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Dickkopf-related protein 3 promotes pathogenic stromal remodeling in benign prostatic hyperplasia and prostate cancer.

Zenzmaier C, Sampson N, Plas E, Berger P - Prostate (2013)

Bottom Line: Knockdown of DKK3 significantly attenuated PrSC proliferation as well as fibroblast-to-myofibroblast differentiation and increased the expression of the vessel stabilizing factor angiopoietin-1.DKK3 knockdown did not affect subcellular localization or levels of β-catenin but attenuated AKT phosphorylation in PrSCs.Consistently the PI3K/AKT inhibitor LY294002 mimicked the effects of DKK3 knockdown.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Aging Research, University of Innsbruck, Innsbruck, Austria. christoph.zenzmaier@i-med.ac.at

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PI3K inhibition mimics the effects of DKK3 knockdown. A: The PI3K inhibitor LY294002 reduced proliferation of PrSCs in a dose-dependent manner, as determined after 72 hr by BrdU-incorporation ELISA (n = 3). B: Western blot analysis of Dkk-3, p21CIP1 and p27KIP1 after 24 hr incubation with LY294002. β-actin served as loading control. C: Effect of PI3K inhibition using 10 µM LY294002 and/or lentiviral-delivered shRNA-mediated DKK3 knockdown (DKK3-shRNA) on fibroblast-to-myofibroblast differentiation of PrSCs as determined by mRNA levels of the marker genes SMA and IGFBP3 after stimulation with 1 ng/ml TGFβ1 (differentiation) or bFGF (control) for 24 hr. Gene expression levels were normalized using the housekeeping gene HMBS and are shown relative to scrambled (SCR-)shRNA and bFGF-treated controls. Bars represent mean ± SEM of three independent experiments. D: LY294002 induced ANGPT1 and ANGPT2 in a dose-dependent manner at mRNA levels within 4 hr of treatment (n = 3). E: Secreted angiopoietin-1 levels as determined in conditioned media of SCR-/DKK3-shRNA-treated PrSCs after incubation without/with 10 µM LY294002 for 72 hr (n = 4).
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fig05: PI3K inhibition mimics the effects of DKK3 knockdown. A: The PI3K inhibitor LY294002 reduced proliferation of PrSCs in a dose-dependent manner, as determined after 72 hr by BrdU-incorporation ELISA (n = 3). B: Western blot analysis of Dkk-3, p21CIP1 and p27KIP1 after 24 hr incubation with LY294002. β-actin served as loading control. C: Effect of PI3K inhibition using 10 µM LY294002 and/or lentiviral-delivered shRNA-mediated DKK3 knockdown (DKK3-shRNA) on fibroblast-to-myofibroblast differentiation of PrSCs as determined by mRNA levels of the marker genes SMA and IGFBP3 after stimulation with 1 ng/ml TGFβ1 (differentiation) or bFGF (control) for 24 hr. Gene expression levels were normalized using the housekeeping gene HMBS and are shown relative to scrambled (SCR-)shRNA and bFGF-treated controls. Bars represent mean ± SEM of three independent experiments. D: LY294002 induced ANGPT1 and ANGPT2 in a dose-dependent manner at mRNA levels within 4 hr of treatment (n = 3). E: Secreted angiopoietin-1 levels as determined in conditioned media of SCR-/DKK3-shRNA-treated PrSCs after incubation without/with 10 µM LY294002 for 72 hr (n = 4).

Mentions: The specific PI3K inhibitor LY294002 was used to investigate whether attenuation of AKT phosphorylation in DKK3-shRNA PrSCs is responsible for the modulatory effects of DKK3 knockdown on angiogenesis and fibroblast-to-myofibroblast differentiation marker expression. PI3K inhibition significantly attenuated cellular proliferation in a dose-dependent manner (Fig. 5A; 0 vs. 10 µM: P = 0.010; 10 vs. 20 µM: P = 0.044). Moreover, similar to DKK3-shRNA, the reduction in proliferation upon PI3K inhibition was associated with elevated CDKN1B/p27KIP1 mRNA and protein levels and reduced p21CIP1 protein levels while CDKN1A mRNA levels were increased (Fig. 5B and Supplemental Fig. 1). Dkk-3 levels were unaffected by LY294002 (Fig. 5B).


Dickkopf-related protein 3 promotes pathogenic stromal remodeling in benign prostatic hyperplasia and prostate cancer.

Zenzmaier C, Sampson N, Plas E, Berger P - Prostate (2013)

PI3K inhibition mimics the effects of DKK3 knockdown. A: The PI3K inhibitor LY294002 reduced proliferation of PrSCs in a dose-dependent manner, as determined after 72 hr by BrdU-incorporation ELISA (n = 3). B: Western blot analysis of Dkk-3, p21CIP1 and p27KIP1 after 24 hr incubation with LY294002. β-actin served as loading control. C: Effect of PI3K inhibition using 10 µM LY294002 and/or lentiviral-delivered shRNA-mediated DKK3 knockdown (DKK3-shRNA) on fibroblast-to-myofibroblast differentiation of PrSCs as determined by mRNA levels of the marker genes SMA and IGFBP3 after stimulation with 1 ng/ml TGFβ1 (differentiation) or bFGF (control) for 24 hr. Gene expression levels were normalized using the housekeeping gene HMBS and are shown relative to scrambled (SCR-)shRNA and bFGF-treated controls. Bars represent mean ± SEM of three independent experiments. D: LY294002 induced ANGPT1 and ANGPT2 in a dose-dependent manner at mRNA levels within 4 hr of treatment (n = 3). E: Secreted angiopoietin-1 levels as determined in conditioned media of SCR-/DKK3-shRNA-treated PrSCs after incubation without/with 10 µM LY294002 for 72 hr (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig05: PI3K inhibition mimics the effects of DKK3 knockdown. A: The PI3K inhibitor LY294002 reduced proliferation of PrSCs in a dose-dependent manner, as determined after 72 hr by BrdU-incorporation ELISA (n = 3). B: Western blot analysis of Dkk-3, p21CIP1 and p27KIP1 after 24 hr incubation with LY294002. β-actin served as loading control. C: Effect of PI3K inhibition using 10 µM LY294002 and/or lentiviral-delivered shRNA-mediated DKK3 knockdown (DKK3-shRNA) on fibroblast-to-myofibroblast differentiation of PrSCs as determined by mRNA levels of the marker genes SMA and IGFBP3 after stimulation with 1 ng/ml TGFβ1 (differentiation) or bFGF (control) for 24 hr. Gene expression levels were normalized using the housekeeping gene HMBS and are shown relative to scrambled (SCR-)shRNA and bFGF-treated controls. Bars represent mean ± SEM of three independent experiments. D: LY294002 induced ANGPT1 and ANGPT2 in a dose-dependent manner at mRNA levels within 4 hr of treatment (n = 3). E: Secreted angiopoietin-1 levels as determined in conditioned media of SCR-/DKK3-shRNA-treated PrSCs after incubation without/with 10 µM LY294002 for 72 hr (n = 4).
Mentions: The specific PI3K inhibitor LY294002 was used to investigate whether attenuation of AKT phosphorylation in DKK3-shRNA PrSCs is responsible for the modulatory effects of DKK3 knockdown on angiogenesis and fibroblast-to-myofibroblast differentiation marker expression. PI3K inhibition significantly attenuated cellular proliferation in a dose-dependent manner (Fig. 5A; 0 vs. 10 µM: P = 0.010; 10 vs. 20 µM: P = 0.044). Moreover, similar to DKK3-shRNA, the reduction in proliferation upon PI3K inhibition was associated with elevated CDKN1B/p27KIP1 mRNA and protein levels and reduced p21CIP1 protein levels while CDKN1A mRNA levels were increased (Fig. 5B and Supplemental Fig. 1). Dkk-3 levels were unaffected by LY294002 (Fig. 5B).

Bottom Line: Knockdown of DKK3 significantly attenuated PrSC proliferation as well as fibroblast-to-myofibroblast differentiation and increased the expression of the vessel stabilizing factor angiopoietin-1.DKK3 knockdown did not affect subcellular localization or levels of β-catenin but attenuated AKT phosphorylation in PrSCs.Consistently the PI3K/AKT inhibitor LY294002 mimicked the effects of DKK3 knockdown.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Aging Research, University of Innsbruck, Innsbruck, Austria. christoph.zenzmaier@i-med.ac.at

Show MeSH
Related in: MedlinePlus