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

Impact of manipulation of NOTCH/HES1 signaling on DTX1 expression. (A) PCR (left panel) and western blot (right panel) analysis of HES1, DTX1 and actin expression in OS187 cells overexpressing ICN1, dnMAM or HES1 compared to vector control. (B) Quantitative PCR analysis of HES1 and DTX1 in the same cells at 3A. Analysis was performed as in figure 1C. (C) COL cells, which have high endogenous DTX1, were manipulated and analyzed as in 3A. Left panel: PCR analysis. Right panel: western blot analysis. (D) Histograms represent matrigel invasion of the COL cells from 3C (*, p<0.05). Assay and analysis were performed as in figure 1E.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Impact of manipulation of NOTCH/HES1 signaling on DTX1 expression. (A) PCR (left panel) and western blot (right panel) analysis of HES1, DTX1 and actin expression in OS187 cells overexpressing ICN1, dnMAM or HES1 compared to vector control. (B) Quantitative PCR analysis of HES1 and DTX1 in the same cells at 3A. Analysis was performed as in figure 1C. (C) COL cells, which have high endogenous DTX1, were manipulated and analyzed as in 3A. Left panel: PCR analysis. Right panel: western blot analysis. (D) Histograms represent matrigel invasion of the COL cells from 3C (*, p<0.05). Assay and analysis were performed as in figure 1E.

Mentions: We wished to understand better the role of Notch pathway genes in regulating DTX1 transcription. In OS187 cells with stable overexpression of intracellular NOTCH1 (ICN1), dominant negative Mastermind (dnMAM, an inhibitor of Notch receptor-CSL interaction) or HES1, HES1 mRNA and protein were upregulated by ICN1 and downregulated by dnMAM (Figure 3A and 3B). ICN1 overexpression did not upregulate DTX1 mRNA or protein expression, while inhibiting Notch/CSL interaction via dnMAM upregulated DTX1 gene and protein expression in OS187 cells. Furthermore, overexpression of HES1 reduced DTX1 protein in OS187 cells, suggesting that HES1 suppresses DTX1 expression. To confirm the inhibition of DTX1 by HES1, we also transduced ICN1 and HES1 into COL cells, which have high endogenous DTX1 expression. Overexpression of ICN1 or HES1 in COL suppressed DTX1 mRNA and protein expression (Figure 3C). Q-PCR confirmed that DTX1 mRNA was reduced by ICN1 or HES1 (data not shown). This observation suggested that ICN1 induced HES1, therefore inhibiting DTX1 mRNA expression in osteosarcoma cells. We also assessed the impact of ICN1 or HES1 transduction on COL invasion (Figure 3D). Compared to vector control, transduction with ICN1 or HES1 significantly increased invasion of COL cells in matrigel, confirming our finding that HES1 is critical for osteosarcoma invasion.


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)

Impact of manipulation of NOTCH/HES1 signaling on DTX1 expression. (A) PCR (left panel) and western blot (right panel) analysis of HES1, DTX1 and actin expression in OS187 cells overexpressing ICN1, dnMAM or HES1 compared to vector control. (B) Quantitative PCR analysis of HES1 and DTX1 in the same cells at 3A. Analysis was performed as in figure 1C. (C) COL cells, which have high endogenous DTX1, were manipulated and analyzed as in 3A. Left panel: PCR analysis. Right panel: western blot analysis. (D) Histograms represent matrigel invasion of the COL cells from 3C (*, p<0.05). Assay and analysis were performed as in figure 1E.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Impact of manipulation of NOTCH/HES1 signaling on DTX1 expression. (A) PCR (left panel) and western blot (right panel) analysis of HES1, DTX1 and actin expression in OS187 cells overexpressing ICN1, dnMAM or HES1 compared to vector control. (B) Quantitative PCR analysis of HES1 and DTX1 in the same cells at 3A. Analysis was performed as in figure 1C. (C) COL cells, which have high endogenous DTX1, were manipulated and analyzed as in 3A. Left panel: PCR analysis. Right panel: western blot analysis. (D) Histograms represent matrigel invasion of the COL cells from 3C (*, p<0.05). Assay and analysis were performed as in figure 1E.
Mentions: We wished to understand better the role of Notch pathway genes in regulating DTX1 transcription. In OS187 cells with stable overexpression of intracellular NOTCH1 (ICN1), dominant negative Mastermind (dnMAM, an inhibitor of Notch receptor-CSL interaction) or HES1, HES1 mRNA and protein were upregulated by ICN1 and downregulated by dnMAM (Figure 3A and 3B). ICN1 overexpression did not upregulate DTX1 mRNA or protein expression, while inhibiting Notch/CSL interaction via dnMAM upregulated DTX1 gene and protein expression in OS187 cells. Furthermore, overexpression of HES1 reduced DTX1 protein in OS187 cells, suggesting that HES1 suppresses DTX1 expression. To confirm the inhibition of DTX1 by HES1, we also transduced ICN1 and HES1 into COL cells, which have high endogenous DTX1 expression. Overexpression of ICN1 or HES1 in COL suppressed DTX1 mRNA and protein expression (Figure 3C). Q-PCR confirmed that DTX1 mRNA was reduced by ICN1 or HES1 (data not shown). This observation suggested that ICN1 induced HES1, therefore inhibiting DTX1 mRNA expression in osteosarcoma cells. We also assessed the impact of ICN1 or HES1 transduction on COL invasion (Figure 3D). Compared to vector control, transduction with ICN1 or HES1 significantly increased invasion of COL cells in matrigel, confirming our finding that HES1 is critical for osteosarcoma invasion.

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