Inflammatory lipid sphingosine-1-phosphate upregulates C-reactive protein via C/EBPβ and potentiates breast cancer progression

Abstract

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.

Introduction

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.

Results

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).

Figure 1
figure1

S1P induces transcriptional activation of CRP in MCF10A cells. (a) Upper, cells were treated with the indicated concentration of S1P for 24 h. Bottom, cells were treated with 10 μM S1P for the indicated time. CRP expression was determined by immunoblot analysis using a CRP antibody. (b) Cells were treated with 10 μM S1P for 24 h with and without 10 μM FTY720. CRP expression was determined by immunoblot analysis using a CRP antibody (*P<0.05). (c) RT–PCR was conducted in cells treated with S1P for 24 h. CRP (440 bp) and β-actin (175 bp) bands were detected (*P<0.05). (d) Luciferase assay was performed to detect the promoter activity of CRP in cells treated with S1P for 24 h (*P<0.05). (e) Left, expressions of Sphk1 and CRP in MCF10A cells and MDA-MB-231 cells were detected by immunoblot analysis. Right, MDA-MB-231 cells were treated with 10 μM S1P for 24 h. CRP expression was determined by immunoblot analysis.

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.

Figure 2
figure2

C/EBPβ is a potential transcription factor responsible for S1P-induced transcriptional activation of CRP. (a) ChIP assay was performed on the DNA–protein complexes of MCF10A cells and MDA-MB-231 cells treated with 10 μM S1P for 24 h. The samples were PCR-amplified using specific primers to the binding sites of C/EBPβ, NF-κB and c-fos in the CRP promoter. A 233-bp PCR product was detected. A quarter of the total input was loaded as a control. IP, immunoprecipitation. (b) Cells were transfected with the wild-type CRP promoter construct (CRP-Luc), mutated constructs (CRPΔC/EBPβ-luc and CRPΔp50NF-κB-luc) and C/EBPβ expressing construct (C/EBPβ). Luciferase assays were performed to detect CRP promoter activity (*P<0.05). (c) Cells were transfected with control siRNA or siRNAs targeting C/EBPβ or c-fos (50 pmol). RT–PCR was performed for cells treated with 10 μM S1P for 24 h (*P<0.05). (d) Interaction of C/EBPβ and c-fos in the nuclear fraction was detected by a co-immunoprecipitation assay on the nuclear fraction of MCF10A cells treated with 10 μM S1P for 24 h (*P<0.05).

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.

Figure 3
figure3figure3

S1P3–Gαq coupling is crucial for S1P-induced CRP expression. (a) Left, cells were transfected with control siRNA or siRNAs targeting S1P3 (50 pmol). Knockdown of S1P3 was confirmed by immunoblot analysis. CRP expression was determined by immunoblot analysis. Right, cells were treated with 10 μM CAY10444. (b) Cells were transfected with control siRNA or siRNAs targeting Gαq (50 pmol). CRP expression in siRNA-transfected cells was detected by immunoblot analysis (*P<0.01). (c and d) Cells were treated with 10 μM S1P for 24 h with or without 5 μM U17322 and 50 μM BAPTA/AM. CRP expression was determined by immunoblot analysis (*P<0.01). (e) Cells were transfected with control siRNA or siRNAs targeting Rac1. Cells were treated with 10 μM S1P for 24 h with or without each inhibitor (50 μM). The inhibition of ERK1/2, p38 and Akt was confirmed by immunoblot analyses. CRP expression was determined by immunoblot analysis using an antibody against CRP (*P<0.01). (f) Left, MDA-MB-231 cells were transfected with control siRNA or siRNAs targeting S1P3 or Gαq (50 pmol). Right, cells were treated with 10 μM S1P for 24 h with or without 50 μM BAPTA/AM and 50 μM PD98059. CRP expression was determined by immunoblot analysis.

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.

Figure 4
figure4

S1P induces CRP expression via the ROS/ERK pathway. (a) Intracellular ROS levels were measured by fluorescence-activated cell sorting analysis after staining with the fluorescent probe DCF-DA in cells treated with 10 μM S1P for 24 h. The control curve corresponds to cells cultured in the presence of methanol. The shift to the right due to the increased fluorescence indicates an increase in the intracellular levels of ROS. (b) Cells were treated with 10 μM S1P for 24 h with or without 200 μg/ml catalase. Activation of ERKs and the expression of CRP were determined by immunoblot analysis. (c) Left, cells were treated with 10 μM S1P for the indicated time. The expression of Nox-4 was determined by immunoblot analysis. Right, cells were transfected with control siRNA or siRNAs targeting Nox-4 (50 pmol). (d) Left, cells were transfected with control siRNA or siRNAs targeting S1P3 (50 pmol). Expression of Nox-4 in siRNA-transfected cells was detected by immunoblot analysis. Right, cells were transfected with dominant-negative Rac1 (N17Rac1). Expression of Nox-4 was determined by immunoblot analysis. (e) Cells were treated with 10 μM S1P for 24 h in the absence or presence of 25, 50, 100 and 200 μg/ml catalase. Gelatin zymogram assay (top) and immunoblot analysis (bottom) were conducted on conditioned media of cells treated with S1P and/or catalase.

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.

Figure 5
figure5

S1P induces transcriptional activation of CRP by C/EBPβ upregulation through the ERK and Ca2+ pathways. Cells were treated with 50 μM PD98059 (a) or 50 μM BAPTA/AM (c) for 24 h in the presence of 10 μM S1P. The expression of C/EBPβ was determined by immunoblot analysis. ChIP assay was performed on the DNA–protein complexes in cells treated 50 μM PD98059 (b) or 50 μM BAPTA/AM (*P<0.05). (d) The samples were PCR-amplified using specific primers of the C/EBPβ binding site in the CRP promoter. A 233-bp PCR product was detected. A quarter of the total input was loaded as a control. IP, immunoprecipitation (*P<0.05).

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.

Figure 6
figure6

CRP expression is crucial for ERK activation, MMP-9 upregulation and cell invasion. (a) Cells were transfected with the control siRNA or siRNAs targeting CRP. The transfected cells were treated with 10 μM of S1P for 24 h and subjected to immunoblot analysis by using phospho-ERK antibodies. (b) The transfected cells were subjected to in vitro invasion assay (*P<0.05). (c) Expression of MMP-9 and MMP-2 in siRNA-transfected cells was detected by immunoblot analysis (*P<0.05). (d) Cells were transfected with the control siRNA or siRNAs targeting CRP. The transfected cells were subjected to luciferase assay (*P<0.01). (e) The transfected cells were subjected to ChIP assay. Samples were PCR-amplified using specific primers of the AP-1 site in the MMP-9 promoter. A 345-bp PCR product was detected. A quarter of the total input was loaded as a control. IP, immunoprecipitation. (f) MCF10A cells were treated with 25 μg CRP for the indicated time. The level of activated ERK1/2 (pERKs) was determined by immunoblot analysis (*P<0.01). (g) Conditioned media of cells treated with CRP for 48 h was subjected to immunoblot analysis by using antibodies against MMP-9 and MMP-2. (h) In vitro invasion assay was performed in MCF10A cells treated with 25 μg CRP for 17 h (*P<0.05).

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.

Figure 7
figure7

S1P induces CRP upregulation in vivo. (a) Immunohistochemical staining was performed for the detection of CRP in the kidney and lung tissues of mice treated with vehicle (control) or S1P. The number of CRP-positive cells in the kidney and lung tissues increased with S1P treatment. Original magnification used was × 400. (b) Left, Balb/c nude mice bearing MDA-MB-231-derived tumor were treated with S1P (5 mg/kg, intraveneously). At 24 h, the tumors were isolated and the lysates were subjected to immunoblot analysis using CRP antibody. The band intensity of CRP from immunoblot analysis was quantified and subjected to statistical analysis (CRP/β-actin, n=8, P<0.01). Right, immunohistochemical staining for CRP, Rac-GTP and phospho-ERK were performed on paraffin-embedded tissues. (c) A proposed model for the signaling networks for the S1P-induced transcriptional activation of CRP leading to breast cell invasion. Bold arrows represent experimentally confirmed pathways and dotted arrows represent the presumed pathway.

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.

Discussion

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

Cell lines

MCF10A cells were cultured as previously described.87, 88 MDA-MB-231 cells were kindly provided by Dr Su-Jae Lee (Hanyang University, Seoul, Korea).

Reagents

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

Immunoblot analysis was performed as previously described.73 Cells were harvested and were subjected to SDS–PAGE and western blotting.

Transfection

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.

siRNA transfection

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).

ChIP assay

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.

Co-immunoprecipitation assay

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).

Immunohistochemistry

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).

References

  1. 1

    Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ . Cancer statistics. CA Cancer J Clin 2007; 57: 43–66.

  2. 2

    Chambers AF, Groom AC, MacDonald IC . Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2002; 2: 563–572.

  3. 3

    Coussens LM, Werb Z . Inflammation and cancer. Nature 2002; 420: 860–867.

  4. 4

    Hojilla CV, Wood GA, Khokha R . Inflammation and breast cancer: metalloproteinases as common effectors of inflammation and extracellular matrix breakdown in breast cancer. Breast Cancer Res 2008; 10: 205.

  5. 5

    Cole SW . Chronic inflammation and breast cancer recurrence. J Clin Oncol 2009; 27: 3418–3419.

  6. 6

    Volanakis JE . Human C-reactive protein: expression, structure, and function. Mol Immunol 2001; 38: 189–197.

  7. 7

    Erlinger TP, Platz EA, Rifai N, Helzlsouer KJ . C-reactive protein and the risk of incident colorectal cancer. JAMA 2004; 291: 585–590.

  8. 8

    Pierce BL, Ballard-Barbash R, Bernstein L, Baumgartner RN, Neuhouser ML, Wener MH et al. Elevated biomarkers of inflammation are associated with reduced survival among breast cancer patients. J Clin Oncol 2009; 27: 3437–3444.

  9. 9

    Allin KH, Nordestgaard BG, Flyger H, Bojesen SE . Elevated pre-treatment levels of plasma C-reactive protein are associated with poor prognosis after breast cancer: a cohort study. Breast Cancer Res 2011; 13: R55.

  10. 10

    Ravishankaran P, Karunanithi R . Clinical significance of preoperative serum interleukin-6 and C-reactive protein level in breast cancer patients. World J Surg Oncol 2011; 9: 18.

  11. 11

    Kessenbrock K, Plaks V, Werb Z . Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 2010; 141: 52–67.

  12. 12

    Lelongt B, Trugnan G, Murphy G, Ronco PM . Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro. J Cell Biol 1997; 136: 1363–1373.

  13. 13

    Sarén P, Welgus HG, PT. Kovanen . TNF-alpha and IL-1beta selectively induce expression of 92-kDa gelatinase by human macrophages. J Immunol 1996; 157: 4159–4165.

  14. 14

    Przybylowska K, Kluczna A, Zadrozny M, Krawczyk T, Kulig A, Rykala J et al. Polymorphisms of the promoter regions of matrix metalloproteinases genes MMP-1 and MMP-9 in breast cancer. Breast Cancer Res Treat 2006; 95: 65–72.

  15. 15

    Montero I, Orbe J, Varo N, Beloqui O, Monreal JI, Rodríguez JA et al. CRP induces matrix metalloproteinase-1 and -10 in human endothelial cells: implications for clinical and subclinical atherosclerosis. J Am Coll Cardiol 2006; 47: 1369–1378.

  16. 16

    Nabata A, Kuroki M, Ueba H, Hashimoto S, Umemoto T, Wada H et al. C-reactive protein induces endothelial cell apoptosis and matrix metalloproteinase-9 production in human mononuclear cells: implications for the destabilization of atherosclerotic plaque. Atherosclerosis 2008; 196: 129–135.

  17. 17

    Doronzo G, Russo I, Mattiello L, Trovati M, Anfossi G . CRP increases matrix metalloproteinase-2 expression and activity in cultured human vascular smooth muscle cells. J Lab Clin Med 2005; 146: 287–298.

  18. 18

    Van Brocklyn JR, Lee MJ, Menzeleev R, Olivera A, Edsall L, Cuvillier O et al. Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation and survival. J Cell Biol 1998; 142: 229–240.

  19. 19

    Ammit AJ, Hastie AT, Edsall LC, Hoffman RK, Amrani Y, Krymskaya VP et al. Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. FASEB J 2001; 15: 1212–1214.

  20. 20

    Hammad SM, Crellin HG, Wu BX, Melton J, Anelli V, Obeid LM . Dual and distinct roles for sphingosine kinase 1 and sphingosine 1 phosphate in the response to inflammatory stimuli in RAW macrophages. Prostaglandins Other Lipid Mediat 2008; 85: 107–114.

  21. 21

    Smicun Y, Reierstad S, Wang FQ, Lee C, Fishman DA . S1P regulation of ovarian carcinoma invasiveness. Gynecol Oncol 2006; 103: 952–959.

  22. 22

    Shida D, Fang X, Kordula T, Takabe K, Lépine S, Alvarez SE et al. Cross-talk between LPA1 and epidermal growth factor receptors mediates up-regulation of sphingosine kinase 1 to promote gastric cancer cell motility and invasion. Cancer Res 2008; 68: 6569–6577.

  23. 23

    Nagahashi M, Ramachandran S, Kim EY, Allegood JC, Rashid OM, Yamada A et al. Sphingosine-1-phosphate produced by sphingosine kinase 1 promotes breast cancer progression by stimulating angiogenesis and lymphangiogenesis. Cancer Res 2012; 72: 726–735.

  24. 24

    Kim ES, Kim JS, Kim SG, Hwang S, Lee CH, Moon A . Sphingosine 1-phosphate regulates matrix metalloproteinase-9 expression and breast cell invasion through S1P3-Gαq coupling. J Cell Sci 2011; 124: 2220–2230.

  25. 25

    Radeke HH, von Wenckstern H, Stoidtner K, Sauer B, Hammer S, Kleuser B . Overlapping signaling pathways of sphingosine 1-phosphate and TGF-beta in the murine Langerhans cell line XS52. J Immunol 2005; 174: 2778–2786.

  26. 26

    Paugh SW, Payne SG, Barbour SE, Milstien S, Spiegel S . The immunosuppressant FTY720 is phosphorylated by sphingosine kinase type 2. FEBS Lett 2003; 554: 189–193.

  27. 27

    French KJ, Schrecengost RS, Lee BD, Zhuang Y, Smith SN, Eberly JL et al. Discovery and evaluation of inhibitors of human sphingosine kinase. Cancer Res 2003; 63: 5962–5969.

  28. 28

    Kleemann R, Gervois PP, Verschuren L, Staels B, Princen HM, Kooistra T . Fibrates down-regulate IL-1-stimulated C-reactive protein gene expression in hepatocytes by reducing nuclear p50-NFkappa B-C/EBP-beta complex formation. Blood 2003; 101: 545–551.

  29. 29

    Young DP, Kushner I, Samols D . Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA. J Immunol 2008; 181: 2420–2427.

  30. 30

    Hurst HC . Transcription factors. 1: bZIP proteins. Protein Profile 1994; 1: 123–168.

  31. 31

    Lekstrom-Himes J, Xanthopoulos KG . Biological role of the CCAAT/enhancer-binding protein family of transcription factors. J Biol Chem 1998; 273: 28545–28548.

  32. 32

    Windh RT, Lee MJ, Hla T, An S, Barr AJ, Manning DR . Differential coupling of the sphingosine-1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the Gi, Gq and G12 families of heterotrimeric G proteins. J Biol Chem 1999; 274: 27351–27358.

  33. 33

    Jongsma M, van Unen J, van Loenen PB, Michel MC, Peters SL, Alewijnse AE . Different response patterns of several ligands at the sphingosine-1-phosphate receptor subtype 3 (S1P(3)). Br J Pharmacol 2009; 156: 1305–1311.

  34. 34

    Koid Y, Hasegawa T, Takahashi A, Endo A, Mochizuki N, Nakagawa M et al. Development of novel EDG3 antagonists using a 3D database search and their structure-activity relationships. J Med Chem 2002; 45: 4629–4638.

  35. 35

    Okajima F, Tomura H, Sho K, Kimura T, Sato K, Im DS et al. Sphingosine 1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca2+ system in FRTL-5 thyroid cells: possible involvement of guanosine triphosphate-binding proteins in the lipid signaling. Endocrinology 1997; 138: 220–229.

  36. 36

    Sato K, Kon J, Tomura H, Osada M, Murata N, Kuwabara A et al. Activation of phospholipase C-Ca2+ system by sphingosine 1-phosphate in CHO cells transfected with Edg-3, a putative lipid receptor. FEBS Lett 1999; 443: 25–30.

  37. 37

    Wilsher NE, Court WJ, Ruddle R, Newbatt YM, Aherne W, Sheldrake PW et al. The phosphoinositide-specific phospholipase C inhibitor U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) spontaneously forms conjugates with common components of cell culture medium. Drug Metab Dispos 2007; 35: 1017–1022.

  38. 38

    Guyton KZ, Liu Y, Gorospe M, Xu Q, Holbrook NJ . Activation of mitogen-activated protein kinase by H2O2. J Biol Chem 1996; 271: 4138–4142.

  39. 39

    Singh P, Hoffmann M, Wolk R, Shamsuzzaman AS, Somers VK . Leptin induces C-reactive protein expression in vascular endothelial cells. Arterioscler Thromb Vasc Biol 2007; 27: e302–e307.

  40. 40

    Chang JW, Kim CS, Kim SB, Park SK, Park JS, Lee SK . C-reactive protein induces NF-kappaB activation through intracellular calcium and ROS in human mesangial cells. Nephron Exp Nephrol 2005; 101: e165–e172.

  41. 41

    Masamune A, Watanabe T, Kikuta K, Satoh K, Shimosegawa T . NADPH oxidase plays a crucial role in the activation of pancreatic stellate cells. Am J Physiol Gastrointest Liver Physiol 2008; 294: G99–G108.

  42. 42

    Price MO, Atkinson SJ, Knaus UG, Dinauer MC . Rac activation induces NADPH oxidase activity in transgenic COSphox cells, and the level of superoxide production is exchange factor-dependent. J Biol Chem 2002; 277: 19220–19228.

  43. 43

    Hu J, Roy SK, Shapiro PS, Rodig SR, Reddy SP, Platanias LC et al. ERK1 and ERK2 activate CCAAAT/enhancer-binding protein-beta-dependent gene transcription in response to interferon-gamma. J Biol Chem 2001; 276: 287–297.

  44. 44

    Salmenperä P, Hämäläinen S, Hukkanen M, Kankuri E . Interferon-gamma induces C/EBP beta expression and activity through MEK/ERK and p38 in T84 colon epithelial cells. Am J Physiol Cell Physiol 2003; 284: C1133–C1139.

  45. 45

    Lewis CE, Hughes R . Inflammation and breast cancer. Microenvironmental factors regulating macrophage function in breast tumours: hypoxia and angiopoietin-2. Breast Cancer Res 2007; 9: 209.

  46. 46

    Goldberg JE, Schwertfeger KL . Proinflammatory cytokines in breast cancer: mechanisms of action and potential targets for therapeutics. Curr Drug Targets 2010; 11: 1133–1146.

  47. 47

    Depraetere S, Willems J, Joniau M . Stimulation of CRP secretion in HepG2 cells: cooperative effect of dexamethasone and interleukin 6. Agents Actions 1991; 34: 369–375.

  48. 48

    Haider DG, Leuchten N, Schaller G, Gouya G, Kolodjaschna J, Schmetterer L et al. C-reactive protein is expressed and secreted by peripheral blood mononuclear cells. Clin Exp Immunol 2006; 146: 533–539.

  49. 49

    Venugopal SK, Devaraj S, Jialal I . Macrophage conditioned medium induces the expression of C-reactive protein in human aortic endothelial cells: potential for paracrine/autocrine effects. Am J Pathol 2005; 166: 1265–1271.

  50. 50

    Lei KJ, Liu T, Zon G, Soravia E, Liu TY, Goldman ND . Genomic DNA sequence for human C-reactive protein. J Biol Chem 1985; 260: 13377–13383.

  51. 51

    Woo P, Korenberg JR, Whitehead AS . Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component. J Biol Chem 1985; 260: 13384–13388.

  52. 52

    Tron K, Manolov DE, Röcker C, Kächele M, Torzewski J, Nienhaus GU . C-reactive protein specifically binds to Fcgamma receptor type I on a macrophage-like cell line. Eur J Immunol 2008; 38: 1414–1422.

  53. 53

    Lu J, Marjon KD, Marnell LL, Wang R, Mold C, Du Clos TW et al. Recognition and functional activation of the human IgA receptor (FcalphaRI) by C-reactive protein. Proc Natl Acad Sci USA 2011; 108: 4974–4979.

  54. 54

    Ganter U, Arcone R, Toniatti C, Morrone G, Ciliberto G . Dual control of C-reactive protein gene expression by interleukin-1 and interleukin-6. EMBO J 1989; 8: 3773–3779.

  55. 55

    Moshage HJ, Roelofs HM, van Pelt JF, Hazenberg BP, van Leeuwen MA et al. The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes. Biochem Biophys Res Commun 1988; 155: 112–117.

  56. 56

    Nishikawa T, Hagihara K, Serada S, Isobe T, Matsumura A, Song J et al. Transcriptional complex formation of c-Fos, STAT3, and hepatocyte NF-1 alpha is essential for cytokine-driven C-reactive protein gene expression. J Immunol 2008; 180: 3492–3501.

  57. 57

    Kilareski EM, Shah S, Nonnemacher MR, Wigdahl B . Regulation of HIV-1 transcription in cells of the monocyte-macrophage lineage. Retrovirology 2009; 6: 118.

  58. 58

    Du Clos TW . Function of C-reactive protein. Ann Med 2000; 32: 274–278.

  59. 59

    Swanson SJ, McPeek MM, Mortensen RF . Characteristics of the binding of human C-reactive protein (CRP) to laminin. J Cell Biochem 1989; 40: 121–132.

  60. 60

    Suresh MV, Singh SK, Agrawal A . Interaction of calcium-bound C-reactive protein with fibronectin is controlled by pH: in vivo implications. J Biol Chem 2004; 279: 52552–52557.

  61. 61

    Patel DN, King CA, Bailey SR, Holt JW, Venkatachalam K, Agrawal A et al. Interleukin-17 stimulates C-reactive protein expression in hepatocytes and smooth muscle cells via p38 MAPK and ERK1/2-dependent NF-kappaB and C/EBPbeta activation. J Biol Chem 2007; 282: 27229–27238.

  62. 62

    Kaur G, Rao LV, Agrawal A, Pendurthi UR . Effect of wine phenolics on cytokine-induced C-reactive protein expression. J Thromb Haemost 2007; 5: 1309–1317.

  63. 63

    Thannickal VJ, Fanburg BL . Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 2000; 279: L1005–L1028.

  64. 64

    Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J . Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007; 39: 44–84.

  65. 65

    Li J, Shao ZH, Xie JT, Wang CZ, Ramachandran S, Yin JJ et al. The effects of ginsenoside Rb1 on JNK in oxidative injury in cardiomyocytes. Arch Pharm Res 2012; 35: 1259–1267.

  66. 66

    Catarzi S, Giannoni E, Favilli F, Meacci E, Iantomasi T, Vincenzini MT . Sphingosine 1-phosphate stimulation of NADPH oxidase activity: relationship with platelet-derived growth factor receptor and c-Src kinase. Biochim Biophys Acta 2007; 1770: 872–883.

  67. 67

    Tanimoto T, Lungu AO, Berk BC . Sphingosine 1-phosphate transactivates the platelet-derived growth factor beta receptor and epidermal growth factor receptor in vascular smooth muscle cells. Circ Res 2004; 94: 1050–1058.

  68. 68

    Dai DF, Chen T, Szeto H, Nieves-Cintrón M, Kutyavin V, Santana LF et al. Mitochondrial targeted antioxidant Peptide ameliorates hypertensive cardiomyopathy. J Am Coll Cardiol 2011; 58: 73–82.

  69. 69

    Tobar N, Guerrero J, Smith PC, Martínez J . NOX4-dependent ROS production by stromal mammary cells modulates epithelial MCF-7 cell migration. Br J Cancer 2010; 103: 1040–1047.

  70. 70

    Devaraj S, Dasu MR, Singh U, Rao LV, Jialal I . C-reactive protein stimulates superoxide anion release and tissue factor activity in vivo. Atherosclerosis 2009; 203: 67–74.

  71. 71

    Wu ZS, Wu Q, Yang JH, Wang HQ, Ding XD, Yang F et al. Prognostic significance of MMP-9 and TIMP-1 serum and tissue expression in breast cancer. Int J Cancer 2008; 122: 2050–2056.

  72. 72

    Gong Y, Hart E, Shchurin A, Hoover-Plow J . Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice. J Clin Invest 2008; 118: 3012–3024.

  73. 73

    Kim MS, Lee EJ, Kim HR, Moon A . p38 kinase is a key signaling molecule for H-Ras-induced cell motility and invasive phenotype in human breast epithelial cells. Cancer Res 2003; 63: 454–5461.

  74. 74

    Shin I, Kim S, Song H, Kim HR, Moon A . H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells. J Biol Che 2005; 280: 4675–14683.

  75. 75

    Kim ES, Jeong JB, Kim S, Lee KM, Ko E, Noh DY et al. The G(12) family proteins upregulate matrix metalloproteinase-2 via p53 leading to human breast cell invasion. Breast Cancer Res Treat 2010; 124: 49–61.

  76. 76

    Yong HY, Hwang JS, Son H, Park HI, Oh ES, Kim HH et al. Identification of H-Ras-specific motif for the activation of invasive signaling program in human breast epithelial cells. Neoplasia 2011; 13: 98–107.

  77. 77

    Nava VE, Hobson JP, Murthy S, Milstien S, Spiegel S . Sphingosine kinase type 1 promotes estrogen-dependent tumorigenesis of breast cancer MCF-7 cells. Exp Cell Res 2002; 281: 115–127.

  78. 78

    Goetzl EJ, Dolezalova H, Kong Y, Zeng L . Dual mechanisms for lysophospholipid induction of proliferation of human breast carcinoma cells. Cancer Res 1999; 59: 4732–4737.

  79. 79

    Long JS, Fujiwara Y, Edwards J, Tannahill CL, Tigyi G, Pyne S et al. Sphingosine 1-phosphate receptor 4 uses HER2 (ERBB2) to regulate extracellular signal regulated kinase-1/2 in MDA-MB-453 breast cancer cells. J Biol Chem 2010; 285: 35957–35966.

  80. 80

    Bodmer B, Siboo R . Isolation of mouse C-reactive protein from liver and serum. J Immunol 1977; 118: 1086–1089.

  81. 81

    Pepys MB, Hirschfield GM . C-reactive protein: a critical update. J Clin Invest 2003; 111: 1805–1812.

  82. 82

    Teoh H, Quan A, Lovren F, Wang G, Tirgari S, Szmitko PE et al. Impaired endothelial function in C-reactive protein overexpressing mice. Atherosclerosis 2008; 201: 318–325.

  83. 83

    Kleemann R, Verschuren L, Morrison M, Zadelaar S, van Erk MJ, Wielinga PY et al. Anti-inflammatory, anti-proliferative and anti-atherosclerotic effects of quercetin in human in vitro and in vivo models. Atherosclerosis 2011; 218: 44–52.

  84. 84

    Azuma H, Takahara S, Ichimaru N, Wang JD, Itoh Y, Otsuki Y et al. Marked prevention of tumor growth and metastasis by a novel immunosuppressive agent, FTY720, in mouse breast cancer models. Cancer Res 2002; 62: 1410–1419.

  85. 85

    Pchejetski D, Bohler T, Brizuela L, Sauer L, Doumerc N, Golzio MV et al. FTY720 (fingolimod) sensitizes prostate cancer cells to radiotherapy by inhibition of sphingosine kinase-1. Cancer Res 2010; 70: 8651–8661.

  86. 86

    Schneider G, Bryndza E, Abdel-Latif A, Ratajczak J, Maj M, Tarnowski M et al. Bioactive lipids S1P and C1P are pro-metastatic factors in human rhabdomyosarcomas cell lines, and their tissue level increases in response to radio/chemotherapy. Mol Cancer Res 2013; 11: 793–807.

  87. 87

    Moon A, Kim MS, Kim TG, Kim SH, Kim HE, Chen YQ et al. H-ras, but not N-ras, induces an invasive phenotype in human breast epithelial cells: a role for MMP-2 in the H-rasinduced invasive phenotype. Int J Cancer 2000; 85: 176–181.

  88. 88

    Cha Y, Kang Y, Moon A . HER2 induces expression of leptin in human breast epithelial cells. BMB Rep 2012; 45: 719–723.

  89. 89

    Han C, Liu J, Liu X, Li M . Angiotensin II induces C-reactive protein expression through ERK1/2 and JNK signaling in human aortic endothelial cells. Atherosclerosis 2010; 212: 206–212.

  90. 90

    Song H, Ki SH, Kim SG, Moon A . Activating transcription factor 2 mediates matrix metalloproteinase-2 transcriptional activation induced by p38 in breast epithelial cells. Cancer Res 2006; 66: 0487–10496.

  91. 91

    Ma Z, Qin H, Benveniste EN . Transcriptional suppression of matrix metalloproteinase-9 gene expression by IFN-gamma and IFN-beta: critical role of STAT-1alpha. J Immunol 2001; 167: 5150–5159.

  92. 92

    Bonnaud S, Niaudet C, Legoux F, Corre I, Delpon G, Saulquin X et al. Sphingosine-1-phosphate activates that AKT pathway to protect small intestines from radiation-induced endothelial apoptosis. Cancer Res 2010; 70: 9905–9915.

Download references

Acknowledgements

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).

Author information

Correspondence to A Moon.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and Permissions

About this article

Keywords

  • C-reactive protein
  • S1P
  • MMP-9
  • invasion
  • breast cell

Further reading