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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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Related in: MedlinePlus

The -154 bp promoter construct retains the ability to respond to IFNβ.(A) Schematic diagram of the deletion constructs. Progressive deletions of MMP-9 promoter were created by PCR amplification with appropriate primers. Various transcription factor binding sites and the TATA box are as indicated. (B) Mapping the promoter region responsible for IFNβ mediated transcriptional repression. HT1080 cells were transiently transfected in 6 well plates in triplicates with MMP-9 promoter deletion constructs as indicated using Effectene transfection reagent (Qiagen). A plasmid encoding Renilla luciferase (pRL-, Promega) was co-transfected for the normalization of transfection efficiencies. 24 hours after transfection, the cells were treated with 30 nM PMA and 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly and Renilla luciferase activities. Black bars indicate untreated values, considered as 1 for each construct. Grey bars indicate PMA treated values represented as fold inductions. White bars indicate PMA and IFNβ treated values also represented as fold inductions. The values represent averages of triplicate samples from four different experiments for each construct and the error bars represent standard deviation. The P-values were calculated using statistical analysis software. As indicated above bars, *, **, ***, and **** symbols indicate P values that indicate significant difference (0.0009, 0.0007, 0.0008, and 0.0009 resp.) and the symbol # indicates a P value of 0.1216 (no significant difference) with significant values being <0.01. Inset: The percent inhibition in response to IFNβ is plotted for each deletion construct. (C) AP-1 binding site is sufficient to confer downregulation by IFNβ on a synthetic reporter construct. HT1080 cells were co-transfected with either consensus AP-1 binding site concatamer (pAP-1-Luc, Stratagene) or MMP-9-specific AP-1 binding site concatamer (AP-1 (MMP9)-Luc) linked to a basal promoter-Luc, and a plasmid encoding Renilla luciferase (pRL-, Promega). 24 hours after transfection, cells were treated with 30 ηM PMA and 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly and Renilla luciferase activities. Black bars indicate untreated cell values, considered as 1 for each construct. Grey bars indicate the PMA treated values represented as fold inductions. White bars indicate the PMA and IFNβ treated values represented as fold inductions. The values are averages from three separate experiments and the error bards represent standard deviation. The P-values were calculated using statistical analysis software. As indicated above the bars, *symbol indicates a P value of 0.0008 (significant difference) and the symbol # indicates a P value of 0.0001 (significant difference), with significant values being <0.01. (D) IFNβ does not inhibit the DNA-binding activity of AP-1. EMSA was carried out using the nuclear extracts prepared from HT1080 cells that were untreated or treated with PMA, PMA plus IFNβ for 30 minutes. 1 µg of nuclear extracts were incubated with the 32P-labeled AP-1 (region −93 to −56 of MMP-9 promoter) probe. Arrows indicate AP-1 complex bound to DNA. Unlabelled specific (SP: AP-1 consensus sequence) and non-specific (NSP: TATA box consensus sequence) competitor oligonucleotides in 100-fold molar excess were utilized to confirm the identity of the bound complex.
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pone-0042152-g001: The -154 bp promoter construct retains the ability to respond to IFNβ.(A) Schematic diagram of the deletion constructs. Progressive deletions of MMP-9 promoter were created by PCR amplification with appropriate primers. Various transcription factor binding sites and the TATA box are as indicated. (B) Mapping the promoter region responsible for IFNβ mediated transcriptional repression. HT1080 cells were transiently transfected in 6 well plates in triplicates with MMP-9 promoter deletion constructs as indicated using Effectene transfection reagent (Qiagen). A plasmid encoding Renilla luciferase (pRL-, Promega) was co-transfected for the normalization of transfection efficiencies. 24 hours after transfection, the cells were treated with 30 nM PMA and 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly and Renilla luciferase activities. Black bars indicate untreated values, considered as 1 for each construct. Grey bars indicate PMA treated values represented as fold inductions. White bars indicate PMA and IFNβ treated values also represented as fold inductions. The values represent averages of triplicate samples from four different experiments for each construct and the error bars represent standard deviation. The P-values were calculated using statistical analysis software. As indicated above bars, *, **, ***, and **** symbols indicate P values that indicate significant difference (0.0009, 0.0007, 0.0008, and 0.0009 resp.) and the symbol # indicates a P value of 0.1216 (no significant difference) with significant values being <0.01. Inset: The percent inhibition in response to IFNβ is plotted for each deletion construct. (C) AP-1 binding site is sufficient to confer downregulation by IFNβ on a synthetic reporter construct. HT1080 cells were co-transfected with either consensus AP-1 binding site concatamer (pAP-1-Luc, Stratagene) or MMP-9-specific AP-1 binding site concatamer (AP-1 (MMP9)-Luc) linked to a basal promoter-Luc, and a plasmid encoding Renilla luciferase (pRL-, Promega). 24 hours after transfection, cells were treated with 30 ηM PMA and 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly and Renilla luciferase activities. Black bars indicate untreated cell values, considered as 1 for each construct. Grey bars indicate the PMA treated values represented as fold inductions. White bars indicate the PMA and IFNβ treated values represented as fold inductions. The values are averages from three separate experiments and the error bards represent standard deviation. The P-values were calculated using statistical analysis software. As indicated above the bars, *symbol indicates a P value of 0.0008 (significant difference) and the symbol # indicates a P value of 0.0001 (significant difference), with significant values being <0.01. (D) IFNβ does not inhibit the DNA-binding activity of AP-1. EMSA was carried out using the nuclear extracts prepared from HT1080 cells that were untreated or treated with PMA, PMA plus IFNβ for 30 minutes. 1 µg of nuclear extracts were incubated with the 32P-labeled AP-1 (region −93 to −56 of MMP-9 promoter) probe. Arrows indicate AP-1 complex bound to DNA. Unlabelled specific (SP: AP-1 consensus sequence) and non-specific (NSP: TATA box consensus sequence) competitor oligonucleotides in 100-fold molar excess were utilized to confirm the identity of the bound complex.

Mentions: IFNβ is known to repress both basal and PMA induced MMP-9 at the transcriptional level [2], [13]. In order to determine the region of the MMP-9 promoter that is mediating IFNβ repression, a promoter deletion series inserted upstream of a firefly luciferase reporter (−1298 bp, −660 bp, −462 bp, −154 bp, and −72 bp) was utilized (Figure 1A). It was previously reported that the recruitment of IRF1 to a IFN-responsive-like element, which overlaps the NF-κB binding site at −615 bp, acts to partially repress MMP-9 expression in response to IFNγ due to IRF1’s competitive inhibition of NF-κB protein binding [13]. However, our results show that IFNβ mediated repression of MMP-9 (between 40% and 50% reduction in luciferase reporter activity, depending on size of MMP-9 promoter deletion) persists until the −154 bp deletion construct (Figure 1B), but is lost in the −72 bp construct. The poor induction of the −72 bp construct is not surprising as this construct does not retain the NF-κB, AP-1 and Sp1 binding sites in the promoter, which are known to be essential for PMA induction of the transcription. Of significance is the fact that the −461 bp and the −154 bp constructs retain strong repressive response to IFNβ (Figure 1B inset), thereby indicating that the loss of NF-κB site does not affect the ability of IFNβ to repress MMP-9 promoter. Based on these results, we concluded that the sequences present within the −154 bp construct are sufficient for mediating IFNβ repression on MMP-9 promoter. It should be noted that PMA induction is partially lost in the −461 bp and all other smaller constructs in the deletion series. This is expected, as PMA induction occurs due to synergistic activation through AP-1, NF-κB, and Sp1 transcription factors, and in the region between −661 and −461 bp of the MMP-9 promoter there exists an NF-κB, Sp1 and the distal AP-1 binding sites.


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)

The -154 bp promoter construct retains the ability to respond to IFNβ.(A) Schematic diagram of the deletion constructs. Progressive deletions of MMP-9 promoter were created by PCR amplification with appropriate primers. Various transcription factor binding sites and the TATA box are as indicated. (B) Mapping the promoter region responsible for IFNβ mediated transcriptional repression. HT1080 cells were transiently transfected in 6 well plates in triplicates with MMP-9 promoter deletion constructs as indicated using Effectene transfection reagent (Qiagen). A plasmid encoding Renilla luciferase (pRL-, Promega) was co-transfected for the normalization of transfection efficiencies. 24 hours after transfection, the cells were treated with 30 nM PMA and 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly and Renilla luciferase activities. Black bars indicate untreated values, considered as 1 for each construct. Grey bars indicate PMA treated values represented as fold inductions. White bars indicate PMA and IFNβ treated values also represented as fold inductions. The values represent averages of triplicate samples from four different experiments for each construct and the error bars represent standard deviation. The P-values were calculated using statistical analysis software. As indicated above bars, *, **, ***, and **** symbols indicate P values that indicate significant difference (0.0009, 0.0007, 0.0008, and 0.0009 resp.) and the symbol # indicates a P value of 0.1216 (no significant difference) with significant values being <0.01. Inset: The percent inhibition in response to IFNβ is plotted for each deletion construct. (C) AP-1 binding site is sufficient to confer downregulation by IFNβ on a synthetic reporter construct. HT1080 cells were co-transfected with either consensus AP-1 binding site concatamer (pAP-1-Luc, Stratagene) or MMP-9-specific AP-1 binding site concatamer (AP-1 (MMP9)-Luc) linked to a basal promoter-Luc, and a plasmid encoding Renilla luciferase (pRL-, Promega). 24 hours after transfection, cells were treated with 30 ηM PMA and 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly and Renilla luciferase activities. Black bars indicate untreated cell values, considered as 1 for each construct. Grey bars indicate the PMA treated values represented as fold inductions. White bars indicate the PMA and IFNβ treated values represented as fold inductions. The values are averages from three separate experiments and the error bards represent standard deviation. The P-values were calculated using statistical analysis software. As indicated above the bars, *symbol indicates a P value of 0.0008 (significant difference) and the symbol # indicates a P value of 0.0001 (significant difference), with significant values being <0.01. (D) IFNβ does not inhibit the DNA-binding activity of AP-1. EMSA was carried out using the nuclear extracts prepared from HT1080 cells that were untreated or treated with PMA, PMA plus IFNβ for 30 minutes. 1 µg of nuclear extracts were incubated with the 32P-labeled AP-1 (region −93 to −56 of MMP-9 promoter) probe. Arrows indicate AP-1 complex bound to DNA. Unlabelled specific (SP: AP-1 consensus sequence) and non-specific (NSP: TATA box consensus sequence) competitor oligonucleotides in 100-fold molar excess were utilized to confirm the identity of the bound complex.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412826&req=5

pone-0042152-g001: The -154 bp promoter construct retains the ability to respond to IFNβ.(A) Schematic diagram of the deletion constructs. Progressive deletions of MMP-9 promoter were created by PCR amplification with appropriate primers. Various transcription factor binding sites and the TATA box are as indicated. (B) Mapping the promoter region responsible for IFNβ mediated transcriptional repression. HT1080 cells were transiently transfected in 6 well plates in triplicates with MMP-9 promoter deletion constructs as indicated using Effectene transfection reagent (Qiagen). A plasmid encoding Renilla luciferase (pRL-, Promega) was co-transfected for the normalization of transfection efficiencies. 24 hours after transfection, the cells were treated with 30 nM PMA and 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly and Renilla luciferase activities. Black bars indicate untreated values, considered as 1 for each construct. Grey bars indicate PMA treated values represented as fold inductions. White bars indicate PMA and IFNβ treated values also represented as fold inductions. The values represent averages of triplicate samples from four different experiments for each construct and the error bars represent standard deviation. The P-values were calculated using statistical analysis software. As indicated above bars, *, **, ***, and **** symbols indicate P values that indicate significant difference (0.0009, 0.0007, 0.0008, and 0.0009 resp.) and the symbol # indicates a P value of 0.1216 (no significant difference) with significant values being <0.01. Inset: The percent inhibition in response to IFNβ is plotted for each deletion construct. (C) AP-1 binding site is sufficient to confer downregulation by IFNβ on a synthetic reporter construct. HT1080 cells were co-transfected with either consensus AP-1 binding site concatamer (pAP-1-Luc, Stratagene) or MMP-9-specific AP-1 binding site concatamer (AP-1 (MMP9)-Luc) linked to a basal promoter-Luc, and a plasmid encoding Renilla luciferase (pRL-, Promega). 24 hours after transfection, cells were treated with 30 ηM PMA and 100 U/mL IFNβ for 18 hours prior to preparation of cell extracts. Cell extracts were assayed for firefly and Renilla luciferase activities. Black bars indicate untreated cell values, considered as 1 for each construct. Grey bars indicate the PMA treated values represented as fold inductions. White bars indicate the PMA and IFNβ treated values represented as fold inductions. The values are averages from three separate experiments and the error bards represent standard deviation. The P-values were calculated using statistical analysis software. As indicated above the bars, *symbol indicates a P value of 0.0008 (significant difference) and the symbol # indicates a P value of 0.0001 (significant difference), with significant values being <0.01. (D) IFNβ does not inhibit the DNA-binding activity of AP-1. EMSA was carried out using the nuclear extracts prepared from HT1080 cells that were untreated or treated with PMA, PMA plus IFNβ for 30 minutes. 1 µg of nuclear extracts were incubated with the 32P-labeled AP-1 (region −93 to −56 of MMP-9 promoter) probe. Arrows indicate AP-1 complex bound to DNA. Unlabelled specific (SP: AP-1 consensus sequence) and non-specific (NSP: TATA box consensus sequence) competitor oligonucleotides in 100-fold molar excess were utilized to confirm the identity of the bound complex.
Mentions: IFNβ is known to repress both basal and PMA induced MMP-9 at the transcriptional level [2], [13]. In order to determine the region of the MMP-9 promoter that is mediating IFNβ repression, a promoter deletion series inserted upstream of a firefly luciferase reporter (−1298 bp, −660 bp, −462 bp, −154 bp, and −72 bp) was utilized (Figure 1A). It was previously reported that the recruitment of IRF1 to a IFN-responsive-like element, which overlaps the NF-κB binding site at −615 bp, acts to partially repress MMP-9 expression in response to IFNγ due to IRF1’s competitive inhibition of NF-κB protein binding [13]. However, our results show that IFNβ mediated repression of MMP-9 (between 40% and 50% reduction in luciferase reporter activity, depending on size of MMP-9 promoter deletion) persists until the −154 bp deletion construct (Figure 1B), but is lost in the −72 bp construct. The poor induction of the −72 bp construct is not surprising as this construct does not retain the NF-κB, AP-1 and Sp1 binding sites in the promoter, which are known to be essential for PMA induction of the transcription. Of significance is the fact that the −461 bp and the −154 bp constructs retain strong repressive response to IFNβ (Figure 1B inset), thereby indicating that the loss of NF-κB site does not affect the ability of IFNβ to repress MMP-9 promoter. Based on these results, we concluded that the sequences present within the −154 bp construct are sufficient for mediating IFNβ repression on MMP-9 promoter. It should be noted that PMA induction is partially lost in the −461 bp and all other smaller constructs in the deletion series. This is expected, as PMA induction occurs due to synergistic activation through AP-1, NF-κB, and Sp1 transcription factors, and in the region between −661 and −461 bp of the MMP-9 promoter there exists an NF-κB, Sp1 and the distal AP-1 binding sites.

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