Báo cáo khoa học: Upregulation of endothelial adhesion molecules by lysophosphatidylcholine pptx

Upregulation of endothelial adhesion molecules by lysophosphatidylcholine Involvement of G protein-coupled receptor GPR4 Yani Zou 1 , Chul H. Kim 1 , Jae H. Chung 1 ,JiY.Kim 1 , Sang W. Chung 1 , Mi K. Kim 2 , Dong S. Im 1 , Jaewon Lee 1 , Byung P. Yu 2,3 and Hae Y. Chung 1,2 1 College of Pharmacy, Pusan National University, Busan, Korea 2 Longevity Life Science & Technology Institute, Pusan National University, Busan, Korea 3 Department of Physiology, University of Texas Health Science Center, San Antonio, TX, USA Lysophosphatidylcholine (LPC), a derivative of phos- phatidylcholine, is generated through the hydrolytic action of phospholipase A 2 at the sn-2 position of phosphatidylcholine. In vivo, LPC is claimed to be the major effective component of oxidized low-density lipoprotein [1,2], and is found in high concentrations Keywords adhesion molecules; cAMP response element-binding protein; G protein-coupled receptor 4; lysophosphatidylcholine; nuclear factor kappaB Correspondence H. Y. Chung, Longevity Life Science & Technology Institute, College of Pharmacy, Pusan National University, 30 Jangjun-dong, Gumjung-gu, Busan 609-735, Korea Fax: +82 51 518 2821 Tel: +82 51 510 2814 E-mail: hyjung@pusan.ac.kr (Received 2 December 2006, revised 11 February 2007, accepted 15 March 2007) doi:10.1111/j.1742-4658.2007.05792.x Lysophosphatidylcholine induces expression of adhesion molecules; how- ever, the underlying molecular mechanisms of this are not well elucidated. In this study, the intracellular signaling by which lysophosphatidylcholine upregulates vascular cell adhesion molecule-1 and P-selectin was delineated using YPEN-1 and HEK293T cells. The results showed that lysophos- phatidylcholine dose-dependently induced expression of vascular cell adhesion molecule-1 and P-selectin, accompanied by the activation of tran- scription factor nuclear factor jB. However, the nuclear factor jB inhibitor caffeic acid phenethyl ester (CAPE) and the antioxidant N-acetylcysteine only partially blocked lysophosphatidylcholine-induced adhesion molecules. Subsequently, we found that the lysophosphatidylcholine receptor G pro- tein-coupled receptor 4 (GPK4) was expressed in YPEN-1 cells and trig- gered the cAMP ⁄ protein kinase A ⁄ cAMP response element-binding protein pathway, resulting in upregulation of adhesion molecules. Further evidence showed that overexpression of human GPK4 enhanced lysophosphatidyl- choline-induced expression of adhesion molecules in YPEN-1 cells, and enabled HEK293T cells to express adhesion molecules in response to lysophosphatidylcholine. In conclusion, the current study suggested two pathways by which lysophosphatidylcholine regulates the expression of adhesion molecules, the lysophosphatidylcholine ⁄ nuclear factor-jB ⁄ adhesion molecule and lysophosphatidylcholine ⁄ GPK4 ⁄ cAMP ⁄ protein kin- ase A ⁄ cAMP response element-binding protein ⁄ adhesion molecule path- ways, emphasizing the importance of the lysophosphatidylcholine receptor in regulating endothelial cell function. Abbreviations AC, adenylyl cyclase; ACREB, dominant-negative mutant CREB protein; AM, adhesion molecule; CAPE, caffeic acid phenethyl ester; CRE, cAMP response element; CREB, cAMP response element-binding protein; EC, endothelial cell; ERK, extracellular signal-related kinase; FSK, forskolin; G2A, G2 accumulation protein; GPR4, G protein-coupled receptor 4; GPR119, G protein-coupled receptor 119; hGPR4, human GPR4 expression vector; LPC, lysophosphatidylcholine; MDL, MDL12330A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAC, N-acetylcysteine; NF-jB, nuclear factor-kappaB; PKA, protein kinase A; PKC, protein kinase C; TNF-a, tumor necrosis factor-a; VCAM-1, vascular cell adhesion molecule-1. FEBS Journal 274 (2007) 2573–2584 ª 2007 The Authors Journal compilation ª 2007 FEBS 2573 in lesional psoriatic skin [3], asthma and rhinitis [4], and atherosclerosis lesions [5]. In addition, LPC has been reported to induce expression of cell adhesion molecules (AMs), which form the framework for leu- kocyte–endothelium binding [6] and subsequent infil- tration of leukocytes across the endothelium, the initial step in atherosclerotic changes. The available data indicate that LPC increases the expression of vascular cell adhesion molecule-1 (VCAM-1) [7], intercellular adhesion molecule-1 [8] and P-selectin [9] both in vitro and in vivo. However, the intracellular signal pathway of these biological effects is still not well characterized. Earlier studies have suggested several possible mech- anisms for LPC’s effects. LPC activates the redox- sensitive transcription factors nuclear factor-kappaB (NF-jB) [10] and activator protein-1 [11] through the mitogen-activated protein kinase and protein kinase C (PKC) pathways. LPC is also known to enhance cAMP response element-binding protein (CREB) ⁄ activating transcription factor activity in endothelial cells (ECs) [12]. However, it is still unclear precisely how LPC triggers these signals. Recent studies demon- strated that low-concentration LPC regulates activa- tion of G protein-coupled receptors [13], including G protein-coupled receptor G2 accumulation protein (G2A), G protein-coupled receptor 4 (GPR4) [14], and G protein-coupled receptor 119 (GPR119) [15], thus providing new molecular insights into the effects of LPC on various signaling activities. In the present study, we attempted to elucidate the mechanism by which LPC triggers the expression of the AMs VCAM-1 and P-selectin in the rat EC line. It was found that YPEN-1 was activated to express AMs by both the NF-jB-mediated and GPR4-mediated path- ways. This finding highlights the biological role of LPC and provides new molecular insights into the activation mechanism of LPC in the cellular signaling pathway. Results Upregulated expression of AMs by LPC Previous reports have shown that LPC induces the expression of AMs in ECs [7]; however, the underlying mechanism was not known. Our earlier study showed that rat endothelial YPEN-1 cells express AMs in response to proinflammatory cytokines, such as tumor necrosis factor-a (TNF-a) [16]. In this study, YPEN-1 was used to explore the underlying mechanisms of LPC-induced expression of AMs. The expression of VCAM-1 and P-selectin induced by LPC in YPEN-1 cells was measured by western blot analysis (Fig. 1). When YPEN-1 was incubated with LPC at increasing concentrations from 3.12 lm to 25 lm for 12 h, expression of VCAM-1 and P-selectin was dose- dependently upregulated, peaking at 12.5 lm (Fig. 1A). Moreover, LPC triggered expression of both AMs in a time-dependent pattern (Fig. 1B). When the transcrip- tional regulation of AMs by LPC was analyzed, mRNA levels of both AMs were found to be increased in a time-dependent fashion, which is comparable to TNF-a-induced activation of AMs (Fig. 1C), confirm- ing the transcriptional regulation of AMs by LPC. To discriminate the cytotoxic effects of LPC, cell viability was examined. The result showed that under the present experimental conditions (i.e. cells were maintained in DMEM medium containing 1% fetal bovine serum when they were challenged by LPC), no significant cell death was observed even at a high con- centration (25 lm) of LPC (Fig. 1D). Contribution of NF-jB to LPC-induced activation of AMs To define the molecular mechanism of LPC-induced upregulation of AMs, the influence of LPC on NF-jB activation was investigated in YPEN-1 cells. This is because the predominant regulatory role of NF-jBis upregulation of VCAM-1 and P-selectin in ECs [17–19]. Western blot analyses were performed to examine the nuclear translocation of NF-jB components p65 and p50. As shown in Fig. 2A, p65 and p50 translocated from the cytosol into nuclei in response to LPC as early as 10 min. This procedure was correlated with increased phosphorylation of cytosolic jB inhibitor (IjBa) (Fig. 2A), implying the activation of NF-jBby LPC. To uncover the interaction between LPC-induced activation of NF-jB and upregulation of AMs, NF-jB luciferase reporter (NF-jB-Luc) and AM gene promo- ters (VCAM-1 gene promoter and P-selectin gene promoter) were transfected into YPEN-1 cells. The luciferase activity was assessed 8 h after treatment of YPEN-1 cells with LPC (12.5 lm), with or without preincubation of the NF-jB inhibitor CAPE (10 lm). As shown in Fig. 2B, LPC increased the activation of NF-jB, as well as the promoter activities of P-selectin and VCAM-1. After preincubation with CAPE (10 lm) for 1 h, activation of NF-jB signaling was significantly blocked (P £ 0.05) and the promoter activity of VCAM-1 was reduced (P £ 0.05). Although the data are not shown, we made certain that these observations did not result from cell death. To verify this finding, western blot analysis was carried out, and showed that increased nuclear translocation of p65 was prevented (Fig. 2C), and also that VCAM-1 expression was parti- ally reduced by preincubation with CAPE (Fig. 2C). GPR4 contributes to LPC-induced AMs Y. Zou et al. 2574 FEBS Journal 274 (2007) 2573–2584 ª 2007 The Authors Journal compilation ª 2007 FEBS Partial blockage of LPC-induced AMs by the antioxidant N-acetylcysteine Given the close association between oxidative stress and the activation of NF-jB, inhibitory effects of antioxi- dants on LPC-induced upregulation of AMs could be expected. Thus, the well-known antioxidant N-acetyl- cysteine (NAC) [20] was used to treat YPEN-1 cells that were transfected with AM promoter reporters or NF-jB-Luc. After 1 h of preincubation with NAC (1 mm), LPC was added to induce activation in YPEN-1 cells. The results showed that although NAC almost completely blocked NF-jB activity (P £ 0.05), it only partially suppressed the activation of VCAM-1 promo- ter (P £ 0.05) (Fig. 3). Considering these results and the above findings on the NF-jB inhibitor CAPE, it was suspected that other signal transduction pathways might participate in LPC-induced AM expression. 0 2 4 6 8 12 (h) VCAM-1 P-selectin β β -Actin LPC (6.25 μ M) B y ti li b ai vl l e C )h0talortnoCfo%( D control 6.25 μ M 12.5 μM 25 μM 0 6 12 (h) 60 100 80 120 140 160 60 80 100 120 140 160 03.126.2512.525 VCA M-1 P-selectin 0talortnoCfo% μ M ( μ M) * * * 60 80 100 120 140 0246812 VCAM-1 P-selectin h0ta l ortn o Cfo% LPC ( μ M) 0 3.12 6.25 12.5 25 VCAM-1 P-selectin β -actin A * ** * * 0 2 4 6 8 8 (h) LPC (25 μ M) β -Actin P-selectin VCAM-1 C TNF- α Fig. 1. LPC-induced expression of AMs in YPEN-1 cells. (A) Expression of AMs induced by LPC in YPEN-1 cells at different concentrations after a 12 h challenge was examined by western blot analysis. The quantitative analysis is shown. (B) Expression of AMs induced by 6.25 l M LPC at different time points. Cells were treated with LPC maintained in 1% fetal bovine serum-containing medium. One representa- tive result is shown from three experiments that yielded similar results, and the quantitative analysis is shown. (C) mRNA of AMs induced by 25 l M LPC was analyzed by RT-PCT at different time points. TNF-a was used as the positive control for monitoring the expression of AMs. One representative result is shown. (D) Cell viability after treatment with LPC at different concentrations was measured by an MTT assay. Data were generated from triplicate experiments, and the results are presented as percentage of control at 0 h. Statistical signifi- cance: *P £ 0.05, **P £ 0.01 versus cells without LPC treatment. Y. Zou et al. GPR4 contributes to LPC-induced AMs FEBS Journal 274 (2007) 2573–2584 ª 2007 The Authors Journal compilation ª 2007 FEBS 2575 Cont. 10 20 40 60 120 240 (min) LPC (6.25 μ μ M) p65 p50 Histone H1 pI κ B α (cytosol) A p65 p50 pIkB nim 0 ta lortnoC fo % Cont. 10 20 40 60 120 240 (min) ** ** * * ** LPC (6.25 μ M) Cont. none CAPE VCAM-1 β -actin p65 (nucleus) C UTC TC LPC LPC + CAPE NF- κ B luciferase reporter P-selectin gene promoter VCAM-1 gene promoter 050100 250200 B Relative RLU (% of TC) 150 * * Fig. 2. LPC-induced activation of NF-jB in YPEN-1 cells. (A) LPC-induced activation of NF-jB was detected by measuring the nuclear translo- cation of NF-jB components p65 and p50, as well as the phosphorylation of the NF-jB inhibitor, IjBa. The levels of p65 and p50 in the nuc- leus were examined by western blot analysis using the cell nuclear fraction after challenge with LPC at different time points. Histone H1 was used as the nuclear fraction internal calibration. The phosphorylation of IjBa was monitored by western blot analysis in the cytosol frac- tion. One representative result is shown, and the quantitative analysis is shown beneath it. *P £ 0.05, **P £ 0.01 versus control. Cont., con- trol. (B) Effects of LPC on NF-jB activation, P-selectin promoter activity and VCAM-1 promoter activity in transfected YPEN-1 ECs were detected by luciferase assay. Cells were transfected with NF-jB luciferase reporter or P-selectin or VCAM gene promoter plasmids, which contain tandem jB-binding site or murine P-selectin or human VCAM-1 promoters, respectively. Twenty-four hours after transfection, cells were treated for 8 h with various reagents, and lysed for determination of luciferase activity. UTC, untransfected control; TC, transfected and untreated cells; LPC, transfected cells challenged with 12.5 l M LPC; LPC + CAPE, transfected cells preincubated with the NF-jB inhib- itor CAPE, 10 l M, for 1 h before challenge with 12.5 lM LPC for an additional 7 h. Each value is expressed as the mean ± SE from three independent experiments. *P £ 0.05 versus TC in the same group. RLU, relative light unit. (C) LPC-induced expression of VCAM-1 through NF-jB activation was detected by applying the NF-jB inhibitor CAPE. YPEN-1 cells were preincubated with CAPE (10 l M) for 1 h before challenge with 6.25 l M LPC. Western blot analysis was carried out to detect the level of VCAM-1 in the cytosol fraction after 12 h of treat- ment with LPC, and the level of p65 in the nucleus was assessed 4 h after treatment with LPC. One representative result is shown. None, without inhibitor. GPR4 contributes to LPC-induced AMs Y. Zou et al. 2576 FEBS Journal 274 (2007) 2573–2584 ª 2007 The Authors Journal compilation ª 2007 FEBS Expression of the LPC receptor GPR4 in YPEN-1 cells To investigate the additional signaling responsible for LPC upregulation of AMs, the involvement of LPC receptors was examined. To date, several G protein- coupled receptors, namely G2A, GPR4, and GPR119, have been suggested as receptors for LPC and to elicit various biological effects. These receptors show more specific functions and trigger biological effects at rela- tively low concentrations of LPC. We analyzed the distribution of LPC receptor pro- teins in YPEN-1 cells and selected rat spleen tissue as the positive control, because it is known to produce both LPC receptors, G2A and GPR4 [21]. As shown in Fig. 4A, an interesting finding was that YPEN-1 ECs expressed GPR4 mRNA but not G2A mRNA. This selectivity for GPR4 expression by ECs is consis- tent with previous reports [21,22]. GPR4 is a GPRC eliciting second messenger cAMP in several cell types [23–25], so the levels of cAMP were determined in LPC-challenged YPEN-1 cells. As indicated in Fig. 4B, a 15-min incubation with LPC at increasing concentra- tions (from 6.25 lm to 12.5 lm) caused dose-depen- dent accumulation of cAMP in YPEN-1 cells. To clarify the downstream signaling of LPC-induced cAMP accumulation, activation of the transcription factor CREB was assessed. It is well known that cAMP binds to specific intracellular regulatory pro- teins such as protein kinase A (PKA) [26], leading to activation of PKA. As a consequence, activated PKA phosphorylates Ser133 of the transcription factor CREB, which then binds to the cAMP response ele- ment (CRE) sequence in targeting genes [26]. In our study, as shown in Fig. 5A, phosphorylation of nuclear CREB was enhanced after challenge with LPC for 30 min; this activation was then sustained for a longer period (Fig. 5A). To delineate LPC-activated CRE signaling, a lucife- rase reporter vector CRE-Luc, containing a CRE site from the human COX-2 gene () 124 bp ⁄ + 59 bp), was applied [27]. Forskolin (FSK) (10 lm), an adenylyl cyclase (AC) activator, served as the positive control. As shown in Fig. 5B, FSK induced significantly high luci- ferase activity in the CRE reporter vector. CRE activity was induced by LPC, and this elevation was reduced by 0 10 20 30 40 50 60 70 80 90 Ⴕ G2A (602 bp) 1 2 M 3 4 GPR4 (546 bp) 1,3. YPEN-1 cDNA; 2,4. Rat spleen cDNA; β -actin A * Control 6.25 12.5 25 LPC ( μ M) gm •lom n( PMAc 1– )nietorp B * Fig. 4. GPR4 expression and LPC-induced cAMP in YPEN-1 cells. (A) Expression patterns of the LPC receptors GPR4 and G2A in YPEN-1 cells were analyzed by RT-PCR, using rat spleen tissue as positive control. Lane 1: YPEN-1 cell cDNA with GPR4 probe. Lane 2: spleen cDNA with GPR4 probe. Lane 3: YPEN-1 cell cDNA with G2A probe. Lane 4: spleen cDNA with G2A probe. M: DNA ladder marker (100 bp). One representative result is shown. (B) LPC- induced accumulation of cAMP was examined by ELISA. YPEN-1 cells were incubated with LPC at different concentrations for 15 min, and cells were then lysed as described in Experimental pro- cedures. cAMP in cell lysate was extracted and detected with a cAMP ELISA kit. Data are presented as average ± SE from tripli- cate experiments. Statistical significance: *P < 0.05 versus control. 0 20 40 60 80 100 120 140 160 180 200 mp1379 VCAM )CT fo %( ULR evitaleR TC LPC LPC + NAC VCAM-1 promoter P-selectin promoter NF- κ B reporter * Fig. 3. Partial blockage of LPC-induced AMs by NAC. Effects of the antioxidant NAC on LPC-induced NF-jB activation, and P-selectin and VCAM-1 promoter activities, in YPEN-1 cells were evaluated by luciferase assay. Cells were transfected with NF-jB-Luc or P-selec- tin or VCAM gene promoter reporters, respectively, treated with reagents for 8 h, and then lysed for determination of luciferase. LPC + NAC, transfected cells preincubated with NAC (1 m M) for 1 h before challenge with 12.5 l M LPC for an additional 7 h. Each value is expressed as the mean ± SE from six independent experi- ments. *P £ 0.05 versus TC in the same group. Y. Zou et al. GPR4 contributes to LPC-induced AMs FEBS Journal 274 (2007) 2573–2584 ª 2007 The Authors Journal compilation ª 2007 FEBS 2577 the AC inhibitor MDL12330A (MDL) (10 lm)(P ¼ 0.069), which prevents the production of cAMP by AC. The PKA inhibitor H89 (10 lm) also reduced the amount of LPC-activated CRE-Luc, implying the involvement of PKA. Thus, the results showed that LPC induced activation of CRE through cAMP and PKA. Upregulation of AMs by LPC through GPR4 signal transduction A subsequent question is whether the GPR4 ⁄ cAMP ⁄ PKA ⁄ CREB pathway contributes to LPC-induced AM upregulation. The effects of the AC inhibitor MDL and the PKA inhibitor H89 on the promoter activities of VCAM-1 and P-selectin in response to LPC were assessed. Pretreatment of YPEN-1 cells with MDL (10 lm) and H89 (10 lm) for 1 h suppressed LPC- induced promoter activities of VCAM-1 and P-selectin as well as CRE signaling (Fig. 6A). Consistent with this result, the expression of VCAM-1 protein was found to be blocked by MDL and H89, as compared to cells treated only with LPC. This result was in line with the reduced phosphorylation of CREB by MDL and H89 (Fig. 6B). )CTfo%(ULRevitaleR B UTC TC LPC LPC+MDLLPC+H89 FSK 0 100 200 300 400 500 600 ** * 0 20 40 60 80 100 120 140 160 nim0talortnoCfo% Cont. 10 30 60 120 240 (min) * * * Histone H1 A Cont. 10 30 60 120 240 (min) LPC (6.25 µ M) pCREB (nucleus) Fig. 5. LPC-induced cAMP ⁄ PKA ⁄ CREB signaling in YPEN-1 cells. (A) LPC-induced downstream signaling of cAMP was examined by the phosphorylation of CREB. YPEN-1 cells were incubated with LPC (6.25 l M) for different times, and nuclear fractions were then used for western blot analysis. The quantitative analysis is shown under the blot. Statistical significance: *P £ 0.05 versus control. (B) The effects of LPC on cAMP ⁄ PKA ⁄ CREB signaling were evaluated by luciferase assay. Cells were transfected with CRE luciferase reporter (CRE-Luc) plasmids that contain the CREB-binding site. LPC + MDL, transfected cells preincubated with MDL (10 l M) for 1 h before challenge with 12.5 l M LPC for an additional 7 h; LPC + H89, transfected cells preincubated with H89 (10 l M) for 1 h before challenge with 12.5 l M LPC for an additional 7 h. FSK, 10 l M, was used as positive control. Each value is expressed as the mean ± SE from six independent experiments. *P £ 0.05 versus TC. β β -Actin pCREB (nucleus) VCAM-1 Cont. none MDL H89 LPC (12.5 μ M ) B TC LPC LPC + MDL Relative RLU (% of TC) LPC + H89 A 60 80 100 120 140 160 180 CRE luciferase reporter P-selectin gene promoter VCAM gene promoter Fig. 6. Activation of AMs by LPC through the cAMP ⁄ PKA pathway. (A) Effects of LPC on CREB activation, P-selectin and VCAM-1 pro- moter activities in transfected YPEN-1 ECs were detected by lucif- erase assay. Cells were transfected with CRE luciferase reporter or P-selectin gene promoter or VCAM gene promoter plasmids, which contain CREB-binding site or murine P-selectin or human VCAM-1 promoters, respectively. LPC + MDL, transfected cells preincubat- ed with MDL (10 l M) for 1 h before challenge with 12.5 lM LPC for an additional 7 h; LPC + H89, transfected cells preincubated with H89 (10 l M) for 1 h before challenge with 12.5 lM LPC for an additional 7 h. Each value is expressed as the mean ± SE from three independent experiments. (B) The contribution of cAMP and PKA activation to LPC-induced CREB activation and expression of VCAM was detected by applying the AC inhibitor MDL and the PKA inhibitor H89. YPEN-1 cells were preincubated with MDL (10 l M) or H89 (10 lM) for 1 h before challenge with 12.5 lM LPC. Western blot analysis was carried out to detect the level of VCAM- 1 in the cytosol fraction after 12 h of treatment with LPC. One rep- resentative result is shown. GPR4 contributes to LPC-induced AMs Y. Zou et al. 2578 FEBS Journal 274 (2007) 2573–2584 ª 2007 The Authors Journal compilation ª 2007 FEBS The role of CREB in LPC-triggered transactivation of AMs was then further examined by utilizing a con- struct encoding a dominant-negative mutant CREB protein, ACREB. ACREB was constructed by fusing a designed acidic amphipathic extension to the N-termi- nus of the CREB leucine zipper domain [28]. The aci- dic extension of ACREB interacts with the basic region of CREB, forming a coiled-coil extension of the leucine zipper, and thus prevents the basic region of wild-type CREB from binding to DNA [28,29]. As shown in Fig. 7, cotransfection of ACREB with the VCAM-Luc and P-selectin gene promoters completely abolished LPC-induced AM promoter activation; in contrast, the ACREB control vector (ACREB con) did not significantly affect the activation of AMs by LPC. The involvement of GPR4 in LPC-induced AM upregulation was confirmed in YPEN-1 cells that were transfected with human GPR4 expression vector (hGPR4). An expression vector for hGPR4 (NM_005282) was prepared using pcDNA3.1(+) ⁄ myc-His [23]. It was found that the basal level of VCAM-1 was significantly increased after transfection with hGPR4, and that it was further elevated by the LPC challenge (Fig. 8A). To avoid the potential effects of endogenous GPR4, the hGPR4 expression vector was further transfected into HEK293T cells. Following transfection of hGPR4, the HEK293T cells gained the ability to induce activation of CRE signaling as well as activation of the VCAM-1 gene promoter, and these activations were further enhanced by LPC challenge (Fig. 8B). LPC ACREB - + + + - - + - + ACREB (con) ** * ULRevitaleR )sllecdetcefsnartnoc-BERCAfo%( 0 40 80 120 160 200 VCAM gene promoter P-selectin gene promoter Fig. 7. Abolition of LPC induction of AMs by cotransfection of ACREB. The involvement of CREB in LPC-induced AM activation was evaluated by cotransfection with the CREB dominant negative vector ACREB. Cells were transfected with ACREB expression vec- tor, ACREB control vector luciferase reporter, P-selectin gene pro- moter or VCAM-1 gene promoter plasmids as indicated. After incubation for 30 h, cells were treated with LPC (12.5 l M) for an additional 8 h, and lysed for determination of luciferase activity. Each value is expressed as the mean ± SE from six independent experiments. Statistical significance: *P<0.05, **P < 0.01 versus cells transfected with ACREB control vector and not challenged with LPC. LPC - + - + + + - - - - + + hGPR4 hGPR4 (con) A ULR evitaleR )lortnoc detcefsnart noc-4RPGh fo %( 0 # ** 200 400 600 800 1000 1200 VCAM gene promoter P-selectin gene promoter B LPC - + - + + + - - - - + + hGPR4 hGPR4 (con) ULR evi taleR )lortnoc detcefsnart no c-4RPGh fo %( ## ** ** 0 50 100 150 200 250 300 350 400 450 VCAM gene promoter CRE luciferase reporter Fig. 8. Enhancement of LPC-induced AM activation by transfection of GPR4. The effect of GPR4 on LPC-induced AM activation was evaluated by transfection of human GPR4 expression vector (hGPR4) to YPEN-1 cells (A) and HEK293T cells (B) before treat- ment with LPC (12.5 l M). Cells were transfected with hGPR4 vec- tor, hGPR4 control vector, P-selectin gene promoter or VCAM-1 gene promoter plasmids as indicated. Thirty hours later, after transfection, cells were treated with LPC (12.5 l M) for an addi- tional 8 h, and lysed for determination of luciferase. Each value is expressed as the mean ± SE from six independent experiments. Statistical significance: **P<0.01 versus cells transfected with hGPR4 control vector without LPC challenge. # P < 0.05 versus cells transfected with hGPR4 expression vector without LPC challenge. Y. Zou et al. GPR4 contributes to LPC-induced AMs FEBS Journal 274 (2007) 2573–2584 ª 2007 The Authors Journal compilation ª 2007 FEBS 2579 Discussion The current study revealed the molecular mechanisms underlying the effects of LPC on the upregulation of AMs. Our data suggested the existence of two path- ways, through NF-jB or through GPR4, emphasizing the involvement of GPR4 in LPC-induced AMs, thereby providing a new insight into the molecular mechanism of LPC’s role in the expression of AMs in ECs. The LPC-induced increase of NF-jB DNA-binding activity was reported previously [30–32]; however, the upstream signal is not known, although PKC [10] is suggested to be involved. From the current data, the onset of NF-jB activation by LPC in ECs seems to be immediate, as indicated by the rapid nuclear transloca- tion of p65, 10 min after challenge with LPC (Fig. 2A); therefore, an early PKC (5 min) response would be expected [33]. Detailed information on the activation of NF-jB signaling by LPC was not sought in the present study, but we were able to confirm the activation of NF-jB in ECs by LPC and its important role in the induction of VCAM-1 and P-selectin by LPC. It is worth mentioning the involvement of react- ive oxygen species in NF-jB activation. NF-jB, being redox-sensitive, has been shown to be influenced by an increase in reactive oxygen species that would disrupt the redox balance [34]. Thus, it remains to be deter- mined whether LPC influences NF-jB activity by increased reactive oxygen species production, e.g. through various oxidases [35–37] or by other pathways such as PKC [10]. What is new in the current study are our data show- ing the involvement of LPC-activated CREB signaling in the upregulation of AMs. LPC-induced activation of CREB [38–40] and an elevation in the level of cAMP in neutrophils [41] have been reported. Rikitake et al. suggest that both p38 and ERK may function as upstream signaling pathways capable of activating CREB and activating transcription factor-1 with subse- quent induction of cyclooxygenase-2 expression by LPC [12]. However, here we did not find that the p38 inhibitor SB 203580 or the ERK inhibitor PD98059 influenced LPC-induced expression of AMs (data not shown). In the current study, we found that GPR4 played an important role in LPC-induced expression of AMs in ECs. Our findings on the expression of GPR4 but not G2A in ECs, shown in Fig. 5, are in agreement with a recent finding of a critical role of GPR4 in endothelial cell function, reported by Kim et al. [22]. As shown in Fig. 8A, the overexpression of GPR4 enhanced the response of YPEN-1 cells to LPC, leading to addi- tional activation of VCAM-1. More convincing evi- dence shows that after transfection with the GPR4 gene, HEK293T cells gain the ability to respond to LPC by expressing VCAM-1 and activating CRE sign- aling, as shown in Fig. 8B. Taken together, these results strongly indicate the existence of receptor-medi- ated signaling in LPC-induced AMs. In a recent study, GPR119 was shown to regulate LPC-induced upregu- lation of cAMP in pancreatic b-cells, resulting in the secretion of insulin [15]. We are currently examining the possible participation of GPR119 in LPC-induced AM expression. The data from the present study indicated that downstream signaling following LPC-induced GPR4 activation was via the cAMP ⁄ PKA ⁄ CRE pathway. These findings are consistent with previous reports showing that GPR4 elicits cAMP in Swiss 3T3 cells [23], activates the cAMP ⁄ PKA pathway in Cryp- tococcus neoformans [25], and stimulates CRE-driven transcription [42]. The role of CREB in LPC-induced AMs was previously unknown. An early study repor- ted the inability of LPC to induce transcription of VCAM-1 in HUVECs [43], probably because the reporter plasmid that the authors used was encoded with a partial promoter region of VCAM-1 (0 ⁄ ) 755 bp) without a CRE () 1420 bp) site, although it did contain NF-jB- and AP-1-binding sites. In contrast, our present study clearly showed that LPC induced AM expression though CREB. The expression of CREB’s dominant-negative control, ACREB, was able to abolish LPC-induced activation of AMs (Fig. 7). Therefore, the participation of LPC ⁄ GPR4 ⁄ cAMP ⁄ PKA signaling in ECs may well contribute to AM expression through activation of the CRE site. One question left unresolved in the present study is whether the LPC ⁄ NF-jB ⁄ AM and LPC⁄ GPR4 ⁄ cAMP ⁄ PKA ⁄ CREB ⁄ AM pathways work equally, one pathway works preferentially over the other, or a there is cross-talk between the two pathways. Further studies should be able to answer this question, and elucidate other factors that may regulate the oxidative stress- induced and receptor-mediated pathways. In conclusion, from the current study, we were able to document two pathways by which LPC regulates the expression of AMs ) the LPC ⁄ NF- jB ⁄ AM and LPC ⁄ GPR4 ⁄ cAMP ⁄ PKA ⁄ CREB ⁄ AM pathways. Our new findings emphasize the import- ance of the LPC receptor in regulating EC function, and highlight the potential critical roles of LPC in modulating many pathophysiologic processes, including atherosclerosis. GPR4 contributes to LPC-induced AMs Y. Zou et al. 2580 FEBS Journal 274 (2007) 2573–2584 ª 2007 The Authors Journal compilation ª 2007 FEBS Experimental procedures Materials LPC (synthetic palmitoyl-LPC 16:0) was purchased from Avanti Polar Lipid Inc. (Alabaster, AL, USA) CAPE was purchased from BioMol (Plymouth Meeting, PA, USA). NAC, MDL, H89, isobutylmethylxanthine and FSK were obtained from Sigma Chemical Co. (St Louis, MO, USA). Cells Cell culture The rat endothelial cell line YPEN-1 was obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in DMEM (Gibco, Grand Island, NY, USA) containing 10% heat-inactive fetal bovine serum (Mediatech, Herndon, VA, USA), glutamine 233.6 mgÆmL )1 , penicillin–streptomycin 72 mgÆmL )1 , and amphotericin B 0.25 mgÆmL )1 , and were adjusted to pH 7.4–7.6 with NaHCO 3 in a CO 2 incubator with an atmosphere of 5% CO 2 at 37 °C. Before treatment, cells were exchanged into fresh DMEM containing 1% fetal bovine serum. For transfection experiments with hGPR4 expression vec- tor, HEK293T cells were obtained from the ATCC and maintained in DMEM supplemented with 10% fetal bovine serum and other supplements as described above. Cell viability assay Cell viability was tested using a 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) assay, which is a sensitive, quantitative colorimetric assay that measures cell viability on the basis of the ability of mitochondrial succinyl dehydrogenase in living cells to convert the yellow substrate MTT into a dark blue formazan product. For the assay, the medium was removed, and a solution containing 0.01% MTT was added to each well; this was followed by incuba- tion at 37 °C for 4 h, and the formazan was dissolved in ethanol ⁄ dimethylsulfoxide (1 : 1, v ⁄ v). The plate was shaken for 5 min, and the absorbance was measured at 560 nm. Cell sample preparation Following incubation for 12 h or where indicated, cells were harvested and lysed in lysis buffer A (10 mm Tris, pH 8.0, 1.5 mm MgCl 2 ,1mm dithiothreitol, 0.1% Nonidet P-40, and protease inhibitors). After centrifugation at 13 000 g for 15 min (Mega 17R, Hanil Science Industrial Co. Ltd., Inchon, Korea, A1.55-24), the supernatant was regarded as the cytosol fraction for assays. The pellets were resuspended in 10 mm Tris (pH 8.0), with 50 mm KCl, 100 mm NaCl, and protease inhibitors, incubated on ice for 30 min, and then centrifuged at 13 000 g at 4 °C for 30 min (Mega 17R, Hanil, A1.55-24). After that, supernatants were used as the nuclear fraction. Western blot For western blotting analysis, cell samples were separated on an SDS ⁄ PAGE mini-gel. The antibodies used in this study were as follows: anti-(phosphorylated CREB) (anti- pCREB) was from Upstate (Charlottesville, VA, USA), and all other antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody labeling was detected using an ECL detection kit (Amersham Life Science, Inc., Arlington Heights, IL, USA). A prestained blue protein marker was used for molecular weight determination. The amount of protein was measured with a Sigma protein assay reagent kit containing bicinchoninic acid. RT-PCR Total RNA from cells or tissue was isolated by a previously described method [44]. Briefly, after treatment, cells were lysed in the presence of RNAzolB (1 mL per 100 mm dish). Chloroform (0.1 mL per 1 mL of lysate) was added to each dish. The samples were shaken vigorously, and then placed on ice for 5 min. After centrifugation twice at 12 000 g at 4 °C for 15 min (Mega 17R, Hanil, A1.55-24), the superna- tant was removed. The RNA pellet was washed with 75% ethanol, dried, and redissolved in diethylpyrocarbonate-trea- ted water. cDNA was synthesized using ImProm-II reverse transcriptase (Promega, Madison, WI). PCR was carried out using a standard protocol. Primers were designed for AMs as follows: P-selectin, sense strand 5¢-CGA CGT GGA CCT ATA ACT AC-3¢ and antisense strand 5¢-CCA CAC TCT TGG ACG TAT TC-3¢; VCAM-1, sense strand 5¢-CTT GGA GAA CCC AGA TAG AC-3¢ and antisense strand 5¢-CAG AAA ATC TCA GGA GCT GG-3¢; GPR4, sense strand 5¢-AGC ATT GCA GAC CTG CTG TA-3¢ and antisense strand 5¢-ATG GTA AGG CGC AAA GCA CA-3¢; G2A, sense strand 5¢-ACC AAT GCA GCA GGA AAC ACC A-3¢ and antisense strand 5¢-AAG CCA AAG GTG AAA CGC AGG T-3¢. Glyceraldehyde-3- phosphate dehydrogenase was used as internal control. Transient transfection and luciferase reporter assay VCAM-1 and P-selectin promoter activity induced by LPC was examined using luciferase plasmid VCAM-Luc (provi- ded by W Aird, Harvard Medical School, MA, USA), which contains a human VCAM-1 promoter region spanning ) 1716 to + 119 bp, and P-selectin -Luc (R P McEver, Uni- versity of Oklahoma Health Sciences Center, OK, USA) containing wild-type murine P-selectin promoter. Activation of NF-jB and CRE was measured using NF-jB luciferase Y. Zou et al. GPR4 contributes to LPC-induced AMs FEBS Journal 274 (2007) 2573–2584 ª 2007 The Authors Journal compilation ª 2007 FEBS 2581 reporter (Invitrogen, Carlsbad, CA, USA) and CRE luci- ferase reporter (H. Inoue, National Cardiovascular Center Research Institute, Osaka, Japan), respectively. Expression vector ACREB (C Vinson, National Cancer Institute, NIH, Rockville, MD, USA) and hGPR4 (K. Seuwen, Novartis Institutes for BioMedical Research, Basel, Switzerland) and their controls were used as described below. Transient transfection was carried out using FuGene 6 (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s instructions. After transfec- tion, cells were treated with reagents as per the experimental design. Briefly, YPEN-1 cells were seeded into 48-well plates (1 · 10 5 cellsÆmL )1 , and 250 lL per well), and cultured in DMEM containing 10% fetal bovine serum overnight. For transfection, the cells should be over 90% confluent. For single transfection, plasmid (0.1 lg per well) was used, and for cotransfection, plasmids were mixed in a 1 : 1 ratio to a total amount of 0.1 lg per well. Following transfection, cells were cultured for 24 h, and then exposed to DMEM con- taining 1% fetal bovine serum with ⁄ without designated rea- gents for an additional 8 h. Luciferase activity was measured with the Steady-Glo Luciferase Assay System (Promega) and detected by luminometer GENios Plus (Tecan Group Ltd, Salzburg, Austria). The obtained raw luciferase activit- ies were normalized by protein concentration per well. Measurement of cAMP The accumulation of cAMP in the YPEN-1 cells was induced by LPC at different concentrations in serum-free medium at 37 °C for 15 min in the presence of 1 mm iso- butylmethylxanthine. The cells were then lysed with 1 m HCl. Quantitative determination of the cAMP concentra- tion in cell lysates was performed using a cAMP (low pH) immunoassay kit (R&D Systems, Minneapolis, MN, USA). All procedures followed the user’s manual provided with the enzyme immunoassay kit. Statistical analysis For western blot analysis or RT-PCR, one representative blot was used from three independent experiments. Image analysis was performed using the imagej (NIH, Bethesda, MD, USA) program. anova was conducted to analyze sig- nificant differences among all groups. Differences among the means of individual groups were assessed by Fischer’s Protected LSD post hoc test. Values of P<0.05 were con- sidered to be statistically significant. Acknowledgements This study was supported by a grant from the Korea Health R&D project, Ministry of Health and Welfare, Republic of Korea (A050166). We thank the Aging Tissue Bank for distributing the aged tissue (R21-2005- 000-10008-0). References 1 Aiyar N, Disa J, Ao Z, Ju H, Nerurkar S, Willette RN, Macphee CH, Johns DG & Douglas SA (2007) Lyso- phosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells. Mol Cell Biochem 295, 113–120. 2 Kougias P, Chai H, Lin PH, Lumsden AB, Yao Q & Chen C (2006) Lysophosphatidylcholine and secretory phospholipase A2 in vascular disease: mediators of endothelial dysfunction and atherosclerosis. Med Sci Monit 12, RA5–RA16. 3 Ryborg AK, Gron B & Kragballe K (1995) Increased lysophosphatidylcholine content in lesional psoriatic skin. Br J Dermatol 133, 398–402. 4 Mehta D, Gupta S, Gaur SN, Gangal SV & Agrawal KP (1990) Increased leukocyte phospholipase A2 activ- ity and plasma lysophosphatidylcholine levels in asthma and rhinitis and their relationship to airway sensitivity to histamine. Am Rev Respir Dis 142, 157–161. 5 Portman OW & Alexander M (1969) Lysophosphatidyl- choline concentrations and metabolism in aortic intima plus inner media: effect of nutritionally induced athero- sclerosis. J Lipid Res 10, 158–165. 6 Vainer B (1997) Role of cell adhesion molecules in inflammatory bowel diseases. Scand J Gastroenterol 32, 401–410. 7 Kume N, Cybulsky MI & Gimbrone MA Jr (1992) Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest 90, 1138–1144. 8 Kita T, Kume N, Ochi H, Nishi E, Sakai A, Ishii K, Nagano Y & Yokode M (1997) Induction of endothelial platelet-derived growth factor-B-chain and intercellular adhesion molecule-1 by lysophosphatidylcholine. Ann NY Acad Sci 811, 70–75. 9 Thukkani AK, McHowat J, Hsu FF, Brennan ML, Hazen SL & Ford DA (2003) Identification of alpha- chloro fatty aldehydes and unsaturated lysophosphati- dylcholine molecular species in human atherosclerotic lesions. Circulation 108, 3128–3133. 10 Sugiyama S, Kugiyama K, Ogata N, Doi H, Ota Y, Ohgushi M, Matsumura T, Oka H & Yasue H (1998) Biphasic regulation of transcription factor nuclear factor-kappaB activity in human endothelial cells by lysophosphatidylcholine through protein kinase C-mediated pathway. Arterioscler Thromb Vasc Biol 18, 568–576. 11 Fang X, Gibson S, Flowers M, Furui T, Bast RC Jr & Mills GB (1997) Lysophosphatidylcholine stimulates GPR4 contributes to LPC-induced AMs Y. Zou et al. 2582 FEBS Journal 274 (2007) 2573–2584 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... (2005) Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor Biochem Biophys Res Commun 326, 744–751 Zou Y, Yoon S, Jung KJ, Kim CH, Son TG, Kim MS, Kim YJ, Lee J, Yu BP & Chung HY (2006) Upregulation of aortic adhesion molecules during aging J Gerontol Biol Sci Med Sci 61, 232–244 Zou Y, Jung KJ, Kim JW, Yu BP & Chung HY (2004) Alteration of soluble... 10068–10077 Minami T & Aird WC (2001) Thrombin stimulation of the vascular cell adhesion molecule-1 promoter in endothelial cells is mediated by tandem nuclear factor-kappa B and GATA motifs J Biol Chem 276, 47632–47641 Spagnuolo G, D’Anto V, Cosentino C, Schmalz G, Schweikl H & Rengo S (2006) Effect of N-acetyl-Lcysteine on ROS production and cell death caused by HEMA in human primary gingival fibroblasts Biomaterials... Ca2+ currents via lysophosphatidylcholine- induced mitochondrial reactive oxygen species (ROS) production Cardiovasc Res 69, 855–864 36 Watanabe N, Zmijewski JW, Takabe W, Umezu-Goto M, Le Goffe C, Sekine A, Landar A, Watanabe A, Aoki J, Arai H et al (2006) Activation of mitogenactivated protein kinases by lysophosphatidylcholineinduced mitochondrial reactive oxygen species generation in endothelial cells... 6299–6303 43 Zhu Y, Lin JH, Liao HL, Verna L & Stemerman MB (1997) Activation of ICAM-1 promoter by lysophosphatidylcholine: possible involvement of protein tyrosine kinases Biochim Biophys Acta 1345, 93–98 44 Kim JW, Zou Y, Yoon S, Lee JH, Kim YK, Yu BP & Chung HY (2004) Vascular aging: molecular modulation of the prostanoid cascade by calorie restriction J Gerontol Biol Sci Med Sci 59, B876–B885 FEBS Journal... Ryborg AK, Johansen C, Iversen L & Kragballe K (2004) Lysophosphatidylcholine induces keratinocyte differentiation and upregulation of AP-1- and NFkappaB DNA-binding activity Acta Derm Venereol 84, 433–438 31 Takeya H, Gabazza EC, Aoki S, Ueno H & Suzuki K (2003) Synergistic effect of sphingosine 1-phosphate on thrombin-induced tissue factor expression in endothelial cells Blood 102, 1693–1700 32 Masamune... 61, 232–244 Zou Y, Jung KJ, Kim JW, Yu BP & Chung HY (2004) Alteration of soluble adhesion molecules during aging and their modulation by calorie restriction FASEB J 18, 320–322 Pan J, Xia L, Yao L & McEver RP (1998) Tumor necrosis factor-alpha- or lipopolysaccharide-induced expression of the murine P-selectin gene in endothelial cells involves novel kappaB sites and a variant activating transcription... endothelial cells Am J Pathol 168, 1737–1748 37 Jia SJ, Jiang DJ, Hu CP, Zhang XH, Deng HW & Li YJ (2006) Lysophosphatidylcholine- induced elevation of asymmetric dimethylarginine level by the NADPH oxidase pathway in endothelial cells Vascul Pharmacol 44, 143–148 38 Sheikh AM, Ochi H & Masuda J (2005) Lysophosphatidylcholine induces tPA gene expression through CREdependent mechanism Biochem Biophys Res... (1999) Lysophosphatidylcholine phosphorylates CREB and activates the jun2TRE site of c-jun promoter in vascular endothelial cells FEBS Lett 457, 241–245 2584 40 Nawa T, Nawa MT, Adachi MT, Uchimura I, Shimokawa R, Fujisawa K, Tanaka A, Numano F & Kitajima S (2002) Expression of transcriptional repressor ATF3 ⁄ LRF1 in human atherosclerosis: colocalization and possible involvement in cell death of vascular... Cryptococcus neoformans Mol Biol Cell 17, 667–679 26 Beavo JA & Brunton LL (2002) Cyclic nucleotide research ) still expanding after half a century Nat Rev Mol Cell Biol 3, 710–718 27 Inoue H, Yokoyama C, Hara S, Tone Y & Tanabe T (1995) Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells Involvement of both nuclear... mechanism underlying COX-2 induction by lysophosphatidylcholine Biochem Biophys Res Commun 281, 1291–1297 Xu Y, Xiao YJ, Zhu K, Baudhuin LM, Lu J, Hong G, Kim KS, Cristina KL, Song LS, Williams F et al (2003) Unfolding the pathophysiological role of bioactive lysophospholipids Curr Drug Targets Immune Endocr Metabol Disord 3, 23–32 Xu Y (2002) Sphingosylphosphorylcholine and lysophosphatidylcholine: G protein-coupled . Upregulation of endothelial adhesion molecules by lysophosphatidylcholine Involvement of G protein-coupled receptor GPR4 Yani Zou 1 , Chul. protein pathway, resulting in upregulation of adhesion molecules. Further evidence showed that overexpression of human GPK4 enhanced lysophosphatidyl- choline-induced expression of adhesion molecules in YPEN-1. express adhesion molecules in response to lysophosphatidylcholine. In conclusion, the current study suggested two pathways by which lysophosphatidylcholine regulates the expression of adhesion molecules,