doi: 10.1161/hq1001.097062
© 2001 American Heart Association, Inc.
Vascular Biology |
Phosphatidylinositol 3-OH Kinase–Akt/Protein Kinase B Pathway Mediates Gas6 Induction of Scavenger Receptor A in Immortalized Human Vascular Smooth Muscle Cell Line
From the First Department of Internal Medicine (W.C., K.M., H.I., M.S., J.T., T.I.), Kagawa Medical University, Kagawa, Japan; Shionogi Research Laboratories (T.N.), Shionogi & Co, Ltd, Osaka, Japan; the Research Center for Advanced Science and Technology (T.K.), University of Tokyo, Tokyo, Japan; the Department of Pathology (Y.S.), University of Occupational and Environment Health, Kitakyushu, Japan; and the Departments of Medicine and Biochemistry & Molecular Biology (N.C.W.W.), Faculty of Medicine, Calgary, Alberta, Canada.
Correspondence to Koji Murao, First Department of Internal Medicine, Kagawa Medical University, 1750-1, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan. E-mail mkoji{at}kms.ac.jp
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Abstract |
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Abstract—— The growth arrest–specific gene 6 encodes a secreted protein, Gas6, which was originally identified as the ligand of a receptor, Axl, with tyrosine kinase activity. The class A scavenger receptor (SRA) mediates lipid uptake into cells, leading to the formation of foam cells, an important step in atherogenesis. Although Gas6 induces SRA expression, the underlying mechanism is not clear. In this report, we show that the Gas6-induced expression of SRA was mediated by the phosphatidylinositol 3-OH kinase (PI3-kinase)–serine/threonine kinase (Akt/protein kinase B [PKB]) pathway involving Akt phosphorylation. This pathway was activated by exposure to Gas6. Furthermore, the effect of Gas6 was abrogated by wortmannin, a specific inhibitor of PI3-kinase. We also demonstrated that the constitutively active form of Akt enhanced activity of the SRA promoter but that the dominant-negative mutant of Akt completely abolished the expression of SRA after treatment with Gas6. These results show that the PI3-kinase–Akt/PKB pathway participates in Gas6-induced SRA expression and suggests that the activation of Akt/PKB plays an important role in Gas6-induced atherosclerosis and foam cell formation in human vascular smooth muscle cells.
Key Words: scavenger receptors • Gas6 • Akt/protein kinase B • smooth muscle cells • Axl
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Introduction |
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Growth arrest–specific gene 6 encodes a secreted protein, Gas6. This protein enhances the proliferation and prevents the death of vascular smooth muscle cells (VSMCs).1,2 Gas6 was originally cloned by using subtractive hybridization to screen for growth arrest–specific genes that were preferentially expressed in the G0 phase of the cell cycle.3 Subsequent studies show that Gas6 is a ligand that binds to a membrane receptor tyrosine kinase, Axl.4
The binding of Gas6 to Axl triggers a chain of cellular events, including the activation of phosphatidylinositol 3-OH kinase (PI3-kinase).5 PI3-kinase is a heterodimeric enzyme directly activated by receptor tyrosine kinase. PI3-kinase is believed to play an important role in the survival response of a number of different cell types.6 The downstream targets of PI3-kinase include phospholipase C,7,8 protein kinase C,9 Rac,10 and the serine/threonine kinase (Akt/protein kinase B [PKB]). Recent studies by Goruppi et al11 have shown that activated Akt/PKB is necessary for Gas6-dependent survival protection.
Akt/PKB is an important mediator of metabolic and survival responses after growth factor stimulation.12 Akt was identified as the product of the oncogene v-akt in the lymphomagenic murine retrovirus, Akt8.13 Other names for Akt include PKB and Rac protein kinase because Akt/PKB shares homologies with protein kinase A and protein kinase C.14 Akt is activated by the binding of 3-phosphoinositides, arising from the actions of PI3-kinase, to its pleckstrin homology domain.15 Activation is followed by translocation of Akt to the plasma membrane.
During atherogenesis, VSMCs within the arterial wall media migrate to the intima. In this new position, proliferating VSMCs accumulate lipid and become foam cells. It is postulated that modified LDL is the ligand for the scavenger receptor on the membrane of macrophages and smooth muscle cells. LDL uptake via this pathway leads to massive accumulation of lipid and thus foam cell formation.16 The uptake of oxidized LDL by macrophages is a key event implicated in the initiation and development of atherosclerotic lesions. Several surface receptors on macrophages, CD36 (a class B scavenger receptor),17 CD68,18 and the macrophage scavenger receptor (a class A scavenger receptor [SRA]),19 are believed to play major roles in the binding and internalization of oxidized LDL. The scavenger receptor has been detected in VSMCs of atherosclerotic lesions in arterial intima but not in normal VSMCs found in the media of the artery. This finding suggests that scavenger receptor activity is upregulated in the smooth muscle cells of atherosclerotic lesions.20,21
We have recently shown that Gas6 upregulated SRA expression in a human VSMC line.22 In the present report, we have extended our previous findings by examining the Gas6-dependent intracellular signaling pathway(s) required to induce SRA expression in VSMCs. The present study shows that the presence of an active Akt/PKB is necessary for Gas6 induction of SRA expression.
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Materials
Wortmannin was purchased from Sigma Chemical Co. A blocking peptide for Axl and a Gas6 antibody were purchased from Santa Cruz Biotechnology. All other reagents were of analytical grade.
Preparation of Recombinant Human Gas6
Chinese hamster ovary cells were transfected with a plasmid that expressed human Gas6. Confluent cells were cultured in protein-free culture medium, PM-1000 (Eiken), in the presence of 4 µmol/L vitamin K2. Recombinant human Gas6 was purified from the culture medium as described previously.3 Maximal cell growth and expression of SRA in ISS10 cells were observed by using 10 ng/mL of recombinant Gas6, as previously described.22
Cell Culture
Primary human aortic smooth muscle cells (HASMCs) were purchased from Kurabo and maintained according to the manufacturer’s instructions. The ISS10 cell line is derived from HASMCs by transfection with origin minus simian virus 40 DNA, as described previously.23 ISS10 cells were plated in DMEM (GIBCO-BRL) supplemented with 10% FCS, 100 µg/mL streptomycin, and 100 U/mL penicillin in a humidified atmosphere containing 5% CO2.
Western Blot Analysis
ISS10 cells were washed, scraped in PBS, and lysed as described previously.24 The proteins were separated by size on a 7.5% SDS–polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane for immunoblotting. The membranes were incubated with 0.1% Tween 20 in PBS (PBS-T) containing anti-SRA antibody (diluted 1:1500 from whole antiserum as previously described19), anti-Axl antibody (diluted 1:500, Santa Cruz Biotechnology), anti-Akt antibody (diluted 1:500, Upstate Biotechnology), or anti–phospho-Akt antibody (diluted 1:500, Upstate Biotechnology). The membranes were then washed with PBS-T and incubated for 1 hour at room temperature in PBS-T containing a second antibody linked to horseradish peroxidase. The signal was visualized by using an enhanced chemiluminescence detection kit (ECL, Amersham Corp).
Uptake of Acetylated LDL in ISS10 Cells
1,1'-Dioctadecyl-1-13,3,3',3'-tetramethylindocarbocyanine perchlorate–labeled acetyl-LDL (DiI-AcLDL, Biomedical Technologies Inc) was added to the culture medium at a final concentration of 10 µg/mL. The cells were incubated for 4 hours at 37°C, washed with PBS, fixed, and then analyzed by using a fluorescence microscope.
Immunoblotting of Akt
Cells were lysed for 10 minutes in ice-cold buffer A (50 mmol/L Tris-HCl [pH 7.5], 1 mmol/L EDTA, 1 mmol/L EGTA, 0.5 mmol/L Na3VO4, 0.1% of 2-mercaptoethanol, 1% Triton X-100, 50 mmol/L NaF, 5 mmol/L sodium pyrophosphate, 10 mmol/L sodium glycerophosphate, 0.1 mmol/L phenylmethylsulfonyl fluoride, 1 µmol/L microcystin, and 1 µg/mL each pepstatin, aprotinin, and leupeptin). The lysates were centrifuged, and the supernatants were collected. The supernatants containing protein concentration of 20 µg/mL were used for immunoblotting according to standard procedures. Akt phosphorylated at Ser473 or Thr308 was detected by using a phospho-specific Akt polyclonal antibody, and total Akt was detected by using phosphorylation-independent antibodies (Upstate Biotechnology). The protein bands were visualized by chemiluminescence.
Transfection of VSMCs and Assaying for Luciferase Activity
To determine whether transcription of the SRA gene was regulated by Akt-mediated signaling, we measured the activity of a reporter construct containing the SRA promoter. A DNA fragment containing 4.26 kb of the human SRA gene upstream from the transcription initiation site fused to the luciferase reporter gene yielded pSRA-LUC, as described previously.25 Purified pSRA-LUC was transfected into ISS10 cells, which were grown to 60% confluence, by using Lipofectamine (Life Technologies). Two micrograms of Rous sarcoma virus–ß-galactosidase was added to all transfections to monitor the efficiency of DNA uptake by ISS10 cells.26 All assays were corrected for ß-galactosidase activity, and the total amount of protein per reaction was identical. Transfected cells were maintained in control media containing 10 ng/mL Gas6 with or without cotransfection of a vector expressing the constitutively active form of Akt (Akt-CA) or a plasmid encoding a dominant-negative mutant of Akt (Akt-DN) for 24 hours as described elsewhere.27,28 Transfected cells were harvested, and ß-galactosidase activity was measured in an aliquot of the cytosol.26 Twenty-microliter aliquots were used for assaying luciferase as outlined in the manufacturer’s instructions (ToyoInk). HASMCs were transfected with Axl expression vector (kindly provided by Dr Liu, National Institutes of Health, Bethesda, Md). After Gas6 treatment for 24 hours, the proteins were extracted, and the level of SRA expression was analyzed by using Western blotting.
Statistical Analysis
Statistical comparisons were made by 1-way ANOVA and the Student t test, with a value of P<0.05 considered to be significant.
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Results |
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Expression of Axl in ISS10 Cells and Axl-Mediated Gas6-Induced SRA Expression
We have previously shown the presence of Axl mRNA in ISS10 cells by using reverse transcription–polymerase chain reaction.22 Whether this mRNA is translated into protein is not known. Therefore, a Western blot containing cytosolic proteins from ISS10 cells was probed with specific antibody against Axl. Figure 1A showed that Axl protein was detected in ISS10 but not in human breast cancer, MCF-7, cells.
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Several lines of evidence show that Gas6 is a ligand for Axl.29 To determine whether Axl played a role in Gas6 induction of SRA expression, we treated ISS10 cells with a blocking peptide that prevents ligand binding to Axl. Results (Figure 1B) showed that Gas6 induced SRA expression (lane 2) and that this induction was blocked in the presence of the peptide (lane 3).
To further demonstrate that induction of SRA was dependent on Axl, we treated HASMCs with Gas6. Axl is not detectable in HASMCs (data not shown). Results (Figure 1C) have shown that exposure of these cells to Gas6 (lane 2) did not induce SRA expression. Furthermore, transfection of these cells with an Axl-expressing plasmid did not increase SRA until the transfected cells were treated with Gas6 (lane 4). Together, these findings showed that Gas6 induction of SRA required the receptor Axl.
Inhibitory Effect of Wortmannin on Gas6-Induced SRA Expression and AcLDL Uptake
To examine the signaling component(s) that follows Axl activation, we tested the effect of wortmannin, a potent PI3-kinase inhibitor, and its ability to affect Gas6-induced SRA expression. Gas6 activation of PI3-kinase is an important component of the intracellular signaling found in the survival mechanism of NIH3T3 cells.5 Thus, ISS10 cells were treated for 0, 6, 12, or 24 hours with control medium, Gas6, Gas6 plus wortmannin, or wortmannin alone, followed by Western blot analysis. Results (Figure 2A) showed that Gas6 induced SRA expression (row 2); in contrast, wortmannin efficiently abolished Gas6-induced expression of SRA (row 3). Wortmannin alone had no effect on SRA expression (row 4).
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If Gas6 induced SRA expression, then it should enhance the uptake of LDL into the cells. To test this hypothesis, we correlated SRA expression with the uptake of AcLDL by measuring DiI-AcLDL internalized into ISS10 cells. The cells were treated with Gas6 in the absence or presence of wortmannin for 24 hours. The confluent monolayers of ISS10 were incubated with DiI-AcLDL, and then fluorescence microscopy was used to assess cellular uptake of DiI-AcLDL. Results (Figure 2B) showed that significant amounts of DiI-AcLDL were internalized in Gas6-treated ISS10 cells (Figure 2B, middle) but that inhibition of PI3-kinase activity by use of wortmannin blocked the uptake of DiI-AcLDL (Figure 2B, right) into the ISS10 cells.
Previous reports have shown abundant expression of Gas6 in the vessel wall.1,2,4 Therefore, it is possible that wortmannin action is exerted by its effects on endogenous Gas6 expression. To examine this possibility, we measured the abundance of Gas6 in HASMCs and ISS10 cells by using Western blot analysis. Results (Figure 2C) showed that Gas6 was easily detected in both cells lines (lanes 1 and 2) but that wortmannin did not affect its expression (lane 3). Together, these results suggest that Gas6 induction of SRA required PI3-kinase activity.
Time Course of Akt Phosphorylation by Gas6
The preceding studies show that PI3-kinase is required for Gas6 induction of SRA. Akt is a potential target of PI3-kinase, thus prompting us to wonder whether Gas6 activates Akt kinase. Therefore, we assessed the kinetics of Akt activation by measuring its ability to autophosphorylate residues Thr308 and Ser473 of the protein. This modification is a prerequisite for catalytic activity of Akt. Results (Figure 3A) showed that Akt autophosphorylation was apparent within 30 minutes after exposure of the ISS10 cells to Gas6, and this activity reached a peak at 60 minutes. Furthermore, autophosphorylation by Akt was dependent on PI3-kinase, as shown by the ability of wortmannin to block this effect of Gas6 (Figure 3B). There are 2 significant findings arising from this set of experiments: (1) Gas6 activates the Akt kinase, and (2) Gas6 induction of Akt autophosphorylation is rapid and attains maximal values at 60 minutes.
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Akt Regulates SRA Promoter Activity
The above findings suggest that activation of the PI3-kinase pathway leading to Akt autophosphorylation is required for Gas6 induction of SRA. To determine whether these events regulate SRA at the promoter level, we asked whether Gas6 affected SRA promoter activity, and if this proved to be the case, we would examine the effects of constitutively active or dominant-negative Akt on promoter activity. In these studies, ISS10 cells were transfected with the luciferase reporter construct, pSRA-LUC. Transfected cells were exposed to Gas6 and then assayed for luciferase activity. Results (Figure 4A) showed a 2.5-fold induction of luciferase activity in response to Gas6.
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Next, we assessed the actions of a Akt-CA on SRA promoter activity. ISS10 cells were cotransfected with either an empty vector or a vector that overexpressed Akt-CA plus pSRA-LUC. Like Gas6 induction, cells expressing Akt-CA showed a 3-fold increase in SRA promoter activity compared with control cells. Further studies confirming the involvement of Akt in Gas6 induction of SRA promoter activity came from ISS10 cells cotransfected with a plasmid encoding Akt-DN or empty vector plus pSRA-LUC. Consistent with our hypothesis, there was a 2.7-fold rise in luciferase activity after Gas6 stimulation in cells containing only pSRA-LUC. As expected, expression of Akt-DN inhibited the actions of Gas6 induction on pSRA-LUC activity (Figure 4B). Together, these findings support the idea that Akt is required for Gas6 induction of SRA expression in ISS10 cells.
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Discussion |
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The ongoing epidemic in cardiovascular disease makes it important to shed new light on the pathogenesis underlying this disease. To combat this deadly disease, the National Institutes of Health recently issued new guidelines for treating hypercholesterolemia that were specifically aimed at lowering LDL cholesterol. There is growing evidence that modified LDL is an atherogenic particle. The uptake of such particles into macrophages and smooth muscle cells is likely mediated by a scavenger receptor. Massive accumulation of lipid in these cells leads to the formation of foam cells, a pathological feature of atherosclerosis.30 In normal VSMCs, the expression of scavenger receptors is weak.31,32 However, in the presence of appropriate stimulation, expression of the receptor is increased. For example, in hypercholesterolemic rabbits undergoing balloon angioplasty, there is increased scavenger receptor expression of aortic smooth muscle cells found in the neointima. In contrast, expression of the receptor in smooth muscle cells of the aortic media did not change. These results clearly demonstrate the induction of scavenger receptor expression in smooth muscle cells of the neointima, which is the location of primordial lesions of atherosclerosis.
Despite the description of changes in the abundance of the scavenger receptor, the factors that regulate its expression in VSMCs in vivo are not known. We22 have shown previously that Gas6 increases SRA expression in a human smooth muscle cell line. Because Gas6 is a ligand that binds to Axl, a receptor tyrosine kinase,29 it is possible that Gas6 induction of SRA is mediated by Axl. Consistent with this idea, Melaragno et al31 have reported increased Axl expression in VSMCs of the neointima after balloon injury. Additionally, Axl expression is also selectively regulated by G protein–coupled receptor agonists in vitro.33
In results summarized in the present study, we tested the possibility that Axl mediated Gas6 induction of SRA in ISS10 cells. These cells expressed Axl, and their exposure to a peptide that blocked the binding of ligand to Axl (Figure 1) abrogated the Gas6 induction of SRA. This finding helped identify Axl as a participant in the induction process. Further support for the role of Axl arose from studies of HASMCs, a cell line that does not express Axl. As expected, the exposure of these cells to Gas6 failed to increase SRA until the cells were transfected with a plasmid that enabled expression of Axl (Figure 1C).
The finding that Axl plays a role in Gas6 induction of SRA in ISS10 cells provided an attractive model to detail the underlying signaling pathways. To address this question, the cells were exposed to wortmannin, an inhibitor of PI3-kinase. Wortmannin inhibited Gas6 induction of SRA expression and also the activity of SRA to take up DiI-AcLDL into the cells (Figure 2B). The inhibitory actions of wortmannin pointed to a role for PI3-kinase in mediating the actions of Gas6. Because Gas6 is abundantly expressed in the cells of the vessel wall,1,2,4 it is not surprising that ISS10 cells have significant endogenous levels of the protein. It is possible that endogenous Gas6 regulates SRA expression in an autocrine or a paracrine manner.
Although PI3-kinase has many potential downstream targets, we focused our attention on Akt because of several reports showing the importance of this kinase in VSMCs. For example, previous studies showed that reactive oxygen species mediated the activation of Akt after exposure to angiotensin II, a hypertrophic or antiapoptotic hormone, in VSMCs.34 PI3-kinase activates the serine/threonine kinase Akt,6 resulting in a block or delay of cell death after induction by several apoptotic stimuli, such as growth factor depletion, matrix detachment, and c-myc activation.27,35 Additionally, Goruppi et al5 noted the participation of PI3-kinase in Gas6/Axl signaling. Furthermore, a recent report showed that activation of Akt was significantly induced after the addition of Gas6, a survival factor to serum-starved cells. Akt may also participate in Gas6-induced protection from cell death after serum deprivation.11 Together, these findings point to Akt as a potential target of PI3-kinase.
That Akt is one of the downstream components of intracellular signaling triggered by Gas6 activation of Axl was demonstrated by Gas6 induction of Akt autophosphorylation. To connect the actions of Akt with SRA expression, we assessed the effects of a constitutively active and a dominant-negative form of Akt on SRA promoter activity (Figure 4). In support of this possibility, the sequence of the SRA promoter contains binding sites for transcription factors shown to participate in "cross talk" between signal transcription and gene regulation.25 Constitutively activated Akt mimics Gas6 induction of SRA promoter activity, and the dominant-negative mutant blocks this effect. Further studies of the role of Akt/PKB in Gas6-mediated SRA stimulation will be necessary to define the pathway by which Axl affects nuclear transcription.
In summary, our findings indicate that a signaling pathway mediated by Akt is necessary for Gas6 induction of SRA gene expression. These findings suggest that Gas6 may play an important role in leading to the formation of foam cells in atherosclerotic lesions.
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Acknowledgments |
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The authors thank R. Kubo, K. Takashima, and K. Yamaji for their excellent technical assistance. The Akt expression vectors used in our studies were kindly provided by Dr M.E. Greenberg, Harvard Medical School, Boston, Mass. The vector containing SRA promoter was kindly provided by Dr C.K. Glass, University of California, San Diego. The vector containing Axl was kindly provided by Dr E.T. Liu, National Institutes of Health, Bethesda, Md.
Received June 6, 2001; accepted July 16, 2001.
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