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+CD49fhigh (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.
Highlights
The Rho family of small GTPases are critical mediators that regulate a plethora of cellular processes including cellular polarity, motility, proliferation and apoptosis [1,2]
Since prostate epithelial cells are detached from their normal environmental cues and manipulated in suspension in this assay, we wondered whether the dissociation-induced, Rho/ ROCK activation-mediated actin-myosin hyperactivation would cause apoptosis of dissociated prostate stem cells, leading to an underestimation of the percentage of prostate stem cells
Our study demonstrates a role of the RhoA/ROCK signaling pathway in the apoptosis of murine prostate stem/progenitor cells upon dissociation from their environmental cues
Summary
The Rho family of small GTPases are critical mediators that regulate a plethora of cellular processes including cellular polarity, motility, proliferation and apoptosis [1,2]. Recent work by Ohgushi et al and Chen et al showed that this dissociation-induced apoptosis is due to the Rho-ROCK pathwaymediated actomyosin hyperactivation [6,7] This explains why the selective ROCK inhibitor Y-27632 is capable of increasing survival and cloning efficiency of dissociated single human embryonic stem cells [8]. It was reported that inhibition of Rho/ROCK pathway by Y-27632 enhances in vitro survival of mouse ES cell derived neural precursors [9], mouse intestinal stem cells [10] and human keratinocytes [11] These studies imply that dissociation-induced Rho/ROCK-mediated apoptosis is a common feature of stem/progenitor cells with an epithelial phenotype, irrespective of their embryonic layer origin
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