A crucial role of the inflammatory lipid sphingosine-1-phosphate (S1P) in breast cancer aggressiveness has been reported. Recent clinical studies have suggested that C-reactive protein (CRP) has a role in breast cancer development. However, limited information is available on the molecular basis for the expression of CRP and its functional significance in breast cell invasion. The present study aimed to elucidate the molecular link between S1P and CRP during the invasive process of breast epithelial cells. This is the first report showing that transcription of CRP was markedly activated by S1P in breast cells. Our data suggest that not only S1P treatment but also the endogenously produced S1P may upregulate CRP in breast carcinoma cells. Transcription factors CCAAT/enhancer-binding protein beta and c-fos were required for S1P-induced CRP expression. Coupling of S1P3 to heterotrimeric Gαq triggered the expression of CRP, utilizing signaling pathways involving reactive oxygen species (ROS), Ca2+ and extracellular signal-related kinases (ERKs). S1P-induced CRP expression was crucial for the transcriptional activation of matrix metalloproteinase-9 through ERKs, ROS and c-fos, leading to breast cell invasion. Using a xenograft mice tumor model, we demonstrated that S1P induced CRP expression both in vitro and in vivo. Taken together, our findings have revealed a molecular basis for S1P-induced transcriptional activation of CRP and its functional significance in the acquisition of the invasive phenotype of human breast epithelial cells under inflammatory conditions. Our findings may provide useful information on the identification of useful therapeutic targets for inflammatory breast cancer.
Breast cancer is one of the most commonly diagnosed types of cancer among women.1 Metastasis of tumor cells is the leading cause of mortality in breast cancer patients.2 Chronic inflammation contributes to cancer development and progression.3 A crucial link between inflammation and breast cancer progression has been suggested. Inflammation is associated with malignant progression and a poor prognosis of breast cancer.4, 5
The inflammatory marker C-reactive protein (CRP) is a major human acute-phase protein that is mainly synthesized by hepatocytes in response to various inflammatory stimuli.6 The risk of cancer is increased when pre-diagnostic CRP levels are high.7 Recent clinical studies suggest a close relationship between CRP and breast cancer. Elevated levels of serum CRP are associated with reduced survival among breast cancer patients8 and a poor breast cancer prognosis.9 Moreover, serum CRP levels correlated well with the extent of breast cancer invasion and metastasis.10 Despite the vast amount of clinical evidence for a possible role of CRP in the pathogenesis of breast cancer, an underlying molecular mechanism has not yet been identified.
A family of extracellular matrix-degrading enzymes, the matrix metalloproteinase (MMP), are implicated in inflammation and cancer.11 In particular, MMP-9 expression is associated with pathological processes, including inflammation, atherosclerosis and tumor-cell invasion and metastasis.12, 13, 14 Effects of CRP on MMPs have been reported in various cell systems. CRP induces the expression of MMP-1, -2, -9, and -10 in human endothelial cells, human vascular smooth muscle cells and mononuclear cells.15, 16, 17
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that binds to one of the G protein-coupled receptors, S1P1–S1P5.18 S1P is a potent mediator of inflammation,19 and its production is enhanced following inflammatory stimuli.20 Increasing evidence suggests a role of S1P in cell invasion and migration.21, 22 Laboratories, including ours, have suggested a crucial role for S1P in breast cancer progression.23, 24
To provide a molecular basis for the clinical importance of CRP in the malignant progression of breast cancer, the present study investigated whether S1P could induce the expression of CRP in breast cells. Here, we show for the first time that S1P induces transcriptional activation of CRP in MCF10A human breast epithelial cells, and that this involves the transcription factors CCAAT/enhancer-binding protein beta (C/EBPβ) and c-fos. We further demonstrate that Ca2+, ERKs and ROS pathways are required for S1P-induced CRP expression, which in turn is crucial for MMP-9 upregulation and breast cell invasion.
S1P induces transcriptional activation of CRP in MCF10A cells
To determine the effect of S1P on CRP expression in MCF10A cells, cells were treated with S1P and subjected to an immunoblot analysis for the detection of CRP. We treated the cells with 1–10 μM of S1P as it was previously shown that cellular responses upon S1P treatment were elicited by 0.1–10 μM of S1P.25 As shown in Figure 1a, the expression of CRP was significantly induced by S1P treatment in a dose-dependent manner. A kinetic study showed that CRP was significantly induced at 24 h after 10 μM S1P treatment. The S1P-induced CRP upregulation was markedly inhibited by treatment with FTY720, an antagonist of S1P.24, 26 S1P induced CRP expression at the transcriptional level as evidenced by RT–PCR analysis (Figure 1c) and luciferase reporter assays (Figure 1d). S1P treatment (up to 10 μM for 24 h) did not affect cell proliferation of MCF10A cells as evidenced by MTT assay (data not shown).
S1P is produced in cells by sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2.27 In particular, SphK1 is overexpressed in human breast cancer and has been implicated in cancer progression.23 We wanted to examine if SphK1 is increased in MDA-MB-231 breast carcinoma cells compared with MCF10A ‘normal’ breast epithelial cells. As shown in Figure 1e (left), the expression of SphK1 was highly detected in the MDA-MB-231 cells, whereas a low level of SphK1 was detected in MCF10A cells. Treatment of S1P also increased the CRP expression in MDA-MB-231 cells (Figure 1e, right). These results suggest that not only S1P treatment but also endogenously produced S1P may upregulate CRP in breast carcinoma cells.
C/EBPβ is a potential transcription factor responsible for S1P-induced transcriptional activation of CRP
To identify the potential transcriptional element(s) in the CRP promoter responsible for the transcriptional regulation by S1P, we examined the DNA-binding affinities of transcription factors C/EBPβ, nuclear factor kappa B (NF-κB) and c-fos, which have been reported to regulate the gene expression of CRP.28, 29 As shown in Figure 2a, S1P markedly increased the binding of C/EBPβ and, to a lesser degree, c-fos to the CRP promoter region in MCF10A cells as well as in MDA-MB-231 cells, whereas the binding of NF-κB was not affected in MCF10A cells. These data suggest that C/EBPβ and c-fos may be potential transcription factors for the S1P-activated transcription of the CRP gene.
The S1P-induced transcriptional activation of CRP was prominently decreased by a mutant construct of the CRP promoter lacking the C/EBPβ binding site at positions −53 to −49 (CRPΔC/EBPβ) (Figure 2b). A mutant construct lacking the p50-NF-κB binding site at positions −45 to −42 (CRPΔp50-NF-κB) did not significantly affect the CRP promoter activity. The overexpression of C/EBPβ resulted in a marked increase in the CRP promoter activity. These data clearly demonstrate that C/EBPβ is a transcription factor responsible for S1P-induced transcriptional activation of CRP.
We next examined the role of C/EBPβ and/or c-fos in S1P-induced CRP transcription. Small interfering RNA (siRNA) knockdown of C/EBPβ or c-fos significantly inhibited the S1P-induced mRNA expression of CRP (Figure 2c), demonstrating the functional significance of C/EBPβ and c-fos in CRP transcription induced by S1P.
C/EBPs often bind to the gene promoter as homo-or heterodimers with other C/EBP family members or other bZIP proteins, including c-fos.30, 31 To test whether C/EBPβ binds to c-fos in the CRP promoter, we performed a co-immunoprecipitation assay by using a nuclear fraction of MCF10A cells treated with S1P. Interaction of phosphorylated-C/EBPβ with c-fos was significantly increased upon S1P treatment, suggesting that S1P activates CRP gene transcription through the formation of C/EBPβ and c-fos complexes (Figure 2d).
S1P3–Gαq coupling is crucial for the S1P-induced CRP expression
S1P exerts cellular responses through binding to one of the S1P receptors, S1P1–S1P5.18 Among these, S1P3 is known to contribute to the activation of the Gαq-coupled signaling cascade by S1P.24, 32, 33 We examined the involvement of S1P3–Gαq coupling in S1P-induced CRP expression in MCF10A cells. As shown in Figure 3a, S1P-induced CRP expression was markedly inhibited by an siRNA targeting S1P3 or by an S1P3 receptor antagonist CAY10444.34 Knockdown of Gαq significantly decreased S1P-induced CRP expression (Figure 3b), demonstrating the crucial role of S1P3 and Gαq in the upregulation of CRP by S1P.
It has been shown that S1P increases intracellular Ca2+ through the activation of phospholipase C (PLC).24, 35, 36 We next examined whether the PLC-Ca2+ pathway participated in the S1P-induced CRP upregulation. When the level of PLC-β4 was inhibited using U73122,37 CRP expression was significantly reduced (Figure 3c). Treatment of MCF10A cells with the intracellular Ca2+ chelator BAPTA/AM significantly inhibited S1P-induced CRP expression (Figure 3d). These data suggest that both PLC-β4 and Ca2+ are involved in the S1P-induced CRP expression in MCF10A cells.
We then investigated the signaling molecules involved in S1P-induced CRP expression. As shown in Figure 3e, Rac1 inhibition by siRNA and ERK inhibition by PD98059, an inhibitor of MEK1, significantly inhibited CRP expression induced by S1P. In contrast, the inhibition of p38 MAPK by SB203580 or Akt signaling by the PI3K inhibitor LY294002, did not significantly affect the S1P-induced CRP expression. The data suggest that S1P-induced CRP expression requires the activation of Rac1 and ERK signaling pathways in MCF10A cells. In MDA-MB-231 cells, blocking of S1P3 and Gαq pathways inhibited the expression of CRP induced by S1P. Treatment of MDA-MB-231 cells with an ERK inhibitor PD98059 or an intracellular Ca2+chelator BAPTA/AM significantly inhibited the S1P-induced CRP expression (Figure 3f). Taken together, we showed that S1P induces CRP expression via activation of Gαq, ERKs and Ca2+ pathways in MDA-MB-231 cells.
S1P induces CRP expression via ROS/ERK pathway
ROS, which are known activators of the MEK/ERK pathways,38 have an important role in CRP expression in several cell systems.39, 40 We next examined whether ROS signaling pathways participated in the S1P-induced CRP expression in MCF10A cells. The intracellular ROS level was increased upon S1P treatment (Figure 4a). Treatment of MCF10A cells with a ROS inhibitor, catalase, inhibited the activation of ERKs as well as CRP expression induced by S1P (Figure 4b). These results show that S1P upregulated CRP expression through ROS generation.
The NADPH oxidase (Nox) is a protein complex that generates ROS in response to various stimuli, including tumor necrosis factor alpha (TNFα), interleukin (IL)-1β and G protein-coupled receptors agonists.41 We examined whether S1P generates ROS by the induction of Nox-4 expression. As shown in Figure 4c (left), Nox-4 expression was induced by S1P treatment in a time-dependent manner. S1P-induced expression of CRP was significantly inhibited following siRNA-mediated knockdown of Nox-4 (Figure 4c, right). The S1P-induced Nox-4 expression was decreased when the S1P3 receptor was knocked down by siRNA (Figure 4d, left), suggesting that S1P generates ROS through binding to the S1P3 receptor. Rac1 is a known regulator of the activity of Nox complexes.42 S1P-induced expression of Nox-4 and CRP was markedly inhibited by a dominant-negative mutant of Rac1 (Figure 4d, right), indicating that upregulation of Nox-4 and CRP by S1P was dependent on Rac1 activity.
We then investigated the functional role of ROS in MMP-2 and/or MMP-9 expression. As shown in Figure 4e, MMP-9, but not MMP-2, was upregulated by S1P, consistent with our previous findings.24 Treatment of cells with catalase significantly decreased S1P-induced MMP-9 upregulation as evidenced by the gelatin zymogram assay (Figure 4e, top) and immunoblot analysis (Figure 4e, bottom). These data demonstrate the involvement of ROS in S1P-mediated upregulation of MMP-9 in MCF10A cells.
S1P induces transcriptional activation of CRP by C/EBPβ upregulation through ERKs and Ca2+ pathways
The effect of S1P on the C/EBPβ expression level was examined by immunoblot analysis. As shown in Figure 5a, S1P treatment markedly increased the expression of C/EBPβ. C/EBPβ was shown to be activated by the ERK pathway;43, 44 hence, we investigated whether ERK signaling is required for C/EBPβ expression induced by S1P. S1P-induced C/EBPβ expression was significantly inhibited by PD98059 (Figure 5a), indicating that ERK signaling has a crucial role in the C/EBPβ expression in S1P-treated cells.
We next investigated whether the increased binding of C/EBPβ to the CRP promoter upon S1P treatment was mediated by the ERK signaling pathways. For this purpose, we performed a ChIP assay on MCF10A cells treated with PD98059 by using an anti-C/EBPβ antibody. The DNA-binding activity of C/EBPβ on the CRP promoter was increased by S1P treatment and it was significantly decreased by PD98059 treatment. These results suggest an important role of ERK signaling in the binding of C/EBPβ to the CRP promoter following S1P treatment (Figure 5b).
The role of Ca2+ in C/EBPβ expression was examined in MCF10A cells treated with S1P. S1P-induced C/EBPβ expression was significantly inhibited by treatment with an intracellular Ca2+ chelator, BAPTA/AM (Figure 5c). The S1P-dependent increase in the DNA binding of C/EBPβ to the CRP promoter was almost completely abolished by treatment with BAPTA/AM (Figure 5d), demonstrating a crucial role of the Ca2+ pathway. Taken together, these results indicate that S1P induced transcriptional activation of CRP by enhancing the C/EBPβ expression, which involved the ERK and Ca2+ signaling pathways.
CRP expression is crucial for ERK activation, MMP-9 upregulation and cell invasion
We next investigated the functional significance of CRP in breast cell invasion promoted by S1P. First, we determined the role of CRP in the activation of ERKs, which are known to mediate S1P-induced invasion and MMP-9 upregulation.24 S1P-induced phosphorylation of ERKs was almost completely abolished by an siRNA targeting CRP (Figure 6a), indicating that enhanced expression of CRP by S1P has an important role in the activation of ERK signaling pathways.
To determine the functional significance of CRP in S1P-induced breast cell invasion, an invasion assay was performed on MCF10A cells following CRP knockdown. The invasive capacity induced by S1P was almost completely inhibited by the knockdown of CRP (Figure 6b), indicating that CRP has an essential role in S1P-induced breast cell invasion.
We next tested the role of CRP in S1P-induced MMP-9 upregulation. Knockdown of CRP significantly attenuated MMP-9 upregulation (Figure 6c). The promoter activity of MMP-9 was significantly inhibited by the knockdown of CRP (Figure 6d), demonstrating that CRP has a crucial role in the transcriptional activation of MMP-9 by S1P. We examined the role of CRP in the DNA-binding affinity of c-fos, a key transcriptional factor for S1P-induced MMP-9 expression.24 A marked reduction of the band representing c-fos binding to the MMP-9 promoter was observed after the knockdown of CRP, indicating an important role of CRP in the S1P-induced transcriptional activation of MMP-9 by c-fos (Figure 6e). Taken together, the results demonstrate that the increased expression of CRP induced by S1P contributes to the activation of ERK signaling pathways. Ultimately, this leads to MMP-9 upregulation, which is associated with breast cell invasion.
It is plausible that the CRP produced upon S1P treatment is secreted and exerts autocrine and/or paracrine effects on MCF10A cells. We next examined the effects of exogenous CRP on ERK signaling and MMP-9 expression in MCF10A cells. A kinetic study showed that ERK phosphorylation was markedly increased 30 min after CRP treatment and decreased to the basal level after 120 min (Figure 6f). A marked induction of MMP-9, but not that of MMP-2, was observed in MCF10A cells treated with CRP (Figure 6g). We next examined the effect of CRP on cell invasion. As shown in Figure 6h, cell invasion was significantly increased by CRP treatment. The data clearly demonstrate that CRP induces the activation of ERK signaling, upregulation of MMP-9 and cell invasion in MCF10A cells.
S1P induces CRP upregulation in mice
To determine whether S1P induces CRP upregulation in vivo, an immunohistochemical analysis was performed on the kidney and lung sections of mice that were treated with a single dose of S1P for 30 min. As shown in Figure 7a, the number of cells expressing CRP was increased in the basal surfaces of tubular epithelial cells in the renal cortex and alveolar macrophages in lung tissues infused with S1P compared with the control. The data demonstrate that S1P induces CRP expression in vivo.
We further investigated the in vivo relevance using a xenograft mice tumor model with an MDA-MB-231 human breast carcinoma cell line. Mice bearing MDA-MB-231 cells were intravenously injected with S1P (5 mg/kg). Mice were killed 24 h after treatment and CRP expression was detected in the tumor tissues. As shown in Figure 7b (left), CRP expression was significantly increased by S1P treatment. To investigate if S1P induced upregulation of CRP in tumor tissues, we performed the immunohistochemical analysis for CRP in paraffin-embedded tissues. As shown in Figure 7b (right), CRP was expressed in most cells treated with S1P and the intensities of CRP staining were increased in tissues infused with S1P compared with the control.
To further validate the biochemical mechanism in vivo, we next examined activation of the signaling molecules for S1P in tumor tissues. As shown in Figure 7b (right), Rac-GTP-positive cells and phospho-ERK-positive cells were increased in tumor tissues treated with S1P compared with control. Taken together, using a xenograft mice tumor model, we demonstrated that S1P induced the expression of CRP and activation of Rac1 and ERKs in vivo.
An inflammatory environment promotes breast cancer progression.45, 46 We have recently provided a direct evidence for the promoting role of the inflammatory lipid S1P in human breast cell invasion.24 Accumulating data support a strong positive correlation between serum levels of CRP and breast cancer development.8 Elevated CRP levels are associated with factors related to poor prognosis such as larger tumor size and the presence of distant metastases.9 These findings led us to hypothesize that S1P induces the expression of CRP, which may contribute to breast cancer aggressiveness. Here, we demonstrated, for the first time, that CRP expression was markedly induced by S1P in breast epithelial cells. On the basis of our previous study24 and the observations in the present study, we propose a model for the signaling networks involved in S1P-induced transcriptional activation of CRP leading to breast cell invasion (Figure 7c).
CRP is known to be secreted by proinflammatory stimuli and acts in an autocrine/paracrine fashion.47, 48, 49 It is secreted as a ∼23 kDa monomer that non-covalently oligomerizes to form the homopentamer.50, 51 Circulating CRP binds and activates receptors such as FcγRI/II and FcαRI on the surface of phagocytic cells, monocytes and macrophages.52, 53 Our data presented in Figure 6f and g suggest that CRP produced upon S1P treatment may be secreted and acts on the plasma membrane of MCF10A cells through a CRP receptor, leading to the activation of ERK signaling and induction of MMP-9 expression. These results suggest that a positive feedback amplification loop exists between S1P and CRP, and thus underlies the invasive process of breast cells, as depicted in Figure 7c.
The transcription factors STAT3, p50-NF-κB, C/EBPβ and C/EBPδ were shown to mediate the expression of CRP by proinflammatory cytokines, interleukin-1 or IL-6 in hepatoma cells and primary human hepatocytes.28, 54, 55 Using a deletion mutant of the CRP promoter lacking the binding site for C/EBPβ, we demonstrated a crucial role of C/EBPβ for transcriptional activation of CRP by S1P in MCF10A cells. It has been shown that complex formation of c-fos, STAT3 and HNF-1 is required for the transcriptional activation of CRP by IL-1β and IL-6.56 AP-1 proteins, including c-fos, bind to the bZIP domain of C/EBPβ to prevent binding by C/EBPβ in the HIV-1 promoter.57 The present study showed that binding of c-fos to C/EBPβ was increased by S1P, suggesting that C/EBPβ binds to the CRP promoter as a heterodimer with c-fos to induce the transcriptional activation of CRP in MCF10A cells. We also examined whether IL-1β treatment induced CRP expression in MCF10A cells. A marked induction of CRP was observed in MCF10A cells treated with 10 ng/ml of IL-1β (data not shown), indicating that IL-1β induced CRP expression in MCF10A cells.
The involvement of Ca2+ in the functions of CRP has been reported. CRP circulates in the body in its Ca2+-bound form. CRP binds to apoptotic cells in a Ca2+ concentration-dependent manner, thus enhancing phagocytosis of apoptotic cells,58 and it binds with laminin in a Ca2+ concentration-dependent manner.59 The Ca2+-bound CRP can interact with fibronectin at inflammation sites and tumors at acidic pH.60 Despite a vast amount of evidence showing the importance of Ca2+ in the function of CRP, a direct role of Ca2+ in CRP expression has not been revealed to date. The present study is the first report showing that signaling pathways involving Ca2+ are crucial for S1P-induced C/EBPβ binding to the CRP promoter, highlighting the fact that Ca2+ pathways can regulate CRP expression. We also showed that the S1P-increased binding of C/EBPβ to the CRP promoter was dependent on ERK signaling pathways. Consistent with our data, ERK-dependent CRP upregulation has been reported in primary human hepatocytes and coronary artery smooth muscle cells upon treatment with IL-1β, IL-6 and IL-17.61, 62
Mounting evidence suggests that ROS has an essential role in inflammation and cancer. ROS generation is stimulated by various inflammatory responses, ionizing radiation and chemotherapeutic drugs.63, 64, 65 ROS induction by S1P is a feature of both human and mouse fibroblast cells.66, 67 Increased ROS production and ERK phosphorylation mediate leptin-induced CRP expression in human coronary artery endothelial cells.39 Recent studies have shown that Nox-4 was increased in a Gαq-overexpressed mouse model,68 and Nox-4-dependent ROS production by stromal mammary cells increased the migration of MCF-7 cells.69 CRP induces oxidative stress in vivo by stimulating NOX via the PKC, ERK and JNK pathways.70 In the present study, we showed that S1P stimulates Rac1/Nox-4/ROS/ERK pathways, which are crucial for the S1P-induced CRP expression and MMP-9 upregulation (Figure 4).
Our data suggest that MMP-9 has a major role in breast cell invasion and that this is triggered by S1P-dependent increases in CRP expression. Similarly, increased levels of serum and tissue expression of MMP-9 are associated with a poor prognosis of breast cancer.71 A high level of MMP-9 expression was detected at sites of inflammation, thus promoting migration of inflammatory cells across the basement membrane.72 Interestingly, we have previously shown that MMP-2, rather than MMP-9, is responsible for the invasive phenotype induced by H-Ras and Gα12/13 in MCF10A human breast epithelial cells,73, 74, 75, 76 suggesting differential regulation of MMP-2 and MMP-9, depending on the type of stimuli.
Laboratories including ours have demonstrated the role of S1P in tumor progression using breast cell lines. S1P level and tumor growth were increased in SphK1-overexpressed MCF-7 cells in mammary fat pads model.77 S1P stimulated cell proliferation in MCF-7 and MDA-MB-453 breast cancer cells.78 Treatment of S1P in MDA-MB-231 cells stimulated the activation of ERKs, suggesting that S1P may have an important role in breast cancer progression.79 S1P induced MMP-9 upregulation and invasiveness in MCF10A breast epithelial cells.24 Our data clearly showed that S1P induced CRP expression by transcription regulation in MCF10A ‘normal’ human breast epithelial cells and in MDA-MB-231 human breast carcinoma cells, providing the molecular basis for the crucial link between S1P and CRP in breast cancer progression.
Using C57BL/6 mice and Balb/c nude mice xenograft tumor model with MDA-MB-231 human breast carcinoma cells, we demonstrated that S1P induced CRP expression leading to breast cancer aggressiveness both in vitro and in vivo. Although CRP is a major acute-phase protein in humans, it is known to be a minor acute-phase reactant in mice.80, 81 For an appropriate animal studies of CRP, transgenic mice for human CRP have been established.82, 83 It would be worthwhile to further investigate the functional significance of CRP in breast cancer progression using these transgenic mice.
Several studies examined the role of S1P in the invasion and metastasis in vivo using an antagonist or a scavenger of S1P. FTY720, a S1P receptor antagonist, significantly decreased the lung, liver and kidney metastasis in mouse mammary cancer model84, and lung and liver metastasis in mouse prostate cancer model.85 Recently, it was shown that the metastasis of rhabdomyosarcoma cell in vivo was effectively inhibited by a S1P-binding scavenger NOX-S93.86 These studies suggest that the inhibition of S1P function may be effective in reducing tumor invasion and metastasis in vivo.
The crucial link between CRP and breast cancer has been the focus of recent clinical studies, however, limited information is available on the molecular mechanism for the increased expression of CRP and its functional role in breast cell invasion. In the present study, we showed that CRP expression could be induced by S1P in breast epithelial cells. A molecular mechanism is provided that links S1P and CRP to the pathologic processes that are relevant to the invasive process of breast cells.
Materials and methods
S1P, catalase (a ROS scavenger), SB203580 (a p38 MAPK inhibitor), LY294002 (a PI3K inhibitor) and BAPTA/AM (an intracellular calcium chelator) were purchased from Sigma-Aldrich (St Louis, MO, USA). S1P was dissolved in methanol. CRP was purchased from Calbiochem (San Diego, CA, USA). PD98059 (an ERK1/2 inhibitor) was purchased from Cell Signaling Technology (Beverly, MA, USA). CAY10444 (an S1P3-selective antagonist) was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). U17322 (a PLC inhibitor) was purchased from Enzo Life Sciences (Plymouth Meeting, PA, USA). FTY720 (2-amino-2-[2-(octyl-phenyl)-ethyl]-1,3-propanediol hydrochloride) was kindly provided by Sanghee Kim (Seoul National University, Seoul, Korea).
Immunoblot analysis was performed as previously described.73 Cells were harvested and were subjected to SDS–PAGE and western blotting.
Transfection was performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The dominant-negative constructs of Rac1 were kindly provided by J.H. Kim of the College of Life Sciences and Biotechnology, University of Korea.
Knockdowns of CRP, S1P3, Rac1, Gαq, C/EBPβ, c-fos and Nox-4 were performed with siRNA molecules targeting these molecules (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were plated in 6-well plates at 1.5 × 105 cells/well, grown for 24 h, then transfected with 50 pmol siRNA for 6 h using Lipofectamine 2000 reagent and OPTI-MEM I (Invitrogen).
Detection of Rac1-GTP
For Rac1 GTPase activity, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in Mg2+lysis/wash buffer. The level of Rac1-GTP was measured by affinity precipitation using the PAK-1 p21-binding domain Rac assay reagent (Upstate Biotechnology, Lake Placid, NY, USA) following the manufacturer’s instructions, as previously described.74
Measurement of ROS
ROS were monitored by measurement of hydrogen peroxide generation. Cells was cultured with 10 μM S1P for 24 h and then harvested. Cells were then washed twice with PBS and incubated with 25 μM 2′-7′-dichlorodihydrofluorescein diacetate (DCF-DA), a ROS indicator, for 15 min at room temperature. The oxidized form of DCF-DA, fluorescent dichlorofluorescein, was measured using the fluorescence-activated cell sorting technique (Beckman Coulter, Fullerton, CA, USA).
Gelatin zymogram assay
Cells were cultured in serum-free DMEM/F12 medium for 48 h. Gelatinolytic activity of the conditioned medium was determined by the gelatin zymogram assay as previously described.87
Reverse transcription PCR
RNA was extracted from cells using Trizol and reverse transcribed with RT-Superscript-III reverse transcriptase (Invitrogen). RT–PCR was performed using primers for CRP.89
The thermocycler conditions used were as previously described.24 Equal volumes of each PCR product were analyzed by means of 2% agarose gel electrophoresis.
Luciferase reporter assay
Luciferase and β-galactosidase activities were assayed using a luciferase assay kit (Promega, Madison, WI, USA) and a Galacto-Light Kit (Tropix Inc., Bedford, MA, USA), respectively, and measured with a luminometer (Tuner Designs, Sunnyvale, CA, USA) as previously described.90 Wild-type human CRP promoter (−300 to −1), CRPΔC/EBPβ and CRPΔp50NF-κB were described previously.28 MMP-9 promoter-luciferase construct91 was kindly provided by Etty N. Benveniste (Department of Cell Biology, University of Alabama, Birmingham, AL, USA).
This assay was conducted as previously described.90 PCR was performed using primers for the AP-1-binding sites (−345 bp) in the MMP-9 promoter24 and the C/EBP and NF-κB binding sites (−233 bp) in the CRP proximal promoter region.29 Equal volumes of each PCR product were analyzed by means of 2% agarose gel electrophoresis.
Nuclear fractions were incubated with 2 μg of an appropriate antibody overnight at 4 °C. Protein G beads were added to the immune complexes and incubated for 3 h under gentle agitation at 4 °C, resolved on SDS polyacrylamide gels, transferred to nitrocellulose filters and immunoblotted with an appropriate antibody.
In vitro invasion assay
In vitro invasion assay was performed, as previously described,73 using a 24-well Transwell unit with polycarbonate filters (Corning Costar, Cambridge, MA, USA).
C57BL/6 mice were anesthetized with ketamine and infused with S1P (0.1 mg/kg) for 30 min via the femoral vein. Control mice were injected with lipopolysaccharide (1 mg/kg). Animals were killed 3 h after treatment, and sections of the kidney and lung were prepared. Briefly, the tissues were dehydrated in graded alcohol and embedded in paraffin. Sections of 6 mm thickness prepared on glass slides were deparaffinized in xylene and rehydrated via ethanol and placed in PBS. Antigen retrieval was performed by boiling the sections for 10 min in citrate buffer (0.1 M, pH 6.0) in a microwave oven. Endogenous peroxidase activity was blocked with 3% H2O2 in PBS. After dewaxing the sections, endogenous peroxidase activity was blocked with 3% H2O2 in PBS. Non-specific adsorption was minimized by pre-incubating the sections in 10% normal donkey serum for 40 min. Expressions of CRP, Rac-GTP and phospho-ERK were detected using specific antibodies. The peroxidase label was detected using diaminobenzidine hydrochloride (Sigma, St Louis, MO, USA). Images were captured using a Leica DMR microscope (Leica, Wetzler, Germany).
Xenograft mice model and tissue isolation
To generate a xenograft tumor model, MDA-MB-231 cells (5 × 106 cells) were subcutaneously injected into the right hind legs of Balb/c nude mice (female, 5-weeks old, SLC, Shizuoka, Japan). When the tumors grew to a size of 100 mm3, the mice were randomly divided into different experimental groups (n=8) and injected intravenously with 5 mg/kg of S1P in solution containing 5% polyethylene glycol 400, 2.5% ethanol, and 0.8% Tween 80.92 Mice were sacrificed 24 h after treatment. Tumor tissues were isolated, frozen in liquid nitrogen and then homogenized in RIPA buffer with 2 mM EDTA, protease inhibitor cocktail and phosphatase inhibitor cocktail solution (Gendepot Co., Barker, TX, USA).
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This work was supported by the National Research Foundation of Korea (NRF) grants (no. ROA-2012-0006262, no R11-2007-0056817, and no. 2013R1A2A2A04013379) and Korea Drug Development Fund (no. A100030101002011).
The authors declare no conflict of interest.
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