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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 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.
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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.

Mentions: TNF receptor l (TNFR1) and TNF receptor-associated factor 2 (TRAF2) generate distinct signals by TNF-α for the induction of differently biological responses in many cell types. Recent evidences suggest that MMP-9 expression was markedly suppressed in TNFR1 KO mice as compared to wild-type mice [37]. Previously, Lee et al. have demonstrated that TNF-α triggered the association between TNFR1 and TRAF2 to induce the MMP-9 expression in A549 cells [38]. In addition, TNFR1 may associate with kinases such as c-Src to engage signaling pathways, activate transcription factors, and modulate gene expression in various cell types [39]. Therefore, to investigate whether TNF-α induces MMP-9 expression via TNFR1, a neutralizing TNFR antibody was used. As shown in Figure 2A, the pretreatment with the TNFR antibody attenuated TNF-α-induced MMP-9 expression in a concentration-dependent manner. Moreover, to demonstrate whether TNFR1-relative proteins are involved in this response, the cell lysates were immunoprecipitated using an anti-TNFR1 antibody and analyzed by Western blot. As shown in Figure 2B, TNF-α stimulated association of TNFR1, TRAF2, and c-Src in a time-dependent manner. There was a significant increase of TRAF2 and c-Src within 3–5 min during the period of observation. Furthermore, the pretreatment with a c-Src inhibitor PP1 attenuated TNF-α-induced MMP-9 expression in a concentration-dependent manner (Figure 2C), confirming that TNF-α-induced MMP-9 expression is mediated through c-Src. Similarly, pretreatment with PP1 also inhibited TNF-α-induced MMP-9 mRNA expression (Figure 2D). Moreover, we investigated whether TNF-α-induced c-Src activation, c-Src phosphorylation was determined by Western blot using anti-phospho-c-Src antibody and transfection with siRNA for TRAF2. As shown in Figure 2E and G, TNF-α time-dependently stimulated c-Src phosphorylation with a significant increase within 10 min and a maximal response within 15 min. Moreover, pretreatment with PP1 (30 μM) and siRNA for TRAF2 significantly attenuated c-Src phosphorylation in response to TNF-α during the period of observation. To further confirm the role of c-Src or TRAF2 in TNF-α-induced MMP-9 expression, cells were transfected with c-Src or TRAF2 siRNA and then incubated with TNF-α for 24 h. Transfection with c-Src or TRAF2 siRNA down-regulated the total c-Src or TRAF2 protein expression and attenuated TNF-α-induced MMP-9 expression (Figure 2F and H). These results suggested that TNF-α-induced MMP-9 expression is mediated through TNFR1-dependent TRAF2 linking to c-Src cascade 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 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.
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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.
Mentions: TNF receptor l (TNFR1) and TNF receptor-associated factor 2 (TRAF2) generate distinct signals by TNF-α for the induction of differently biological responses in many cell types. Recent evidences suggest that MMP-9 expression was markedly suppressed in TNFR1 KO mice as compared to wild-type mice [37]. Previously, Lee et al. have demonstrated that TNF-α triggered the association between TNFR1 and TRAF2 to induce the MMP-9 expression in A549 cells [38]. In addition, TNFR1 may associate with kinases such as c-Src to engage signaling pathways, activate transcription factors, and modulate gene expression in various cell types [39]. Therefore, to investigate whether TNF-α induces MMP-9 expression via TNFR1, a neutralizing TNFR antibody was used. As shown in Figure 2A, the pretreatment with the TNFR antibody attenuated TNF-α-induced MMP-9 expression in a concentration-dependent manner. Moreover, to demonstrate whether TNFR1-relative proteins are involved in this response, the cell lysates were immunoprecipitated using an anti-TNFR1 antibody and analyzed by Western blot. As shown in Figure 2B, TNF-α stimulated association of TNFR1, TRAF2, and c-Src in a time-dependent manner. There was a significant increase of TRAF2 and c-Src within 3–5 min during the period of observation. Furthermore, the pretreatment with a c-Src inhibitor PP1 attenuated TNF-α-induced MMP-9 expression in a concentration-dependent manner (Figure 2C), confirming that TNF-α-induced MMP-9 expression is mediated through c-Src. Similarly, pretreatment with PP1 also inhibited TNF-α-induced MMP-9 mRNA expression (Figure 2D). Moreover, we investigated whether TNF-α-induced c-Src activation, c-Src phosphorylation was determined by Western blot using anti-phospho-c-Src antibody and transfection with siRNA for TRAF2. As shown in Figure 2E and G, TNF-α time-dependently stimulated c-Src phosphorylation with a significant increase within 10 min and a maximal response within 15 min. Moreover, pretreatment with PP1 (30 μM) and siRNA for TRAF2 significantly attenuated c-Src phosphorylation in response to TNF-α during the period of observation. To further confirm the role of c-Src or TRAF2 in TNF-α-induced MMP-9 expression, cells were transfected with c-Src or TRAF2 siRNA and then incubated with TNF-α for 24 h. Transfection with c-Src or TRAF2 siRNA down-regulated the total c-Src or TRAF2 protein expression and attenuated TNF-α-induced MMP-9 expression (Figure 2F and H). These results suggested that TNF-α-induced MMP-9 expression is mediated through TNFR1-dependent TRAF2 linking to c-Src cascade 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