Activation function 1 of progesterone receptor is required for mammary development and regulation of RANKL during pregnancy

Abstract

Progesterone receptor (PGR) is a member of the nuclear receptor superfamily of transcription factors. It is critical for mammary stem cells expansion, mammary ductal branching and alveologenesis. The transcriptional activity of PGR is mainly mediated by activation functions AF1 and AF2. Although the discovery of AF1 and AF2 propelled the understanding of the mechanism of gene regulation by nuclear receptors, their physiological roles are still poorly understood. This is largely due to the lack of suitable genetic models. The present study reports gain or loss of AF1 function mutant mouse models in the study of mammary development. The gain of function mutant AF1_QQQ exhibits hyperactivity while the loss of function mutant AF1_FFF shows hypoactivity on mammary development. However, the involvement of AF1 is context dependent. Whereas the AF1_FFF mutation causes significant impairment in mammary development during pregnancy or in response to estrogen and progesterone, it has no effect on mammary development in nulliparous mice. Furthermore, Rankl, but not Wnt4 and Areg is a major target gene of AF1. In conclusion, PGR AF1 is a pivotal ligand-dependent activation domain critical for mammary development during pregnancy and it exerts gene specific effect on PGR regulated genes.

Figure 1

AF1_QQQ mutations promote mammary development in response to estrogen and progesterone. (a) Domain structure of PGR and CRISPR-Cas9 gene editing strategy. The red bar indicates exon 1 of Pgr gene (1–1671 bp). Cas9 mRNA, 2 Pgr gRNAs and the homologous single-stranded oligonucleotide (OLN) containing the 3 mutations were injected into the pronuclei for mutant generation. (b) AF1_QQQ mutations did not significantly affected PGR levels in estrogen-treated ovariectomized mice. (c,d) AF1_QQQ mice show greater number of TEB and ductal branching in response to EBP. Mice were ovariectomized at 3.5 weeks old. After 2 weeks, the mice were treated with control vehicle (Ctrl) or EBP for 16 days. (c) Representative whole mount images of mammary gland. Rudimentary mammary ducts in Ctrl are indicated by arrows. (d) Quantification of TEBs, mammary ductal branches and ductal length from the whole mount images of EBP-treated mammary gland (mean ± SEM, *p < 0.05, n = 6). The vehicle-treated mammary gland only contains a few rudimentary ducts and were therefore not quantified. (e,f) AF1_QQQ mice at dioestrus also show greater number of mammary ductal branching than the WT mice. (e) Representative whole mount mammary images with enlarged insets to show greater details. (f) Quantification of ductal branching (results are expressed as mean ± SEM, n = 6, *p < 0.05).

Figure 2

AF1_FFF mutations result in reduced mammary development in response to EBP but have no effect on intact nulliparous mice. (a,b) Mice ovariectomizd at 6 weeks old were treated with EBP for two weeks before whole mount analysis of the 4th mammary gland. (a) Representative whole mount images; (b) quantification of the TEB, ductal branching and ductal length by image analysis (n = 5 for WT and n = 6 for AF1_FFF). (c,d) No difference in mammary development between the WT and AF_FFF mutant virgin mice at 12 weeks old. (c) Representative whole mount images; (d) quantification of the ductal branching and length (n = 4 for each genotype). All numeric data are expressed as mean ± SEM, *p < 0.05; **p < 0.01, ns not significant.

Figure 3

AF1_FFF mutations lead to reduced mammary growth during pregnancy. (a,b) Mammary development on GD 3 is similar between the WT and AF1_FFF mice. (a) Representative whole mount images on GD3; (b) quantitative data of alveolar buds, ductal branching and ductal length (n = 7 for WT, n = 8 for AF1_FFF, ns, not significant). (c,d) Significant less MaSC (Lin⁻CD49f^hiCD24⁺) in AF1_FFF mice on GD3 (*p < 0.05). (c) FACS dot blots showing Lin⁻CD24⁺CD49f^lo and Lin⁻ CD24⁺CD49f^hi cell gating. (d) Percentage of Lin⁻CD24⁺CD49f^lo and Lin⁻CD24⁺CD49f^hi cells relative to Lin⁻ cell populations (WT, n = 4; AF1_FFF, n = 4, *p < 0.05). (e,f) Mammary development on GD7.5 in AF1_FFF mice is significantly impaired. (e) Representative whole mount images; (f) quantitative data of the alveolar buds, ductal branching and ductal length (WT, n = 10; AF1_FFF, n = 11, ****p < 0.001). All numeric data are expressed as mean ± SEM.

Figure 4

Effect of AF1_FFF mutations on the expression of Pgr and PGR target genes important for MG development. Total RNA from the mammary gland on GD3, GD4.5 and GD7.5 were analyzed for gene expression by RT-qPCR using 36B4 primers as internal control. The results are expressed relative to 36B4 gene (mean ± SEM, n = 6 for all groups). *Denotes statistically significant difference between the WT and the mutant.

Figure 5

RANKL protein was significantly reduced in AF1_FFF mammary tissues in early pregnancy. (a) Protein levels of PR, ERα, WNT4 and RANKL in the mammary tissue were analysed by Western blotting analysis. Lanes indicated by X followed by a line is a defective sample. There is a upshift of PR protein bands on GD7.5 as compared to that on GD3. The major RANKL protein bands were detected are ~ 27 kDa and 22 kDa (indicated by black arrow heads) which correspond to soluble RANKL proteins. The level of GAPDH was used as a loading control. (b) Quantification of protein levels by densitometry. The results are expressed as mean ± SEM. **p < 0.01; ***p < 0.001. (c) Representative images of WT and AF1_FFF mammary sections probed with RANKL antibody and detected using VECTASTAIN Elite ABC HRP Kit.