Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflammation via adhesion molecules induction, and then causes lung injury. However, the mechanisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these responses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was mediated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrated that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation.


Introduction
Lung inflammation is a pivotal event in the pathogenesis of chronic obstructive pulmonary disease and asthma. These inflammatory responses are mediated by complex interactions between both circulating polymorphonuclear cells (PMNs) and the vascular endothelium. Several studies indicate that expression of adhesion molecules on the cell surface of endothelial cells plays a critical role in the inflammatory responses [1]. Raised levels of adhesion molecules might contribute to the recruitment of PMNs to the regions of inflammatory tissue. These adhesion molecules are classified into two major families: the Ig superfamily (e.g., ICAM-1 and VCAM-1) and the selectins (e.g., P-selectin and E-selectin) [2]. ICAM-1 is an inducible cell surface glycoprotein on several cell types, which mediates the tight adhesiveness of PMNs and thus facilitates PMNs migration across the vascular endothelium barrier and then interacts with lung epithelium [3].
Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite that plays important roles in allergic responses, including asthma and anaphylaxis [4]. S1P regulates numerous cellular responses, including motility and cytoskeletal rearrangements, formation of adherens junctions, proliferation, survival, angiogenesis, and the trafficking of immune cells [5]. These myriad effects are partly elicited by binding of S1P to a family of five G protein-coupled receptors (S1PRs), termed S1PR1-5. Moreover, S1P has been shown to induce lung injury and inflammation [6]. In addition, S1P has been also shown to induce ICAM-1 or VCAM-1 expression in various cell types [7,8]. However, the mechanisms of S1P-regulated ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) are not completely understood. Thus, to clarify the mechanisms of ICAM-1 induction by S1P in lung epithelium was recognized as a new therapeutic approach in the management of respiratory diseases.
c-Src, a common modular participating in the crosstalk between the cytoplasmic protein tyrosine kinases and receptors, has been shown to mediate ICAM-1 expression in various cell types [9,10]. On the other hand, previous studies indicated that c-Src regulates platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) transactivation [11], which further promotes inflammatory responses. Mitogen-activated protein kinases (MAPKs) are important components of signaling modules activated by neurotransmitters, cytokines, and growth factors, as well as chemical and mechanical stressors. MAPKs are also implicated in S1P-induced inflammatory responses [12,13]. Recent studies suggested that numerous components of the PI3K/Akt pathway play a crucial role in the expression and activation of inflammatory mediators, inflammatory cell recruitment, immune cell function, airway remodeling, and corticosteroid insensitivity in chronic inflammatory respiratory diseases [2]. Indeed, previous studies indicated that PI3K/Akt regulates the expression of adhesion molecules in various cell types [10,14]. S1P has been shown to enhance Akt activation [15,16]. Although these studies have demonstrated that ICAM-1 induction was regulated via various signaling components, whether these signalings also participated in ICAM-1 expression and monocyte adhesion on HPAEpiCs challenged with S1P remains unknown.
The ICAM-1 promoter has been shown to contain several binding sequences for various transcription factors, including AP-1 [17]. AP-1 is a heterogeneous collection of dimeric transcription factors comprising Jun, Fos, and ATF subunits. Among AP-1 subunits, c-Jun is the most important transcriptional activator in inflammatory status [2]. AP-1 activity is regulated by multiple mechanisms, including phosphorylation by various MAPKs [18]. Thus, in this study, we also investigated the role of AP-1 in ICAM-1 expression in HPAEpiCs challenged with S1P.
In addressing these questions, experiments were undertaken to investigate the effects of S1P on expression of ICAM-1 and monocyte adhesion on HPAEpiCs. These findings suggest that the increased expression of ICAM-1 and monocyte adhesion on S1P-challenged HPAEpiCs are mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation. These results provide new insights into the mechanisms of S1P action on HPAEpiCs to regulate the expression of ICAM-1 and thus exaggerate the inflammation responses.

Western blot
Growth-arrested cells were incubated with S1P at 37°C for the indicated time intervals. The cells were washed, scraped, collected, and centrifuged at 45000 × g at 4°C for 1 h to yield the whole cell extract, as previously described [19]. Samples were denatured, subjected to SDS-PAGE using a 10% running gel, and transferred to nitrocellulose membrane. Membranes were incubated with an anti-ICAM-1 antibody for 24 h, and then membranes were incubated with an anti-rabbit horseradish peroxidase antibody for 1 h. The immunoreactive bands were detected by ECL reagents.

Real-time PCR
Total RNA was extracted using TRIzol reagent. mRNA was reverse-transcribed into cDNA and analyzed by real-time PCR using SYBR Green PCR reagents (Applied Biosystems, Branchburg, NJ) with primers specific for ICAM-1, c-Fos, c-Jun, and GAPDH. The levels of ICAM-1, c-Fos, and c-Jun expression were determined by normalizing to that of GAPDH expression.

RT-PCR analysis
Total RNA was isolated using TRIzol according to the protocol of the manufacturer. The power field well using a fluorescence microscope (Zeiss, Axiovert 200M). Experiments were performed in triplicate and repeated at least three times.

Co-immunoprecipitation assay
Cell lysates containing 1 mg of protein were incubated with 2 μg of an anti-c-Src antibody at 4°C for 24 h, and then 10 μl of 50% protein A-agarose beads was added and mixed at 4°C for 24 h. The immunoprecipitates were collected and washed thrice with a lysis buffer without Triton X-100. 5X Laemmli buffer was added and subjected to electrophoresis on SDS-PAGE, and then blotted using an anti-EGFR, anti-PDGFR, or anti-c-Src antibody.

Luciferase activity assay
The human ICAM-1 (pIC-339) firefly luciferase was kindly provided by Dr. P. T. van der Saag (Hubrecht Laboratory, Utrecht, The Netherlands). All plasmids were prepared by using QIA-GEN plasmid DNA preparation kits. ICAM-1-luc activity was determined as previously described using a luciferase assay system (Promega, Madison, WI) [3].
Analysis of data. All the data were expressed as the mean or mean±S.E.M. The data were analyzed using a GraphPad Prism Program (GraphPad, San Diego, CA) by one-way analysis of variance (ANOVA) followed with Tukey's post-hoc test. A P<0.05 value was considered significant.

S1P induces ICAM-1-dependent monocyte adhesion
To investigate the effect of S1P on ICAM-1 expression, HPAEpiCs were incubated with various concentrations of S1P for the indicated time intervals. As shown in Fig. 1A, S1P induced ICAM-1 expression in a time-and concentration-dependent manner. In addition, S1P also enhanced ICAM-1 mRNA expression and promoter activity in a time-dependent manner in HPAEpiCs ( Fig. 1B and C). Finally, we demonstrated that adhesion of THP-1 to HPAEpiCs challenged with S1P was enhanced, which was inhibited by an ICAM-1 neutralizing antibody but not by anti-IgG Ab (Fig. 1D). Taken together, we suggest that S1P induces monocyte adhesion via an ICAM-1-dependent pathway in HPAEpiCs. S1PR1 and S1PR3 play key roles in S1P-induced ICAM-1 expression S1PR1, S1PR2, and S1PR3 are ubiquitously expressed, whereas the levels of S1PR4 and S1PR5 expression are predominantly existed in immune cells, CNS, and some organs [5]. Which types of S1P receptors expressed on HPAEpiCs are still unclear. Therefore, we identified the expression of S1P receptors on HPAEpiCs. As shown in Fig. 2A, S1PR1, S1PR2, and S1PR3 are expressed on HPAEpiCs, determined by RT-PCR. On the other hand, we observed that pretreatment with the selective inhibitor of S1PR1 (W123) or S1PR3 (CAY10444) markedly reduced S1P-induced ICAM-1 protein levels (Fig. 2B). Indeed, pretreatment with the inhibitor of S1PR2 (JTE-013) had no effect on S1P-induced ICAM-1 expression (data not shown). In addition, pretreatment with W123 or CAY10444 markedly inhibited S1P-induced ICAM-1 mRNA levels and promoter activity (Fig. 2C). These two inhibitors also attenuated the monocyte adhesion to HPAEpiCs challenged with S1P (Fig. 2D). To further confirm the roles of S1PR1 and S1PR3 in S1P-induced ICAM-1 expression, siRNA of S1PR1, S1PR2, or S1PR3 was used. As shown in Fig. 2E, transfection with S1PR1 and S1PR3 but not S1PR2 siRNA significantly reduced S1P-induced ICAM-1 expression in HPAEpiCs. Thus, we demonstrate that S1PR1 and S1PR3 play critical roles in S1P-induced ICAM-1 expression in HPAEpiCs and monocyte adhesion.
c-Src plays a critical role in S1P-induced ICAM-1 expression c-Src, a common modulator participating in the crosstalk between the cytoplasmic protein tyrosine kinases and receptors, has been shown to mediate ICAM-1 expression in various cell types [9,10]. Thus, we investigated whether c-Src was involved in S1P-induced ICAM-1 expression in HPAEpiCs. As shown in Fig. 3A and B, pretreatment with the inhibitor of c-Src (PP1) markedly reduced S1P-induced ICAM-1 protein and mRNA expression and promoter activity. We confirmed the role of c-Src in S1P-induced ICAM-1 expression in HPAEpiCs by using c-Src siRNA. As shown in Fig. 3C, transfection with c-Src siRNA markedly reduced the c-Src protein expression, and then inhibited S1P-induced ICAM-1 expression. In addition, c-Src inhibition by PP1 also attenuated monocyte adhesion to HPAEpiCs challenged with S1P ( Fig. 3D). Finally, we observed that S1P could enhance c-Src phosphorylation in a time-  scrambled, S1PR1, S1PR2, or S1PR3, and then incubated with S1P (10 μM) for 16 h. The levels of S1PR1, S1PR2, S1PR3, and ICAM-1 proteins were determined by Western blot. Data are expressed as mean (E) or mean±S.E.M. (B, C, and D) of three independent experiments. *P<0.05; # P<0.01, as compared with the cells exposed to S1P alone (B, C, and D) or transfected with siRNA of scrambled+S1P (E).

S1P induces ICAM-1 expression via EGFR and PDGFR activation
Previous studies indicated that c-Src regulates platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) transactivation [11], which further promotes the expression of inflammatory genes. Thus, we investigated whether EGFR or PDGFR was involved in S1P-induced ICAM-1 expression in HPAEpiCs. As shown in Fig. 4A and B, pretreatment with the inhibitor of EGFR (AG1478) or PDGFR (AG1296) markedly reduced S1P-induced ICAM-1 protein expression, mRNA levels, and promoter activity. We confirmed the role of EGFR or PDGFR in S1P-induced ICAM-1 expression in HPAEpiCs by using siRNA of EGFR or PDGFR. As shown in Fig. 4C, transfection with EGFR or PDGFR siRNA markedly reduced the respective protein expression, and then inhibited S1P-induced ICAM-1 expression. We observed that S1P could enhance EGFR or PDGFR phosphorylation in a timedependent manner in these cells, which was inhibited by AG1478 or AG1296, respectively. We further investigated the relationship of c-Src, EGFR, and PDGFR in S1P-stimulated HPAE-piCs. As shown in Fig. 4E, we found that pretreatment with PP1 markedly reduced S1P-stimulated EGFR and PDGFR phosphorylation, suggesting that c-Src plays as an upstream molecule in regulating S1P-stimulated EGFR and PDGFR phosphorylation. Finally, we investigated the physical association of EGFR, PDGFR, and c-Src in S1P-stimulated HPAEpiCs by immunoprecipitation using an anti-c-Src, anti-EGFR, or anti-PDGFR antibody and Western blot. As shown in Fig. 4F, we observed that S1P time-dependently induced the formation of a c-Src/ EGFR/PDGFR complex in these cells. Taken together, we suggest that S1P-induced ICAM-1 expression is mediated through a c-Src/EGFR or a c-Src/PDGFR pathway in HPAEpiCs.

S1P induces ICAM-1 expression via MAPKs
MAPK cascades are highly conserved signaling modules downstream of receptors/sensors that relay extracellular stimuli into intracellular responses in eukaryotes. MAPKs also have been shown to regulate S1P-induced inflammatory responses [12,13]. In this study, we found that pretreatment with the inhibitor of p38 MAPK (SB202190), MEK1/2 (U0126), or JNK1/2 (SP600125) significantly reduced S1P-induced ICAM-1 protein and mRNA expression and promoter activity ( Fig. 5A and B). In addition, all these three inhibitors could attenuate monocyte adhesion to HPAEpiCs challenged with S1P (Fig. 5C). We further used siRNA of p38, p42, or JNK1 to confirm the roles of MAPKs in S1P-induced ICAM-1 expression in HPAEpiCs. As shown in Fig. 5D, transfection with siRNA of p38, p42, or JNK1 down-regulated the expression of respective proteins and markedly reduced S1P-induced ICAM-1 expression in HPAEpiCs. To investigate whether S1P-induced ICAM-1 expression was mediated through MAPKs activation, the phosphorylation of p42/p44 MAPK, JNK1/2, or p38 MAPK was observed in S1Pstimulated HPAEpiCs. As shown in Fig. 5E, S1P markedly stimulated the phosphorylation of these three MAPKs in a time-dependent manner, which was reduced by their respective inhibitors. Thus, we demonstrated that S1P-induced ICAM-1 expression was mediated through a MAPKs signaling pathway in HPAEpiCs.

S1P induces c-Src/EGFR, PDGFR-dependent p42/p44 MAPK and p38 MAPK activation
Here, we investigated the relationship of c-Src, EGFR, PDGFR, and MAPKs in S1P-stimulated HPAEpiCs. As shown in Fig. 6A and B, S1P markedly induced p42/p44 MAPK and p38 MAPK phosphorylation in a time-dependent manner, which was inhibited by PP1, AG1478,  or AG1296 in HPAEpiCs. However, we found that pretreatment with PP1 was attenuated S1Pinduced JNK1/2 activation, but not AG1296 and AG1478 in these cells (Fig. 6C). To further confirm the roles of c-Src-dependent EGFR/PDGFR in S1P-induced MAPKs activation, siR-NAs of c-Src, EGFR, and PDGFR were used. As shown in Fig. 6D, transfection with siRNA of c-Src was significantly reduced S1P-induced MAPKs activation. However, S1P-induced phosphorylation of p42/p44 MAPK and p38 MAPK but not JNK was significantly reduced by transfection with siRNA of EGFR, or PDGFR. Thus, we demonstrated that S1P stimulated p38 MAPK and p42/p44 MAPK phosphorylation via a c-Src/EGFR and PDGFRdependent pathway. S1P induces ICAM-1 expression via PI3K/Akt in HPAEpiCs Previous studies indicated that PI3K/Akt regulates the expression of adhesion molecules in various cell types [10,14]. S1P has been shown to enhance Akt activation [15,16]. Thus, we investigated whether PI3K/Akt were involved in S1P-induced ICAM-1 expression in HPAEpiCs. In this study, we found that pretreatment with the inhibitor of PI3K (LY294002) significantly reduced S1P-enhanced ICAM-1 protein and mRNA expression and promoter activity ( Fig. 7A  and B). In addition, monocyte adhesion to HPAEpiCs challenged with S1P was also reduced by AG1478, AG1296, or LY294002 (Fig. 7C). We found that S1P markedly stimulated Akt phosphorylation in a time-dependent manner, which was inhibited by LY294002 (Fig. 7D). We further investigated the relationship of c-Src, EGFR, PDGFR, and Akt in S1P-stimulated HPAEpiCs. As shown in Fig. 7E, S1P-stimulated Akt phosphorylation was reduced by Genistein (an inhibitor of tyrosine protein kinases), PP1, AG1478, or AG1296 in these cells. On the other hand, we also observed the relationship between MAPKs and Akt in S1P-stimulated HPAEpiCs. As shown in Fig. 7F, we found that S1P-stimulated Akt phosphorylation was inhibited by SB202190 or U0126, but not SP600125. Indeed, we found that pretreatment with LY294002 had no effects on S1P-stimulated p42/p44 MAPK, JNK1/2, and p38 MAPK phosphorylation (data not shown). Thus, we suggested that S1P-stimulated Akt phosphorylation is mediated through p42/p44 MAPK and p38 MAPK in HPAEpiCs.

S1P induces ICAM-1 expression via AP-1 in HPAEpiCs
The ICAM-1 promoter has been shown to contain several binding sequences for various transcription factors, including AP-1 [17]. To investigate whether AP-1 was involved in S1P-induced ICAM-1 expression, the inhibitor of AP-1 (Tanshinone IIA) was used. As shown in Fig. 8A and B, pretreatment with Tanshinone IIA markedly reduced S1P-induced ICAM-1 protein and mRNA expression and promoter activity. In addition, Tanshinone IIA also attenuated monocyte adhesion to HPAEpiCs challenged with S1P (Fig. 8C). AP-1 is a heterogeneous collection of dimeric transcription factors comprising Jun, Fos, and ATF subunits. Among AP-1 subunits, c-Jun and c-Fos are the most important transcriptional activators in inflammatory status. In this study, we found that S1P markedly induced c-Fos, but not c-Jun mRNA expression in these cells (Fig. 8D). To further confirm the roles of c-Jun and c-Fos in S1P-induced ICAM-1 expression in HPAEpiCs, as shown in Fig. 8E, transfection with c-Jun or c-Fos siRNA significantly reduced c-Jun or c-Fos protein expression, and then inhibited S1P-induced ICAM-1 expression in HPAEpiCs. Finally, we observed that S1P time-dependently stimulated c-Jun phosphorylation, which was reduced by PP1, AG1296, AG1478, U0126, SP600125, h. The levels of p38, p42, JNK1, and ICAM-1 proteins were determined by Western blot. (E) Cells were pretreated without or with U0126 (1 μM), SB202190 (10 M), or SP600125 (10 M) for 1 h, and then incubated with S1P (10 M) for the indicated time intervals. The levels of phospho-p42/p44 MAPK, phospho-p38 MAPK, and phospho-JNK1/2 were determined by Western blot. Data are expressed as mean (A, D) or mean±S.E.M (B, C) of three independent experiments. *P<0.05; # P<0.01, as compared with the cells exposed to S1P alone (A, B, C) or transfected with siRNA of scrambled+S1P (D).   Several studies have reported that c-Src is an essential component for cytokine-stimulated PDGFR or EGFR transactivation via the phosphorylation of cytoplasmic domains of EGFR or PDGFR [11,18]. The PDGF family of growth factors consists of five different disulphide-linked dimers built up of four different polypeptide chains encoded by four different genes. These isoforms, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD, act via two receptor tyrosine kinases, PDGF receptors α and β [18]. The classic PDGFs, PDGF-A and PDGF-B, undergo intracellular activation during transport in the exocytic pathway for subsequent secretion, while the novel PDGFs, PDGF-C and PDGF-D, are secreted as latent factors that require activation by extracellular proteases [18]. EGFR exists on the cell surface and is activated by binding of its specific ligands, including EGF and transforming growth factor-α (TGF-α). Moreover, activation of EGFR and PDGFR has been shown to induce respiratory system inflammation [1,2]. Here, we established that S1P In addition, we also demonstrated that S1P could induce the formation of a c-Src/EGFR/PDGFR complex in these cells. Although the detailed protein-protein interactions among c-Src, EGFR, and PDGFR are not known, our results are the first time to show a novel role of c-Src/EGFR/PDGFR complex formation in S1P-induced ICAM-1 expression in HPAE-piCs. In the future, we will further determine which domains of c-Src, EGFR, and PDGFR are involved in protein-protein interactions caused by S1P. The MAPKs regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, including p42/p44 MAPK, JNK1/2, and p38 MAPK [2]. MAPKs also have been shown to regulate S1P-induced inflammatory responses [12,13]. In addition, MAPKs also have been shown to regulate ICAM-1 induction and monocyte adhesion in response to various stimuli [3,17]. Indeed, in HPAEpiCs, we found that all these three MAPKs were involved in ICAM-1 expression and monocyte adhesion induced by S1P. Thus, we suggest that MAPKs play key roles in S1P-induced inflammatory responses. Lin et al. indicated that thrombininduced NF-κB activation is mediated by a c-Src-dependent p42/p44 MAPK pathway in lung epithelial cells [25]. Our group also showed that c-Src-dependent MAPKs/AP-1 activation is Fig 10. Proposed model to illustrate the signaling pathways involved in ICAM-1 expression and monocyte adhesion in HPAEpiCs challenged with S1P. S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR and PDGFR/p38 MAPK and p42/p44 MAPK/Akt-or S1PR1/3/JNK1/2-dependent AP-1 activation.
The PI3Ks are a conserved family of signal transduction enzymes that are involved in cellular activation, inflammatory responses, chemotaxis, and apoptosis. PI3K/Akt have been shown to be a downstream component of EGFR or PDGFR activated by different stimuli in various cell types [11,18]. This is confirmed by our observation that S1P-induced Akt phosphorylation was reduced through the inhibition of c-Src, EGFR, or PDGFR. On the other hand, we found that pretreatment with LY294002 inhibited S1P-induced ICAM-1 expression, consistent with the results obtained with that ICAM-1 expression was mediated via a PI3K/Akt cascade in IL-1β-challenged A549 cells [10]. PI3K/Akt has been shown to regulate MAPKs activation in response to various stimuli, such as Japanese encephalitis virus and TNF-α [18,26]. In this study, we found that S1P-induced MAPKs activation was not regulated via PI3K/Akt (data not shown). Interestingly, we observed that S1P-induced Akt phosphorylation was reduced by SB202190 or U0126, but not SP600125, suggesting that S1P induced p38 MAPK-or p42/p44 MAPK-dependent Akt activation in HPAEpiCs. Indeed, Takahashi et al. showed that VEGF may stimulate PI3K/Akt through activation of the PKC and p42/p44 MAPK pathway in hepatic stellate cells [27]. Shi et al. also indicated that inhibition of p38 MAPK decreases Akt phosphorylation in proteasome inhibitors-stimulated breast carcinoma cells [28]. Thus, we suggest that in HPAEpiCs, S1P-stimulated p38 MAPK and p42/p44 MAPK phosphorylation plays key roles in mediating Akt activation leading to ICAM-1 expression.
It has been well established that inflammatory responses following exposure to extracellular stimuli are highly dependent on activation of AP-1, which plays an important role in the expression of several target genes. The ICAM-1 promoter has been shown to contain several binding sequences for various transcription factors, including AP-1 [17]. These studies suggest that AP-1 plays a critical role in the regulation of ICAM-1 expression in the inflammatory responses. Moreover, we found that AP-1 inhibition could reduce S1P-induced ICAM-1 expression. In S1P-stimulated HPAEpiCs, c-Fos mRNA expression was up-regulated. In addition, S1P also stimulated c-Jun phosphorylation in these cells. However, S1P had no effects on c-Jun mRNA levels. Previous studies indicated that AP-1 is regulated by various signaling components, such as c-Src and MAPKs [17,18,26], consistent with our results indicating that in HPAEpiCs, S1P stimulated c-Jun phosphorylation via a c-Src/EGFR and PDGFR/p42/p44 MAPK and p38 MAPK/PI3K/Akt-or JNK1/2-dependent pathway.
In summary, as depicted in Fig. 10, our results showed that in HPAEpiCs, S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR and PDGFR/p38 MAPK and p42/p44 MAPK/Akt-or S1PR1/3/c-Src/JNK1/2-dependent AP-1 activation. These results provide new insights into the mechanisms of S1P-induced the expression of ICAM-1 and monocyte adhesion and thus exaggerate the inflammatory responses. Increased understanding of signaling mechanisms underlying ICAM-1 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.

Author Contributions
Conceived and designed the experiments: CCL ITL CKH CMY. Performed the experiments: CHH PLC CKH LDH. Analyzed the data: CCL ITL CKH. Contributed reagents/materials/ analysis tools: CHH PLC CKH LDH. Wrote the paper: CCL ITL CMY.