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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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A) Western blot demonstrating cycloheximide effects of Wnt-3A mediated accumulation of β-catenin. NCCIT cells were stimulated for 4 hours with CCM (C) or Wnt-3A CM (Wnt) in the presence (20 microgram/ml) or absence (0) of cycloheximide. Note that β-catenin does not accumulate in response to Wnt-3A CM when cycloheximide is present. B) Northern analysis demonstrating that MSX2 induction by Wnt-3A is abolished in the presence of cycloheximide. Cells were stimulated for 4 hours with CCM (C), BMP-4 or Wnt-3A CM (Wnt) in the presence (20 microgram/ml media) or absence of cycloheximide. As expected, BMP-4 mediated induction of MSX2 is not affected by cycloheximide which contrasts with the inhibition of Wnt-3A mediated induction of MSX2 in the presence of cycloheximide. GAPDH was used to verify equal loading. The inhibitory effect was also seen for MSX1, ID2, Follistatin, Versican and Cyclin D1 (not shown).
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Figure 4: A) Western blot demonstrating cycloheximide effects of Wnt-3A mediated accumulation of β-catenin. NCCIT cells were stimulated for 4 hours with CCM (C) or Wnt-3A CM (Wnt) in the presence (20 microgram/ml) or absence (0) of cycloheximide. Note that β-catenin does not accumulate in response to Wnt-3A CM when cycloheximide is present. B) Northern analysis demonstrating that MSX2 induction by Wnt-3A is abolished in the presence of cycloheximide. Cells were stimulated for 4 hours with CCM (C), BMP-4 or Wnt-3A CM (Wnt) in the presence (20 microgram/ml media) or absence of cycloheximide. As expected, BMP-4 mediated induction of MSX2 is not affected by cycloheximide which contrasts with the inhibition of Wnt-3A mediated induction of MSX2 in the presence of cycloheximide. GAPDH was used to verify equal loading. The inhibitory effect was also seen for MSX1, ID2, Follistatin, Versican and Cyclin D1 (not shown).

Mentions: We tested human teratocarcinoma cells (NCCIT) cells for a Wnt response. Despite the cancerous origin of these cells, they share many properties with embryonic stem cells [7]. These cells express several members of the Frizzled family (including FZD7), one of the receptors for Wnt (data not shown; Figure 2A). To stimulate the NCCIT cells with Wnt, we used tissue culture medium containing active Wnt-3A protein produced by mouse L cells (Wnt-3A CM [8]), in comparison to control conditioned medium (CCM). In initial experiments, we found that Wnt-3A protein elevates the levels of β-catenin 5–10 fold (Figure 4); and that a transiently transfected TCF reporter construct is activated 3–4 fold (not shown).


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)

A) Western blot demonstrating cycloheximide effects of Wnt-3A mediated accumulation of β-catenin. NCCIT cells were stimulated for 4 hours with CCM (C) or Wnt-3A CM (Wnt) in the presence (20 microgram/ml) or absence (0) of cycloheximide. Note that β-catenin does not accumulate in response to Wnt-3A CM when cycloheximide is present. B) Northern analysis demonstrating that MSX2 induction by Wnt-3A is abolished in the presence of cycloheximide. Cells were stimulated for 4 hours with CCM (C), BMP-4 or Wnt-3A CM (Wnt) in the presence (20 microgram/ml media) or absence of cycloheximide. As expected, BMP-4 mediated induction of MSX2 is not affected by cycloheximide which contrasts with the inhibition of Wnt-3A mediated induction of MSX2 in the presence of cycloheximide. GAPDH was used to verify equal loading. The inhibitory effect was also seen for MSX1, ID2, Follistatin, Versican and Cyclin D1 (not shown).
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Related In: Results  -  Collection

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Figure 4: A) Western blot demonstrating cycloheximide effects of Wnt-3A mediated accumulation of β-catenin. NCCIT cells were stimulated for 4 hours with CCM (C) or Wnt-3A CM (Wnt) in the presence (20 microgram/ml) or absence (0) of cycloheximide. Note that β-catenin does not accumulate in response to Wnt-3A CM when cycloheximide is present. B) Northern analysis demonstrating that MSX2 induction by Wnt-3A is abolished in the presence of cycloheximide. Cells were stimulated for 4 hours with CCM (C), BMP-4 or Wnt-3A CM (Wnt) in the presence (20 microgram/ml media) or absence of cycloheximide. As expected, BMP-4 mediated induction of MSX2 is not affected by cycloheximide which contrasts with the inhibition of Wnt-3A mediated induction of MSX2 in the presence of cycloheximide. GAPDH was used to verify equal loading. The inhibitory effect was also seen for MSX1, ID2, Follistatin, Versican and Cyclin D1 (not shown).
Mentions: We tested human teratocarcinoma cells (NCCIT) cells for a Wnt response. Despite the cancerous origin of these cells, they share many properties with embryonic stem cells [7]. These cells express several members of the Frizzled family (including FZD7), one of the receptors for Wnt (data not shown; Figure 2A). To stimulate the NCCIT cells with Wnt, we used tissue culture medium containing active Wnt-3A protein produced by mouse L cells (Wnt-3A CM [8]), in comparison to control conditioned medium (CCM). In initial experiments, we found that Wnt-3A protein elevates the levels of β-catenin 5–10 fold (Figure 4); and that a transiently transfected TCF reporter construct is activated 3–4 fold (not shown).

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