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AP-1 mediated transcriptional repression of matrix metalloproteinase-9 by recruitment of histone deacetylase 1 in response to interferon β.

Mittelstadt ML, Patel RC - PLoS ONE (2012)

Bottom Line: Using reporter assays with promoter deletion constructs we show that IFNβ's inhibitory effects require a region of the promoter between -154 and -72, which contains an AP-1 binding site.Chromatin immunoprecipitation (ChIP) studies indicate that IFNβ increases histone deacetylase (HDAC)-1 recruitment to the MMP-9 promoter and reduces histone H3 acetylation, in addition to reduced NF-κB recruitment.Altogether, our results establish that the repression of MMP-9 transcription in response to IFNβ occurs by the recruitment of HDAC1 via the proximal AP-1 binding site.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America.

ABSTRACT
Matrix metalloproteinase-9 (MMP-9) is a 92 kDa zinc-dependant endopeptidase that degrades components of the extracellular matrix. Increased expression of MMP-9 is implicated in many pathological conditions including metastatic cancer, multiple sclerosis, and atherosclerosis. Although it has been widely noted that interferon-β (IFNβ) downregulates both the basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression at the transcriptional level, the molecular mechanism of this repression is poorly understood. In the present study we identify a novel mechanism for repression of MMP-9 transcription by IFNβ in HT1080 fibrosarcoma cells. Using reporter assays with promoter deletion constructs we show that IFNβ's inhibitory effects require a region of the promoter between -154 and -72, which contains an AP-1 binding site. Chromatin immunoprecipitation (ChIP) studies indicate that IFNβ increases histone deacetylase (HDAC)-1 recruitment to the MMP-9 promoter and reduces histone H3 acetylation, in addition to reduced NF-κB recruitment. ChIP analysis shows that IFNβ induced HDAC1 recruitment to the MMP-9 promoter and IFNβ mediated transcriptional repression is lost when the AP-1 binding site is inactivated by a point mutation. Altogether, our results establish that the repression of MMP-9 transcription in response to IFNβ occurs by the recruitment of HDAC1 via the proximal AP-1 binding site.

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Loss of IFNβ mediated repression of MMP-9 promoter when the binding of AP-1 to the proximal site is abrogated.HT1080 cells were transfected with −154 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional. Cells were prepared as described previously and were treated with 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly luciferase activities, and normalized by protein concentration to account for any cell concentration differences between samples. Black bars: untreated values considered as 1 for each construct, white bars: IFNβ treated. The experiment was repeated three times and the error bars represent standard deviation calculated from three separate experiments. The P-values were calculated using statistical analysis software. As indicated above the bars, * symbol indicates a P value of 1.8×10−4 (significant difference) and the symbol # indicates a P value of 0.092 (no significant difference), with significant values being <0.01.
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pone-0042152-g002: Loss of IFNβ mediated repression of MMP-9 promoter when the binding of AP-1 to the proximal site is abrogated.HT1080 cells were transfected with −154 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional. Cells were prepared as described previously and were treated with 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly luciferase activities, and normalized by protein concentration to account for any cell concentration differences between samples. Black bars: untreated values considered as 1 for each construct, white bars: IFNβ treated. The experiment was repeated three times and the error bars represent standard deviation calculated from three separate experiments. The P-values were calculated using statistical analysis software. As indicated above the bars, * symbol indicates a P value of 1.8×10−4 (significant difference) and the symbol # indicates a P value of 0.092 (no significant difference), with significant values being <0.01.

Mentions: Since AP-1 is sufficient to confer IFNβ mediated repression on a heterologous promoter (Figure 1C) and IFNβ did not prevent AP-1’s DNA binding ability in response to IFNβ (Figure 1D, and [14], we next examined whether AP-1 binding could in fact be required for IFNβ mediated transcriptional repression of MMP-9 promoter. An MMP-9 expression construct was utilized which contained a mutated proximal AP-1 site, and had been previously described to disrupt binding of AP-1 subunits, rendering the AP-1 binding site non-functional [15]. This construct was transfected into HT1080 cells alongside the wt −154 bp MMP-9 construct and luciferase activities were compared after treatment with IFNβ. Promoter activity of the wt −154 bp promoter was downregulated 45% under IFNβ treatment conditions, similar to the amount of repression seen in the −660 bp promoter (Fig. 1 B) under IFNβ treatment conditions. Importantly, this repression is nearly absent in the −154 bp promoter containing the AP-1 site point mutation (Figure 2). Treatment with PMA does not result in induction of the −154 AP-1 mutant promoter piece, as this promoter segment does not contain any of the cis-acting binding sites known to mediate PMA induction of MMP-9 (NF-κB, Sp1, or AP-1) (data not shown). Because of this striking alteration in the capacity for downregulation under IFNβ treatment conditions, we concluded that binding of AP-1 to the proximal AP-1 binding site of the MMP-9 promoter is essential for IFNβ repression. Additionally, because the binding of AP-1 is required for IFNβ repression, it is possible that AP-1 may be recruiting a repressor to the MMP-9 promoter under IFNβ treatment conditions to bring about downregulation of MMP-9 transcription.


AP-1 mediated transcriptional repression of matrix metalloproteinase-9 by recruitment of histone deacetylase 1 in response to interferon β.

Mittelstadt ML, Patel RC - PLoS ONE (2012)

Loss of IFNβ mediated repression of MMP-9 promoter when the binding of AP-1 to the proximal site is abrogated.HT1080 cells were transfected with −154 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional. Cells were prepared as described previously and were treated with 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly luciferase activities, and normalized by protein concentration to account for any cell concentration differences between samples. Black bars: untreated values considered as 1 for each construct, white bars: IFNβ treated. The experiment was repeated three times and the error bars represent standard deviation calculated from three separate experiments. The P-values were calculated using statistical analysis software. As indicated above the bars, * symbol indicates a P value of 1.8×10−4 (significant difference) and the symbol # indicates a P value of 0.092 (no significant difference), with significant values being <0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412826&req=5

pone-0042152-g002: Loss of IFNβ mediated repression of MMP-9 promoter when the binding of AP-1 to the proximal site is abrogated.HT1080 cells were transfected with −154 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional. Cells were prepared as described previously and were treated with 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly luciferase activities, and normalized by protein concentration to account for any cell concentration differences between samples. Black bars: untreated values considered as 1 for each construct, white bars: IFNβ treated. The experiment was repeated three times and the error bars represent standard deviation calculated from three separate experiments. The P-values were calculated using statistical analysis software. As indicated above the bars, * symbol indicates a P value of 1.8×10−4 (significant difference) and the symbol # indicates a P value of 0.092 (no significant difference), with significant values being <0.01.
Mentions: Since AP-1 is sufficient to confer IFNβ mediated repression on a heterologous promoter (Figure 1C) and IFNβ did not prevent AP-1’s DNA binding ability in response to IFNβ (Figure 1D, and [14], we next examined whether AP-1 binding could in fact be required for IFNβ mediated transcriptional repression of MMP-9 promoter. An MMP-9 expression construct was utilized which contained a mutated proximal AP-1 site, and had been previously described to disrupt binding of AP-1 subunits, rendering the AP-1 binding site non-functional [15]. This construct was transfected into HT1080 cells alongside the wt −154 bp MMP-9 construct and luciferase activities were compared after treatment with IFNβ. Promoter activity of the wt −154 bp promoter was downregulated 45% under IFNβ treatment conditions, similar to the amount of repression seen in the −660 bp promoter (Fig. 1 B) under IFNβ treatment conditions. Importantly, this repression is nearly absent in the −154 bp promoter containing the AP-1 site point mutation (Figure 2). Treatment with PMA does not result in induction of the −154 AP-1 mutant promoter piece, as this promoter segment does not contain any of the cis-acting binding sites known to mediate PMA induction of MMP-9 (NF-κB, Sp1, or AP-1) (data not shown). Because of this striking alteration in the capacity for downregulation under IFNβ treatment conditions, we concluded that binding of AP-1 to the proximal AP-1 binding site of the MMP-9 promoter is essential for IFNβ repression. Additionally, because the binding of AP-1 is required for IFNβ repression, it is possible that AP-1 may be recruiting a repressor to the MMP-9 promoter under IFNβ treatment conditions to bring about downregulation of MMP-9 transcription.

Bottom Line: Using reporter assays with promoter deletion constructs we show that IFNβ's inhibitory effects require a region of the promoter between -154 and -72, which contains an AP-1 binding site.Chromatin immunoprecipitation (ChIP) studies indicate that IFNβ increases histone deacetylase (HDAC)-1 recruitment to the MMP-9 promoter and reduces histone H3 acetylation, in addition to reduced NF-κB recruitment.Altogether, our results establish that the repression of MMP-9 transcription in response to IFNβ occurs by the recruitment of HDAC1 via the proximal AP-1 binding site.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of South Carolina, Columbia, South Carolina, United States of America.

ABSTRACT
Matrix metalloproteinase-9 (MMP-9) is a 92 kDa zinc-dependant endopeptidase that degrades components of the extracellular matrix. Increased expression of MMP-9 is implicated in many pathological conditions including metastatic cancer, multiple sclerosis, and atherosclerosis. Although it has been widely noted that interferon-β (IFNβ) downregulates both the basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression at the transcriptional level, the molecular mechanism of this repression is poorly understood. In the present study we identify a novel mechanism for repression of MMP-9 transcription by IFNβ in HT1080 fibrosarcoma cells. Using reporter assays with promoter deletion constructs we show that IFNβ's inhibitory effects require a region of the promoter between -154 and -72, which contains an AP-1 binding site. Chromatin immunoprecipitation (ChIP) studies indicate that IFNβ increases histone deacetylase (HDAC)-1 recruitment to the MMP-9 promoter and reduces histone H3 acetylation, in addition to reduced NF-κB recruitment. ChIP analysis shows that IFNβ induced HDAC1 recruitment to the MMP-9 promoter and IFNβ mediated transcriptional repression is lost when the AP-1 binding site is inactivated by a point mutation. Altogether, our results establish that the repression of MMP-9 transcription in response to IFNβ occurs by the recruitment of HDAC1 via the proximal AP-1 binding site.

Show MeSH
Related in: MedlinePlus