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ABSTRACT: 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 in basal cell number and luminal cell hyperproliferation. TGFβ dominates over Notch signaling and overrides Notch ablation-induced proliferation of prostate basal cells. However, Notch confers sensitivity and positive feedback by upregulating a plethora of TGFβ signaling components including TgfβR1. These findings reveal crucial roles of the self-enforced positive reciprocal regulatory loop between TGFβ and Notch in maintaining prostate basal stem cell dormancy.
Cell stem cell 11/2012; 11(5):676-88. · 23.56 Impact Factor
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ABSTRACT: Keratin 8 (K8) is a type II keratin that is associated with the type I keratins K18 or K19 in single layered epithelia. We generated a bacterial artificial chromosome (BAC) transgenic mouse line that expresses the tamoxifen inducible CreER(T2) inserted into the endogenous murine K8 gene. The transgenic mouse line contains two copies of the BAC transgene. To determine the expression specificity and inducibility of CreER(T2), the K8-CreER(T2) mice were bred with a Gt(ROSA 26)( ACTB-tdTomato-EGFP ) fluorescent protein-based reporter transgenic mouse line. We demonstrated that CreER(T2) and the endogenous K8 gene share the same patterns of expression and that the enzymatic activity of CreER(T2) can be efficiently induced by tamoxifen in all K8-expressing tissues. This mouse line will be useful for studying gene function in development and homeostasis of simple epithelia, and investigating both tissue lineage hierarchy and the identity of the cells of origin for epithelial cancers.
Transgenic Research 02/2012; 21(5):1117-23. · 2.75 Impact Factor
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ABSTRACT: The prostate epithelial lineage hierarchy and the cellular origin for prostate cancer remain inadequately defined. Using a lineage-tracing approach, we show that adult rodent prostate basal and luminal cells are independently self-sustained in vivo. Disrupting the tumor suppressor Pten in either lineage led to prostate cancer initiation. However, the cellular composition and onset dynamics of the resulting tumors are distinctive. Prostate luminal cells are more responsive to Pten null-induced mitogenic signaling. In contrast, basal cells are resistant to direct transformation. Instead, loss of Pten activity induces the capability of basal cells to differentiate into transformation-competent luminal cells. Our study suggests that deregulation of epithelial differentiation is a critical step for the initiation of prostate cancers of basal cell origin.
Cancer cell 02/2012; 21(2):253-65. · 25.29 Impact Factor
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ABSTRACT: Activation of the RhoA/ROCK signaling pathway has been shown to contribute to dissociation-induced apoptosis of embryonic and neural stem cells. We previously demonstrated that approximately 1 out of 40 Lin(-)Sca-1(+)CD49f(high) (LSC) prostate basal epithelial cells possess the capacities of stem cells for self-renewal and multi-lineage differentiation. We show here that treating LSC cells with the ROCK kinase inhibitor Y-27632 increases their cloning efficiency by 8 fold in an in vitro prostate colony assay. Y-27632 treatment allows prostate colony cells to replate efficiently, which does not occur otherwise. Y-27632 also increases the cloning efficiency of prostate stem cells in a prostate sphere assay and a dissociated prostate cell regeneration assay. The increased cloning efficiency is due to the suppression of the dissociation-induced, RhoA/ROCK activation-mediated apoptosis of prostate stem cells. Dissociation of prostate epithelial cells from extracellular matrix increases PTEN activity and attenuates AKT activity. Y-27632 treatment alone is sufficient to suppress cell dissociation-induced activation of PTEN activity. However, this does not contribute to the increased cloning efficiency, because Y-27632 treatment increases the sphere-forming unit of wild type and Pten null prostate cells to a similar extent. Finally, knocking down expression of both ROCK kinases slightly increases the replating efficiency of prostate colony cells, corroborating that they play a major role in the Y-27632 mediated increase in cloning efficiency. Our study implies that the numbers of prostate cells with stem/progenitor activity may be underestimated based on currently employed assays, supports that dissociation-induced apoptosis is a common feature of embryonic and somatic stem cells with an epithelial phenotype, and highlights the significance of environmental cues for the maintenance of stem cells.
PLoS ONE 01/2011; 6(3):e18271. · 4.09 Impact Factor
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ABSTRACT: Dicer is an RNase III enzyme essential for microRNA maturation. Dicer ablation in diverse tissues has been shown to block tissue differentiation, induce cell apoptosis, impair specialized cellular function, and perturb organ structures. To gain insight into the role of microRNAs in prostate tissue function and homeostasis, we conditionally disrupted Dicer activity in the mouse prostate using an ARR2PB-Cre. We demonstrated that Dicer activity is disrupted in both prostatic basal/stem cells and differentiated luminal cells. Dicer knockout murine prostates are smaller in size and mass and develop epithelial hypotrophy in ventral prostates by 4 months. Dicer ablation induces increased apoptosis in the prostate, predominantly in the differentiated luminal cells. Paradoxically, a concurrent increase in proliferation is observed in both basal/stem cells and luminal cells, presumably due to compensatory growth of the cells devoid of homologous recombination in response to the elevated cellular apoptosis. We have previously shown that Lin(CD31CD45Ter119)(-)Sca-1(+)CD49f(high) (LSC) cells enrich for prostate stem cell activity. Through proliferation and differentiation, some LSC cells are capable of forming prostate spheres composed of cells at various stages of differentiation. Although LSC cells were expanded by threefold in Dicer knockout mice, the sphere-forming units of Dicer knockout prostate cells decreased by more than half compared with wild-type cells. In addition, most prostate spheres in the Dicer knockout culture were derived from cells that did not undergo homologous recombination. Our results demonstrate a critical role of microRNAs for the proliferative capacity of prostate stem cells and the maintenance of prostate homeostasis.
Stem Cells 07/2010; 28(7):1260-9. · 7.78 Impact Factor
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ABSTRACT: miRNAs are a class of naturally occurring small RNAs that generally repress gene expression. They have been shown to actively control diverse biological processes including stem cell differentiation and lineage commitment.
Fluorescence-activated cell sorting was utilized to isolate murine prostate stem cells and differentiated luminal cells. The expression levels of Drosha and Dicer1, the two key RNAseIII enzymes for miRNA maturation, were evaluated by quantitative RT-PCR. Low-density Taqman miRNA array analyses were also performed to identify miRNAs that are differentially expressed in individual lineages.
Drosha and Dicer1 are expressed at comparable transcriptional levels in murine prostate stem cells and differentiated luminal cells. Twenty-nine miRNAs were discovered to be differentially expressed in prostate stem cells and luminal cells. Many of these miRNAs are coded in clusters, suggesting a cell-specific transcriptional regulation. Some of these differentially expressed miRNAs have been reported to regulate genes relevant to the molecular and phenotypic features of each lineage.
miRNAs may play a potentially critical role in fine regulation of prostatic lineage identity.
The Prostate 10/2009; 70(3):297-304. · 3.48 Impact Factor
