Sex-Dependent, Osteoblast Stage-Specific Effects of Progesterone Receptor on Bone Acquisition

Abstract

The role of the progesterone receptor (PR) in the regulation of sexual dimorphism in bone has yet to be determined. Here we utilized genetic fate mapping and Western blotting to demonstrate age-dependent PR expression in the mouse femoral metaphysis and diaphysis. To define sex-dependent and osteoblast stage-specific effects of PR on bone acquisition, we selectively deleted PR at different stages of osteoblast differentiation. We found that when Prx1-Cre mice were crossed with PR floxed mice to generate a mesenchymal stem cell (MSC) conditional KO model (Prx1; PRcKO), the mutant mice developed greater trabecular bone volume with higher mineral apposition rate and bone formation. This may be explained by increased number of MSCs and greater osteogenic potential, particularly in males. Age-related trabecular bone loss was similar between the Prx1; PRcKO mice and their WT littermates in both sexes. Hormone deficiency during the period of rapid bone growth induced rapid trabecular bone loss in both the WT and the Prx1; PRcKO mice in both sexes. No differences in trabecular bone mass was observed when PR was deleted in mature osteoblasts using osteocalcin-Cre (Bglap-Cre). Also, there were no differences in cortical bone mass in all three PRcKO mice. In conclusion, PR inactivation in early osteoprogenitor cells but not in mature osteoblasts influenced trabecular bone accrual in a sex-dependent manner. PR deletion in osteoblast lineage cells did not affect cortical bone mass. © 2017 American Society for Bone and Mineral Research.

Keywords: BONE ACCRUAL; CONDITIONAL GENE KNOCKOUT; MESENCHYMAL STROMAL CELLS; OSTEOBLASTS; OSTEOCYTES; PROGESTERONE RECEPTOR.

Figure 1. Characterization of PR expression in vivo and in vitro

PR-Cre mice were crossed with Ai14D, red fluorescent protein tdTomato expression is activated following the stop codon removal by Cre, and crossed with Col1a1-GFP, where the green fluorescent GFP is driven by 2.3 kb rat procollagen, type 1, alpha 1(Col1a1) promoter, which is known to be active in mature osteoblasts. Representative fluorescent images of distal femur from 5 (A) or 8-week-old (B) male PR-Cre; Ai14D; Col1a1-GFP mice. Green fluorescence indicates mature osteoblast, while red fluorescence indicates PR expression. The rectangle region was magnified in to show the merged fluorescence pictures. Cell nuclei were stained blue with DAPI. (C), Anti-PR staining were performed on the Col1a1-GFP mouse, 8 weeks of age. White arrows illustrated co-localizations of PR and Col1a1-GFP+ cells. (D). PR protein level in bone decreases with age. Protein was extracted from distal femurs of 1-, 2-, and 6-month-old male and female mice, PR protein and internal control protein β-actin were detected with corresponding antibodies. Only isoform PR-B (118 kDa) was detected. (E). Bone marrow stromal cells were differentiated into osteoblasts with osteogenic media. qRT-PCR was performed to detect PR expression at 1, 3 and 6 days. Result was presented as fold-changes from day 0. (F). Calvarial cells or bone marrow stromal cells were differentiated with osteogenic medium for indicated time. RNA was extracted for PR real-time qPCR controlled with internal β-actin mRNA level. Ovary, uterus and oviduct tissues from a 3-month-old female mouse were used as positive controls. (G). Bone marrow stromal cells were differentiated in osteogenic medium for indicated time. Total cell lysate was prepared for PR western blot. Oviduct protein was used as positive control and GAPDH was used as internal control.

Figure 2. Construction of Prx1; PRcKO conditional knockout mouse model

(A). A diagram shows Prx1-Cre, mT/mG, and PR-flox cassettes. (B). A representative fluorescent image of distal femur from 5-week-old Prx1-Cre; mT/mG mice. Green fluorescence indicates Cre activity in both osteoblasts and chondrocytes, while red fluorescence indicates all other cells without Cre activity. The rectangle region was magnified to show the detailed structure in growth plate region. Cell nuclei were stained blue with DAPI. (C). Protein was extracted from epiphysis (Epi) or femoral middle shaft (Shaft) of wild-type (WT), heterozygous ((PR-flox/+; Het), or homozygous (PR-flox/flox; Hom) Prx1; PRcKO 1-month-old male mice. PR protein and internal control protein GAPDH were detected with corresponding antibodies. Only isoform PR-B (118 kDa) was detected. (D). Serum level of progesterone measured at two and four months of age WT and Prx1; PRcKO animals. (E). Bone marrow stromal cells were collected from 2-month-old wild type or mutant Prx1; PRcKO mice and were cultured for a week before flow cytometric analyses of MSC markers.

Figure 3. Prx1; PRcKO mice developed high bone mass at the distal femurs in both sexes

(A). Distal femurs from 2- and 6-month-old female and male Prx1; PRcKO mice were scanned with microCT. Bone volume (BV), total volume (TV), BV/TV, trabecular number (Tb. N), and trabecular thickness (Tb. Th) were compared between wild type and mutant mice. (B). Three-dimensional images were reconstructed for trabeculae at the distal femurs of 2-month-old mice.

Figure 4. Prx1; PRcKO mice displayed high bone formation in both sexes

Mice were given two fluorescents labeling at −7 and −1 day(s) before sacrifice. Dynamic bone histomorphometry was performed at the distal femurs from wild type and Prx1; PRcKO mice of both sexes at 2 or 6 months of age. Representative images of double calcein labeling (white arrowheads) at the trabeculae obtained from 6-month-old female (A) and male (B) WT or Prx1; PRcKO mice. (C). Quantitative measurements for mineralizing surface (MS/BS), mineral apposition rate, bone formation rate and osteoclast surface at the distal femoral metaphysis.

Figure 5. Prx1; PRcKO mice had similar rate of trabecular bone loss following gonadectomy

Two-month-old mice were sham-operated, ovariectomized (OVX) or orchiectomized (ORX) and euthanized 4 weeks later (n=5–10/group). Repeated in vivo microCT was performed before surgeries and prior to euthanization. Trabecular bone volume was obtained from the distal femurs when at 2 months (baseline) and 3 months (one-month post-surgeries) in female (A) and male (B mice. Three-dimensional image registrations were reconstructed for baseline (red) and one-month post-surgery (green), with overlapping regions colored purple. (C), Cortical bone volume was measured at the middle femurs when at 2 months (baseline) and 3 months (one-month post-surgeries) in both female and male mice.

Figure 6. Dmp1 expression in bone and surface-based histomorphometric analysis of Dmp1; PRcKO mice

(A). A representative fluorescent image of distal femur from 4-week-old Dmp1-Cre; mT/mG mice. Green fluorescence indicated Cre activity was being observed at the trabecular bone surface. (B). Trabecular bone volume/total volume (BV/TV) was obtained from the distal femurs and cortical bone volume was obtained at the middle femurs from 2- and 4-month-old female and male Dmp1; PRcKO mice by microCT scans. (C). Surface-based bone formation was measured at the distal femurs. (D). Maximum load and work-to-failure were measured at the femurs.