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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 Miz1 inhibits cell proliferation and tumorigenesis in PZp53MED1 medulloblastoma cells.(A) and (B) The rate of cell proliferation was measured in stable scrambled control (Scr) and Miz1 knockdown (#3 and #5) PZp53MED1 cells using WST-1 (A) and 3H-thymidine incorporation assays (B). Data shown in the graphs represent the mean ± SEM (n = 3; **: p<0.001, compared to the Scr sample, t test). (C) and (D) The levels of mRNA expression for Gli1 (C) and Ptch1 (D) were measured using qRT-PCR in control and Miz1 knockdown PZp53MED1 cells. Data shown in the graphs represent the mean ± SEM (n = 4 for Gli1 and n = 3 for Ptch1; *: p<0.05; and **: p<0.001, compared to the Scr sample, t test). (E) SCID mice were inoculated subcutaneously with stable control and Miz1 knockdown PZp53MED1 cells. Image shows the tumors dissected from mice at the end of the experiment. ND represents non-detectable tumor growth around the injection sites. (F) Quantitative representation of tumor weights measured. Data in the graph represent the mean ± SEM (n = 5 for each group; *: p<0.05, compared to the Scr sample, t test).
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pone-0063353-g006: Knockdown of Miz1 inhibits cell proliferation and tumorigenesis in PZp53MED1 medulloblastoma cells.(A) and (B) The rate of cell proliferation was measured in stable scrambled control (Scr) and Miz1 knockdown (#3 and #5) PZp53MED1 cells using WST-1 (A) and 3H-thymidine incorporation assays (B). Data shown in the graphs represent the mean ± SEM (n = 3; **: p<0.001, compared to the Scr sample, t test). (C) and (D) The levels of mRNA expression for Gli1 (C) and Ptch1 (D) were measured using qRT-PCR in control and Miz1 knockdown PZp53MED1 cells. Data shown in the graphs represent the mean ± SEM (n = 4 for Gli1 and n = 3 for Ptch1; *: p<0.05; and **: p<0.001, compared to the Scr sample, t test). (E) SCID mice were inoculated subcutaneously with stable control and Miz1 knockdown PZp53MED1 cells. Image shows the tumors dissected from mice at the end of the experiment. ND represents non-detectable tumor growth around the injection sites. (F) Quantitative representation of tumor weights measured. Data in the graph represent the mean ± SEM (n = 5 for each group; *: p<0.05, compared to the Scr sample, t test).

Mentions: Aberrant activation of Hh signaling has been implicated in many types of cancer [3], [4], [5], [6]. Mutations in Ptch1 and Smo occur in patients with medulloblastoma (MB), an aggressive childhood cancer arising from cerebellar GNPs [27]. To test the function of Miz1, we stably knocked down the endogenous expression of Miz1 in PZp53MED1 cells generated from a murine Hh-driven medulloblastoma allograft model [24]. Loss of Miz1 significantly reduced cell proliferation as assessed using WST-1 assays (Figure 6A, Scr, 0.35±0.03; versus #3, 0.13±0.01 and #5, 0.14±0.01) and 3H-thymidine incorporation assays (Figure 6B, Scr, 6326±457; versus #3, 824±40 and #5, 1193±64). The suppression of cell growth coincides with a reduction in the mRNA levels of Hh target genes Gli1 and Ptch1 (Figure 6C and 6D, for Gli1 mRNA, Scr, 4.09±0.32, versus #3, 0.57±0.15 and #5, 0.61±0.09; and for Ptch1 mRNA, Scr, 7.97±2.31, versus #3, 2.82±0.86 and #5, 2.85±1.49), suggesting decreased Hh signaling is responsible for the growth inhibition observed in Miz1 knockdown cells.


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 Miz1 inhibits cell proliferation and tumorigenesis in PZp53MED1 medulloblastoma cells.(A) and (B) The rate of cell proliferation was measured in stable scrambled control (Scr) and Miz1 knockdown (#3 and #5) PZp53MED1 cells using WST-1 (A) and 3H-thymidine incorporation assays (B). Data shown in the graphs represent the mean ± SEM (n = 3; **: p<0.001, compared to the Scr sample, t test). (C) and (D) The levels of mRNA expression for Gli1 (C) and Ptch1 (D) were measured using qRT-PCR in control and Miz1 knockdown PZp53MED1 cells. Data shown in the graphs represent the mean ± SEM (n = 4 for Gli1 and n = 3 for Ptch1; *: p<0.05; and **: p<0.001, compared to the Scr sample, t test). (E) SCID mice were inoculated subcutaneously with stable control and Miz1 knockdown PZp53MED1 cells. Image shows the tumors dissected from mice at the end of the experiment. ND represents non-detectable tumor growth around the injection sites. (F) Quantitative representation of tumor weights measured. Data in the graph represent the mean ± SEM (n = 5 for each group; *: p<0.05, compared to the Scr sample, t test).
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pone-0063353-g006: Knockdown of Miz1 inhibits cell proliferation and tumorigenesis in PZp53MED1 medulloblastoma cells.(A) and (B) The rate of cell proliferation was measured in stable scrambled control (Scr) and Miz1 knockdown (#3 and #5) PZp53MED1 cells using WST-1 (A) and 3H-thymidine incorporation assays (B). Data shown in the graphs represent the mean ± SEM (n = 3; **: p<0.001, compared to the Scr sample, t test). (C) and (D) The levels of mRNA expression for Gli1 (C) and Ptch1 (D) were measured using qRT-PCR in control and Miz1 knockdown PZp53MED1 cells. Data shown in the graphs represent the mean ± SEM (n = 4 for Gli1 and n = 3 for Ptch1; *: p<0.05; and **: p<0.001, compared to the Scr sample, t test). (E) SCID mice were inoculated subcutaneously with stable control and Miz1 knockdown PZp53MED1 cells. Image shows the tumors dissected from mice at the end of the experiment. ND represents non-detectable tumor growth around the injection sites. (F) Quantitative representation of tumor weights measured. Data in the graph represent the mean ± SEM (n = 5 for each group; *: p<0.05, compared to the Scr sample, t test).
Mentions: Aberrant activation of Hh signaling has been implicated in many types of cancer [3], [4], [5], [6]. Mutations in Ptch1 and Smo occur in patients with medulloblastoma (MB), an aggressive childhood cancer arising from cerebellar GNPs [27]. To test the function of Miz1, we stably knocked down the endogenous expression of Miz1 in PZp53MED1 cells generated from a murine Hh-driven medulloblastoma allograft model [24]. Loss of Miz1 significantly reduced cell proliferation as assessed using WST-1 assays (Figure 6A, Scr, 0.35±0.03; versus #3, 0.13±0.01 and #5, 0.14±0.01) and 3H-thymidine incorporation assays (Figure 6B, Scr, 6326±457; versus #3, 824±40 and #5, 1193±64). The suppression of cell growth coincides with a reduction in the mRNA levels of Hh target genes Gli1 and Ptch1 (Figure 6C and 6D, for Gli1 mRNA, Scr, 4.09±0.32, versus #3, 0.57±0.15 and #5, 0.61±0.09; and for Ptch1 mRNA, Scr, 7.97±2.31, versus #3, 2.82±0.86 and #5, 2.85±1.49), suggesting decreased Hh signaling is responsible for the growth inhibition observed in Miz1 knockdown cells.

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