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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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DKK3 knockdown reduces PrSC proliferation and induces p27KIP1 levels. A: Western blot analysis of total cell lysates of primary prostatic stromal (PrSC) and epithelial (PrEC) cells isolated from three individual donors revealed significant Dkk-3 expression in both cell types. β-actin served as loading control. B: DKK3 mRNA levels after lentiviral-delivered overexpression (OE DKK3) compared with empty vector control virus (OE VEC) and lentiviral-delivered DKK3 specific shRNA (DKK3 shRNA) compared with scrambled control (SCR shRNA) as determined by qPCR 72h post-transduction of PrSCs (overexpression: n = 4; shRNA: n = 5). DKK3 gene expression levels were normalized using the housekeeping gene HMBS and are shown relative to controls. C: Secreted Dkk-3 protein levels in PrSCs after overexpression and knockdown of DKK3 (n = 4). D: DKK3-shRNA significantly reduced cellular proliferation of PrSCs determined by BrdU-incorporation ELISA at day 6 post-transduction (n = 5). E: DKK3 mRNA levels of PC3 (n = 3) and HT-29 cells (n = 3) compared with PrSCs (n = 5). F: Cellular proliferation of PC3 and HT-29 cells as determined by BrdU-incorporation ELISA at day 6 post-transduction with DKK3-shRNA compared with SCR-shRNA, respectively (n = 3). G: Western blot analysis of apoptosis-related proteins (BAX, phospho-p53) and the cyclin-dependent kinase inhibitors p27KIP1 and p21CIP1 in DKK3-shRNA and SCR-shRNA PrSCs 72 hr post-transduction. GAPDH served as loading control.
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fig01: DKK3 knockdown reduces PrSC proliferation and induces p27KIP1 levels. A: Western blot analysis of total cell lysates of primary prostatic stromal (PrSC) and epithelial (PrEC) cells isolated from three individual donors revealed significant Dkk-3 expression in both cell types. β-actin served as loading control. B: DKK3 mRNA levels after lentiviral-delivered overexpression (OE DKK3) compared with empty vector control virus (OE VEC) and lentiviral-delivered DKK3 specific shRNA (DKK3 shRNA) compared with scrambled control (SCR shRNA) as determined by qPCR 72h post-transduction of PrSCs (overexpression: n = 4; shRNA: n = 5). DKK3 gene expression levels were normalized using the housekeeping gene HMBS and are shown relative to controls. C: Secreted Dkk-3 protein levels in PrSCs after overexpression and knockdown of DKK3 (n = 4). D: DKK3-shRNA significantly reduced cellular proliferation of PrSCs determined by BrdU-incorporation ELISA at day 6 post-transduction (n = 5). E: DKK3 mRNA levels of PC3 (n = 3) and HT-29 cells (n = 3) compared with PrSCs (n = 5). F: Cellular proliferation of PC3 and HT-29 cells as determined by BrdU-incorporation ELISA at day 6 post-transduction with DKK3-shRNA compared with SCR-shRNA, respectively (n = 3). G: Western blot analysis of apoptosis-related proteins (BAX, phospho-p53) and the cyclin-dependent kinase inhibitors p27KIP1 and p21CIP1 in DKK3-shRNA and SCR-shRNA PrSCs 72 hr post-transduction. GAPDH served as loading control.

Mentions: Primary prostatic stromal cells (PrSCs) were used to investigate the functional significance of Dkk-3 in the stromal compartment in vitro. Consistent with the predominant expression of Dkk-3 in the epithelial compartment of the benign prostate 1, Dkk-3 was more abundant in cell lysates from primary prostatic epithelial cells (PrECs) than PrSCs at the protein level as determined by Western blot analysis (Fig. 1A), however PrSCs secreted Dkk-3 at significant levels (Fig. 1C).


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)

DKK3 knockdown reduces PrSC proliferation and induces p27KIP1 levels. A: Western blot analysis of total cell lysates of primary prostatic stromal (PrSC) and epithelial (PrEC) cells isolated from three individual donors revealed significant Dkk-3 expression in both cell types. β-actin served as loading control. B: DKK3 mRNA levels after lentiviral-delivered overexpression (OE DKK3) compared with empty vector control virus (OE VEC) and lentiviral-delivered DKK3 specific shRNA (DKK3 shRNA) compared with scrambled control (SCR shRNA) as determined by qPCR 72h post-transduction of PrSCs (overexpression: n = 4; shRNA: n = 5). DKK3 gene expression levels were normalized using the housekeeping gene HMBS and are shown relative to controls. C: Secreted Dkk-3 protein levels in PrSCs after overexpression and knockdown of DKK3 (n = 4). D: DKK3-shRNA significantly reduced cellular proliferation of PrSCs determined by BrdU-incorporation ELISA at day 6 post-transduction (n = 5). E: DKK3 mRNA levels of PC3 (n = 3) and HT-29 cells (n = 3) compared with PrSCs (n = 5). F: Cellular proliferation of PC3 and HT-29 cells as determined by BrdU-incorporation ELISA at day 6 post-transduction with DKK3-shRNA compared with SCR-shRNA, respectively (n = 3). G: Western blot analysis of apoptosis-related proteins (BAX, phospho-p53) and the cyclin-dependent kinase inhibitors p27KIP1 and p21CIP1 in DKK3-shRNA and SCR-shRNA PrSCs 72 hr post-transduction. GAPDH served as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842835&req=5

fig01: DKK3 knockdown reduces PrSC proliferation and induces p27KIP1 levels. A: Western blot analysis of total cell lysates of primary prostatic stromal (PrSC) and epithelial (PrEC) cells isolated from three individual donors revealed significant Dkk-3 expression in both cell types. β-actin served as loading control. B: DKK3 mRNA levels after lentiviral-delivered overexpression (OE DKK3) compared with empty vector control virus (OE VEC) and lentiviral-delivered DKK3 specific shRNA (DKK3 shRNA) compared with scrambled control (SCR shRNA) as determined by qPCR 72h post-transduction of PrSCs (overexpression: n = 4; shRNA: n = 5). DKK3 gene expression levels were normalized using the housekeeping gene HMBS and are shown relative to controls. C: Secreted Dkk-3 protein levels in PrSCs after overexpression and knockdown of DKK3 (n = 4). D: DKK3-shRNA significantly reduced cellular proliferation of PrSCs determined by BrdU-incorporation ELISA at day 6 post-transduction (n = 5). E: DKK3 mRNA levels of PC3 (n = 3) and HT-29 cells (n = 3) compared with PrSCs (n = 5). F: Cellular proliferation of PC3 and HT-29 cells as determined by BrdU-incorporation ELISA at day 6 post-transduction with DKK3-shRNA compared with SCR-shRNA, respectively (n = 3). G: Western blot analysis of apoptosis-related proteins (BAX, phospho-p53) and the cyclin-dependent kinase inhibitors p27KIP1 and p21CIP1 in DKK3-shRNA and SCR-shRNA PrSCs 72 hr post-transduction. GAPDH served as loading control.
Mentions: Primary prostatic stromal cells (PrSCs) were used to investigate the functional significance of Dkk-3 in the stromal compartment in vitro. Consistent with the predominant expression of Dkk-3 in the epithelial compartment of the benign prostate 1, Dkk-3 was more abundant in cell lysates from primary prostatic epithelial cells (PrECs) than PrSCs at the protein level as determined by Western blot analysis (Fig. 1A), however PrSCs secreted Dkk-3 at significant levels (Fig. 1C).

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