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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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Knockdown of endogenous Miz1 attenuates Hh signaling.(A) Knock-down of Miz1 diminishes SAG-dependent activation of Gli activity. Stable NIH 3T3 cells infected with either a scrambled shRNA (Scr) or Miz1 shRNA lentiviruses (#3 and #5) were transfected with the Hh-responsive reporter and treated with DMSO or SAG. The Gli reporter activity was determined using the ratio of firefly/Renilla luciferase light-units. The value in Scr control cells treated with DMSO was set to 1, and relative Gli activities for all other conditions was normalized accordingly. Data shown in the graph represent the mean ± SEM (n = 3, **: p<0.001, compared to the SAG-stimulated Scr sample, t test). The knockdown efficiency of Miz1 was verified and shown in upper panels. Specifically, endogenous Miz1 was immunoprecipitated from cell lysates and subsequently detected by immunoblotting using the Miz1 antibody. β-actin was used as a loading control. (B) and (C) Loss of Miz1 decreases the mRNA expression of Hh target genes. The scrambled control (Scr) and Miz1 knockdown (#3 and #5) cells were treated with DMSO or SAG for 24 hours. The mRNA levels of Gli1 (B) and Ptc1 (C) were analyzed using qRT-PCR. Data shown in both graphs represent the mean ± SEM (n = 3, **, p<0.001, compared to the Scr sample, t test).
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pone-0063353-g002: Knockdown of endogenous Miz1 attenuates Hh signaling.(A) Knock-down of Miz1 diminishes SAG-dependent activation of Gli activity. Stable NIH 3T3 cells infected with either a scrambled shRNA (Scr) or Miz1 shRNA lentiviruses (#3 and #5) were transfected with the Hh-responsive reporter and treated with DMSO or SAG. The Gli reporter activity was determined using the ratio of firefly/Renilla luciferase light-units. The value in Scr control cells treated with DMSO was set to 1, and relative Gli activities for all other conditions was normalized accordingly. Data shown in the graph represent the mean ± SEM (n = 3, **: p<0.001, compared to the SAG-stimulated Scr sample, t test). The knockdown efficiency of Miz1 was verified and shown in upper panels. Specifically, endogenous Miz1 was immunoprecipitated from cell lysates and subsequently detected by immunoblotting using the Miz1 antibody. β-actin was used as a loading control. (B) and (C) Loss of Miz1 decreases the mRNA expression of Hh target genes. The scrambled control (Scr) and Miz1 knockdown (#3 and #5) cells were treated with DMSO or SAG for 24 hours. The mRNA levels of Gli1 (B) and Ptc1 (C) were analyzed using qRT-PCR. Data shown in both graphs represent the mean ± SEM (n = 3, **, p<0.001, compared to the Scr sample, t test).

Mentions: To determine the role of endogenous Miz1 in regulating Hh signaling, we utilized lentivius-based shRNA to knockdown Miz1 levels and measured Gli luciferase reporter activity in the presence of a potent Smo agonist, SAG, in NIH 3T3 cells. We identified two shRNA targeting sequences (Miz1 shRNA #3 and shRNA #5, and indicated as #3 and #5, respectively) specific for knocking-down endogenous Miz1 (Figure 2A). In control cells exposed to scrambled shRNA, SAG activated the Gli reporter 16.2±1.6 fold. In each of the Miz1 knockdown cell lines, SAG-induced activation was substantially decreased (6.72±0.46 and 4.16±0.60 in #3 and #5 respectively, Figure 2A), indicating that Miz1 is necessary for the optimal activation of Hh signaling. Furthermore, since the activation of the Hh pathway leads to elevated Gli1 and Ptch1 mRNA expression, we assessed changes in mRNA level of these Hh target genes in response to Miz1 depletion. Control and Miz1 knockdown cells were treated with DMSO or SAG. In scrambled control cells, SAG treatment greatly increased Gli1 and Ptch1 mRNA levels by 1854.0±121.5 and 181.7±6.5 fold, respectively, whereas the level of Gli1 and Ptch1 mRNA induction was significantly inhibited in Miz1 knockdown cells (Figure 2B, Gli1 mRNA induction fold, #3, 386.3±1.7 and #5, 414.6±5.7, and Figure 2C, Ptch1 mRNA induction fold, #3, 36.7±15.0 and #5, 15.8±1.2). Collectively, these results confirmed that Miz1 plays a positive role in controlling Hh signaling.


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)

Knockdown of endogenous Miz1 attenuates Hh signaling.(A) Knock-down of Miz1 diminishes SAG-dependent activation of Gli activity. Stable NIH 3T3 cells infected with either a scrambled shRNA (Scr) or Miz1 shRNA lentiviruses (#3 and #5) were transfected with the Hh-responsive reporter and treated with DMSO or SAG. The Gli reporter activity was determined using the ratio of firefly/Renilla luciferase light-units. The value in Scr control cells treated with DMSO was set to 1, and relative Gli activities for all other conditions was normalized accordingly. Data shown in the graph represent the mean ± SEM (n = 3, **: p<0.001, compared to the SAG-stimulated Scr sample, t test). The knockdown efficiency of Miz1 was verified and shown in upper panels. Specifically, endogenous Miz1 was immunoprecipitated from cell lysates and subsequently detected by immunoblotting using the Miz1 antibody. β-actin was used as a loading control. (B) and (C) Loss of Miz1 decreases the mRNA expression of Hh target genes. The scrambled control (Scr) and Miz1 knockdown (#3 and #5) cells were treated with DMSO or SAG for 24 hours. The mRNA levels of Gli1 (B) and Ptc1 (C) were analyzed using qRT-PCR. Data shown in both graphs represent the mean ± SEM (n = 3, **, p<0.001, compared to the Scr sample, t test).
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pone-0063353-g002: Knockdown of endogenous Miz1 attenuates Hh signaling.(A) Knock-down of Miz1 diminishes SAG-dependent activation of Gli activity. Stable NIH 3T3 cells infected with either a scrambled shRNA (Scr) or Miz1 shRNA lentiviruses (#3 and #5) were transfected with the Hh-responsive reporter and treated with DMSO or SAG. The Gli reporter activity was determined using the ratio of firefly/Renilla luciferase light-units. The value in Scr control cells treated with DMSO was set to 1, and relative Gli activities for all other conditions was normalized accordingly. Data shown in the graph represent the mean ± SEM (n = 3, **: p<0.001, compared to the SAG-stimulated Scr sample, t test). The knockdown efficiency of Miz1 was verified and shown in upper panels. Specifically, endogenous Miz1 was immunoprecipitated from cell lysates and subsequently detected by immunoblotting using the Miz1 antibody. β-actin was used as a loading control. (B) and (C) Loss of Miz1 decreases the mRNA expression of Hh target genes. The scrambled control (Scr) and Miz1 knockdown (#3 and #5) cells were treated with DMSO or SAG for 24 hours. The mRNA levels of Gli1 (B) and Ptc1 (C) were analyzed using qRT-PCR. Data shown in both graphs represent the mean ± SEM (n = 3, **, p<0.001, compared to the Scr sample, t test).
Mentions: To determine the role of endogenous Miz1 in regulating Hh signaling, we utilized lentivius-based shRNA to knockdown Miz1 levels and measured Gli luciferase reporter activity in the presence of a potent Smo agonist, SAG, in NIH 3T3 cells. We identified two shRNA targeting sequences (Miz1 shRNA #3 and shRNA #5, and indicated as #3 and #5, respectively) specific for knocking-down endogenous Miz1 (Figure 2A). In control cells exposed to scrambled shRNA, SAG activated the Gli reporter 16.2±1.6 fold. In each of the Miz1 knockdown cell lines, SAG-induced activation was substantially decreased (6.72±0.46 and 4.16±0.60 in #3 and #5 respectively, Figure 2A), indicating that Miz1 is necessary for the optimal activation of Hh signaling. Furthermore, since the activation of the Hh pathway leads to elevated Gli1 and Ptch1 mRNA expression, we assessed changes in mRNA level of these Hh target genes in response to Miz1 depletion. Control and Miz1 knockdown cells were treated with DMSO or SAG. In scrambled control cells, SAG treatment greatly increased Gli1 and Ptch1 mRNA levels by 1854.0±121.5 and 181.7±6.5 fold, respectively, whereas the level of Gli1 and Ptch1 mRNA induction was significantly inhibited in Miz1 knockdown cells (Figure 2B, Gli1 mRNA induction fold, #3, 386.3±1.7 and #5, 414.6±5.7, and Figure 2C, Ptch1 mRNA induction fold, #3, 36.7±15.0 and #5, 15.8±1.2). Collectively, these results confirmed that Miz1 plays a positive role in controlling Hh signaling.

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