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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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Involvement of HDAC in IFNβ mediated repression of MMP-9 promoter.(A) Response of various promoter deletion constructs to a HDAC inhibitor. HT1080 cells were transiently co-transfected with MMP-9 promoter constructs. 24 hours after transfection, cells were pooled and redistributed amongst the wells, so that transfection efficiency was the same for all samples. 48 hours after transfection, the cells were treated with 220 nM TSA, 30 nM PMA and 100 U/mL IFNβ as indicated 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. Grey bars: untreated, white bars: PMA treated, grey stippled bars: PMA + TSA, hatched bars: IFNβ, and black bars: PMA + IFNβ + TSA. The P-values were calculated using statistical analysis software. As indicated above bars, *, **, ***, and **** symbols indicate P values that indicate significant difference (0.0011, 0.0009, 0.0007, and 0.0008 resp.) and the symbol # indicates a P value of 0.0874 (no significant difference) with significant values being <0.01. (B) AP-1 binding to the proximal site is required for HDAC1 recruitment to MMP-9 promoter in response to IFNβ. HT1080 cells were transfected in 6-well dishes in triplicates with −660 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional, or with wild type −660 MMP-9 construct. Cells were prepared as described previously, including treatments with 220 nM TSA, 30 nM PMA and 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, dark grey bars: PMA treated, white bars: PMA + IFNβ, and light grey bars: PMA + IFNβ + TSA. 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 0.0004 (significant difference) and the symbol # indicates a P value of 0.47 (no significant difference), with significant values being <0.01.
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pone-0042152-g003: Involvement of HDAC in IFNβ mediated repression of MMP-9 promoter.(A) Response of various promoter deletion constructs to a HDAC inhibitor. HT1080 cells were transiently co-transfected with MMP-9 promoter constructs. 24 hours after transfection, cells were pooled and redistributed amongst the wells, so that transfection efficiency was the same for all samples. 48 hours after transfection, the cells were treated with 220 nM TSA, 30 nM PMA and 100 U/mL IFNβ as indicated 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. Grey bars: untreated, white bars: PMA treated, grey stippled bars: PMA + TSA, hatched bars: IFNβ, and black bars: PMA + IFNβ + TSA. The P-values were calculated using statistical analysis software. As indicated above bars, *, **, ***, and **** symbols indicate P values that indicate significant difference (0.0011, 0.0009, 0.0007, and 0.0008 resp.) and the symbol # indicates a P value of 0.0874 (no significant difference) with significant values being <0.01. (B) AP-1 binding to the proximal site is required for HDAC1 recruitment to MMP-9 promoter in response to IFNβ. HT1080 cells were transfected in 6-well dishes in triplicates with −660 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional, or with wild type −660 MMP-9 construct. Cells were prepared as described previously, including treatments with 220 nM TSA, 30 nM PMA and 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, dark grey bars: PMA treated, white bars: PMA + IFNβ, and light grey bars: PMA + IFNβ + TSA. 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 0.0004 (significant difference) and the symbol # indicates a P value of 0.47 (no significant difference), with significant values being <0.01.

Mentions: It has been demonstrated that metastasis associated gene MTA1, which associates with the NuRD complex, reduces both basal and PMA induced MMP-9 protein and mRNA levels in HT1080 cells. MTA1’s ability to repress MMP-9 is partially dependent upon HDAC2 recruitment by MTA1 and reduced histone H3 and H4 acetylation at the MMP-9 promoter [11]. In order to investigate if HDAC recruitment could be a possible mechanism of repression of MMP-9 transcription by IFNβ, we utilized a broad spectrum HDAC inhibitor, trichostatin A (TSA). Strikingly, our results show that TSA treatment rescued the IFNβ mediated downregulation of PMA induced MMP-9 transcription in −1288 bp through −154 bp promoter constructs (Figure 3A). Both IFNβ mediated downregulation as well as the TSA rescue of MMP-9 transcription is lost in the −72 bp deletion construct (Figure 3A). Since the −154 bp construct retains the capacity to respond to TSA mediated rescue, the region between −154 and −72 bp mediates HDAC involvement in transcriptional regulation of MMP-9. Thus, the proximal AP-1 site located within this region may mediate the recruitment of HDAC.


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)

Involvement of HDAC in IFNβ mediated repression of MMP-9 promoter.(A) Response of various promoter deletion constructs to a HDAC inhibitor. HT1080 cells were transiently co-transfected with MMP-9 promoter constructs. 24 hours after transfection, cells were pooled and redistributed amongst the wells, so that transfection efficiency was the same for all samples. 48 hours after transfection, the cells were treated with 220 nM TSA, 30 nM PMA and 100 U/mL IFNβ as indicated 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. Grey bars: untreated, white bars: PMA treated, grey stippled bars: PMA + TSA, hatched bars: IFNβ, and black bars: PMA + IFNβ + TSA. The P-values were calculated using statistical analysis software. As indicated above bars, *, **, ***, and **** symbols indicate P values that indicate significant difference (0.0011, 0.0009, 0.0007, and 0.0008 resp.) and the symbol # indicates a P value of 0.0874 (no significant difference) with significant values being <0.01. (B) AP-1 binding to the proximal site is required for HDAC1 recruitment to MMP-9 promoter in response to IFNβ. HT1080 cells were transfected in 6-well dishes in triplicates with −660 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional, or with wild type −660 MMP-9 construct. Cells were prepared as described previously, including treatments with 220 nM TSA, 30 nM PMA and 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, dark grey bars: PMA treated, white bars: PMA + IFNβ, and light grey bars: PMA + IFNβ + TSA. 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 0.0004 (significant difference) and the symbol # indicates a P value of 0.47 (no significant difference), with significant values being <0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412826&req=5

pone-0042152-g003: Involvement of HDAC in IFNβ mediated repression of MMP-9 promoter.(A) Response of various promoter deletion constructs to a HDAC inhibitor. HT1080 cells were transiently co-transfected with MMP-9 promoter constructs. 24 hours after transfection, cells were pooled and redistributed amongst the wells, so that transfection efficiency was the same for all samples. 48 hours after transfection, the cells were treated with 220 nM TSA, 30 nM PMA and 100 U/mL IFNβ as indicated 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. Grey bars: untreated, white bars: PMA treated, grey stippled bars: PMA + TSA, hatched bars: IFNβ, and black bars: PMA + IFNβ + TSA. The P-values were calculated using statistical analysis software. As indicated above bars, *, **, ***, and **** symbols indicate P values that indicate significant difference (0.0011, 0.0009, 0.0007, and 0.0008 resp.) and the symbol # indicates a P value of 0.0874 (no significant difference) with significant values being <0.01. (B) AP-1 binding to the proximal site is required for HDAC1 recruitment to MMP-9 promoter in response to IFNβ. HT1080 cells were transfected in 6-well dishes in triplicates with −660 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional, or with wild type −660 MMP-9 construct. Cells were prepared as described previously, including treatments with 220 nM TSA, 30 nM PMA and 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, dark grey bars: PMA treated, white bars: PMA + IFNβ, and light grey bars: PMA + IFNβ + TSA. 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 0.0004 (significant difference) and the symbol # indicates a P value of 0.47 (no significant difference), with significant values being <0.01.
Mentions: It has been demonstrated that metastasis associated gene MTA1, which associates with the NuRD complex, reduces both basal and PMA induced MMP-9 protein and mRNA levels in HT1080 cells. MTA1’s ability to repress MMP-9 is partially dependent upon HDAC2 recruitment by MTA1 and reduced histone H3 and H4 acetylation at the MMP-9 promoter [11]. In order to investigate if HDAC recruitment could be a possible mechanism of repression of MMP-9 transcription by IFNβ, we utilized a broad spectrum HDAC inhibitor, trichostatin A (TSA). Strikingly, our results show that TSA treatment rescued the IFNβ mediated downregulation of PMA induced MMP-9 transcription in −1288 bp through −154 bp promoter constructs (Figure 3A). Both IFNβ mediated downregulation as well as the TSA rescue of MMP-9 transcription is lost in the −72 bp deletion construct (Figure 3A). Since the −154 bp construct retains the capacity to respond to TSA mediated rescue, the region between −154 and −72 bp mediates HDAC involvement in transcriptional regulation of MMP-9. Thus, the proximal AP-1 site located within this region may mediate the recruitment of HDAC.

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