Estrogen-triggered delays in mammary gland gene expression during the estrous cycle: evidence for a novel timing system.

During the estrous cycle and beginning in estrus, the mammary gland undergoes pregnancy-like development that depends on transcriptional regulation by the estrogen and progesterone receptors (ER, PR) and Pax-2 as well as the action of the growth factors Wnt-4 and RANKL. In this report, we first describe the decay and delayed expression of ERalpha, PR, and Pax-2 proteins as well as depression of Wnt-4 and RANKL mRNA coincident with the strong estrogen surge in proestrus. In time-course studies using ovari-ectomized mice, a single estrogen injection replicated these delays and caused an 18 h delay in Wnt-4 expression. Molecular time-delay systems are at the core of cellular cycles, most notably the circadian clock, and depend on proteasome degradation of transcriptional regulators that exhibit dedicated timing functions. The cytoplasmic dynamics of these regulators govern delay duration through negative transcription/translation feedback loops. A proteasome inhibitor, PS-341, blocked estrogen-stimulated ERalpha, PR, and Pax-2 decay and proteasome chymotryptic activity, assayed using a fluorogenic substrate, was elevated in proestrus correlating with the depletion of the transcription factors. The 18-h delay in Wnt-4 induction corresponded to the turnover time of Pax-2 protein in the cytoplasm and was eliminated in Pax-2 knockout mammary tissue, demonstrating that Pax-2 has a unique timing function. The patterns of estrogen-triggered ERalpha, PR, and Pax-2 turnover were consistent with a negative transcriptional feedback. Retarding the expression of ERalpha, PR, and Pax-2 may optimize preparations for pregnancy by coordinating expression of critical receptors and transcription factors with rising estrogen and progesterone levels in estrus. The estrogen surge in proestrus has no defined mammotropic function. This study provides the first evidence that it is a synchronizing signal triggering proteasome-dependent turnover of mammary gland ERalpha, PR, and Pax-2. We hypothesize that the delays reflect a previously unrecognized timing system, which is present in all ovarian target tissues.

Expression of the PAX2 oncogene in human breast cancer and its role in progesterone-dependent mammary growth.

In this study, we first describe expression of the paired domain transcription factor PAX2 in the normal and cancerous human breast, then demonstrate in a murine model a novel function for PAX2 in the regulation of progesterone stimulation of secondary ductal growth. In human mammary tissue, PAX2 expression was coincident with sub-populations of mammary ductal cells, some of which possessed an undifferentiated histiotype, and was also found in >50% of the human breast tumors surveyed (n=38). In the mouse, mammary parenchyma with a targeted deletion of PAX2 developed normal ductal systems when grafted into wild-type host mammary fat pads, but failed to undergo higher order side-branching and lobular development in response to progesterone. A previously unsuspected PAX2/WT1 (Wilms' tumor suppressor gene) regulatory axis in the mammary gland was also indicated. Using RT-PCR, a significant reduction in WT1 mRNA expression was detected in the PAX2 mutant glands compared to wild-type counterparts and double-antibody immunohistochemistry detected the co-localization of PAX2 and WT1 in the nuclei of normal and cancerous breast cells. These data indicate a role for PAX2 (and possibly WT1) in the regulation of the progesterone response of the mature mammary gland. The potential contribution of PAX2 to breast tumor pathogenesis is discussed.