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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-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.
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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.

Mentions: Several studies have shown that up-regulation of MMP-9 mRNA is mediated through an NF-κB-dependent pathway [35]. MMP-9 promoter also contains NF-κB binding sites [41]. To determine whether NF-κB element is essential for TNF-α-induced MMP-9 gene regulation, the MMP-9 promoter was constructed into a pGL3-Basic vector containing a luciferase reporter system (as illustrated in Figure 8A, upper part; pGL-MMP-9-Luc), which contains several putative recognition elements for a variety of transcriptional factors such as NF-κB. Next, to determine the effect of TNF-α on the MMP-9 promoter activity, cells were transfected with a pGL-MMP-9-Luc construct and then incubated with TNF-α for the indicated time intervals. As shown in Figure 8A (lower panel), TNF-α increased the MMP-9 promoter activity in a time-dependent manner. A maximal response was obtained within 10 h. The increasing of MMP-9 promoter activity stimulated by TNF-α was significantly inhibited by pretreatment with the TNFR antibody or the inhibitor of c-Src (PP1), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or NF-κB (Bay11-7082) (Figure 8B). To further ensure that NF-κB indeed mediated TNF-α-induced MMP-9 promoter activity through binding to NF-κB element on the MMP-9 promoter region, a wild-type (WT) MMP-9 promoter mutated by a single-point mutation of the NF-κB binding site (mt-κB) was constructed (as indicated in Figure 8C, upper part), TNF-α-stimulated MMP-9 promoter activity was significantly blocked in MC3T3-E1 cells transfected with a mt-κB-MMP-9 reporter construct (Figure 8C, lower part), indicating that NF-κB binding element was required for TNF-α-induced MMP-9 promoter activity. These results demonstrated that TNF-α-induced MMP-9 promoter activity is mediated through an NF-κB binding domain of the MMP-9 promoter region 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-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.
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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.
Mentions: Several studies have shown that up-regulation of MMP-9 mRNA is mediated through an NF-κB-dependent pathway [35]. MMP-9 promoter also contains NF-κB binding sites [41]. To determine whether NF-κB element is essential for TNF-α-induced MMP-9 gene regulation, the MMP-9 promoter was constructed into a pGL3-Basic vector containing a luciferase reporter system (as illustrated in Figure 8A, upper part; pGL-MMP-9-Luc), which contains several putative recognition elements for a variety of transcriptional factors such as NF-κB. Next, to determine the effect of TNF-α on the MMP-9 promoter activity, cells were transfected with a pGL-MMP-9-Luc construct and then incubated with TNF-α for the indicated time intervals. As shown in Figure 8A (lower panel), TNF-α increased the MMP-9 promoter activity in a time-dependent manner. A maximal response was obtained within 10 h. The increasing of MMP-9 promoter activity stimulated by TNF-α was significantly inhibited by pretreatment with the TNFR antibody or the inhibitor of c-Src (PP1), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or NF-κB (Bay11-7082) (Figure 8B). To further ensure that NF-κB indeed mediated TNF-α-induced MMP-9 promoter activity through binding to NF-κB element on the MMP-9 promoter region, a wild-type (WT) MMP-9 promoter mutated by a single-point mutation of the NF-κB binding site (mt-κB) was constructed (as indicated in Figure 8C, upper part), TNF-α-stimulated MMP-9 promoter activity was significantly blocked in MC3T3-E1 cells transfected with a mt-κB-MMP-9 reporter construct (Figure 8C, lower part), indicating that NF-κB binding element was required for TNF-α-induced MMP-9 promoter activity. These results demonstrated that TNF-α-induced MMP-9 promoter activity is mediated through an NF-κB binding domain of the MMP-9 promoter region 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