Wnt and Notch pathways have interrelated opposing roles on prostate progenitor cell proliferation and differentiation

Abstract

Tissue stem cells are capable of both self-renewal and differentiation to maintain a constant stem cell population and give rise to the plurality of cells within a tissue. Wnt signaling has been previously identified as a key mediator for the maintenance of tissue stem cells; however, possible cross-regulation with other developmentally critical signaling pathways involved in adult tissue homeostasis, such as Notch, is not well understood. By using an in vitro prostate stem cell colony ("prostasphere") formation assay and in vivo prostate reconstitution experiments, we demonstrate that Wnt pathway induction on Sca-1(+) CD49f(+) basal/stem cells (B/SCs) promotes expansion of the basal epithelial compartment with noticeable increases in "triple positive" (cytokeratin [CK] 5(+), CK8(+), p63(+)) prostate progenitor cells, concomitant with upregulation of known Wnt target genes involved in cell-cycle induction. Moreover, Wnt induction affects expression of epithelial-to-mesenchymal transition signature genes, suggesting a possible mechanism for priming B/SC to act as potential tumor-initiating cells. Interestingly, induction of Wnt signaling in B/SCs results in downregulation of Notch1 transcripts, consistent with its postulated antiproliferative role in prostate cells. In contrast, induction of Notch signaling in prostate progenitors inhibits their proliferation and disrupts prostasphere formation. In vivo prostate reconstitution assays further demonstrate that induction of Notch in B/SCs disrupts proper acini formation in cells expressing the activated Notch1 allele, Notch-1 intracellular domain. These data emphasize the importance of Wnt/Notch cross-regulation in adult stem cell biology and suggest that Wnt signaling controls the proliferation and/or maintenance of epithelial progenitors via modulation of Notch signaling.

Figure 1

Lin⁻Sca1⁺CD49f⁺ cells (LSCs) cultured in Matrigel. (A): Induction of the Wnt pathway via Wnt3a-conditioned media promotes stabilization and nuclear localization of β-catenin. Arrow, nuclear β-catenin. Scale bar = 25 µm. (B): LSCs cultured in Matrigel for 10 days. Wnt induction leads to increase in the number of p63⁺ cells. Scale bar =100 µm. (C): p63, red; cytokeratin 5 (CK5), blue; and CK8, green staining of prostaspheres. Arrowheads, triple positive cells. Scale bar = 50 µm. (D): Prolonged Wnt pathway induction (15 days) promotes enlargement of prostaspheres. Scale bar = 100 µm. Abbreviations: CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; L-CM, L cell-conditioned media; Wnt3a-CM, Wnt3a-conditioned media.

Figure 2

Lin⁻Sca1⁺CD49f⁺ cells cultured in Matrigel. (A): Bright-field image of prostasphere infected with either control lentivirus (LV) or LV-mediated transduction of constitutively active β-catenin. Scale bar = 100 µm. (B): Analysis of β-catenin expression via immunostaining. Scale bar, 25 µm. (C): β-Catenin, green; p63, red; DAPI, blue. Scale bar = 25 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; LV, lentivirus; β-Cat*-LV, LV-mediated transduction of constitutively active β-catenin.

Figure 3

Analysis of Lin⁻Sca1⁺CD49f⁺cell (LSC) grafts for β-catenin and prostate epithelial markers. (A): β-Catenin immunostaining. Arrow, nuclear β-catenin. Scale bar = 50 µm. (B): p63 immunostaining. Arrows, p63⁺ cells in control acini. Graph: average of four fields. Scale bar =100 µm. (C): LSC grafts stained with cytokeratin 5 (CK5) and CK8 antibodies. Arrows, CK5/CK8 double positive cells. Scale bar = 50 µm. Abbreviations: CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; LV, lentivirus; β-Cat*-LV, LV-mediated transduction of constitutively active β-catenin.

Figure 4

qRT-PCR analysis of Wnt3a-induced Lin⁻Sca1⁺CD49f⁺ cells (LSCs). Proliferation analysis of control and lentivirus-mediated transduction of constitutively active β-catenin (β-cat*-LV)-infected LSC grafts. (A): qRT-PCR analysis of selected Wnt target genes. (B): qRT analysis of epithelial-to-mesenchymal transition (EMT)-associated candidate genes. (C): qRT analysis of Wnt target genes and candidate genes involved in EMT in LSCs expressing β-Cat*-LV. (D): Prostasphere colony formation analysis. LSCs were cultured in two separate experiments each containing four replicates. (E): Immunostaining of LSC grafts with Ki-67 and analysis of Ki-67⁺ cells in control and β-cat*-LV-infected grafts. Scale bar = 100 µm. Abbreviations: β-cat*-LV, lentivirus-mediated transduction of constitutively active β-catenin; LSCs, Lin⁻Sca1⁺CD49f⁺ cells.

Figure 5

Analysis of Notch1 mRNA and protein expression in Lin⁻Sca1⁺CD49f⁺ cell (LSC)-derived prostaspheres. Manipulation of the Notch pathway in LSCs. (A): Real-time analysis of Notch1 and Jagged1. Real-time data was obtained from three separate in vitro Matrigel experiments. Individual qRT-PCR experiments were performed in triplicate. (B): Western blot analysis of control and Wnt-induced LSC protein fractions via anti-Notch1 antibody. Antibody detects cleaved Notch1 fragment. (C): Notch1 immunostaining in prostaspheres. Scale bar = 50 µm. (D): LSCs infected with control lentivirus (LV) or LV-encoding the intracellular domain of Notch1 (NICD-LV) cultured in Matrigel. Arrow, NICD-LV-infected cells. Two separate experiments, each repeated in triplicates were performed to accumulate Notch1 induction data. Scale bar = 100 µm. (E): Inhibition of Notch signaling in LSCs using 5 µM DAPT. Notch pathway inhibition was repeated in three separate experiments, and each experiment was performed in triplicates. (F): Immunostaining of control and DAPT-treated prostaspheres. DAPI, blue; p63, red. Scale bar = 100 µm. (G): H&E stain of LSC transplants using control LV or NICD-LV-infected LSCs. Scale bar = 100 µm. (H): Immunostaining of LSC transplants using control LV or NICD-LV-infected LSCs. Arrows, nuclear staining of NICD. Scale bar = 50 µm. Abbreviations: DAPT, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester; DAPI, 4′,6-diamidino-2-phenylindole; NICD-LV: LV-encoding the intracellular domain of Notch1; RFP: red fluorescence protein; Wnt3a-CM, Wnt3a-conditioned media.