br Results br Discussion In this study we

Results
Discussion In this study, we identified the existence of non-neuronal ACS in developing prostate epithelium. Activation of ACS promoted the proliferation of epithelial progenitor hif inhibitor and inhibited their differentiation to luminal cells. On the contrary, blockade of CHRM3 effectively inhibited the proliferation and promoted the differentiation of epithelial progenitor cells both in vitro and in vivo. In addition, we proved that ACS regulated prostate development and epithelial progenitor maintenance in a Ca2+/calmodulin-signaling-dependent manner. Importantly, we found that CHRM3 was upregulated in a large subgroup of BPH human prostate samples. We further demonstrated that activation of CHRM3 promoted BPH cell proliferation through Ca2+/calmodulin-signaling-mediated phosphorylation of AKT. Taken together, our study demonstrates an important role of non-neuronal ACS in the regulation of proliferation of prostate epithelial progenitor cells and BPH cells. Although several signaling pathways have been reported to regulate postnatal prostate epithelial development, including Notch, Hedgehog, Wnt, and FGF signaling (Shen and Abate-Shen, 2010; Wang et al., 2003, 2006b, 2008, 2015a), the present study add ACS to the list. On the one hand, similar to other pathways such as Wnt (Wang et al., 2008), activation of ACS increases the percentage of basal epithelial cells by promoting proliferation and inhibiting differentiation of basal cells. On the other hand, different from other pathways, activation of ACS also promotes the proliferation of luminal cells. We further explored the mechanism of ACS in regulating prostatic development and found that ACS likely promotes proliferation of epithelial cells via calcium-signaling-mediated phosphorylation of AKT. It would be interesting in future studies to determine whether there are interactions between the ACS and these other signaling pathways. It is important to point out that we performed ex vivo cell lineage tracing experiments with CK14-CreERT/Rosa26-RFP mice to study the differentiation of epithelial progenitor cells. Initially, activation of Cre by 4-OHT only labeled CK5+ basal cells. However, after 2 days in culture, there appeared RFP+CK5− differentiated luminal cells that originated from the RFP+ basal cells. Then we analyzed the percentage of RFP+CK5− cells over the total RFP-labeled cells under different culture conditions. We found that carbachol decreased the percentage of RFP+CK5− cells over the total RFP+ cells, indicating that carbachol inhibited the differentiation of epithelial progenitor cells. On the contrary, darifenacin and W-7 increased the percentage of RFP+CK5− differentiated cells. These findings reinforce the notion that ACS regulates the differentiation of prostate epithelial progenitor cells. BPH, an important clinical prostate disease, is a common prostatic disorder in elderly men. An estimated 40% of men aged ≥50 years and >90% of men aged ≥80 years have microscopic histopathological evidence of BPH (McNicholas and Kirby, 2011). However, at present, the cellular and molecular pathology of BPH is still poorly understood, and better clinical treatment is desired. In the present study, our findings provided a potential molecular mechanism for hyperplasia of the epithelial components of BPH. We found that CHRM3 was upregulated in a large subset of BPH samples. Activation of CHRM3 promoted BPH1 cell proliferation through promoting Ca2+/calmodulin-signaling-dependent phosphorylation of AKT. Together with our previous study reporting an involvement of ACS in prostate cancer growth and castration resistance (Wang et al., 2015b), these findings indicate that aberrant activation of ACS also promotes BPH cell proliferation and suggest a potential application of a selective CHRM3 antagonist in the treatment of prostate diseases, including not only prostate cancer but also BPH.
Experimental Procedures
Author Contributions