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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 required for nuclear translocation of Gli2.(A) NIH 3T3 cells were stably infected with lentivirus encoding either the scrambled control shRNA (Scr) or shRNAs against Miz1 (#3 and #5). Cells treated with DMSO or 0.25 μM SAG for 24 hours were stained with the Gli2 antibody. Cell nuclei were stained with DAPI. Red dashed circles adapted from corresponding DAPI images indicate the regions of nuclei. (B) Graphic representation of data shown in panel (A). The amount of Gli2 in the nucleus was measured as the intensity of Gli2 fluorescence staining within the DAPI-positive nuclear regions [circled areas in images shown in (A)]. Data represent the mean ± SEM (n = 203, 127, 211, 210, 200 and 241 cells from left to right, respectively. **: p<0.001, t test).
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pone-0063353-g005: Miz1 is required for nuclear translocation of Gli2.(A) NIH 3T3 cells were stably infected with lentivirus encoding either the scrambled control shRNA (Scr) or shRNAs against Miz1 (#3 and #5). Cells treated with DMSO or 0.25 μM SAG for 24 hours were stained with the Gli2 antibody. Cell nuclei were stained with DAPI. Red dashed circles adapted from corresponding DAPI images indicate the regions of nuclei. (B) Graphic representation of data shown in panel (A). The amount of Gli2 in the nucleus was measured as the intensity of Gli2 fluorescence staining within the DAPI-positive nuclear regions [circled areas in images shown in (A)]. Data represent the mean ± SEM (n = 203, 127, 211, 210, 200 and 241 cells from left to right, respectively. **: p<0.001, t test).

Mentions: Studies have shown that the Gli2 accumulation at the tip of primary cilia within minutes of Hh stimulation is followed by its trafficking to the nucleus [12], [13]. However, it is not clear how this process is regulated. We demonstrated above that Miz1 is translocated to primary cilia (Figure 3) and subsequently accumulated in the nucleus upon activation of Smo (Figure 4). To examine whether Miz1 facilitates Gli2 nuclear translocation, we quantified the intensity of Gli2 in the nucleus by immunofluorescence staining in the presence or absence of SAG treatment (Figure 5A). Approximately a 1.5 fold increase of Gli2 nuclear localization was observed after SAG stimulation in scrambled control cells (in Scr, DMSO, 341.8±9.0, and SAG, 499.5±15.9), whereas knockdown of Miz1 completely prevented Gli2 nuclear recruitment (Figure 5B, in #3, DMSO, 331.3±3.9 and SAG, 336.8±3.9; and in #5, DMSO, 312.5±7.4 and SAG, 305.4±7.0). These results indicate that Miz1 regulates Hh signaling by facilitating the nuclear recruitment of Gli2.


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 required for nuclear translocation of Gli2.(A) NIH 3T3 cells were stably infected with lentivirus encoding either the scrambled control shRNA (Scr) or shRNAs against Miz1 (#3 and #5). Cells treated with DMSO or 0.25 μM SAG for 24 hours were stained with the Gli2 antibody. Cell nuclei were stained with DAPI. Red dashed circles adapted from corresponding DAPI images indicate the regions of nuclei. (B) Graphic representation of data shown in panel (A). The amount of Gli2 in the nucleus was measured as the intensity of Gli2 fluorescence staining within the DAPI-positive nuclear regions [circled areas in images shown in (A)]. Data represent the mean ± SEM (n = 203, 127, 211, 210, 200 and 241 cells from left to right, respectively. **: p<0.001, t test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643979&req=5

pone-0063353-g005: Miz1 is required for nuclear translocation of Gli2.(A) NIH 3T3 cells were stably infected with lentivirus encoding either the scrambled control shRNA (Scr) or shRNAs against Miz1 (#3 and #5). Cells treated with DMSO or 0.25 μM SAG for 24 hours were stained with the Gli2 antibody. Cell nuclei were stained with DAPI. Red dashed circles adapted from corresponding DAPI images indicate the regions of nuclei. (B) Graphic representation of data shown in panel (A). The amount of Gli2 in the nucleus was measured as the intensity of Gli2 fluorescence staining within the DAPI-positive nuclear regions [circled areas in images shown in (A)]. Data represent the mean ± SEM (n = 203, 127, 211, 210, 200 and 241 cells from left to right, respectively. **: p<0.001, t test).
Mentions: Studies have shown that the Gli2 accumulation at the tip of primary cilia within minutes of Hh stimulation is followed by its trafficking to the nucleus [12], [13]. However, it is not clear how this process is regulated. We demonstrated above that Miz1 is translocated to primary cilia (Figure 3) and subsequently accumulated in the nucleus upon activation of Smo (Figure 4). To examine whether Miz1 facilitates Gli2 nuclear translocation, we quantified the intensity of Gli2 in the nucleus by immunofluorescence staining in the presence or absence of SAG treatment (Figure 5A). Approximately a 1.5 fold increase of Gli2 nuclear localization was observed after SAG stimulation in scrambled control cells (in Scr, DMSO, 341.8±9.0, and SAG, 499.5±15.9), whereas knockdown of Miz1 completely prevented Gli2 nuclear recruitment (Figure 5B, in #3, DMSO, 331.3±3.9 and SAG, 336.8±3.9; and in #5, DMSO, 312.5±7.4 and SAG, 305.4±7.0). These results indicate that Miz1 regulates Hh signaling by facilitating the nuclear recruitment of Gli2.

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