Our in vivo studies in intact and developed mammary glands demonstrate that mammary epithelial cells exhibit a relatively long half life, and that STAT5a activation suppresses their normal turnover and causes rapid cell expansion, alveolar differentiation, and persistent lactogenesis. Unexpectedly, the entire mammary gland can be replaced by the progeny of approximately 0.3% of the cells that were initially transduced with retrovirus expressing caSTAT5a. Importantly, caSTAT5a-induced differentiation and lactogenesis do not require ovarian functions, although the caSTAT5a-induced cell proliferation partly depends on ovarian hormones.
Traditional studies of cell turnover in the mammary gland have only provided the net gain or loss of all cells or a subset of cells in whole tissue. There are a paucity of tools for cell fate tracking of individual cells in intact mammary glands, although mosaic analysis with double markers (MADM) and other genetic recombination methods have been used to mark specific cells in other epithelial tissues. Our approach provides the opportunity to introduce genetic markers or potential cell fate regulators into individual ductal epithelial cells, so that the cell fate of individual cells in the context of a normal surrounding environment can be examined. With this approach, we demonstrated a slow turnover of the ductal epithelial cell population. Of note, the MMTV LTR, which directs the tva expression and thus the spectrum of the cells that are susceptible to infection in our mice, is most strongly active in differentiated ductal epithelial cells. While these more differentiated infected cells turn over relatively quickly; the less differentiated cell population that is not targeted by this virus may remain for a much longer time.
With this cell-tracking approach, we discovered a master role of STAT5a activation in driving both proliferation and alveolar differentiation of ductal epithelial cells. These results also reveal the extraordinary potential of ductal epithelial cells in proliferation and bipotential differentiation into both alveolar cells and myoepithelial cells. Our results are consistent with previous reports concluding an essential role for STAT5a in regulating mammary alveolar differentiation, and a new report that transplantation of mammary cells infected by a lentivirus expressing an activated version of STAT5 can induce alveoli-like expansion in cleared fat pads in a virgin host.
Approximately 0.3% of the mammary epithelial cells are infected in this viral system. This pool of cells diminished over time (based on the result using RCAS-β-actin), suggesting little progenitor potential among them and consistent with the characteristics of the MMTV promoter which defines the expression of tva and thus the cells susceptible to infection. However, this small pool of cells, after gaining caSTAT5a, expanded and replaced the entire mammary gland after one year. The positive staining of caSTAT5 was even observed in the basal layer. These data suggest that STAT5a activation can induce a bipotential progenitor potential in this RCAS-infected subset of relatively quiescent and differentiated mammary ductal epithelial cells; although the infected cells were likely heterogenous and only a subset of them might have the potential to undergo bipotential expansion and differentiation. Nevertheless, our results are consistent with previous reports of a role of STAT5 in regulating alveolar progenitors and stem cells, and provide a potential molecular mechanism for the generation of the so called “parity-induced mammary cells”, which have been found to have stem and progenitor properties.
Alveolar cells are reported to be produced and quickly turned over during the estrus cycle in virgin mice, and lactogenic cells undergo involution rapidly especially after the cessation of suckling. Therefore, it is a surprise that these caStat5-induced highly differentiated and lactogenic cells did not disappear over time, but rather expanded and persisted in virgin animals. This observation may be due to reported antiapoptotic roles of STAT5a, which transcriptionally activates Bcl2 and BclXL. Persistent STAT5a signaling may also interfere with STAT3 activation, which is known to occur at the end of lactation to cause mammary cell apoptosis through transcriptional activation of the genes encoding suppressor of cytokine signaling 3 (Socs3) and CCAAT/enhancer binding protein delta (C/EBPδ). Indeed, we did not detect activated STAT3 in these infected glands by immunohistochemical staining (Supplemental figure 6
), and nor did we detect an increase in the level of apoptosis over a low baseline level in the non-infected glands based on a TUNEL assay (data not shown). However, we do not know whether involution may activate STAT3 in these caSTAT5a
-expressing cells and cause them to undergo apoptosis if the infected mice were allowed to progress through a reproductive cycle.
Like prolactin-Jak2-STAT5 signaling, estrogen and progesterone signaling have also been linked to both mammary cell proliferation and differentiation. These pathways also appear to interact with each other. For example, acute treatment of ovariectomized mice with estrogen and progesterone has been shown to induce STAT5a expression; conversely, STAT5a can induce ER transcription. However, it was not previously known whether STAT5-regulated mammary cell proliferation or differentiation actually requires these ovarian hormones. Our results provide strong evidence that in the absence of any ovarian function, STAT5a activation is sufficient to cause ductal epithelial cells to undergo alveolar differentiation and lactogenesis as well as a modest increase in cell proliferation. However, ovarian hormones can significantly increase the proliferative response to STAT5a activation.
Transgenic overexpression of wild-type STAT5a or an activated version of STAT5a (which is a fusion of part of STAT5a with the transcriptional activation domain of STAT6 and the kinase domain of Jak2), or even a dominant-negative version of STAT5a has been reported to occasionally cause tumors with a long latency (8–12 months) after repeated reproductive cycles. Occasional tumors also arose after the first or second round of pregnancy and lactation in mice that have been transplanted with mammary cells ex vivo infected with a lentivirus expressing STAT5a(S711F). Only one tumor was observed in twenty RCAS-caSTAT5a-infected mice followed for over one year, and at necropsy two glands were found to have a focal hyperplasia based on whole mount and H&E staining. The rare progression of caSTAT5a+ cells in our study may be due to a smaller pool of STAT5a-activated cells, a different subset of cells with STAT5a activation, a different expression level of activated STAT5a, a different version of activated STAT5a, or the tumor-inhibitory effects of the surrounding normal and intact virgin ductal epithelium.
In conclusion, using a retrovirus-mediated in vivo gene expression approach in developmentally normal mammary glands, we discovered that the ductal epithelial cells in the adult virgins turned over slowly, that STAT5a activation leads to rapid cell expansion with concurrent alveolar differentiation and persistent lactogenesis, and that these effects are largely independent of ovarian functions.