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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

Cell morphology of homogeneous GCT stromal cells induced by IL-1β. Representative picture was taken with light microscope at magnification ×400.
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fig1: Cell morphology of homogeneous GCT stromal cells induced by IL-1β. Representative picture was taken with light microscope at magnification ×400.

Mentions: To identify which members of the AP-1 family are present in GCT stromal cells, an AP-1 screening assay was used to detect specific transcription factor DNA binding activity in the nuclear extracts. GCT stromal cells stimulated with IL-1β were isolated and passaged from primary patient tissue sample into homogeneous mesenchymal stromal cells, as shown in Figure 1, before extracting the lysate. The AP-1 screening assay examines both the Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) families in AP-1. The specific competitor double-stranded oligonucleotide was included as an important control for verifying the specificity of the colorimetric signal resulting from protein binding to the labeled AP-1 probe. Only c-Fos and c-Jun were shown to be significantly present in GCT stromal cells (Figure 2). Both were reduced when the specific competitor was added to the lysate, indicating specific binding.


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)

Cell morphology of homogeneous GCT stromal cells induced by IL-1β. Representative picture was taken with light microscope at magnification ×400.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Cell morphology of homogeneous GCT stromal cells induced by IL-1β. Representative picture was taken with light microscope at magnification ×400.
Mentions: To identify which members of the AP-1 family are present in GCT stromal cells, an AP-1 screening assay was used to detect specific transcription factor DNA binding activity in the nuclear extracts. GCT stromal cells stimulated with IL-1β were isolated and passaged from primary patient tissue sample into homogeneous mesenchymal stromal cells, as shown in Figure 1, before extracting the lysate. The AP-1 screening assay examines both the Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) families in AP-1. The specific competitor double-stranded oligonucleotide was included as an important control for verifying the specificity of the colorimetric signal resulting from protein binding to the labeled AP-1 probe. Only c-Fos and c-Jun were shown to be significantly present in GCT stromal cells (Figure 2). Both were reduced when the specific competitor was added to the lysate, indicating specific binding.

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