PR inactivation in the Mx1+ calvarial cells and calvariae <i>in vitro</i>.

<p>(<b>A</b>) A schematic diagram showing that PR-flox is crossed to Mx1-Cre. Mx1-Cre can be activated by IFNα to delete exon 2 of the PR gene to generate PR mutants (ΔPR). (<b>B</b>) Mx1-Cre;PR-flox/flox calvarial cells were treated with IFNα (500 units/mL) or without IFNα (control) for three days. The cells were then differentiated into osteoblasts in osteogenic medium without IFNα for 14 days. The relative expressions of RUNX2, Osteocalcin (Ocn) and DMP1 were evaluated by real-time PCR at day 14 and normalized to endogenous β-actin. (<b>C</b>) The cells were collected for alkaline phosphatase (ALP) activity assays on day 10 and alizarin red staining (AR) on day 21. The optical density (OD) values were normalized to the corresponding total protein concentrations. Calvarial cells from the PR-flox/flox (without Cre) mice were used as a negative control to exclude the effect of INFα itself. (<b>D</b>) The calvariae obtained from Mx1-Cre;mT/mG double transgenic pups exhibited significant numbers (~40%) of cells that became GFP-positive after three days of IFNα (500 units/mL) treatment, and significantly more cells (~80%) became GFP-positive after an additional six days of culture without IFNα. (<b>E</b>) Genomic DNA was isolated from PR-flox/flox calvarial cells three days after IFNα treatment, and subjected to PR allele-specific PCR. The deleted PR band (ΔPR) indicated Cre-mediated DNA recombination. (<b>F</b>) Five-weeks-old PR-flox/flox or Mx1-Cre;PR-flox/flox mice were injected with pI-pC. Calvariae were collected five months later for microCT analysis for bone volume and (G) representative calvarial images from microCT scans.</p

Skeletal phenotypes of Mx1-Cre-driven PR inactivation <i>in vivo</i>.

<p>(A) A distal femur from non-pI-PC treated Mx1-Cre;mT/mG mouse. (B—C) Mx1-Cre;mT/mG mice were injected with pI-pC intraperitoneally to induce Cre at one month of age and then sacrificed one (B) or two (C) months later. The distal femurs (D.F.) were collected and sectioned to observe the GFP (green) and tdTomato (red) fluorescence. Green indicates the Mx1+ cells that expressed Cre, and red indicates the Cre-negative cells. The nuclei were stained with DAPI (blue). The femoral trabecular bones from the Col1a1-CreERT2;mT/mG mice that received 4 days of tamoxifen injections were used as a positive control and are shown in the insert in (B). The white arrows indicate the green osteocytes that were observed in the trabecular bone in the Col1a1-mT/mG mice but were absent in the trabecular or cortical bone of the Mx1-mT/mG mice (the white arrowheads indicate the GFP-/tdTomato+ osteocytes). There were no green osteocytes observed in the Mx1-mT/mG bones on either day 30 or 60. (D) Bone marrow cells were collected from 1-month-old Mx1-Cre;mT/mG mice. Some multinuclear cells turned green indicating Mx1-Cre activation in these cells (yellow arrows). (E) Serum osteocalcin and (F) serum CTX1 levels were measured by ELISA two months post pI-pC injection (n = 8/group). (G) Five-week-old male and female Mx1-Cre;PR-flox/flox or PR-flox/flox (control) mice were injected with pI-pC intraperitoneally to induce Cre activity. Baseline microCT scans (BL) of the distal femurs were performed on five-week-old mice without receiving the pI-pC injection. The animals were sacrificed at one, two or five months post-pI-pC injection, and the hind limbs were collected for microCT analysis (n = 8/group).</p

Characterization of Mx1-Cre in the long bones and calvariae <i>in vivo</i>.

<p><b>(A)</b> A schematic diagram showing that mT/mG is crossed to Mx1-Cre and that IFNα can activate Cre expression to delete the tdTomato (red fluorescent) cassette and initiate GFP (green fluorescent, “mG”) expression. <b>(B)</b> Mx1-Cre;mT/mG mice at the age of 5 weeks were injected intraperitoneally with PBS (control) or pI-pC (activated). The distal femurs and calvariae were collected for cryosection after three days. The nuclei were stained with DAPI (blue). The arrows indicate bone surface GFP expression in the pI-pC-treated bones. The dotted lines circle the cartilage regions in the distal femur. GP, growth plate cartilage; Ct, cortical bone.</p

Comparison of BMSCs and calvarial cells in terms of Mx1-Cre activation and PR expression <i>in vitro</i>.

<p>(<b>A</b>) BMSCs or calvarial cells were obtained from Mx1-Cre;mT/mG double transgenic mice and treated with IFNα (500 units/mL) for three days. Fluorescent images were taken three days (BMSCs) or 14 days (Calvarial cells) after the IFNα treatment. The image of calvarial cells three days post IFNα treatment is shown in the insert. The levels of Cre activation were quantitated by GFP (green) expression. (<b>B</b>) A quantitative histogram showing the induction rates (the GFP+ cells versus the total cells) of the BMSCs and calvarial cells shown in Figure A. CONT, control; ACT, activated. (<b>C</b>) Western blotting was performed to detect GFP protein in the total cell lysates from the calvarial cells with (activated) or without (control) IFNα treatment on day 3. β-actin was used as an internal control. (<b>D</b>) Mx1-Cre;mT/mG calvarial cells were treated with different concentrations of IFNα for three days. The ratios of GFP+ cells to total cells under each IFNα concentration were quantified (A). A PR-Cre;tdTomato schematic diagram showing that the Cre gene is inserted downstream of the endogenous PR gene after an internal ribosome entry site (IRES) sequence such that Cre is expressed simultaneously with PR. When Cre is expressed (together with PR), it recombines loxp sites to remove the stop codon before the tdTomato cassette and activates tdTomato (red fluorescence) expression. (E) A diagram of PR-Cre; tdTomato. The tdTomato expression can be activated following the upstream stop codon removal by Cre, which is expressed simultaneously with endogenous PR gene. So that PR+ cells will express tdTomato (red fluorescent). (<b>F</b>) PR-Cre;tdTomato calvarial cells were differentiated into osteoblasts with osteogenic media or chondrocytes with chondrogenic media for 14 days. The majority of the cells cultured in the osteogenic medium were red. However, when the cells were cultured in the chondrogenic medium, only a small percentage of the fibroblast-like cells (arrows) turned red. The nuclei were stained with DAPI (blue). (G) Distal femurs or calvariae (inserts) from 3-week-old or 4-week-old PR-Cre;tdTomato mice were sectioned for fluorescent microscopy. The nuclei were stained with DAPI (blue). The red fluorescence indicated PR promoter activity.</p

OVX-induced bone remodeling in lumbar vertebra and cortical bone.

<p>Six weeks after operations, the sixth lumbar vertebra (LVB6), and tibia were processed and analyzed by µCT, as described in Materials and Methods. Trabecular bone volume/tissue volume (A), trabecular thickness (B), and trabecular number (C) of LVB6, and cortical thickness of tibia at distal tibiofibular junction (D) are shown. WT: wild-type mice; DKO: mice lacking both DAP12 and FcRγ. N = 5 in each group. *: p<0.05; **: p<0.01; ***: p<0.001; when compared to SHAM groups, respectively.</p

Osteoblast differentiation and apoptosis-related gene expressions in osteoblastic cultures.

<p>Bone marrow cells were extracted from 3-month-old mice and cultured with ascorbic acid and β-glycerophosphate from WT and PRKO mice to monitor osteoblastogenesis. RNA was extracted from the cultures on day 14. Real-time PCR was performed to monitor gene expression for osteoblast differentiation (Runx2, Osterix, Osteocalcin, OPG and RANKL) or apoptosis (Foxo1 and FasL). ^*<i>p</i><0.05 compared with WT of the same sex.</p

Bone loss in the trabecular region of the distal femur.

<p>Measurements acquired at six weeks after OVX (N = 5).</p

Bone mass and bone formation changes with RU486 treatment.

<p>One month-old female WT mice were treated with vehicle or RU486 (500 µg/d, 5x/week for two months). MicroCT was performed at the distal femurs to measure the cancellous bone volume (A). Bone formation was measured in either serum (P1NP, B) or at the distal femurs by surface-based bone histomorphometry (bone formation rate/bone surface, BFR/BS; C). Bone resorption was measured in the urine (DPD/Cr, D), at the distal femurs by surface-based bone histomorphometry (osteoclast surface, Oc.S/BS; E) and from bone marrow osteoclastic cultures (TRAP+, F). ^*<i>p</i><0.05 compared with WT.</p

Bone formation parameters measured in the three-month-old WT and PRKO mice.

<p>Markers for bone formation, P1NP (A) and osteocalcin (B) that were measured from serum. Surfaced-based bone histomorphometry was performed at the distal femoral metaphysis that included (C), mineralized surface (MS/BS), and (D), bone formation rate/bone surface (BFR/BS). Data was presented as mean ± SD.</p

Expression of the progesterone nuclear receptor (PR-A and PR-B) in osteoblast and osteoclast.

<p>Bone marrow cells were derived from WT mice and differentiated into osteoblasts in an osteogenic medium with ascorbic acid and β-glycerophosphate or into osteoclasts with mCSF and RANKL stimulation. PR-A and PR-B were detected by immunofluoresence using PR (C19) primary antibody against both PR-A and PR-B for mouse and FITC–Conjugated secondary antibody for osteoblasts (A, yellow arrow) or Texas-red conjugated secondary antibody for osteoclasts (B, white arrows). DAPI was used to stain nucleus in the osteoclast culture (bright blue staining). C, osteoclast culture from the PRKO mice. Both PR-A and PR-B were expressed by WT osteoblast (A, yellow arrow) and the WT osteoclast (B, white arrows) but not by the PRKO osteoclast (C). In osteoclasts, the PR-A and PR-B expression was mainly observed around the nucleus, but weak expression was detected in the cytoplasm as well. Original magnification, 100×. B, Western blot analysis of the expression of PR-A (molecular weight 96 kDa) and PR-B (molecular weight about 120 kDa) proteins in osteoblasts and osteoclasts. Whole cell lysates were obtained from osteoblast or osteoclast cultures and probed with PR (C19) antibody. Uterus tissue lysates from the WT were used as a positive control while the uterus tissue lysates from the PRKO were used as a negative control. GAPDH was used as a housekeeping control. All the experiments were repeated three times.</p