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(Circulation Research. 2004;95:684.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Flow-Dependent Regulation of Endothelial Toll-Like Receptor 2 Expression Through Inhibition of SP1 Activity

Stefan Dunzendorfer, Hyun-Ku Lee, Peter S. Tobias

From the Scripps Research Institute, Department of Immunology, La Jolla, Calif.

Correspondence to Peter S. Tobias, The Scripps Research Institute, Department of Immunology IMM-12, 10550 North Torrey Pines Rd, La Jolla, CA 92037. E-mail tobias{at}scripps.edu

	Abstract

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Innate immune system activation is associated with atherosclerotic lesion development. The specific sites of lesion development are believed to be defined by the shear stress of blood flow. Consequently, we investigated the responsiveness of human coronary artery endothelial cells (HCAECs) to Toll-like receptor (TLR) 2 and 4 agonists in an in vitro model of chronic laminar flow. HCAECs under chronic laminar flow were found to be normally responsive to lipopolysaccharide (and tumor necrosis factor) in terms of E-selectin expression but were found to be hyporesponsive to stimulation with the specific TLR2 ligands macrophage activating lipopeptide-2, PAM₂-Cys, and Lip19; this was observed to be attributable to downregulation of TLR2 transcription and protein expression. We found that laminar flow induced SP1 serine phosphorylation by protein kinase CK2 and thereby blocked SP1 binding to the TLR2 promoter, which is required for TLR2 expression. This regulatory mechanism also blocked lipopolysaccharide- and tumor necrosis factor–induced TLR2 upregulation in HCAECs and could be important for suppression of other flow-sensitive endothelial proteins. These results extend the role of flow in controlling endothelial responsiveness. Given the current evidence that TLRs are proatherogenic, flow suppression of TLR2 expression may be atheroprotective.

Key Words: endothelial cell • shear stress • innate immunity • atherosclerosis • TLR2 • TLR4

	Introduction

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Atherosclerosis, the leading cause of death in Westernized societies, is a progressive degenerative disease of the large and medium-sized arteries.¹ Beside hypercholesterolemia, infection and activation of the innate immune system have been implicated in the pathogenesis of this chronic inflammatory disease.^2–5 The recruitment of leukocytes is essential for lesion formation,⁶ and in this context, endothelial dysfunction and adhesion molecule expression seems to play a crucial role.¹

Despite the systemic nature of its associated risk factors, atherosclerosis is a geometrically focal disease that has a propensity to distinct susceptible areas.⁷ The specific arterial sites, such as branches, bifurcations, and curvatures, cause characteristic alterations in the flow of blood, including decreased shear stress and increased turbulence.^8,9 The nature of flow appears to be important in determining whether lesions occur at these vascular sites; thus blood flow alterations determine which arterial sites are prone to have lesions.^2,10 As initially proposed by Caro et al,¹¹ research in the last 3 decades has validated the low-shear hypothesis of atherosclerosis. Physiological levels of shear stress (1.0 to 7.0 N/m2 in the arterial system) shield against atherosclerosis via effects on the endothelium.⁷ Decreasing shear stress at branches, bifurcations, and curvatures results in endothelial activation, adhesion molecule expression, and greater monocyte transmigration.¹²

The family of Toll-like receptors (TLRs) has recently been defined as a key component of the pathogen-associated molecular pattern recognition machinery.¹³ Currently, 10 TLRs have been reported in mammalian species.¹⁴ Some of these receptors have been suggested to be involved in atherosclerotic plaque activation¹⁵ and two very recent publications directly linked atherogenic serum cholesterol levels to activation of a TLR signaling pathway.^5,16

We investigated the effects of microbial products in human coronary artery endothelial cells under chronic physiological laminar shear stress and found that laminar flow at 0.5 N/m² and above inhibits endothelial TLR2 expression via protein kinase CK2- and SP1-dependent mechanisms. This mechanism also inhibited TLR2 upregulation in human endothelial cells by lipopolysaccharide (LPS) and tumor necrosis factor (TNF). Given a role for TLR signaling in atherosclerotic lesion development, flow-regulated expression of TLR2 may be atheroprotective.

	Materials and Methods

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Materials
Escherichia coli LPS (O55:B5) was purchased from List Biological Laboratories (Campbell, Calif) and recombinant human TNF was from Chemicon. Macrophage-activating lipopeptide 2 (MALP-2) was from Alexis Biochemicals and PAM₂-Cys-SKKK (PAM₂) was from EMC Microcollections. Lip19 was produced in our laboratory.¹⁷ The selective protein kinase CK2 (formerly casein kinase II) inhibitor 4,5,6,7-Tetrabromobenzotriazole (TBB) was purchased from EMD Biosciences. Human interferon (IFN) was from Sigma.

Cell Culture
Human coronary artery endothelial cells (HCAECs) were purchased from Clonetics and grown to confluence in full growth medium (EGM-2-MV) from the same company. Cells were from young donors and were used for experiments from passage 4 to 8. There was no difference in TLR2 surface expression or responsiveness to various stimuli between cells of different passages. For flow experiments, cells were cultured on fibronectin (100 µmol/L) (Sigma) coated cover glasses (Corning) that were transferred to the flow chamber apparatus right before cells reached confluence.

Flow Chamber Model
HCAECs were exposed to laminar or disturbed flow using a parallel plate flow chamber apparatus connected to a recirculating flow circuit similar to that described in detail elsewhere.¹⁸ Shear stress was calculated as follows: [N/m²]=0.6µ [mPa sec]xU [mL/sec]/w [cm]xh² [cm]. 1.0 N/m² (Pascal) equals 10 dyne/cm². The medium used in experiments (EM) (EGM w/o hydrocortisone; Clonetics) was maintained at 37°C, and gassed continuously with a humidified mixture of 5% CO₂ in air. The stimulatory capacity of agonist containing media derived from the flow chamber circuit after a stimulation experiment was found to be unchanged over the course of an experiment. Maintaining medium sterility during an experiment was never problematic and was assessed by culture for 2 days after the flow experiment. Control experiments revealed no differences in cell stimulation under static conditions between cells that were stimulated in the flow chamber apparatus or in 6-well plates.

Cell Stimulation Experiments
With the exception of the time-course experiments (0 to 12 hours), all cell stimulation experiments started after a 12-hour period of adaptation to laminar flow. Within this time period, the cells in the laminar region of flow acquired an elongated morphology. Medium was also changed to EM in static control cells 12 hours before the experiments. HCAEC activation was investigated after 6 hours, and TLR2 expression after 12 hours, of cell stimulation after adaptation to flow. Cells were then either stained for FACS analyses using PE-labeled anti-human E-selectin (PharMingen) or anti-human TLR2 monoclonal antibody 2932 (gift from P. Godowski, Genentech, South San Francisco, Calif), or cells were subjected to mRNA, nuclear extract, or whole cell lysate preparation. Cell activation was determined by measuring E-selectin surface expression. This adhesion molecule is barely expressed on resting HCAECs but rapidly and highly induced on stimulation. Moreover, E-selectin expression was not changed by laminar flow itself, as previously found by others.^19,20

Fluorescence Microscopy
HCAECs from the laminar or disturbed flow region in the chamber were stained with anti-TLR2 (mAb 2932) or with anti-CD144 (VE-cadherin), and fluorescence microscopy was performed as previously described.²¹

RT-PCR
RNA was purified from HCAECs using the Absolutely RNA^TM RT-PCR Miniprep Kit from Stratagene. mRNA expression of TLR2, TLR4, and SP1 were determined by RT-PCR. Briefly, the first-strand cDNA was reverse-transcribed from 1 µg total RNA with random primers using the SuperScript first-strand synthesis system from Invitrogen. The cDNA product was amplified by 30 cycles of 30 seconds at 94°C, 45 seconds at 55°C, and 2 minutes extension at 72°C using specific primer pairs (SP1 forward 5'-TCACAAGCCAGTTCCAGCTCC-3' and reverse 5'-GGGTGCACCTGGATTCCTGAA- 3' and TLR2, TLR4, and GAPDH as previously described²¹). The RT-PCR products were separated in 1.2% (wt/vol) agarose gels and visualized with ethidium bromide.

Electrophoretic Mobility Shift Assay
Nuclear proteins were isolated from static or flow stressed HCAECs as recently described,²² with the exception that none of the buffers used contained EDTA or EGTA. In our experiments, both chelators inhibited zinc-dependent SP1 DNA binding,²³ which is what is measured in an electrophoretic mobility shift assay (EMSA) for SP1. The SP1 consensus sequence oligonucleotide probe (Promega) and a 22-mer oligonucleotide with the sequence 5'-CCGTGGAAGGG-GCGGTTCCCG C-3' that spans the putative proximal SP1/Ets binding site in the TLR2 promoter²⁴ were labeled using T4 polynucleotide kinase (New England BioLabs) and [-³²P]ATP (Amersham Biosciences). NF-B nuclear translocation was detected with the oligonucleotide probe from Promega. Isolated nuclear proteins (2 µg) were incubated with labeled probes in binding buffer (10 mmol/L Tris-HCl, pH=8.0, 150 mmol/L KCl, 0.1% Triton-X 100, 12.5% glycerol, and 0.2 mmol/L DTT) at room temperature for 30 minutes. In supershift assays, 1 µg of anti-SP1 or anti-SP3 (both from Upstate) were added 10 minutes before the addition of the labeled probe. The nucleotide-protein complexes were then electrophoresed in a 5% polyacrylamide gel, dried, and autoradiographed.

Western Blot Analyses
Whole cell lysates (RIPA buffer) or nuclear extracts²² from HCAECs under static or flow conditions were electrophoresed in NuPAGE 4% to 12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes (BioRad). After blocking in Tris-buffered saline with 5% nonfat dry milk and 0.1% Tween 20, the membranes were incubated with either anti-human SP1 or anti-human SP3 for 2 hour at RT, followed by washing and incubation with secondary horseradish-peroxidase-linked antibodies (Sigma). In some experiments, antibodies were stripped from the membranes, which were then reprobed with an anti-phosphoserine (PS) antibody (Sigma). Proteins were detected using SuperSignal Chemiluminescent Substrate from Pierce.

	Results

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HCAECs Under Laminar Flow Are Hyporesponsive to TLR2 Ligands
Endothelial cells were adapted to flow conditions for 12 hours before they were stimulated with various concentrations of LPS, TNF, or selective TLR2 ligands for a further 6 hours. Thereafter, HCAEC activation status was measured by FACS analyses of E-selectin surface expression and compared with static control cells. Flow-adapted cells responded slightly, but significantly, better to LPS stimulation at 1 ng/mL and 10 ng/mL (Figure 1A), whereas no differences were found in the responsiveness to TNF stimulation at 0.1 pg/mL to 10 ng/mL (Figure 1B). MALP-2 (10 to 100 ng/mL), PAM₂, or Lip19 (both at 100 ng/mL) activated cells under static conditions, whereas flow-adapted cells were highly resistant to these stimuli (Figure 1C).

View larger version (21K): [in this window] [in a new window]   Figure 1. HCAECs were adapted to chronic laminar flow (1.5 N/m2) or kept under static conditions for 12 hours. Thereafter, cells were stimulated (static or under flow) for a further 6 hours. Cell activation was determined by measuring cell surface E-selectin expression in FACS analyses. Data are given as mean±SEM of the phycoerythrin-mean channel fluorescence (PE-MCF). n=6. *P<0.05; **P<0.01 vs flow conditions. #P<0.05 vs medium control (Mann Whitney U test).

Laminar, but not Disturbed Flow, Affects HCAEC TLR2 Expression
HCAECs were exposed to flow for 12 hours in a chamber with a barrier to create regions of laminar (LF) or disturbed flow (DF). Cells in the LF region aligned in the direction of flow, which was not observed in the DF region (Figure 2A). Fluorescence microscopy after staining for TLR2 clearly showed less TLR2 protein expression in aligned HCAEC of the LF region, compared with unaligned cells of the DF region (Figure 2B). Cells under these conditions were exposed to MALP-2, LPS, or medium alone for 6 hours after 12-hour adaptation to flow and then harvested from the DF and LF regions for FACS analysis of E-selectin expression. Cells from the DF region were MALP-2 responsive, whereas cells from the LF region were not (Figure 2C). Thus disturbed flow and static conditions are mimetic of each other insofar as MALP-2 reactivity and TLR2 expression is concerned.

View larger version (38K): [in this window] [in a new window]   Figure 2. A flow barrier in the chamber created regions of disturbed flow (DF) or laminar flow (LF). VE-cadherin (CD144) staining after 12-hour flow shows aligned and nonaligned HCAECs (A). Cells were stained for TLR2 (red) and analyzed by fluorescence microscopy (blue; DAPI nuclear stain) (B) or stimulated for further 6 hours with MALP-2 (100 ng/mL) or LPS (10 ng/mL). Thereafter, HCAECs from the DF or LF regions were harvested and activation was determined by measuring E-selectin surface expression. Static conditions served as control (C). n=3. *P<0.05 vs DF or static conditions.

Laminar Flow Reduces Basal and Inducible HCAEC TLR2 Protein Surface Expression
Under no-flow culture conditions, LPS (100 ng/mL) and TNF (10 ng/mL) can enhance TLR2 expression in a time-dependent manner. This is seen at the protein and mRNA levels (Figure 3A). When cells were adapted to flow for 12 hours and further kept under flow for 12 hours either with medium, LPS (100 ng/mL), or TNF (10 ng/mL), shear stress not only reduced baseline TLR2 surface expression but also prevented LPS- or TNF-induced receptor upregulation. IFN (200 U/mL) also induced TLR2 and added to the LPS and TNF effects under static conditions, but it was ineffective on cells adapted to flow (Figure 3B). Flow-mediated downregulation of basal TLR2 protein expression and inhibition of LPS- or TNF-triggered TLR2 upregulation was found to be a function of shear stress (0 to 2.5 N/m²) (Figure 3C).

View larger version (25K): [in this window] [in a new window]   Figure 3. A, HCAECs under static conditions were incubated with LPS (100 ng/mL) or TNF (10 ng/mL) for various time periods. Thereafter, cells were analyzed by FACS for TLR2 surface expression or TLR2 mRNA was quantitated by RT-PCR. Time-dependent TLR2 mRNA upregulation is shown for stimulation with LPS. Bottom row represents GAPDH mRNA that served as control. B, Cells were under static conditions or adapted to flow (1.5 N/m2) for 12 hours. They were then incubated with medium, LPS (100 ng/mL), or TNF (10 ng/mL) with or without IFN (200 U/mL) for a further 12 hours under static or flow conditions. TLR2 surface expression was quantitated by FACS. C, Cells were kept under static conditions or exposed to flow (0.5 to 2.5 N/m2) for 12 hours and then LPS (100 ng/mL) or TNF (10 ng/mL) was added for a further 12 hours. TLR2 surface expression was quantitated by FACS. FACS data are given as mean±SEM of the phycoerythrin-mean channel fluorescence (PE-MCF). n=3. *P<0.05 vs static conditions (Mann Whitney U test).

TLR2 Surface Expression Determines Susceptibility to MALP-2 Activation
TLR2 was upregulated in HCAECs under static conditions with LPS (100 ng/mL), TNF (10 ng/mL), or IFN (200 U/mL) for 12 hours. Thereafter, responsiveness to a suboptimal concentration of MALP-2 (10 ng/mL) was tested for 6 hours. Upregulation of TLR2 (Figure 3) was clearly correlated to MALP-2 activation of the cells (Figure 4). This effect was seen best with IFN, which induces TLR2 in static cells (Figure 3B) but does not itself stimulate the cells (Figure 4).

View larger version (39K): [in this window] [in a new window]   Figure 4. HCAEC were incubated in medium, LPS (100 ng/mL), TNF (10 ng/mL), or IFN (200U/mL) for 12 hour under static conditions. Thereafter, cells were stimulated with MALP-2 (10 ng/mL) for 6 hour and cell activation was determined by measuring E-selectin surface expression. Data are given as mean±SEM of the phycoerythrin-mean channel fluorescence (PE-MCF). n=3. *P<0.05 versus respective black bar (Mann Whitney U test).

Laminar Flow Reduces TLR2 mRNA Expression
HCAECs were adapted to flow for 12 hours and shear stress was maintained for a further 12 hours with medium, LPS (100 ng/mL), or TNF (10 ng/mL). Thereafter, mRNA expression was assessed by semiquantitative RT-PCR (Figure 5). The pattern observed for TLR2 mRNA levels under various conditions clearly correlated with TLR2 surface protein expression (Figure 3B). No change was noticed in the expression levels of TLR4 mRNA (Figure 5).

View larger version (67K): [in this window] [in a new window]   Figure 5. HCAECs were under static conditions or adapted to flow (1.5 N/m2) for 12 hours. Then they were incubated with medium (s-Med; f-Med), LPS (100 ng/mL) (s-LPS; f-LPS), or TNF (10 ng/mL) (s-TNF; f-TNF) for further 12 hours under static or flow conditions. TLR2 mRNA and TLR4 mRNA were analyzed by RT-PCR. GAPDH mRNA served as control. Shown is one representative experiment of three.

SP1 DNA Binding Activity Is Altered by Laminar Flow
After flow adaptation, HCAECs were kept under flow for another 12 hours in medium alone or medium containing LPS (100 ng/mL) or TNF (10 ng/mL). Control cells under static conditions were treated the same way. Thereafter, DNA binding of nuclear proteins was investigated by EMSA. Neither LPS nor TNF treatment showed any effect on the DNA binding activity of SP1 compared with medium control. But, when SP1 derived from flow-adapted cells was compared with SP1 from static control cells, its DNA binding activity was found to be markedly decreased, no matter whether cells were stimulated or not. This effect was also seen for SP3 (Figure 6, left). LPS (100 ng/mL) or TNF (10 ng/mL) induced NF-B translocation. Chronic laminar flow did not influence NF-B translocation (Figure 6, right).

View larger version (66K): [in this window] [in a new window]   Figure 6. HCAECs were under static conditions or adapted to flow (1.5 N/m2) for 12 hours. Then they were incubated with medium (s-Med; f-Med), LPS (100 ng/mL) (s-LPS; f-LPS), or TNF (10 ng/mL) (s-TNF; f-TNF) for a further 12 hours under static or flow conditions. Nuclear extracts were analyzed in EMSAs. For supershift assays, 1 µg of specific antibodies (anti-SP1; anti-SP3) and for competition assays a 100-fold concentration of unlabeled oligonucleotide was added 10 minutes before the addition of the labeled probe. Shown is one representative experiment of three.

Chronic Laminar Flow Induces SP1 Phosphorylation in HCAECs
SP1 was further investigated in HCAECs under flow in time course experiments ranging from 0 to 12 hours of shear stress to determine the cause of the flow-induced loss of SP1 activity. SP1 mRNA analyses revealed no quantitative change within this period of flow (Figure 7A), which was confirmed also for SP1 protein levels in cell lysates (Figure 7B, CL). Although flow slightly increased SP1 protein in nuclear extracts, no major changes in the abundance of the protein during the time of flow could be observed (Figure 7B, NE). When the blotting membranes were stripped and reprobed with an anti-phosphoserine antibody, it turned out that flow induces serine phosphorylation of SP1 in a time-dependent manner (Figure 7B, NE). Phosphorylation was inversely correlated to the DNA binding activity of SP1 and SP3 (Figure 7C). A labeled oligonucleotide spanning the putative SP1/Ets binding site of the TLR2 promoter showed a weaker signal, but the same correlation with SP1 phosphorylation as the standard SP1 oligonucleotide. Phosphorylation of SP3 could not be detected in Western blots in our experimental setting.

View larger version (38K): [in this window] [in a new window]   Figure 7. HCAECs were kept under static conditions or exposed to laminar shear stress (1.5 N/m2) up to 12 hours. A, SP1 mRNA was analyzed by RT-PCR. GAPDH mRNA served as control. B, Cell lysates (CL) or nuclear extracts (NE) were subjected to Western blot analyzes. Blots were probed with an anti-SP1 antibody (SP1), stripped, and reprobed with an anti-phosphoserine antibody (PS). C, Nuclear extracts were incubated with a SP1 consensus oligonucleotide probe (SP1, SP3) or with a probe spanning the putative SP1 bindings site in the TLR2 promoter (SP1/Ets) and analyzed by EMSA. Shown is one representative experiment of three.

Inhibition of Protein Kinase CK2 Restores SP1/SP3 DNA Binding Activity
HCAECs were adapted to laminar flow and kept under flow conditions for a further 12 hours in the absence or presence of TBB (10 µmol/L), which is a very specific CK2 inhibitor.²⁵ TBB had no effect on SP1/SP3 DNA binding in the static control cells. As expected, laminar flow reduced SP1/SP3 binding, which was restored by TBB treatment (Figure 8A). TLR2 protein expression was investigated under the same conditions. TBB treatment counteracted flow-induced inhibition of TLR2 expression, but it did not enable LPS to stimulate additional TLR2 expression (Figure 8B). Using TBB to abolish the flow effect on basal TLR2 expression, HCAECs regained the ability to respond to MALP-2 with E-selectin expression (Figure 8C).

View larger version (26K): [in this window] [in a new window]   Figure 8. HCAECs were kept under static conditions or exposed to laminar shear stress (1.5 N/m2) for 12 hours with or without the continuous presence of a protein kinase CK2 inhibitor (TBB; 10µmol/L). A, Nuclear extracts were analyzed in an EMSA using a SP1 consensus oligonucleotide probe. Shown is one representative experiment of three. B, Cell surface expression of TLR2 protein was analyzed by FACS. Data are given as mean±SEM of the phycoerythrin-mean channel fluorescence (PE-MCF). n=3. *P<0.05 (Mann Whitney U test). C, Cells were stimulated for further 6 hours with MALP-2 (100 ng/mL) and activation was determined by measuring E-selectin surface expression.

	Discussion

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Hemodynamic forces play a fundamental role in the regulation of endothelial cell functions.⁷ One of these important physical forces acting on endothelial cells is fluid shear stress. Areas of the endothelium that are exposed to disturbed flow and lower shear stress, such as arterial branching or curvature, are preferential sites for atherosclerotic lesion formation.^1,2 Evidence is emerging that there is a link between the innate immune system and atherogenesis^5,16 and recent in vivo work demonstrates augmented expression of some TLRs in endothelial cells of atherosclerotic lesions. This was also correlated to cell activation.¹⁵ Thus, we were curious whether hemodynamic forces would alter endothelial activation by bacterial products.

Our in vitro flow model simulates a chronic continuous laminar flow, as it occurs in vivo in straight tubular arterial sections, and uses human arterial endothelial cells of midsized arteries (coronary artery). Endothelial cells in the tubular regions of arteries, where blood flow is uniform and laminar, are ellipsoid in shape and aligned in the direction of flow. This was also observed in our model; on adaptation to flow, fusiform endothelial cells were aligned in the flow direction. Our in vitro no-flow model is perhaps less representative of turbulent flow as it occurs at arterial branches or curvature. However, using a model of turbulent flow, we found that TLR2 expression and reactivity in disturbed flow and no-flow environments were similar.

HCAECs aligned and shaped by flow were found to be almost unresponsive to activation by the selective TLR2 ligands MALP-2, PAM₂, or Lip19, whereas responsiveness to the TLR4 ligand LPS and the cytokine TNF, as measured by E-selectin expression, remained intact. Our search for the mechanism causing this specific phenomenon led us first to investigate surface receptor expression. Cells under chronic laminar flow expressed little TLR2 on the membrane and induction of TLR2 expression by LPS or TNF was completely suppressed. Flow suppression was not attributable to a diminished NF-B response to LPS or TNF stimulation. In agreement with previous findings,²⁶ chronic shear stress did not alter LPS- or TNF-induced NF-B translocation. Experiments further revealed that the amount of TLR2 expressed correlated with MALP-2 inducible cell activation and thus the hyporesponsiveness observed could be linked to reduced receptor protein expression.

Reduced TLR2 expression was found to be attributable to reduced abundance of TLR2 mRNA under flow conditions. No change could be observed for TLR4 expression, although cells seemed slightly more susceptible to LPS under flow. However, TLR4 functions intracellularly in these cells and needs cooperation with scavenger receptors to take up LPS²¹; thus several factors could be responsible for the slight change in TLR4 signaling. Flow had no effect on the E-selectin response to TNF. Thus, we pursued further the basis for altered TLR2 expression.

In view of the report that SP1 and SP3 elements regulate TLR2 promoter activity in the human myeloid THP-1 cell line,²⁴ we explored whether SP1/SP3 could be involved in flow-regulated expression of TLR2 in primary human endothelial cells. We found that laminar flow markedly decreased SP1 and SP3 DNA binding activity, which was not changed whether LPS or TNF were present. Time course experiments starting at the point of flow onset revealed no change in SP1 mRNA or protein in the cells, but clearly demonstrate an inverse correlation of SP1 serine-phosphorylation and DNA binding. SP1 can be phosphorylated by a DNA-dependent kinase,²⁷ cyclin A-cyclin–dependent kinase complexes,²⁸ protein kinase A,²⁹ and by cell cycle–regulated SP1-associated kinase activity,³⁰ but none of these inhibits the SP1 DNA binding activity. The DNA binding domains of SP1 and SP3 contain three zinc fingers that recognize DNA binding sites with similar binding affinities.³¹ This domain is the most highly conserved part of the SP-family members.³² Important for our study is SP1 phosphorylation by protein kinase CK2, which mainly phosphorylates serine in the zinc finger domain.³³ This inhibits SP1 DNA binding activity. CK2 has also been reported to be involved in SP3 phosphorylation.³⁴ SP1 and SP3 have been thought to compete for their binding site(s), but this seems not to be the case in promoters containing multiple binding sites,³² as it is in the TLR2 promoter.

When we treated HCAECs during flow with TBB, a highly selective inhibitor of protein kinase CK2,²⁵ the SP1 and SP3 binding activity and TLR2 receptor expression were restored, suggesting that laminar shear stress–induced SP1 serine-phosphorylation is mediated by protein kinase CK2. Why it did not restore LPS-induced TLR2 upregulation, such as occurs under static conditions, remains speculative, but suggests that another mechanism is responsible for LPS-induced TLR2 induction. This machinery is apparently overruled by the strength of the shear stress mechanism. Because the diminished response to TLR2 ligands under flow was attributable to reduced TLR2 expression and TBB restored surface TLR2 to levels observed under static conditions, it was not surprising that TBB treatment of flow cells also enabled full activation by MALP-2.

In the context of atherosclerosis and flow-regulated genes, expression of PDGF-A/B, MCP-1, VCAM, and TM have also been investigated under chronic flow and were found to be suppressed.⁷ The promoter regions of these major functional human endothelial proteins all contain SP1 binding sites and require SP1 besides other transcription factors for enhanced expression.^35–39 We observed that LPS-inducible VCAM expression in HCAECs was reduced >80% under flow conditions (data not shown). The SP1/SP3 mechanism described in this study is flow and endothelial cell specific. It is not present in HeLa cells (data not shown) and it is not general silencing of the cells, because LPS- and TNF-induced E-selectin expression stayed intact (see Figure 1).

Using intravital microscopy, Yipp et al⁴⁰ recently showed that lipoteichoic acid, a TLR2 agonist, was ineffective at inducing leukocyte endothelial adhesion, whereas LPS was very effective. Their results are probably explained by our finding that flow inhibits TLR2 expression and thus also responses to lipoteichoic acid. However, that the precise CK2/SP1 regulatory mechanism we describe for HCAECs is also functional in murine cells is not so clear: in murine cells, TLR2 expression may be regulated somewhat differently.^41–43

Atherosclerotic lesions appear first at lesion-prone sites, where specific molecules form on the activated endothelium that are responsible for the recruitment of monocytes and T cells.² Our results suggest one mechanism for the observed regional expression of those molecules; a flow-specific susceptibility to TLR2 expression. Innate immune system activation and bacterial infection have been linked to atherogenesis.^3,15 Recently, atherosclerotic lesion formation in hyperlipidemic ApoE^–/– mice was found to be dependent on functional MyD88^5,16 and to a lesser extent on TLR4. The results herein suggest the hypothesis of an important role of TLR2. Signaling from both TLRs 2 and 4 passes through MyD88. It is not yet clear exactly how these molecules promote atherosclerosis in response to endogenous (eg, oxLDL) or exogenous (eg, LPS and other microbial products) ligands. Our results suggest the hypothesis that flow suppression of TLR2 expression could play a role in the observed regiospecificity of atherosclerotic lesion progression. Experiments to test this hypothesis are underway.

	Acknowledgments

 
This work was supported in part by grants from the California Tobacco-Related Disease Research Program (11-RT-0073) to Peter S. Tobias, the Max Kade Foundation, NY, and the Austrian Science Fund (FWF project number J2310-B05) to S. Dunzendorfer. This is publication no. 16612-IMM from the Scripps Research Institute. We thank Z.M. Ruggeri and J. Mathison (The Scripps Research Institute) for assistance in setting up the flow apparatus.

	Footnotes

 
Original received June 25, 2004; resubmission received July 23, 2004; accepted August 19, 2004.

	References

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