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Regulation of mechanical stress-induced MMP-13 and ADAMTS-5 expression by RUNX-2 transcriptional factor in SW1353 chondrocyte-like cells  T. Tetsunaga,

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Presentation on theme: "Regulation of mechanical stress-induced MMP-13 and ADAMTS-5 expression by RUNX-2 transcriptional factor in SW1353 chondrocyte-like cells  T. Tetsunaga,"— Presentation transcript:

1 Regulation of mechanical stress-induced MMP-13 and ADAMTS-5 expression by RUNX-2 transcriptional factor in SW1353 chondrocyte-like cells  T. Tetsunaga, K. Nishida, T. Furumatsu, K. Naruse, S. Hirohata, A. Yoshida, T. Saito, T. Ozaki  Osteoarthritis and Cartilage  Volume 19, Issue 2, Pages (February 2011) DOI: /j.joca Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

2 Fig. 1 The change of COL2A1 mRNA expression after 5% and 10% mechanical stretch in SW1353 cells. In real-time PCR (n=6 per experimental group and time point) analyses, COL2A1 mRNA expression decreased temporarily at 15h by 10% stretch and returned to baseline 30h later. Relative mRNA levels of COL2A1 gene were normalized using G3PDH controls and plotted relative to CTS (−) at time 0. “Time 0” represents the time finished CTS. SW1353 cells were cultured for 30h after CTS. The data were presented as mean with 95% CI of triplicate determinations. ∗P= relative to CTS (−), ∗∗P= relative to 5%. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

3 Fig. 2 Effects of CTS on RUNX-2, MMP-13 and aggrecanase expressions in SW1353 cells. Results of Western blotting (A) show the RUNX-2 phosphorylation 15min after CTS in SW1353 cells. Total protein extracts were subjected to PAGE (15μg of total protein/lane), and Western blot analysis was performed with total and phosphorylated RUNX-2 (p-RUNX-2) antibodies. RT-PCR (B) and real-time PCR (C–G, n=6 per experimental group and time point) analyses were performed using specific primers. After CTS loading, the expression of RUNX-2 was up-regulated (B, C). Twenty-four hours after CTS, the expression of MMP-13 (B, D), ADAMTS-4 (B, E), and ADAMTS-9 (B, G) was increased. Up-regulation of the ADAMTS-5 gene expression was biphasic (B, F). Relative mRNA levels of each gene were normalized using G3PDH controls and plotted relative to CTS (−) at time 0. “Time 0” represents the time finished CTS. The data were presented as mean with 95% CI of triplicate determinations. ∗P<0.0001, ∗∗P=0.0001, †P=0.0002, ‡P=0.0009, §P= relative to CTS (−), ○=CTS (−), ■=CTS (+). Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

4 Fig. 3 Effect of RUNX-2 transfection on MMP-13 and aggrecanase mRNA expressions by SW1353 cells with or without CTS. RUNX-2 overexpression-mediated changes in aggrecanase levels were investigated by RT-PCR (A) and real-time PCR (B–F, n=6 per experimental group) analyses. Mechanical stretch increased the expression of RUNX-2, MMP-13, ADAMTS-4, -5 and -9 genes in SW1353 cells. The expressions of MMP-13 (A, C) and ADAMTS-5 (A, E) genes were up-regulated after RUNX-2 transfection. The ADAMTS-4 and -9 genes were not stimulated by RUNX-2 overexpression (A, D, F). All genes were normalized to G3PDH and plotted relative to CTS (−), transfection (−). The data were presented as mean with 95% CI of triplicate determinations. ∗P<0.0001, ∗∗P=0.0062, †P=0.0001, ‡P=0.0103, §P= relative to TF, (−), TF; transfection. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

5 Fig. 4 RUNX-2 siRNA inhibits MMP-13 and ADAMTS-5 gene expression in SW1353 cells. Results of real-time PCR analyses showed that relative RUNX-2 expression in SW1353 cells was decreased by siRUNX-2 to 10% the level of non-transfected cells (A). RUNX-2 siRNA down-regulated MMP-13 (B) and ADAMTS-5 (C) expression to 70% and 40%, respectively, the level of non-transfected-cells. CTS-induced MMP-13 and ADAMTS-5 expressions were not detected after RUNX-2 siRNA induction (B, C), n=6 per experimental group. All genes were normalized to G3PDH and plotted relative to CTS (−), siRUNX-2 (−). The data were presented as mean with 95% CI of triplicate determinations. ∗P<0.0001, ∗∗P=0.003. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

6 Fig. 5 Mechanical stretch modulates stress fiber formation and increases the expressions of RUNX-2 and ADAMTS-5 in SW1353 cells. Rearrangements of stress fibers, detected by phalloidin reagents, were induced by CTS (red signals). The expression of RUNX-2 was localized in the nucleus of SW1353 cells (RUNX-2, upper panel, green signals). The expression of ADAMTS-5 was localized to the cytoplasm in SW1353 cells (ADAMTS-5, middle panel, green signals). Merged images are shown. Cells incubated in the absence of primary antibodies were used as controls (lower panel). Arrows denote the direction of stretch. Stress fibers were randomly aligned. Bar; 100μm. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

7 Fig. 6 p38 MAPK modulated stretch-induced RUNX-2, MMP-13 and ADAMTS-5 induction. (A) The blots were probed for total p38, p44/42, JNK or phosphorylated p38 (p-p38), p44/42 (p-p44/42), JNK (p-JNK). (B) Fold increase in phosphorylation (CTS: − vs +) based on densitometric analyses of Western blots. Results of Western blotting and densitometric analyses show the protein phosphorylation of p38, but not of p44/42 and JNK, in SW1353 cells after CTS for 30min. Total protein extracts were subjected to PAGE (10μg of total protein/lane). SW1353 cells cultured in serum-free medium were pretreated with SB for 30min then stimulated with CTS for 30min. Real-time PCR analyses showed that the expression levels of RUNX-2 (C), MMP-13 (D) and ADAMTS-5 mRNA (E) were attenuated by SB in a dose-dependent manner (n=6 per experimental group). All genes were normalized to G3PDH and plotted relative to CTS (−), SB (−). The data were presented as mean with 95% CI of triplicate determinations. ∗P<0.0001, ∗∗P=0.0313, †P=0.0451, ‡P=0.0152, §P= relative to TF (−). Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

8 Fig. 7 Possible signaling pathways involved in MMP-13 and collagenase gene regulation by CTS in SW1353 cells. The mechanism of up-regulation of ADAMTS-4, -9 expressions by CTS was not investigated in the current study, but cytokines such as IL-1might be involved in this process 20. Thick arrow; demonstrated in the current study, thin arrow; demonstrated previously in the literature19,20,22,38,41,42,44–46,48,49. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2010 Osteoarthritis Research Society International Terms and Conditions

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