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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 c-Fos, c-Jun, Runx2, MMP-13, and GAPDH mRNA expression in the IL-1β -induced mesenchymal stromal cells of GCT. GCT stromal cells were transfected by electroporation with corresponding siRNA for 48 h. Treated samples were examined using real-time PCR. The ΔΔCT method was used to calculate the real-time RT-PCR fold change using RPS18 mRNA as an endogenous control. Three independent experiments were performed. mRNA expression of (a) c-Jun, (b) Runx2, (c) MMP-13, and (d) GAPDH upon treatment with combinations of siRNA. *P < .05 is versus random nonspecific siRNA control. Statistical comparison by analysis of variance with post hoc Tukey's tests.
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fig4: The effect of siRNA knockdown on c-Fos, c-Jun, Runx2, MMP-13, and GAPDH mRNA expression in the IL-1β -induced mesenchymal stromal cells of GCT. GCT stromal cells were transfected by electroporation with corresponding siRNA for 48 h. Treated samples were examined using real-time PCR. The ΔΔCT method was used to calculate the real-time RT-PCR fold change using RPS18 mRNA as an endogenous control. Three independent experiments were performed. mRNA expression of (a) c-Jun, (b) Runx2, (c) MMP-13, and (d) GAPDH upon treatment with combinations of siRNA. *P < .05 is versus random nonspecific siRNA control. Statistical comparison by analysis of variance with post hoc Tukey's tests.

Mentions: Next, to further elucidate the role of AP-1 and Runx2 in MMP-13 transcriptional regulation, we depleted various combinations of c-Fos, c-Jun, and Runx2 in the mesenchymal stromal cells of GCT by using RNA interference. Random siRNA served as the negative control. All siRNA treatments (c-Fos, c-Jun, Runx2, and c-Fos + c-Jun, c-Fos + c-Jun + Runx2) were normalized to the random siRNA negative control. The expression of c-Jun was 40–60% suppressed when treated with c-Jun, and c-Fos + c-Jun, c-Fos + c-Jun + Runx2 siRNA, respectively (Figure 4(a)). More than 70% of Runx2 expression was depleted in both Runx2 and c-Fos + c-Jun + Runx2 siRNA conditions, as detected by real-time PCR (Figure 4(b)). Importantly, MMP-13 expression was decreased by 40–50% following knockdown of c-Jun alone, Runx2 alone, c-Fos and c-Jun in combination, and c-Fos, c-Jun and Runx2 in combination (Figure 4(c)). Yet, the lack of effect in MMP-13 suppression may be due to the low efficiency of the cFos knockdown. As a test of transfection efficiency, GAPDH mRNA was decreased by 70% when treated with the GAPDH siRNA but unaffected in the random and other siRNA conditions (Figure 4(d)).


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 c-Fos, c-Jun, Runx2, MMP-13, and GAPDH mRNA expression in the IL-1β -induced mesenchymal stromal cells of GCT. GCT stromal cells were transfected by electroporation with corresponding siRNA for 48 h. Treated samples were examined using real-time PCR. The ΔΔCT method was used to calculate the real-time RT-PCR fold change using RPS18 mRNA as an endogenous control. Three independent experiments were performed. mRNA expression of (a) c-Jun, (b) Runx2, (c) MMP-13, and (d) GAPDH upon treatment with combinations of siRNA. *P < .05 is versus random nonspecific siRNA control. Statistical comparison by analysis of variance with post hoc Tukey's tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: The effect of siRNA knockdown on c-Fos, c-Jun, Runx2, MMP-13, and GAPDH mRNA expression in the IL-1β -induced mesenchymal stromal cells of GCT. GCT stromal cells were transfected by electroporation with corresponding siRNA for 48 h. Treated samples were examined using real-time PCR. The ΔΔCT method was used to calculate the real-time RT-PCR fold change using RPS18 mRNA as an endogenous control. Three independent experiments were performed. mRNA expression of (a) c-Jun, (b) Runx2, (c) MMP-13, and (d) GAPDH upon treatment with combinations of siRNA. *P < .05 is versus random nonspecific siRNA control. Statistical comparison by analysis of variance with post hoc Tukey's tests.
Mentions: Next, to further elucidate the role of AP-1 and Runx2 in MMP-13 transcriptional regulation, we depleted various combinations of c-Fos, c-Jun, and Runx2 in the mesenchymal stromal cells of GCT by using RNA interference. Random siRNA served as the negative control. All siRNA treatments (c-Fos, c-Jun, Runx2, and c-Fos + c-Jun, c-Fos + c-Jun + Runx2) were normalized to the random siRNA negative control. The expression of c-Jun was 40–60% suppressed when treated with c-Jun, and c-Fos + c-Jun, c-Fos + c-Jun + Runx2 siRNA, respectively (Figure 4(a)). More than 70% of Runx2 expression was depleted in both Runx2 and c-Fos + c-Jun + Runx2 siRNA conditions, as detected by real-time PCR (Figure 4(b)). Importantly, MMP-13 expression was decreased by 40–50% following knockdown of c-Jun alone, Runx2 alone, c-Fos and c-Jun in combination, and c-Fos, c-Jun and Runx2 in combination (Figure 4(c)). Yet, the lack of effect in MMP-13 suppression may be due to the low efficiency of the cFos knockdown. As a test of transfection efficiency, GAPDH mRNA was decreased by 70% when treated with the GAPDH siRNA but unaffected in the random and other siRNA conditions (Figure 4(d)).

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