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AP-1 as a Regulator of MMP-13 in the Stromal Cell of Giant Cell Tumor of Bone.

Mak IW, Turcotte RE, Popovic S, Singh G, Ghert M - Biochem Res Int (2011)

Bottom Line: We have previously shown that MMP-13 expression in these cells is regulated, at least in part, by the RUNX2 transcription factor.We then used siRNA gene knockdown to determine that these elements, in particular c-Jun, are upstream regulators of MMP-13 expression and activity in GCT stromal cells.We conclude that there was no synergy found between RUNX2 and AP-1 in the regulation of the MMP13 expression and that these transcription factors may be independently regulated in these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, McMaster University, Hamilton, ON, Canada L8S 4L8.

ABSTRACT
Matrix-metalloproteinase-13 (MMP-13) has been shown to be an important protease in inflammatory and neoplastic conditions of the skeletal system. In particular, the stromal cells of giant cell tumor of bone (GCT) express very high levels of MMP-13 in response to the cytokine-rich environment of the tumor. We have previously shown that MMP-13 expression in these cells is regulated, at least in part, by the RUNX2 transcription factor. In the current study, we identify the expression of the c-Fos and c-Jun elements of the AP-1 transcription factor in these cells by protein screening assays and real-time PCR. We then used siRNA gene knockdown to determine that these elements, in particular c-Jun, are upstream regulators of MMP-13 expression and activity in GCT stromal cells. We conclude that there was no synergy found between RUNX2 and AP-1 in the regulation of the MMP13 expression and that these transcription factors may be independently regulated in these cells.

No MeSH data available.


Related in: MedlinePlus

The effect of siRNA knockdown on the level of MMP-13 activity in IL-1β-stimulated GCT cells. Filtered and concentrated conditioned media from siRNA silenced GCT stromal cells were analyzed using the MMP-13 activity kit as per optimization in our lab. Results of the ELISA activity assay are shown in triplicate with error bars. The MMP-13 activity levels were normalized to the amount of total protein for each condition.
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fig5: The effect of siRNA knockdown on the level of MMP-13 activity in IL-1β-stimulated GCT cells. Filtered and concentrated conditioned media from siRNA silenced GCT stromal cells were analyzed using the MMP-13 activity kit as per optimization in our lab. Results of the ELISA activity assay are shown in triplicate with error bars. The MMP-13 activity levels were normalized to the amount of total protein for each condition.

Mentions: To validate the effect of silencing c-Fos, c-Jun, and Runx2 on their downstream target MMP-13 at the translational level, MMP-13 enzyme activity was measured from culture medium collected from the siRNA treatments. The percentage of MMP-13 activity (Figure 5) exhibited a similar trend to measurements from the MMP-13 real-time PCR data (Figure 4(c)). However, c-Fos siRNA demonstrated a 30% knockdown of MMP-13 activity as shown in Figure 5, as compared to its insignificant effect on MMP-13 transcription. c-Jun and Runx2 gene silencing resulted in 40% and 55% knockdown in MMP-13 activity, respectively. Interestingly, AP-1 knockdown resulted in greater MMP-13 activity silencing than did Runx2 knockdown, indicating that both transcription factors play a role in MMP-13 expression and activity in these cells.


AP-1 as a Regulator of MMP-13 in the Stromal Cell of Giant Cell Tumor of Bone.

Mak IW, Turcotte RE, Popovic S, Singh G, Ghert M - Biochem Res Int (2011)

The effect of siRNA knockdown on the level of MMP-13 activity in IL-1β-stimulated GCT cells. Filtered and concentrated conditioned media from siRNA silenced GCT stromal cells were analyzed using the MMP-13 activity kit as per optimization in our lab. Results of the ELISA activity assay are shown in triplicate with error bars. The MMP-13 activity levels were normalized to the amount of total protein for each condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3065034&req=5

fig5: The effect of siRNA knockdown on the level of MMP-13 activity in IL-1β-stimulated GCT cells. Filtered and concentrated conditioned media from siRNA silenced GCT stromal cells were analyzed using the MMP-13 activity kit as per optimization in our lab. Results of the ELISA activity assay are shown in triplicate with error bars. The MMP-13 activity levels were normalized to the amount of total protein for each condition.
Mentions: To validate the effect of silencing c-Fos, c-Jun, and Runx2 on their downstream target MMP-13 at the translational level, MMP-13 enzyme activity was measured from culture medium collected from the siRNA treatments. The percentage of MMP-13 activity (Figure 5) exhibited a similar trend to measurements from the MMP-13 real-time PCR data (Figure 4(c)). However, c-Fos siRNA demonstrated a 30% knockdown of MMP-13 activity as shown in Figure 5, as compared to its insignificant effect on MMP-13 transcription. c-Jun and Runx2 gene silencing resulted in 40% and 55% knockdown in MMP-13 activity, respectively. Interestingly, AP-1 knockdown resulted in greater MMP-13 activity silencing than did Runx2 knockdown, indicating that both transcription factors play a role in MMP-13 expression and activity in these cells.

Bottom Line: We have previously shown that MMP-13 expression in these cells is regulated, at least in part, by the RUNX2 transcription factor.We then used siRNA gene knockdown to determine that these elements, in particular c-Jun, are upstream regulators of MMP-13 expression and activity in GCT stromal cells.We conclude that there was no synergy found between RUNX2 and AP-1 in the regulation of the MMP13 expression and that these transcription factors may be independently regulated in these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, McMaster University, Hamilton, ON, Canada L8S 4L8.

ABSTRACT
Matrix-metalloproteinase-13 (MMP-13) has been shown to be an important protease in inflammatory and neoplastic conditions of the skeletal system. In particular, the stromal cells of giant cell tumor of bone (GCT) express very high levels of MMP-13 in response to the cytokine-rich environment of the tumor. We have previously shown that MMP-13 expression in these cells is regulated, at least in part, by the RUNX2 transcription factor. In the current study, we identify the expression of the c-Fos and c-Jun elements of the AP-1 transcription factor in these cells by protein screening assays and real-time PCR. We then used siRNA gene knockdown to determine that these elements, in particular c-Jun, are upstream regulators of MMP-13 expression and activity in GCT stromal cells. We conclude that there was no synergy found between RUNX2 and AP-1 in the regulation of the MMP13 expression and that these transcription factors may be independently regulated in these cells.

No MeSH data available.


Related in: MedlinePlus