p44 Functions in Prostate Epithelial Cells Figure 5. Nuclear p44 promotes differentiation of prostate epithelial cells. (A) Cytoplasmic p44 is required for proliferation of prostate epithelial cells. P44-null prostate epithelial cells infected with control lentivirus or lentivirus expressing p44, NES- p44, or NLS-p44 were allowed to grow in the presence of BrdU and immunostained with an anti-BrdU antibody (shown brown). (B) Nuclear p44 enhanced CK18 expression. P44-null prostate epithelial cells infected with control lentivirus or lentivirus expressing p44, NES-p44, or NLS-p44 were immunostained for CK18. (C) Nuclear p44 (NLS-p44) enhanced expression of prostate-secreted proteins by quantitative RT-PCR analysis of expression of prostate-secreted proteins. Total RNAs were isolated from p44-null prostate epithelial cells infected with control lentivirus (Og2) or lentivirus expressing p44, NES-p44, or NLS-p44 and amounts of RNAs were measured by real-time PCR. Relative expression indicated specific mRNA levels divided by b-actin mRNA levels. One-way ANOVA analysis: for PSP94 gene, F = 1,297, p,0.0001, R square = 0.9870; for probasin gene, F = 1,198, p,0.0001, R square = 0.9965; for Calr gene, F = 1,140, p,0.0001, R square = 0.9938; for PDI gene, F = 1,149, p,0.0001, R square = 0.9927; for PLCa gene, F = 1,385, p,0.0001, R square = 0.9917; and for SBP gene, F = 1,203, p,0.0001, R square = 0.9974. The p value of unpaired Student’s t-Test of data between Og2 and NES-p44 or NLS-p44 epithelial cells: 0.0103 or ,0.0001 (PSP94), ,0.0001 or ,0.0001 (Probasin), 0.0381 or ,0.0001 (Calr). 0.0005 or ,0.0001 (PDI-1), 0.126 or ,0.0001 (PLCa), and 0.5019 or ,0.0001 (SBP). doi:10.1371/journal.pone.0049173.g005 To examine cell proliferation, we treated mice with a bromodeoxyuridine (BrdU) pulse for 3 days to label proliferating cells. DLP sections were prepared, and cells that had BrdU incorporation (i.e., cells in S-phase) were identified by immuno- histochemistry with an anti-BrdU antibody (Fig. 2A). Proliferating epithelial cells were quantified (Fig. 2B), and this quantification showed that the proliferation rate of epithelial cells was dramatically decreased during mouse development between 7 and 60 days after birth. The proliferation rate reached a low level (0.8–1.0%) in mice 60 days old and stayed at this level until 510 days. Thus, there is a good correlation between p44 nuclear translocation and decreased proliferation rate in prostate epithelial cells. Cytoplasmic p44 is Essential for the Growth of Prostate Epithelial Cells Epithelial cells were isolated from the whole prostate glands of the mice carrying a floxed (loxP-flanked) p44 gene locus (p44loxp/ loxp) at the age of 21 days [18] and then immortalized with large T antigen (Fig. 3A). The immortalized mouse prostate epithelial cells (MPECs) expressed CK5, CK18 (Fig. 3B), AR, p63, and p44 (Fig. 3C). MPECs were then infected with control adenovirus (Ad- GFP) or adenovirus harboring the Cre recombinase (Ad-Cre) to delete the p44 gene. Western blotting and immunostaining indicated loss of p44 expression in MPECs upon infection with Ad-Cre (Fig. 4A, lane 1 versus lane 5; Fig. 4B, 1st versus 2nd panels). Expression of p44 could be partially restored by infecting p44-null MPECs with lentivirus harboring the p44 cDNA (Fig. 4A, lane 2; Fig. 4B, 3rd panel). The p44 protein mainly localized to the cytoplasm in MPECs (Fig. 4B, top and 3rd panels). Deletion of the p44 gene completely abolished MPEC growth (Fig. 4C, blue line) and resulted in complete cell death within 2 weeks. Exogenous expression of p44 (Fig. 4A, lane 2; Fig. 4B, 3rd panel) or nuclear exclusion signal-tagged p44 (NES-p44, localized in the cytoplasm) [18,19] (Fig. 4A, lane 3; Fig. 4B, 4th panel) restored growth of the p44- MPECs (Fig. 4C, pink and yellow lines, respectively). In contrast, expression of nuclear localization signal (NLS)-tagged p44 (NLS-p44) [18,19] (Fig. 4A, lane 4; Fig. 4B, 5th panel) did not have much effect on the growth of p44- MPECs (Fig. 4C, green line). Dual cytoplasmic and nuclear localization of the NLS-p44 protein in some MPECs may explain a slight increase in cell numbers of NLS-p44-expressing p44-null MPECs. Thus, cytoplasmic p44 is essential for growth of prostate epithelial cells. We used a BrdU incorporation assay to measure the prolifer- ation of MPECs (Fig. 5A). The percentages of BrdU-positive p44loxP/loxP MPECs (83% 67%) were much higher than those of p44-null MPECs (2.5% 61%), indicating that the loss of p44 expression inhibited the proliferation of MPECs. Exogenous expression of p44 (78% 68%) or NES-p44 (82% 66%) restored proliferation of p44-null MPECs, but expression of NLS-p44 only slightly enhanced it (5% 61%). Thus, cytoplasmic p44 is essential for proliferation of prostate epithelial cells. Nuclear p44 Promotes Cell Differentiation To directly test the role of nuclear p44 in MPECs, we expressed NLS-p44 to force p44 nuclear translocation in the p44-null MPECs (Fig. 4B, 5th panel). NLS-p44 expression dramatically increased expression of CK18 (Fig. 5B) and six prostate secretory proteins (Fig. 5C). In contrast, expression of NES-p44 inhibited expression of these proteins (Fig. 5C). These data confirm that nuclear p44 is required for the differentiation and functionality of prostate epithelial cells. NLS-p44 also extended the survival time (.30 days) of the p44-null MPECs in tissue culture (Fig. 4C). Proliferation and Differentiation of Prostate Epithelial Cells in a Temperature- Dependent Manner The transgenic mouse strain H-2Kb-tsA58, whose tissues harbor a temperature-sensitive simian virus 40 (SV40) large tumor (LT) antigen, has been used to isolate and propagate primary cells from various organs for long-term culture [32,33]. We used this mouse strain to generate a culture of mouse prostate epithelial cells. LTts- MPECs were isolated from the whole prostate of mouse at the age of 21 days and cultured under permissive temperature (33uC), that is, a temperature at which the LT antigen was functional (Fig. 6A, lane 1); the LTts-MPECs were immortal and grew normally in tissue culture with a doubling time of about 20 hr (Fig. 6B). Transfer of the cultured LTts-MPECs to non-permissive temper- ature (37uC) resulted in the induction of a quiescent state due to inactivation of the LT antigen (Fig. 6A, lanes 2–5; Fig. 5B). Additional dihydrotestosterone (DHT) added into the medium further enhanced the induction of the quiescent state (Fig. 6B). Cell sizes increased when LTts-MPECs were grown at 37uC (Fig. 6C). The basal cell marker p63 was expressed in LTts-MPECs cultured at 33uC but not in those grown at 37uC (Fig. 6D). Almost all the LTts-MPECs (.95%) expressed epithelial cell markers AR and CK18 (Fig. 7, white arrowheads in panels b and c indicate a cell without CK18 expression). Androgen-driven AR nuclear translocation was observed in LTts-MPECs (Fig. 7, panels j-l versus panels g-i). The cultured LTts-MPECs also expressed low levels of the basal cell marker CK5 (Fig. 7, 2nd panel). Thus, the isolated LTts-MPECs resembled prostate epithelial cells at the intermediate differentiation stage. The dramatic increase in CK18 expression in LTts-MPECs cultured at 37uC (Fig. 8, panels d-f) relative to those cultured at 33uC (Fig. 8, panels a-c) indicated differentiation of the LTts- MPECs. Expression of five prostate secretory proteins [34] increased in LTts- MPECs cultured at 37uC compared to those grown at 33uC (Fig. 9A), also indicating the functional differen- tiation of LTts-MPECs cultured at 37uC. PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49173