Limits...
Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness.

Zhang P, Yang Y, Nolo R, Zweidler-McKay PA, Hughes DP - Oncogene (2010)

Bottom Line: The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors.An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1.HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics Research, Children's Cancer Hospital, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT
The highly conserved NOTCH signaling pathway has many essential functions in the development of diverse cells, tissues and organs from Drosophila to humans, and dysregulated NOTCH signaling contributes to several disorders, including vascular and bone defects, as well as several cancers. Here we describe a novel mechanism of NOTCH regulation by reciprocal inhibition of two NOTCH downstream effectors: Deltex1 and HES1. This mechanism appears to regulate invasion of osteosarcoma cells, as Deltex1 blocks osteosarcoma invasiveness by downregulating NOTCH/HES1 signaling. The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors. Conversely, we show that the NOTCH target gene HES1 causes transcriptional inhibition of Deltex1 by directly binding to the promoter of Deltex1. An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1. HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors. Taken together, we define a molecular mechanism regulating NOTCH signaling by reciprocal inhibition of the NOTCH target genes HES1 and Deltex1 in mammalian cells. This mechanism may have important clinical implications for targeting NOTCH signaling in osteosarcoma and other cancers.

Show MeSH

Related in: MedlinePlus

DTX1 physically interacts with ICN1 and induces ubiquitination and proteasome-mediated degradation of ICN1. (A) COL cells transduced with DTX1 or empty vector were treated with 5mM EDTA for 15 minutes to increase NOTCH1 cleavage and total ICN1 level. Immunoprecipitation (IP) of DTX1 from DTX1-transduced cells yielded both DTX1 and ICN1, as shown by immunoblot (IB). (B) Endogenous DTX1 was knocked down with either of 2 specific shRNA constructs (1) or (2); Immunoblot (IB) shows ICN1, HES1 and DTX1 protein from shRNA transduced cells compared to vector (V). (C) Histograms represent matrigel invasion of the COL cells shown in figure 1B. 1×104 COL cells with DTX1 shRNAs were seeded in each transwell and invasion was quantified after 48 hours; histograms shown the mean of three wells, and error bars show standard deviation. (*, p<0.05). (D) HA-ICN1 or DTX1, alone or in combination, were transiently overexpressed in 293T cells for 48 hours. After treatment with proteasome inhibitor PS341 (0.1μM) for 6 hours or no treatment, total cell lysates were prepared. Immunoblots (IB) of ICN1, DTX1 and actin in the whole cell extracts are shown (upper panels). After immunoprecipitation of ICN1 with anti-HA beads, ubiquitination of ICN1 was measured using anti-ubiquitin antibody (lower panel). Ubiquitination level of ICN1 was quantified by densitometry (bottom panel); histograms represent relative ubiquitination level.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2874642&req=5

Figure 2: DTX1 physically interacts with ICN1 and induces ubiquitination and proteasome-mediated degradation of ICN1. (A) COL cells transduced with DTX1 or empty vector were treated with 5mM EDTA for 15 minutes to increase NOTCH1 cleavage and total ICN1 level. Immunoprecipitation (IP) of DTX1 from DTX1-transduced cells yielded both DTX1 and ICN1, as shown by immunoblot (IB). (B) Endogenous DTX1 was knocked down with either of 2 specific shRNA constructs (1) or (2); Immunoblot (IB) shows ICN1, HES1 and DTX1 protein from shRNA transduced cells compared to vector (V). (C) Histograms represent matrigel invasion of the COL cells shown in figure 1B. 1×104 COL cells with DTX1 shRNAs were seeded in each transwell and invasion was quantified after 48 hours; histograms shown the mean of three wells, and error bars show standard deviation. (*, p<0.05). (D) HA-ICN1 or DTX1, alone or in combination, were transiently overexpressed in 293T cells for 48 hours. After treatment with proteasome inhibitor PS341 (0.1μM) for 6 hours or no treatment, total cell lysates were prepared. Immunoblots (IB) of ICN1, DTX1 and actin in the whole cell extracts are shown (upper panels). After immunoprecipitation of ICN1 with anti-HA beads, ubiquitination of ICN1 was measured using anti-ubiquitin antibody (lower panel). Ubiquitination level of ICN1 was quantified by densitometry (bottom panel); histograms represent relative ubiquitination level.

Mentions: DTX1 contains a RING-H2 domain and two WWE domain structures, suggesting a E3 ubiquitin ligase function (Matsuno et al., 2002). To assess DTX1 regulatory function, we measured DTX1 interaction with ICN1 and induction of proteasome-dependent ICN1 downregulation. We treated COL cells, which have high endogenous DTX1, with 5mM EDTA for 15 minutes to induce NOTCH1 cleavage and ICN internalization, then immunoprecipated DTX1 from the treated cells (Figure 2A). Immunoprecipitation of DTX1 also brought down ICN1 protein, confirming physical interaction of endogenous DTX1 and ICN1.


Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness.

Zhang P, Yang Y, Nolo R, Zweidler-McKay PA, Hughes DP - Oncogene (2010)

DTX1 physically interacts with ICN1 and induces ubiquitination and proteasome-mediated degradation of ICN1. (A) COL cells transduced with DTX1 or empty vector were treated with 5mM EDTA for 15 minutes to increase NOTCH1 cleavage and total ICN1 level. Immunoprecipitation (IP) of DTX1 from DTX1-transduced cells yielded both DTX1 and ICN1, as shown by immunoblot (IB). (B) Endogenous DTX1 was knocked down with either of 2 specific shRNA constructs (1) or (2); Immunoblot (IB) shows ICN1, HES1 and DTX1 protein from shRNA transduced cells compared to vector (V). (C) Histograms represent matrigel invasion of the COL cells shown in figure 1B. 1×104 COL cells with DTX1 shRNAs were seeded in each transwell and invasion was quantified after 48 hours; histograms shown the mean of three wells, and error bars show standard deviation. (*, p<0.05). (D) HA-ICN1 or DTX1, alone or in combination, were transiently overexpressed in 293T cells for 48 hours. After treatment with proteasome inhibitor PS341 (0.1μM) for 6 hours or no treatment, total cell lysates were prepared. Immunoblots (IB) of ICN1, DTX1 and actin in the whole cell extracts are shown (upper panels). After immunoprecipitation of ICN1 with anti-HA beads, ubiquitination of ICN1 was measured using anti-ubiquitin antibody (lower panel). Ubiquitination level of ICN1 was quantified by densitometry (bottom panel); histograms represent relative ubiquitination level.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874642&req=5

Figure 2: DTX1 physically interacts with ICN1 and induces ubiquitination and proteasome-mediated degradation of ICN1. (A) COL cells transduced with DTX1 or empty vector were treated with 5mM EDTA for 15 minutes to increase NOTCH1 cleavage and total ICN1 level. Immunoprecipitation (IP) of DTX1 from DTX1-transduced cells yielded both DTX1 and ICN1, as shown by immunoblot (IB). (B) Endogenous DTX1 was knocked down with either of 2 specific shRNA constructs (1) or (2); Immunoblot (IB) shows ICN1, HES1 and DTX1 protein from shRNA transduced cells compared to vector (V). (C) Histograms represent matrigel invasion of the COL cells shown in figure 1B. 1×104 COL cells with DTX1 shRNAs were seeded in each transwell and invasion was quantified after 48 hours; histograms shown the mean of three wells, and error bars show standard deviation. (*, p<0.05). (D) HA-ICN1 or DTX1, alone or in combination, were transiently overexpressed in 293T cells for 48 hours. After treatment with proteasome inhibitor PS341 (0.1μM) for 6 hours or no treatment, total cell lysates were prepared. Immunoblots (IB) of ICN1, DTX1 and actin in the whole cell extracts are shown (upper panels). After immunoprecipitation of ICN1 with anti-HA beads, ubiquitination of ICN1 was measured using anti-ubiquitin antibody (lower panel). Ubiquitination level of ICN1 was quantified by densitometry (bottom panel); histograms represent relative ubiquitination level.
Mentions: DTX1 contains a RING-H2 domain and two WWE domain structures, suggesting a E3 ubiquitin ligase function (Matsuno et al., 2002). To assess DTX1 regulatory function, we measured DTX1 interaction with ICN1 and induction of proteasome-dependent ICN1 downregulation. We treated COL cells, which have high endogenous DTX1, with 5mM EDTA for 15 minutes to induce NOTCH1 cleavage and ICN internalization, then immunoprecipated DTX1 from the treated cells (Figure 2A). Immunoprecipitation of DTX1 also brought down ICN1 protein, confirming physical interaction of endogenous DTX1 and ICN1.

Bottom Line: The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors.An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1.HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics Research, Children's Cancer Hospital, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT
The highly conserved NOTCH signaling pathway has many essential functions in the development of diverse cells, tissues and organs from Drosophila to humans, and dysregulated NOTCH signaling contributes to several disorders, including vascular and bone defects, as well as several cancers. Here we describe a novel mechanism of NOTCH regulation by reciprocal inhibition of two NOTCH downstream effectors: Deltex1 and HES1. This mechanism appears to regulate invasion of osteosarcoma cells, as Deltex1 blocks osteosarcoma invasiveness by downregulating NOTCH/HES1 signaling. The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors. Conversely, we show that the NOTCH target gene HES1 causes transcriptional inhibition of Deltex1 by directly binding to the promoter of Deltex1. An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1. HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors. Taken together, we define a molecular mechanism regulating NOTCH signaling by reciprocal inhibition of the NOTCH target genes HES1 and Deltex1 in mammalian cells. This mechanism may have important clinical implications for targeting NOTCH signaling in osteosarcoma and other cancers.

Show MeSH
Related in: MedlinePlus