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Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members.

Malik N, Vollmer S, Nanda SK, Lopez-Pelaez M, Prescott A, Gray N, Cohen P - Biochem. J. (2015)

Bottom Line: In the present study, we found that BI-2536 is likely to exert this effect by preventing the interaction of the transcription factors IRF3 (interferon-regulatory factor 3) and c-Jun with the IFNB promoter, but without affecting the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}-catalysed phosphorylation of IRF3 at Ser³⁹⁶, the dimerization and nuclear translocation of IRF3 or the phosphorylation of c-Jun and ATF2 (activating transcription factor 2).We found that BET inhibitors that do not inhibit PLKs phenocopied the effect of BI-2536 on IFNB gene transcription.We found that the BET family member BRD4 (bromodomain-containing protein 4) was associated with the IFNB promoter and that this interaction was enhanced by TLR3- or TLR4-ligation and prevented by BI-2536 and other BET inhibitors.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, Dow Street, University of Dundee, Dundee DD1 5EH, Scotland, U.K.

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BI-2536 and JQ1 do not impair the poly(I:C)- or LPS -stimulated activation of TBK1 or the phosphorylation and dimerization of IRF3(A and B) RAW cells were incubated for 1 h with the indicated concentrations of BI-2536, and then stimulated for 2 h without (−) or with (+) poly(I:C) (10 μg/ml) (A) or LPS (100 ng/ml) (B). Cell lysates (25 μg of protein) were subjected to SDS/PAGE, transferred on to PVDF membranes and immunoblotted with antibodies that recognize TBK1 phosphorylated at Ser172, IRF3 phosphorylated at Ser396 and GAPDH as loading control. (C and D) RAW cells were incubated for 1 h without (−) or with (+) 2.0 μM of the TBK1 inhibitor MRT67307, 1.0 μM BI-2536 or 1.0 μM JQ1, and then stimulated for 1 h with poly(I:C) (10 μg/ml) (C) or LPS (100 ng/ml) (D). Cell extracts were immunoblotted with the anti-IRF3 and anti-GAPDH antibodies used in (A) and (B). Similar results were obtained in two other independent experiments for (A)–(D). (E and F) RAW cells were incubated for 1 h without (−) or with (+) 2.0 μM MRT67307, 1.0 μM BI-2536 or 1.0 μM JQ1 and then stimulated with poly(I:C) (10 μg/ml) (E) or LPS (100 ng/ml) (F) for the times indicated. The cell lysates (10 μg of protein) were subjected to native PAGE to separate the monomeric and dimeric forms of IRF3, which were detected by immunoblotting with an antibody that recognizes all forms of IRF3.
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Figure 1: BI-2536 and JQ1 do not impair the poly(I:C)- or LPS -stimulated activation of TBK1 or the phosphorylation and dimerization of IRF3(A and B) RAW cells were incubated for 1 h with the indicated concentrations of BI-2536, and then stimulated for 2 h without (−) or with (+) poly(I:C) (10 μg/ml) (A) or LPS (100 ng/ml) (B). Cell lysates (25 μg of protein) were subjected to SDS/PAGE, transferred on to PVDF membranes and immunoblotted with antibodies that recognize TBK1 phosphorylated at Ser172, IRF3 phosphorylated at Ser396 and GAPDH as loading control. (C and D) RAW cells were incubated for 1 h without (−) or with (+) 2.0 μM of the TBK1 inhibitor MRT67307, 1.0 μM BI-2536 or 1.0 μM JQ1, and then stimulated for 1 h with poly(I:C) (10 μg/ml) (C) or LPS (100 ng/ml) (D). Cell extracts were immunoblotted with the anti-IRF3 and anti-GAPDH antibodies used in (A) and (B). Similar results were obtained in two other independent experiments for (A)–(D). (E and F) RAW cells were incubated for 1 h without (−) or with (+) 2.0 μM MRT67307, 1.0 μM BI-2536 or 1.0 μM JQ1 and then stimulated with poly(I:C) (10 μg/ml) (E) or LPS (100 ng/ml) (F) for the times indicated. The cell lysates (10 μg of protein) were subjected to native PAGE to separate the monomeric and dimeric forms of IRF3, which were detected by immunoblotting with an antibody that recognizes all forms of IRF3.

Mentions: We confirmed earlier observations that BI-2536 prevents the LPS-stimulated secretion of IFNβ when included in the cell culture medium at a concentration of 1.0 μM or higher (Supplementary Figure S1A). Consistent with these observations, the production of Ifnb mRNA induced by either poly(I:C) (Supplementary Figure S1B) or LPS (Supplementary Figure S1C) was also prevented by the inclusion of BI-2536 (1.0 μM). In contrast, BI-2536 did not suppress the poly(I:C)-stimulated (Figure 1A) or LPS-stimulated (Figure 1B) activation of TBK1, as judged by the phosphorylation of its activation loop at Ser172 [32], or the phosphorylation (Figures 1A–1D), dimerization (Figures 1E and 1F) and nuclear translocation (Supplementary Figures S1D and S1E) of IRF3. In contrast, MRT67307, a potent and relatively specific inhibitor of TBK1 [33], blocked IRF3 phosphorylation (Figures 1C and 1D) and dimerization (Figures 1E and 1F) as expected. Our results disagree with the previous study in which BI-2536 was reported to prevent the nuclear translocation of IRF3 [23]. The compound JQ1, an inhibitor of the BET family of bromodomain inhibitors, which was included in these experiments for reasons discussed below, phenocopied the effects of BI-2536 (Figure 1).


Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members.

Malik N, Vollmer S, Nanda SK, Lopez-Pelaez M, Prescott A, Gray N, Cohen P - Biochem. J. (2015)

BI-2536 and JQ1 do not impair the poly(I:C)- or LPS -stimulated activation of TBK1 or the phosphorylation and dimerization of IRF3(A and B) RAW cells were incubated for 1 h with the indicated concentrations of BI-2536, and then stimulated for 2 h without (−) or with (+) poly(I:C) (10 μg/ml) (A) or LPS (100 ng/ml) (B). Cell lysates (25 μg of protein) were subjected to SDS/PAGE, transferred on to PVDF membranes and immunoblotted with antibodies that recognize TBK1 phosphorylated at Ser172, IRF3 phosphorylated at Ser396 and GAPDH as loading control. (C and D) RAW cells were incubated for 1 h without (−) or with (+) 2.0 μM of the TBK1 inhibitor MRT67307, 1.0 μM BI-2536 or 1.0 μM JQ1, and then stimulated for 1 h with poly(I:C) (10 μg/ml) (C) or LPS (100 ng/ml) (D). Cell extracts were immunoblotted with the anti-IRF3 and anti-GAPDH antibodies used in (A) and (B). Similar results were obtained in two other independent experiments for (A)–(D). (E and F) RAW cells were incubated for 1 h without (−) or with (+) 2.0 μM MRT67307, 1.0 μM BI-2536 or 1.0 μM JQ1 and then stimulated with poly(I:C) (10 μg/ml) (E) or LPS (100 ng/ml) (F) for the times indicated. The cell lysates (10 μg of protein) were subjected to native PAGE to separate the monomeric and dimeric forms of IRF3, which were detected by immunoblotting with an antibody that recognizes all forms of IRF3.
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Figure 1: BI-2536 and JQ1 do not impair the poly(I:C)- or LPS -stimulated activation of TBK1 or the phosphorylation and dimerization of IRF3(A and B) RAW cells were incubated for 1 h with the indicated concentrations of BI-2536, and then stimulated for 2 h without (−) or with (+) poly(I:C) (10 μg/ml) (A) or LPS (100 ng/ml) (B). Cell lysates (25 μg of protein) were subjected to SDS/PAGE, transferred on to PVDF membranes and immunoblotted with antibodies that recognize TBK1 phosphorylated at Ser172, IRF3 phosphorylated at Ser396 and GAPDH as loading control. (C and D) RAW cells were incubated for 1 h without (−) or with (+) 2.0 μM of the TBK1 inhibitor MRT67307, 1.0 μM BI-2536 or 1.0 μM JQ1, and then stimulated for 1 h with poly(I:C) (10 μg/ml) (C) or LPS (100 ng/ml) (D). Cell extracts were immunoblotted with the anti-IRF3 and anti-GAPDH antibodies used in (A) and (B). Similar results were obtained in two other independent experiments for (A)–(D). (E and F) RAW cells were incubated for 1 h without (−) or with (+) 2.0 μM MRT67307, 1.0 μM BI-2536 or 1.0 μM JQ1 and then stimulated with poly(I:C) (10 μg/ml) (E) or LPS (100 ng/ml) (F) for the times indicated. The cell lysates (10 μg of protein) were subjected to native PAGE to separate the monomeric and dimeric forms of IRF3, which were detected by immunoblotting with an antibody that recognizes all forms of IRF3.
Mentions: We confirmed earlier observations that BI-2536 prevents the LPS-stimulated secretion of IFNβ when included in the cell culture medium at a concentration of 1.0 μM or higher (Supplementary Figure S1A). Consistent with these observations, the production of Ifnb mRNA induced by either poly(I:C) (Supplementary Figure S1B) or LPS (Supplementary Figure S1C) was also prevented by the inclusion of BI-2536 (1.0 μM). In contrast, BI-2536 did not suppress the poly(I:C)-stimulated (Figure 1A) or LPS-stimulated (Figure 1B) activation of TBK1, as judged by the phosphorylation of its activation loop at Ser172 [32], or the phosphorylation (Figures 1A–1D), dimerization (Figures 1E and 1F) and nuclear translocation (Supplementary Figures S1D and S1E) of IRF3. In contrast, MRT67307, a potent and relatively specific inhibitor of TBK1 [33], blocked IRF3 phosphorylation (Figures 1C and 1D) and dimerization (Figures 1E and 1F) as expected. Our results disagree with the previous study in which BI-2536 was reported to prevent the nuclear translocation of IRF3 [23]. The compound JQ1, an inhibitor of the BET family of bromodomain inhibitors, which was included in these experiments for reasons discussed below, phenocopied the effects of BI-2536 (Figure 1).

Bottom Line: In the present study, we found that BI-2536 is likely to exert this effect by preventing the interaction of the transcription factors IRF3 (interferon-regulatory factor 3) and c-Jun with the IFNB promoter, but without affecting the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}-catalysed phosphorylation of IRF3 at Ser³⁹⁶, the dimerization and nuclear translocation of IRF3 or the phosphorylation of c-Jun and ATF2 (activating transcription factor 2).We found that BET inhibitors that do not inhibit PLKs phenocopied the effect of BI-2536 on IFNB gene transcription.We found that the BET family member BRD4 (bromodomain-containing protein 4) was associated with the IFNB promoter and that this interaction was enhanced by TLR3- or TLR4-ligation and prevented by BI-2536 and other BET inhibitors.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, Dow Street, University of Dundee, Dundee DD1 5EH, Scotland, U.K.

Show MeSH
Related in: MedlinePlus