Original Article

Critical role of p42/44^MAPK activation in anisomycin and hepatocyte growth factor-induced LDL receptor expression: activation of Raf-1/MEK-1/p42/44^MAPK cascade alone is sufficient to induce LDL receptor expression

Correspondence to: Kamal D. Mehta

	ABSTRACT

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The protein synthesis inhibitor anisomycin activates stress-related mitogen-activated protein kinases (MAPKs), namely, c-jun NH₂-terminal kinase (p46/54^JNK) and p38^MAPK in mammalian cells. In this paper, we show that although exposure to anisomycin resulted in rapid and strong activation of p46/54^JNK and p38^MAPK, with a delayed low level dual-phosphorylation of mitogen/extracellular protein kinase (p42/44^MAPK), low density lipoprotein (LDL) receptor induction depends solely on the mild activation of p42/44^MAPK signaling cascade in HepG2 cells. Unlike hepatocyte growth factor (HGF) which caused LDL receptor induction via rapid, strong, and Ras-dependent p42/44^MAPK activation, anisomycin-induced p42/44^MAPK activity and increased LDL receptor expression in a Ras-independent manner. Finally, we examined the role of the p42/44^MAPK signaling cascade in LDL receptor induction by activating this kinase independently of anisomycin or HGF. By using estrogen-dependent human Raf-1 protein kinase in transient transfection assays, we show that the exclusive activation of the Raf-1/MEK-1/p42/44^MAPK signaling cascade with antiestrogen ICI 182,780 caused induction of LDL receptor expression to the same level as observed with either HGF or anisomycin. Consistent with the role of p42/44^MAPK, induction was strongly inhibited by pretreatment with the MEK-1/2 inhibitor PD98059.

Our observation that anisomycin can use p42/44^MAPK signaling cascade is a departure from established thinking, and the results presented shows that activation of the p42/44^MAPK alone is sufficient to fully induce LDL receptor transcription.—Dhawan, P., A. Bell, A. Kumar, C. Golden, and K. D. Mehta. Critical role of p42/44^MAPK activation in anisomycin and hepatocyte growth factor-induced LDL receptor expression: activation of Raf-1/MEK-1/p42/44^MAPK cascade alone is sufficient to induce LDL receptor expression. J. Lipid Res. 1999. 40: 1911;–1919.

Supplementary key words: anisomycin, hepatocyte growth factor, mitogen-activated protein kinase, LDL receptor induction

	INTRODUCTION

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Mitogen-activated protein kinases (MAPKs) are proline-directed serine;–threonine kinases that have important functions as mediators of cellular responses to a variety of extracellular stimuli (reviewed in references (1) (2) (3) (4)). The MAPKs comprise a family of kinases, the mitogen/extracellular protein kinase (p42/44^MAPK), the c-Jun NH₂ terminal kinases (p46/54 ^JNK), and the p38^MAPK. Whereas p42/44^MAPK are characteristically associated with cell proliferation and protection from apoptosis, p46/54 ^JNK and p38^MAPK can promote apoptosis in many systems. Members of the p46/54 ^JNK and p38^MAPK subfamilies are strongly activated in response to stress stimuli such as UV radiation, heat shock, major proinflammatory cytokines, and hyperosmolarity. The most potent protein synthesis inhibitor, anisomycin, has been shown to elicit highly specific and strong activation of stress-related p46/54 ^JNK and p38^MAPK (5). Due to reported selectivity in its action, there has been a recent resurgence in the use of anisomycin, particularly as an agent that selectively activates these stress-related MAPKs (6) (7) (8) (9) (10) (11) (12) (13) (14). The MAPKs are tightly regulated by multicomponent signal transduction cascades consisting of small GTP binding proteins (Ras, Cdc42, and Rac), MAPK kinase kinases (Raf and MEKK), and MAPK kinases (MEKs, MAPKKs, and SEK) (reviewed in references (1), (15)). Several downstream targets for MAPKs have also been characterized (3). For example, the p42/44^MAPK and one downstream target, the protein kinase RSK, translocates to the nucleus on activation and indirect evidence suggests that in the nucleus, these protein kinases may phosphorylate and regulate the activities of several transcription factors, including Jun, Fos, Elk-1, cyclic AMP responsive element binding protein, and serum response factor (reviewed in reference 4). Similarly, p38^MAPK and p46/54^JNK also regulate transcription factors like ATF2, Elk-1, and Jun (4).

Anisomycin strongly induces transcription of several immediate early (IE) genes, including c-fos, fos B, c-jun, jun B, and jun D, and the activation of the stress-related kinase has been correlated with its ability to induce their expression (5) (16) (17). Anisomycin has also been shown to induce low density lipoprotein (LDL) receptor transcription in Jurkat cells (18), as it does for the normally labile IE genes (17). Treatment of human fibroblasts with other protein synthesis inhibitors has been shown to induce LDL receptor expression at the transcriptional level (19). The induction of LDL receptor expression in response to protein synthesis inhibitors has been proposed to be a secondary effect of translational arrest that results in the disappearance of a labile repressor (18) (19). Although the existence of such a factor has been postulated for years (20) (21), this labile repressor has not yet been biochemically characterized in eukaryotic cells.

In view of the documented ability of anisomycin to specifically activate p46/54^JNK and p38^MAPK, we set out to investigate the role of these MAPKs in regulating LDL receptor expression. Here we report that anisomycin, unlike puromycin and cycloheximide, specifically induces LDL receptor expression over a wide range of concentrations in HepG2 cells. Although anisomycin strongly activates p46/54^JNK and p38^MAPK, LDL receptor induction actually depends on the mild activation of p42/44^MAPK cascade. We also show that not only is the p42/44^MAPK cascade required for anisomycin-induced LDL receptor transcription, but activation of this signaling cascade alone is sufficient for induction of LDL receptor transcription. These results highlight the role of p42/44^MAPK cascade as an obligatory step in anisomycin signaling and underscore the importance of performing specific controls when assessing roles of the stress-related MAPKs by using anisomycin.

	MATERIALS AND METHODS

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Materials
Anisomycin, cycloheximide, puromycin, 12-O-tetradecanoylphorbol-13-acetate (TPA), calphostin C, PD98059, and SB202190 were purchased from Calbiochem (San Diego, CA). PD98059 from Research Biochemicals Inc. (Natick, MA) was also used in some experiments. ICI 182,780 was purchased from Tocris (Ballwin, MO). BZA-5B, a specific inhibitor of Ras farnesylation, was a kind gift from Genentech Inc. (South San Francisco, CA). Phospho-specific antibodies to the activated forms of p42/44^MAPK (Thr 202/Tyr 204), p46/54^JNK (Thr 183/Tyr 188), p38^MAPK (Thr 180/Tyr 182), and MAPK/ERK kinase kinase, MEK-1/2 (Ser 217/221) were purchased from New England Biolabs (Beverly, MA). Antibodies to p42/44^MAPK, 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. (Gaithersburg, MD). Zeta probe blotting membrane and the protein assay reagent were purchased from Bio-Rad (Hercules, CA). [-³²P]dCTP (3000 Ci/mmol) was obtained from DuPont (Boston, MA) and the enhanced chemiluminescence (ECL) detection kit was obtained from Amersham International (Arlington Heights, IL). Interleukin-1ß (IL-1ß) and hepatocyte growth factor (HGF) were purchased from R & D Systems (Minneapolis, MN). The chemiluminescent reporter gene assay system for the detection of luciferase was purchased from TROPIX, Inc. (Bedford, MA).

Cell culture
HepG2 cells were maintained as monolayer cultures in a humidified 5% CO₂ atmosphere at 37°C in Eagle's minimum essential medium (EMEM) (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.

[³⁵S]methionine labeling of HepG2 cells
Cells were grown in a 24-well plate, starved for 16 h in EMEM supplemented with 0.5% fetal calf serum, and washed twice with PBS and once with methionine-free medium. After incubation in this medium at 37°C for 10 min, the medium was replaced by 1 ml of medium containing [³⁵S]methionine (15 µCi/ml) and either anisomycin, cycloheximide, or puromycin at the indicated concentrations. Cells were incubated for 1 h at 37°C, washed twice with EMEM, and then lysed in 200 µl of whole cell lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 5 mM MgCl₂, 1% Triton X-100, 0.1% deoxycholate, protease inhibitor cocktail). Debris were pelleted for 10 min at 10,000 g, and 100 µl of the supernatant was precipitated with trichloroacetic acid and collected onto glass filters (Whatman). The filters were washed with 10 ml of 5% trichloroacetic acid followed by 3 ml of 100% ethanol, and then counted.

Immunoblot analysis
Proteins were fractionated by SDS-PAGE with a 10% acrylamide separation gel, and the separated proteins were transferred to nitrocellulose and processed as described previously (22) (23) (24). 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 x 5 min), and incubated for 1 h with primary antibody in Tris/NaCl/Tween-20 containing 1% milk at room temperature for non-phospho-specific antibodies and overnight at 4°C for phospho-specific antibodies. The following dilutions were used for individual antibodies against different proteins: p42/44^MAPK (1:1600); phospho-p42/44^MAPK (1:1000); phospho-p46/54^JNK (1:1000); phospho-p38^MAPK (1:1600); phospho-MEK-1/2 (1:1000); 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 densitometric scanning of the autoradiograms and are representative of 3 or more independent experiments.

Northern analysis
Total RNA was isolated using TRIzol, and Northern blotting was done essentially as described earlier (22) (23) (24). Briefly, 20 µg total cellular RNA was fractionated on 1% formaldehyde agarose gel and transferred to Zeta Probe membrane by capillary blotting. RNA blots were hybridized with LDL receptor and squalene synthase-specific single-stranded M13 probes labeled with [-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 the film on a model 300A laser densitometer (Molecular Dyanamics, CA) with Image Quant software. LDL receptor mRNA was normalized to squalene synthase (SS) (included in this study) or to ß-actin (data not shown) mRNA level, and data for each point were plotted as the percentage of LDL receptor mRNA as compared to controls.

Expression vectors and reporter constructs
The human LDL receptor promoter construct used for luciferase assays contained the human LDL receptor promoter from residues -273 kb to +35 cloned upstream of the luciferase gene as described previously (25). Construction of the estradiol-regulated forms of oncogenic Raf-1 (Raf-1:ER) was described earlier (26) (27), and contained the kinase domain of oncogenic Raf-1 fused to the hormone binding domain of the human estrogen receptor. These constructs were provided by Dr. Martin McMahon, Cancer Center, University of California. The dominant-negative (D.N.) and constitutively active (C.A.) mutants of Raf-1:ER contained substitution of tyrosines (Y340 and Y341) with phenylalanine (FF) and aspartic acid (DD), respectively. The expression vectors for MEK1, C.A. MEK1 mutant (MKK1-SS/DD), and D.N. MEK1 (MKK1-S222A) were obtained from Dr. Jacques Pouysse'gur, Centre de Biochimie, CNRS, Nice Cedex, France. Dominant-negative Ras and MKP-1 were obtained from Dr. Jeffrey Pessin, Dept. of Physiology, University of Iowa.

Transient transfection and luciferase assay
HepG2 cells were transfected by the Lipofectamine method as described previously (25). Briefly, HepG2 cells were seeded at a density of 1 x 10⁶ cells per 6-well plates and co-transfected with human LDL receptor promoter-luciferase reporter plasmid (0.5 µg) and 0.25 µg of relevant expression vector or the corresponding empty vector. After transfection, cells were washed three times and maintained in 0.5% FCS with or without IL-1ß (5 ng/ml), HGF (10 ng/ml), anisomycin (50 ng/ml), or ICI 182,780 (1 µM) for 8;–12 h. Luciferase assays were performed as described before [25].

	RESULTS

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Anisomycin induces LDL receptor expression in HepG2 cells
We examined the effects of three protein synthesis inhibitors, that are structurally and mechanistically different in the manner in which they inhibit protein synthesis (5), on LDL receptor expression in HepG2 cells. Northern blot analysis of HepG2 cells treated with different concentrations of various inhibitors is shown in Figure 1. At concentrations that completely blocked protein synthesis ( Figure 2), puromycin (150 µg/ml) and cycloheximide (5 µg/ml) did not significantly induce LDL receptor transcript, whereas anisomycin strongly induced at all concentrations tested (0.01;–5 µg/ml) in medium containing either 0.5% serum (Figure 1) or 10% serum (results not shown). Anisomycin treatments led to a significant increase in LDL receptor mRNA at concentrations which are insufficient to block protein synthesis. The maximal level of LDL receptor mRNA was reached at a concentration of 50 ng/ml (Figure 1) which still allowed translation to proceed at 60;–70% of its efficiency in the absence of the drug (Figure 2). Anisomycin was unable to induce expression of another sterol-responsive SS gene (Figure 1). Induction of LDL receptor expression, even at concentrations of anisomycin below that which inhibits protein synthesis, and the lack of the effect of cycloheximide and puromycin suggest that the inhibition of protein synthesis per se is not sufficient for anisomycin-induced LDL receptor expression in HepG2 cells. These results clearly rule out the involvement of a continuously synthesized labile protein in significantly repressing LDL receptor gene expression in HepG2 cells.

View larger version (41K): [in this window] [in a new window]   Figure 1. LDL receptor levels in HepG2 cells treated with puromycin, anisomycin, or cycloheximide at varying concentrations. HepG2 cells (1 x 106/25 cm2 flask) were plated on day 0 in EMEM supplemented with 10% fetal bovine serum. On day 2, cells were refed with fresh media. On day 4, media containing 0.5% serum and the given concentration of the indicated inhibitor were added to the cells. Total RNA was subjected to Northern blot analysis, and the filter was hybridized with 32P-labeled LDL receptor, SS, and actin cDNA probes. LDL receptor mRNA levels were normalized to actin mRNA levels. The experiment was repeated two times with similar results. Cyclohex, cycloheximide.

View larger version (16K): [in this window] [in a new window]   Figure 2. Protein synthesis levels in HepG2 cells in response to different protein synthesis inhibitors. To determine the inhibitory effects of various concentrations of protein synthesis inhibitors, HepG2 cells were treated for 60 min without (0) or with the indicated concentrations of protein synthesis inhibitors. Incorporation of [35S]methionine was determined in quadriplicate, and the average results were normalized to control values. The results are presented as means ± standard errors and represent at least three individual experiments.

Anisomycin activates all the three MAPKs in HepG2 cells
In view of numerous recent reports showing that anisomycin acts as a signaling agonist at sub-inhibitory concentrations due to potent and specific activation of p46/54^JNK and p38^MAPK (6) (7) (8) (9) (10) (11) (12) (13) (14), we investigated the activation of three MAPKs by anisomycin in HepG2 cells. We first evaluated the temporal characteristics of MAPKs activation in response to 50 ng/ml of anisomycin in HepG2 cells by using antibodies specific for phosphorylated (activated) forms of the p42/44^MAPK, p46/54^JNK, p38^MAPK, and MEK-1/2. As shown in Figure 3A, anisomycin treatment resulted in rapid and strong activation of the p46/54^JNK and p38^MAPK with a slight activation of the p42/44^MAPK. The magnitude and time course of the activation between p42/44^MAPK and p46/54^JNK or p38^MAPK are different; both p46/54^JNK and p38^MAPK showed peak activities 30 min post-stimulation, whereas p42/44^MAPK was most active at approximately 4 h. The activity of p42/44^MAPK was sustained for at least up to 8 h. The increase in activity of p42/44^MAPK does not result from an increase in expression of p42/44^MAPK protein, as determined by Western blotting with phosphorylation-independent p42/44^MAPK antibody (Figure 3A).

View larger version (36K): [in this window] [in a new window]   Figure 3. Kinetics of activation of p42/44MAPK, p46/54JNK, p38MAPK, and MEK-1/2 in HepG2 cells treated with anisomycin. (A) HepG2 cells were grown as described above, and treated with 50 ng/ml of anisomycin for various time intervals in media containing 0.5% serum. After indicated times, cells were washed with phosphate-buffered saline and lysed with 1 x SDS sample buffer. Equal amounts of cell lysates were analyzed by Western blotting using the indicated phospho-specific antibodies. Phosphorylation-independent p42/44MAPK antibody was used to measure expression levels of p42/44MAPK protein. (B) Effects of anisomycin on MEK-1/2 phosphorylation and MKP-1 expression level in HepG2 cells. The cell lysates prepared above were probed with phospho-specific anti-MEK-1/2 or phosphorylation-independent MKP1 antibody. Results shown are indicative of three separate experiments.

Anisomycin treatment also induced MEK-1/2 phosphorylation (Figure 3B) with kinetics similar to p42/44^MAPK activation, suggesting that anisomycin acts upstream of the p42/44^MAPK phosphorylation, at least at the level of MEK-1/2 activation. At the same time, anisomycin did not cause any significant change in the expression of MKP-1 at those time points where maximum activation of MEK-1/2 or MAPKs was observed (Figure 3B), indicating that the increase in p42/44^MAPK activity is not due to a decrease in MKP-1 expression. The above results are consistent with the notion that activation of p42/44^MAPK is mediated by MEK-1/2 activation.

Anisomycin-induced LDL receptor expression is mediated through p42/44^MAPK
The activation of three MAPKs raised the question of which pathway is required for anisomycin-induced LDL receptor expression. To determine the ability of specific inhibitors of MEK and p38^MAPK to affect anisomycin-induced LDL receptor expression, HepG2 cells were treated with varying concentrations of PD98059 (a specific MEK inhibitor) (22) (23) (24) (28) or SB202190 (a specific p38^MAPK inhibitor) (24) (29) (30). Interestingly, induction was completely prevented by PD98059, but not by SB202190 ( Figure 4), thereby providing evidence for the crucial involvement of p42/44^MAPK activation in anisomycin-induced LDL receptor expression. The role of the p42/44^MAPK in anisomycin-induced signaling pathway was surprising in view of only a slight activation of this enzyme, an enzyme characteristically associated with cell proliferation and protection from apoptosis. In contrast, inhibition of p38^MAPK signaling pathway by SB202190 resulted in the super-induction of LDL receptor expression in a dose-dependent manner (Figure 4) without affecting the expression of SS gene. It is likely that this effect is due to cross-talk between the p38^MAPK and the p42/44^MAPK signaling pathways (24).

View larger version (38K): [in this window] [in a new window]   Figure 4. Effects of PD98059 and SB202190 inhibitors on anisomycin-induced LDL receptor expression in HepG2 cells. Cells pretreated for 30 min with the indicated concentrations of inhibitor were either untreated or treated with anisomycin (50 ng/ml) for 4 h in medium containing 0.5% serum. Total RNA was subjected to Northern blot analysis. Autoradiographs were quantified densitometrically within the linear range of the film on a model 300A laser densitometer (Molecular Dyanamics, CA) and Image Quant software. A representative autoradiogram is shown above, and the results of the densitometric analysis of LDL receptor mRNA levels normalized to actin mRNA (not shown) or to SS are shown below (fold induction over untreated cells (zero time point)).

Comparison of the effects of BZA-5B, a selective Ras farnesyltransferase inhibitor, on LDL receptor induction and p42/44^MAPK activation by anisomycin, IL-1ß, and HGF
To study the role of farnesylated Ras in anisomycin-induced LDL receptor expression, we used an inhibitor of Ras farnesyltransferase, the benzodiazepine peptidomimetic BZA-5B (31). BZA-5B has been shown to block attachment of a farnesyl moiety to some Ras isoforms, among other proteins, thereby effectively suppressing Ras biological functions and downstream signaling. In order to deplete cells of farnesylated Ras proteins, cells were pretreated with 50 µM BZA-5B for 3;–4 days and then stimulated with either anisomycin, IL-1ß, or HGF. HGF was included as a positive control because stimulation of receptor tyrosine kinases usually results in the activation of the Raf-1/MEK/p42/44^MAPK through Ras in many cell types (32). As expected, HGF caused rapid and strong activation of p42/44^MAPK through MEK-1/2 ( Figure 5A), and PD98059 treatment prevented HGF-induced p42/44^MAPK activation and an increase in LDL receptor expression (Figure 5B) demonstrating that the LDL receptor induction by HGF is mainly mediated by the p42/44^MAPK ( Figure 6). IL-1ß was also included for comparative study because we showed previously that this agent induces LDL receptor expression through p42/44^MAPK activation (23). Treatment with BZA-5B completely suppressed HGF-induced p42/44^MAPK activation and LDL receptor induction ( Figure 7A and Figure 7B). This is perhaps expected as functional Ras is required for transduction of the mitogenic signals from activated receptor tyrosine kinases to p42/44^MAPK. On the other hand, IL-1ß and anisomycin-induced p42/44^MAPK activation and induction of LDL receptor expression were not blocked by BZA-5B treatments (Figure 7A and Figure B). Similarly, BZA-5B treatments did not significantly reduce p46/54^JNK activation by anisomycin (Figure 7B). From these results, we conclude that unlike HGF, anisomycin may activate p42/44^MAPK and induce LDL receptor expression through Ras-independent pathways in HepG2 cells.

View larger version (29K): [in this window] [in a new window]   Figure 5. HGF-induced LDL receptor expression is mediated through p42/44MAPK in HepG2 cells. (A) HepG2 cells were treated with 10 ng/ml HGF in media containing 0.5% serum. Cell extracts were made at the indicated times, and equal amounts of cell extracts were separated by SDS-PAGE and then transferred onto nitrocellulose. Phosphorylation levels of p42/44MAPK and MEK-1/2, and expression levels of p42/44MAPK were determined as described above. In the last lane, cell extracts from HepG2 cells pretreated with 20 µM PD98059 for 30 min were subjected to HGF (10 ng/ml) treatment (20 min). (B) Inhibition of HGF-induced LDL receptor expression by PD98059. HepG2 cells were either untreated or treated with HGF (10 ng/ml) for 4 h either in the absence or presence of indicated concentrations of PD98059 added 30 min prior to 10 ng/ml HGF addition. Ethidium bromide staining of RNA gel before blotting onto a nitrocellulose is also shown to demonstrate equal loading of RNA in all lanes. Results shown are indicative of three separate experiments.

View larger version (42K): [in this window] [in a new window]   Figure 6. Effects of BZA-5B on anisomycin-, IL-1ß-, and HGF-induced LDL receptor expression and p42/44MAPK activation. (A) HepG2 cells were seeded on day 0. On day 1, 50 µM BZA-5B was added to the cells for 3 days. On day 4, fresh media containing 0.5% serum and 50 µM BZA-5B along with anisomycin (50 ng/ml), IL-1ß (5 ng/ml), or HGF (10 ng/ml) were added for 4 h. RNA was isolated and Northern blotting was performed as described above using the LDL receptor and SS cDNA probes. Equal loading of RNA in each lane was confirmed by staining of the RNA gel before blotting onto nitrocellulose. (B) HepG2 cells pretreated with BZA-5B (50 µM) for 5 days were treated with HGF (10 ng/ml) for 15 min, or with anisomycin (50 ng/ml) for 1.5 h in media supplemented with 0.5% serum. Total cell extracts were subjected to Western blotting with phospho-specific p42/44MAPK, phosphorylation-independent p42/44MAPK, or phospho-specific p46/54JNK antibody.

View larger version (21K): [in this window] [in a new window]   Figure 7. Effects of dominant negative and constitutively active regulatory enzymes in the Raf-1/MAPK pathway on LDL receptor expression. HepG2 cells were cotransfected with a human LDL receptor-luciferase reporter plasmid (1 µg/plate) with or without the indicated constructs (500 ng each) in the presence or absence of ICI 182,780 as shown. Where indicated, the following were added at the concentration indicated for 8;–12 h: IL-1ß (5 ng/ml), HGF (10 ng/ml), anisomycin (100 ng/ml), ICI 182,780 (1 µM). After treatment, cells were harvested, and luciferase activity was determined and normalized to the protein content of each extract. The fold increase in luciferase activity on treatment with IL-1ß, HGF, or anisomycin was calculated relative to the basal expression level of the untreated cells. For cotransfection experiments, luciferase activity expressed by cells transfected with Raf-1:ER plasmids in the absence of ICI 182,780 was given an arbitrary value of 1 and was corrected for empty vector effects. The results are presented means ± standard errors (error bars) and represent four individual experiments.

Activation of the p42/44^MAPK cascade alone is sufficient to induce LDL receptor expression in HepG2 cells
The above results in conjunction with our earlier findings (22) (23) (24) have revealed a critical role for p42/44^MAPK activation during induction of LDL receptor expression by a variety of extracellular stimuli. In view of these observations, we wished to examine the contribution of the activation of p42/44^MAPK cascade alone on LDL receptor expression in HepG2 cells. We used previously characterized conditionally active Raf-1 expression constructs (Raf-1:ER) in which the kinase domain of oncogenic Raf-1 is fused to the hormone-binding domain of the human estrogen receptor. Estradiol or antiestrogen ICI 182,780 treatments of cells transfected with this construct resulted in a rapid and near-complete activation of the MEK and p42/44^MAPK in different cell types, including HepG2 (26) (27) (33). By using transient transfection assays, we determined whether activation of the Raf-1/MEK/p42/44^MAPK cascade by Raf-1:ER fusion protein mimics anisomycin or HGF with respect to LDL receptor induction. We cotransfected HepG2 cells with human LDL receptor reporter plasmid A and a vector encoding either C.A. or D.N. form of Raf-1:ER hybrid protein. After 24 h, transfected cells were treated with ICI 182,780 for 15 h at 1 µM, a concentration of hormone that is sufficient to fully activate both Raf:ER fusion proteins (26) (27). A large increase in luciferase reporter gene expression was observed in an ICI-dependent manner in cells transfected with a C.A. Raf-1:ER construct when compared to luciferase expression in cells transfected with either a control vector or with a D.N. Raf-1:ER construct (Figure 7). Treatment with PD98059 blocked ICI-dependent induction, further confirming that activation of the Raf-1/MEK-1/p42/44^MAPK cascade is indeed responsible for increased LDL receptor promoter activity. Similar increase in LDL receptor promoter activity was observed when a constitutively activated form of MEK-1 (MEK-1-SS/DD) was coexpressed in HepG2 cells (Figure 7). This up-regulation resulted from higher p42/44^MAPK activity, as co-expression of MKP-1, a dual specificity phosphatase shown to inactivate p42/44^MAPK or PD98059 treatment, totally abolished an increase in LDL receptor promoter-luciferase expression (Figure 7). Identical results were observed in at least four independent transfection experiments. Taken together, these results demonstrate that activation of the Raf-1/MEK/p42/44^MAPK cascade alone initiates positive regulatory signals responsible for LDL receptor expression.

	DISCUSSION

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Anisomycin can possibly induce LDL receptor expression through different mechanisms. By virtue of its activity as a stimulator of signal transduction cascades, it can cause direct transcriptional, activation. Alternatively, due to its ability to inhibit translation, it might cause the loss of a labile transcriptional repressor or ribonucleases, thereby inducing transcription in an indirect manner or cause mRNA stabilization, respectively. From the studies presented in this paper, we conclude that anisomycin-induced LDL receptor expression cannot be the consequence of translational arrest, first, because neither puromycin nor cycloheximide produced these effects, and second, because induction was observed even at anisomycin concentrations much below that required for efficient inhibition of protein synthesis. Anisomycin led to significant accumulation of LDL receptor mRNA at concentrations that are insufficient to block protein synthesis and the maximal level of LDL receptor mRNA was reached at a concentration of 50 ng/ml which still allowed translation to proceed at about 60;–70% of its efficiency in the absence of drugs. These results strongly suggest that anisomycin induces LDL receptor expression by a mechanism independent of protein synthesis.

Results presented herein clearly show that not only is the p42/44^MAPK required for LDL receptor transcription, but its activation alone is sufficient for induction of LDL receptor expression. By transfecting estrogen-dependent human Raf-1 protein kinase (Raf-1:ER), we demonstrate that exclusive activation of the Raf-1/MEK-1/p42/44^MAPK cascade with ICI 182,780 induces LDL receptor expression to the same levels as observed with either HGF or anisomycin. Furthermore, consistent with the role of this kinase cascade, expression of a constitutively active MEK-1 produced an increase in LDL receptor transcription in the absence of any other growth signal, which was approximately equivalent to the expression level measured in cells stimulated with anisomycin or HGF. Inhibition of Raf- and anisomycin-induced LDL receptor expression by PD98059 further supports the conclusion that the positive signal is mediated through the p42/44^MAPK for the induction of LDL receptor expression in HepG2 cells. The mechanism by which p42/44MAPK activation results in LDL receptor induction is not known and is under investigation. Our observation that anisomycin-induced LDL receptor expression at a concentration that inhibited protein synthesis suggests that the transcription factors mediating the induction of LDL receptor gene expression must have been present before the onset of stimulation with anisomycin. By this criterion, LDL receptor can be classified as an IE gene, not requiring de novo synthesis of the transcriptional stimulators. Establishment of a link between the p42/44^MAPK and anisomycin-induced LDL receptor expression could potentially explain super-induction of IE genes by anismycin via direct promoter activation without the existence of a labile repressor. For example, by prolonging the normally transient p42/44^MAPK activation in response to cytokines, hormones, and TPA, anisomycin may make an additional contribution to the prolonged transcriptional activation of IE genes under superinducing conditions. In addition, involvement of the p42/44^MAPK in anisomycin signaling can also account for the identical phosphorylation of non-histone mobility-group protein HMG-14 on treatment with either anisomycin or mitogen (34).

The role of p42/44^MAPK in anisomycin signaling cascade in HepG2 cells is somewhat unexpected, due to numerous reports suggesting that anisomycin-induced changes in gene expression are mediated through the p46/54^JNK and/or p38^MAPK in other cell types. Although the profile of anisomycin-activated stress-related MAPKs in HepG2 cells is similar to that reported earlier for various non-hepatic cells, activation of the p42/44^MAPK by anisomycin may be due to significant heterogeneity between cell types. Such differences have been observed earlier between neuronal and non-neuronal cell types. For example, cAMP has been reported to inhibit p42/44^MAPK activation in fibroblasts, and in contrast, activates this kinase in PC-12 cells (35). It appears that differential expression of B-Raf in PC-12 cells contributes to cell type-specific differences in the regulation of p42/44^MAPK signaling cascade. Similar differences have been observed in the upstream regulation of stress-related MAPK cascades between the PC-12 and KB cell types (36). Thus, it is likely that the differences observed in the present study may reflect differences in the relative regulation of the p42/44^MAPK and the stress-related MAPK pathways between hepatic and non-hepatic cell types. Furthermore, activation of all three MAPKs by anisomycin should not be surprising because the specificities of activating stimuli for the three subfamilies of MAPKs are not absolute; for example, inflammatory cytokines IL-1ß and tumor necrosis factor- are known to activate p42/44^MAPK in many cell types, including HepG2 cells (23), and some growth factors can produce a weak activation of the p46/54^JNK and p38^MAPK (37). Based on the above results, we propose that one should exercise a great deal of caution in extrapolating results using anisomycin.

To elucidate whether Ras is required for anisomycin-induced activation of p42/44^MAPK and LDL receptor induction, we performed experiments using BZA-5B, an inhibitor of Ras farnesyltransferase. We found that although HGF-induced p42/44^MAPK activation and LDL receptor induction was completely suppressed by treatment with BZA-5B, anisomycin-induced activation was not attenuated by this manipulation, suggesting that the anisomycin-induces LDL receptor expression in a Ras-independent manner. Therefore, the upstream signaling pathways resulting in the activation of the MEK are different for HGF and anisomycin. This observation is consistent with our results showing that anisomycin and HGF induce p42/44^MAPK activity with different kinetics. HGF-induced p42/44^MAPK activation is rapid and strong, whereas anisomycin induce activation is slow and mild. The delay in anisomycin-induced p42/44^MAPK is most likely due to the late appearance of an intermediate signaling component through de novo synthesis, post-translational modification, or localization. The involvement of Ras-independent pathway(s) in the activation of p42/44^MAPK and p46/54^JNK in HepG2 cells is interesting in view of a recent report that shows anisomycin-induced activation of the p46/54^JNK cascade requires Ras in neuronal PC-12 cells (36). This type of difference further supports the existence of cell type-specific differences in the regulation of MAPKs.

In conclusion, results presented in this paper emphasize a general point of significance, namely, although one specific signaling pathway may appear to dominate a response, another mildly activated pathway can be critical, and thus should not be overlooked or ignored. The requirement of p42/44^MAPK cascade for TPA (22), cytokines (23), HGF, and anisomycin (this paper)-induced LDL receptor expression suggests that a diverse array of extracellular signals utilize p42/44^MAPK signaling cascade to induce LDL receptor expression in HepG2 cells. Promoter dissection studies in progress should lead us to the identification of the crucial DNA sequence motives involved in LDL receptor induction in response to the above agents. The transcription factor(s) interacting with such sequence motives are expected to be the critical targets of the p42/44^MAPK signal transduction pathway. From the above studies, it appears that the LDL receptor gene is a very useful tool to investigate the regulation of gene expression by the p42/44^MAPK signaling pathway. Moreover, differential activation of the p42/44^MAPK cascade may alter cellular events that contribute to differences in expression and responses of LDL receptor gene in a cell-specific manner.

	FOOTNOTES

¹ Present address: Molecular and Cellular Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892.

	ACKNOWLEDGMENTS

We warmly thank all investigators who supplied the plasmids indicated under Experimental Procedures. We thank R.P. Singh and G. Kapoor for their help with some of the experiments. We also thank Caroline Simmons for critically reading the manuscript. We acknowledge Genentech Inc. for their generous gift of BZA-5B. This work was supported by research grants from the National Institutes of Health (HL-51592-04), Established Investigator Grant from the American Heart Association (9940163N), and UAMS Hornick Endowment award to K. D. M.

Manuscript received February 11, 1999; and in revised form June 3, 1999

Abbreviations: MAPK, mitogen-activated protein kinase; p42/44^MAPK, mitogen/extracellular protein kinase; p46/54^JNK, c-jun NH₂ terminal kinases; HGF, hepatocyte growth factor; MEK, MAPK/ERK kinase; MEKK, MAPK kinase kinase; IL-1ß, interleukin-1ß; FCS, fetal calf serum; TPA, 12-O-tetradecanoylphorbol-13-acetate; C.A., constitutively active; D.N., dominant-negative; LDL, low density lipoprotein; SS, squalene synthase; Raf-1:ER, conditionally active form of Raf kinase

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