International Immunology

International Immunology Advance Access originally published online on March 22, 2007
International Immunology 2007 19(5):609-619; doi:10.1093/intimm/dxm026

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An RNA-binding protein CP-1 is involved in the STAT3-mediated suppression of NF-B transcriptional activity

Hitomi Nishinakamura¹, Yasumasa Minoda¹, Kazuko Saeki¹, Keiko Koga¹, Giichi Takaesu¹, Masafumi Onodera², Akihiko Yoshimura¹ and Takashi Kobayashi¹

¹ Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
² Department of Hematology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba 305-8575, Japan

Correspondence to: T. Kobayashi; E-mail: takashik{at}bioreg.kyushu-u.ac.jp

	Abstract

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Signal transducer and activator of transcription 3 (STAT3) has been shown to mediate the anti-inflammatory effect of IL-10. Activated STAT3 suppresses LPS-induced IL-6, tumor necrosis factor- and IL-12 gene expression in macrophages and dendritic cells. However, the mechanism of Toll-like receptor (TLR) signal suppression by STAT3 has not been clarified. In this study, we investigated the effect of constitutively activated STAT3 (STAT3C) on LPS-induced nuclear factor-B (NF-B) activation. The forced expression of STAT3C in HEK293/TLR4 cells, but neither wild-type STAT3 nor dominant-negative form of STAT3, suppressed LPS–TLR4-mediated NF-B reporter activation. The over-expression of STAT3C did not affect the signal transduction of TLR4, such as the phosphorylation of inhibitory nuclear factor-B and mitogen-activated protein kinases and the DNA-binding activity of NF-B. Thus, STAT3C could suppress the transcriptional and/or translational activity of NF-B. To define the molecular mechanism, we searched STAT3C-binding proteins by using a proteomic approach and found that a novel RNA-binding protein, CP-1, interacted with STAT3C. CP-1 is a K-homology domain-containing RNA-binding protein with specificity for C-rich pyrimidine tracts. Such proteins play pivotal roles in a broad-spectrum of transcriptional and translational events. The over-expression of CP-1 augmented the suppressive effect of STAT3C on NF-B activation in HEK293/TLR4 cells. Furthermore, the forced expression of CP-1 enhanced the antagonistic effect of IL-10 on IL-6 production in RAW264.7 cells, while small interfering RNA against CP-1 reduced it. These data suggest that CP-1 is involved in the STAT3-mediated suppression of NF-B activity.

Keywords: gene regulation, IL-10, lipopolysaccharide, NF-B, signal transduction, STAT3, Toll-like receptor 4

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
IL-10 is a potent immunoregulatory cytokine with numerous effects on antigen-presenting cells (APCs), such as the down-regulation of pro-inflammatory cytokines, chemokines and co-stimulatory molecules. However, the mechanisms by which IL-10 exerts these effects remain largely unknown. Several mechanisms have been proposed for the IL-10-mediated inhibition of LPS-induced pro-inflammatory gene expression (1), including the activation of the heme oxygenase-1 carbon monoxide pathway (2) and the nuclear factor-B (NF-B) pathway (3), the inhibition of Akt activity (4) and the induction of B cell lymphoma-3 (Bcl-3) (5). However, the precise mechanisms remain controversial; specifically, the ability of IL-10 to inhibit LPS-induced gene expression has been shown to be transcriptionally mediated via the inhibition of the NF-B pathway or a post-transcriptional mechanism via destabilizing mRNA. In the case of tumor necrosis factor- (TNF-), the latter effect requires the AU-rich elements in the 3'-untranslated region (6) and the de novo protein production responsible for mediating the stabilization of mRNA. IL-10 mediates its inhibitory effects by binding to its receptor complex, which induces the activation of the cytoplasmic receptor-associated tyrosine kinases, JAK1 and Tyk2 (1), followed by signal transducer and activator of transcription 3 (STAT3) phosphorylation, homodimerization and translocation to the nucleus, where it binds to STAT-binding elements in the promoters of various IL-10-inducible genes. The essential role of STAT3 in the effect of IL-10 has been clearly demonstrated by studies using a mouse model with the genetically deleted STAT3 gene in macrophages (7). The over-expression of a dominant-negative form of STAT3 (dnSTAT3) completely reversed the ability of IL-10 to inhibit LPS-mediated TNF- and IL-6 production (8).

The obligate role of STAT3 in IL-10 signaling raises the vexing issue of pathway redundancy and specificity as many receptors utilize STAT3. For example, IL-6 signaling also activates the JAK1–STAT3 pathway, which is incapable of activating the anti-inflammatory response. Previously, we demonstrated that the suppressor of cytokine signaling 3 (SOCS3) causes the differences between IL-10 and IL-6 (9). SOCS3 is required to regulate STAT3 signaling from receptors such as gp130 (e.g. the IL-6 receptor), the leptin receptor and the G-CSF-R. In macrophages stimulated with either IL-6 or IL-10, SOCS3 expression is strongly induced. However, the inhibitory effects of SOCS3 are restricted to the gp130 subunit of the IL-6R, SOCS3 binds to phosphorylated Tyr757 of gp130 and thereby blocks signaling. In contrast, IL-10R appears refractory to the effects of SOCS3 because the SOCS3 SH2 domain does not bind to the IL-10R subunits (9). The results of our study suggest that the prolonged activation of STAT3 is necessary for the suppression of Toll-like receptor (TLR) signaling, while the transient activation of STAT3 is not sufficient for the suppression of TLR signaling. A similar result was recently obtained by using mutant STAT3-activating receptors lacking SOCS3-binding sites (10). This idea is also supported by the fact that constitutively activated STAT3 in many tumor cells suppresses pro-inflammatory cytokine and chemokine synthesis by suppressing NF-B activity (11). Toxoplasma gondii activates STAT3 rapidly and independently of secreted factors that activate STAT3, such as IL-10 itself (12). The activation of STAT3 suppresses the ability of macrophages to produce the pro-inflammatory cytokines necessary to kill and control the parasite and presumably forms one element in T. gondii’s repertoire of survival tools. This information raises the possibility that prolonged, constitutively activated STAT3 is sufficient for the suppression of TLR signals. However, there is no direct evidence that activated STAT3 is sufficient for the suppression of LPS-induced gene regulation.

Thus, we examined the effect of STAT3C, a mutant form of constitutively activated STAT3, on TLR signaling and NF-B activity. We found that STAT3C inhibits NF-B transcriptional activity without affecting signaling pathways such as inhibitory nuclear factor-B (IB) phosphorylation and mitogen-activated protein kinases (MAPKs) activation. We sought STAT3C-binding proteins by a proteomic approach and identified several proteins, including an RNA-binding protein, CP-1, which could augment the suppressive effect of STAT3C and IL-10. Our data suggest a novel mechanism for the transcriptional repressor activity of STAT3 for NF-B activation and give rise to a novel approach to the suppression of inflammation.

	Methods

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Cells, antibodies and reagents
HEK293 cells stably expressing human TLR4, MD2 and CD14 genes (HEK293/TLR4) were purchased from Invivogen (San Diego, CA, USA). Bone marrow-derived dendritic cells (BMDCs) were prepared from mouse bone marrow cells as described (13). Culture supernatants containing recombinant mouse IL-10 were prepared from HEK293T cells transfected with mouse IL-10 cDNA in pME18S. The amount of IL-10 was determined by ELISA, and the activity of IL-10 was confirmed as the suppression of surface molecules on LPS-treated RAW264.7 or BMDC by FACS (data not shown). The anti-extracellular signal-regulated kinase (ERK)-2 (C-14), anti-Myc (9E10) antibodies and anti-CP-1 goat polyclonal antibody (T-18) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-phospho-ERK1/2 (9106), anti-FLAG (M2), anti-phospho-c-Jun N-terminal kinase (JNK) (9255), anti-phospho-p38 (9216), anti-JNK (9252), anti-p38 (9212), anti-phospho-IB (9246) and anti-IB (9242) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). LPS (Escherichia coli serotype 055:B5) was from Sigma Chemical (St Louis, MO, USA).

Plasmids
The expression plasmid of STAT3C with mutations at A661C and N663C was kindly provided by J.E. Darnell (14). A dnSTAT3 vector carrying a mutation at Y705F was described previously (15). The human and mouse CP-1 cDNAs were amplified by reverse transcription (RT)–PCR using primers human CP-1: 5'-aagcttatggatgccggtgtg-3' and 5'-gaattcgctgcaccccatgccctt-3' and mouse CP-1: 5'-gcggccgcatggacgccggtgtga-3' and 5'-gtcgacctagctgcaccccat-3', respectively. The cDNA was inserted into pCMV14 (Sigma Chemical) for FLAG-tag, pcDNA4 (Invitrogen, Carlsbad, CA, USA) for Myc-tag and pGEX-4T1 (Amersham Pharmacia Biotech, Piscataway, NJ, USA) for glutathione S-transferase (GST)-tag. The pMX-IRES-GFP and pGCDNsamI/E vectors were used for retroviral transduction (16). The psiRNA-hH1G2 or psiRNA-hH1 neo expression vector (Invivogen) was used for small interfering RNA (siRNA) knockdown (KD) experiments.

Retroviral constructs and transduction
pMX-STAT3C-IRES-GFP or pMX-dnSTAT3-IRES-GFP was retrovirally transduced in BMDCs according to the process presented in previous papers (17, 18). Briefly, PlatE, a packaging cell line, was transfected with pMX-STAT3C-IRES-GFP or pMX-dnSTAT3-IRES-GFP using FuGENE HD (Roche, Basel, Switzerland). GFP-positive PlatE cells were sorted by a cell sorter (FACSAria, BD Biosciences, San Jose, CA, USA). Stable virus producers were established by repeated sorting. The viruses were collected from the culture supernatants of the virus producers and used for the transduction of BMDCs. The mouse CP-1-pGCDNsamI/E vector-containing IRES-GFP cassette was transfected into 293GPG-packaging cells as described previously (16). RAW264.7 cells were retrovirally transduced with the virus supernatants. Thirty-two hours later, GFP-positive cells (40% GFP⁺ cells) were sorted by a cell sorter and used for the assays.

Intracellular staining for cytokines and FACS
Retrovirally transduced BMDCs were stimulated with 10 ng ml^–1 of LPS for 8 h in the presence of Brefeldin A (3 µg ml^–1, eBioscience, San Diego, CA, USA). The cells were stained with anti-CD11c–Allophycocyanin antibody (eBioscience), fixed and permeabilized using a Fixation and Permeabilization kit (eBioscience) followed by intracellular staining using anti-TNF-–PE or anti-IL-6–PE antibodies (eBioscience). The cells were analyzed by a FACSCalibur (BD Biosciences).

Reporter gene analysis and EMSA
The NF-B-responsive promoter luciferase reporter gene, a generous gift from T. Fujita (Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Japan), has been described (19). Reporter gene assay and Electrophoretic Mobility Shift Assay (EMSA) were performed as described previously (20, 21).

Immunoprecipitation assay, GST pull-down assay and western blot analysis
For immunoprecipitation, equal amounts of cellular proteins were incubated with anti-FLAG M2 affinity gel (Sigma Chemical) or protein G sepharose (Amersham Pharmacia Biotech) beads for 2 h at 4°C. The immunoprecipitates were collected by centrifugation and washed five times in washing buffer (1% NP-40 and 25 mM HEPES). Eluted samples were resolved by SDS–PAGE. The proteins were transferred to polyvinylidene difluoride membranes, and the membranes were immunoblotted with specific antibodies and visualized with appropriate HRP-conjugated secondary antibodies using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL, USA).

For GST pull-down assay, E. coli strain BL21 was transformed with human CP-1-pGEX-4T1. GST and GST-CP-1 proteins were purified by affinity chromatography. Transiently transfected HEK293T cells with FLAG-STAT3C plasmid were lysed and incubated with a purified control GST protein or GST-CP-1 protein along with the suspension of glutathione–sepharose beads (Amersham Pharmacia Biotech) at 4°C for 2 h. The beads were washed and the precipitated proteins were analyzed by western blotting as previously described (22).

Preparation of nuclear extract, large-scale pull-down assay and mass spectrometric analysis
HEK293/TLR4 cells (1 x 10⁷) were lysed in 5 ml of Buffer A [10 mM HEPES–HCl pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 1 mM dithiothreitol (DTT) and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan)] on ice for 10 min. Then 10% NP-40 was added to a final concentration of 0.2%, and the mixture was vortexed for 10 sec and centrifuged at 10 000 r.p.m. for 1 min at 4°C. One milliliter volume of Buffer C [50 mM HEPES–HCl pH 7.9, 1.5 mM MgCl₂, 420 mM KCl, 1 mM EDTA, 20% glycerol, DTT (to 1 mM) and protease inhibitor cocktail] was added, and the pellets were left on ice for 30 min, vortexed for 10 sec and centrifuged at 15 000 r.p.m. for 10 min at 4°C. For the proteome-based analysis, the nuclear protein was incubated with anti-FLAG M2 affinity gel (Sigma Chemical) for 1 h at 4°C. The precipitated immune complexes were washed five times with washing buffer [0.25% NP-40 in Tris-buffered saline (TBS)], the bound proteins were eluted with FLAG peptide (Sigma Chemical) and the eluted fractions were concentrated with an Ultrafree-MC 10 000 NMW Filter Unit (Millipore, Billerica, MA, USA). The proteins were separated by SDS–PAGE and visualized by silver staining. For mass spectrometric analysis, proteins were cut from gels, digested with modified trypsin (sequencing grade, Promega, Madison, WI, USA) (23) and loaded into an automated nanoflow liquid chromatography system for tandem mass spectrometry (Finnigan LCQ Deca, Thermo Fisher Scientific, Waltham, MA, USA). The peptide masses obtained by LC-MS/MS analysis were searched against the non-redundant protein sequence database of the National Center for Biotechnology Information using the Mascot search engine (Matrix Science, Boston, MA, USA).

Measurement of cytokines
mRNA expression levels and protein levels for IL-6 and TNF- were determined by RT–PCR and ELISA (eBioscience) as described previously (24, 25).

Generation of mouse CP-1 KD cells
siRNA sequences that target the human CP-1 position at 991 bp (5'-ggctctgctgccagtattagt-3') and the mouse CP-1 position at 11 bp (5'-gtgtgactgaaagcggactca-3') were determined by Invivogen siRNA Wizard software. The annealed nucleotides were inserted into the psiRNA-hH1G2 or psiRNA-hH1 neo expression vector. RAW264.7 cells were transfected with the control or psiRNA-hH1neo-CP-1 vector using FuGENE HD. Stable RAW264.7 cell transformants were selected with 0.8 mg ml^–1 of G418 (26). Several G418-resistant clones were isolated and assayed.

RNA co-immunoprecipitation assay
RAW264.7 cells were untreated or stimulated with LPS (100 ng ml^–1) for 3 h. Then the cells were lysed with the lysis buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 12.5 mM ß-glycerophosphate, 1.5 mM MgCl₂, 2 mM EGTA, 10 mM NaF, 2 mM DTT, 1 mM Na₃VO₄, 1 mM phenylmethylsulphonylfluoride, 20 µM aprotinin and 0.5% Triton X-100), and the cell lysates were incubated with anti-CP-1 antibody, control goat IgG or no antibody for 30 min at 4°C, followed by 45 min of further incubation along with protein G sepharose beads. The beads were washed with TBS containing 0.05% Tween 20, re-suspended with 20 µl of the same buffer and boiled for 5 min. The supernatants were subjected to RT–PCR as described previously (24, 25).

	Results

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STAT3C suppresses NF-B activity without any effects on signal transduction in LPS signaling
Although STAT3 is indispensable for IL-10 function, it is still unclear whether STAT3 activation is sufficient for the suppression of pro-inflammatory cytokines. We first examined the effect of STAT3C, a constitutively activated form of STAT3, on LPS-induced IL-6 and TNF- production in BMDCs. The over-expression of STAT3C, but not an empty control or dnSTAT3, suppressed the protein production of IL-6 and TNF- (Fig. 1). This result suggests that activated STAT3 is sufficient for the suppression of pro-inflammatory cytokine production in BMDCs.

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Constitutive activation of STAT3 results in reduced LPS-induced IL-6 and TNF-. BMDCs were retrovirally transduced with constitutively activated STAT3 (STAT3C)-IRES-GFP, dnSTAT3-IRES-GFP or an empty GFP vector. The BMDCs were left untreated (filled histogram) or stimulated with LPS (open histogram) for 8 h, followed by surface staining with an anti-CD11c antibody and intracellular staining with an anti-IL-6 or anti-TNF- antibody. CD11c+ GFP+ cells were analyzed by FACS.

 
Then we examined whether the over-expression of STAT3C in HEK293/TLR4 cells is sufficient for the suppression of NF-B transcriptional activity (Fig. 2A). STAT3C, but neither wild-type STAT3 nor dnSTAT3, suppressed LPS–TLR4-mediated NF-B reporter activation. The suppressive effect of STAT3C was even greater than that of IL-10 (Fig. 2A). The inhibitory effect of STAT3C on NF-B activation was also observed in the cells in which NF-B was activated by the forced expression of signaling molecules downstream of TLR4, such as MyD88, TRAF6, TAB1/TAK1, IKKß and the heterodimeric complex of NF-B subunits, p65/p50 (Fig. 2B). The inhibitory effect of STAT3C was not non-specific transcriptional repression since STAT3C enhanced APRE reporter promoter activity (data not shown). This result suggests that STAT3C inhibits NF-B transcriptional activity but not signaling intermediates.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 STAT3C suppresses the LPS-induced activation of NF-B. (A) The indicated amounts of wild-type STAT3, STAT3C and dnSTAT3 cDNA were transfected into HEK293/TLR4 cells along with an NF-B luciferase reporter. Twenty-four hours later, the cells were pre-treated with IL-10 stimulated with LPS, and then luciferase activity was measured and normalized based on ß-galactosidase activity. (B) HEK293T cells were transfected with the indicated amount of STAT3C or dnSTAT3 together with an NF-B luciferase reporter and MyD88, TRAF6, TAB1/TAK1, IKKß or p65/p50 cDNA. Luciferase activity was determined as described in (A). Means ± SD of triplicate samples of one representative experiment out of three independent experiments are shown.

 
To investigate this possibility further, we examined the effects of STAT3C on the activation of signaling molecules involved in LPS signaling. HEK293/TLR4 cells were transfected with either STAT3C or an empty vector and then stimulated with LPS. The phosphorylation of IB, ERK, p38 and JNK, as well as the degradation of IB, was measured by western blotting (Fig. 3A). Similar activation of MAPKs, as well as IB phosphorylation and degradation, was observed between empty and STAT3C vector transfected cells. Furthermore, EMSA assay revealed that DNA-binding activity of NF-B was similarly activated by LPS in the presence or absence of STAT3C (Fig. 3B). These results suggest that STAT3C does not affect proximal signaling events of TLR4, including the DNA-binding activity of NF-B, but rather affects the transcriptional activity of NF-B.

View larger version (65K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 STAT3C does not affect LPS signaling. (A) HEK293/TLR4 cells were transfected with an empty vector or STAT3C and then stimulated with LPS. The cells were harvested and lysed at the indicated time points. Western blot analysis was performed with the indicated antibodies. One representative experiment out of three independent experiments is shown. (B) HEK293/TLR4 cells were transfected with the indicated amount of empty vector or STAT3C and then stimulated with LPS for 30 min. The NF-B DNA-binding activity in nuclear extracts was determined by EMSA. One representative experiment out of two independent experiments is shown.

 
Identification of CP-1 as a STAT3C-binding protein
To define the molecular mechanism of transcriptional repression by STAT3C, we first examined the direct interaction between STAT3C and NF-B subunits. We could not show direct co-immunoprecipitation of STAT3C with a p65 subunit of NF-B (data not shown). STAT3 has been shown to interact with transcriptional co-activator p300 (27). Thus, we examined the effect of the over-expression of p300 to examine the possibility of sequestration of p300 by STAT3C. However, the over-expression of p300 did not reverse the suppressive effect of STAT3C (data not shown). Because transcriptional repression is often mediated by co-repressor complexes with histone deacetylase (HDAC) activity, we examined the effect of various HDAC inhibitors. However, none of the HDAC inhibitors reversed the effect of STAT3C (data not shown), leading us to suspect that STAT3C recruits a novel transcriptional/translational repressor.

To identify the proteins interacting with STAT3C, FLAG-STAT3C was over-expressed in HEK293/TLR4 cells and immunoprecipitated with FLAG M2 beads from nuclear extracts. The proteins eluted were resolved by SDS–PAGE and then visualized with silver staining (Fig. 4A). The bands co-precipitated with STAT3C were identified using automated nanoflow liquid chromatography. Among several proteins identified, an 40 kDa protein was identified as CP-1 [hnRNP E1 and poly(C)-binding protein (PCBP)-1] by tryptic fragmentation and subsequent MS/MS analysis (Fig. 4B). We further characterized this molecule because CP-1 is reported to suppress the expression of several genes (28, 29).

View larger version (41K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 STAT3C interacts with an RNA-binding protein, CP-1. (A) HEK293/TLR4 cells were transfected with empty or FLAG-STAT3C plasmid. Thirty-six hours later, the cells were lysed and dissolved by SDS–PAGE. The silver staining of the gel is shown. Distinct bands in the STAT3C-transfected, LPS-stimulated condition were subjected to LC-ESI-MS/MS mass spectrometric analysis. The locations of STAT3C and CP-1 are indicated. (B) A schematic diagram of the CP-1 protein. KH represents the K-homology domain. (C) HEK293T cells were transfected with FLAG-STAT3C and Myc-CP-1 vectors. Twenty-four hours later, the whole-cell extracts (WCE) were immunoprecipitated (IP) with anti-FLAG or anti-Myc antibody and blotted with antibodies as indicated (WB). (D) GST-CP-1 fusion protein was expressed in BL21 Escherichia coli, extracted and visualized by Coomassie brilliant blue staining (upper panel). The WCE from HEK293T cells transfected with FLAG-STAT3C or an empty vector (lower left panel) were incubated with control GST or GST-CP-1, precipitated with glutathione–sepharose beads and blotted with anti-FLAG antibody (lower right panel).

 
The CP-1 is a K-homology domain containing an RNA-binding protein and a member of the PCBP family, which consists of five PCBPs (CP-1-4 and hnRNP K). First, we confirmed that CP-1 was constitutively expressed in the cytoplasm and nucleus and was not induced by IL-10 (data not shown). We obtained the full-length CP-1 cDNA by RT–PCR and sub-cloned it into an expression vector. Then we confirmed the interaction between STAT3C and CP-1 by the co-immunoprecipitation assay (Fig. 4C). When FLAG-tagged STAT3C and Myc-tagged CP-1 were expressed in HEK293T cells, STAT3C and CP-1 were clearly co-immunoprecipitated with opponent antibodies. Furthermore, we confirmed the interaction between STAT3C and GST-CP-1 in vitro using recombinant GST-CP-1 (Fig. 4D). Purified GST-CP-1 beads were incubated with HEK293T cell extracts containing FLAG-STAT3C. As shown in Fig. 4(D), GST-CP-1, but not GST, precipitated STAT3C. These data indicate that CP-1 physically interacted with STAT3C.

Role of CP-1 in LPS-induced NF-B activation
To examine the effect of CP-1 on LPS-mediated NF-B activation, we over-expressed various amounts of CP-1 and STAT3C in HEK293/TLR4 cells. As shown in Fig. 5(A), CP-1 alone suppressed LPS-induced NF-B activity in a dose-dependent manner. Moreover, when CP-1 and STAT3C were simultaneously expressed in HEK293/TLR4 cells, further suppression of NF-B activity was observed.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 CP-1 suppresses LPS-induced NF-B activation. (A) HEK293/TLR4 cells were transfected with the indicated amounts of STAT3C, CP-1 or both cDNA and an NF-B luciferase reporter. Twenty-four hours later, the cells were stimulated with LPS for 6 h, and then luciferase activities were measured. Means ± SD of triplicate samples of one representative experiment out of three independent experiments are shown. (B) HEK293/TLR4 cells were transfected with STAT3C, siRNA-CP-1 or an empty vector along with an NF-B luciferase reporter. Luciferase activity was determined as described in (A).

 
Next, we examined the effect of the reduced expression of endogenous CP-1 by siRNA. The siRNA expression reduced the level of CP-1 mRNA (data not shown) and partly reversed NF-B repression by STAT3C (Fig. 5B). These results suggest that CP-1 is involved in the STAT3-mediated suppression of LPS-induced NF-B activation. CP-1, in particular, could be a partial transcriptional repressor of NF-B because of its suppressive effect on NF-B transcriptional activity.

Effect of forced expression of CP-1 on LPS-induced pro-inflammatory cytokines
To elucidate the in vitro function of CP-1 in the context of pro-inflammatory cytokine induction in macrophages, we first generated macrophage-like cells expressing elevated levels of CP-1. RAW264.7 cells were transduced with CP-1-IRES-GFP or a control GFP retrovirus vector, and the GFP-positive fraction was sorted by FACS. As shown in Fig. 6(A), an 2-fold increase in CP-1 expression levels was detected by western blotting with anti-CP-1 antibody in CP-1-transduced cells. In our condition, IL-6 was slightly produced in RAW264.7 cells infected with an empty vector without LPS stimulation, and IL-10 had little effect on the basal level of IL-6 production in the cells (Fig. 6B). IL-10 strongly reduced the LPS-induced production of IL-6 and TNF- in the cells with the empty vector. Although LPS-induced IL-6 levels were similar between the cells infected with empty and CP-1 vectors, IL-10 suppressed LPS-induced IL-6 production more profoundly in the cells with CP-1. Thus, the suppressive effect of IL-10 on IL-6 was significantly enhanced by the forced expression of CP-1. However, the forced expression of CP-1 did not affect IL-10-mediated TNF- suppression (Fig. 6B), indicating an IL-6-specific manner.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Forced expression of CP-1 enhances the suppressive effect of IL-10 on LPS-induced IL-6 production. (A–C) RAW264.7 cells were retrovirally transduced with empty GFP or CP-1 GFP, and the GFP-positive cells were sorted. (A) The whole-cell extracts were analyzed by western blotting with the indicated antibodies. The relative intensities of each CP-1 band normalized by STAT5 expression levels are shown above. (B and C) Retrovirally transduced RAW264.7 cells were pre-treated with IL-10 for 30 min and then stimulated with LPS for 12 h (B) or for the indicated times (C). The concentrations of IL-6 and TNF- in the culture supernatants were measured by ELISA (B). Asterisk indicates statistically significant differences between the groups (P < 0.005). The IL-6 mRNA was analyzed by RT–PCR (C).

 
To further confirm the role of CP-1 in the suppressive effect of IL-10 on IL-6 production, we investigated the mRNA expression of IL-6 in the RAW264.7 cells infected with an empty or CP-1 vector. As shown in Fig. 6(C), the elevation of IL-6 mRNA levels by LPS was suppressed in IL-10 pre-treated cells infected with an empty vector. The reduction of IL-6 mRNA levels by IL-10 was prominent in the cells infected with CP-1 and showed no effect on the kinetics (Fig. 6C). In contrast, the reduction of TNF- mRNA levels by IL-10 was not affected by CP-1 (data not shown). These results suggest that CP-1 is involved in the IL-10–STAT3-mediated reduction of mRNA levels of pro-inflammatory cytokine, particularly IL-6, but not TNF-.

Effect of the knockdown of CP-1 on LPS-induced pro-inflammatory cytokines
In order to confirm the role of CP-1 in suppressing IL-6 production in macrophages, we established several CP-1-KD RAW264.7 cell clones. As shown in Fig. 7(A), the expression levels were decreased by 50% in the KD cell line, clone KD-1, compared with the endogenous CP-1 level. Consistent with the results shown in Fig. 6(B), IL-6 was slightly produced in the clone with the empty vector without LPS stimulation, and IL-10 had little effect on the basal level of IL-6 production in the cells (Fig. 7B). The basal level of IL-6 in KD-1 was slightly increased by IL-10 treatment. As expected, the suppressive effect of IL-10 on IL-6 secretion was attenuated significantly in KD-1. Similar results were also observed in the different clones (data not shown). On the other hand, the ratio of suppression of TNF- production by IL-10 (80% suppression) was comparable between the control clone and KD-1 (Fig. 7B). Interestingly, TNF- production by LPS was enhanced in KD-1 (Fig. 7B).

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Knockdown of CP-1 cancels the suppressive effect of IL-10 on LPS-induced IL-6 production. (A–C) The expression of CP-1 was down-regulated by siRNA in RAW264.7 cells. (A) The whole-cell extracts from RAW264.7 cells transfected with the control vector (control) and an CP-1 KD clone (KD-1) were analyzed by western blotting with the indicated antibodies. The relative intensities of each CP-1 band normalized by STAT5 expression levels are shown above. (B and C) Control and KD-1 RAW264.7 cells were pre-treated with IL-10 for 30 min and then stimulated with LPS for 12 h (B) or for the indicated times (C). The concentrations of IL-6 and TNF- in the culture supernatants were measured by ELISA (B). Asterisk indicates statistically significant differences between the groups (P < 0.005). The IL-6 mRNA was analyzed by RT–PCR (C).

 
A reduced IL-10 effect was also confirmed by RT–PCR. As shown in Fig. 7(C), IL-10 failed to suppress IL-6 mRNA expression in KD-1, especially at 3 h stimulation. However, there was no difference in the TNF- mRNA levels of the control and KD-1 cells (data not shown). These findings further support a strong link between the CP-1 and IL-10–STAT3-mediated suppression of IL-6.

Selective interaction between CP-1 protein and IL-6 mRNA
CP-1 is a member of the PCBP family, which interacts with RNA. Thus, we investigated whether the CP-1 protein can bind to pro-inflammatory cytokine mRNA. RAW264.7 cells were stimulated with or without LPS, and then the CP-1 protein was immunoprecipitated with an anti-CP-1 antibody. IL-6 or TNF- mRNA was detected by RT–PCR using RNA co-immunoprecipitated with an anti-CP-1 antibody as a template. As shown in Fig. 8, IL-6 mRNA, but not TNF- mRNA, was detected in the anti-CP-1 antibody immunoprecipitates. These results suggest that the CP-1 protein selectively binds to IL-6 mRNA but not to TNF- mRNA, which may account for the suppressive effect of CP-1 on IL-6 but not on TNF-, as shown in Figs. 6 and 7.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 CP-1 protein binds to IL-6 mRNA but not to TNF- mRNA. RAW264.7 cells were untreated (–) or treated with LPS for 3 h (+) and then lysed. IL-6, TNF- and G3PDH Glyceraldehyde-3-phosphate dehydrogenase mRNAs were amplified by RT–PCR using the lysates of whole-cell extracts (input) or the lysates of immunoprecipitates (IP) with an anti-CP-1 antibody, control goat IgG or no antibody (none) along with protein G sepharose beads.

 

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The IL-10–STAT3-signaling pathway plays a critical role in anti-inflammatory effects on the immune system. For example, IL-10-deficient mice as well as conditional knockout mice deficient for STAT3 in APCs show the spontaneous development of chronic enterocolitis (7, 30). IL-10 strongly suppresses various aspects of inflammatory responses induced by LPS on APCs, such as macrophages and dendritic cells. While the signaling pathways emanated from TLR to activate APCs are well characterized, the mechanism by which IL-10 suppresses TLR-mediated NF-B activation is poorly understood. Numerous groups have searched for the IL-10-regulated genes responsible for the anti-inflammatory effect because anti-inflammatory effects of IL-10 has been shown to require de novo protein synthesis. However, none of the target genes has been able to replace the anti-inflammatory effect of IL-10 alone. For example, the over-expression of Bcl-3, an IL-10-inducible gene, inhibits LPS-induced TNF- production, but IL-10 still shows the suppression of IL-6 production in macrophages lacking Bcl-3 (5). Bcl-3 is shown to interact with NF-B p50 subunit, which may interfere with the recruitment of the NF-B, p65/p50 heterodimer to the TNF- promoter. On the other hand, the over-expression of IBNS, an IL-10-dependent colonic lamina propria macrophage-specific gene, inhibits LPS-induced IL-6 production but not TNF- production (31). Therefore, it is possible that anti-inflammatory effects of IL-10 could be a sum of the effects of IL-10-inducible, anti-inflammatory genes.

In addition, we suspected that activated STAT3 itself may have an anti-inflammatory effect because we and others have demonstrated that the prolonged activation of STAT3 is necessary to elicit an anti-inflammatory effect (9, 10). To further demonstrate this notion, we examined whether STAT3C, a constitutively activated form of STAT3, directly suppresses TLR4 signaling and NF-B activity. We have clearly demonstrated that STAT3 can directly suppress NF-B transcriptional activity without affecting the signal transduction pathways of TLR4. We then hypothesized that a novel factor associated with STAT3C links STAT3 activation to NF-B suppression.

To evaluate this hypothesis, we sought the factors associated with STAT3C and found an RNA-binding protein, CP-1. In this study, we propose that this novel STAT3C-binding protein, CP-1, is involved in the IL-10–STAT3-mediated suppression of NF-B activation. CPs have been implicated in a wide range of post-transcriptional regulatory pathways, including not only mRNA stabilization but also translation silencing (32, 33). hnRNP K has been reported to directly interact in vitro and in vivo with zinc finger transcriptional repressor Zik-1 and with other structurally related transcriptional repressors such as Kid-1 and MZF-1 (32). Since CP-1 over-expression with STAT3C suppressed NF-B reporter gene activity (Fig. 5), CP-1 may directly or indirectly inhibit NF-B transcriptional activity. Thus, CP-1 may function as a transcriptional repressor. However, the molecular mechanism for this function is unclear at present.

We found that CP-1 bound to IL-6 mRNA but not to TNF- mRNA (Fig. 8), which may explain why the effect of CP-1 is stronger for IL-6 than for TNF- (Figs 6 and 7). Although CP-1 has been shown to stabilize certain mRNAs by binding to a 3'-untranslated region C-rich motif (33), this mechanism is not at work the case because the forced expression of CP-1 reduced the mRNA levels of IL-6 in RAW264.7 cells treated with IL-10 (Fig. 6C). The MAPK, p38 pathway, mediates the stabilization of TNF- mRNA via HuR and Tristetraprolin (TTP) in myeloid cells stimulated with LPS (6, 34). One of the effects of IL-10 is the destabilization of the mRNA of TNF- by the suppression of p38 activation and the inhibition of HuR expression (6). There is a possibility that CP-1 interacts with IL-6 mRNA 3'-untranslated region and blocks stabilization by HuR and TTP. However, we did not see any differences in IL-6 mRNA stability by the forced expression of CP-1 (data not shown). Thus, CP-1 may affect the transcription, splicing or nuclear transport, rather than the stability, of IL-6 mRNA.

CP-1 may affect translation. CP-1, CP-2 and hnRNP K were shown to specifically and efficiently inhibit the translation of the HPV-16 L2 mRNA in vitro (35). CP-2 has been shown to inhibit the translation of CCAAT/enhancer binding protein mRNA, which leads to the suppression of the expression of C/EBP and G-CSF-R and leads to rapid cell death in the breakpoint cluster region-Abelson-expressing myeloid cells (36). It has also been shown that hnRNP K and CP-1 induce the translational silencing of 15-lipoxygenase (37). Thus, STAT3–CP-1 may suppress the translation of pro-inflammatory genes, particularly IL-6, in addition to transcriptional repression. Further investigation is necessary to reveal the precise mechanisms suppressing IL-6 transcription by STAT3–CP-1. A comprehensive analysis using microarray is required to identify other genes regulated by CP-1. In addition, the loss or gain of function analysis of CP-1 in vivo using animal models is critical to clarify the physiological role of this molecule.

Our data provide a novel mechanism employing CP-1 for the IL-10–STAT3-mediated suppression of NF-B activation and IL-6 production. Therefore, the up-regulation of CP-1 may represent a potential therapeutic pathway for the treatment of inflammatory diseases by enhancing the effect of IL-10.

	Acknowledgements

 
We thank Yuuki Kawabata and Tomoko Yoshioka for general assistance. Emiko Fujimoto and Masumi Ohtsu (Technical Support Center, Medical Institute of Bioregulation) for conducting mass spectrometry and DNA sequencing, Makoto Matsumoto (Hyogo College Of Medicine) for EMSA assay and Yukiko Nishi for preparing the manuscript. This work was supported by special grants-in-aid from the Ministry of Education, Science, Technology, Sports and Culture of Japan and from the Japan Diabetes Foundation, the Uehara Memorial Foundation, the Kato Memorial Bioscience Foundation, the Takeda Science Foundation and the Haraguchi Memorial Foundation.

	Abbreviations

 

APC, antigen-presenting cell

Bcl-3, B cell lymphoma-3

BMDC, bone marrow-derived dendritic cell

C/EBP, CCAAT/enhancer binding protein

dnSTAT3, dominant-negative form of signal transducer and activator of transcription 3

DTT, dithiothreitol

EMSA, electrophoretic mobility shift assay

ERK, extracellular signal-regulated kinase

G3PDH, glyceraldehyde-3-phosphate dehydrogenase

GST, glutathione S-transferase

HDAC, histone deacetylase

IB, inhibitory nuclear factor-B

IPTG, isopropyl ß-D-thiogalactoside

JNK, c-Jun N-terminal kinase

KD, knockdown

MAPK, mitogen-activated protein kinase

NF-B, nuclear factor-B

PCBP, poly(C)-binding protein

RT, reverse transcription

siRNA, small interfering RNA

SOCS3, suppressor of cytokine signaling 3

STAT3, signal transducer and activator of transcription 3

TBS, Tris-buffered saline

TLR, Toll-like receptor

TNF-, tumor necrosis factor-

TTP, Tristetraprolin

	Notes

 
Transmitting editor: T. Watanabe

Received 4 January 2007, accepted 15 February 2007.

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