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Sphingosine kinase-1 mediates TNF--induced MCP-1 gene expression in endothelial cells: upregulation by oscillatory flow

Discovery Research, AtheroGenics, Alpharetta, Georgia 30004

Submitted 8 November 2003 ; accepted in final form 7 June 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Atherosclerosis is a focal inflammatory disease and preferentially occurs in areas of low fluid shear stress and oscillatory flow, whereas the risk of atherosclerosis is decreased in regions of high fluid shear stress and steady laminar flow. Sphingosine kinase-1 (SphK1) catalyzes the conversion of sphingosine to sphingosine-1 phosphate (S1P), a sphingolipid metabolite that plays important roles in angiogenesis, inflammation, and cell growth. In the present study, we demonstrated that exposure of human aortic endothelial cells to oscillatory flow (shear stress, ±5 dyn/cm² for 48 h) resulted in a marked increase in SphK1 mRNA levels compared with endothelial cells kept in static culture. In contrast, laminar flow (shear stress, 20 dyn/cm² for 48 h) decreased SphK1 mRNA levels. We further investigated the role of SphK1 in TNF--induced expression of inflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1) and VCAM-1 by using small interfering RNA (siRNA) specifically for SphK1. Treatment of endothelial cells with SphK1 siRNA suppressed TNF--induced increase in MCP-1 mRNA levels, MCP-1 protein secretion, and activation of p38 MAPK. SphK1 siRNA also inhibited TNF--induced cell surface expression of VCAM-1, but not ICAM-1, protein. Exposure of endothelial cells to S1P led to an increase in MCP-1 protein secretion and MCP-1 mRNA levels and activation of NF-B-mediated transcriptional activity. Treatment of endothelial cells with the p38 MAPK inhibitor SB-203580 suppressed S1P-induced MCP-1 protein secretion. These data suggest that SphK1 mediates TNF--induced MCP-1 gene expression through a p38 MAPK-dependent pathway and may participate in oscillatory flow-mediated proinflammatory signaling pathway in the vasculature.

p38 MAPK

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes (25, 31). Recently, it was reported (35) that sphingosine kinase (SphK), the enzyme that catalyzes the formation of S1P from sphingosine, is involved in the signaling pathway of proinflammatory cytokines such as TNF-. Exposure of endothelial cells to TNF- resulted in a rapid activation of SphK activity and release of S1P from endothelial cells. With the use of a putative SphK inhibitor N,N-dimethylsphingosine (DMS), it was demonstrated that SphK is involved in the TNF--induced expression of E-selectin and VCAM-1 in endothelial cells (35). The SphK enzymes are encoded by at least two gene products, sphingosine kinase-1 and -2 (SphK1 and SphK2) (19). Vascular endothelial cells express the gene encoding the SphK1 isoform of the enzyme (2).

Monocyte chemoattractant protein-1 (MCP-1) plays a critical role in the initiation and progression of atherosclerosis (23). Absence of MCP-1 expression reduces macrophage recruitment and provides sustained protection from foam cell formation and atherosclerotic lesion development in several atherosclerosis models (11, 12). In the present study, we demonstrated that SphK1 gene expression is upregulated by oscillatory flow compared with laminar flow or static culture. With the use of a small interfering RNA (siRNA) to inhibit the expression of SphK1, we demonstrate that SphK1 is involved in TNF--induced MCP-1 expression and activation of the p38 MAPK pathway in endothelial cells. These data suggest that SphK1 mediates TNF--induced MCP-1 gene expression through a p38 MAPK-dependent pathway and may participate in oscillatory flow-mediated proinflammatory signaling pathways in the vasculature.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Cell culture and DNA plasmids. Human aortic endothelial cells (HAEC) were obtained from Cambrex (San Diego, CA) and cultured in EGM-2 growth medium. Cells were used between passages 5 and 9. Human dermal microvascular endothelial cells (HMEC) have been described previously (40) and were cultured in modified MCDB-131 (GIBCO-BRL) supplemented with 10% fetal bovine serum and EGM SingleQuotes (Cambrex). Cells were maintained at 37°C in a 5% CO₂ incubator. Enh.MCP-1/CAT, a chimeric reporter gene containing coordinates –2704 to –2081 linked to –319 to +61 of the human MCP-1 promoter and fused to the chloramphenicol acetyl transferase (CAT) reporter gene, was a generous gift of Dr. Atsuhisa Ueda, (Yokohama University, Yokohama, Japan) (34). p(HIVB)₄-CAT contains four tandem copies of HIV long-terminal repeat B-DNA sequences linked to a CAT reporter gene (17). Sphingosine-1-phosphate was obtained from Calbiochem, and p38 MAPK inhibitor SB-203580 was purchased from Sigma.

Flow system. The flow system used has been previously described (6, 7). Briefly, HAECs were seeded onto gelatin-coated glass slides and grown overnight before exposure to flow. Cells were either kept in static conditions or exposed to flow conditions. The glass plate containing the monolayer was inserted into a parallel-plate flow chamber that was installed in a closed-loop flow system. For laminar flow conditions, endothelial monolayers were subjected to laminar flow with a shear stress of 20 dynes/cm². For oscillatory flow experiments, the endothelial monolayers were exposed to a very low mean shear stress (<0.5 dyn/cm²) with a superimposed instantaneous oscillatory shear stress that cycled between +5 and –5 dyn/cm² at a periodicity of 1 Hz. This small mean flow component was included to ensure adequate medium exchange over the endothelial monolayer. After indicated times, RNA samples were collected.

Preparation of RNA and real-time PCR analysis. Total RNA samples were isolated by the TRIzol method (Life Technologies, Grand Island, NY). First-strand cDNA templates were generated with oligo(dT) with the Reverse Transcription Kit (Invitrogen, Carlsbad, CA). The amount of mRNA for each sample was quantitatively determined by running a SYBRgreen (Qiagen, Valencia, CA)-based real-time PCR using the iCycler IQ Optical System (Bio-Rad, Hercules, CA). Cycling conditions were as follows: initial denaturation at 95°C for 15 min, 40 cycles of denaturation at 95°C for 15 s, annealing and elongation at 50°C for 1 min. The primers for SphK1 are 5'-GTATGAATGCCCCTACTTGG-3' (forward primer) and 5'-AACACACCTTTCCCATCCT-3' (reverse primer). The primers for MCP-1 are 5'-CCCAGTCACCTGCTGTTAT-3' (forward primer) and 5'-TGCTGCTGGTGATTCTTCT-3' (reverse primer). The primers for GAPDH are 5'-CCCAGTCACCTGCTGTTAT-3' (forward primer) and 5'-TGCTGCTGGTGATTCTTCT-3' (reverse primer).

Inhibition of SphK1 expression by siRNA. Inhibition of SphK1 expression in HMECs was performed by using specific siRNA reagents as previously described (2). Human SphK1-specific 21-nucleotide siRNAs (Dharmacon Research, Lafayette, CO) 5'-GAGCUGCAAGGCCUUGCCCdTdT-3' and 5'-GGGCAAGGCCUUGCAGCUCdTdT-3' were targeted 70 nucleotides downstream of the start codon. This siRNA for SphK1 has been shown to be strongly suppressed SphK activity in endothelial cells (2). Scrambled siRNA was used as negative control. HMECs were transfected with the 21-nucleotide duplexes using Oligofectamine (Invitrogen) according to the manufacturer's instruction.

ELISA for MCP-1 protein. HMECs growing in 24-well plates were treated with S1P (1–5 µM) or TNF- (100 U/ml) for 16 h. Cell culture supernatants were collected and assayed for MCP-1 protein levels by ELISA using the Quantikine Colorimetric Sandwich ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Western blot analysis. HMECs were lysed for 30 min on ice in 1 ml of a lysis buffer as previously described (7). Protein samples (15 µg) were subjected to electrophoresis on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. Antibody-bound protein bands were then visualized via horseradish peroxidase-dependent chemiluminescence (Amersham Biosciences, Piscataway, NJ). Phosphorylated-p38 MAPK and p38 MAPK antibodies were obtained from Cell Signaling Technology (Beverly MA).

ELISA for phosphorylated and total p38 MAPK. HMECs growing in 60-mm plates were transfected with SphK1 or scrambled (negative control) siRNA for 48 h. Cells were then exposed to TNF- (100 U/ml) for 20 min. Phosphorylated and total p38 MAPK levels were determined by ELISA (BioSource, Camarillo, CA) according to the manufacturer's instructions.

Transfection and promoter activity assays. HMECs were grown to 60 to 70% confluence in six-well plates and transfected with various plasmids as indicated in the figure legends using SuperFect transfection reagent according to the manufacturer's instructions (Qiagen). CAT activities were determined as previously described (7) by using [¹⁴C]chloramphenicol (Amersham Bioscience) and n-butyryl coenzyme A. The plasmid pRL-TK (Renilla luciferase constitutively expressed under the control of the thymidine kinase promoter) was cotransfected in all samples and was used to normalize for transfection efficiency. Renilla luciferase activity was measured by using a luciferase reporter assay system according to the manufacturer's instructions (Promega, Madison, WI). All CAT activities were normalized to the Renilla luciferase activity.

Statistical analyses. Data are presented as means ± SD. Statistical comparisons between groups of data were carried out by using the Student's t-test, and values were considered significantly different at the 95% confidence level.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Oscillatory flow and TNF- increases expression of SphK1. Atherosclerosis is a focal inflammatory disease and preferentially occurs at branched and/or curved regions of the vasculature associated with low and/or disturbed fluid shear stress. Conversely, the frequency of early atherosclerosis lesions is decreased in the relatively straight and nonbranching regions of the vasculature that experience high levels of fluid shear stress and steady laminar flow. To identify endothelial genes that were differentially regulated by exposure to fluid shear stress, we used cDNA microarray (Incyte Genomics, Wilmington, DE) analysis to evaluate global gene expression profiles. We compared RNA expression profiles from HAECs exposed in vitro to either static, oscillatory flow (shear stress, ±5 dyn/cm²) or laminar flow (shear stress, 20 dyn/cm²). Many of the genes whose expression was induced by oscillatory flow compared with laminar flow are known to have a role in regulating vascular inflammatory responses. SphK1 is one of the genes upregulated by oscillatory flow that has been previously shown to play a role in regulating endothelial cell adhesion molecule expression. The relative expression levels for SphK1 mRNA were increased more than twofold in oscillatory flow compared with laminar flow. To confirm that SphK1 was differentially regulated by hemodynamic stress, mRNA was collected from several different independent experiments in which HAECs were subjected to laminar flow, oscillatory flow, or kept in static culture and subjected to quantitative real-time PCR analysis. As shown in Fig. 1, oscillatory flow induced a threefold increase in mRNA levels for SphK1 compared with cells kept in static condition. In contrast, laminar flow treatment resulted a 40% decrease in SphK1 mRNA levels compared with cells kept in static conditions. These data demonstrated that SphK1 mRNA levels are differentially regulated by hemodynamic stress and confirm our initial results from the cDNA expression arrays. These observations are consistent with the notion that low levels of shear stress and/or disturbed fluid flow induce a more atherogenic and/or inflammatory phenotype to the endothelial cell, whereas laminar flow has an atheroprotective phenotype (7, 33).

View larger version (12K): [in this window] [in a new window]   Fig. 1. Oscillatory flow upregulates and laminar flow downregulates sphingosine kinase-1 (SphK1) gene expression in human aortic endothelial cells (HAECs). HAECs grown on glass plates were kept in static condition or subjected to laminar flow (LF) with shear stress at 20 dyn/cm2 or oscillatory flow (OF) with shear stress at ±5 dyn/cm2 for 48 h. Relative SphK-1 mRNA levels were determined by quantitative real-time RT-PCR and normalized to -actin levels. Values are means ± SE, n = 4. *P < 0.05 compared with static culture.

View larger version (8K): [in this window] [in a new window]   Fig. 2. TNF- upregulates SphK1 gene expression in human dermal microvascular endothelial cells (HMECs). HMECs were exposed to TNF- (100 U/ml) for 4 h. Relative SphK1 mRNA levels were determined by quantitative real-time RT-PCR and normalized to -actin levels. Values are means ± SE, n = 4. *P < 0.05 compared with control group.

View larger version (12K): [in this window] [in a new window]   Fig. 3. SphK1-specific small interfering RNA (siRNA) inhibits SphK1 gene expression. HMECs were transfected with small interfering SphK1 (siSphK1), scrambled siRNA (siScrambled; 200 nM) or no siRNA (mock) for 48 h. Relative SphK1 mRNA levels were determined by quantitative real-time RT-PCR and normalized to -actin levels. Values are means ± SE, n = 4. *P < 0.05 compared with control group.

View larger version (18K): [in this window] [in a new window]   Fig. 4. SphK1-specific siRNA inhibits TNF--induced myocyte chemoattractant protein-1 (MCP-1) protein secretion, mRNA accumulation and gene transcription. A: HMECs were transfected with SphK1, scrambled siRNA (200 nM), or no siRNA (mock) for 48 h and then exposed to TNF- (100 U/ml) for 4 h. Conditioned medium was collected, and MCP-1 protein levels were determined by ELISA. B: relative MCP-1 mRNA levels were determined by real-time RT-PCR and normalized to -actin levels. C: reporter gene Enh-MCP1-CAT was transiently transfected alone with 200 nM SphK-1 or scramble siRNA for 48 h and then exposed to TNF- (100 U/ml) for 16 h. Cell extracts were collected 16 h later, and CAT activity was determined. Values are means ± SE, n = 4. *P < 0.05 compared with mock-transfected cells treated with TNF-.

SphK1 siRNA suppresses TNF--induced cell surface expression of VCAM-1 but not ICAM-1 protein expression. VCAM-1 and ICAM-1 are two cytokine-inducible adhesion molecules that play critical roles in regulation of the vascular inflammatory response. To investigate whether SphK1 may play a role in TNF--induced endothelial cell expression of these genes as well, HMECs were treated with SphK1 or scrambled siRNA for 48 h, exposed to TNF- (100 U/ml) for 16 h, and then levels of cell-surface VCAM-1 and ICAM-1 were measured by ELISA. Treatment with siRNA specific for SphK1, but not scrambled siRNA, suppressed TNF--induced cell surface protein expression of VCAM-1 by 40% (Fig. 5A). SphK1 siRNA only slightly (statistically insignificant) inhibited TNF--induced ICAM-1 levels (Fig. 5B).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Effects of SphK1 siRNA on TNF--induced cell surface expression of VCAM-1 and ICAM-1 protein expression. HMECs were transfected with SphK-1, scrambled siRNA (200 nM), or no siRNA (mock) for 48 h and then exposed to TNF- (100 U/ml) for 16 h. Cell surface expression of VCAM-1 (A) and ICAM-1 (B) was determined by ELISA. Values are means ± SE, n = 4. *P < 0.05 compared with mock transfected cells treated with TNF-. OD, optical density.

SphK1 siRNA inhibits TNF--induced activation of p38 MAPK.

View larger version (22K): [in this window] [in a new window]   Fig. 6. SphK1 siRNA suppresses TNF--induced activation of p38 MAPK. HMEC were transfected with SphK-1, scrambled siRNA (200 nM), or no siRNA (mock) for 48 h and exposed to TNF- (100 U/ml) for 20 min. p38 MAPK activity was determined by assaying for the level of the total or phosphorylated form of p38 MAPK by Western blot analysis with antibodies for phosphorylated p38 MAPK or total p38 (A) and by ELISA as described in MATERIALS AND METHODS (B and C). Values are means ± SE, n = 4. *P < 0.05 compared with mock siRNA transfected cells treated with TNF-.

S1P increases MCP-1 protein secretion and MCP-1 mRNA accumulation and activates NF-B-driven promoter activity.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Sphingosine-1-phosphate (S1P) stimulates MCP-1 secretion, MCP-1 mRNA accumulation, and NF-B-driven promoter activity. A: HMECs were treated with S1P (0.5 to 5 µM) for 16 h. Medium was collected and assayed for MCP-1 protein secretion using ELISA. B: HMECs were treated with S1P (5 µM) or TNF- (100 U/ml) for 4 h. Relative MCP-1 mRNA levels were determined by real-time PCR and normalized to -actin gene levels. C: HMECs were transfected with a NF-B-driven promoter construct p(HIVB)4-CAT and treated with S1P (5 µM) or TNF- (100 U/ml) for 16 h. Cell extracts were collected, and CAT activity was determined as described in MATERIALS AND METHODS. Values are means ± SD, n = 4. *P < 0.05 compared with the untreated group.

View larger version (9K): [in this window] [in a new window]   Fig. 8. p38 MAPK mediates S1P-induced MCP-1 secretion. HAECs were pretreated with p38 MAPK inhibitor SB-203580 (10 µM) for 1 h and then exposed to S1P (5 µM) for 16 h. Medium was collected and assayed for MCP-1 protein secretion using ELISA. Values are means ± SD, n = 4. *P < 0.05 compared with the TNF--treated group.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

SphK plays an important role in the regulation of MAPKs. ERK1/2 activation by TNF- and platelet-derived growth factor is at least partially dependent on SphK (28, 35). Also, S1P stimulates ERK1/2 MAPK in airway smooth muscle cells (27). The p38 MAPK regulates expression of many inflammatory genes, such as MCP-1 (10), IL-6 (5), VCAM-1 (26), and TNF- (39). With the use of specific p38 MAPK inhibitors or a dominant-negative mutant of MKK6 (the upstream kinase activator of p38) it has been shown that p38 MAPK is required for the activation of MCP-1 gene expression in response to IL- and TNF- (10, 30). The present study demonstrates that SphK1 is, at least partially, involved in TNF--mediated activation of the p38 MAPK pathway in endothelial cells. This is the first report demonstrating regulation of p38 MAPK by SphK in TNF- activation. These data are consistent with an earlier study (16) in osteoblasts in which S1P activates the p38 MAPK. In addition, S1P-induced heat shock protein 27 gene expression is dependent on the p38 MAPK activation (16). Our results suggest that p38 MAPK may be involved in the modulation of TNF--induced MCP-1 expression by SphK1.

S1P is unique in that it can function both as an extracellular ligand and as an intracellular second messenger (31). It is well established that S1P is the ligand for the EDG-1, EDG-3, and EDG-5 family of GPCR receptors that couple to a variety of G proteins to regulate diverse biological functions (18, 29). In this study, we found that exogenous S1P stimulates MCP-1 expression and NF-B activation in endothelial cells. These data are consistent with an earlier study that demonstrated activation of E-selectin and VCAM-1 gene expression by S1P (35), suggesting that S1P may serve as proinflammatory stimuli in the vasculature.

Oscillatory flow enhances monocyte adhesion and stimulates VCAM-1, ICAM-1, and E-selectin gene expression (6). The present study demonstrates that SphK1 gene expression is upregulated by oscillatory flow and downregulated by laminar flow in endothelial cells. In addition, SphK1 gene expression was also induced by TNF-. Increased expression of SphK-1 by these proinflammatory stimuli will enhance the capacity of endothelial cells to generate S1P. Increased production of S1P may contribute to activation of NF-B and stimulation of MCP-1, VCAM-1, and E-selectin gene expression through autocrine and paracrine mechanisms. These observations are consistent with the evidence that areas of presumed oscillatory flow in vivo exhibit recruitment of circulating monocytes that may contribute to plaque formation (6). From the present study, it is clear that oscillatory flow represents a stimulus distinct from that of laminar flow with respect to SphK1 gene expression. These findings parallel earlier studies (6) demonstrating that laminar flow suppresses VCAM-1 gene expression, whereas oscillatory flow increases VCAM-1 gene expression. These data support the conclusion that prolonged laminar flow impart anti-inflammatory and atheroprotective phenotype, whereas oscillatory flow may impart a proatherogenic and proinflammatory phenotype. These data provide further evidence that oscillatory flow is a proinflammatory stimulus to endothelial cells.

Cumulatively, the studies reported here extend previous observations on the involvement of the SphK1/S1P pathway in regulation of cytokine-activated inflammatory gene expression in the endothelium. We provide evidence that inflammatory stimuli including TNF- activation and exposure to proinflammatory hemodynamic stress, activate expression of the SphK1 gene in endothelial cells. Furthermore, we suggest that modulation of p38 MAPK via the SphK1/S1P pathway may constitute a molecular link in the signaling pathway that mediates TNF--induced expression of inflammatory gene expression. These studies begin to associate diverse signaling pathways into a common mechanism for the cytokine-mediated activation of inflammatory genes in the vasculature. Further understanding of the role of SphK1 in TNF--mediated gene expression may reveal new opportunities for therapeutic intervention for the treatment of vascular inflammatory diseases.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
All authors are employees of AtheroGenics, Inc., Alpharetta, GA.

	ACKNOWLEDGMENTS

 
We thank Dr. Signe E. Varner and Anjali Rao for contributions in flow experiments and RNA preparations used for the cDNA microarray analysis.

	FOOTNOTES

 

Address for reprint requests and other correspondence: X.-L. Chen, Discovery Research, AtheroGenics, Inc., 8995 Westside Pkwy., Alpharetta, GA 30004 (E-mail: xchen{at}atherogenics.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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