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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1667-1674.)
© 1997 American Heart Association, Inc.

Articles

High Density Lipoproteins Stimulate Mitogen-Activated Protein Kinases in Human Skin Fibroblasts

Mark A. Deeg; Rosario F. Bowen; John F. Oram; ; Edwin L. Bierman

From the Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, University of Washington, Seattle, Wash (J.F.O., E.L.B.) and the Division of Endocrinology and Metabolism, Department of Medicine, Indiana University and the Richard L. Roudebush Veteran Affairs Medical Center, Indianapolis, Ind (M.A.D., R.F.B.).

Correspondence to John F. Oram, PhD, Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, University of Washington, Box 356426, Seattle, WA 98195-6426. E-mail joram{at}u.washington.edu

	Abstract

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Abstract Protein kinase C (PKC) seems to play an important role in many of HDL effects on cells, including removal of excess cholesterol. HDL removes cholesterol by at least two mechanisms. One mechanism involves desorption/diffusion of cholesterol from the plasma membrane onto the acceptor particle, whereas the second is mediated by apolipoproteins and may involve intracellular translocation of cholesterol to the plasma membrane for subsequent efflux. In this report, we examined the possibility that mitogen-activated protein (MAP) kinase is one of the downstream events from HDL activation of PKC. Using a gel kinase assay with myelin basic protein incorporated into the gel, HDL (50 µg protein/mL) stimulated multiple kinases of 42, 50, 52, 58, and 60 kDa. The 42-kDa protein kinase, corresponding to the unresolved MAP kinases ERK1 and ERK2 based on immunoblotting, was activated over 2-fold by HDL. HDL activated all identified kinases in a concentration- and time-dependent manner, which became maximal within 5 to 10 minutes and remained activated for at least 60 minutes. HDL activation of MAP kinase seems to be partially mediated by PKC, because down-regulation of PKC and known PKC inhibitors inhibited the HDL effect by 40 to 50%. Free apolipoproteins A-I (10 µg/mL) and A-II (10 µg/mL) had no significant effect on MAP kinase activation. Moreover, modifying HDL with trypsin or tetranitromethane, which abolishes apolipoprotein-mediated cholesterol efflux, had no effect on HDL activation of MAP kinase. These results suggest that HDL activates MAP kinase via multiple signal transduction pathways that are likely involved in an HDL effect unrelated to apolipoprotein-mediated cholesterol translocation and efflux.

Key Words: high density lipoprotein • MAP kinase • cholesterol transport • apolipoprotein A-I

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
HDL is a heterogeneous mixture of lipoprotein particles that differ in size and composition.¹ The levels of HDL cholesterol correlate inversely with risk for coronary artery disease.^2,3 HDL has been postulated to be involved in removing cholesterol from the periphery for delivery to the liver for excretion from the bile, a process termed reverse cholesterol transport.⁴ In addition, HDL has been shown to have additional cellular effects depending on the cell type. These include stimulation of lipid secretion,^5-8 protein secretion,^9,10 and mitogenesis.^11,12 PKC seems to play a role in most of these effects, including HDL-stimulated cholesterol efflux.⁶ Events subsequent to HDL activation of PKC are unknown. We recently determined that HDL stimulates increased phosphorylation of three acidic proteins including MARCKS (Myristylated Alanine-Rich C-Kinase Substrate), a well-known PKC substrate, in cholesterol-loaded human skin fibroblasts.¹³ What role these proteins and PKC play in HDL signal transduction and cholesterol efflux is unknown at this time.

HDL seems to stimulate cholesterol efflux from cells by at least two distinct mechanisms. One mechanism involves desorption/diffusion of cholesterol from the plasma membrane to the HDL particle and is independent of cellular binding.¹⁴ The second mechanism is mediated by apolipoprotein interaction, either on HDL or as lipid-poor apolipoproteins, with cell surface binding sites which in turn directly removes cholesterol and phospholipid from the plasma membrane^15-19 and stimulates translocation of newly synthesized sterol to the plasma membrane and its subsequent efflux.^6,7,20-23 This second mechanism seems to involve activation of PKC.^6,7,23 Modification of HDL with trypsin or tetranitromethane (TNM) abolishes cholesterol efflux mediated by the second mechanism but not by the first.^6,21,24 Apolipoprotein-mediated cholesterol and phospholipid efflux is impaired in fibroblasts from subjects with Tangier disease.^22,23,25

One downstream event of PKC activation in many cells is stimulation of MAP kinases. MAP kinases are a family of serine/threonine kinases uniquely activated by dual phosphorylation of threonine and tyrosine residues. Tyrosine kinase receptor activation of MAP kinase is the best understood pathway.²⁶ This pathway utilizes the autophosphorylation of the tyrosine kinase receptor to initiate a cascade of events involving activation of Ras, which in turn activates Raf. A cascade of protein kinase activation occurs, which leads to MAP kinase activation. PKC regulates the MAP kinase cascade in a number of different ways. In some cells, PKC regulation of MAP kinase is Ras dependent, whereas in fibroblasts it is Ras independent.²⁷

Because PKC has been implicated in a number of HDL-stimulated events, we undertook these studies to determine whether HDL activation of PKC was associated with MAP kinase activation and whether the MAP kinase activation may be related to cholesterol efflux. Results show that HDL activated MAP kinase (ERK1 and ERK2) in a concentration- and time-dependent fashion. In addition, HDL activated additional kinases of 50, 52, 58, and 60 kDa (referred to as pk50, pk52, pk58, and pk60), the identities of which are unknown. Lipid-free apolipoprotein (apo) A-I activated MAP kinase to a small, nonsignificant extent but did not activate the other kinases. In contrast, lipid-free apoA-II had no effect on any of the kinases examined. Trypsin- or TNM-treated HDL also activated MAP kinase. Activation of MAP kinase by HDL or these modified HDL particles was partially blocked with down-regulation of PKC or by treatment of cells with bisindolylmaleimide, a PKC inhibitor. These results indicate that HDL may activate MAP kinase by more than one signal transduction pathway, including at least one involving PKC. However, HDL activation of MAP kinase does not seem to be related to the cholesterol efflux mechanism mediated by the interaction of HDL apolipoproteins with cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
-[³²P]ATP was purchased from Amersham. Anti-ERK1 and ERK2 were purchased from Santa Cruz Biotechnology. Bisindolylmaleimide and PD98059 were purchased from Calbiochem and New England Biolabs, respectively. Myelin basic protein (MBP) was from Sigma. Epidermal growth factor (EGF) was purchased from Upstate Biological Inc. Phorbol 12-myristate 13-acetate (PMA) was purchased from LC Services. All other materials were of analytical grade or better.

Preparation of HDL, ApoA-I, and Trypsin-Modified HDL
HDL₃ (herein referred to as HDL) was isolated from plasma of healthy male volunteers by standard sequential ultracentrifugation techniques at d=1.125 to 1.210 g/mL. HDL was subjected to heparin-agarose affinity chromatography to remove apoB- and apoE-containing particles.²⁸ Proteolytically digested HDL particles were generated by treating HDL with trypsin for 10 minutes at 37°C at an HDL/trypsin ratio of 40/1.²¹ Lipid-free apoA-I and apoA-II were purified from HDL as described.⁶ TNM-modified HDL was prepared as described previously.²⁴ Cross-linking of apoA-I was verified by Western blotting.

Cell Culture
Human skin fibroblasts obtained from normal individuals were plated (5x10⁵ cells) in 60-mm plates and grown to confluence (5 to 7 days) in DMEM supplemented with 10% fetal calf serum. On reaching confluence, the cells were washed twice in PBS containing 2 mg/mL fatty acid-free BSA and incubated for 24 hours in DMEM containing 2 mg/mL BSA with 30 µg/mL cholesterol to load cells with sterol.^6,21 Cells then were washed twice with PBS/BSA and incubated overnight in DMEM containing 1 mg/mL BSA to equilibrate cellular sterol pools.

After the overnight equilibration, the cells were washed twice with DMEM without BSA and incubated for 2 hours in DMEM without BSA to remove the BSA and allow the cells to rest. This period of rest was required to return MAP kinase activity to baseline after media washes. Cells were stimulated by adding a concentrated stock solution of stimulant to the media and incubating at 37°C for the times indicated. The incubation was terminated by removing the media and washing the cells with ice-cold PBS, and the cells were lysed by adding 250 µL lysis buffer (10 mmol/L HEPES, pH=7.4, 50 mmol/L Na pyrophosphate, 50 mmol/L NaF, 50 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 2 mmol/L Na₃VO₄, 0.1% Triton X-100, 0.5 mmol/L PMSF, and 10 µg/mL leupeptin) and frozen in a dry ice/ethanol bath. After thawing, the dishes were scraped with a rubber policeman, and the cell extracts were sonicated for 5 seconds and centrifuged for 30 minutes at 16,000g at 4°C. Protein concentration was determined using the Bradford protein assay (Bio-Rad).²⁹

MAP Kinase Assay
MAP kinase activity was determined by a gel kinase assay as described previously.³⁰ Briefly, equal amounts of lysate protein (10 µg) from each sample were separated by SDS polyacrylamide gel electrophoresis (PAGE) using a 12% polyacrylamide gel, 7 cm in length, containing MBP at 0.4 mg/mL. SDS was removed from the gel, and the proteins were denatured and renatured. The gel was incubated in 50 mmol/L HEPES, pH=7.4, 5 mmol/L ß-mercaptoethanol, 100 µmol/L Na₃VO₄, 10 mmol/L MgCl₂, 50 µmol/L ATP, and 50 µCi -[³²P]ATP for 1 hour at 30°C. The reaction was terminated by washing in 10 mmol/L Na pyrophosphate and 5% trichloroacetic acid. The gel was dried and subjected to autoradiography. The activity was quantified by densitometry.

MAP Kinase Immunoblotting
Fibroblasts were treated with stimulants and harvested as described above. Equal amounts of protein (10 µg) were separated by SDS-PAGE (12%, 16 cm in length) and transferred to nitrocellulose. The longer gel length was required to resolve ERK1 from ERK2. Western blot analysis was performed with polyclonal ERK1 and ERK2 antibodies. After incubation with horseradish peroxidase-conjugated secondary antibody, the blots were developed using enhanced chemiluminescence (Amersham).

Statistics
Statistical significance was determined by a two-tailed Student's t test. Results were considered significant for P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
HDL Stimulates Multiple Myelin Basic Protein Kinases
HDL has been shown to activate PKC in multiple cell types, including fibroblasts. Because MAP kinase is one of the downstream events of PKC activation, we examined the hypothesis that HDL might activate MAP kinase in a PKC-dependent manner. To determine if HDL activates MAP kinase, cholesterol-loaded fibroblasts were stimulated with HDL (50 µg protein/mL) for 5 minutes and MAP kinase activity in the cell lysates was determined with a gel kinase assay that uses MBP as the substrate. This concentration of HDL has been shown previously to cause maximal binding to specific cellular HDL binding sites with minimal nonspecific binding.^31,32 PMA (100 nmol/L) and EGF (50 µg/mL) were used as positive controls.

Under basal conditions, numerous kinases that phosphorylate MBP were observed (Fig 1A). The most prominent had molecular masses of 42, 50, 52, 58, and 60 kDa and are referred to herein as pk42, pk50, pk52, pk58, and pk60, respectively. Occasionally, pk42 was resolved into two bands. Higher molecular weight kinases could be observed with longer exposures, but HDL had no effect on these kinases (data not shown). Autophosphorylation of kinases did not account for the radiolabeling observed, because kinase assays performed with MBP absent from the gel did not produce any radiolabeled bands.

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of HDL, PMA, and EGF on MBP kinases and ERK1/2 mobility on SDS-PAGE. Human skin fibroblasts were grown to confluence (Con) and cholesterol loaded as described in "Materials and Methods." Cells were treated with HDL (50 µg protein/mL), PMA (100 nmol/L), or EGF (50 µg/mL), and cell lysates were analyzed for MBP kinase activity (panel A). Molecular masses (kDa) of the kinases are indicated. The results (mean±SEM) for HDL (n=7), PMA (n=5), and EGF (n=3) effects on MBP kinases are shown in panel B (*P<.05). The effect of HDL, PMA, and EGF on ERK1 and ERK2 mobility is shown in panel C. Cell lysates were prepared as described for panel A, and proteins were separated on a 12% SDS-PAGE gel. Immunoblotting for ERK1 and ERK2 was performed as described in "Materials and Methods." The phosphorylated forms of ERK1 (ppk44) and ERK2 (ppk42) are indicated.

HDL stimulated the activity of several kinases including pk42, pk50, pk52, and pk60 (Fig 1A). Using densitometry to quantify the kinase activity, HDL increased pk42 activity more than 2-fold (Fig 1B). Because pk50 could not be resolved from pk52 and pk58 from pk60 by the densitometer, pk50 and pk52 were quantified together (pk50/52), and pk58 and pk60 were quantified together (pk58/60). HDL increased the activity of pk50/52 by 2-fold and pk58/60 by 60% (Fig 1B).

Compared with HDL, both PMA (100 nmol/L) and EGF (50 µg/mL) increased pk42 activity 2.9- and 3.5-fold, respectively (Fig 1A and 1B). However, in contrast to HDL, PMA and EGF had different effects on the other kinases. PMA also increased activity of pk50/52 but not pk58/60 (Fig 1B). EGF had no significant effect on pk50/52. However, EGF increased pk58/60 to a similar extent as HDL (Fig 1B), which did not approach statistical significance (P=.09, n=3). These results suggest that PMA mimics some but not all of the HDL effects on MBP kinases and that different transduction systems may be regulating pk50/52 and pk58/60 activities.

To determine whether the observed pk42 activity corresponds to the ERK family of MAP kinases, in particular ERK1 (44 kDa) and ERK2 (42 kDa), ERK1 and ERK2 were identified in fibroblast lysates by Western blotting. Lysates were probed with anti-ERK1 and anti-ERK2 under basal and stimulated conditions. Fibroblasts contain both ERK1 and ERK2 (Fig 1C). When these kinases are phosphorylated (with either 1 or 2 phosphate groups, the latter being required for increased kinase activity), a shift in the mobility of ERK1 and ERK2 on SDS-PAGE occurs.³³ Under basal conditions, a significant amount of ERK2 is phosphorylated.

Stimulating cholesterol-loaded fibroblasts with HDL, PMA, or EGF caused a decrease in the amount of nonphosphorylated ERK1 and ERK2 with concomitant increases in the phosphorylated forms. The degree of shifting of ERK1 and ERK2 corresponded to the increase in pk42 activity (compare Fig 1C and 1B). These results are consistent with HDL-induced phosphorylation of ERK1 and ERK2 and suggest that the increased pk42 kinase observed by the gel kinase assay corresponds to ERK1 and ERK2. This is supported by the observation that PD98059, a MEK inhibitor^34,35(MEK phosphorylates MAP kinase), inhibits HDL activation of pk42 by 50, 60, and 80%, at 0.2, 2.0, and 20 µmol/L, respectively (data not shown). Herein, the pk42 kinase activity will be referred to as MAP kinase.

Time and Concentration Dependence of HDL Activation of MBP Kinases
To further characterize HDL activation of MAP kinase, pk50/52, and pk58/60, the time and concentration dependence of HDL activation of these kinases was determined. HDL (50 µg/mL) rapidly activated these kinases, with maximal activation occurring at 5 to 10 minutes and remaining elevated for 60 to 120 minutes (Fig 2). MAP kinase activity returned to baseline by 120 minutes. HDL activated MAP kinase, pk50/52, and pk58/60 in a concentration-dependent manner. HDL activated MAP kinase, pk50/52, and pk58/60 at a concentration as low as 2.5 µg/mL. HDL activation of MAP kinase seemed to reach a saturation at 25 to 100 µg/mL (Fig 3). HDL activated pk50/52 and pk58/60 without an apparent saturation at the highest concentration tested, 100 µg protein/mL (Fig 3).

View larger version (16K): [in this window] [in a new window]   Figure 2. Time course of effect of HDL on MBP kinases. Fibroblasts were prepared and treated with HDL (50 µg protein/mL) for the times indicated, and MBP kinase activity in cell lysates was determined as described in Fig 1. Values represent mean±SEM for 3 to 5 experiments.

View larger version (15K): [in this window] [in a new window]   Figure 3. Concentration-dependence of effect of HDL on MBP kinases. Fibroblasts were prepared and treated with HDL (1 to 100 µg protein/mL) for 5 minutes, and MBP kinase activity in cell lysates was determined as described in Fig 1. Values represent mean±SEM from 3 experiments.

HDL Activation of MAP Kinase Is Partially PKC Dependent
PKC is involved in activating the MAP kinase pathway for a variety of receptors in a number of different cell types. To determine whether MAP kinase activation might be downstream from HDL activation of PKC, cells were pretreated with PMA for 24 hours to down-regulate PKC. Fibroblasts contain PKC , , and .³⁶ PMA treatment resulted in down-regulation of PKC and in these cells (data not shown) without affecting basal MAP kinase activity (Table 1). Acute treatment (5 minutes) with PMA increased MAP kinase activity but did not activate MAP kinase (Table 1), pk50/52, or pk58/60 (data not shown) in PMA down-regulated cells. Chronic PMA treatment inhibited HDL activation of MAP kinase by 55% (Table 1). Similar results were obtained with bisindolylmaleimide, a PKC inhibitor.³⁷ Preincubating cholesterol-loaded fibroblasts for 30 minutes with bisindolylmaleimide (5 µmol/L) inhibited HDL and PMA activation of MAP kinase by 39 and 79%, respectively (Table 1). As with chronic PMA treatment, preincubating cells with bisindolylmaleimide had no significant effect on basal MAP kinase activity (Table 1). These results indicate that HDL activation of PKC may be one but not the exclusive pathway for activating MAP kinase in human skin fibroblasts.

View this table: [in this window] [in a new window]   Table 1. Effect of Inhibiting PKC on HDL Activation of MAP Kinase

Apolipoprotein Regulation of MAP Kinase
HDL particles are a heterogeneous mixture of particles differing in protein and lipid composition. To determine whether the two major apolipoproteins in HDL, apoA-I and apoA-II, are responsible for the HDL effect on MAP kinase, cells were treated with lipid-free apolipoproteins for 5 minutes, and MBP kinase activities were determined. Free apoA-I and apoA-II stimulate cholesterol efflux in cholesterol-loaded cells.^15,19,25,38 ApoA-I (10 µg protein/mL) stimulated MAP kinase to a small, nonsignificant (P=.07) extent, with no significant effect on pk50/52 or pk58/60 (Fig 4). This concentration is above the maximum for stimulating cholesterol efflux.^15,19,25 ApoA-II (10 µg protein/mL) had no effect on any kinase (Fig 4). These results suggest that apoA-I may account for at least a portion of the ability of HDL to stimulate MAP kinase.

View larger version (38K): [in this window] [in a new window]   Figure 4. Effect of apoA-I (AI) and apoA-II (AII) on MBP kinase activity. Fibroblasts were prepared and incubated with apoA-I (10 µg/mL) or apoA-II (10 µg/mL) for 5 minutes, and cell lysate MBP kinase was determined as described in Fig 1. Results from 3 to 5 independent experiments are shown as mean±SEM (*P<.05).

Effect of Modified HDL on MAP Kinase Activation
HDL-stimulated intracellular cholesterol translocation and efflux can be abolished by short-term treatment of HDL with trypsin^6,21 or tetranitromethane (TNM).²⁴ These treatments inhibit the apolipoprotein-mediated cholesterol efflux but not the passive desorption of cholesterol from the plasma membrane.^6,21,24 We reasoned that if MAP kinase activation by HDL is related to cholesterol translocation, then these modifications of HDL should also abolish HDL-mediated MAP kinase activation.

HDL (50 µg/mL), trypsin-modified HDL (trHDL), and TNM-modified HDL (TNM-HDL) (equivalent concentration based on phospholipid content) stimulated MAP kinase by 2.2-, 3.5-, and 2.9-fold (combined data from Tables 1 and 2). PKC also seems to play a role in activating MAP kinase by these modified HDL particles, because chronic PMA or acute bisindolylmaleimide treatments partially inhibited trHDL and TNM-HDL stimulation of MAP kinase (Table 1), although the number of samples was too small to obtain significance.

View this table: [in this window] [in a new window]   Table 2. Effect of Cholesterol Loading on MAP Kinase Activation in Fibroblasts

Effect of Cholesterol Loading on HDL Activation of MAP Kinase
The above results indicate that HDL activation of MAP kinase may be unrelated to apolipoprotein-mediated cholesterol efflux. To explore further this hypothesis, the effect of cholesterol loading on HDL activation of MAP kinase was examined. Cholesterol loading of human skin fibroblasts is associated with an increase in HDL binding^31,32 and HDL- and apolipoprotein-mediated cholesterol efflux.^16,21,32 Incubation of human skin fibroblasts with free cholesterol (30 µg/mL) is associated with an approximate 3- and 10-fold increase in the cellular content of total cholesterol and cholesterol esters, respectively (data not shown). Cholesterol loading did not significantly affect the basal MAP kinase activity (Table 2). PMA, EGF, HDL, and TNM-HDL activated MAP kinase in both nonloaded and cholesterol-loaded fibroblasts. There was no significant difference in the MAP kinase activation in cholesterol-loaded versus nonloaded cells for any of the agents tested. HDL activation of MAP kinase peaked at 5 minutes in nonloaded cells (data not shown), comparable to what was observed for cholesterol-loaded cells (see Fig 2).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
HDL has a multitude of cellular effects and activates a number of signal transduction systems, including PKC. In this report, we demonstrate that HDL treatment of cholesterol-loaded fibroblasts stimulates MBP kinases of 42, 50, 52, and 60 kDa. Multiple lines of evidence suggest that the 42-kDa protein activated by HDL corresponds to the ERK family of MAP kinases. First, PMA and EGF, known activators of these MAP kinases, also stimulate the pk42 activity. Second, the pk42 activity co-migrates with ERK1/2 by Western blotting. Third, HDL stimulation of fibroblasts results in band shifting of ERK1 and ERK2 on SDS-PAGE, consistent with phosphorylation of these kinases, an event required for activation. Fourth, a MEK inhibitor blocks HDL activation of pk42.

HDL activation of MAP kinase seems to be partially but not completely PKC dependent. Prolonged treatment of fibroblasts with PMA, which down-regulates PKC and but not in these cells (M. Deeg, unpublished data), abolishes PMA activation of MAP kinase but only inhibits HDL activation of MAP kinase by 40 to 50%. This conclusion is supported by the observation that bisindolylmaleimide also inhibits HDL activation of MAP kinase by approximately 40%. It is unclear what other pathways may be involved in HDL activation of MAP kinase, but participation of tyrosine kinases is one possibility. TNM-HDL and trHDL activation of MAP kinase also seems to involve PKC to some extent, because PMA down-regulation of PKC seemed to partially inhibit activation of MAP kinase with these modified HDL particles. This seems to contradict previously published reports that HDL particles modified with trypsin or TNM do not activate PKC based on translocation of either PKC activity or mass from a cytosolic to membrane fraction.^6,39,40 PKC activation, however, does not require PKC translocation.⁴¹ That HDL, trHDL, and TNM-HDL all activate PKC was shown by their abilities to increase ³²P labeling of MARCKS¹³, a well-known PKC substrate in intact cells. In contrast, apoA-I stimulates phosphorylation of another protein, pp18, in a PMA-sensitive manner despite an insignificant ability to activate MAP kinase or increase MARCKS phosphorylation.¹³ These results suggest that HDL and apoA-I may both activate PKC, but differences may occur with respect to the PKC isoforms activated or subcellular location of enzyme translocation, as has been described for PKC activation in endothelial cells.⁴² ApoA-I mimics some but not all of the signal transduction systems activated by HDL (see below), some of which may be involved in cholesterol translocation and efflux.

The components of HDL which may be responsible for stimulating MAP kinase are unknown. From the data presented here, it seems unlikely that an apolipoprotein component in HDL is responsible for HDL activation of MAP kinase. ApoA-I and apoA-II, the major protein components of HDL, had no significant effect on the kinases measured, suggesting that these proteins are minimally involved, if at all. In addition, modifying the proteins in HDL by trypsinization or TNM treatment had no effect on HDL activation of MAP kinase. However, involvement of a relatively trypsin-resistant protein in HDL cannot be entirely eliminated because the proteolytic procedure used in these studies removes <30% of the protein present in HDL.²¹ Alternatively, because the conformation of apoA-I and apoA-II are dependent on the lipid composition of the particle^43,44 and lipid-free apolipoproteins were used in these studies, it is conceivable that different conformations of apoA-I and apoA-II present in HDL have different potencies in activating MAP kinases. No systematic study has been published looking at the effect of apoA-I or A-II conformation on stimulating signal transduction systems. Addition of lipid-free apoA-I to cells results in the formation of apoA-I:phospholipid particles,^15-17 but the time frame for this effect (hours) is relatively slow compared to the apoA-I activation of MAP kinase that occurs within minutes.

Another possibility is that there are at least two agonist activities present in HDL. Recently published data³⁹ demonstrated that apoA-I and apoA-II proteoliposomes mimicked some but not all of the effects of HDL on phosphatidylcholine phospholipase D activation. In addition, HDL but not apoA-I or apoA-II proteoliposomes activated phosphatidylinositol- or phosphatidylcholine-phospholipase C activities in these same studies. A likely second agonist activity may be a lipid component of HDL. Lysophosphatidylcholine or phosphatidic acid, which are present in HDL, have been shown to activate MAP kinases in other cells^12,45 and may be responsible for HDL activation of some or all of the kinases examined. This possibility would explain the lack of an effect of modifying HDL proteins with trypsin or TNM on HDL activation of MAP kinase. A lipid component in HDL has been implicated in HDL-stimulated mobilization of intracellular Ca²⁺.¹²

HDL also activates other kinases that phosphorylate MBP, but the identity of these proteins is unknown. Potential candidates include other members of the MAP kinase family, including the recently described JNK or stress-activated protein (SAP) kinases (54 kDa) and the p57 MAP kinase.⁴⁶ Treatment of cells with cycloheximide increases JNK/SAP kinase activity⁴⁷ but did not increase the activity of these proteins in cholesterol-loaded fibroblasts (data not shown), suggesting that these proteins do not correspond to the SAP kinases. Based on immunoblotting, none of these kinases correspond to the known PKC isoforms (, , ) present in fibroblasts (data not shown). In addition, because it was difficult to resolve all of these proteins, it was not possible to draw conclusions on the specific regulation of these kinases by HDL, PMA, or EGF. However, it is interesting to note that pk50/52 was activated by HDL and PMA but not by EGF, suggesting that this kinase may be regulated in a PKC-dependent manner.

Like insulin, which utilizes a variety of signal transduction systems to mediate the plethora of cellular responses, HDL seems to initiate a number of intracellular signal transduction systems, some of which seem to be related to cholesterol translocation and efflux. What role might MAP kinase play in mediating HDL action? It is tempting to speculate that HDL activation of MAP kinase may be involved in mediating HDL-stimulated mitogenesis.^11,12 Mitogen activation of MAP kinase related to cell growth typically results in prolonged activation of MAP kinase (>120 minutes) or a second phase of activation.⁴⁸ HDL stimulation does result in a prolonged activation of MAP kinase (60 to 120 minutes), although not nearly as potent as other mitogens such as EGF, which is consistent with the weak mitogenic effects of HDL observed in fibroblasts¹² and other cell types.¹¹

MAP kinase activation by HDL is clearly not exclusively related to cholesterol translocation and efflux based on the observation that there is no correlation between cholesterol efflux and MAP kinase activation with apolipoproteins, HDL, TNM-HDL, or trHDL. This is further supported by the observation that HDL activation of MAP kinase did not differ between cholesterol loaded and nonloaded cells. Cholesterol loading is associated with increased HDL binding and HDL-mediated cholesterol efflux.³² These results are consistent with the conclusion that specific MAP kinase-independent signal transduction pathways exist for regulating cholesterol translocation and efflux.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein EGF = epidermal growth factor MAP = mitogen-activated protein MBP = myelin basic protein PKC = protein kinase C SAP = stress-activated protein TNM = tetranitromethane MARCKS = myristylated alanine-rich C-kinase substrate

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-18645 (J.F.O.), DK02456 (J.F.O.), and DK49010 (M.A.D.). A portion of this work was conducted while M.A.D. was a Pfizer Postdoctoral Fellow at the University of Washington under the direction of Edwin L. Bierman.

Received January 23, 1996; accepted January 3, 1997.

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