br Experimental Procedures br Author

Experimental Procedures
Author Contributions
Acknowledgments We thank Cory Abate-Shen, Maho Shibata, and Flaminia Talos for insightful comments on the manuscript. This work was supported by NIHR01DK076602. A.M. and R.T. were supported by post-doctoral fellowships from the DOD Prostate Cancer Research Program.
Introduction Regulation of proliferation and differentiation of prostate epithelial progenitor cells is important for the prostate development process of branching morphogenesis and prostate diseases. In the normal prostate, there are three epithelial cell types: basal cells (p63+ and CK5+), luminal cells (CK8+), and neuroendocrine cells (Synapthophysin+) (Leong et al., 2008). Even though several studies have identified the multipotent potential of luminal cells under castration conditions (Wang et al., 2009), in organoid cultures or in renal capsule recombination assays (Chua et al., 2014; Karthaus et al., 2014), it is also believed that multipotent epithelial progenitor cells are located in the basal cell compartment (Goldstein et al., 2010; Lawson et al., 2007; Leong et al., 2008; Ousset et al., 2012), which can differentiate into not only basal cells but also luminal cells and neuroendocrine cells. Previous studies, including the work from our group (Shou et al., 2001; Wang et al., 2003, 2006b, 2008), have shown that various signaling pathways can regulate epithelial progenitor cell proliferation and differentiation and, in turn, affect the formation of prostate diseases (Shen and Abate-Shen, 2010). However, whether or not there are additional important molecules that regulate prostate epithelial progenitor cells is still unclear. Muscarinic receptors belong to the family of G-protein coupled receptors. There are five members: CHRM1–CHRM5 (Spindel, 2012). Activation of muscarinic receptors by adrenergic receptors usually stimulates Ca2+ influx, glandular secretion, and smooth muscle contraction (Wessler and Kirkpatrick, 2012). It was traditionally believed that acetylcholine was predominantly synthesized in the neuronal system. However, besides the neuronal cholinergic system, there is also a widespread cholinergic system in non-neuronal tissues, which has been identified in airway epithelial cells, hematopoietic stem cells, small intestine epithelial cells, colon epithelial cells, mesenchymal stem cells, and embryonic stem cells (Wessler and Kirkpatrick, 2012). The non-neuronal cholinergic signaling functions in regulating the differentiation and proliferation of embryonic stem cells (Landgraf et al., 2010), hematopoietic stem cells (Serobyan et al., 2007), and small intestine stem cells (Takahashi et al., 2014). In particular, our previous study demonstrated a role of autocrine cholinergic signaling (ACS) in promoting prostate cancer growth and castration resistance (Wang et al., 2015b). However, whether or not such ACS also plays a role in regulating proliferation and differentiation of prostate epithelial progenitor cells is unknown. In the present study, we discovered the existence of ACS in the developing mouse epithelium. We used an organotypic culture system, which is free of functional nerve fibers, to study the roles of ACS in regulating prostate development. We further confirmed the results by lineage tracing and renal capsule tissue recombination assay. We found that activation of ACS promoted prostate development by enhancing the proliferation of epithelial progenitor cells and preventing these progenitor cells from differentiation. In addition, we demonstrated that these effects were achieved through Ca2+/calmodulin signaling, which could be blocked or reversed by a specific calmodulin inhibitor, W-7. More importantly, we found that CHRM3 was upregulated in a large subset of BPH tissues compared with normal tissues. ACS promoted BPH cell proliferation through Ca2+/calmodulin-signaling-mediated phosphorylation of AKT. Taken together, our findings identify ACS as another important component that keeps prostate epithelial progenitor cells in the proliferating state, and blockade of ACS may have clinical implications for the management of BPH.