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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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Related in: MedlinePlus

Miz1 regulates Hh signaling in primary cilia.(A) Confocal images showing the localization of Miz1 in primary cilia. NIH 3T3 cells treated with DMSO or SAG were co-stained with antibodies for Miz1 (green color) and acetylated-tubulin (red color, to indicate primary cilia). Cell nuclei were stained with DAPI (blue color). Co-localization is shown in the overlay panel. Middle and bottom panels correspond to the regions in the dashed white boxes in the upper panels. (B) Quantification of the average intensity of Miz1 in primary cilia. Total 101 and 103 cilia were quantified for DMSO and SAG treated group, respectively (**, p<0.001, compared to the DMSO sample, t test). (C) Knockdown of Miz1 expression inhibits activation-dependent recruitment of Smo into primary cilia. NIH 3T3 cells expressing GFP-Smo were stably infected with scrambled (Scr) control or Miz1 shRNAs (#3 and #5). Cells treated with DMSO or SAG were stained with anti-acetylated tubulin (red color, to indicate primary cilia), and cell nuclei were indicated by DAPI staining (blue color). GFP-Smo was shown in green color. Overlay images are shown in upper panels while the single-channel GFP-Smo images are shown directly below. Arrowheads in each image indicate primary cilia. (D) Quantification of the average intensity of GFP-Smo in primary cilia. Data shown in the graph represent the mean ± SEM (n = 37, 204, 267, 268, 215, and 212 cilia from left to right; **: p<0.001, t test). (E) Knockdown of Miz1 inhibits the accumulation of Gli2 at the tip of primary cilia upon Hh activation. NIH 3T3 cells were stably infected with lentiviral particles encoding either scrambled control shRNA (Scr) or shRNAs against Miz1 (#3 and #5). Cells were treated with DMSO or SAG for 24 hours and subsequently co-stained with antibodies for Gli2 (green color) and detyrosinated-tubulin (GluTu, red color, to indicate primary cilia). Cell nuclei were stained with DAPI (blue color). Overlay images are shown in upper panels for each condition, and the enlarged images of areas enclosed by the dashed white boxes are shown in the panels directly below. (F) Quantification of the percentage Gli2-containing primary cilia. Data shown in the graph represent the mean ± SEM (n = 276, 406, 214, 239, 246, and 307 cilia from left to right. **: p<0.001, t test).
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pone-0063353-g003: Miz1 regulates Hh signaling in primary cilia.(A) Confocal images showing the localization of Miz1 in primary cilia. NIH 3T3 cells treated with DMSO or SAG were co-stained with antibodies for Miz1 (green color) and acetylated-tubulin (red color, to indicate primary cilia). Cell nuclei were stained with DAPI (blue color). Co-localization is shown in the overlay panel. Middle and bottom panels correspond to the regions in the dashed white boxes in the upper panels. (B) Quantification of the average intensity of Miz1 in primary cilia. Total 101 and 103 cilia were quantified for DMSO and SAG treated group, respectively (**, p<0.001, compared to the DMSO sample, t test). (C) Knockdown of Miz1 expression inhibits activation-dependent recruitment of Smo into primary cilia. NIH 3T3 cells expressing GFP-Smo were stably infected with scrambled (Scr) control or Miz1 shRNAs (#3 and #5). Cells treated with DMSO or SAG were stained with anti-acetylated tubulin (red color, to indicate primary cilia), and cell nuclei were indicated by DAPI staining (blue color). GFP-Smo was shown in green color. Overlay images are shown in upper panels while the single-channel GFP-Smo images are shown directly below. Arrowheads in each image indicate primary cilia. (D) Quantification of the average intensity of GFP-Smo in primary cilia. Data shown in the graph represent the mean ± SEM (n = 37, 204, 267, 268, 215, and 212 cilia from left to right; **: p<0.001, t test). (E) Knockdown of Miz1 inhibits the accumulation of Gli2 at the tip of primary cilia upon Hh activation. NIH 3T3 cells were stably infected with lentiviral particles encoding either scrambled control shRNA (Scr) or shRNAs against Miz1 (#3 and #5). Cells were treated with DMSO or SAG for 24 hours and subsequently co-stained with antibodies for Gli2 (green color) and detyrosinated-tubulin (GluTu, red color, to indicate primary cilia). Cell nuclei were stained with DAPI (blue color). Overlay images are shown in upper panels for each condition, and the enlarged images of areas enclosed by the dashed white boxes are shown in the panels directly below. (F) Quantification of the percentage Gli2-containing primary cilia. Data shown in the graph represent the mean ± SEM (n = 276, 406, 214, 239, 246, and 307 cilia from left to right. **: p<0.001, t test).

Mentions: The primary cilium plays a critical role in Hh signaling pathway [7]. Many Hh key components have been found to localize in primary cilium upon Hh activation [10], [11], [12], [13], [14], [15]. To determine whether Miz1 is involved in primary cilium-dependent Hh activation, we first examined the subcellular localization of endogenous Miz1 in primary cilia by confocal microscopy in NIH3T3 cells. Cells treated with either DMSO or 0.25 μM SAG were stained with the anti-Miz1 antibody and an antibody for the primary cilium marker acetylated tubulin. A significant increase of Miz1 was observed in primary cilia after SAG stimulation (DMSO 1.00±0.08, SAG 1.65±0.16) (Figure 3A and 3B). To verify whether the transport of Miz1 into primary cilia depends on Smo activity, we performed the same experiment on Smo−/− MEF. We did not observe any ciliary localization of Miz1 in Smo−/− MEF (DMSO, 1.00±0.16 and SAG, 1.18±0.19). These results indicate that Smo activation is essential for the trafficking of Miz1 into cilia.


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 regulates Hh signaling in primary cilia.(A) Confocal images showing the localization of Miz1 in primary cilia. NIH 3T3 cells treated with DMSO or SAG were co-stained with antibodies for Miz1 (green color) and acetylated-tubulin (red color, to indicate primary cilia). Cell nuclei were stained with DAPI (blue color). Co-localization is shown in the overlay panel. Middle and bottom panels correspond to the regions in the dashed white boxes in the upper panels. (B) Quantification of the average intensity of Miz1 in primary cilia. Total 101 and 103 cilia were quantified for DMSO and SAG treated group, respectively (**, p<0.001, compared to the DMSO sample, t test). (C) Knockdown of Miz1 expression inhibits activation-dependent recruitment of Smo into primary cilia. NIH 3T3 cells expressing GFP-Smo were stably infected with scrambled (Scr) control or Miz1 shRNAs (#3 and #5). Cells treated with DMSO or SAG were stained with anti-acetylated tubulin (red color, to indicate primary cilia), and cell nuclei were indicated by DAPI staining (blue color). GFP-Smo was shown in green color. Overlay images are shown in upper panels while the single-channel GFP-Smo images are shown directly below. Arrowheads in each image indicate primary cilia. (D) Quantification of the average intensity of GFP-Smo in primary cilia. Data shown in the graph represent the mean ± SEM (n = 37, 204, 267, 268, 215, and 212 cilia from left to right; **: p<0.001, t test). (E) Knockdown of Miz1 inhibits the accumulation of Gli2 at the tip of primary cilia upon Hh activation. NIH 3T3 cells were stably infected with lentiviral particles encoding either scrambled control shRNA (Scr) or shRNAs against Miz1 (#3 and #5). Cells were treated with DMSO or SAG for 24 hours and subsequently co-stained with antibodies for Gli2 (green color) and detyrosinated-tubulin (GluTu, red color, to indicate primary cilia). Cell nuclei were stained with DAPI (blue color). Overlay images are shown in upper panels for each condition, and the enlarged images of areas enclosed by the dashed white boxes are shown in the panels directly below. (F) Quantification of the percentage Gli2-containing primary cilia. Data shown in the graph represent the mean ± SEM (n = 276, 406, 214, 239, 246, and 307 cilia from left to right. **: p<0.001, t test).
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pone-0063353-g003: Miz1 regulates Hh signaling in primary cilia.(A) Confocal images showing the localization of Miz1 in primary cilia. NIH 3T3 cells treated with DMSO or SAG were co-stained with antibodies for Miz1 (green color) and acetylated-tubulin (red color, to indicate primary cilia). Cell nuclei were stained with DAPI (blue color). Co-localization is shown in the overlay panel. Middle and bottom panels correspond to the regions in the dashed white boxes in the upper panels. (B) Quantification of the average intensity of Miz1 in primary cilia. Total 101 and 103 cilia were quantified for DMSO and SAG treated group, respectively (**, p<0.001, compared to the DMSO sample, t test). (C) Knockdown of Miz1 expression inhibits activation-dependent recruitment of Smo into primary cilia. NIH 3T3 cells expressing GFP-Smo were stably infected with scrambled (Scr) control or Miz1 shRNAs (#3 and #5). Cells treated with DMSO or SAG were stained with anti-acetylated tubulin (red color, to indicate primary cilia), and cell nuclei were indicated by DAPI staining (blue color). GFP-Smo was shown in green color. Overlay images are shown in upper panels while the single-channel GFP-Smo images are shown directly below. Arrowheads in each image indicate primary cilia. (D) Quantification of the average intensity of GFP-Smo in primary cilia. Data shown in the graph represent the mean ± SEM (n = 37, 204, 267, 268, 215, and 212 cilia from left to right; **: p<0.001, t test). (E) Knockdown of Miz1 inhibits the accumulation of Gli2 at the tip of primary cilia upon Hh activation. NIH 3T3 cells were stably infected with lentiviral particles encoding either scrambled control shRNA (Scr) or shRNAs against Miz1 (#3 and #5). Cells were treated with DMSO or SAG for 24 hours and subsequently co-stained with antibodies for Gli2 (green color) and detyrosinated-tubulin (GluTu, red color, to indicate primary cilia). Cell nuclei were stained with DAPI (blue color). Overlay images are shown in upper panels for each condition, and the enlarged images of areas enclosed by the dashed white boxes are shown in the panels directly below. (F) Quantification of the percentage Gli2-containing primary cilia. Data shown in the graph represent the mean ± SEM (n = 276, 406, 214, 239, 246, and 307 cilia from left to right. **: p<0.001, t test).
Mentions: The primary cilium plays a critical role in Hh signaling pathway [7]. Many Hh key components have been found to localize in primary cilium upon Hh activation [10], [11], [12], [13], [14], [15]. To determine whether Miz1 is involved in primary cilium-dependent Hh activation, we first examined the subcellular localization of endogenous Miz1 in primary cilia by confocal microscopy in NIH3T3 cells. Cells treated with either DMSO or 0.25 μM SAG were stained with the anti-Miz1 antibody and an antibody for the primary cilium marker acetylated tubulin. A significant increase of Miz1 was observed in primary cilia after SAG stimulation (DMSO 1.00±0.08, SAG 1.65±0.16) (Figure 3A and 3B). To verify whether the transport of Miz1 into primary cilia depends on Smo activity, we performed the same experiment on Smo−/− MEF. We did not observe any ciliary localization of Miz1 in Smo−/− MEF (DMSO, 1.00±0.16 and SAG, 1.18±0.19). These results indicate that Smo activation is essential for the trafficking of Miz1 into cilia.

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