p44 Functions in Prostate Epithelial Cells Figure 6. The growth and differentiation of prostate epithelial cells are temperature-dependent. (A) Temperature-dependent inactivation of the large T antigen (LT). Western blot of whole cell lysates derived from temperature sensitive LT prostate epithelial cells (LTts-ECs) grown at 33uC (lane 1) or 37uC (lanes 2–5) with anti-LTA or -actin antibody as indicated. (B) Non-permissive temperature (37uC) induced prostate epithelial cells to enter a quiescent state. Growth curves of LTAts-ECs at 33 or 37uC without or with additional androgen (DHT, 10 nM). One-way ANOVA analysis: F = 83.02, p = 0.0006, R square = 0.9722. The p value of unpaired Student’s t-Test of data between prostate epithelial cells at 33uC and 37uC or 37uC plus DHT is 0.0005 or 0.004. (C) The non-permissive temperature altered the cellular structure of prostate epithelial cells. Prostate epithelial cells were cultured at 33 or 37uC for 3 or 7 days and photographed. (D) P63 expression was lost when prostate cells were grown at 37uC. Western blot analysis of whole cell lysates (10 mg) made from prostate epithelial cells grown at 33uC (lane 1) or 37uC (lane 2) with anti-p63 (top) or b- actin (bottom) antibody. doi:10.1371/journal.pone.0049173.g006 P44 Nuclear Translocation is Essential for Differentiation of MPECs The p44 protein localized in the cytoplasm when LTts-MPECs were proliferating (at 33uC) (Fig. 8, panels g-i) and in the nucleus when LTts-MPECs were quiescent and differentiated (at 37uC; Fig. 8, panels j-l). Thus, similar to p44 in epithelial cells in the prostate gland, p44 nuclear translocation was associated with cell differentiation. Similar to that in prostate cancer cells, expression of NES-p44 in LTts-MPECs further enhanced the cells’ growth, whereas expression of NLS-p44 inhibited the growth of LTts- MPECs cultured at 33uC (Fig. 9B). To our surprise, expression of NES-p44 promoted growth of LTts-MPECs cultured at 37uC (Fig. 9B), indicating that p44 in the cytoplasm can drive the growth of quiescent epithelial cells. Expression of NES-p44 blocked temper- ature-induced p44 nuclear translocation (Fig. 8, panels m-o) and CK18 expression (Fig. 8, panels p-r). Thus, nuclear p44 is essential for differentiation of prostate epithelial cells. We used a BrdU incorporation assay to measure the prolifer- ation of LTts-MPECs (Fig. 9C). The percentages of BrdU-positive LTts-MPECs cultured at 33uC (83% 68%) were significant higher than those of LTts-MPECs cultured at 37uC (5% 61%), indicating that the loss of proliferation of LTts-MPECs at non-permissive temperature. Exogenous expression of NES-p44 significantly increased proliferation of LTts-MPECs cultured at both 33uC (95% 66%) and 37uC (66% 66%). In contrast, expression of NLS-p44 inhibited proliferation (22% 63%) of LTts-MPECs cultured at 33uC. These results suggest that the quiescence that occurred at 37uC was largely due to blockage of cell proliferation induced by p44’s translocation from the cytoplasm to the nucleus. Discussion We have demonstrated that cytoplasmic p44 is essential for proliferation whereas nuclear p44 is required for differentiation of MPECs. These findings suggest a novel mechanism by which p44 controls the proliferation and differentiation of epithelial cells during prostate development and tumorigenesis. The results of the present study suggest that p44 plays essential roles in cell proliferation in the prostate. Specifically, we found that p44 cytoplasmic localization was associated with the proliferation of MPECs during development. P44 is in abundance in the cytoplasm of prostate epithelial cells harvested from mice during the early stages of development, when cells are proliferating, and ts in growing immortalized temperature-sensitive lung epithelial cells (LT - MPECs). In contrast, p44 expression was limited in the nucleus of adult MPECs and of immortalized growth-arrested LTts-MPECs. In addition, we found that p44 expression was sufficient to promote proliferation of LT- immortalized growth-arrested LTts-MPECs at a non-permissive temperature. Finally, we found that deletion of the p44 gene abolished the growth of MPECs, and expression of NES-p44, but not NLS-p44, completely restored this growth deficiency. These results strongly indicate that cytoplasmic p44 is essential for the growth of prostate epithelial cells. During the development of a multicellular organism, cells proliferate for a defined length of time before they begin to differentiate functionally [1,2,3]. Terminal cell division must be precisely regulated to ensure that the proper numbers of differentiated cells are produced at the appropriate times. Although the control of proliferation and differentiation is highly coordinated, certain differentiation decisions are not compatible with continued proliferation. One essential component of these processes in the prostate gland is p44. The presence of p44 in the cytoplasm of prostate epithelial cells (PECs) during the early stages of prostate development is essential for their proliferation. When proper numbers of prostate epithelial cells are generated, p44 translocates into the nucleus, and proliferation stops. Nuclear p44 is an AR cofactor and drives expression of a set of AR target genes to promote PEC differentiation [17,18]. In the prostate, p44 plays roles in proliferation or differentiation, depending on its subcel- lular localization. We have also found that p44 is essential for epithelial cells to progress through the G1 phase [17,18]. The process of differentiation of primitive cells into more specialized cells involves an increasing restriction in proliferation capacity culminating in cell-cycle exit (34.35). Maintenance of cell-cycle arrest in terminally differentiated cells is important for the ultimate architecture and function of specific tissues [36,37,38]. Whether a cell will differentiate into one or another cell type is usually determined at the G1 phase of the cell cycle [36,39] Consistent with these studies, loss of p44 expression inhibited the proliferation of PECs and prostate cancer cells by means of arrest at the G1 phase. Our findings suggest that p44 also plays an important role in prostate cancer. Tumor cells do not differentiate normally and have an unlimited capacity to proliferate. It is likely that p44 exerts the same biological activities during tumorigenesis as it does during normal prostate development. We detected the cytoplasmic localization of p44 in prostate cancer samples [17,18], which suggests that cytoplasmic p44 has an important role in the proliferation of prostate cancer cells. Indeed, p44 silencing or expression of NLS-p44 dramatically inhibited prostate cancer cell growth in tissue culture and abolished the growth of prostate tumor xenografts in nude mice [17,18]. Thus, p44 cytoplasmic expression is also essential for prostate tumor growth. The molecular events that cause terminally differentiated PECs to re- enter the cell cycle remain unidentified; however, our findings suggest that the cytoplasmic translocation of p44 is essential to this process. Our findings also suggest that the p44 cytoplasmic translocation event is a novel target for the prevention and treatment of prostate cancer. However, the ways in which this developmental program is reactivated in prostate cancer cells remains unknown. Our current work is focused on identifying the regulatory networks controlling p44 subcellular translocation during prostate development, which may give us some clues about prostate tumorigenesis. PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49173