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ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.

Yoshikawa T, Wu J, Otsuka M, Kishikawa T, Ohno M, Shibata C, Takata A, Han F, Kang YJ, Chen CY, Shyu AB, Han J, Koike K - Nucleic Acids Res. (2015)

Bottom Line: Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors.Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription.ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

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ROCK inhibition enhances the interaction between HNF4A and ROCK1. (A) ROCK1 binds HNF4A. HCT116 cells were transfected with flag-tagged HNF4A expressing plasmids. Cells were treated or non-treated with Y27632 for 6 h followed by immunoprecipitation using mouse anti-flag antibody. Mouse control IgG was used as a negative control. Precipitated lysates were subjected to western blotting. Five percent of the total cell lysate before immunoprecipitation was used as an internal control (‘input’). The results are representative of four independent experiments. (B) ROCK inhibition enhances the interaction between ROCK1 and HNF4A. HCT116 cells were transfected with flag-tagged HNF4A and Halo-tagged ROCK1-expressing plasmids as indicated. Cells were treated with Y27632 for 6 h before immunoprecipitation. Cell lysates were immunoprecipitated with an anti-flag antibody. Precipitated lysates were subjected to western blotting with anti-ROCK1 antibodies and anti-flag antibodies. Five percent of total cell lysate was used as ‘input.’ The results shown are representative of four independent experiments. (C) Deletion constructs of HNF4A. Plasmids expressing C terminal domain-deleted and flag-tagged HNF4A were constructed and named as indicated. (D) HCT116 cells were transfected with flag-tagged HNF4A expressing plasmids as indicated and subjected to immunoprecipitation using anti-flag antibodies. Y27632 was used for 6 h before immunoprecipitation in indicated cases. Endogenous ROCK1 was detected in the precipitated lysates. The results are representative of four independent experiments. (E) Cytoplasmic and nuclear fractions of Huh7 cells treated or non-treated with Y27632 for 6 h were blotted with anti-ROCK1 and anti-HNF4A antibodies. GAPDH (cytoplasmic marker) and Lamin B (nucleus marker) were blotted to confirm appropriate fractionation. The results are representative of four independent experiments. (F) Flag-tagged HNF4A and ROCK1 proteins were purified using 293T cell lysates transfected with flag-tagged HNF4A and Halo-tagged ROCK1 expressing plasmids by immunoprecipitation with anti-flag antibody and halo-tagged beads, followed by the elution using flag peptides and endoprotease, respectively. Equal amounts of protein were mixed in vitro and immunoprecipitated (IP) using anti-flag antibodies. Y27632 was added in the mixture for 6 h before the precipitation. Precipitated proteins, with 5% of purified proteins as an expression check, were subjected to western blotting using anti-ROCK1 and anti-flag antibodies. The results shown are representative of four independent experiments. (G) Y27632 increased the binding of HNF4A protein to the PAIP2 promoter. A gel-shift assay was performed by mixing DNA probes with purified HNF4A and ROCK1 proteins. 1, no protein; 2, HNF4A protein only; 3, HNF4A protein only with Y27632 treatment for 30 min; 4, HNF4A with ROCK1 protein; 5, as in (4), plus Y27632 treatment for 30 min; 6, as in (5), plus anti-ROCK1 antibody; 7, as in (4), with an excess amount of cold probes. * indicates the position of protein-bound DNA probe. n.s.; non-specific bands. DNA probes derived from –992 to –965 of the PAIP2 promoter were used as described in Figure 4. The results shown are representative of four independent experiments.
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Figure 5: ROCK inhibition enhances the interaction between HNF4A and ROCK1. (A) ROCK1 binds HNF4A. HCT116 cells were transfected with flag-tagged HNF4A expressing plasmids. Cells were treated or non-treated with Y27632 for 6 h followed by immunoprecipitation using mouse anti-flag antibody. Mouse control IgG was used as a negative control. Precipitated lysates were subjected to western blotting. Five percent of the total cell lysate before immunoprecipitation was used as an internal control (‘input’). The results are representative of four independent experiments. (B) ROCK inhibition enhances the interaction between ROCK1 and HNF4A. HCT116 cells were transfected with flag-tagged HNF4A and Halo-tagged ROCK1-expressing plasmids as indicated. Cells were treated with Y27632 for 6 h before immunoprecipitation. Cell lysates were immunoprecipitated with an anti-flag antibody. Precipitated lysates were subjected to western blotting with anti-ROCK1 antibodies and anti-flag antibodies. Five percent of total cell lysate was used as ‘input.’ The results shown are representative of four independent experiments. (C) Deletion constructs of HNF4A. Plasmids expressing C terminal domain-deleted and flag-tagged HNF4A were constructed and named as indicated. (D) HCT116 cells were transfected with flag-tagged HNF4A expressing plasmids as indicated and subjected to immunoprecipitation using anti-flag antibodies. Y27632 was used for 6 h before immunoprecipitation in indicated cases. Endogenous ROCK1 was detected in the precipitated lysates. The results are representative of four independent experiments. (E) Cytoplasmic and nuclear fractions of Huh7 cells treated or non-treated with Y27632 for 6 h were blotted with anti-ROCK1 and anti-HNF4A antibodies. GAPDH (cytoplasmic marker) and Lamin B (nucleus marker) were blotted to confirm appropriate fractionation. The results are representative of four independent experiments. (F) Flag-tagged HNF4A and ROCK1 proteins were purified using 293T cell lysates transfected with flag-tagged HNF4A and Halo-tagged ROCK1 expressing plasmids by immunoprecipitation with anti-flag antibody and halo-tagged beads, followed by the elution using flag peptides and endoprotease, respectively. Equal amounts of protein were mixed in vitro and immunoprecipitated (IP) using anti-flag antibodies. Y27632 was added in the mixture for 6 h before the precipitation. Precipitated proteins, with 5% of purified proteins as an expression check, were subjected to western blotting using anti-ROCK1 and anti-flag antibodies. The results shown are representative of four independent experiments. (G) Y27632 increased the binding of HNF4A protein to the PAIP2 promoter. A gel-shift assay was performed by mixing DNA probes with purified HNF4A and ROCK1 proteins. 1, no protein; 2, HNF4A protein only; 3, HNF4A protein only with Y27632 treatment for 30 min; 4, HNF4A with ROCK1 protein; 5, as in (4), plus Y27632 treatment for 30 min; 6, as in (5), plus anti-ROCK1 antibody; 7, as in (4), with an excess amount of cold probes. * indicates the position of protein-bound DNA probe. n.s.; non-specific bands. DNA probes derived from –992 to –965 of the PAIP2 promoter were used as described in Figure 4. The results shown are representative of four independent experiments.

Mentions: Both phosphorylation and lysine acetylation of HNF4A affect its transcriptional activity (31–33). Therefore, we first examined changes in post-translational modifications of HNF4A after ROCK inhibition, but detected no changes in such modifications under our experimental conditions (Supplementary Figure S6A). Next, to determine the mechanism by which the transcriptional ability of HNF4A is enhanced by ROCK inhibition, we investigated the interaction between HNF4A and two ROCK paralogs by immunoprecipitation of overexpressing flag-tagged HNF4A. There was a clear interaction between HNF4A and cellular endogenous ROCK1 and not ROCK2 (Figure 5A). Moreover, the interaction between HNF4A and ROCK1 was significantly enhanced upon treatment with the ROCK inhibitor (Figure 5A). To confirm the binding between HNF4A and ROCK1, we used HCT116 cells transfected with a halo-tagged ROCK1-expressing plasmid and flag-tagged HNF4A-expressing plasmid. Endogenous ROCK1 and halo-tagged ROCK1 proteins were co-precipitated by immunoprecipitation of the flag-tagged HNF4A protein. Consistent with the data shown in Figure 5A, the interaction between HNF4A and ROCK1 was enhanced upon treatment with the ROCK inhibitor (Figure 5B).


ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.

Yoshikawa T, Wu J, Otsuka M, Kishikawa T, Ohno M, Shibata C, Takata A, Han F, Kang YJ, Chen CY, Shyu AB, Han J, Koike K - Nucleic Acids Res. (2015)

ROCK inhibition enhances the interaction between HNF4A and ROCK1. (A) ROCK1 binds HNF4A. HCT116 cells were transfected with flag-tagged HNF4A expressing plasmids. Cells were treated or non-treated with Y27632 for 6 h followed by immunoprecipitation using mouse anti-flag antibody. Mouse control IgG was used as a negative control. Precipitated lysates were subjected to western blotting. Five percent of the total cell lysate before immunoprecipitation was used as an internal control (‘input’). The results are representative of four independent experiments. (B) ROCK inhibition enhances the interaction between ROCK1 and HNF4A. HCT116 cells were transfected with flag-tagged HNF4A and Halo-tagged ROCK1-expressing plasmids as indicated. Cells were treated with Y27632 for 6 h before immunoprecipitation. Cell lysates were immunoprecipitated with an anti-flag antibody. Precipitated lysates were subjected to western blotting with anti-ROCK1 antibodies and anti-flag antibodies. Five percent of total cell lysate was used as ‘input.’ The results shown are representative of four independent experiments. (C) Deletion constructs of HNF4A. Plasmids expressing C terminal domain-deleted and flag-tagged HNF4A were constructed and named as indicated. (D) HCT116 cells were transfected with flag-tagged HNF4A expressing plasmids as indicated and subjected to immunoprecipitation using anti-flag antibodies. Y27632 was used for 6 h before immunoprecipitation in indicated cases. Endogenous ROCK1 was detected in the precipitated lysates. The results are representative of four independent experiments. (E) Cytoplasmic and nuclear fractions of Huh7 cells treated or non-treated with Y27632 for 6 h were blotted with anti-ROCK1 and anti-HNF4A antibodies. GAPDH (cytoplasmic marker) and Lamin B (nucleus marker) were blotted to confirm appropriate fractionation. The results are representative of four independent experiments. (F) Flag-tagged HNF4A and ROCK1 proteins were purified using 293T cell lysates transfected with flag-tagged HNF4A and Halo-tagged ROCK1 expressing plasmids by immunoprecipitation with anti-flag antibody and halo-tagged beads, followed by the elution using flag peptides and endoprotease, respectively. Equal amounts of protein were mixed in vitro and immunoprecipitated (IP) using anti-flag antibodies. Y27632 was added in the mixture for 6 h before the precipitation. Precipitated proteins, with 5% of purified proteins as an expression check, were subjected to western blotting using anti-ROCK1 and anti-flag antibodies. The results shown are representative of four independent experiments. (G) Y27632 increased the binding of HNF4A protein to the PAIP2 promoter. A gel-shift assay was performed by mixing DNA probes with purified HNF4A and ROCK1 proteins. 1, no protein; 2, HNF4A protein only; 3, HNF4A protein only with Y27632 treatment for 30 min; 4, HNF4A with ROCK1 protein; 5, as in (4), plus Y27632 treatment for 30 min; 6, as in (5), plus anti-ROCK1 antibody; 7, as in (4), with an excess amount of cold probes. * indicates the position of protein-bound DNA probe. n.s.; non-specific bands. DNA probes derived from –992 to –965 of the PAIP2 promoter were used as described in Figure 4. The results shown are representative of four independent experiments.
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Figure 5: ROCK inhibition enhances the interaction between HNF4A and ROCK1. (A) ROCK1 binds HNF4A. HCT116 cells were transfected with flag-tagged HNF4A expressing plasmids. Cells were treated or non-treated with Y27632 for 6 h followed by immunoprecipitation using mouse anti-flag antibody. Mouse control IgG was used as a negative control. Precipitated lysates were subjected to western blotting. Five percent of the total cell lysate before immunoprecipitation was used as an internal control (‘input’). The results are representative of four independent experiments. (B) ROCK inhibition enhances the interaction between ROCK1 and HNF4A. HCT116 cells were transfected with flag-tagged HNF4A and Halo-tagged ROCK1-expressing plasmids as indicated. Cells were treated with Y27632 for 6 h before immunoprecipitation. Cell lysates were immunoprecipitated with an anti-flag antibody. Precipitated lysates were subjected to western blotting with anti-ROCK1 antibodies and anti-flag antibodies. Five percent of total cell lysate was used as ‘input.’ The results shown are representative of four independent experiments. (C) Deletion constructs of HNF4A. Plasmids expressing C terminal domain-deleted and flag-tagged HNF4A were constructed and named as indicated. (D) HCT116 cells were transfected with flag-tagged HNF4A expressing plasmids as indicated and subjected to immunoprecipitation using anti-flag antibodies. Y27632 was used for 6 h before immunoprecipitation in indicated cases. Endogenous ROCK1 was detected in the precipitated lysates. The results are representative of four independent experiments. (E) Cytoplasmic and nuclear fractions of Huh7 cells treated or non-treated with Y27632 for 6 h were blotted with anti-ROCK1 and anti-HNF4A antibodies. GAPDH (cytoplasmic marker) and Lamin B (nucleus marker) were blotted to confirm appropriate fractionation. The results are representative of four independent experiments. (F) Flag-tagged HNF4A and ROCK1 proteins were purified using 293T cell lysates transfected with flag-tagged HNF4A and Halo-tagged ROCK1 expressing plasmids by immunoprecipitation with anti-flag antibody and halo-tagged beads, followed by the elution using flag peptides and endoprotease, respectively. Equal amounts of protein were mixed in vitro and immunoprecipitated (IP) using anti-flag antibodies. Y27632 was added in the mixture for 6 h before the precipitation. Precipitated proteins, with 5% of purified proteins as an expression check, were subjected to western blotting using anti-ROCK1 and anti-flag antibodies. The results shown are representative of four independent experiments. (G) Y27632 increased the binding of HNF4A protein to the PAIP2 promoter. A gel-shift assay was performed by mixing DNA probes with purified HNF4A and ROCK1 proteins. 1, no protein; 2, HNF4A protein only; 3, HNF4A protein only with Y27632 treatment for 30 min; 4, HNF4A with ROCK1 protein; 5, as in (4), plus Y27632 treatment for 30 min; 6, as in (5), plus anti-ROCK1 antibody; 7, as in (4), with an excess amount of cold probes. * indicates the position of protein-bound DNA probe. n.s.; non-specific bands. DNA probes derived from –992 to –965 of the PAIP2 promoter were used as described in Figure 4. The results shown are representative of four independent experiments.
Mentions: Both phosphorylation and lysine acetylation of HNF4A affect its transcriptional activity (31–33). Therefore, we first examined changes in post-translational modifications of HNF4A after ROCK inhibition, but detected no changes in such modifications under our experimental conditions (Supplementary Figure S6A). Next, to determine the mechanism by which the transcriptional ability of HNF4A is enhanced by ROCK inhibition, we investigated the interaction between HNF4A and two ROCK paralogs by immunoprecipitation of overexpressing flag-tagged HNF4A. There was a clear interaction between HNF4A and cellular endogenous ROCK1 and not ROCK2 (Figure 5A). Moreover, the interaction between HNF4A and ROCK1 was significantly enhanced upon treatment with the ROCK inhibitor (Figure 5A). To confirm the binding between HNF4A and ROCK1, we used HCT116 cells transfected with a halo-tagged ROCK1-expressing plasmid and flag-tagged HNF4A-expressing plasmid. Endogenous ROCK1 and halo-tagged ROCK1 proteins were co-precipitated by immunoprecipitation of the flag-tagged HNF4A protein. Consistent with the data shown in Figure 5A, the interaction between HNF4A and ROCK1 was enhanced upon treatment with the ROCK inhibitor (Figure 5B).

Bottom Line: Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors.Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription.ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Show MeSH
Related in: MedlinePlus