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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 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).
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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).

Mentions: TNF-α has been shown to induce the expression of MMP-9 in human osteoblasts, osteoprogenitors, and mesenchymal stem cells [13,36]. To determine the effect of TNF-α on MMP-9 expression, MC3T3-E1 cells were incubated with various concentrations of TNF-α for the indicated time intervals. The conditioned media were collected to determine the MMP-9 expression activity by gelatin zymography. As shown in Figure 1A, the conditioned media from MC3T3-E1 cells displayed proteolytic activity at 110 kDa, corresponding to the pro-form of mouse MMP-9, and TNF-α induced proMMP-9 expression in a time- and concentration-dependent manner. There was a significant increase within 16 h and a maximal increase was achieved by 36–48 h during the period of observation. In contrast, TNF-α had no effect on MMP-2 expression which served as an internal control. To further examine whether the increase in MMP-9 expression induced by TNF-α results from an increase of MMP-9 mRNA expression, MC3T3-E1 cells were incubated with 15 ng/ml TNF-α for the indicated time intervals. The levels of MMP-9 mRNA were determined by RT-PCR and real-time PCR. As shown in Figure 1B, TNF-α time-dependently induced the expression of MMP-9 mRNA, a significant increase within 4 h and reached a peak by 6 h. These data suggested that TNF-α induces MMP-9 expression via increasing mRNA and protein levels in MC3T3-E1 cells. We further investigated whether TNF-α-induced MMP-9 expression is mediated through transcription and translation, a transcription inhibitor Act.D and a translation inhibitor CHI were used for these purposes. The data showed that the pretreatment with either Act.D or CHI concentration-dependently blocked TNF-α-induced MMP-9 expression determined by gelatin zymography (Figure 1C), suggesting that TNF-α-induced proMMP-9 expression occurs at both transcriptional and translational levels. Additionally, TNF-α-induced MMP-9 mRNA expression was inhibited by Act.D, but not CHI, revealed by real-time PCR (Figure 1D). These results indicated that TNF-α induces MMP-9 expression via both transcription and translation levels 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 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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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).
Mentions: TNF-α has been shown to induce the expression of MMP-9 in human osteoblasts, osteoprogenitors, and mesenchymal stem cells [13,36]. To determine the effect of TNF-α on MMP-9 expression, MC3T3-E1 cells were incubated with various concentrations of TNF-α for the indicated time intervals. The conditioned media were collected to determine the MMP-9 expression activity by gelatin zymography. As shown in Figure 1A, the conditioned media from MC3T3-E1 cells displayed proteolytic activity at 110 kDa, corresponding to the pro-form of mouse MMP-9, and TNF-α induced proMMP-9 expression in a time- and concentration-dependent manner. There was a significant increase within 16 h and a maximal increase was achieved by 36–48 h during the period of observation. In contrast, TNF-α had no effect on MMP-2 expression which served as an internal control. To further examine whether the increase in MMP-9 expression induced by TNF-α results from an increase of MMP-9 mRNA expression, MC3T3-E1 cells were incubated with 15 ng/ml TNF-α for the indicated time intervals. The levels of MMP-9 mRNA were determined by RT-PCR and real-time PCR. As shown in Figure 1B, TNF-α time-dependently induced the expression of MMP-9 mRNA, a significant increase within 4 h and reached a peak by 6 h. These data suggested that TNF-α induces MMP-9 expression via increasing mRNA and protein levels in MC3T3-E1 cells. We further investigated whether TNF-α-induced MMP-9 expression is mediated through transcription and translation, a transcription inhibitor Act.D and a translation inhibitor CHI were used for these purposes. The data showed that the pretreatment with either Act.D or CHI concentration-dependently blocked TNF-α-induced MMP-9 expression determined by gelatin zymography (Figure 1C), suggesting that TNF-α-induced proMMP-9 expression occurs at both transcriptional and translational levels. Additionally, TNF-α-induced MMP-9 mRNA expression was inhibited by Act.D, but not CHI, revealed by real-time PCR (Figure 1D). These results indicated that TNF-α induces MMP-9 expression via both transcription and translation levels 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