REGULATION OF THE PROGESTERONE RECEPTOR IN THE MOUSE MAMMARY GLAND: CHARACTERIZATION OF THE TRANSCRIPTION UNIT, THE ROLE OF ACTIVATING PROTEIN-1 (AP-1), AND THE INFLUENCE OF OVARIAN HORMONES ByEmily Elizabeth FlynnThe mouse progesterone receptor (Pgr) locus is comprised of tandem distal and proximal promoters that give rise to PR(B) and PR(A) transcripts encoding PRB and PRA proteins, respectively. This locus also forms a cis -sense/antisense pair with a naturally occurring, non-coding antisense transcript (PR antisense ). The PR antisense transcript overlaps the majority of sense exon 1, with transcription initiating near the 3'-end of exon 1 driven by an antisense PR promoter. All three promoters responded to activating protein-1 (AP-1), but the sense PRA and PRB promoters were up-regulated primarily in response to c-Jun while the antisense PR promoter was more responsive to JunD or JunB, and to a lesser extent to c-Jun. Both in cells gown in culture and in tissues in vivo , the PR antisense transcript was localized to the same cells that expressed PR sense message and PRA protein. The PR sense and PR antisense transcripts showed co-regulation, rather than anti-regulation, and were co-expressed across mouse mammary gland development. Expression of the mouse Pgr gene during mammary gland development is regulated by complex interplay between hormones and growth factors that affect growth and differentiation of the mammary gland. Many of these factors ultimately influence the activity of AP-1 family members and other transcription factors. We examined the effect of steroid hormones and co-expression of Jun and Fos subunits on the activity of the mouse PR promoters and on their respective mRNA transcripts. We hypothesized that differential regulation of PR isoform expression occurs at a transcriptional level and there are independent mechanisms such as estrogen receptor (ER) acting alone or together with specificity protein 1 (Sp1) and AP-1 to separately regulate the distal and proximal promoters. Although the hormonal milieu of pregnancy supports increased PRB expression in the mouse, these studies did not support a prominent role of ER, Sp1, or AP-1 in this regulation. Additional experiments utilized an immunofluorescence approach with isoform-specific antibodies to examine the relationship between AP-1 and PR expression across development in ovary-intact mice and after hormone treatment in ovariectomized mice. The underlying hypothesis for these studies is that a change in the composition of AP-1 subunits is, at least in part, responsible for the shift from PRA to PRB expression during alveologenesis, perhaps due to the ability of different AP-1 isoforms to differentially regulate the two promoters. These experiments establish that AP-1 alone cannot account for differential regulation of the PR promoters, however, the composition of AP-1 subunits did change across development and, as noted above, AP-1 promotes transcription from the PR promoters. c-Jun AP-1 nonetheless correlated most highly with the overall expression of PRA, and PRA expression invariably accompanied the expression of one Jun isoform or another. In the ovariectomized state, the expression of c-Fos disappeared entirely, while the effects of ovariectomy on PRA were relatively modest and only apparent in the intensity of PRA staining.In summary, these studies characterized the role of hormone and phorbol ester treatment along with ER, Sp1, and AP-1 in transcriptional regulation of the mouse Pgr locus. The expression of AP-1 and PR antisense correlated positively with PRA expression across development in the mouse mammary gland. These correlations persisted in the pregnant mammary gland, where a subset of c-Jun-positive/PRB-positive cells maintained their expression of PRA. Additionally, the ability of steroid hormones to regulate AP-1 expression was examined in the ovariectomized mouse mammary gland, with the interesting finding that P as well as E2 can stimulate expression of c-Fos and c-Jun.
Read
