CCAAT/enhancer binding protein β regulates the expression of tumor necrosis factor-α in the nucleus pulposus cells

Reagents and Plasmids

K3-luc (TNF-α promoter element; #11110), pcDNA 3.1 (−) rat C/EBP beta (LAP) (#12558), pcDNA 3.1 (−) rat C/EBP beta (LIP) (#12562), pcDNA3-T7- extracellular signal-regulated protein kinases (ERKs) 1 (#14440), and pcDNA3-HA-ERK2 WT (#8974) were purchased from the Addgene repository (Cambridge, MA). FLAG-tagged wild type (WT) and dominant-negative forms (AF) of human p38α, p38β, p38γ, and p38δ were provided by Dr. Jiahuai Han (Xiamen University, China).25 We used the vector pGL4.74 (Promega, Madison, WI) containing the Renilla reniformis luciferase gene as an internal transfection control. The ERK inhibitor (PD98059, #9900) and p38/mitogen-activated protein kinase (MAPK) inhibitor (SB202190, #8158) were obtained from Cell Signaling Technology (Danvers, MA). The c-Jun N-terminal kinase (JNK) inhibitor (SP600125, #S5567) was obtained from Sigma–Aldrich (St. Louis, MO).

Human NP Tissue Specimens

We obtained informed consent from the patients for the use of their IVD tissues. The participants’ written consent was obtained according to the Declaration of Helsinki. Ethical approval was obtained from the Institutional Ethics Review Board of the Tokai University School of Medicine. Seven patients undergoing surgery for lumbar disc herniation were enrolled in this study. Written informed consent was obtained from the next-of-kin, caretakers, or guardians on behalf of the minor/child participants involved in the study. The patients’ ages ranged from 16 to 66 years (mean age, 41.5 years). We harvested the NP tissue from the disc attached to the intact bottom end plate.

Cell Isolation and Culture

Human NP tissues were digested with TrypLE Express (Gibco, Carlsbad, CA) and then collagenase-P (Roche Diagnostics, Mannheim, Germany), washed, and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY). Rat NP cells and AF cells were isolated from multiple levels of the lumbar discs of 11-week-old Sprague Dawley rats (n = 32). Primary rat NP and AF cells were isolated as described.9 The NP and AF tissues obtained from the same animal were pooled together. The isolated cells were maintained in DMEM supplemented with 10% FBS and antibiotics, at 37°C in a humidified atmosphere of 5% CO₂. Confluent NP and AF cells were harvested and subcultured in 10-cm dishes. Low-passage (<3) cells cultured in monolayers were used for all experiments, because cells obtained from the rat IVD tissues exhibited variable morphology until passage 2 or 3.

Immunofluorescence Staining

Rat NP cells were plated in flat-bottom 96-well plates (3 × 10³ cells/well) and incubated for 24 h. Cells were cotransfected with LAP plasmids or the backbone vector. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used as the transfection reagent. Subsequently, rat NP cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (vol/vol) in phosphate-buffered saline (PBS) for 8 ms, blocked with PBS containing 10% FBS, and incubated overnight at 4°C with antibodies against TNF-α (1:100, Cell Signaling Technology; #3707). The cells were washed and incubated with an anti-rabbit Alexa Fluor 488 (green) antibody (Invitrogen) at 1:200 and with 10 μM 4′, 6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature, for nuclear staining. The samples were observed under a fluorescence microscope interfaced with a digital imaging system. Cells treated with normal IgG (Cell Signaling Technology) at equal protein concentrations were used as negative controls.

Immunohistological Studies

To gain insight into the expression of C/EBP β in the IVD, freshly isolated spines from 3- (young) and 11 (mature) -week-old rats were fixed in 4% paraformaldehyde in PBS, decalcified, and then embedded in paraffin wax. Sagittal sections were deparaffinized in xylene, rehydrated through a graded ethanol series, and stained with hematoxylin. Sections were incubated with antibodies to C/EBP β (Santa Cruz Biotechnology) in 2% bovine serum albumin in PBS at 1:100 overnight at 4°C. Nonimmune IgG was used as a negative control (Cell Signaling Technology) and rat kidney was used as positive control. The sections were washed thoroughly and incubated with a biotinylated universal secondary antibody (Vector Laboratories Canada, Burlington, Ontario, Canada) at 1:20 for 10 min at room temperature. Sections were incubated with a streptavidin—peroxidase complex for 5 min and washed with PBS, and color was developed using 3, 3′-diaminobenzidine (Vectastain Universal Quick Kit; Vector Laboratories) and examined under a fluorescence microscope. The number of positively immunolabeled cells and the total number of cells per high-power field in each section were determined, and the percentage of positively labeled cells was calculated.

Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis

After treatment, total RNA was extracted from the cells using the TRIzol RNA isolation protocol (Invitrogen). RNA was treated with RNase-free DNAse I. Total RNA (100 ng) was used as the template for the RT-PCR analyses. The cDNA was synthesized via the reverse transcription of mRNA, as described previously.9 Reactions were arranged in triplicate in 96-well plates using 1 μl of cDNA with SYBR Green PCR Master Mix (Applied Biosystems), to which gene-specific forward and reverse PCR primers for TNF-α were added. The primers were synthesized by Takara Bio Inc. (Tokyo, Japan) or FASMAC Corp. (Tokyo, Japan) and are shown in Table 1 (rat- C/EBP α, β, δ, ϵ, γ, and ζ and TNF-α) and Table 2 (human- C/EBP α, β, δ, ϵ, γ, and ζ). PCR reactions were carried out in an Applied Biosystems 7,500 Fast system, according to the manufacturer's instructions. The expression scores were obtained using the ΔΔC_t calculation method. The relative quantification of gene expression in treatment groups vs. controls (cells isolated freshly before culture) was performed using the comparative threshold cycle method, 2^[(Ct_GAPDH— Ct_Gene)_treatment—(Ct_GAPDH—Ct_Gene)_control], where glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping control gene.

Western Blot Analysis

Treated rat NP cells were immediately placed on ice and washed with cold PBS. To prepare the total cellular proteins, the cells were lysed with lysis buffer containing 10 mM Tris–HCl (pH7.6), 50 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, complete protease inhibitor cocktail (Roche, IN), 1 mM NaF, and 1 mM Na₃VO₄. Heat-denatured samples were separated on sodium dodecyl sulfate polyacrylamide gels and electrotransferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore, MA). The membranes were then blocked with blocking buffer (5% BSA and 0.1% NaN₃ in PBS), and subsequently incubated overnight at 4°C with the anti- TNF-α (1:1,000, Cell Signaling Technology) and anti-C/EBP β (1:200, Santa Cruz Biotechnology) antibodies diluted in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Tokyo, Japan). Chemiluminescent signals were visualized with an Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA), and scanned using an Ez-Capture MG imaging system (ATTO, Tokyo, Japan). The western blot data were quantified using Image J pixel analysis (NIH Image software). Western blot data are presented as band intensities normalized to that of the loading control (β-actin). To measure the band intensity, the data shown are representative of at least three independent experiments. The previous study demonstrated that TNF-α protein levels as detected in the supernatants of phorbol 12-myristate 13-acetate (PMA) -stimulated human promyelocytic leukemia cells (HL-60 cells).26 Thus we used as positive controls.

Sequence Analyses

To identify transcriptional regulatory sequences and potential transcription factor binding sites on the TNF-α promoter region, the sequences of human, mouse, and rat were obtained from GenBank. We used the program Alibaba 2.1 provided by TESS (Transcription Element Search System) (http://labmom.com/link/alibaba_2_1_tf_binding_prediction) and an online server TFSEARCH [Searching Transcription Factor Binding Sites (ver. 1.3)]).

Transient Transfection

Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. For the siRNA transfection experiment, rat NP cells grown on a 12-well plate were cotransfected with a control or LAP expression vector together with either of the following small interfering (si) RNAs (Signal Silence, Cell Signaling); control (#6568) or p42/44 MAPK(ERK1/2) (#6560) (100–200 nM) using Lipofectamine 2000. Six hours after transfection, the medium was replaced with complete growth medium, and the cells were allowed to recover for 18 h. Cells were then cultured for 24 h, and luciferase activity was measured.

Transfections and Dual-Luciferase™ Assay

Rat NP cells were transferred to 24-well plates at 3 × 10⁴ cells/well 1 day before transfection. Cells were cotransfected with 100–500 ng of expression plasmids or the backbone vector together with the reporter plasmids. Lipofectamine 2000 (Invitrogen) was used as the transfection reagent. Reporter activity was measured 48 h after transfection using the Dual-Luciferase™ reporter assay system (Promega) for the sequential measurements of firefly and Renilla luciferase activities. The results were normalized regarding transfection efficiency and are expressed as the ratio of luciferase to pGL4.74 activity (denoted as “relative activity”). NP cells were transfected with a plasmid encoding green fluorescent protein to check the transfection efficiency, which was 60–70% in NP cells. Luciferase activities and relative ratios were quantified using a Turner Designs Luminometer Model TD-20/20 instrument (Promega).

Statistical Analysis

All experiments were used for same isolated cells. Moreover, all experiments were performed in triplicates and each experiment was done at least twice independently, and data are presented as the mean ± SD. Student's t-test or one-way ANOVA was used to investigate significant differences in expression levels between the study groups. While the t-test is used to compare the means between two groups, one-way ANOVA is used to compare means between three or more groups. Values of p < 0.05 were considered significant.

Expression of C/EBP Subtypes in IVD Cells

The expression of C/EBP family members in mature rat cells was studied using real-time PCR. We measured the mRNA expression of the C/EBP subtypes C/EBP α, β, δ, ϵ, γ, and ζ in rat NP and AF cells (Fig. 1A). C/EBP α, β, and δ mRNA expression was significantly higher in rat AF cells than it was in rat NP cells. Moreover, the expression of the C/EBP β mRNA was significantly higher than that of the mRNA of other C/EBP family members, both in rat NP and AF cells.

Next, we measured the mRNA levels of the members of the C/EBP family in the human samples. Real-time PCR showed a higher level of the C/EBP δ mRNA compared with the C/EBP β mRNA in the human samples. Different expression patterns of the C/EBP δ and C/EBP β mRNAs were observed between rat and human NP cells (Fig. 1B). In addition, expression of C/EBP β in IVD cultured cells was studied using western blot analysis. Figure 1C indicates that both NP and AF cells expressed a prominent 45 kDa C/EBP β band. Moreover, the expression level of C/EBP β in AF cells was higher than that observed in NP cells.

To determine whether altered C/EBP β expression contributes to the age-related increase in rat NP cells, we measured C/EBP β expression levels in IVDs from sagittal sections of skeletally young (3 weeks) and mature (11 weeks) rat discs. The percentage of cells that were immunopositive for C/EBP β in the rat NP increased significantly from 3 to 11 weeks. The percentage of cells in the rat NP that were immunopositive for C/EBP β also increased significantly with age. C/EBP β was robustly expressed in cells of the NP and weakly expressed in cells of the AF and cartilaginous end plate in mature rat discs. In all cases, staining was localized to the cytosol to a greater extent compared with the nucleus (Fig. 2).

C/EBP β Induced TNF-α Expression in NP Cells

To explore the premise that C/EBP β associated with IVD degeneration regulated TNF-α expression, we first assessed whether TNF-α expression is modulated by C/EBP β in rat NP cells. We investigated the regulation of TNF-α expression to determine whether a relation exists between TNF-α and the C/EBP β of NP cells by analyzing the 0.83 kb promoter sequence of rat TNF-α. Sequence analysis shows that the TNF-α promoter contains two C/EBPβ-binding motifs (198-CCAACAACTG-207, 613-AACAGTTGCG-622) (data not shown). We cotransfected NP cells with a plasmid expressing C/EBP β (LAP) and mutated plasmid (LIP), the results showed an increase in the activity of the TNF-α promoter 24 h after LAP expression vector treatment, whereas TNF-α promoter activity was not affected by cotransfection with LIP expression vector (Fig. 3A). These results suggest that C/EBP β regulates the expression of TNF-α at the transcriptional level. To confirm the reporter assay data, we cotransfected rat NP cells with a plasmid expressing LAP and performed a real-time PCR analysis of TNF-α mRNA expression. This analysis demonstrated that cotransfection with LAP expression vector increased the expression of the TNF-α mRNA (3.66 ± 0.70) (Fig. 3B). A densitometric analysis of western blots also confirmed that in rat NP cells, the accumulation of the TNF-α protein was increased significantly after cotransfection with LAP expression vector compared with what was observed in unstimulated control cells, whereas the expression of the TNF-α protein was not affected by cotransfection with LIP expression vector (Fig. 3C).

Induction of TNF-α by cotransfection with LAP expression vector was also demonstrated by immunofluorescence staining. As shown in Figure 3D, TNF-α expression was weak in the basal state, but was apparent in the cytosol and nuclear envelope after transfection with LAP expression vector. On the other hands, we have examined whether C/EBP β exposure increased expression of C/EBP β gene and protein. The results showed that there was not changed the expression of C/EBP β gene and protein compared to the control cells (Fig. S1).

TNF-α Was Activated by C/EBP β Via the MAPK Pathways in NP Cells

The role of C/EBP β in the TNF-α promoter in mediating the MAPK pathway was investigated in rat NP cells. To identify which MAPK pathways are necessary for C/EBP β-induced activation of TNF-α, the ERK1/2 pathway was selectively blocked with PD98059, the p38 pathway was blocked with SB202190, and the JNK pathway was blocked with SP600125. The C/EBP β-induced TNF-α promoter activity in the presence of MAPK inhibitors was measured to confirm that C/EBP β-dependent transient activation of the MAPK pathways was required for TNF-α activation. Figure 4 shows that cotransfection with LAP expression vector significantly increased the activity of the TNF-α promoter; this activity was suppressed completely by the MAPK inhibitors PD98059 and SB202190 but not by SP600125. Rat NP cells were transfected with LAP expression vector and treated with inhibitors of ERK (PD98059) and p38 (SB202190) signaling individually. Suppression of the TNF-α mRNA and TNF-α protein was also partially blocked by the individual inhibitors (Fig. 5A and B). To investigate the mechanism of MAPK pathway (ERK1/2 and p38)-mediated regulation of TNF-α expression, we transfected rat NP cells with LAP expression vector together with WT and AF expression plasmids, and measured TNF-α promoter activity. TNF-α promoter activity was induced after transfection with a high dose of WT-ERK1. However, no induction of TNF-α promoter activity was observed after transfection with a high dose of WT-ERK2 (Fig. 6A). In a further approach, we transfected rat NP cells with an LAP expression vector together with a control or p44/42MAPK siRNA at 24 h prior to the experiments. Treatment with the p44/42 MAPK siRNA significantly reduced LAP-induced TNF-α promoter activity compared with the control siRNA. Subsequently, rat NP cells were cotransfected with LAP expression vector and WT and AF-p38 plasmids. The results of both the gain- and loss-of-function analyses showed that cotransfection with p38γ regulated TNF-α promoter activity.