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Knee loading reduces MMP13 activity in the mouse cartilage.

Hamamura K, Zhang P, Zhao L, Shim JW, Chen A, Dodge TR, Wan Q, Shih H, Na S, Lin CC, Sun HB, Yokota H - BMC Musculoskelet Disord (2013)

Bottom Line: Load-driven reduction in MMP13 was associated with a decrease in the phosphorylation level of p38 MAPK (p-p38) and NFκB (p-NFκB).Silencing Rac1 GTPase significantly reduced MMP13 expression and p-p38 but not p-NFκB.Knee loading reduces MMP13 activity at least in part through Rac1-mediated p38 MAPK signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Engineering, Indiana University Purdue University Indianapolis, SL220C, 723 West Michigan Street, Indianapolis, IN 46202, USA. hyokota@iupui.edu.

ABSTRACT

Background: Moderate loads with knee loading enhance bone formation, but its effects on the maintenance of the knee are not well understood. In this study, we examined the effects of knee loading on the activity of matrix metalloproteinase13 (MMP13) and evaluated the role of p38 MAPK and Rac1 GTPase in the regulation of MMP13.

Methods: Knee loading (0.5-3 N for 5 min) was applied to the right knee of surgically-induced osteoarthritis (OA) mice as well as normal (non-OA) mice, and MMP13 activity in the femoral cartilage was examined. The sham-loaded knee was used as a non-loading control. We also employed primary non-OA and OA human chondrocytes as well as C28/I2 chondrocyte cells, and examined MMP13 activity and molecular signaling in response to shear at 2-20 dyn/cm².

Results: Daily knee loading at 1 N for 2 weeks suppressed cartilage destruction in the knee of OA mice. Induction of OA elevated MMP13 activity and knee loading at 1 N suppressed this elevation. MMP13 activity was also increased in primary OA chondrocytes, and this increase was attenuated by applying shear at 10 dyn/cm². Load-driven reduction in MMP13 was associated with a decrease in the phosphorylation level of p38 MAPK (p-p38) and NFκB (p-NFκB). Molecular imaging using a fluorescence resonance energy transfer (FRET) technique showed that Rac1 activity was reduced by shear at 10 dyn/cm² and elevated by it at 20 dyn/cm². Silencing Rac1 GTPase significantly reduced MMP13 expression and p-p38 but not p-NFκB. Transfection of a constitutively active Rac1 GTPase mutant increased MMP13 activity, while a dominant negative mutant decreased it.

Conclusions: Knee loading reduces MMP13 activity at least in part through Rac1-mediated p38 MAPK signaling. This study suggests the possibility of knee loading as a therapy not only for strengthening bone but also preventing tissue degradation of the femoral cartilage.

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FRET fluorescent images using the Rac1 GTPase biosensor in C28/I2 chondrocyte cells. The red color indicates the activation level of Rac1. (A-D) Rac1 activity in response to fluid flow shear at 2, 5, 10, and 20 dyn/cm2 for 60 min. (E) Reduced Rac1 activity in response to fluid flow shear at 10 dyn/cm2 [zoom of the white boxes in (C)]. (F) Elevated Rac1 activity in response to fluid flow shear at 20 dyn/cm2 [zoom of the white boxes in (D)]. n = 7 to 17 (A-F).
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Figure 7: FRET fluorescent images using the Rac1 GTPase biosensor in C28/I2 chondrocyte cells. The red color indicates the activation level of Rac1. (A-D) Rac1 activity in response to fluid flow shear at 2, 5, 10, and 20 dyn/cm2 for 60 min. (E) Reduced Rac1 activity in response to fluid flow shear at 10 dyn/cm2 [zoom of the white boxes in (C)]. (F) Elevated Rac1 activity in response to fluid flow shear at 20 dyn/cm2 [zoom of the white boxes in (D)]. n = 7 to 17 (A-F).

Mentions: To evaluate the role of Rac1 GTPase in mechanical regulation of MMP13, Rac1 activity of C28/I2 chondrocyte cells was monitored using the Rac1 biosensor and FRET imaging. The cells were subjected to flow-induced shear stress at 2, 5, 10, or 20 dyn/cm2, and changes in the emission ratio of YFP/CFP of the Rac1 biosensor were determined. The result presented shear intensity-dependent regulation of Rac1 activity. At low fluid flow shear of 2 and 5 dyn/cm2, Rac1 activity was not significantly altered (Figure 7A-B). When shear intensity was raised to 10 dyn/cm2, however, it was continuously lowered in a 60-min observation period (Figure 7C). When shear intensity was further elevated to 20 dyn/cm2, Rac1 activity was significantly increased 20–50 min after the onset of fluid flow (Figure 7D). The spatial distribution of Rac1 activity was not uniform within individual cells. Depicted are the regions with high sensitivity (Figure 7E-F) for the responses to 10 and 20 dyn/cm2, respectively, where the red spots indicate activation of Rac1 GTPase.


Knee loading reduces MMP13 activity in the mouse cartilage.

Hamamura K, Zhang P, Zhao L, Shim JW, Chen A, Dodge TR, Wan Q, Shih H, Na S, Lin CC, Sun HB, Yokota H - BMC Musculoskelet Disord (2013)

FRET fluorescent images using the Rac1 GTPase biosensor in C28/I2 chondrocyte cells. The red color indicates the activation level of Rac1. (A-D) Rac1 activity in response to fluid flow shear at 2, 5, 10, and 20 dyn/cm2 for 60 min. (E) Reduced Rac1 activity in response to fluid flow shear at 10 dyn/cm2 [zoom of the white boxes in (C)]. (F) Elevated Rac1 activity in response to fluid flow shear at 20 dyn/cm2 [zoom of the white boxes in (D)]. n = 7 to 17 (A-F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924329&req=5

Figure 7: FRET fluorescent images using the Rac1 GTPase biosensor in C28/I2 chondrocyte cells. The red color indicates the activation level of Rac1. (A-D) Rac1 activity in response to fluid flow shear at 2, 5, 10, and 20 dyn/cm2 for 60 min. (E) Reduced Rac1 activity in response to fluid flow shear at 10 dyn/cm2 [zoom of the white boxes in (C)]. (F) Elevated Rac1 activity in response to fluid flow shear at 20 dyn/cm2 [zoom of the white boxes in (D)]. n = 7 to 17 (A-F).
Mentions: To evaluate the role of Rac1 GTPase in mechanical regulation of MMP13, Rac1 activity of C28/I2 chondrocyte cells was monitored using the Rac1 biosensor and FRET imaging. The cells were subjected to flow-induced shear stress at 2, 5, 10, or 20 dyn/cm2, and changes in the emission ratio of YFP/CFP of the Rac1 biosensor were determined. The result presented shear intensity-dependent regulation of Rac1 activity. At low fluid flow shear of 2 and 5 dyn/cm2, Rac1 activity was not significantly altered (Figure 7A-B). When shear intensity was raised to 10 dyn/cm2, however, it was continuously lowered in a 60-min observation period (Figure 7C). When shear intensity was further elevated to 20 dyn/cm2, Rac1 activity was significantly increased 20–50 min after the onset of fluid flow (Figure 7D). The spatial distribution of Rac1 activity was not uniform within individual cells. Depicted are the regions with high sensitivity (Figure 7E-F) for the responses to 10 and 20 dyn/cm2, respectively, where the red spots indicate activation of Rac1 GTPase.

Bottom Line: Load-driven reduction in MMP13 was associated with a decrease in the phosphorylation level of p38 MAPK (p-p38) and NFκB (p-NFκB).Silencing Rac1 GTPase significantly reduced MMP13 expression and p-p38 but not p-NFκB.Knee loading reduces MMP13 activity at least in part through Rac1-mediated p38 MAPK signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Engineering, Indiana University Purdue University Indianapolis, SL220C, 723 West Michigan Street, Indianapolis, IN 46202, USA. hyokota@iupui.edu.

ABSTRACT

Background: Moderate loads with knee loading enhance bone formation, but its effects on the maintenance of the knee are not well understood. In this study, we examined the effects of knee loading on the activity of matrix metalloproteinase13 (MMP13) and evaluated the role of p38 MAPK and Rac1 GTPase in the regulation of MMP13.

Methods: Knee loading (0.5-3 N for 5 min) was applied to the right knee of surgically-induced osteoarthritis (OA) mice as well as normal (non-OA) mice, and MMP13 activity in the femoral cartilage was examined. The sham-loaded knee was used as a non-loading control. We also employed primary non-OA and OA human chondrocytes as well as C28/I2 chondrocyte cells, and examined MMP13 activity and molecular signaling in response to shear at 2-20 dyn/cm².

Results: Daily knee loading at 1 N for 2 weeks suppressed cartilage destruction in the knee of OA mice. Induction of OA elevated MMP13 activity and knee loading at 1 N suppressed this elevation. MMP13 activity was also increased in primary OA chondrocytes, and this increase was attenuated by applying shear at 10 dyn/cm². Load-driven reduction in MMP13 was associated with a decrease in the phosphorylation level of p38 MAPK (p-p38) and NFκB (p-NFκB). Molecular imaging using a fluorescence resonance energy transfer (FRET) technique showed that Rac1 activity was reduced by shear at 10 dyn/cm² and elevated by it at 20 dyn/cm². Silencing Rac1 GTPase significantly reduced MMP13 expression and p-p38 but not p-NFκB. Transfection of a constitutively active Rac1 GTPase mutant increased MMP13 activity, while a dominant negative mutant decreased it.

Conclusions: Knee loading reduces MMP13 activity at least in part through Rac1-mediated p38 MAPK signaling. This study suggests the possibility of knee loading as a therapy not only for strengthening bone but also preventing tissue degradation of the femoral cartilage.

Show MeSH
Related in: MedlinePlus