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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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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.
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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.

Mentions: To determine whether p38 MAPK is involved in TNF-α-induced MMP-9 expression, a p38 MAPK inhibitor (SB202190) was used. As shown in Figures 4A and B, the pretreatment with SB202190 significantly attenuated TNF-α-induced MMP-9 expression in a concentration-dependent manner and mRNA expression revealed by gelatin zymography and real-time PCR, respectively. To further determine whether TNF-α stimulates p38 MAPK activation, the phosphorylation of p38 MAPK was assayed by Western blot using an antibody specific for the phosphorylated, active form of p38 MAPK. As shown in Figure 4C, TNF-α time-dependently stimulated phosphorylation of p38 MAPK in MC3T3-E1 cells. A maximal response was obtained within 10 min and declined to the basal level within 30 min. Moreover, pretreatment with SB202190 (particularly 30 μM) attenuated TNF-α-stimulated p38 MAPK phosphorylation during the period of observation. These results suggested a link between phosphorylation of p38 MAPK and up-regulation of MMP-9 induced by TNF-α in MC3T3-E1 cells. To further ensure the involvement of p38 MAPK in TNF-α-induced MMP-9 expression, cells were transfected with p38 MAPK siRNA. The results showed that transfection with p38 MAPK siRNA down-regulated the total p38 protein expression and attenuated TNF-α-induced MMP-9 expression (Figure 4D). These data suggested that TNF-α-induced MMP-9 expression is mediated through a p38 MAPK 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)

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.
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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.
Mentions: To determine whether p38 MAPK is involved in TNF-α-induced MMP-9 expression, a p38 MAPK inhibitor (SB202190) was used. As shown in Figures 4A and B, the pretreatment with SB202190 significantly attenuated TNF-α-induced MMP-9 expression in a concentration-dependent manner and mRNA expression revealed by gelatin zymography and real-time PCR, respectively. To further determine whether TNF-α stimulates p38 MAPK activation, the phosphorylation of p38 MAPK was assayed by Western blot using an antibody specific for the phosphorylated, active form of p38 MAPK. As shown in Figure 4C, TNF-α time-dependently stimulated phosphorylation of p38 MAPK in MC3T3-E1 cells. A maximal response was obtained within 10 min and declined to the basal level within 30 min. Moreover, pretreatment with SB202190 (particularly 30 μM) attenuated TNF-α-stimulated p38 MAPK phosphorylation during the period of observation. These results suggested a link between phosphorylation of p38 MAPK and up-regulation of MMP-9 induced by TNF-α in MC3T3-E1 cells. To further ensure the involvement of p38 MAPK in TNF-α-induced MMP-9 expression, cells were transfected with p38 MAPK siRNA. The results showed that transfection with p38 MAPK siRNA down-regulated the total p38 protein expression and attenuated TNF-α-induced MMP-9 expression (Figure 4D). These data suggested that TNF-α-induced MMP-9 expression is mediated through a p38 MAPK 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