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A transcriptional response to Wnt protein in human embryonic carcinoma cells.

Willert J, Epping M, Pollack JR, Brown PO, Nusse R - BMC Dev. Biol. (2002)

Bottom Line: The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin.Several of the target genes have a cooperative response to a combination of Wnt and BMP.Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305 USA. jennifer.willert@cox.net

ABSTRACT

Background: Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway.

Results: We have identified target genes of Wnt signaling using microarray technology and human embryonic carcinoma cells stimulated with active Wnt protein. The ~50 genes upregulated early after Wnt addition include the previously known Wnt targets Cyclin D1, MYC, ID2 and betaTRCP. The newly identified targets, which include MSX1, MSX2, Nucleophosmin, Follistatin, TLE/Groucho, Ubc4/5E2, CBP/P300, Frizzled and REST/NRSF, have important implications for understanding the roles of Wnts in development and cancer. The protein synthesis inhibitor cycloheximide blocks induction by Wnt, consistent with a requirement for newly synthesized beta-catenin protein prior to target gene activation. The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin. Several of the target genes have a cooperative response to a combination of Wnt and BMP.

Conclusions: Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

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Related in: MedlinePlus

Time course of Wnt induced gene expression and cooperation with BMP. NCCIT cells were exposed to Wnt-3A CM, BMP-4 (10 ng/ml final concentration) or the combination of Wnt-3A and BMP-4 for the specified number of hours. Shown here are MSX1, MSX2 and ID2 demonstrating that Wnt-3A induced effects are not seen until 2 hours (which corresponds to β-catenin accumulation). BMP-4 mediated induction of MSX1, MSX2 and ID2 occurs rapidly, as early as 30 minutes and stays relatively constant. Cooperative effects of Wnt-3A and BMP-4 are not evident until approximately 2 hours. The height of the bars presents the ratio induced/non-induced, quantified by phosphoimager analysis of Northern blots and normalized to GAPDH expression measured in the same experiment. Each bar represents average values obtained from 3–5 experiments for each mRNA tested.
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Figure 3: Time course of Wnt induced gene expression and cooperation with BMP. NCCIT cells were exposed to Wnt-3A CM, BMP-4 (10 ng/ml final concentration) or the combination of Wnt-3A and BMP-4 for the specified number of hours. Shown here are MSX1, MSX2 and ID2 demonstrating that Wnt-3A induced effects are not seen until 2 hours (which corresponds to β-catenin accumulation). BMP-4 mediated induction of MSX1, MSX2 and ID2 occurs rapidly, as early as 30 minutes and stays relatively constant. Cooperative effects of Wnt-3A and BMP-4 are not evident until approximately 2 hours. The height of the bars presents the ratio induced/non-induced, quantified by phosphoimager analysis of Northern blots and normalized to GAPDH expression measured in the same experiment. Each bar represents average values obtained from 3–5 experiments for each mRNA tested.

Mentions: Several of the Wnt target genes had been identified previously as targets of BMP signaling [11]. We were therefore interested in comparing the effects of BMP and Wnt-3A and examining their combined effects. As has been reported before for other cells [11], MSX1, MSX2 and ID2 are elevated by BMP-4 in NCCIT cells (Figure 3). When Wnt-3A and BMP-4 were combined, gene expression was increased to yet higher levels. MSX1 expression was markedly elevated by the combination of BMP-4 and Wnt-3A. Similar but less dramatic effects were found for ID2 and MSX2 (Figure 3). Hence, BMP-4 and Wnt-3A appear to have an additive or perhaps synergistic effect on target gene expression. However, when the time courses of Wnt- and BMP-induced gene expression were compared, it appeared that BMP-4 acts as soon as at 30 minutes, but that Wnt takes 2 hours to have an effect (Figure 3). Hence, Wnt-induced changes in gene expression are slower than BMP, even on the same target genes and in the same cell type.


A transcriptional response to Wnt protein in human embryonic carcinoma cells.

Willert J, Epping M, Pollack JR, Brown PO, Nusse R - BMC Dev. Biol. (2002)

Time course of Wnt induced gene expression and cooperation with BMP. NCCIT cells were exposed to Wnt-3A CM, BMP-4 (10 ng/ml final concentration) or the combination of Wnt-3A and BMP-4 for the specified number of hours. Shown here are MSX1, MSX2 and ID2 demonstrating that Wnt-3A induced effects are not seen until 2 hours (which corresponds to β-catenin accumulation). BMP-4 mediated induction of MSX1, MSX2 and ID2 occurs rapidly, as early as 30 minutes and stays relatively constant. Cooperative effects of Wnt-3A and BMP-4 are not evident until approximately 2 hours. The height of the bars presents the ratio induced/non-induced, quantified by phosphoimager analysis of Northern blots and normalized to GAPDH expression measured in the same experiment. Each bar represents average values obtained from 3–5 experiments for each mRNA tested.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117803&req=5

Figure 3: Time course of Wnt induced gene expression and cooperation with BMP. NCCIT cells were exposed to Wnt-3A CM, BMP-4 (10 ng/ml final concentration) or the combination of Wnt-3A and BMP-4 for the specified number of hours. Shown here are MSX1, MSX2 and ID2 demonstrating that Wnt-3A induced effects are not seen until 2 hours (which corresponds to β-catenin accumulation). BMP-4 mediated induction of MSX1, MSX2 and ID2 occurs rapidly, as early as 30 minutes and stays relatively constant. Cooperative effects of Wnt-3A and BMP-4 are not evident until approximately 2 hours. The height of the bars presents the ratio induced/non-induced, quantified by phosphoimager analysis of Northern blots and normalized to GAPDH expression measured in the same experiment. Each bar represents average values obtained from 3–5 experiments for each mRNA tested.
Mentions: Several of the Wnt target genes had been identified previously as targets of BMP signaling [11]. We were therefore interested in comparing the effects of BMP and Wnt-3A and examining their combined effects. As has been reported before for other cells [11], MSX1, MSX2 and ID2 are elevated by BMP-4 in NCCIT cells (Figure 3). When Wnt-3A and BMP-4 were combined, gene expression was increased to yet higher levels. MSX1 expression was markedly elevated by the combination of BMP-4 and Wnt-3A. Similar but less dramatic effects were found for ID2 and MSX2 (Figure 3). Hence, BMP-4 and Wnt-3A appear to have an additive or perhaps synergistic effect on target gene expression. However, when the time courses of Wnt- and BMP-induced gene expression were compared, it appeared that BMP-4 acts as soon as at 30 minutes, but that Wnt takes 2 hours to have an effect (Figure 3). Hence, Wnt-induced changes in gene expression are slower than BMP, even on the same target genes and in the same cell type.

Bottom Line: The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin.Several of the target genes have a cooperative response to a combination of Wnt and BMP.Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305 USA. jennifer.willert@cox.net

ABSTRACT

Background: Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway.

Results: We have identified target genes of Wnt signaling using microarray technology and human embryonic carcinoma cells stimulated with active Wnt protein. The ~50 genes upregulated early after Wnt addition include the previously known Wnt targets Cyclin D1, MYC, ID2 and betaTRCP. The newly identified targets, which include MSX1, MSX2, Nucleophosmin, Follistatin, TLE/Groucho, Ubc4/5E2, CBP/P300, Frizzled and REST/NRSF, have important implications for understanding the roles of Wnts in development and cancer. The protein synthesis inhibitor cycloheximide blocks induction by Wnt, consistent with a requirement for newly synthesized beta-catenin protein prior to target gene activation. The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin. Several of the target genes have a cooperative response to a combination of Wnt and BMP.

Conclusions: Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

Show MeSH
Related in: MedlinePlus