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. Author manuscript; available in PMC: 2013 Nov 2.
Published in final edited form as: Cell Stem Cell. 2012 Nov 2;11(5):676–688. doi: 10.1016/j.stem.2012.07.003

Notch and TGFβ Form a Reciprocal Positive Regulatory Loop that Suppresses Murine Prostate Basal Stem/Progenitor Cell Activity

Joseph M Valdez 1, Li Zhang 1, Qingtai Su 1, Olga Dakhova 2, Yiqun Zhang 3, Payam Shahi 2,3, David M Spencer 2,3, Chad J Creighton 3, Michael M Ittmann 2,3, Li Xin 1,2,3,4
PMCID: PMC3490134  NIHMSID: NIHMS396378  PMID: 23122291

Summary

The role of Notch signaling in the maintenance of adult murine prostate epithelial homeostasis remains unclear. We found that Notch ligands are mainly expressed within the basal cell lineage, while active Notch signaling is detected in both the prostate basal and luminal cell lineages. Disrupting the canonical Notch effector RBP-J impairs the differentiation of prostate basal stem cells and increases their proliferation in vitro and in vivo, but does not affect luminal cell biology. Conversely, ectopic Notch activation in adult prostates results in a decrease of basal cell number and luminal cell hyper-proliferation. TGFβ dominates over Notch signaling and overrides Notch ablation-induced proliferation of prostate basal cells. However, Notch confers sensitivity and positive feedback by up-regulating a plethora of TGFβ signaling components including TGFβRI. These findings reveal crucial roles of the self-enforced positive reciprocal regulatory loop between TGFβ and Notch in maintaining prostate basal stem cell dormancy.

Keywords: prostate stem cells, Notch, TGFβ, RBP-J

Introduction

The Notch pathway is evolutionarily conserved and is crucial for the proper development and homeostasis of a number of tissues in multicellular species (Kopan and Ilagan, 2009). Canonical Notch signaling involves activation of the Notch transmembrane receptor (Notch1–4 in mammals) by binding to one of its ligands (Dll1,3,4 and Jagged1,2 in mammals) which are expressed on the cell surface of adjacent cells. Upon binding, Notch receptor is sequentially cleaved by an ADAM family metalloprotease and the γ-secretase complex resulting in the release of the Notch intracellular domain (NICD). NICD translocates into the nucleus and binds the transcription factor RBP-J, releasing repressive cofactors and recruiting transcription machinery to activate RBP-J target genes.

Notch plays a complicated role during organogenesis and tissue homeostasis (Chiba, 2006). Notch activation increases the self-renewal capacity of hematopoietic, neural and muscle stem cells (Androutsellis-Theotokis et al., 2006; Conboy et al., 2003; Duncan et al., 2005), but induces differentiation of stem cells in skin and breast (Bouras et al., 2008; Okuyama et al., 2004). To add to the complexity, the biological consequence of Notch activation can be distinct at different developmental stages. For example, Notch activation suppresses the differentiation of murine intestinal stem cells in embryos, but inhibits secretory fates of intestinal stem cells in the adult (Stanger et al., 2005).

The majority of Notch-related studies in the prostate focused on its function during embryonic and neonatal stages. Developmentally, the prostate originates from a hindgut-derived endodermal tube termed urogenital sinus (UGS) (Staack et al., 2003). Prostatic morphogenesis starts at E15 in mice when the UGS epithelia (UGSE) protrude into the surrounding mesenchyme. The UGSE cells are the putative embryonic prostate epithelial stem cells and express both Keratin 5 and Keratin 8. Subsequently, the UGSE modules canalize to form lumens. UGSE cells undergo lineage commitment by selectively losing one of the keratins to form K8-expressing luminal cells surrounding the lumen and K5-expressing basal cells that reside between the basement membrane and luminal cells (Wang et al., 2001). Pharmacological suppression of Notch signaling in in vitro cultured UGS inhibits UGSE differentiation and blocks basal and luminal cell lineage commitment (Orr et al., 2009; Wang et al., 2006). Similar conclusions were made when Notch1 was ablated in neonatal prostate tissues (Wang et al., 2006). In another study, disrupting RBP-J in embryonic prostate epithelial cells caused defects in epithelial stratification and a reduction in basal cells (Wu et al., 2011).

The function of Notch in adult prostate epithelial homeostasis has not been as well characterized. Studies in recent years demonstrated that adult prostate basal and luminal cells are mainly self-sustained independently in vivo, but basal cells display prominent multi-potent stem cell activity in a transplantation-based regeneration assay (Burger et al., 2005; Choi et al., 2012; Lawson et al., 2007). Notch-1 expressing cells are indispensable for functional regeneration of adult prostate, which implies a potential role for Notch signaling in the regenerative capacity of prostate epithelia (Wang et al., 2004). We previously showed that Wnt signaling induces prostate basal cell proliferation by down-regulating Notch signaling (Shahi et al., 2011). Ectopic Notch activation has also been shown to result in a benign prostate hyperplasia phenotype in vivo (Wu et al., 2011). However, the expression pattern of Notch signaling components and the functional role of Notch in different prostate epithelial lineages in adults still remain understudied.

Prostate epithelial cells turn over very slowly. The quiescence of prostate epithelia is the overall net outcome of a variety of signaling networks that are often regulated by stromal-epithelial interactions (Marker et al., 2003). Among those signaling networks, TGFβ is one of the most extensively investigated signal transduction pathways. TGFβ has been shown to play a critical role in maintaining prostatic basal stem cell quiescence. Prostatic stem cells are enriched in the proximal region of the prostate that are anatomically adjacent to the urethra (Burger et al., 2005; Lawson et al., 2007). A large band of smooth muscle that secretes high levels of TGFβ envelops this proximal region (Nemeth and Lee, 1996). Salm et al. have shown that TGFβ can inhibit the proliferation of prostatic epithelial cells, and that the concentration and activity of TGFβ is higher in the proximal region, which contributes to the replicative quiescent nature of prostate stem cells (Salm et al., 2005). TGFβ signaling has been specifically disrupted in prostate epithelia. Surprisingly, no distinctive changes in cellular proliferation were observed when TGFβRII (Placencio et al., 2008) or Smad4 (Ding et al., 2011) were ablated in the prostate, while only a very mild increase in prostate epithelial proliferation was reported when a dominant negative TGFβRII was ectopically expressed (Kundu et al., 2000). This implies the existence of other signaling pathways that are redundant to TGFβ, among which Notch is one of them (Kluppel and Wrana, 2005). This study aims to investigate the role of Notch in adult prostate epithelial maintenance and differentiation, and to unveil how Notch and TGFβ coordinate together to regulate these biological processes.

Results

Expression of Notch signaling components in prostate epithelium

To gain insight into how Notch signaling is activated in the prostate, we determined the expression pattern of a comprehensive collection of Notch signaling components in prostate basal and luminal cell lineages by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. Individual adult murine prostate cell lineages were separated by fluorescence activated cell sorting (FACS) based on their surface antigenic expression profiles as described previously (Lawson et al., 2007)(Fig. 1A). QRT-PCR analyses showed that transcripts for the basal cell marker P63, the luminal cell marker Nkx3.1, and the stromal cell marker Vimentin were enriched in their corresponding cell fractions, demonstrating successful cell fractionation (Fig. 1B).

Fig. 1. Expression of Notch signaling components in prostate epithelia.

Fig. 1

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(A) FACS plot of prostate lineage fractionation. Prostate basal, luminal and stromal cells are Lin−Sca-1+CD49fHi, Lin−Sca-1−CD49fLow, and Lin−ScaI+CD49f-, respectively. (B) Quantitative RT-PCR analysis of markers for basal (P63), luminal (Nkx3.1), and stromal (Vimentin) cells in FACS-sorted prostate cell lineages. (C) Expression of Notch signaling components in prostate basal and luminal cell lineages. Data represent means and SD from 3 independent experiments. *: p < 0.05.

A pathway-focused PCR array was utilized to determine the expression of 84 Notch-signaling related genes in the prostate basal and luminal epithelial cell lineages (Fig. 1C). Notch1 and 2 are the most highly expressed receptors in both prostate basal and luminal cells. Jagged1 is the most highly expressed Notch ligand and is highly enriched in basal cells. Interestingly, although Delta1 and Jagged2 were expressed at lower levels they are also preferentially expressed in basal cells. Dll3 and 4 were undetectable (data not shown). Notch processing components such as ADAM metalloproteases and γ-Secretase complex members as well as signal modulators such as Numb, NCoR2, and the Fringe family members were widely expressed in both cell lineages. Lastly, downstream canonical Notch target genes Hes1, Hey1, and HeyL and the transcription factor Rbp-j were expressed in both basal and luminal compartments. Collectively, these results suggest that Notch signaling is active in at least a fraction of cells in both basal and luminal epithelial cells of the prostate, and that basal cells express ligands that may activate Notch signaling in adjacent basal and luminal cells.

Loss of Notch signaling induces basal cell expansion during regeneration

To investigate whether Notch signaling plays a role in the maintenance of adult murine prostate epithelial homeostasis, we ablated Notch signaling specifically in the prostate by breeding RBP-Jf/f mice (Han et al., 2002)(hereafter referred to as WT mice) with ARR2PB-Cre mice (Jin et al., 2003) to generate ARR2PB-Cre;RBP-Jf/f mice (hereafter referred to as KO mice). Using a fluorescence reporter-based strategy, we demonstrated that ARR2PB-Cre mediates homologous recombination in both luminal and basal epithelial cells in the prostate (Fig. S1A). Expression of Rbp-j in KO mouse prostates was decreased by half as determined by qRT-PCR (Fig. 2A). Genomic analyses of the floxed exon 6 of Rbp-j revealed approximately 30% deletion in both basal and luminal lineages (Fig. 2B). Consistently, qRT-PCR analyses also confirmed a 65% and 96%decrease in Rbp-j transcript in FACS -sorted KO prostate basal cells and in prostate spheres derived from KO mouse prostate basal cells, respectively(Fig. 2C). Collectively, these data demonstrate that Rbp-j was deleted in both the prostate basal and luminal cell lineages.

Fig. 2. Loss of Notch signaling induces basal cell expansion during regeneration.

Fig. 2

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(A) Analysis of Rbp-j transcript in WT and KO prostate by qRT-PCR. (B) Quantitative PCR analysis of the floxed exon 6 of Rbp-j in FACS-sorted basal and luminal cells of KO mice. (C) Analysis of Rbp-j transcript in FACS-sorted basal cells from WT and KO mice or from basal cell-derived prostate spheres. (D) Transillumination images of urogenital organs and H&E staining of prostates from WT and KO mice. (E–G) IHC analyses of K5 and P63 (E), Ki67 and P63 (F), and Ki67 and K14 (G) in WT and KO mouse prostates that have undergone two cycles of involution-regeneration. Bar graphs show quantifications. Data represent means and SD from 2 experiments per group (A–C) or 3 mice each group (E–G). *: p < 0.05. See also Figure S1.

No defects in morphogenesis, lineage composition and cellular turnover were observed in adult KO mice, suggesting that RBP-J mediated canonical Notch signaling is dispensable for the maintenance of prostate epithelial homeostasis (Fig. 2D and S1B–C). We then investigated whether Notch regulates lineage proliferation and differentiation during tissue regeneration. To induce extensive cellular turnover in the prostate, androgen was deprived and replaced for two cycles in adult WT and KO mice as described in the Supplemental Experimental Procedures. KO mouse prostates were able to involute and regenerate similarly to WT prostates in response to fluctuating serum testosterone levels, suggesting that RBP-J mediated Notch signaling is not essential for the regenerative capacity of prostate epithelia (Fig. S1D). Alternatively, recombination-escaped cells are sufficient to maintain the homeostasis and the regenerative potential of KO prostates.

However, we observed a 1.5- to 2-fold increase in the number of K5+ or P63+ basal cells in the KO mice (Fig. 2E). This expansion in basal cells is due to an increase in proliferation as measured by Ki67 (Fig. 2F–G), and not a decrease in apoptosis (data not shown). In contrast, there is only a slight but not statistically significant increase in proliferation in the P63− cells (Fig. 2F). This minor increase is probably due to the inclusion of the proliferation of P63− basal cells because no change in proliferation is observed in the non-basal cells when another more inclusive basal cell marker K14 was used in the analysis (Fig. 2G). Taken together these data demonstrate that in the absence of canonical Notch signaling basal cells over-proliferate during prostate regeneration.

Loss of Notch signaling results in large hyper-budded spheres

We employed an in vitro prostate sphere assay (Xin et al., 2007) to further investigate how Notch signaling regulates basal cell proliferation and differentiation. FACS isolated adult murine prostate basal cells were cultured in the prostate sphere assay with or without N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT), an inhibitor of the γ-Secretase complex. DAPT effectively suppressed the generation of NICD and the expression of Notch downstream targets Hes-1 and Hey-1 in prostate sphere cells (Fig 3A–B). Higher concentrations of DAPT are not able to further decrease NICD formation suggesting that this reduction may be maximal for this assay (Fig. S2A). Spheres treated with DAPT are larger and appear hyper-budded (Fig 3C and Fig S2B) (Shahi et al., 2011). In addition, prostate cells from RBP-J KO animals (Fig 3D) and prostate cells expressing a dominant negative mastermind-like protein (dnMAML) (Maillard et al., 2008) that blocks Notch signaling (Fig 3E and S2C) both show a similar sphere phenotype, confirming that the large hyper-budded phenotype is due to the suppression of Notch and not other DAPT targets. Lastly, overexpressing NICD in prostate spheres suppressed the DAPT-induced phenotype (Fig 3F and S2D). Collectively, these results suggest that the large hyper-budding phenotype is a direct consequence of inhibition of Notch signaling.

Fig. 3. Loss of Notch signaling results in large hyper-budded spheres.

Fig. 3

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(A) Western blot analysis of Notch intracellular domain (NICD) in vehicle- and DAPT-treated prostate spheres. (B) Quantitative RT-PCR analysis of Hes1 and Hey1 expression in prostate spheres in response to DAPT. (C–E) Transillumination images of DMSO vehicle- and DAPT-treated prostate spheres (C), prostate spheres derived from WT and RBP-J KO mouse cells (D), and prostate spheres expressing GFP and dnMAML (E). (F) ICN overexpression suppresses DAPT-induced hyper-budding phenotype. Bar graphs show quantifications. Data represent means and SD from 2 experiments per group. *: p < 0.05. See also Figure S2.

Suppressing Notch signaling increases the proliferation of prostate basal cells

Gene expression profiling of prostate spheres reveals that the cell cycle gene ontology group is significantly altered between vehicle- and DAPT-treated prostate spheres, of which several major targets were verified by qRT-PCR (Fig 4A–B and S3A). Cell cycle analysis confirmed that cells treated with DAPT were enriched in S and G2/M phases compared to the vehicle control (Figure 4C). To investigate the biological consequence of Notch activation in prostate basal cells in vivo, we bred ARR2PB-Cre mice with ROSA(N1IC) transgenic mice (Stanger et al., 2005) to express the constitutively active Notch1 intracellular domain in prostate epithelium (Fig. S3B). As shown in Fig. 4D, while Notch activation induces a hyperplastic growth of luminal cells, the number of basal cells is decreased, as quantified by FACS analysis in Fig. 4E. Ectopic expression of NICD in FACS isolated adult mouse prostate basal cells by lentiviral infection did not induce their luminal differentiation (Fig. S3C). This suggests that the skewed lineage composition in the ARR2PB-ROSA(N1IC) model is not due to a luminal fate bias of prostate stem cells by Notch activation. Instead, it suggests that Notch activation elicits distinctive growth outcomes in prostate basal and luminal cell lineages. Taken together these data suggest that Notch signaling negatively regulates the proliferation of prostate basal cells.

Fig. 4. Suppressing Notch signaling increases the proliferation of prostate basal cells.

Fig. 4

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(A) Heatmaps from microarray data show that DAPT treatment increases expression of genes promoting cell cycle and represses CDKIs. (B) Validation of expression changes of several representative genes in microarray analysis by qRT-PCR. (C) FACS plots of cell cycle analysis for DMSO vehicle- and DAPT-treated prostate sphere cells. Bar graphs show quantifications. Data represent means and SD from 2 independent experiments. *: p < 0.05. (D) Images of anterior and ventral prostate of ROSA(N1IC) and ARR2PB-ROSA(N1IC) mice stained with K5 (red) and K8 (green) antibodies. Note the decrease in K5+ cells in the ARR2PB-ROSA(N1IC) model. (E) FACS plots for analysis of lineage composition in ROSA(N1IC) and ARR2PB-ROSA(N1IC) mice. Data represent means and SD from 4 mice each group. *: p < 0.05. See also Figure S3.

Loss of Notch Signaling Inhibits Basal Stem Cell Differentiation

Cells in the outermost layers of prostate spheres express P63, possess a higher replating capacity, and represent more primitive progenitor cells (Xin et al., 2007). Surprisingly, DAPT-treated spheres express P63 throughout the entirety of the sphere (Fig 5A). We reasoned that inhibiting Notch signaling in prostate spheres not only increases their proliferation, but also attenuates their differentiation. QRT-PCR analysis confirmed that PSCA and AR, two markers for differentiation, were markedly decreased in the DAPT treated spheres (Fig 5B). Di-hydro-testosterone (DHT) has been shown to further induce differentiation of prostate sphere cells (Xin et al., 2007). DHT induces dilation of the sphere lumen, resulting in morphologically hollow prostate spheres (Fig. 5C). In addition, while in DHT-free prostate sphere culture most spheres are composed predominantly of K5+K8− cells (Type I), DHT increases the proportion of spheres that consist of nests of K5−K8+ cells in the center (Type II) or spheres that are mainly composed of K5−K8+ cells (Type III) (Fig. 5D). DAPT treatment effectively suppresses the formation of DHT-induced lumen formation and the formation of Type II and III spheres (Fig. 5C–D). These results suggest that inhibiting Notch signaling suppresses the differentiation of prostate basal stem/progenitor cells.

Fig. 5. Loss of Notch signaling inhibits differentiation.

Fig. 5

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(A) IHC analysis of P63 in control- and DAPT-treated prostate spheres. (B) QRT-PCR analysis of PSCA and AR in spheres treated with DAPT and DMSO vehicle. (C–D) Transillumination images of prostate spheres in the presence of DHT alone or DHT in combination with DAPT. Bar graphs show quantification of spheres with hollow and solid morphologies. (D) IHC analysis of K5 and K8 reveals three types of prostate spheres at different differentiation stages in vehicle and DAPT-treated sphere culture. Bar graphs show quantification. (E–F) bar graphs show quantification of sphere-forming units in DMSO- and DAPT-treated primary (E) and secondary (F) sphere culture. (G) Dot graph shows quantification of sphere-forming cells from single prostate spheres in DMSO- and DAPT-treated culture. Error bars in B–G represent means and SD from 2 independent experiments. *: p < 0.05. (H) IHC analysis of K5 and K8 in prostate tissues regenerated from WT and RBP-J KO prostate basal stem cells. See also Figure S4.

We then performed serial passaging experiments to functionally test whether inhibition of Notch signaling would increase the percentage of progenitor cells in prostate spheres. Treating dissociated primary prostate epithelial cells with DAPT did not increase the sphere-forming unit, indicating that DAPT treatment per se does not confer sphere forming activity (Fig. 5E). No significant differences in biology were noticed when higher concentrations of DAPT were used (Fig. S4). The control and DAPT-treated primary spheres were then dissociated and passaged in bulk in the absence of DAPT. DAPT-treated spheres generated more secondary spheres compared to the control spheres, suggesting that there are more sphere-forming cells in the DAPT-treated primary culture (Fig 5F). We also dissociated single spheres and passaged them separately and found that the average sphere-forming unit within single DAPT-treated spheres is approximately 3-fold higher than that of the control spheres (Fig 5G). These results demonstrate that ablating Notch activity results in the expansion of sphere-forming progenitor cells. Finally, FACS sorted prostate basal cells from WT and RBP-J KO mice were mixed with urogenital sinus mesenchyme and injected subcutaneously into SCID mice to regenerate prostatic tissues (Xin et al., 2003). The KO basal cells formed prostate glands containing more K5+ cells (Fig. 5H); further corroborating that Notch ablation induces expansion of basal cells in vivo during regeneration.

Notch inhibition attenuates but does not override TGFβ induced cytostasis

Since Notch ablation only induced prostate basal cell proliferation when basal cells were cultured alone in vitro or when they underwent regeneration due to changes in their environmental cues, we reasoned that Notch coordinates with signaling mediated by prostate stromal cells to regulate basal cell homeostasis. Our expression microarray data show that the TGFβ signaling pathway ontology group is significantly down-regulated in the DAPT-treated group, which was verified by qRT-PCR (Fig. 6A–B and S5A). TGFβ signaling plays a critical role in prostate epithelial quiescence (Salm et al., 2005) and its signaling strength is negatively regulated by androgen (Nantermet et al., 2004). Additionally, Notch and Tgfβ signaling have been shown to interact in regulating cellular quiescence in a variety of tissues (Blokzijl et al., 2003; Carlson et al., 2008; Niimi et al., 2007). Therefore, we hypothesized that Notch and TGFβ signaling coordinate together in regulating prostate basal cell homeostasis.

Fig. 6. Notch inhibition attenuates but does not override TGFβ-induced cytostasis.

Fig. 6

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(A) Heatmap from microarray analysis shows down-regulation of genes involved in TGFβ signaling. (B) Validation of expression changes of several representative genes in microarray analysis by qRT-PCR. (C) Inhibition of TGFβ signaling by SB431542 produces spheres similar to DAPT treatment. (D) Treatment of prostate spheres with TGFβ leads to a reduction in size which can be partially blocked by DAPT. (E) Sphere size is controlled by TGFβ in a dose-dependent fashion. KO cells are relatively resistant to TGFβ but also decrease in size in response to increasing TGFβ. (F) QRT-PCR analysis shows that expression of multiple CDKIs is induced by treatment with TGFβ which can be blocked by DAPT. (G) CDKI KO prostate spheres are differentially protected from the suppressive effects of TGFβ. (H) Quantitative RT-PCR shows that spheres treated with TGFβ have increased expression of Jag1, Hes1, and Hey1, which can be suppressed by DAPT treatment. (I) Smad4f/f prostate spheres infected with Cre adenovirus (AdCre) generate a heterogeneous mixture of spheres in response to Tgfβ as compared to cells infected with the control empty adenovirus (AdEmpty) which is suppressed to sizes under 50 μm. Large spheres express lower levels of Smad4, Jag1, Hes1, and Hey1 as determined by qRT-PCR. (J) Jag1f/f prostate spheres treated with AdenoCre mimic Smad4f/f spheres treated with AdenoCre and display reduced Jag1 and Hes1. Error bars represent means and SD from 3 independent experiments (B, E, F, and H) or 2 independent experiments (G, I, and J). *: p < 0.05. See also Figure S5.

Prostate sphere cells respond to TGFβ since prostate basal cells express both TGFβRI and TGFβRII (Fig. S5B). We found that both the Notch inhibitor DAPT and the TgfβRI inhibitor SB431542 caused the same hyper-budding phenotype in prostate sphere culture (Fig. 6C and S5C). No synergistic effect was noticed when both inhibitors were used (Fig. S5D). These results imply that Notch and Tgfβ act together in a linear pathway. DAPT-treated cells or RBP-J null cells are relatively resistant to the growth inhibitory effects of Tgfβ (Fig. 6D–E), suggesting that TGFβ signaling acts upstream of Notch signaling (Zavadil et al., 2004). Nevertheless, average sizes of KO prostate spheres decrease in response to TGFβ (Fig. 6E), which indicates that Notch is only one of the downstream effectors of TGFβ signaling, and that Notch inhibition is not sufficient to abolish the TGFβ-induced cytostatic program. This conclusion is also supported by our observation that ablating canonical Notch signaling in the prostate at physiological conditions does not override the homeostatic program safeguarded by TGFβ (Fig. 2D and S1B–C).

To determine the molecular mechanism through which loss of Notch attenuates TGFβ-induced cytostasis, we evaluated the expression of 5 CDKIs upregulated by TGFβ (Niimi et al., 2007) in control DMSO- and DAPT-treated prostate sphere cells. Upregulation of p15, p16, p19, and p21 by Tgfβ requires activation of Notch signaling, while p27 and cMyc do not seem to be affected (Fig. 6F). Similar changes were obtained using WT and KO prostate sphere cells though to a lesser extent (Fig. S5E). In order to determine the biological relevance of CDKI upregulation we performed prostate sphere assays using 4 available CDKI KO mice. While the resistance to Tgfβ signaling varied for prostate sphere cells from the different CDKI KO mice, it interestingly correlated with the qRT-PCR data where p16, which was most strongly upregulated, was also the most resistant KO line to Tgfβ signaling (Fig. 6G).

TGFβ has been shown to induce Jagged1 expression in mammary epithelial cells to activate downstream Notch signaling (Zavadil et al., 2004). As shown in Fig. 6H and S5F, TGFβ treatment induces the expression of Jagged1, Hes1, and Hey1 and this induction is suppressed by loss of Notch signaling. To determine whether Tgfβ induces Jagged1 expression through Smad4, we infected prostate basal cells from Smad4fl/fl mice with Cre adenovirus or the control empty adenovirus and evaluated their responses to Tgfβ in the sphere culture. As shown in Fig. 6I, while the empty adenovirus-infected cells formed predominantly small spheres with diameters less than 50μm, the size of spheres derived from Cre adenovirus-infected cells was heterogeneous, ranging from 30 to 200μm. The larger spheres adopted the same hyper-budded phenotype as those in the DAPT treated prostate sphere culture (Fig. 6I). QRT-PCR analysis of size-fractionated prostate spheres revealed that Smad4 is expressed at lower levels in bigger spheres (>50μm) compared to smaller spheres (<50μm). This result suggests that the heterogeneous sizes of prostate spheres reflect an incomplete infection of Smad4fl/fl cells by Cre adenovirus. The larger and smaller spheres were mainly derived from Smad4fl/fl cells that were infected and uninfected by Cre adenovirus, respectively. The expression levels of Jagged1 and downstream Notch signaling targets are also decreased in bigger spheres, indicating that Smad4 plays a critical role in Tgfβ-induced Jagged1 expression (Fig. 6I). Similarly, disrupting Jagged1 expression by infecting Jagged1fl/fl mouse prostate basal cells with Cre adenovirus also resulted in the formation of relatively bigger prostate spheres despite the presence of TGFβ (Fig. 6J), demonstrating the critical role of Jagged1 in Tgfβ induced cytostasis of prostate sphere cells.

Notch activation mediates a positive feedback loop by upregulating TGFβ ligands and receptors

Notch activation has previously been shown to induce the expression of Tgfβ ligands, proposing a positive feedback loop between Tgfβ and Notch signaling (Lee et al., 2007; Niranjan et al., 2008). Our results showed that Notch inhibition mitigates TGFβ-mediated signaling strength (Fig. 6A–B) and biology (Fig. 6D–E), possibly by abolishing this positive feedback. Additionally, overexpressing NICD in prostate spheres enhances Smad3 activation (Fig. 7A). We reasoned that treating prostate sphere cells with TGFβ would lead to positive feedback by upregulation of TGFβ signaling components through activation of Notch. As shown in Fig. 7B and Fig. S6A, qRT-PCR analyses demonstrate that treating prostate sphere cells with Tgfβ increases expression of not only Tgfβ1, but also additional components of the Tgfβ signaling pathway including Tgfβ3, TgfβR1, and LTBPs. However, these inductions were blocked in DAPT-treated cells where Notch signaling was abolished (Fig. 7B and S6A). Similar observations were made using RBP-J KO cells, suggesting that the induction of these TGFβ signaling components is RBP-J dependent (Fig. S6B). Western blot analysis further corroborated that DAPT treatment suppresses the induction of TgfβR1 in prostate spheres (Fig. 7C). There are several RBP-J consensus binding sites (G/CTGGGAA) (Zhao et al., 2006) in the TgfβR1 promoter (Fig 7D). ChIP analysis using RWPE cells, a human prostate epithelial cell line, demonstrates that NICD directly binds to 2 of these putative binding sites closest to the translational start codon, corroborating that TgfβR1 is a direct Notch target (Fig 7D). Additionally, a luciferase reporter assay showed that Notch intracellular domain enhances the transcriptional activity of a 3.5 kb fragment of TGFβRI promoter containing these two putative RBP-J binding sites and single nucleotide mutations in these consensus sites abolished reporter activity (Fig. 7E and S6C). These data demonstrate a positive feedback loop between Notch and Tgfβ and suggest that Notch and Tgfβ are redundant pathways that coordinate to safeguard basal cell homeostasis. To test this hypothesis, we perturbed both pathways by treating WT and Rbp-J KO mice with the TGFβRI inhibitor SB431542. While the percentage of basal cells in WT mouse prostate epithelia did not change in response to SB431542, the RBP-J null basal cells expanded by 2 fold (Fig. 7F). These results demonstrate that Notch serves not only as a downstream effector but also an amplifier for the TGFβ induced cytostatic program in basal cells and that these pathways overlap to inhibit basal cell proliferation in vivo (Fig. 7G).

Fig. 7. Notch activation mediates a positive feedback loop by upregulating TGFβ ligands and receptors.

Fig. 7

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(A) Western blot shows that pSmad3 expression is increased in prostate sphere cells overexpressing Notch1 intracellular domain (NICD). (B) Quantitative RT-PCR shows that Tgfβ ligands and receptors are increased in response to exogenous TGFβ. This induction can be suppressed by DAPT. (C) Western blot shows that DAPT suppresses TGFβ-upregulated TgfβRI. Bar graphs show quantifications. (D) ChIP analysis of the TgfβRI promoter demonstrates endogenous Notch binding at 2 RBP-J consensus sequences. (E) Luciferase reporter containing TgfβRI promoter region with two intact RBP-J binding sites is activated by ICN. Data in A–E represent means and SD from 2–3 independent experiments. *: p < 0.05. (F) IHC analysis of expression of K5 and K8 in anterior and ventral prostate lobes of WT and Rbp-j KO mice that have been treated with SB431542 or the control DMSO vehicle. Bar graphs show quantification. Data represent means and SD from 2–3 independent mice in each group. *: p < 0.05. (G) Schematic illustration of the reciprocal positive regulatory loop between TGFβ and Notch in prostate epithelia. See also Figure S6.

Discussion

The significance of a homeostatic prostate basal cell lineage

Our study revealed how Notch and TGFβ cooperate to regulate the homeostasis of the prostate basal cell lineage. This is important because deregulation of the basal cell lineage is a critical biological event during prostate cancer initiation and progression. A traditional view for the function of prostate basal cells is that they serve as barriers to protect luminal cells from oncogenic insults (El-Alfy et al., 2000). Loss of prostate basal cells is a diagnostic marker for prostate cancer. Basal cell loss could be partly due to suppression of proliferation by increased local TGFβ concentration during the progression of precancerous lesions. Our study showed that Notch is important for basal cells to aptly sense TGFβ signaling strength. It is tempting to hypothesize that suppressing Notch signaling may delay progression of PIN lesions by sustaining an intact basal cell layer. Alternatively, we recently demonstrated that prostate basal cells can also serve as the cellular origin for prostate cancer, which requires the differentiation of basal cells into luminal cells. (Choi et al., 2012). We showed here that although increased Notch activity is not sufficient to promote differentiation of basal cells into luminal cells, it does contribute to this process. Therefore, suppressing Notch signaling may also delay initiation of prostate cancer with a basal cell origin by inhibiting differentiation of basal cells into luminal cells. In summary, regardless of the role of basal cells in prostate cancer initiation, our study provides rationale for suppressing prostate cancer progression by targeting Notch signaling. Unfortunately, systemic and long-term Notch inhibition often causes intestinal toxicity and vascular neoplasms. Therefore, defining molecular and cellular mechanisms that dictate lineage-specific biological functions of Notch will inspire novel therapeutic avenues to target Notch signaling in a more cell lineage-specific manner.

Basal cells are in direct contact with each other in prostate spheres. In contrast, basal cells in mouse prostate are not always closely associated with each other and tend to form a punctuated layer. It has been shown that ligand/receptor cis-interactions inhibit, rather than activate, Notch signaling (del Alamo et al., 2011). Therefore, our conclusion from the in vitro sphere assay that TGFβ-induced cytostasis occurs partly through upregulation of Jagged1 may not be extended to the in vivo situation in mice. Nevertheless, human prostate basal cells form a continuous layer and adjacent basal cells are always in direct contact with each other. Therefore, our conclusions derived from the prostate sphere system may be adapted well in human prostate in vivo. Pertinent to this discussion, it has been observed that human and mouse prostate basal cells also differ by surface marker expression and possess differential proliferation indices. Therefore, it should be noted that not all conclusions derived from murine cells will apply to human cells.

The role of the reciprocal regulatory loop between Notch and TGFβ in prostate epithelial homeostasis

Crosstalk between Notch and TGFβ has been extensively studied in many organ systems (Blokzijl et al., 2003; Carlson et al., 2008; Lee et al., 2007; Niimi et al., 2007; Zavadil et al., 2004). Our study not only corroborated some of these findings, but also identified novel interactions between Notch and TGFβ in the prostate. Notch signaling acts downstream of the TGFβ program and is partially responsible for inhibiting proliferation of prostate basal cells. Furthermore, Notch signaling positively regulates multiple members of the TGFβ family and allows it to amplify the TGFβ-mediated cytostatic program by a positive feedback mechanism. Specifically, we identified TGFβR1 as a novel direct Notch target. Our findings suggest that this positive feedback loop increases the sensitivity of prostate basal cells to respond to TGFβ stimulation promptly, and fortifies the cytostatic effect of TGFβ signaling to balance the stimulatory signaling mediated by growth factors to safeguard basal cell homeostasis (Fig. 7G). This model nicely explains why a distinctive proliferating phenotype of the RBP-J KO prostate basal cells can only be observed in experimental models where TGFβ signaling is also diminished (Figs. 2E–G) or inhibited (Fig. 7F). In the sphere assay, TGFβ concentration is lower than that in vivo due to the absence of prostate stromal cells which are major sources of active TGFβs in vivo (Fig. S5B); in the castration-regeneration model, expression of TGFβ in the prostate is significantly reduced when androgen is replaced in castrated mice (Nantermet et al., 2004).

Paradoxically, in the muscle where TGFβ also promotes stem cell quiescence, Notch activation has an opposing role and can reverse this pathway, allowing myoblasts to proliferate and regenerate (Carlson et al., 2008). This suggests that the functional outcome of the interaction between Notch and TGFβ is tissue context dependent, which is in agreement with the fact that both SMADs and the Notch/RBP-J complex are promiscuous in joining with other complexes and are recruited to distinct genomic loci under different cellular contexts (Blokzijl et al., 2003). This may partially account for the tissue-dependent regulation of self-renewal of stem cells by Notch. Notch activation increases the self-renewal capacity of hematopoietic, neural and muscle stem cells, but induces differentiation of epithelial stem cells in skin, breast, lung, and thymic epithelia. A common feature of these epithelial stem cells and the prostate basal stem cells is that they all express P63, a transcription factor that maintains the proliferative potential of epithelial stem cells (Mills et al., 1999). Interestingly, P63 is negatively regulated by Notch at the transcriptional level through the interferon signaling pathway (Nguyen et al., 2006). In addition, P63 functionally antagonizes TGFβ during the epithelial-mesenchymal transition (Adorno et al., 2009). These facts suggest that the balance between the signaling mediated by P63, Notch and Tgfβ can be a major determinant for the overall biological outcome in those epithelial systems.

The multifaceted functions of Notch in different prostate epithelial lineages

Our study also revealed distinctive biological consequences of Notch activation in the prostate basal and luminal cell lineages. While Notch activation suppresses prostate basal cells, it induces proliferation in the luminal cell lineage. This cellular context-dependent biological function of Notch is conserved in the mammary gland. The mammary gland myoepithelial cells are anatomically and functionally reminiscent of the prostate basal cells. Suppressing Notch signaling in myoepithelial cells leads to their expansion, while ectopically activating Notch signaling in luminal cells results in hyperplastic growth (Bouras et al., 2008; Buono et al., 2006). Interestingly, Notch activation in mammary repopulating stem cells drives their commitment towards the luminal cell lineage (Bouras et al., 2008). This conclusion does not seem to directly carry over to the prostate. We showed that ectopic expression of NICD is not sufficient to induce differentiation of prostate basal cells into luminal cells (Fig. S3C). This is also consistent with our previous study showing that adult prostate basal and luminal cell lineages are mainly self-sustained independently (Choi et al., 2012). However, in vitro Notch plays a partial role during DHT mediated differentiation. In conclusion, our study demonstrated clearly that Notch activation confers distinct growth outcomes in the prostate basal and luminal cell lineages, but does not support a major role for Notch in cell fate choice for adult prostate basal stem cells in vivo and in vitro.

Though luminal cells express relatively high levels of Notch receptors, the machinery for Notch activation, and its downstream targets, we did not detect any changes in luminal cell biology when the Notch pathway was ablated. This result suggests that the contribution of Notch/Rbp-j signaling to prostate luminal development is completed before the activation of the probasin promoter. It is possible that Notch is only activated in a small fraction of adult luminal cells, some of which have escaped homologous recombination mediated by ARR2PB-Cre. The recombination-escaped cell population may be sufficient to maintain luminal epithelial homeostasis. Alternatively, RBP-J independent non-canonical Notch signaling (Heitzler, 2010) is still intact in the KO mice and may play an even more crucial, yet unknown, role in prostate homeostasis.

It was previously reported that human prostate transit-amplifying basal cells require Notch for their survival when cultured in low calcium medium in 2-dimensional culture (Litvinov et al., 2006). We found here that Notch is not essential for the survival of basal cells. These different results could be due to experimental conditions such as our 3-dimensional culture inside matrigel with a high-concentration of calcium, or transplantation in vivo. Under these experimental conditions, signals provided by growth factors in matrigel in vitro or in basement membrane in vivo may be redundant to Notch-mediated survival signaling, hence making Notch dispensable for survival. On the other hand, these previous studies suggested that calcium-induced E-Cadherin mediated interaction between transit-amplifying cells activates Notch signaling and promotes cell differentiation, which is in agreement with our conclusion that Notch activation suppresses prostate basal cell proliferation and induces differentiation.

Finally, Wu et al. recently also activated Notch signaling in the prostate using a PB-Cre4 model (Wu et al., 2011). Paradoxically, they observed an expansion of not only the luminal but also the basal cells. The potential mechanisms underlying the distinct phenotypes between the two studies are unknown. It is possible that the PB-Cre4 mouse model possesses higher stromal Notch signaling due to leaky activation of the PB promoter in the stromal layer (Wu et al., 2011), which indirectly confers survival and proliferative potential to basal cells.

Experimental Procedures

Mouse strains and procedures

The sources of experimental mice and the genotyping strategies are described in Supplementary Experimental Procedures. Castration, androgen replacement, dissociation of prostate, prostate sphere assay, and prostate regeneration assay were performed using standard techniques described previously (Xin et al., 2005; Xin et al., 2007). All of the mice were housed and bred under the regulation of The Center for Comparative Medicine at the Baylor College of Medicine.

Superarray and expression microarray

Analysis of Notch pathway members was performed on RT2 Profiler PCR Array Mouse Notch Signaling Pathway plates following the manufacturer’s instruction (SABiosciences, Frederick, MD). Expression microarray assays were performed using 4×44K Whole Mouse Genome Oligo Microarray chip (Agilent Technologies, Santa Clara, CA) and the Feature Extraction Software v9.1.3.1 (Agilent Technologies) was used to extract and analyze the signals. Array data have been deposited on GEO (GSE34067).

Pharmacological manipulation of Notch and TGFβ pathways

The γ-Secretase inhibitor N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) (Tocris Bioscience, Ellisville, MO) and the TgfβR antagonist 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) (Tocris Bioscience, Ellisville, MO) were dissolved in DMSO and used at a concentration of 5μM and 10μM in vitro, respectively. Cell culture media were replaced every 24 hours. For mouse studies, SB431542 (10mg/kg) or vehicle (DMSO) was injected i.p. every 24 hours for 10 days. Recombinant Tgfβ1 (R&D Systems, Minneapolis, MN) was reconstituted at 10μg/ml in sterile 4mM HCl with 0.1% BSA and stored at −80°C.

Western blot, histology and IHC analyses

Quantification of Western blots was performed by Odyssey infrared imaging system (Li-Cor Biosciences) or traditional chemiluminescent imagers. Paraffin embedded sections were stained and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO). Secondary antibodies were labeled with Alexa Fluro 488 or 594 (Invitrogen, Carlsbad, CA). Immuno-fluorescence staining was imaged using an Olympus BX60 fluorescence microscope or a Leica EL6000 confocal microscope. Cell counting was performed either manually or via ImagePro Software.

ChIP assay and TgfβRI promoter reporter assay

The chromatin immuno-precipitation (ChIP) was performed using the ChIP-IT Express kit (Active Motif, Carlsbad, CA).The luciferase reporter assay was performed using a 3.5 Kb genomic sequence upstream of the transcriptional start codon of the human TgfβRI. The procedures are described in detail in Supplemental Experimental Procedures.

Statistics

All experiments were performed using 2–10 mice in independent experiments. Data are presented as mean ± SD. Student’s t test was used to determine significance between groups. For all statistical tests, the 0.05 level of confidence was accepted for statistical significance.

Supplementary Material

01

Highlights.

  • Notch ablation results in prostate basal cell expansion during regeneration

  • Notch activation suppresses the prostate basal cell lineage in vivo

  • Notch induces lineage-specific biological outcomes in the prostate

  • A positive feedback loop between Notch and TGFβ maintains basal stem cell dormancy

Acknowledgments

We thank Dr. Tasuku Honjo for the Rbp-J conditional mice, Dr. Jon Aster for the dnMAML construct, the Cytometry and Cell Sorting Facility at the Baylor College of Medicine for technical support, Drs. Francesco DeMayo, Yi Li, Brendan Lee, and Michael Lewis for sharing mouse colonies, and Drs. Andy Groves and Jeffrey Rosen for critical comments. J.M.V. is supported by a Research Supplement to Promote Diversity in Health-Related Research (NIH CA125937S1). This work is supported by NIH R00CA125937 (L.X.), R01DK092202 (L.X.) and U01CA141497 (M.M.I.).

Footnotes

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