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Krüppel-like factor 4 interacts with p300 to activate mitofusin 2 gene expression induced by all-trans retinoic acid in VSMCs.

Zhang R, Han M, Zheng B, Li YJ, Shu YN, Wen JK - Acta Pharmacol. Sin. (2010)

Bottom Line: Adenoviral vector of KLF4-mediated overexpression and Western blot analysis were used to determine the effect of KLF4 on mfn-2 expression.KLF4 acetylation by p300 increased its activity to transactivate the mfn-2 promoter.ATRA induces KLF4 acetylation by p300 and increases the ability of KLF4 to transactivate the mfn-2 promoter in VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Hebei Medical University, Shijiazhuang, China.

ABSTRACT

Aim: To elucidate how krüppel-like factor 4 (KLF4) activates mitofusin 2 (mfn-2) expression in all-trans retinoic acid (ATRA)-induced vascular smooth muscle cell (VSMC) differentiation.

Methods: The mfn-2 promoter-reporter constructs and the KLF4 acetylation-deficient or phosphorylation-deficient mutants were constructed. Adenoviral vector of KLF4-mediated overexpression and Western blot analysis were used to determine the effect of KLF4 on mfn-2 expression. The luciferase assay and chromatin immunoprecipitation were used to detect the transactivation of KLF4 on mfn-2 gene expression. Co-immunoprecipitation and GST pull-down assays were used to determine the modification of KLF4 and interaction of KLF4 with p300 in VSMCs.

Results: KLF4 mediated ATRA-induced mfn-2 expression in VSMCs. KLF4 bound directly to the mfn-2 promoter and activated its transcription. ATRA increased the interaction of KLF4 with p300 by inducing KLF4 phosphorylation via activation of JNK and p38 MAPK signaling. KLF4 acetylation by p300 increased its activity to transactivate the mfn-2 promoter.

Conclusion: ATRA induces KLF4 acetylation by p300 and increases the ability of KLF4 to transactivate the mfn-2 promoter in VSMCs.

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Acetylation of KLF4 by p300 enhances its binding to the mfn-2 promoter. (A) Identification of anti-Ac-lys antibody. VSMCs were treated with 10 μmol/L ATRA for 2 h, cell lysates were immunoprecipitated using an irrelevant antibody (nonimmune IgG) or the antibodies against KLF4 and acetylated lysine, the precipitates were detected via Western blotting using anti-KLF4 or anti-acetylated lysine antibodies. (B) ATRA induced KLF4 acetylation. VSMCs were treated with 10 μmol/L ATRA for the indicated times. Cell lysates were immunoprecipitated with anti-KLF4 antibody, and acetylated KLF4 was detected via Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (C) ATRA increased the interaction of KLF4 with p300. VSMCs were treated with ATRA for 1 h. Cell lysates were immunoprecipitated with anti-KLF4 or anti-p300 antibodies as indicated (IP). The precipitates were analyzed by Western blotting (IB) with anti-p300 and anti-KLF4 antibodies, respectively. (D) KLF4 was acetylated by p300 in vitro. KLF4 or its acetylation-deficient mutants (300 ng) and p300 (100 ng) were incubated with acetyl-CoA for 30 min at 30 °C, and reaction products were separated by SDS-PAGE, and then acetylated KLF4 was determined by Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (E) The interaction of p300 with KLF4 and its acetylation-deficient mutants. The lysates of VSMCs treated with or without ATRA were incubated with GST, GST-KLF4, GST-KLF4 (K225R), GST-KLF4 (K229R), or GST-KLF4 (K225/229R). After pull down with GST beads, p300 was detected via Western blotting using anti-p300 antibody, and KLF4 was detected with anti-GST antibody. (F) Acetylation of KLF4 enhanced the mfn-2 promoter activity. Luciferase assay was performed in A293 cells transfected with the mfn-2 promoter-reporter plasmid (containing nucleotides −441 to +15 of the mfn-2 promoter), along with different combinations of expression plasmids for KLF4 or its acetylation-deficient mutant (K225/229R), p300, and deacetylase HDAC2. The bars represent the means±SE from three independent experiments. bP<0.05 vs the control.
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fig3: Acetylation of KLF4 by p300 enhances its binding to the mfn-2 promoter. (A) Identification of anti-Ac-lys antibody. VSMCs were treated with 10 μmol/L ATRA for 2 h, cell lysates were immunoprecipitated using an irrelevant antibody (nonimmune IgG) or the antibodies against KLF4 and acetylated lysine, the precipitates were detected via Western blotting using anti-KLF4 or anti-acetylated lysine antibodies. (B) ATRA induced KLF4 acetylation. VSMCs were treated with 10 μmol/L ATRA for the indicated times. Cell lysates were immunoprecipitated with anti-KLF4 antibody, and acetylated KLF4 was detected via Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (C) ATRA increased the interaction of KLF4 with p300. VSMCs were treated with ATRA for 1 h. Cell lysates were immunoprecipitated with anti-KLF4 or anti-p300 antibodies as indicated (IP). The precipitates were analyzed by Western blotting (IB) with anti-p300 and anti-KLF4 antibodies, respectively. (D) KLF4 was acetylated by p300 in vitro. KLF4 or its acetylation-deficient mutants (300 ng) and p300 (100 ng) were incubated with acetyl-CoA for 30 min at 30 °C, and reaction products were separated by SDS-PAGE, and then acetylated KLF4 was determined by Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (E) The interaction of p300 with KLF4 and its acetylation-deficient mutants. The lysates of VSMCs treated with or without ATRA were incubated with GST, GST-KLF4, GST-KLF4 (K225R), GST-KLF4 (K229R), or GST-KLF4 (K225/229R). After pull down with GST beads, p300 was detected via Western blotting using anti-p300 antibody, and KLF4 was detected with anti-GST antibody. (F) Acetylation of KLF4 enhanced the mfn-2 promoter activity. Luciferase assay was performed in A293 cells transfected with the mfn-2 promoter-reporter plasmid (containing nucleotides −441 to +15 of the mfn-2 promoter), along with different combinations of expression plasmids for KLF4 or its acetylation-deficient mutant (K225/229R), p300, and deacetylase HDAC2. The bars represent the means±SE from three independent experiments. bP<0.05 vs the control.

Mentions: To determine the specificity of anti-Ac-lys antibody, we used cross-CoIP to detect the the level of acetylated-KLF4 in ATRA-induced VSMCs. The immunoprecipitates pulled down with anti-KLF4 antibody were detected by anti-Ac-lys antibody and the immunoprecipitates pulled down with anti-Ac-lys antibody were detected by anti-KLF4 antibody. The results showed that the acetylated KLF4 was detected by both methods at the predicted molecular mass, which proved the specificity of anti-Ac-lys antibody (Figure 3A). Previous study has shown that KLF4 is acetylated by p300, which is associated with regulation of its transactivation function 26. We speculated that acetylation of KLF4 could also be important for its binding to the mfn-2 promoter. To test the hypothesis, we determined the levels of KLF4 acetylation in VSMCs treated with ATRA compared to treatment with ethanol (as a control). The result revealed that ATRA enhanced the levels of KLF4 acetylation within 24 h after ATRA treatment in a time-dependent manner, quantitative results showing a 2.2-fold increase at 1 h and a 5-fold increase at 24 h after ATRA treatment (Figure 3B), suggesting ATRA treatment induced KLF4 acetylation. We further sought to determine whether ATRA-induced KLF4 acetylation is caused by the interaction of KLF4 with p300. Co-IP assay showed that treatment with ATRA for 1 h enhanced p300 levels in the precipitates pulled down with anti-KLF4 antibody. Likewise, the level of KLF4 pulled down with anti-p300 antibody also markedly increased in cells treated with ATRA (Figure 3C), suggesting that ATRA treatment promotes the interaction of KLF4 with p300. To determine whether the increase in KLF4 acetylation induced by ATRA results from its interaction with p300, we performed an in vitro acetylation assay. Recombinant p300-HAT and GST-KLF4 or its acetylation-deficient mutants were incubated with cold acetyl-CoA, and KLF4 acetylation was measured by Western blotting with an anti-Ac-lys antibody. As shown in Figure 3D, the acetylation of KLF4 was strongly increased in the presence of p300, but acetylation-deficient mutants KLF4-K225R, KLF4-K229R, and KLF4-K225/229R could not be acetylated by p300. To identify whether these acetylation site mutations affect the ability of KLF4 to interact with p300, we incubated the lysates from VSMCs treated with and without ATRA with GST-KLF4 or its acetylation-deficient mutants, and found that both single point mutants K225R, K229R and double point mutant K225/229R could still interact with p300 in ATRA-treated cells (Figure 3E), consistent with previous findings26. To further determine the effect of KLF4 acetylation on the mfn-2 promoter activation, A293 cells were co-transfected with the mfn-2 promoter reporter plasmids, along with different combinations of expression plasmids for KLF4, KLF4 mutant, p300, or deacetylase HDAC2, and luciferase activity was detected. As expected, the strongest activation of the mfn-2 promoter was observed when KLF4 expression plasmids and p300 were co-transfected into the cells, with an approximately 10-fold luciferase activity relative to baseline. When deacetylase HDAC2, which makes KLF4 deacetylated, was co-expressed with KLF4, the mfn-2 promoter activity was reduced to 10% of that transfected with KLF4 alone. Meanwhile, acetylation-deficient mutant of KLF4 (K225/229R) could not enhance the promoter activity, indicating that KLF4 acetylation by p300 is required for the activation of the mfn-2 promoter (Figure 3F).


Krüppel-like factor 4 interacts with p300 to activate mitofusin 2 gene expression induced by all-trans retinoic acid in VSMCs.

Zhang R, Han M, Zheng B, Li YJ, Shu YN, Wen JK - Acta Pharmacol. Sin. (2010)

Acetylation of KLF4 by p300 enhances its binding to the mfn-2 promoter. (A) Identification of anti-Ac-lys antibody. VSMCs were treated with 10 μmol/L ATRA for 2 h, cell lysates were immunoprecipitated using an irrelevant antibody (nonimmune IgG) or the antibodies against KLF4 and acetylated lysine, the precipitates were detected via Western blotting using anti-KLF4 or anti-acetylated lysine antibodies. (B) ATRA induced KLF4 acetylation. VSMCs were treated with 10 μmol/L ATRA for the indicated times. Cell lysates were immunoprecipitated with anti-KLF4 antibody, and acetylated KLF4 was detected via Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (C) ATRA increased the interaction of KLF4 with p300. VSMCs were treated with ATRA for 1 h. Cell lysates were immunoprecipitated with anti-KLF4 or anti-p300 antibodies as indicated (IP). The precipitates were analyzed by Western blotting (IB) with anti-p300 and anti-KLF4 antibodies, respectively. (D) KLF4 was acetylated by p300 in vitro. KLF4 or its acetylation-deficient mutants (300 ng) and p300 (100 ng) were incubated with acetyl-CoA for 30 min at 30 °C, and reaction products were separated by SDS-PAGE, and then acetylated KLF4 was determined by Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (E) The interaction of p300 with KLF4 and its acetylation-deficient mutants. The lysates of VSMCs treated with or without ATRA were incubated with GST, GST-KLF4, GST-KLF4 (K225R), GST-KLF4 (K229R), or GST-KLF4 (K225/229R). After pull down with GST beads, p300 was detected via Western blotting using anti-p300 antibody, and KLF4 was detected with anti-GST antibody. (F) Acetylation of KLF4 enhanced the mfn-2 promoter activity. Luciferase assay was performed in A293 cells transfected with the mfn-2 promoter-reporter plasmid (containing nucleotides −441 to +15 of the mfn-2 promoter), along with different combinations of expression plasmids for KLF4 or its acetylation-deficient mutant (K225/229R), p300, and deacetylase HDAC2. The bars represent the means±SE from three independent experiments. bP<0.05 vs the control.
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fig3: Acetylation of KLF4 by p300 enhances its binding to the mfn-2 promoter. (A) Identification of anti-Ac-lys antibody. VSMCs were treated with 10 μmol/L ATRA for 2 h, cell lysates were immunoprecipitated using an irrelevant antibody (nonimmune IgG) or the antibodies against KLF4 and acetylated lysine, the precipitates were detected via Western blotting using anti-KLF4 or anti-acetylated lysine antibodies. (B) ATRA induced KLF4 acetylation. VSMCs were treated with 10 μmol/L ATRA for the indicated times. Cell lysates were immunoprecipitated with anti-KLF4 antibody, and acetylated KLF4 was detected via Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (C) ATRA increased the interaction of KLF4 with p300. VSMCs were treated with ATRA for 1 h. Cell lysates were immunoprecipitated with anti-KLF4 or anti-p300 antibodies as indicated (IP). The precipitates were analyzed by Western blotting (IB) with anti-p300 and anti-KLF4 antibodies, respectively. (D) KLF4 was acetylated by p300 in vitro. KLF4 or its acetylation-deficient mutants (300 ng) and p300 (100 ng) were incubated with acetyl-CoA for 30 min at 30 °C, and reaction products were separated by SDS-PAGE, and then acetylated KLF4 was determined by Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (E) The interaction of p300 with KLF4 and its acetylation-deficient mutants. The lysates of VSMCs treated with or without ATRA were incubated with GST, GST-KLF4, GST-KLF4 (K225R), GST-KLF4 (K229R), or GST-KLF4 (K225/229R). After pull down with GST beads, p300 was detected via Western blotting using anti-p300 antibody, and KLF4 was detected with anti-GST antibody. (F) Acetylation of KLF4 enhanced the mfn-2 promoter activity. Luciferase assay was performed in A293 cells transfected with the mfn-2 promoter-reporter plasmid (containing nucleotides −441 to +15 of the mfn-2 promoter), along with different combinations of expression plasmids for KLF4 or its acetylation-deficient mutant (K225/229R), p300, and deacetylase HDAC2. The bars represent the means±SE from three independent experiments. bP<0.05 vs the control.
Mentions: To determine the specificity of anti-Ac-lys antibody, we used cross-CoIP to detect the the level of acetylated-KLF4 in ATRA-induced VSMCs. The immunoprecipitates pulled down with anti-KLF4 antibody were detected by anti-Ac-lys antibody and the immunoprecipitates pulled down with anti-Ac-lys antibody were detected by anti-KLF4 antibody. The results showed that the acetylated KLF4 was detected by both methods at the predicted molecular mass, which proved the specificity of anti-Ac-lys antibody (Figure 3A). Previous study has shown that KLF4 is acetylated by p300, which is associated with regulation of its transactivation function 26. We speculated that acetylation of KLF4 could also be important for its binding to the mfn-2 promoter. To test the hypothesis, we determined the levels of KLF4 acetylation in VSMCs treated with ATRA compared to treatment with ethanol (as a control). The result revealed that ATRA enhanced the levels of KLF4 acetylation within 24 h after ATRA treatment in a time-dependent manner, quantitative results showing a 2.2-fold increase at 1 h and a 5-fold increase at 24 h after ATRA treatment (Figure 3B), suggesting ATRA treatment induced KLF4 acetylation. We further sought to determine whether ATRA-induced KLF4 acetylation is caused by the interaction of KLF4 with p300. Co-IP assay showed that treatment with ATRA for 1 h enhanced p300 levels in the precipitates pulled down with anti-KLF4 antibody. Likewise, the level of KLF4 pulled down with anti-p300 antibody also markedly increased in cells treated with ATRA (Figure 3C), suggesting that ATRA treatment promotes the interaction of KLF4 with p300. To determine whether the increase in KLF4 acetylation induced by ATRA results from its interaction with p300, we performed an in vitro acetylation assay. Recombinant p300-HAT and GST-KLF4 or its acetylation-deficient mutants were incubated with cold acetyl-CoA, and KLF4 acetylation was measured by Western blotting with an anti-Ac-lys antibody. As shown in Figure 3D, the acetylation of KLF4 was strongly increased in the presence of p300, but acetylation-deficient mutants KLF4-K225R, KLF4-K229R, and KLF4-K225/229R could not be acetylated by p300. To identify whether these acetylation site mutations affect the ability of KLF4 to interact with p300, we incubated the lysates from VSMCs treated with and without ATRA with GST-KLF4 or its acetylation-deficient mutants, and found that both single point mutants K225R, K229R and double point mutant K225/229R could still interact with p300 in ATRA-treated cells (Figure 3E), consistent with previous findings26. To further determine the effect of KLF4 acetylation on the mfn-2 promoter activation, A293 cells were co-transfected with the mfn-2 promoter reporter plasmids, along with different combinations of expression plasmids for KLF4, KLF4 mutant, p300, or deacetylase HDAC2, and luciferase activity was detected. As expected, the strongest activation of the mfn-2 promoter was observed when KLF4 expression plasmids and p300 were co-transfected into the cells, with an approximately 10-fold luciferase activity relative to baseline. When deacetylase HDAC2, which makes KLF4 deacetylated, was co-expressed with KLF4, the mfn-2 promoter activity was reduced to 10% of that transfected with KLF4 alone. Meanwhile, acetylation-deficient mutant of KLF4 (K225/229R) could not enhance the promoter activity, indicating that KLF4 acetylation by p300 is required for the activation of the mfn-2 promoter (Figure 3F).

Bottom Line: Adenoviral vector of KLF4-mediated overexpression and Western blot analysis were used to determine the effect of KLF4 on mfn-2 expression.KLF4 acetylation by p300 increased its activity to transactivate the mfn-2 promoter.ATRA induces KLF4 acetylation by p300 and increases the ability of KLF4 to transactivate the mfn-2 promoter in VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Hebei Medical University, Shijiazhuang, China.

ABSTRACT

Aim: To elucidate how krüppel-like factor 4 (KLF4) activates mitofusin 2 (mfn-2) expression in all-trans retinoic acid (ATRA)-induced vascular smooth muscle cell (VSMC) differentiation.

Methods: The mfn-2 promoter-reporter constructs and the KLF4 acetylation-deficient or phosphorylation-deficient mutants were constructed. Adenoviral vector of KLF4-mediated overexpression and Western blot analysis were used to determine the effect of KLF4 on mfn-2 expression. The luciferase assay and chromatin immunoprecipitation were used to detect the transactivation of KLF4 on mfn-2 gene expression. Co-immunoprecipitation and GST pull-down assays were used to determine the modification of KLF4 and interaction of KLF4 with p300 in VSMCs.

Results: KLF4 mediated ATRA-induced mfn-2 expression in VSMCs. KLF4 bound directly to the mfn-2 promoter and activated its transcription. ATRA increased the interaction of KLF4 with p300 by inducing KLF4 phosphorylation via activation of JNK and p38 MAPK signaling. KLF4 acetylation by p300 increased its activity to transactivate the mfn-2 promoter.

Conclusion: ATRA induces KLF4 acetylation by p300 and increases the ability of KLF4 to transactivate the mfn-2 promoter in VSMCs.

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