FOXOs attenuate bone formation by suppressing Wnt signaling (original) (raw)
Combined deletion of Foxo1, -3, and -4 in osteoprogenitors increases bone mass. Foxo1, -3, and -4 were deleted in osteoblast precursors using transgenic mice in which a Cre-GFP fusion protein is under the control of Osx1 regulatory elements (8). The Osx1-Cre transgene is expressed in committed osteoblast progenitors present in the bone-forming regions of the perichondrium and primary spongiosa as well as in hypertrophic chondrocytes. In addition, _Osx1/GFP_-expressing cells are present in the thin periosteal layer overlaying the cortical bone surface (28). Mice lacking Foxo1, -3, and -4 in _Osx1-Cre_–expressing cells, hereafter referred to as Foxo1,-3,-4f/f;Osx1-Cre mice, were born at the expected Mendelian ratios, and their growth plates were indistinguishable from those of control Foxo1,-3,-4f/f littermates (Figure 1A). Female, but not male, mice exhibited a modest decrease in body weight (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI68049DS1). Foxo1, -3, and -4 mRNA levels were reduced by almost 90% in Osx1-GFP–positive calvaria cells from Foxo1,-3,-4f/f;Osx1-Cre mice as compared with cells from Osx1-Cre mice, isolated by FACS (Figure 1B). As expected, Foxo mRNA was unaltered in spleen and liver from Foxo1,-3,-4f/f;Osx1-Cre mice (Supplemental Figure 1B).
Deletion of Foxos in _Osx1-Cre_–expressing cells increases bone mass. (A) Histological sections of the distal femurs of 4-week-old mice stained with Safranine-O (cartilage stains red). Scale bar: 500 μm. (B) Foxo mRNA levels in Osx1-GFP calvaria cell cultures (triplicates) determined by quantitative RT-PCR (qRT-PCR). (C) BMDs determined by DXA in 3-month-old male (n = 15–20/group) and female (n = 23–30/group) mice. (D) Micro-CT measurements of the distal femur of 3-month-old males (n = 7–10/group). Representative images of the cancellous bone region (red box). BV/TV, bone volume per tissue volume; Tb, trabecular. (E) Cortical measurements determined by micro-CT in femoral diaphysis of the samples described in D and representative images of the region analyzed. Pm, perimeter. Bars represent mean + SD. *P < 0.05 versus Osx1-Cre or Foxo1,-3,-4f/f by Student’s t test.
Male and female Foxo1,-3,-4f/f;Osx1-Cre mice exhibited increased spinal and femoral bone mineral density (BMD) at 12 weeks of age, as measured by DXA (Figure 1C). The increased BMD was due to the loss of Foxos and not to unspecific actions of Cre recombinase, as indicated by the lack of a skeletal phenotype in Osx1-Cre mice as compared with wild-type controls obtained following a breeding strategy similar to that used to generate the Foxo1,-3,-4f/f;Osx1-Cre and Foxo1,-3,-4f/f mice (Supplemental Figure 1C). Nonetheless, Osx1-Cre mice exhibited decreased body weight (Supplemental Figure 1D), in line with previous evidence that the Osx1-Cre transgene decreases body size (29).
Micro-CT analysis of the femur (Figure 1, D and E) and vertebra (Supplemental Figure 1E) revealed that the deletion of Foxos in _Osx1_-expressing cells led to an increase in both cancellous and cortical bone mass. The former was detected as early as 4 weeks of age in the femur (Supplemental Table 1), and it was associated with an increase in trabecular number and connectivity and with a decrease in trabecular spacing. The higher cortical thickness of the Foxo1,-3,-4f/f;Osx1-Cre mice was detected as early as 7 weeks of age (Supplemental Table 1). This change resulted from an enlargement of the outer perimeter (Figure 1E), indicating that Foxo deletion from osteoprogenitor cells promotes periosteal apposition. No changes were detected in the inner perimeter of the femur.
We had previously shown that Foxo3 was expressed at higher levels than Foxo1 and Foxo4 in osteoblastic cells (18). To determine whether deletion of Foxo3 alone could recapitulate the skeletal effect seen with the triple Foxo deletion, we generated Foxo3f/f;Osx1-Cre mice and the respective wild-type, Foxo3f/f, and Osx1-Cre littermate controls. Foxo3 mRNA expression in cultured bone marrow–derived osteoblastic cells from Foxo3f/f;Osx1-Cre was decreased by 60%, while the expression of FOXO1 or FOXO4 was not affected (Supplemental Figure 2A). Body weight and femur BMD in Osx1-Cre or Foxo3f/f;Osx1-Cre mice were lower as compared with those in wild-type or Foxo3f/f littermate control mice (Supplemental Figure 2, B and C). Despite the smaller body size and the reduced femoral BMD in both Osx1-Cre and Foxo3f/f;Osx1-Cre mice, these 2 measures were indistinguishable between Osx1-Cre and Foxo3f/f;Osx1-Cre mice. This result indicates that, whereas Osx1-Cre expression in and of itself had an effect in decreasing body size and femoral BMD, Foxo3 deletion per se did not affect any of these measurements. Furthermore, spine BMD was indistinguishable among the 4 genotypes (Supplemental Figure 2D). These results are in agreement with the published evidence that Foxo1, -3, and -4 have redundant functions (14, 20, 21).
The high bone mass phenotype of the Foxo-deficient mice is maintained throughout life. To determine whether the effect of Foxo deletion on bone mass could be elicited in an adult animal, we induced the triple Foxo deletion after most of the growth phase was completed (at 3 months of age), taking advantage of the Tet-off system incorporated into the Osx1-Cre transgene. As expected, Foxo1, -3, and -4 transcripts were significantly decreased in bone marrow–derived osteoblasts from the Foxo1,-3,-4f/f;Osx1-Cre mice (Supplemental Figure 3A). Longitudinal BMD measurements in these mice showed a significant increase in bone mass at the spine and femur 12 weeks following the deletion (Figure 2A). Moreover, micro-CT measurements revealed that deletion of Foxos at 3 months of age caused identical microarchitectural changes to those seen when Foxos were deleted from conception, including increased cancellous bone mass, trabecular number, and connectivity as well as decreased trabecular spacing at both the femur and spine (Figure 2B and Supplemental Figure 3B). Likewise, femoral cortical thickness was increased (Figure 2B). Therefore, the effect of the deletion on bone mass was not caused by changes imparted during development and growth.
The high bone mass of Foxo1,-3,-4f/f;Osx1-Cre mice is maintained throughout life. (A and B) Female mice from both genotypes were maintained in a doxycycline-containing diet from conception until 3 months of age, when the diet was changed to regular chow to activate Foxo deletion (n = 10–11/group). (A) Longitudinal BMD measurements were determined by DXA. Data represent mean + SD. *P < 0.05 based on comparisons of least squares means from a mixed effects model including genotype, age, and their interaction. The interaction term was not statistically significant. (B) Micro-CT measurements in 7.5-month-old mice. Bars represent mean + SD. *P < 0.05 by Student’s t test. (C) Longitudinal DXA BMD measurements in female mice in which Foxos were deleted since conception (initial n = 24–33/group; final n = 12–18/group). Data represent mean + SD. *P < 0.05 based on comparisons of least squares means from a mixed effects model including genotype, age, age2, and the interactions of genotype with age and age2. All factors, including both interactions were statistically significant.
Age-related bone loss is associated with increased oxidative stress and decreased growth factor levels, 2 conditions that can stimulate FOXOs (24, 30). We, therefore, determined whether FOXOs in osteoprogenitors could influence bone mass during aging. To do this, we aged a cohort of female Foxo1,-3,-4f/f;Osx1-Cre mice. The high bone mass of Foxo1,-3,-4f/f;Osx1-Cre mice was maintained up to 24 months of age (Figure 2C), as determined by longitudinal DXA BMD measurements.
The high bone mass phenotype of the Foxo-deficient mice is independent of ROS. We had previously shown that global combined Foxo1, -3, and -4 deletion increases oxidative stress in bone (18). We therefore sought to determine whether targeted deletion of Foxos in osteoprogenitors had a similar effect. The mRNA levels of known antioxidant FOXO target genes were not affected in Osx1-GFP–positive calvaria cells from Foxo1,-3,-4f/f;Osx1-Cre mice (Supplemental Table 2). In contrast, the mRNA encoding glutathione S–transferase α 4 (Gsta4) — a protein involved in the generation of the antioxidant glutathione (GSH) — was increased 4-fold. N-Acetylcysteine (NAC) administration for 12 weeks had no effect on BMD in Foxo1,-3,-4f/f control mice nor did it affect the increase in BMD at the spine and femur seen in the Foxo1,-3,-4f/f;Osx1-Cre mice (Supplemental Figure 4A). NAC, however, decreased ROS and increased GSH levels in the bone marrow (Supplemental Figure 4B) and the phosphorylation of p66shc — a marker of oxidative stress — in the vertebra (Supplemental Figure 4C). In line with the increase of Gsta4 mRNA levels, ROS and p66shc phosphorylation were decreased in the bone of Foxo1,-3,-4f/f;Osx1-Cre mice; the decrease of ROS was maintained at 24 months of age (Supplemental Figure 4D). Taken together, these results suggest that FOXOS do not attenuate ROS in osteoblast progenitors and that the increase in bone mass resulting from Foxo deletion in Osx1 cells could not be accounted for by altered redox balance.
Deletion of Foxos increases bone formation. In agreement with the high–bone mass phenotype, the bone-formation rate (BFR) in cancellous bone of 7-week-old Foxo1,-3,-4f/f;Osx1-Cre mice was increased by 40% as a result of elevated mineralizing perimeter and mineral apposition rate (MAR) (Figure 3A). These changes were accompanied by increased osteoblast numbers (Figure 3B) and expression of the osteoblast specific gene Ocn in bone (Figure 3C). At 24 months of age, BFR was 10-fold lower in both littermate controls and Foxo1,-3,-4f/f;Osx1-Cre mice (Supplemental Figure 5), as compared with the 7-week-old mice, due to reduced mineralizing perimeter and MAR. Nonetheless, in spite of the persistence of higher bone mass in the aged Foxo1,-3,-4f/f;Osx1-Cre mice as compared with the littermate controls, we could no longer detect a difference in bone formation, suggesting that aging-related mechanisms overrode the positive effect of the Foxo deletion seen at 7 weeks, albeit bone mass remained higher in the Foxo1,-3,-4f/f;Osx1-Cre mice, probably because the gain made earlier in life was preserved after a steady-state remodeling rate was achieved.
Foxo deletion in _Osx1-Cre_–expressing cells increases bone formation. (A) MAR, mineralizing surface (MS), and BFR as determined by tetracycline labels, shown in the photomicrographs, in the cancellous bone of longitudinal undecalcified vertebral (L1–L3) sections from 7-week-old mice (n = 8/9 per group). Scale bar: 20 μm. (B) Osteoblast (Ob) and (C) osteoclast (Oc) perimeter and number (N) per mm cancellous bone surface in the vertebral sections described in A (n = 4/group). (D) Opg mRNA levels in sorted Osx1-GFP calvaria cells (triplicates) and in calvaria bone from 1-month-old mice (n = 4–5/group) determined by qRT-PCR. Bars represent mean + SD; *P < 0.05 versus Foxo1,-3,-4f/f or Osx1-Cre by Student’s t test.
In line with earlier findings by us and others in mice with loss or gain of FOXO function in mature osteoblasts (18, 27), the number of osteoclasts was higher in the Foxo1,-3,-4f/f;Osx1-Cre mice (Figure 3D). However, the high bone mass phenotype of the latter indicates that the effect of the increased osteoclast number was overridden by the increase in osteoblastogenesis. FOXO1 activation in osteoblasts increases the expression of the anti-osteoclastogenic cytokine Opg (31). Opg mRNA was decreased in whole bone and in Osx1-GFP–positive calvaria cells from our Foxo1,-3,-4f/f;Osx1-Cre mice (Figure 3D); but Rankl and M-csf were unaffected (Supplemental Table 3), suggesting that a decrease in Opg may contribute to the higher osteoclast number seen in these mice.
Deletion of Foxos increases the proliferation of osteoprogenitor cells. Deletion of Foxos in Osx1 cells did not affect the number of mesenchymal progenitors present in the bone marrow capable of initiating osteoblastic colonies, as indicated by the CFU osteoblast (CFU-OB) assay (Figure 4A). However, there was an increase in the number of BrdU-positive osteoblasts in cancellous bone (Figure 4B). Similarly, the rate of proliferation of cultured periosteal cells from femurs was increased (Figure 4C), consistent with an increase in periosteal apposition and the larger outer perimeter of the femur. As expected, the cultured periosteal cells displayed lower expression of Foxo1, -3, and -4 (Supplemental Figure 6A). An increased rate of proliferation was also observed in Osx1-GFP calvaria cells from the Foxo1,-3,-4f/f;Osx1-Cre mice (Figure 4C). Apoptosis was not affected in _Foxo_-deleted Osx1-GFP calvaria cells (Figure 4C). However, in line with our previous findings (18), calvaria-derived osteoblasts from Foxo1,-3,-4f/f;Osx1-Cre mice cultured for 10 days in ascorbic acid exhibited increased apoptosis (Supplemental Figure 6B). Nevertheless, the increased osteoprogenitor proliferation apparently overrode the increase in mature osteoblast apoptosis in Foxo1,-3,-4f/f;Osx1-Cre mice.
Foxo deletion in _Osx1-Cre_–expressing cells increases proliferation. (A) Number of CFU-OB in the bone marrow from femurs of 3-month-old mice cultured for 21 days with 1% ascorbate (triplicates). (B) Osteoblast proliferation as determined by BrdU incorporation in decalcified paraffin-embedded vertebral (L4–L5) sections from 7-week-old mice (n = 9–11/group). (C) BrdU incorporation in cultures of periosteal cells from 7-week-old mice (left) and sorted Osx1-GFP calvaria cells (right) (triplicate wells each). (D) Caspase 3 activity in sorted Osx1-GFP calvaria cells (triplicates). (E) ALP activity and (F) Alizarin red staining (triplicate) of high-density bone marrow stromal cells cultured with 1% ascorbate for 3 and 21 days, respectively. (G) mRNA levels by qRT-PCR of osteoblast differentiation markers, in the cultures described in E. Bars represent mean + SD; *P < 0.05 versus Foxo1,-3,-4f/f or Osx1-Cre by Student’s t test.
Alkaline phosphatase (ALP) activity and mineralization were higher in bone marrow–derived osteoblastic cells from Foxo1,-3,-4f/f;Osx1-Cre mice (Figure 4, E and F). However, the expression of genes involved in osteoblast differentiation, such as Alpl, Runx2, and Col1a1, was unaffected (Figure 4G). Osx1 was modestly increased in the bone marrow–derived osteoblastic cells, but Osx1 or any of the other differentiation-associated genes was unaffected in Osx1-GFP calvaria cells (Supplemental Table 3). The lack of an effect of Foxo deletion on osteoblast differentiation in both bone marrow and calvaria cells suggests that the increases in ALP activity and mineralization observed in the bone marrow–derived osteoblastic cells from Foxo1,-3,-4f/f;Osx1-Cre mice were the result of increased osteoblast number.
The higher proliferation of Foxo-deficient osteoprogenitors is due to increased β-catenin/TCF activation. We next focused on elucidating whether the restraining effect of FOXOs on osteoblastogenesis was the result of attenuation of Wnt signaling, as our earlier work had suggested (24). Osx1-GFP calvaria cells from Foxo1,-3,-4f/f;Osx1-Cre mice exhibited increased expression of several Wnt-target genes including cyclin D1, Cnx43, Col6a1, and Mmp16 (Figure 5A). Recombinant Wnt3a increased the expression of the same genes in cells from wild-type mice, establishing that these are indeed bona fide Wnt-target genes in osteoblastic cells (Figure 5B). The increase in cyclin D1 mRNA was confirmed in bone as well as in periosteal and bone marrow cell cultures from Foxo1,-3,-4f/f;Osx1-Cre mice (Figure 5C).
Wnt/TCF signaling is responsible for increased osteoprogenitor proliferation. (A) Wnt-target genes determined by microarray analysis in Osx1-GFP calvaria cells. (B) mRNA levels by qRT-PCR in calvaria cells treated with vehicle (veh) or 50 ng/ml Wnt3a overnight (triplicates). (C) mRNA levels by qRT-PCR in femoral shafts from 3-month-old mice (n = 8–9/group), and in periosteal and bone marrow osteoblastic cell cultures described in Figure 4, C and G. (D) mRNA by qRT-PCR (left, triplicates) and protein levels by Western blot (right) in calvaria-derived cells infected with Adeno-Cre to induce Foxo deletion. (E) BrdU incorporation in cells, described in D, treated with veh (–) or 50 ng/ml Wnt3a (triplicates). (F) Lysates from nuclear extracts of calvaria cells, described in D, treated with vehicle or 50 ng/ml Wnt3a, immunoprecipitated with an anti–TCF-4 or anti-IgG antibody, and probed with an anti–β-catenin antibody. (G) mRNA levels by qRT-PCR and (H) BrdU incorporation in calvaria cells infected with Adeno-Cre and with lentivirus expressing shRNA directed against β-catenin or nontargeted shRNA followed by treatment with vehicle or 50 ng/ml Wnt3a overnight (triplicates). #P < 0.05 by Student’s t test; *P < 0.05 by ANOVA with Bonferroni’s test. Bars represent mean + SD.
As was the case with cells from Foxo1,-3,-4f/f;Osx1-Cre mice, the expression of cyclin D1 was increased following Foxo deletion using Adeno-Cre (Ad-Cre) in vitro (Figure 5D). The Cre recombinase efficiently reduced Foxo1, -3, and -4 mRNA and protein levels by more than 60%. Deletion of Foxos with Ad-Cre or treatment with Wnt3a also increased the proliferation of calvaria cells (Figure 5E). Having established that Ad-Cre deletion recapitulated the effects of the in vivo triple Foxo deletion on cyclin D1 and proliferation, we determined whether the availability of β-catenin for TCF binding increased in the absence of FOXOs. To do this, we immunoprecipitated β-catenin from control or _Foxo_-deleted calvaria cells and assessed the amount of TCF-4 bound to β-catenin in nuclear extracts (Figure 5F) or in total cell lysates (Supplemental Figure 7). The abundance of total β-catenin and TCF-4 did not differ between the 2 genotypes. However, the amount of β-catenin associated with TCF-4 increased in the absence of Foxos. As expected, Wnt3a promoted the association of β-catenin with TCF-4 in cells from wild-type mice. We then examined the contribution of β-catenin activity to the increased expression of cyclin D1 and proliferation by silencing β-catenin (Supplemental Figure 8A). shRNA-mediated knockdown of β-catenin prevented the Wnt3a-induced increase in cyclin D1 (Figure 5G). Silencing of β-catenin also abrogated the increased cyclin D1 expression in _Foxo_-deleted cells. In agreement with these findings, silencing of β-catenin prevented Wnt3A-induced proliferation as well as the increased proliferation seen in _Foxo_-deleted osteoblastic cells (Figure 5H). These results, nonetheless, could not exclude the possibility that FOXOs attenuated Wnt signaling by upregulating the expression of Wnt-signaling inhibitors, such as DKK1, SOST, SFRP1, and SFRP2 (21, 32). To address this alternative mechanism, we searched for and found that the expression of all these Wnt-signaling inhibitors was unaffected in bone or calvaria cells from Foxo1,-3,-4f/f;Osx1-Cre mice (Supplemental Figure 8B and Supplemental Table 3). Similarly, deletion of Foxos did not alter the expression of several members of the Wnt protein family, except for a decrease in Wnt5a (Supplemental Table 3). Taken together, these results support the idea that FOXOs inhibit osteoprogenitor cell proliferation by attenuating the expression of the β-catenin/TCF-target cyclin D1.
Deletion of Foxos increases marrow adiposity in old age. Finally, consistent with the evidence that Wnt/β-catenin signaling is a potent suppressor of adipogenesis (6) and that the Osx1-Cre transgene is active in cells capable of becoming either osteoblasts or adipocytes (33), the number of adipocytes present in the bone marrow was decreased in 24-month-old Foxo1,-3,-4f/f;Osx1-Cre mice as compared with the littermate controls (Figure 6A). In addition, the number of adipocytes formed in response to rosiglitazone in cultured bone marrow stromal cells from Foxo1,-3,-4f/f;Osx1-Cre mice was reduced (Figure 6B) as was the expression of the adipocyte markers _Ppar_γ and Fabp4 (Figure 6C).
Foxo deletion decreases bone marrow adipogenesis. (A) Adipocytes (arrow) in distal femurs of 24 month-old mice (n = 6/group) were quantified by histomorphometry of decalcified sections stained with H&E. Scale bar: 20 μm. (B) Number of adipocytes, stained with oil red O, in bone marrow stromal cells, cultured for 6 days with 1 μM rosiglitazone (triplicates). (C) mRNA levels of the indicated genes by qRT-PCR in cells cultured as described in B. Bars represent mean + SD. *P < 0.05 by Student’s t test.