MKP-1 expression in HepG2 cells. Total cell extracts from HepG2 cell treated for different time periods with IL-1beta were immunoblotted with anti-MKP-1 antibody.

MAPKs phosphorylation is a reversible process, indicating that protein phosphatases play a crucial role in controlling enzymatic activity (39). The dual specificity phosphatase family is exemplified by mitogen-activated protein kinase phosphatase-1 (MKP-1), which dephosphorylates these kinases at both the Thr and Tyr residues necessary for enzymatic activity (40, 41). In view of recent demonstrations (42-44) that expression and activity of MKP-1 are regulated in response to stress and insulin, the expression of MKP-1 was measured in HepG2 cells that had been incubated in the absence and presence of 5 ng/ml IL-1beta for identical periods of time. As shown in Fig. 3, IL-1beta did not induce any significant changes in MKP-1 expression at those time points where maximum activation of MEK-1/2, or MAPKs was observed, suggesting that the increases in MAPKs activity are not due to changes in MKP-1 expression level. A slight increase in MKP-1 expression was observed at later time points, suggesting the possibility that this increase may account for the decrease in ERK-1/2 phosphorylation. The above results are consistent with the notion that activation of ERK-1/2 is mediated by MEK-1/2 activation.

Next, we examined the role of MEK-1/2 and its downstream effectors, ERK-1/2, in mediating IL-1beta -induced expression of the LDL receptor by use of a selective inhibitor of MEK activation, PD98059 (45, 46). Fig. 4 shows that PD98059 (5-50 µM) completely blocked IL-1beta -induced ERK-1/2 activation without affecting activation of p46/54JNK or p38MAPK. Neither IL-1beta nor PD98059 affected ERK-1/2 protein expression, as indicated by immunoblotting of the same extracts with a phosphorylation-independent ERK antibody (Fig. 4). Having established these conditions, the effect of 5-50 µM PD98059 on IL-1beta -dependent activation of LDL receptor expression was next examined in HepG2 cells. As shown in Fig. 5, 80% inhibition of IL-1beta -dependent LDL receptor expression was observed in HepG2 cells pretreated with PD98059, suggesting an absolute requirement of this signaling cascade in IL-1beta -induced LDL receptor expression. Furthermore, transfection of HepG2 cells with human LDL receptor promoter-reporter plasmids followed by IL-1beta treatment resulted in an increased luciferase gene expression, and this effect was blocked by the PD98059.2 We have repeated the above experiments using PD98059 and IL-1beta from two different sources with similar results in at least four different studies.


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Fig. 4.   Effects of PD98059 and SB202190 inhibitors on phosphorylation of IL-1beta induced ERK-1/2, p46/54JNK and p38MAPK in HepG2 cells. Cells were grown as described in Fig. 2 and were treated with IL-1beta (5 ng/ml) for 15 min in the absence or presence of the indicated concentrations of PD98059 or SB202190. The inhibitor was added 30 min prior to IL-1beta . Cell extracts were prepared, and equal amounts were subjected to SDS-PAGE and immunoblotting with anti-phospho-MEK1/2, anti-phospho-ERK-1/2, phosphorylation-independent anti-ERK-1/2, anti-phospho-p46/54JNK, or anti-phospho-p38MAPK antibodies. Autoradiographs were quantified densitometrically.


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Fig. 5.   Effect of PD98059 and SB202190 on IL-1beta - induced LDL receptor expression. HepG2 cells were grown as described in Fig. 1. They were either untreated or treated with IL-1beta (5 ng/ml) for 4 h either in the absence or presence of indicated concentrations of PD98059 or SB202190 added 30 min prior to IL-1beta addition. Total RNA (20 µg) was subjected to Northern blot analysis. The filter was hybridized with a 32P-labeled LDL receptor probe. A representative autoradiogram is shown above, and the results of the densitometric analysis of LDL receptor mRNA levels normalized to actin mRNA (not shown) are shown below (fold induction over untreated cells (zero time point)).

Collectively, these results suggest that, although IL-1beta potently activates and exerts most of its physiological effects through p38MAPK and p46/54JNK, it is the slight activation of the ERK-1/2 cascade which is mainly responsible for the LDL receptor induction by IL-1beta in HepG2 cells.

The p38MAPK Cascade Antagonizes IL-1beta -induced LDL Receptor Expression in HepG2 Cells-- As many of the effects of IL-1beta have been shown to be mediated through p38MAPK pathway (2), we tested the effect of SB202190, a potent p38MAPK inhibitor (47, 48), on IL-1beta -mediated LDL receptor induction. SB202190 is known to inhibit p38MAPK with an IC50 of 0.6 µM and even at 100 µM had no effect on activities of 12 other protein kinases tested, including ERK-1/2 or p46/54JNK (48). We also confirmed that 5 µM SB202190 did not inhibit the phosphorylation levels of p38MAPK and p46/54JNK (Fig. 4). Pretreatment of HepG2 cells for 30 min with or without 0.5 to 5 µm SB202190, followed by incubation with IL-1beta for 4 h reproducibly enhanced LDL receptor expression (Fig. 5). Interestingly, this induction was accompanied by a concomitant increase in ERK-1/2 activity without affecting phosphorylation levels of the p38MAPK and P46/54JNK (Fig. 4). Furthermore, enhanced LDL receptor expression and increased ERK-1/2 activity was not observed with SB202190 in the presence of PD98059 (data not shown). These results strongly suggests that the p38MAPK negatively regulates IL-1beta -induced ERK-1/2 activity and LDL receptor induction.

Sterols Block IL-1beta -induced LDL Receptor Expression at a Point Downstream of ERK-1/2-- Treatment of HepG2 cells with IL-1beta in the presence of sterols (25-hydroxycholesterol and cholesterol) resulted in loss of IL-1beta -induced LDL receptor expression (Fig. 6A), an observation consistent with an earlier study (18). The reduced expression of squalene synthase in the presence of sterols (lane 3) is consistent with its well established negative regulation by sterols in HepG2 cells (49). To test whether the induction is lost due to lack of activation of ERK-1/2, we evaluated the effects of sterols on IL-1beta -dependent activation of this kinase in HepG2 cells. IL-1beta was added to the cells pretreated with sterols, and ERK-1/2 activation was measured. As shown in Fig. 6B, irrespective of sterol concentrations used, IL-1beta induced ERK-1/2 activity, suggesting that the loss of an IL-1beta -dependent increase in LDL receptor induction is not due to lack of activation of ERK-1/2 in the presence of sterols in HepG2 cells.


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Fig. 6.   Sterols do not affect IL-1beta -induced ERK-1/2 activation in HepG2 cells. A, IL-1beta can not induce LDL receptor expression in the presence of sterols in HepG2 cells. HepG2 cells either untreated or pretreated with sterols (2 µg/ml 25-hydroxycholesterol and 10 µg/ml cholesterol) were induced with 5 ng/ml IL-1beta for 4 h. Total RNA was subjected to Northern blotting to determine mRNA levels of LDL receptor and squalene synthase. Ethidium bromide staining of northern gel before blotting onto a nitrocellulose to demonstrate equal loading of RNA in three lanes. B, HepG2 cells were either untreated or pretreated with sterols followed by treatment with IL-1beta for 15 min. Lysates were subjected to SDS-PAGE followed by immunoblotting with anti-phospho-ERK-1/2. Concentrations of 25-hydroxycholesterol were 2, 5, and 10 µg/ml.

TNF-induced LDL Receptor Expression Is Mediated by ERK-1/2 Cascade-- Earlier studies had proposed that IL-1beta and TNF possibly enhance LDL receptor expression by different mechanisms in HepG2 cells (18). Therefore, we tested the involvement of ERK-1/2 in TNF-dependent activation of LDL receptor expression in this cell line. TNF (100 ng/ml) treatment for 4 h increased LDL receptor expression in HepG2 cells (Fig. 7A). Like IL-1beta , TNF strongly activated p46/54JNK and p38MAPK (data not shown) with a slight activation of the ERK-1/2 (Fig. 7B). Pretreatment with 50 µM PD98059 for 30 min blocked TNF induced LDL receptor expression (Fig. 7A), thus supporting the involvement of the ERK-1/2 signaling in the induction process. Furthermore, similar to IL-1beta , treatment with 5 µM SB202190 enhanced TNF induced LDL receptor expression (Fig. 7A), with a parallel increase in ERK-1/2 activation (Fig. 7B).


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Fig. 7.   TNF induced LDL receptor expression is mediated by ERK-1/2 activation. HepG2 cells were either untreated or treated with TNF (100 ng/ml) for 4 h either in the absence or presence of 50 µM PD98059 or 5 µM SB202190 added 30 min prior to TNF treatments. A, total RNA was subjected to Northern blotting and was probed with 32P-labeled LDL receptor probe, or with 32P-labeled squalene synthase probe. Autoradiographs were quantified densitometrically. In the bottom panel, values are means of at least three separate experiments ± S.D. B, cell extracts under identical conditions were subjected to immunoblotting with non-phospho- as well as phospho-ERK-1/2 antibodies.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The results presented in this study establish a role for the ERK-1/2 activation in IL-1beta -induced LDL receptor expression in HepG2 cells. First, IL-1beta treatment leads to an activation of ERK-1/2 in a time-dependent manner (Fig. 2). The kinetics of ERK-1/2 activation and LDL receptor induction are closely related, thereby providing the first in vivo evidence linking them kinetically. Second, PD98059, a specific inhibitor of MEK activity, inhibits both ERK-1/2 activation and LDL receptor expression induced by IL-1beta , without affecting phosphorylation levels of p46/54JNK and p38MAPK. Although IL-1beta treatment resulted in activation of other MAPKs, the complete loss of IL-1beta induced LDL receptor expression on treatment with PD98059 rules out involvement of other MAPKs in LDL receptor induction by IL-1beta . Furthermore, IL-1beta -induced phosphorylation of MEK-1/2 correlates with the ERKs activation, which is consistent with the notion that ERK-1/2 induction is mediated through MEK activation. These results show that IL-1beta signal acts upstream of ERK-1/2 phosphorylation, at least at the level of MEK-1/2 activation. The ERK cascade is normally required for the essential function as a positive regulator of cell proliferation in response to growth signals, and strong correlations between ERKs activation and DNA synthesis have been shown in earlier studies (27, 50, 51). In view of an earlier demonstration that IL-1beta -induced activation of LDL receptor expression in HepG2 cells is not a consequence of increased DNA synthesis, involvement of ERK-1/2 in IL-1beta -dependent LDL receptor induction is surprising, and suggests that these isoforms can be coordinately activated in response to a wide range of mitogenic as well as nonmitogenic stimuli. Furthermore, although physiological concentrations of IL-1beta strongly activated p46/54JNK and p38MAPK, the lessor induction of ERK-1/2 proved to be the crucial mediator of the cytokine's action in LDL receptor induction. These results emphasize a general point of significance, namely, that although a specific signaling pathway may appear to dominate a response, other mildly activated pathways can be critical, and thus should not be overlooked or ignored.

The present study has focused on the role of MAPKs in IL-1beta -induced LDL receptor expression. Results presented here along with an earlier report (18) are consistent with the notion that IL-1beta induces LDL receptor expression at the transcriptional level. In most cell types, the mitogenic signal is relayed from the cytoplasm into the nucleus by the nuclear translocation of the phosphorylated ERK-1/2, resulting in phosphorylation and activation of a range of transcription factors (52). The precise role of phosphorylation in transcriptional activation of LDL receptor expression in response to IL-1beta is unclear but may involve phosphorylation of transcription factors critical for LDL receptor expression (53-55), or modification of co-activators such as the cAMP-response element binding (CREB) protein (56). Given that ERK-1/2 activation occurs rapidly and transiently (Fig. 2), and LDL receptor induction follows relatively slow kinetics (Fig. 1), the mechanism likely involves this protein kinase cascade in the initial step and does not require it for sustained activation of LDL receptor expression by IL-1beta in HepG2 cells. Our demonstration that IL-1beta can regulate gene expression through selective activation of ERK-1/2 may explain the lack of effect of p38MAPK inhibitor on IL-1beta induction of E-selectin expression on vascular endothelial cells, or IL-1-induced IL-6 production in gingival fibroblasts (57). Furthermore, the lack of activation of the squalene synthase gene by IL-1beta suggests that the members of the MAPK family selectively regulate expression of specific genes involved in cholesterol homeostasis.

Although p38MAPK has been found to be activated by several forms of environmental stress and cytokines, the physiological and pathophysiological function of this kinase in mammalian cells is still unclear. Recently, roles for p38MAPK in the regulation of cell quiescence and in the promotion of programmed cell death in differentiated cellular systems have been reported (58-63). Our demonstration that treatment with SB202190 resulted in superinduction of the LDL receptor by IL-1beta suggests that the p38MAPK signaling cascade exerted a negative effect on LDL receptor expression through negative regulation of the ERK-1/2. Although the precise role for this level of regulation is not yet clear, such a mechanism may provide cells a means to counteract the actions of cytokines and growth factors on LDL receptor expression.

It has been demonstrated earlier that, unlike IL-1beta , TNF requires protein synthesis for induction of LDL receptor transcription in HepG2 cells (18). Based on this observation, it was suggested that biologically redundant cytokines, TNF and IL-1beta , may act via different mechanisms to increase LDL receptor expression in HepG2 cells. In the present study, we have shown that both cytokines require ERK-1/2 for induction of LDL receptor expression. PD98059 was found to block the IL-1beta - and TNF-dependent induction of LDL receptor expression at a concentration shown to completely inhibit ERKs in HepG2 cells. It is safe to conclude from the above studies that whatever differences exist in their upstream signal transduction pathways, both cytokines activate ERK-1/2 and this step is critically required for LDL receptor induction in response to them. Furthermore, an observation that IL-1beta -dependent induction of LDL receptor expression is sensitive to cellular cholesterol levels allowed us to test whether sterols exert their effect upstream or downstream of ERKs activation. The lack of effect of sterols on ERK-1/2 activation under conditions shown to inhibit IL-1beta induced LDL receptor expression suggests that sterols acts at a step downstream of this protein kinase pathway. On the other hand, lack of activation of the sterol-sensitive squalene synthase gene by IL-1beta (Fig. 1), which is known to contain a functional SRE-1 (44), suggests that SREBP alone may not be solely responsible for LDL receptor induction by these cytokines.

In conclusion, we have provided evidence that MAPK signal transduction pathways play essential and differential roles in the induction of LDL receptor transcription by IL-1beta , thus establishing a link between MAPK-mediated intracellular signaling and regulation of LDL receptor gene expression. LDL receptor induction by IL-1beta occurs via a complex chain of events initiated after ligand binding that leads to the activation of certain kinases that promote MAPKs phosphorylation. Our results support the possibility that p38MAPK negatively regulates ERK-1/2 activation and some of the responses mediated by this kinase in the presence of IL-1beta . The cross-talk between various signaling pathways involving protein phosphorylation is an emerging theme in intracellular communication. Based on the above results, we propose that interplay between these pathways probably has a central role in the processing circuits that direct the transcription of LDL receptor gene. Results presented here will help form a framework for further investigation of the signaling pathways responsible for LDL receptor transcriptional regulation by mitogenic and nonmitogenic stimuli.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Research Grant HL-51592-04), American Heart Association Grant 94012580, and the University of Arkansas for Medical Sciences Hornick Endowment Award (to K. D. M.).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.

Dagger Present address: Molecular and Cellular Endocrinology Branch, NIDDK, Bethesda, MD 20892.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205. Tel.: 501-686-8053; Fax: 501-686-8169; E-mail: mehtakamald{at}exchange.uams.edu.

1 The abbreviations used are: IL, interleukin; TNF-alpha , tumor necrosis factor; LDL, low density lipoprotein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen/extracellular-regulated protein kinase kinase; MKP, MAPK phosphatase; TPA, 12-O-tetradecanoylphorbol-13-acetate; PAGE, polyacrylamide gel electrophoresis.

2 A. Kumar and K. D. Mehta, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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