A. General strategy used. Exon 2 (E2; encoding the first zinc finger in the DNA binding domain) of the ERRα gene was flanked by loxP sites in ERRα^fl/fl mice (here after referred to as control [c] mice). Crossing these mice with Col1a-Cre animals resulted in ERRαΔ^OBΔ^OB,Col1a^Cre/+ mice (hereafter referred to as conditional knock-out [cKO] animals), which display E2 deletion specifically in maturing osteoblasts. Location of PCR primers (see sequences in Material and Methods section) for the detection of the various alleles is depicted. Note that the gene structure is not drawn to scale. B. Genotyping of ERRα alleles. PCR using the indicated primers were performed using DNA extracted from the indicated organs. Upper panel, detection of the floxed allele (506 bp) in all organs tested; lower panel, detection of the recombinant allele (386 bp) in skull vault and long bone. M: size marker; −: blank PCR (no DNA).

Pre-osteoblasts from control (c) or conditional knock-out (cKO) mice were isolated from calvaria and allowed to differentiate ex vivo. A. Expression of ERRα detected in western blot after the indicated differentiation period. Hsp90 was used as a loading control. B. Lack of ERRα expression in Col1a-expressing cKO cells. After 6 days of differentiation, cells were fixed and stained with Dapi, anti-Col1a or anti-ERRα antibodies as indicated. C. Alkaline phosphatase activity was detected by whole cell cultures staining after 10 days of differentiation (left panel), or by in vitro enzymatic assay relative to protein content (right panel) after the indicated days of differentiation. The latter panel depicts a typical experiment (out of three) performed in triplicate. Data are expressed as average with error bars representing S.D. D. Mineralization activity was detected by von Kossa staining after 15 days of differentiation. Whole culture wells are presented on the left panels, field enlargements on the right ones.

A. Evaluation of the expression of osteoblast differentiation markers by real time PCR at the indicated time after differentiation of mouse calvarial cells. ALP: alkaline phosphatase; Ocn: osteocalcin; Opn: osteopontin. A single representative experiment (out of three, performed in triplicate) is shown. Data are expressed relative to differentiation day 1 with error bars representing S.D. B. Reintroduction of ERRα in cKO cells normalizes the expression of differentiation parameters. Lentivirus encoding human ERRα was used to infect mouse calvarial cells induced in osteoblastic differentiation. RNAs were extracted after differentiation day 5. Data are expressed relative to control-infected cells (empty lentivirus: c) as mean +/− s.e.m. Differences for each gene are significant (p<0.005) except where indicated. Experiments were performed in triplicate with n = 4. C. ERRα represses Runx2 transcriptional activities. C3H10T1/2 cells were transfected with OSE6-Luc (Runx2-responsive) or ERRE-Luc (ERRα-responsive) plasmid together with the indicated amount of Runx2, and fixed amounts of ERRα or ERRγ, as indicated. CMV-renillaLuc was added as a transfection efficiency control and PGC-1α-encoding plasmid was added together with ERR for the right panel as indicated. Results are expressed as fold activation over transfection with activator and expressed as the average of a typical triplicate experiment (out of three) with error bars representing S.D. *: p<0.05; **: p<0.01; ***: p<0.005; ns: non significant.

Bone volume fraction (BV/TV; A), bone mineral density (BMD; B), trabecular number (Tb N; C), trabecular spacing (Tb Sp; D) and trabecular thickness (Tb Th; E) were determined by microCT-scan at 14 (white bars) and 24 wk (black bars) in the femur of female control (c) and conditional ERRα knock-out (cKO) mice (n = 6 to 11 per group). Error bars represent s.e.m. *: p<0.05; ***: p<0.005; ns: non significant.

A. Photograph of uteri in sham-operated or ovariectomized (ovx) control (c) or cKO mice. B–F. Same parameters as in Fig. 3 were determined in females either sham-operated (white bars) or two weeks after ovariectomy (ovx; black bars). BV/TV (G) and BMD (H) were determined in male mice either sham-operated (white bars) or four weeks after orchidectomy (orx; black bars). All mice (n = 6 to 10 per group) were 14 wk old at the end of the experiment. Error bars represent s.e.m. *: p<0.05; ***: p<0.005; ns: non significant.

ERRα impairs the later steps of osteoblastic differentiation. The results presented here show that these activities are involved in bone loss induced by estrogen deficiency and may involve the repression of Runx2- driven transcriptional activities. In contrast, these activities are not involved in bone sensitivity to ageing. Data published by other show that ERRα also promotes the early commitment of mesenchymal stem cells (MSCs) toward the adipocytic pathway while restricting the osteoblastic one. We hypothesize that these early activities are responsible, at least in part, for bone loss induced by ageing. See text for details and references.