TNF-α induces matrix metalloproteinase-9-dependent soluble intercellular adhesion molecule-1 release via TRAF2-mediated MAPKs and NF-κB activation in osteoblast-like MC3T3-E1 cells

Abstract

Background: Matrix metalloproteinase-9 (MMP-9) has been shown to be induced by cytokines including TNF-α and may contribute to bone inflammatory diseases. However, the mechanisms underlying MMP-9 expression induced by TNF-α in MC3T3-E1 cells remain unclear.

Results: We applied gelatin zymography, Western blot, RT-PCR, real-time PCR, selective pharmacological inhibitors of transcription (actinomycin D, Act.D), translation (cycloheximide, CHI), c-Src (PP1), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), and NF-κB (Bay11-7082), respective siRNAs transfection, promoter assay, immunofluorescence staining, and ELISA to investigate the MMP-9 expression and soluble ICAM-1 (sICAM-1) release induced by TNF-α in MC3T3-E1 cells. Here we demonstrated that TNF-α-induced MMP-9 expression was attenuated by Act.D, CHI, PP1, U0126, SB202190, SP600125, and Bay11-7082, and by the transfection with siRNAs for ERK2, p38 MAPK, and JNK2. TNF-α-stimulated TNFR1, TRAF2, and c-Src complex formation was revealed by immunoprecipitation and Western blot. Furthermore, TNF-α-stimulated NF-κB phosphorylation and translocation were blocked by Bay11-7082, but not by PP1, U0126, SB202190, or SP600125. TNF-α time-dependently induced MMP-9 promoter activity which was also inhibited by PP1, U0126, SB202190, SP600125, or Bay11-7082. Up-regulation of MMP-9 was associated with the release of sICAM-1 into the cultured medium, which was attenuated by the pretreatment with MMP-2/9i, an MMP-9 inhibitor.

Conclusions: In this study, we demonstrated that TNF-α up-regulates MMP-9 expression via c-Src, MAPKs, and NF-κB pathways. In addition, TNF-α-induced MMP-9 expression may contribute to the production of sICAM-1 by MC3T3-E1 cells. The interplay between MMP-9 expression and sICAM-1 release may exert an important role in the regulation of bone inflammatory diseases.

Figure 1

TNF-α induces MMP-9 expression through transcription and translation levels. (A) MC3T3-E1 cells were incubated with various concentrations of TNF-α for the indicated time intervals. The conditioned media were collected and analyzed by gelatin zymography. The proteolytic activities of MMP-9 and MMP-2 were manifested as horizontal white bands on a blue background, and the expression of MMP-2 served as an internal control. (B) Cells were treated with TNF-α (15 ng/ml) for various time intervals. The total RNA were collected and analyzed by RT-PCR and real-time PCR. (C) Growth-arrested cells were pretreated with various concentrations of either Act. D or CHI for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. The conditioned media were collected and assayed by gelatin zymography. The cell lysates were analyzed by Western blot to determine the expression of GAPDH as an internal control. (D) Cells were pretreated with Act.D (1 nM) or CHI (100 nM) for 1 h and then incubated with TNF-α (15 ng/ml) for 6 h. The isolated RNA samples were analyzed for the level of MMP-9 mRNA by real-time PCR. Data are expressed as mean±SEM of three independent experiments. ^*P < 0.05, ^#P <0.01, as compared to the cells treated with vehicle (A,B) and TNF-α alone (C,D).

Figure 2

TNF-α induces MMP-9 expression via TNFR1-dependent c-Src cascade. (A) Cells were pretreated with various concentrations of TNF-α receptor antibody (TNFR Ab) for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (B) MC3T3-E1 cells were incubated with TNF-α (15 ng/ml) for the indicated time intervals, and the protein-protein interaction was determined by immunoprecipitation (IP) and Western blot using the antibodies as indicated. (C,D) Cells were treated with TNF-α (15 ng/ml) for (C) 24 h or (D) 6 h in the absence or presence of PP1. (D) The isolated RNA samples were analyzed for the levels of MMP-9 mRNA by real-time PCR. Data are expressed as mean±SEM of three independent experiments. ^*P < 0.05; ^#P < 0.01, as compared to the cells incubated with TNF-α alone. (E,G) Cells were pretreated with or without PP1 (30 μM) for 1 h or transfected with TRAF2 siRNA and then stimulated with TNF-α for the indicated time intervals. Phosphorylation of c-Src was determined by Western blot using an anti-phospho-c-Src antibody. (F,G) Cells were transfected with c-Src siRNA or TRAF2 siRNA for 24 h and then incubated with TNF-α (15 ng/ml) for 24 h. (A,C,F,G) MMP-9 expression was determined as described in Figure 1. The cell lysates were determined by Western blot using an anti-c-Src, anti-TRAF2 or anti-GAPDH antibody.

Figure 3

TNF-α induces MMP-9 expression via ERK1/2 phosphorylation. (A) Cells were pretreated with U0126 for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (B) Cells were pretreated with U0126 (3 μM) for 1 h and then incubated with TNF-α (15 ng/ml) for 6 h. The isolated RNA samples were analyzed for the levels of MMP-9 mRNA by real-time PCR. (C) Cells were pretreated with or without U0126 (3 μM) for 1 h and then stimulated with TNF-α for the indicated time intervals. Phosphorylation of ERK1/2 was determined by Western blot using an anti-phospho-ERK1/2 antibody. Data are expressed as mean±SEM of three independent experiments. ^#P < 0.01, as compare with the cells treated with TNF-α alone. (D) Cells were transfected with ERK2 siRNA for 24 h and then incubated with TNF-α (15 ng/ml) for 24 h. (A,D) MMP-9 expression was determined as described in Figure 1. The cell lysates were determined by Western blot using an anti-ERK2 or anti-GAPDH antibody.

Figure 4

Involvement of p38 MAPK phosphorylation in TNF-α-induced MMP-9 expression. (A) Cells were pretreated with SB202190 for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (B) Cells were pretreated with SB202190 (30 μM) for 1 h and then incubated with TNF-α (15 ng/ml) for 6 h. The isolated RNA samples were analyzed for the levels of MMP-9 mRNA by real-time PCR. Data are expressed as mean±SEM of three independent experiments. ^*P < 0.05; ^#P < 0.01, as compare to the cells treated with TNF-α alone. (C) Cells were pretreated with or without SB202190 (30 μM) for 1 h and then stimulated with TNF-α for the indicated time intervals. Phosphorylation of p38 MAPK was determined by Western blot using an anti-phospho-p38 MAPK antibody. (D) Cells were transfected with p38 siRNA for 24 h and then incubated with TNF-α (15 ng/ml) for 24 h. (A,D) MMP-9 expression was determined as described in Figure 1. The cell lysates were determined by Western blot using an anti-p38 MAPK or anti-GAPDH antibody.

Figure 5

TNF-α induces MMP-9 expression via JNK1/2 phosphorylation. (A) Cells were pretreated with SP600125 for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (B) Cells were pretreated with SP600125 (3 μM) for 1 h and then incubated with TNF-α (15 ng/ml) for 6 h. The isolated RNA samples were analyzed for the levels of MMP-9 mRNA by real-time PCR. (C) Cells were pretreated without or with SP600125 for 1 h and then incubated with TNF-α (15 ng/ml) for various time intervals. The cell lysates were analyzed by Western blot using an anti-phospho-JNK1/2 antibody or anti-GAPDH (as an internal control) antibody. Data are expressed as mean±SEM of three independent experiments. ^#P < 0.01, as compare to the cells treated with TNF-α alone. *P < 0.05, as compare to the cells treated with TNF-α alone (A) or vehicle (C). (D) Cells were transfected with JNK2 siRNA for 24 h and then incubated with TNF-α (15 ng/ml) for 24 h. (A,D) MMP-9 expression was determined as described in Figure 1. The cell lysates were determined by Western blot using an anti-JNK2 or anti-GAPDH antibody.

Figure 6

NF-κB is required for TNF-α-induced MMP-9 expression. (A) Cells were pretreated with Bay11-7082 for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. MMP-9 expression was determined as described in Figure 1. (B) Cells were pretreated with Bay11-7082 (10 μM) for 1 h and then incubated with TNF-α (15 ng/ml) for 6 h. The isolated RNA samples were analyzed for the levels of MMP-9 mRNA by real-time PCR. (C) Cells were incubated with TNF-α (15 ng/ml) for the indicated time intervals. The cell lysates were analyzed by Western blot using an anti-phospho-IKKα/β, anti-phospho-p65, or anti-β-actin antibody (left part). The cytosolic and nuclear fractions were analyzed by Western blot using an anti-NF-κB (p65), anti-Lamin A, and anti-GAPDH antibody (right part). (D) Cells were pretreated without or with Bay11-7082 (10 μM) for 1 h and then stimulated with TNF-α (15 ng/ml) for 15 min. The nuclear fraction was analyzed by Western blot using an anti-NF-κB (p65) and anti-Lamin A antibody. The translocation of p65 NF-κB was also observed by immunofluorescence staining (middle part) and the histogram of p65 translocation (lower part). (E) Cells were transiently transfected with NF-κB-Luc construct and then incubated with TNF-α (15 ng/ml) for the indicated time intervals in the absence or presence of Bay11-7082 (10 μM). The cell lysates were collected and determined the luciferase activity. Data are expressed as mean±SEM of three independent experiments. ^#P < 0.01, as compare to the cells treated with TNF-α alone. *P < 0.05, as compare to the cells treated with TNF-α (A,B,E) or vehicle alone (E).

Figure 7

TNF-α stimulates c-Src-dependent MAPKs and NF-κB-dependent cascades in MC3T3-E1 cells. MC3T3-E1 cells were pretreated with 3 μM PP1 (A) transfected with c-Src siRNA (B) or pretreated with (C) U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then stimulated with TNF-α (15 ng/ml) for the indicated time intervals. The cell lysates were analyzed by Western blot using an anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-IKKα/β, anti-phospho-p65, or anti-GAPDH (as a control) antibody. (D) Cells were pretreated with U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), or TNF-α receptor 1 neutralized antibody (TNFR nAb) for 1 h and then stimulated with TNF-α (15 ng/ml) for 15 min. The translocation of p65 NF-κB was observed by immunofluorescence staining. (E) Cells were transfected with NF-κB-Luc construct, pretreated with U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then incubated with TNF-α (15 ng/ml) for 4 h. The cell lysates were collected and determined NF-κB-Luc activity. Similar results were obtained in three independent experiments.

Figure 8

TNF-α-induced MMP-9 expression is mediated through the NF-κB element in MMP-9 promoter leading to soluble ICAM-1 release. (A) Cells were transiently transfected with a wild-type MMP-9 promoter-luciferase reporter construct (WT-MMP9), and then incubated with TNF-α for the indicated time intervals. (B) The WT-MMP9 transfected cells were pretreated with anti-TNFR1 neutralizing antibody (TNFR-Ab, 10 μg/ml), PP1 (3 μM), U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), and Bay11-7082 (10 μM) for 1 h and then incubated with 15 ng/ml TNF-α for 6 h. (C) Cells were transfected with WT-MMP9 or mt-κB-MMP9 for 24 h and then incubated with TNF-α (15 ng/ml) for 6 h. The cell lysates were collected and determined the luciferase activity. (D,E) Cells were pretreated with GM6001 or MMP2/9i for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (D, upper panel) Conditioned media were collected and analyzed by gelatin zymography to determine the MMP-9 expression. The conditioned media were analyzed by trichloroacetic acid-protein precipitation and Western blot using an anti-sICAM-1 antibody. (E) The cell lysates were analyzed by Western blot to determine the expression of ICAM-1. (D, lower panel) Cells were pretreated with MMP-2/9i (10 μM), PP1 (10 μM), U0126 (10 μM), SB202190 (10 μM), SP600125 (10 μM), or Bay11-7082 (10 μM) for 1 h and then incubated with TNF-α (15 ng/ml) for the indicated time intervals. The levels of sICAM-1 were determined in conditioned media using an sICAM-1 ELISA kit. Data are expressed as mean±SEM of three independent experiments. ^#P < 0.01, as compare to the cells exposed to vehicle (A,D) or TNF-α alone (B-D). (F) Schematic representation of signaling pathways involved in TNF-α-induced MMP-9 expression and sICAM-1 release in MC3T3-E1 cells. TNF-α stimulates two independent pathways through TNFR1/TRAF2 activates both c-Src-dependent MAPKs and c-Src-independent IKK/NF-κB pathways.