Fluid Shear Stress Transcriptionally Induces Lectin-like Oxidized LDL Receptor-1 in Vascular Endothelial Cells
Abstract—Fluid shear stress has been shown to modulate various endothelial functions, including gene expression. In this study, we examined the effect of fluid shear stress on the expression of lectin-like oxidized LDL receptor-1 (LOX-1), a novel receptor for atherogenic oxidized LDL in cultured bovine aortic endothelial cells (BAECs). Exposure of BAECs to the physiological range of shear stress (1 to 15 dyne/cm2) upregulated LOX-1 protein and mRNA in a time-dependent fashion. LOX-1 mRNA levels peaked at 4 hours, and LOX-1 protein levels peaked at 8 hours. Inhibition of de novo RNA synthesis by actinomycin D totally abolished shear stress–induced LOX-1 mRNA expression. Furthermore, nuclear runoff assay showed that shear stress directly stimulates transcription of the LOX-1 gene. Chelation of intracellular Ca2+ with quin 2-AM completely reduced shear stress–induced LOX-1 mRNA expression; furthermore, the treatment of BAECs with ionomycin upregulated LOX-1 mRNA levels in a dose-dependent manner. Taken together, physiological levels of fluid shear stress can regulate LOX-1 expression by a mechanism dependent on intracellular Ca2+ mobilization. Inducible expression of LOX-1 by fluid mechanics may play a role in localized expression of LOX-1 and atherosclerotic lesion formation in vivo.
Endothelial cells lining the inner surface of vessel walls are in direct contact with blood flow, which generates a hemodynamic shear stress acting on the apical surface of vascular endothelium.1 2 Fluid shear stress has been shown to modulate a variety of endothelial functions, including the expression of a variety of genes, such as endothelin-1,3 4 5 cyclooxygenase-2,6 NO synthase,6 7 tissue factor,8 transforming growth factor-β,9 platelet-derived growth factor (PDGF),10 11 basic fibroblast growth factor,11 heparin-binding epidermal growth factor–like growth factor,12 monocyte chemotactic protein-1,13 intercellular adhesion molecule-1,14 15 and vascular cell adhesion molecule-1,16 17 as well as its effects on cytoskeletal organization and cell morphology.4 18 19 These biological effects elicited by fluid shear stress appear to be mediated by intracellular signal transduction cascades, including intracellular Ca2+ mobilization,20 21 22 inositol trisphosphate,23 K+ channel,24 G protein,25 mitogen-activated protein kinases,26 27 N-terminal Jun kinase,28 29 and platelet endothelial cell adhesion molecule-1 tyrosine phosphorylation,30 and by the subsequent activation of transcription factors, such as activator protein-1 (AP-1),31 32 nuclear factor-κB (NF-κB),31 33 and Egr-1 (an early growth response gene product),34 35 and may potentially affect vascular tone, thrombus formation, and atherogenesis.1 36
On the other hand, oxidatively modified LDL (Ox-LDL) has been suggested to play key roles in atherogenesis. In particular, modulation of endothelial functions elicited by Ox-LDL and its lipid constituents has been implicated in the initiation of this complex disease process.36 37 We have recently identified a novel endothelial receptor for Ox-LDL, designated lectin-like Ox-LDL receptor-1 (LOX-1), whose structure belongs to the C-type lectin family.38 LOX-1 is expressed in vascular endothelium in vivo in arterial and aortic endothelial cells, including atherosclerotic lesions.38 Functional analysis revealed that LOX-1 expressed on the surface of endothelial cells supports binding, internalization, and proteolytic degradation of Ox-LDL. With regard to transcriptional regulation of LOX-1, an inflammatory stimulus, such as tumor necrosis factor-α, appears to stimulate transcription of the LOX-1 gene.38A LOX-1 expression in vivo also has been shown to be upregulated in the aortas and veins of hypertensive rats.39 Therefore, we have tested the hypothesis that expression of LOX-1 can be modulated by fluid mechanical stimuli in vascular endothelial cells.
In the present study, we provide evidence that fluid shear stress is a potent stimulus to transcriptionally induce the expression of LOX-1 in cultured bovine aortic endothelial cells (BAECs).
Materials and Methods
DMEM was obtained from Nissui. FCS was obtained from Boehringer Mannheim. [32P]UTP, the Gene Image DNA labeling and detection system, ECL Western blotting detection reagents, and Hybond-N+ membranes were obtained from Amersham. Quin 2-AM, Isogen, and Isogen-LS were from Wako Pure Chemical. PVDF transfer membranes were obtained from Millipore.
BAECs were isolated by scraping the inner surface of bovine aortas with a glass coverslip and cultured in DMEM containing 10% heat-inactivated FCS in an atmosphere of 95% air/5% CO2 at 37°C as previously described.38 For shear experiments, BAECs were seeded on collagen-coated glass slides (70×100 mm) in DMEM with 10% FCS. Before experiments, BAECs on the glass slides were incubated with serum-free DMEM for 24 hours.
Shear Stress Apparatus
A parallel-plate type of flow chamber was used to produce laminar flow as previously described.15 16 17 20 In brief, BAECs were cultured on glass plates and loaded into a rectangular parallel-plate flow chamber. Shear stress was increased suddenly from 0 (static) to 15 dyne/cm2 (or the indicated intensities of shear stress) and was maintained at the same level for the time periods indicated for each experiment. Culture media were recirculated into the chamber and kept at a constant temperature of 37°C with a gas mixture of 95% air/5% CO2.
Northern Blot Analysis
BAECs were washed in PBS, and total RNA was isolated using Isogen. Equal amounts (10 μg) of total RNA were subjected to electrophoresis through 1% agarose/formamide gels and blotted onto Hybond-N+ membranes. Hybridization and detection were performed by fluorescein-labeled cDNA using the Gene Image random prime labeling and detection system (Amersham) according to the manufacturer’s instructions. In brief, the blots were prehybridized for 6 hours at 65°C in a solution containing 5% (wt/vol) dextran sulfate, 5× SSC, 0.1% (wt/vol) SDS, and blocking reagent and subsequently hybridized with a fluorescein-labeled cDNA probe at 65°C overnight. The membranes were washed with 1× SSC and 0.1% (wt/vol) SDS for 15 minutes and then washed with 0.1× SSC and 0.1% (wt/vol) SDS for 15 minutes at 65°C. After nonspecific binding of antibodies was blocked, the membranes were incubated with alkaline phosphatase–labeled anti-fluorescein antibody for 1 hour and then detected by chemiluminescence (CDP-star reagent, Amersham). Densitometric scanning was performed to quantify the amounts of mRNA using NIH Image. Amounts of LOX-1 mRNA were normalized with the amounts of GAPDH mRNA.
Western Blot Analysis
BAECs were washed in PBS and lysed in buffer containing 62.5 mmol/L Tris-HCl, 2% (wt/vol) SDS, and 10% (vol/vol) glycerol. Equal protein concentrations of the lysates were subjected to SDS-polyacrylamide (10%) gel electrophoresis, followed by electroblotting onto an Immobilon PVDF transfer membrane. Determination of protein concentrations was carried out by the method of Lowry. Blotted membranes were probed with a mouse monoclonal antibody directed to bovine LOX-1,38 incubated with horseradish peroxidase–labeled-anti-mouse immunoglobulin for 1 hour, washed with PBS containing 0.1% (vol/vol) Tween 20, and then detected by ECL Western blotting detection reagents. Densitometric scanning was performed to quantify the amounts of LOX-1 protein using NIH Image.
Nuclear Runoff Assay
Nuclear runoff assay was performed as previously described,40 with minor modification. Briefly, the cells were washed with ice-cold PBS and lysed with 0.5% Nonidet P-40 solution (10 mmol/L Tris-HCl, 10 mmol/L NaCl, 3 mmol/L MgCl2, and 0.5% NP-40 [vol/vol], pH 7.4). The nuclei were isolated by centrifugation and resuspended in a 40% glycerol buffer (50 mmol/L Tris-HCl, 40% [vol/vol] glycerol, 5 mmol/L MgCl2, and 0.1 mmol/L EDTA, pH 8.3). Nascent transcription in vitro was performed with [32P]UTP and other unlabeled nucleotides at 30°C for 30 minutes. Transcribed RNA was isolated by Isogen-LS, followed by denaturation with sodium hydroxide and ethanol precipitation. Linearized target cDNAs (5 μg in plasmid form) were alkali-denatured and immobilized onto Hybond-N+ membranes using a slot blot apparatus (Schleicher & Shuell Inc). The membranes were hybridized with transcribed RNAs containing an equal amount of radioactivity in a solution containing 50% (vol/vol) formamide, 5× SSPE (1× SSPE consists of 0.15 mol/L NaCl, 10 mmol/L NaH2PO3, and 1 mmol/L EDTA, pH 7.4), 0.1% (wt/vol) SDS, 10% (vol/vol) Denhardt’s solution, and denatured salmon sperm DNA at 42°C for 36 hours. Filters were washed in 1× SSC with 0.1% (wt/vol) SDS for 15 minutes at room temperature, washed with 0.2× SSC supplemented with 0.1% (wt/vol) SDS for 10 minutes at 42°C, and then autoradiographed with Fujix Bioimage Analyzer BAS2000 (Fuji Photo Film).
Shear Stress Induces LOX-1 Expression in Cultured Endothelial Cells
To test whether shear stress induces LOX-1 expression at the protein level, confluent BAECs in a flow chamber were subjected to a steady level of laminar shear stress of 15 dyne/cm2 for various periods of time. Time course of shear stress–induced LOX-1 protein expression showed that elevated levels of LOX-1 protein were detectable within 4 hours and reached to the maximal level at 8 hours in response to 15 dyne/cm2 of shear stress (Figure 1A⇓). Densitometric analysis showed that exposure to shear stress resulted in 2.4-fold, 3.7-fold, and 2.9-fold increases in the amounts of LOX-1 protein at 4, 8, and 12 hours, respectively, compared with the levels in BAECs kept in a static condition. To determine whether shear stress–induced expression of LOX-1 depends on increased expression of LOX-1 mRNA, Northern blot analyses were performed. As shown in Figure 1B⇓, confluent BAECs constitutively expressed modest levels of LOX-1 mRNA. Exposure of BAECs to 15 dyne/cm2 of shear stress led to a rapid and transient increase in the amount of LOX-1 mRNA. The LOX-1 mRNA levels peaked at 4 hours (5.6-fold increase) and were reduced after 8 hours.
To examine shear-force dependence in LOX-1 expression, confluent BAECs were exposed to a broad range of shear stresses (1 to 15 dyne/cm2) for 8 hours. These levels of shear stress appear to be within the physiological range of shear stress in vivo and have been shown to regulate a variety of endothelial genes in vitro. As shown in Figure 2A⇓ and 2B⇓, shear stress force-dependently induced the expression of LOX-1 protein; increased amounts of LOX-1 protein were detectable in BAECs exposed to shear stress as low as 1 to 2 dyne/cm2. Elevated levels of LOX-1 mRNA were observed in BAECs stimulated with shear stress as low as 2 dyne/cm2 (Figure 2C⇓).
To explore whether continuous exposure to shear stress is needed in induced expression of LOX-1, BAECs that had been exposed to shear stress for the indicated time periods were switched to a static condition for the residual time periods. BAECs that were exposed to shear stress for an initial 30 minutes and then cultured in a static condition showed elevated levels of LOX-1 mRNA expression after 4 hours that were almost equal to those in BAECs exposed continuously to shear stress for 4 hours (Figure 3⇓). These results indicate that the initial changes in fluid shear stress may be crucial in the induced expression of LOX-1.
Shear Stress–Induced LOX-1 Expression Does Not Require De Novo Protein Synthesis
To test whether de novo protein synthesis is necessary for the shear stress–induced LOX-1 mRNA expression, cycloheximide was added to BAECs 1 hour before the application of shear stress. The inhibition of the cellular protein synthesis by cycloheximide was accompanied by increased basal LOX-1 levels, thought to be due to “superinduction,” and increased LOX-1 mRNA levels in the static cells were the same as the levels in cells subjected to shear stress of 15 dyne/cm2 (Figure 4⇓). When cycloheximide-treated cells were subjected to shear stress, levels for LOX-1 mRNA in these cells were again higher than those in cycloheximide-treated cells. Taken together, de novo protein synthesis is not necessary for shear stress–induced LOX-1 gene expression, but protein synthesis inhibition superinduced LOX-1 mRNA.
Shear Stress Stimulates Transcription of LOX-1 Gene
To examine whether shear stress–induced expression of LOX-1 depends on enhanced transcription of the LOX-1 gene, actinomycin D, an inhibitor of de novo mRNA synthesis, was added to BAECs 30 minutes before the application of shear stress. Pretreatment with actinomycin D completely abolished LOX-1 mRNA induction elicited by shear stress (Figure 5⇓). These results suggest that shear stress can stimulate transcription of the LOX-1 gene.
To obtain direct evidence that LOX-1 mRNA expression was regulated at the transcriptional level, nuclear runoff assays were performed with the use of nuclei isolated from BAECs stimulated with or without fluid shear stress. Enhanced transcription of LOX-1 was observed in nuclear extracts from shear stress–treated cells compared with those kept in a static condition (Figure 6⇓). Transcription of the GAPDH gene, in contrast, was not significantly altered by fluid shear stress. Thus, these results demonstrate that fluid shear stress stimulates transcription of the LOX-1 gene.
Intracellular Ca2+ Regulates Shear Stress–Induced Expression of LOX-1
Fluid shear stress can rapidly elevate intracellular Ca2+ levels; this Ca2+ elevation has been implicated in shear stress–induced gene expression, such as induction of NO synthase7 and heparin-binding epidermal growth factor–like growth factor.12 To determine whether shear stress–mediated increases in intracellular Ca2+ were crucial for shear stress–induced LOX-1 expression, effects of quin 2-AM, a chelator of intracellular Ca2+, were examined. Pretreatment of BAECs with quin 2-AM (10 to 20 μmol/L) for 2 hours completely inhibited shear stress–induced expression of LOX-1 mRNA (Figure 7⇓).
We also tested whether Ca2+ mobilization alone is sufficient for LOX-1 induction in BAECs. Ionomycin, a Ca2+ ionophore, markedly induced LOX-1 mRNA in a dose-dependent manner (Figure 8⇓). The LOX-1 mRNA level in BAECs treated with 10 μmol/L of ionomycin for 4 hours was 5.7 times higher than that in untreated BAECs. These results indicate that shear stress–induced LOX-1 expression appears to depend on intracellular Ca2+ mobilization.
Vascular endothelial cells are located as an interface between the bloodstream and vessel wall cells. Endothelial functions can be dynamically modulated in response to a variety of pathophysiological stimuli, including mechanical forces. In the present study, we have explored the possibility that LOX-1, a novel endothelial receptor for Ox-LDL, is also a shear stress–inducible molecule.
Inducible expression of LOX-1 by shear stress is demonstrated at both the protein and the mRNA levels in BAECs (Figures 1⇑ and 2⇑). Furthermore, Northern blot analyses using actinomycin D, as well as nuclear runoff assays, have revealed that shear stress can stimulate transcription of the LOX-1 gene (Figures 5⇑ and 6⇑). Induction of LOX-1 by fluid shear stress was relatively transient; shear stress–induced LOX-1 mRNA levels peaked at 4 hours and had decreased by 50% after 12 hours compared with the peak level. LOX-1 protein levels peaked at 8 hours and were reduced by 30% after 12 hours. These time courses of LOX-1 expression induced by shear stress are different from those observed with tumor necrosis factor-α, which induced sustained levels of LOX-1 expression (data not shown), suggesting that distinct signal transduction pathways and transcriptional regulatory mechanisms are involved.
Our studies involving the transient application of shear stress in BAECs (Figure 3⇑) show that initial stimulation with shear stress is sufficient for induced expression of LOX-1 and that sustained application of shear stress is not necessarily required. This appears to suggest that early signal transduction events elicited by shear stress may play a crucial role in LOX-1 induction. Therefore, we have focused on early events in signal transduction pathways activated by shear stress. Previous publications have shown that shear stress can stimulate phosphatidylinositol turnover, generating inositol 1,4,5-trisphosphate and 1,2-diacylglycerol23 in cultured vascular endothelial cells. Inositol 1,4,5-trisphosphate can promote the rapid release of Ca2+ from endoplasmic reticulum and, thereby, raise the cytosolic free Ca2+ levels. Thus, the rapid increases in intracellular Ca2+ levels elicited by shear stress have been proposed as one of the earliest events in shear stress–induced signal transduction pathways in endothelial cells.20 21 22 As we have shown in Figure 7⇑, induction of LOX-1 mRNA by shear stress was completely inhibited by chelation of intracellular Ca2+ with quin 2-AM. In addition, ionomycin, which raises intracellular Ca2+ levels, by itself increased LOX-1 mRNA levels (Figure 8⇑). Taken together, elevated levels of intracellular Ca2+ were necessary and sufficient for LOX-1 gene expression elicited by shear stress.
On the other hand, 1,2-diacylglycerol, an activator of protein kinase C (PKC), also appears to be responsible for a variety of cellular responses. In fact, shear stress–induced expression of certain genes, such as PDGF,10 heparin-binding epidermal growth factor–like growth factor,12 and c-fos,41 were shown to be mediated by PKC activation. Although both Ca2+ mobilization and PKC activation have been shown to be induced by fluid shear stress, our preliminary studies have shown that GF109203X, a specific inhibitor of PKC, failed to inhibit shear stress–induced LOX-1 expression (data not shown), suggesting that PKC may not play significant roles in shear stress–induced LOX-1 expression.
Fluid shear stress appears to stimulate transcription of the LOX-1 gene. Since cycloheximide did not inhibit shear stress–induced expression of LOX-1 mRNA (Figure 4⇑), de novo synthesis of proteins, such as transcription factors, is not required in this process. NF-κB can be rapidly activated, without de novo protein synthesis, by phosphorylation of inhibitor κB and the subsequent dissociation from the p50/p65 complex, which are followed by nuclear translocation of p50/p65. The NF-κB p50/p65 heterodimer has been shown to bind to the shear stress responsive element (SSRE) and thereby activates the SSRE-dependent gene expression, such as PDGF-B chain, in response to shear stress.33 42 In addition, a previous report has also indicated that AP-1, as well as NF-κB, can be activated by shear stress.31 33 Activation of AP-1 has been shown to be responsible for shear stress–induced transcription of the monocyte chemotactic protein-1 gene.32 Involvement of AP-1 and TPA responsive element are also implicated in transcriptional downregulation of the vascular cell adhesion molecule-1 gene.17 43 In transcriptional regulation of PDGF-A chain by shear stress, interactions between transcription factors Sp-1 and Egr-1 have also been demonstrated.34 35 In the 5′ flanking region of the LOX-1 gene, both consensus NF-κB–like sequence and SSRE have been identified (data not shown). Further studies are necessary to elucidate the transcriptional regulatory mechanisms involved in shear stress–induced LOX-1 gene expression.
In summary, the present study provides evidence, for the first time, that physiological levels of laminar fluid flow shear stress transcriptionally induce LOX-1 expression in BAECs by a mechanism dependent on intracellular Ca2+ mobilization. Endothelial expression of LOX-1, a novel receptor for Ox-LDL, may also be dynamically modulated, in vivo, in response to dynamic changes in blood flow. Although pathophysiological consequences of Ox-LDL uptake by vascular endothelium through LOX-1 remain to be fully clarified, modulated expression of this Ox-LDL receptor by fluid mechanical stimuli may play an important role in the localized formation of atherosclerotic lesions in vivo.
This study was supported by Grants-in-Aid for Scientific Research (Nos. 08407026, 08670788, and 09044293), for Scientific Research on Priority Areas (09281103 and 09281104), and for Creative Basic Research (09NP0601) from the Japanese Ministry of Education, Science, Sports, and Culture and by Research Grants for Cardiovascular Diseases (A8-1) from the Ministry of Health and Welfare of Japan.
- Received March 5, 1998.
- Accepted June 16, 1998.
- © 1998 American Heart Association, Inc.
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