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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 stimulates c-Src-dependent MAPKs and NF-κB-dependent cascades in MC3T3-E1 cells. MC3T3-E1 cells were pretreated with 3 μM PP1 (A) transfected with c-Src siRNA (B) or pretreated with (C) U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then stimulated with TNF-α (15 ng/ml) for the indicated time intervals. The cell lysates were analyzed by Western blot using an anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-IKKα/β, anti-phospho-p65, or anti-GAPDH (as a control) antibody. (D) Cells were pretreated with U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), or TNF-α receptor 1 neutralized antibody (TNFR nAb) for 1 h and then stimulated with TNF-α (15 ng/ml) for 15 min. The translocation of p65 NF-κB was observed by immunofluorescence staining. (E) Cells were transfected with NF-κB-Luc construct, pretreated with U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then incubated with TNF-α (15 ng/ml) for 4 h. The cell lysates were collected and determined NF-κB-Luc activity. Similar results were obtained in three independent experiments.
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Figure 7: TNF-α stimulates c-Src-dependent MAPKs and NF-κB-dependent cascades in MC3T3-E1 cells. MC3T3-E1 cells were pretreated with 3 μM PP1 (A) transfected with c-Src siRNA (B) or pretreated with (C) U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then stimulated with TNF-α (15 ng/ml) for the indicated time intervals. The cell lysates were analyzed by Western blot using an anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-IKKα/β, anti-phospho-p65, or anti-GAPDH (as a control) antibody. (D) Cells were pretreated with U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), or TNF-α receptor 1 neutralized antibody (TNFR nAb) for 1 h and then stimulated with TNF-α (15 ng/ml) for 15 min. The translocation of p65 NF-κB was observed by immunofluorescence staining. (E) Cells were transfected with NF-κB-Luc construct, pretreated with U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then incubated with TNF-α (15 ng/ml) for 4 h. The cell lysates were collected and determined NF-κB-Luc activity. Similar results were obtained in three independent experiments.

Mentions: According to the above data, we have demonstrated that TNF-α induced MMP-9 expression via activation of c-Src, ERK1/2, p38 MAPK, JNK1/2, and NF-κB in MC3T3-E1 cells. It would be important to determine the relationship among these molecules, including c-Src, MAPKs, and NF-κB in the responses. Cells were pretreated with the inhibitor of c-Src (PP1), MEK1/2 (U0126), p38 MAPK (SB202190), or JNK1/2 (SP600125) for 1 h and then stimulated with TNF-α for the indicated time intervals. Phosphorylation of ERK1/2, p38 MAPK, JNK1/2, IKKα/β and p65 was assayed by Western blotting. As shown in Figure 7A, TNF-α-stimulated phosphorylation of ERK1/2, p38 MAPK, and JNK1/2, but not IKKα/β and p65 was significantly attenuated by the pretreatment with PP1 during the period of observation. Moreover, PP1 has inhibitory effects on not only c-Src but also other Src family kinases. Therefore, MC3T3-E1 cells were transfected with c-Src siRNA to confirm whether MAPKs and the IKK/NF-κB pathway are inhibited by c-Src knockdown. The data were correlated with Figure 7A, TNF-α-stimulated phosphorylation of ERK1/2, p38 MAPK, and JNK1/2, but not IKKα/β and p65 was significantly attenuated by transfection with siRNA of c-Src during the period of observation (Figure 7B). The data demonstrated that TNF-α-stimulated phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 is dependent on c-Src activation. TNF-α-stimulated p65 NF-κB activation was independent of c-Src. Moreover, we found that TNF-α-stimulated p65 phosphorylation and translocation was not significantly inhibited by the pretreatment with U0126, SB202190, or SP600125 determined by Western blotting during the period of observation (Figure 7C) and immunofluorescence staining of p65 NF-κB (Figure 7D). Subsequently, we also demonstrated that TNF-α-stimulated NF-κB transcriptional activity is independent of these MAPKs, revealed by NF-κB-luciferase reporter assay (Figure 7E). These data demonstrated that TNF-α induced MMP-9 expression via two independent pathways, including c-Src-dependent MAPKs and c-Src-independent IKK/NF-κB cascades 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 stimulates c-Src-dependent MAPKs and NF-κB-dependent cascades in MC3T3-E1 cells. MC3T3-E1 cells were pretreated with 3 μM PP1 (A) transfected with c-Src siRNA (B) or pretreated with (C) U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then stimulated with TNF-α (15 ng/ml) for the indicated time intervals. The cell lysates were analyzed by Western blot using an anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-IKKα/β, anti-phospho-p65, or anti-GAPDH (as a control) antibody. (D) Cells were pretreated with U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), or TNF-α receptor 1 neutralized antibody (TNFR nAb) for 1 h and then stimulated with TNF-α (15 ng/ml) for 15 min. The translocation of p65 NF-κB was observed by immunofluorescence staining. (E) Cells were transfected with NF-κB-Luc construct, pretreated with U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then incubated with TNF-α (15 ng/ml) for 4 h. The cell lysates were collected and determined NF-κB-Luc activity. Similar results were obtained in three independent experiments.
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Figure 7: TNF-α stimulates c-Src-dependent MAPKs and NF-κB-dependent cascades in MC3T3-E1 cells. MC3T3-E1 cells were pretreated with 3 μM PP1 (A) transfected with c-Src siRNA (B) or pretreated with (C) U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then stimulated with TNF-α (15 ng/ml) for the indicated time intervals. The cell lysates were analyzed by Western blot using an anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-IKKα/β, anti-phospho-p65, or anti-GAPDH (as a control) antibody. (D) Cells were pretreated with U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), or TNF-α receptor 1 neutralized antibody (TNFR nAb) for 1 h and then stimulated with TNF-α (15 ng/ml) for 15 min. The translocation of p65 NF-κB was observed by immunofluorescence staining. (E) Cells were transfected with NF-κB-Luc construct, pretreated with U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then incubated with TNF-α (15 ng/ml) for 4 h. The cell lysates were collected and determined NF-κB-Luc activity. Similar results were obtained in three independent experiments.
Mentions: According to the above data, we have demonstrated that TNF-α induced MMP-9 expression via activation of c-Src, ERK1/2, p38 MAPK, JNK1/2, and NF-κB in MC3T3-E1 cells. It would be important to determine the relationship among these molecules, including c-Src, MAPKs, and NF-κB in the responses. Cells were pretreated with the inhibitor of c-Src (PP1), MEK1/2 (U0126), p38 MAPK (SB202190), or JNK1/2 (SP600125) for 1 h and then stimulated with TNF-α for the indicated time intervals. Phosphorylation of ERK1/2, p38 MAPK, JNK1/2, IKKα/β and p65 was assayed by Western blotting. As shown in Figure 7A, TNF-α-stimulated phosphorylation of ERK1/2, p38 MAPK, and JNK1/2, but not IKKα/β and p65 was significantly attenuated by the pretreatment with PP1 during the period of observation. Moreover, PP1 has inhibitory effects on not only c-Src but also other Src family kinases. Therefore, MC3T3-E1 cells were transfected with c-Src siRNA to confirm whether MAPKs and the IKK/NF-κB pathway are inhibited by c-Src knockdown. The data were correlated with Figure 7A, TNF-α-stimulated phosphorylation of ERK1/2, p38 MAPK, and JNK1/2, but not IKKα/β and p65 was significantly attenuated by transfection with siRNA of c-Src during the period of observation (Figure 7B). The data demonstrated that TNF-α-stimulated phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 is dependent on c-Src activation. TNF-α-stimulated p65 NF-κB activation was independent of c-Src. Moreover, we found that TNF-α-stimulated p65 phosphorylation and translocation was not significantly inhibited by the pretreatment with U0126, SB202190, or SP600125 determined by Western blotting during the period of observation (Figure 7C) and immunofluorescence staining of p65 NF-κB (Figure 7D). Subsequently, we also demonstrated that TNF-α-stimulated NF-κB transcriptional activity is independent of these MAPKs, revealed by NF-κB-luciferase reporter assay (Figure 7E). These data demonstrated that TNF-α induced MMP-9 expression via two independent pathways, including c-Src-dependent MAPKs and c-Src-independent IKK/NF-κB cascades 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