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Rac1 GTPase and the Rac1 exchange factor Tiam1 associate with Wnt-responsive promoters to enhance beta-catenin/TCF-dependent transcription in colorectal cancer cells.

Buongiorno P, Pethe VV, Charames GS, Esufali S, Bapat B - Mol. Cancer (2008)

Bottom Line: In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway.Furthermore, endogenous Tiam1 associated with endogenous beta-catenin, and this interaction was enhanced in response to Wnt3a stimulation.Taken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active beta-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription.

View Article: PubMed Central - HTML - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5T 3L9, Canada. pinella.buongiorno@utoronto.ca

ABSTRACT

Background: beta-catenin is a key mediator of the canonical Wnt pathway as it associates with members of the T-cell factor (TCF) family at Wnt-responsive promoters to drive the transcription of Wnt target genes. Recently, we showed that Rac1 GTPase synergizes with beta-catenin to increase the activity of a TCF-responsive reporter. This synergy was dependent on the nuclear presence of Rac1, since inhibition of its nuclear localization effectively abolished the stimulatory effect of Rac1 on TCF-responsive reporter activity. We hypothesised that Rac1 plays a direct role in enhancing the transcription of endogenous Wnt target genes by modulating the beta-catenin/TCF transcription factor complex.

Results: We employed chromatin immunoprecipitation studies to demonstrate that Rac1 associates with the beta-catenin/TCF complex at Wnt-responsive promoters of target genes. This association served to facilitate transcription, since overexpression of active Rac1 augmented Wnt target gene activation, whereas depletion of endogenous Rac1 by RNA interference abrogated this effect. In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway. Tiam1 promoted the formation of a complex containing Rac1 and beta-catenin. Furthermore, endogenous Tiam1 associated with endogenous beta-catenin, and this interaction was enhanced in response to Wnt3a stimulation. Intriguingly, Tiam1 was recruited to Wnt-responsive promoters upon Wnt3a stimulation, whereas Rac1 was tethered to TCF binding elements in a Wnt-independent manner.

Conclusion: Taken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active beta-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription. Our results demonstrate a novel functional mechanism underlying the cross-talk between Rac1 and the canonical Wnt signalling pathway.

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Tiam1 lies upstream of Rac1-mediated activation of the canonical Wnt pathway. (A) HCT116 cells were co-transfected with pTOPFlash or pFOPFlash reporter plasmids and pCMVβ-galactosidase, in addition to the indicated expression plasmids: wild-type (wt) Rac1 alone (0.4 μg), Tiam1 alone (0.5 μg), wt Rac1 and Tiam1 in combination (0.4 μg and 0.5 μg, respectively), and N17Rac1 and Tiam1 in combination (0.4 μg and 0.5 μg, respectively). Empty vector was transfected to establish basal TOPFlash activity. Luciferase activity was normalized to β-galactosidase expression, and is expressed as total relative light units (RLU). Results shown are representative for three independent experiments performed in triplicate. Bars represent mean ± S.E. and *p < 0.05. (B) HCT116 cells were transiently co-transfected with either Tiam1-specific siRNA or control, and with either pTOPFlash or pFOPFlash reporter plasmids for 64 hours. Total RNA was analyzed by real time RT-PCR for the detection of Tiam1 transcript levels, which were normalized to transcript levels of β-actin (left). Luciferase activity was measured and normalized to β-galactosidase expression, and is expressed as total relative light units (right). Bars represent mean ± S.D. and *p < 0.05. (C) HCT116 cells were transfected with indicated plasmids and immunoprecipitated (IP) with β-catenin antibody. Immunoblotting (IB) was performed using β-catenin and myc-tag antibodies to detect myc-tagged wild-type (wt) Rac1. Whole cell lysates (WCL) were immunoblotted with β-catenin, myc-tag, or HA-tag antibodies to confirm expression of β-catenin, wild-type Rac1, or Tiam1, respectively. (D) Whole cell extracts of HCT116 cells were immunoprecipitated (IP) with Tiam1 antibody and analyzed by Western blotting (IB) using Tiam1, β-catenin, or Rac1 antibodies. Endogenous expression of these proteins was confirmed by immunoblotting whole cell lysates with indicated antibodies.
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Figure 3: Tiam1 lies upstream of Rac1-mediated activation of the canonical Wnt pathway. (A) HCT116 cells were co-transfected with pTOPFlash or pFOPFlash reporter plasmids and pCMVβ-galactosidase, in addition to the indicated expression plasmids: wild-type (wt) Rac1 alone (0.4 μg), Tiam1 alone (0.5 μg), wt Rac1 and Tiam1 in combination (0.4 μg and 0.5 μg, respectively), and N17Rac1 and Tiam1 in combination (0.4 μg and 0.5 μg, respectively). Empty vector was transfected to establish basal TOPFlash activity. Luciferase activity was normalized to β-galactosidase expression, and is expressed as total relative light units (RLU). Results shown are representative for three independent experiments performed in triplicate. Bars represent mean ± S.E. and *p < 0.05. (B) HCT116 cells were transiently co-transfected with either Tiam1-specific siRNA or control, and with either pTOPFlash or pFOPFlash reporter plasmids for 64 hours. Total RNA was analyzed by real time RT-PCR for the detection of Tiam1 transcript levels, which were normalized to transcript levels of β-actin (left). Luciferase activity was measured and normalized to β-galactosidase expression, and is expressed as total relative light units (right). Bars represent mean ± S.D. and *p < 0.05. (C) HCT116 cells were transfected with indicated plasmids and immunoprecipitated (IP) with β-catenin antibody. Immunoblotting (IB) was performed using β-catenin and myc-tag antibodies to detect myc-tagged wild-type (wt) Rac1. Whole cell lysates (WCL) were immunoblotted with β-catenin, myc-tag, or HA-tag antibodies to confirm expression of β-catenin, wild-type Rac1, or Tiam1, respectively. (D) Whole cell extracts of HCT116 cells were immunoprecipitated (IP) with Tiam1 antibody and analyzed by Western blotting (IB) using Tiam1, β-catenin, or Rac1 antibodies. Endogenous expression of these proteins was confirmed by immunoblotting whole cell lysates with indicated antibodies.

Mentions: Our previous work has shown that Rac1 must be in its active, GTP-bound state in order to synergize functionally with β-catenin. We demonstrated that only GTP-bound active Rac1, and not the inactive GDP-bound form, could stimulate TOPFlash activity [34]. The requirement for Rac1 activation prompted us to investigate the potential involvement of known regulators of Rac1 activity. Tiam1 is a Rac1-specific GEF [39] that is overexpressed in many colorectal cancers [6,5]. To investigate the possibility that Tiam1 plays a role in Rac1-mediated stimulation of β-catenin/TCF-dependent transcription, we conducted a TOPFlash reporter assay. HCT116 cells transiently transfected with wild-type Rac1 alone had no effect on TOPFlash activity compared to baseline, while Tiam1 alone caused a modest, 1.5-fold increase in TOPFlash activity (Figure 3A). This effect was likely modest due to the inherently high level of Rac1 activation in these cells [34]. When Tiam1 was co-transfected with wild-type Rac1, TOPFlash reporter activity increased by approximately 2.5-fold compared to baseline, similar to the effect of V12Rac1 [see Additional file 3]. In contrast, Tiam1 failed to activate the TOPFlash reporter in the presence of a dominant negative Rac1 mutant that is constitutively inactive (N17Rac1). To further explore the role of Tiam1 in canonical Wnt signalling, we analyzed the effect of Tiam1 knock-down on β-catenin/TCF-dependent transcription. Transfection of HCT116 cells with Tiam1 siRNA caused a reduction in Tiam1 transcript levels by approximately 50% (Figure 3B, left), and significantly diminished TOPFlash reporter activity (p < 0.05) compared to control (Figure 3B, right). This effect was likely modest due to incomplete knock-down of Tiam1 and due to inherently high levels of Rac1 activation in HCT116 cells, as noted above. Taken together, these results suggest that Tiam1 may act upstream of Rac1-mediated stimulation of the canonical Wnt pathway.


Rac1 GTPase and the Rac1 exchange factor Tiam1 associate with Wnt-responsive promoters to enhance beta-catenin/TCF-dependent transcription in colorectal cancer cells.

Buongiorno P, Pethe VV, Charames GS, Esufali S, Bapat B - Mol. Cancer (2008)

Tiam1 lies upstream of Rac1-mediated activation of the canonical Wnt pathway. (A) HCT116 cells were co-transfected with pTOPFlash or pFOPFlash reporter plasmids and pCMVβ-galactosidase, in addition to the indicated expression plasmids: wild-type (wt) Rac1 alone (0.4 μg), Tiam1 alone (0.5 μg), wt Rac1 and Tiam1 in combination (0.4 μg and 0.5 μg, respectively), and N17Rac1 and Tiam1 in combination (0.4 μg and 0.5 μg, respectively). Empty vector was transfected to establish basal TOPFlash activity. Luciferase activity was normalized to β-galactosidase expression, and is expressed as total relative light units (RLU). Results shown are representative for three independent experiments performed in triplicate. Bars represent mean ± S.E. and *p < 0.05. (B) HCT116 cells were transiently co-transfected with either Tiam1-specific siRNA or control, and with either pTOPFlash or pFOPFlash reporter plasmids for 64 hours. Total RNA was analyzed by real time RT-PCR for the detection of Tiam1 transcript levels, which were normalized to transcript levels of β-actin (left). Luciferase activity was measured and normalized to β-galactosidase expression, and is expressed as total relative light units (right). Bars represent mean ± S.D. and *p < 0.05. (C) HCT116 cells were transfected with indicated plasmids and immunoprecipitated (IP) with β-catenin antibody. Immunoblotting (IB) was performed using β-catenin and myc-tag antibodies to detect myc-tagged wild-type (wt) Rac1. Whole cell lysates (WCL) were immunoblotted with β-catenin, myc-tag, or HA-tag antibodies to confirm expression of β-catenin, wild-type Rac1, or Tiam1, respectively. (D) Whole cell extracts of HCT116 cells were immunoprecipitated (IP) with Tiam1 antibody and analyzed by Western blotting (IB) using Tiam1, β-catenin, or Rac1 antibodies. Endogenous expression of these proteins was confirmed by immunoblotting whole cell lysates with indicated antibodies.
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Related In: Results  -  Collection

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Figure 3: Tiam1 lies upstream of Rac1-mediated activation of the canonical Wnt pathway. (A) HCT116 cells were co-transfected with pTOPFlash or pFOPFlash reporter plasmids and pCMVβ-galactosidase, in addition to the indicated expression plasmids: wild-type (wt) Rac1 alone (0.4 μg), Tiam1 alone (0.5 μg), wt Rac1 and Tiam1 in combination (0.4 μg and 0.5 μg, respectively), and N17Rac1 and Tiam1 in combination (0.4 μg and 0.5 μg, respectively). Empty vector was transfected to establish basal TOPFlash activity. Luciferase activity was normalized to β-galactosidase expression, and is expressed as total relative light units (RLU). Results shown are representative for three independent experiments performed in triplicate. Bars represent mean ± S.E. and *p < 0.05. (B) HCT116 cells were transiently co-transfected with either Tiam1-specific siRNA or control, and with either pTOPFlash or pFOPFlash reporter plasmids for 64 hours. Total RNA was analyzed by real time RT-PCR for the detection of Tiam1 transcript levels, which were normalized to transcript levels of β-actin (left). Luciferase activity was measured and normalized to β-galactosidase expression, and is expressed as total relative light units (right). Bars represent mean ± S.D. and *p < 0.05. (C) HCT116 cells were transfected with indicated plasmids and immunoprecipitated (IP) with β-catenin antibody. Immunoblotting (IB) was performed using β-catenin and myc-tag antibodies to detect myc-tagged wild-type (wt) Rac1. Whole cell lysates (WCL) were immunoblotted with β-catenin, myc-tag, or HA-tag antibodies to confirm expression of β-catenin, wild-type Rac1, or Tiam1, respectively. (D) Whole cell extracts of HCT116 cells were immunoprecipitated (IP) with Tiam1 antibody and analyzed by Western blotting (IB) using Tiam1, β-catenin, or Rac1 antibodies. Endogenous expression of these proteins was confirmed by immunoblotting whole cell lysates with indicated antibodies.
Mentions: Our previous work has shown that Rac1 must be in its active, GTP-bound state in order to synergize functionally with β-catenin. We demonstrated that only GTP-bound active Rac1, and not the inactive GDP-bound form, could stimulate TOPFlash activity [34]. The requirement for Rac1 activation prompted us to investigate the potential involvement of known regulators of Rac1 activity. Tiam1 is a Rac1-specific GEF [39] that is overexpressed in many colorectal cancers [6,5]. To investigate the possibility that Tiam1 plays a role in Rac1-mediated stimulation of β-catenin/TCF-dependent transcription, we conducted a TOPFlash reporter assay. HCT116 cells transiently transfected with wild-type Rac1 alone had no effect on TOPFlash activity compared to baseline, while Tiam1 alone caused a modest, 1.5-fold increase in TOPFlash activity (Figure 3A). This effect was likely modest due to the inherently high level of Rac1 activation in these cells [34]. When Tiam1 was co-transfected with wild-type Rac1, TOPFlash reporter activity increased by approximately 2.5-fold compared to baseline, similar to the effect of V12Rac1 [see Additional file 3]. In contrast, Tiam1 failed to activate the TOPFlash reporter in the presence of a dominant negative Rac1 mutant that is constitutively inactive (N17Rac1). To further explore the role of Tiam1 in canonical Wnt signalling, we analyzed the effect of Tiam1 knock-down on β-catenin/TCF-dependent transcription. Transfection of HCT116 cells with Tiam1 siRNA caused a reduction in Tiam1 transcript levels by approximately 50% (Figure 3B, left), and significantly diminished TOPFlash reporter activity (p < 0.05) compared to control (Figure 3B, right). This effect was likely modest due to incomplete knock-down of Tiam1 and due to inherently high levels of Rac1 activation in HCT116 cells, as noted above. Taken together, these results suggest that Tiam1 may act upstream of Rac1-mediated stimulation of the canonical Wnt pathway.

Bottom Line: In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway.Furthermore, endogenous Tiam1 associated with endogenous beta-catenin, and this interaction was enhanced in response to Wnt3a stimulation.Taken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active beta-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription.

View Article: PubMed Central - HTML - PubMed

Affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5T 3L9, Canada. pinella.buongiorno@utoronto.ca

ABSTRACT

Background: beta-catenin is a key mediator of the canonical Wnt pathway as it associates with members of the T-cell factor (TCF) family at Wnt-responsive promoters to drive the transcription of Wnt target genes. Recently, we showed that Rac1 GTPase synergizes with beta-catenin to increase the activity of a TCF-responsive reporter. This synergy was dependent on the nuclear presence of Rac1, since inhibition of its nuclear localization effectively abolished the stimulatory effect of Rac1 on TCF-responsive reporter activity. We hypothesised that Rac1 plays a direct role in enhancing the transcription of endogenous Wnt target genes by modulating the beta-catenin/TCF transcription factor complex.

Results: We employed chromatin immunoprecipitation studies to demonstrate that Rac1 associates with the beta-catenin/TCF complex at Wnt-responsive promoters of target genes. This association served to facilitate transcription, since overexpression of active Rac1 augmented Wnt target gene activation, whereas depletion of endogenous Rac1 by RNA interference abrogated this effect. In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway. Tiam1 promoted the formation of a complex containing Rac1 and beta-catenin. Furthermore, endogenous Tiam1 associated with endogenous beta-catenin, and this interaction was enhanced in response to Wnt3a stimulation. Intriguingly, Tiam1 was recruited to Wnt-responsive promoters upon Wnt3a stimulation, whereas Rac1 was tethered to TCF binding elements in a Wnt-independent manner.

Conclusion: Taken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active beta-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription. Our results demonstrate a novel functional mechanism underlying the cross-talk between Rac1 and the canonical Wnt signalling pathway.

Show MeSH
Related in: MedlinePlus