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Regulation of hedgehog signaling by Myc-interacting zinc finger protein 1, Miz1.

Lu J, Chen M, Ren XR, Wang J, Lyerly HK, Barak L, Chen W - PLoS ONE (2013)

Bottom Line: Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect.Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2.Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Duke University, Durham, North Carolina, USA.

ABSTRACT
Smoothened (Smo) mediated Hedgehog (Hh) signaling plays an essential role in regulating embryonic development and postnatal tissue homeostasis. Aberrant activation of the Hh pathway contributes to the formation and progression of various cancers. In vertebrates, however, key regulatory mechanisms responsible for transducing signals from Smo to the nucleus remain to be delineated. Here, we report the identification of Myc-interacting Zinc finger protein 1 (Miz1) as a Smo and Gli2 binding protein that positively regulates Hh signaling. Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect. Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2. Furthermore, Miz1 is localized to the nucleus upon Hh activation in a Smo-dependent manner, and loss of Miz1 prevents the nuclear translocation of Gli2. More importantly, silencing Miz1 expression inhibits cell proliferation in vitro and the growth of Hh-driven medulloblastoma tumors allografted in SCID mice. Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

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Miz1 is transported into nucleus upon Hh activation.(A) NIH 3T3 cells treated with DMSO, 0.25 μM SAG, or 0.25 μM SAG plus 1 μM GDC-0449 (SAG+GDC) were fractionated into cytosol (Cyto) and nuclear (Nu) fractions. Endogenous Miz1 was immunoprecipitated from each fraction first and analyzed using immunoblotting. The presence of Miz1 was detected using the Miz1 antibody. Lactate dehydrogenase (LDH) and Histone H2B (H2B) were used as protein markers of cytosol and nuclear fractions, respectively. β-actin was used as the loading control. (B) Graphic representation of data shown in panel (A). The amount of Miz1 detected in the nuclear fraction was normalized to that of cytosol, and this number (Nu/Cyto) for the DMSO treated group was set to 1. Data shown in the graph represent the mean ± SEM (n = 3; **: p<0.001, t test). (C) Smo is required for Miz1 translocation into the nucleus. Smo−/− MEF cells treated with DMSO or SAG were fractionated into cytosol (Cyto) and nuclear (Nu) fractions. Endogenous Miz1 was immunoprecipitated from each fraction first and analyzed using immunoblotting. Note that the amount of Miz1 in the cytosol or nucleus is not changed upon SAG treatment in Smo−/− MEF.
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pone-0063353-g004: Miz1 is transported into nucleus upon Hh activation.(A) NIH 3T3 cells treated with DMSO, 0.25 μM SAG, or 0.25 μM SAG plus 1 μM GDC-0449 (SAG+GDC) were fractionated into cytosol (Cyto) and nuclear (Nu) fractions. Endogenous Miz1 was immunoprecipitated from each fraction first and analyzed using immunoblotting. The presence of Miz1 was detected using the Miz1 antibody. Lactate dehydrogenase (LDH) and Histone H2B (H2B) were used as protein markers of cytosol and nuclear fractions, respectively. β-actin was used as the loading control. (B) Graphic representation of data shown in panel (A). The amount of Miz1 detected in the nuclear fraction was normalized to that of cytosol, and this number (Nu/Cyto) for the DMSO treated group was set to 1. Data shown in the graph represent the mean ± SEM (n = 3; **: p<0.001, t test). (C) Smo is required for Miz1 translocation into the nucleus. Smo−/− MEF cells treated with DMSO or SAG were fractionated into cytosol (Cyto) and nuclear (Nu) fractions. Endogenous Miz1 was immunoprecipitated from each fraction first and analyzed using immunoblotting. Note that the amount of Miz1 in the cytosol or nucleus is not changed upon SAG treatment in Smo−/− MEF.

Mentions: Miz1 does not possess a nuclear localization signal, but Miz1 can locate in both cytosol and nucleus [17]. To study the distribution of Miz1 upon Hh activation, NIH 3T3 cells were fractionated into cytoslic and nuclear components, and lactate dehydrogenase (LDH) and Histone 2B (H2B) were used as markers for cytosol and nucleus, respectively. Activation of Smo resulted in a 6-fold increase in the amount of Miz1 accumulated in the nuclear fraction in SAG treated cells, and this accumulation was greatly blocked by the Smo antagonist GDC-0449 (Figure 4A and 4B, SAG, 6.00±0.91 and SAG+GDC-0449, 1.62±0.13). The total Miz1 protein level was not changed by the treatment, indicating that the increase of Miz1 in the nuclear fraction is not due to an increase of Miz1 expression. In contrast, the amount of Miz1 in the nuclear fraction was not altered by the SAG treatment in Smo−/− MEF cells, suggesting that the nuclear translocation of Miz1 is a Smo activation-dependent process (Figure 4C).


Regulation of hedgehog signaling by Myc-interacting zinc finger protein 1, Miz1.

Lu J, Chen M, Ren XR, Wang J, Lyerly HK, Barak L, Chen W - PLoS ONE (2013)

Miz1 is transported into nucleus upon Hh activation.(A) NIH 3T3 cells treated with DMSO, 0.25 μM SAG, or 0.25 μM SAG plus 1 μM GDC-0449 (SAG+GDC) were fractionated into cytosol (Cyto) and nuclear (Nu) fractions. Endogenous Miz1 was immunoprecipitated from each fraction first and analyzed using immunoblotting. The presence of Miz1 was detected using the Miz1 antibody. Lactate dehydrogenase (LDH) and Histone H2B (H2B) were used as protein markers of cytosol and nuclear fractions, respectively. β-actin was used as the loading control. (B) Graphic representation of data shown in panel (A). The amount of Miz1 detected in the nuclear fraction was normalized to that of cytosol, and this number (Nu/Cyto) for the DMSO treated group was set to 1. Data shown in the graph represent the mean ± SEM (n = 3; **: p<0.001, t test). (C) Smo is required for Miz1 translocation into the nucleus. Smo−/− MEF cells treated with DMSO or SAG were fractionated into cytosol (Cyto) and nuclear (Nu) fractions. Endogenous Miz1 was immunoprecipitated from each fraction first and analyzed using immunoblotting. Note that the amount of Miz1 in the cytosol or nucleus is not changed upon SAG treatment in Smo−/− MEF.
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Related In: Results  -  Collection

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pone-0063353-g004: Miz1 is transported into nucleus upon Hh activation.(A) NIH 3T3 cells treated with DMSO, 0.25 μM SAG, or 0.25 μM SAG plus 1 μM GDC-0449 (SAG+GDC) were fractionated into cytosol (Cyto) and nuclear (Nu) fractions. Endogenous Miz1 was immunoprecipitated from each fraction first and analyzed using immunoblotting. The presence of Miz1 was detected using the Miz1 antibody. Lactate dehydrogenase (LDH) and Histone H2B (H2B) were used as protein markers of cytosol and nuclear fractions, respectively. β-actin was used as the loading control. (B) Graphic representation of data shown in panel (A). The amount of Miz1 detected in the nuclear fraction was normalized to that of cytosol, and this number (Nu/Cyto) for the DMSO treated group was set to 1. Data shown in the graph represent the mean ± SEM (n = 3; **: p<0.001, t test). (C) Smo is required for Miz1 translocation into the nucleus. Smo−/− MEF cells treated with DMSO or SAG were fractionated into cytosol (Cyto) and nuclear (Nu) fractions. Endogenous Miz1 was immunoprecipitated from each fraction first and analyzed using immunoblotting. Note that the amount of Miz1 in the cytosol or nucleus is not changed upon SAG treatment in Smo−/− MEF.
Mentions: Miz1 does not possess a nuclear localization signal, but Miz1 can locate in both cytosol and nucleus [17]. To study the distribution of Miz1 upon Hh activation, NIH 3T3 cells were fractionated into cytoslic and nuclear components, and lactate dehydrogenase (LDH) and Histone 2B (H2B) were used as markers for cytosol and nucleus, respectively. Activation of Smo resulted in a 6-fold increase in the amount of Miz1 accumulated in the nuclear fraction in SAG treated cells, and this accumulation was greatly blocked by the Smo antagonist GDC-0449 (Figure 4A and 4B, SAG, 6.00±0.91 and SAG+GDC-0449, 1.62±0.13). The total Miz1 protein level was not changed by the treatment, indicating that the increase of Miz1 in the nuclear fraction is not due to an increase of Miz1 expression. In contrast, the amount of Miz1 in the nuclear fraction was not altered by the SAG treatment in Smo−/− MEF cells, suggesting that the nuclear translocation of Miz1 is a Smo activation-dependent process (Figure 4C).

Bottom Line: Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect.Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2.Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Duke University, Durham, North Carolina, USA.

ABSTRACT
Smoothened (Smo) mediated Hedgehog (Hh) signaling plays an essential role in regulating embryonic development and postnatal tissue homeostasis. Aberrant activation of the Hh pathway contributes to the formation and progression of various cancers. In vertebrates, however, key regulatory mechanisms responsible for transducing signals from Smo to the nucleus remain to be delineated. Here, we report the identification of Myc-interacting Zinc finger protein 1 (Miz1) as a Smo and Gli2 binding protein that positively regulates Hh signaling. Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect. Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2. Furthermore, Miz1 is localized to the nucleus upon Hh activation in a Smo-dependent manner, and loss of Miz1 prevents the nuclear translocation of Gli2. More importantly, silencing Miz1 expression inhibits cell proliferation in vitro and the growth of Hh-driven medulloblastoma tumors allografted in SCID mice. Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

Show MeSH
Related in: MedlinePlus