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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.

Tsai CL, Chen WC, Hsieh HL, Chi PL, Hsiao LD, Yang CM - J. Biomed. Sci. (2014)

Bottom Line: Furthermore, TNF-α-stimulated NF-κB phosphorylation and translocation were blocked by Bay11-7082, but not by PP1, U0126, SB202190, or SP600125.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.In addition, TNF-α-induced MMP-9 expression may contribute to the production of sICAM-1 by MC3T3-E1 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan. chuenmao@mail.cgu.edu.tw.

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.

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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.
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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.

Mentions: In addition, to determine whether the activation of JNK1/2 is also involved in TNF-α-induced MMP-9 expression, a pharmacological inhibitor of JNK1/2 SP600125 was used. As shown in Figures 5A and B, the pretreatment with SP600125 attenuated TNF-α-induced MMP-9 expression in a concentration-dependent manner and mRNA expression, revealed by zymography and real-time PCR. We further investigated whether JNK1/2 phosphorylation participates in TNF-α-induced MMP-9 expression in MC3T3-E1 cells, activation of JNK1/2 was assayed by Western blotting using an antibody specific for the phosphorylated, active forms of JNK1/2. We found that TNF-α time-dependently stimulated JNK1/2 phosphorylation with a significant increase within 10 min and a maximal response within 60 min in MC3T3-E1 cells (Figure 5C), which was attenuated by the pretreatment with SP600125 during the period of observation. Moreover, we confirmd the role of JNK1/2 in TNF-α-induced MMP-9 expression, cells were transfected with a JNK2 siRNA. The data showed that transfection with JNK2 siRNA down-regulated the total JNK2 protein expression and attenuated TNF-α-induced MMP-9 expression (Figure 5D). These data suggested that TNF-α-induced MMP-9 expression is mediated through a JNK1/2 pathway in MC3T3-E1 cells.


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.

Tsai CL, Chen WC, Hsieh HL, Chi PL, Hsiao LD, Yang CM - J. Biomed. Sci. (2014)

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.
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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.
Mentions: In addition, to determine whether the activation of JNK1/2 is also involved in TNF-α-induced MMP-9 expression, a pharmacological inhibitor of JNK1/2 SP600125 was used. As shown in Figures 5A and B, the pretreatment with SP600125 attenuated TNF-α-induced MMP-9 expression in a concentration-dependent manner and mRNA expression, revealed by zymography and real-time PCR. We further investigated whether JNK1/2 phosphorylation participates in TNF-α-induced MMP-9 expression in MC3T3-E1 cells, activation of JNK1/2 was assayed by Western blotting using an antibody specific for the phosphorylated, active forms of JNK1/2. We found that TNF-α time-dependently stimulated JNK1/2 phosphorylation with a significant increase within 10 min and a maximal response within 60 min in MC3T3-E1 cells (Figure 5C), which was attenuated by the pretreatment with SP600125 during the period of observation. Moreover, we confirmd the role of JNK1/2 in TNF-α-induced MMP-9 expression, cells were transfected with a JNK2 siRNA. The data showed that transfection with JNK2 siRNA down-regulated the total JNK2 protein expression and attenuated TNF-α-induced MMP-9 expression (Figure 5D). These data suggested that TNF-α-induced MMP-9 expression is mediated through a JNK1/2 pathway in MC3T3-E1 cells.

Bottom Line: Furthermore, TNF-α-stimulated NF-κB phosphorylation and translocation were blocked by Bay11-7082, but not by PP1, U0126, SB202190, or SP600125.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.In addition, TNF-α-induced MMP-9 expression may contribute to the production of sICAM-1 by MC3T3-E1 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Pharmacology and Health Ageing Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan. chuenmao@mail.cgu.edu.tw.

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.

Show MeSH
Related in: MedlinePlus