Wnt4 signaling prevents skeletal aging and inflammation by inhibiting nuclear factor-κB

Abstract

Aging-related bone loss and osteoporosis affect millions of people worldwide. Chronic inflammation associated with aging promotes bone resorption and impairs bone formation. Here we show that Wnt4 attenuates bone loss in osteoporosis and skeletal aging mouse models by inhibiting nuclear factor-κB (NF-κB) via noncanonical Wnt signaling. Transgenic mice expressing Wnt4 from osteoblasts were significantly protected from bone loss and chronic inflammation induced by ovariectomy, tumor necrosis factor or natural aging. In addition to promoting bone formation, Wnt4 inhibited osteoclast formation and bone resorption. Mechanistically, Wnt4 inhibited NF-κB activation mediated by transforming growth factor-β-activated kinase-1 (Tak1) in macrophages and osteoclast precursors independently of β-catenin. Moreover, recombinant Wnt4 alleviated bone loss and inflammation by inhibiting NF-κB in vivo in mouse models of bone disease. Given its dual role in promoting bone formation and inhibiting bone resorption, our results suggest that Wnt4 signaling could be an attractive therapeutic target for treating osteoporosis and preventing skeletal aging.

Figure 1

Wnt4 promotes postnatal bone formation in vivo. (a) Western blot showing Wnt4 expression in primary calvarial cells extracted from WT and Wnt4 mice following osteogenic induction. (b) RT-PCR analysis of Wnt4 mRNA expression in various tissues and organs. (c–e) µCT reconstruction (c), BMD (d) as well as BV/TV (e) of metaphysis regions of distal femurs from 1, 2, and 3-month-old WT and Wnt4 mice. Scale bars, 200 µm; n = 12 per group. (f) H&E staining of femur sections from 1, 2, and 3-month-old WT (n = 8 per group) and Wnt4 mice (n = 10 per group). Scale bars, 300 µm. Ob.S, osteoblast surface. Ob.N, osteoblast number. BS, bone surface. (g) Histomorphometric analysis of osteoblast counts in 3-month-old Wnt4 (n = 10) vs WT mice (n = 8). (h) BFR and MAR measurements from dual-fluorescent calcein labeling of 3-month-old Wnt4 (n = 10) vs WT mice (n = 8). (i) ALP staining of femur bone marrow MSCs from Wnt4 vs WT mice, after osteogenic induction. (j) ARS of MSCs from Wnt4 vs WT mice after osteogenic induction. * P < 0.05, unpaired two-tailed t-test.

Figure 2

Wnt4 attenuates osteoporosis induced by OVX. (a,b) µCT reconstruction (a) of metaphysis of distal femurs, as well as BMD and BV/TV (b) in WT vs Wnt4 mice at two months post OVX. Scale bars, 200 µm. (c) BFR measurement of calcein dual labeling in WT vs Wnt4 mice two months after OVX or sham operation. (d, e) Morphometric analysis of osteoblast counts (d) and osteoclast counts (e) in WT vs Wnt4 mice after OVX or sham operation. (f) TRAP staining of femur sections from WT and Wnt4 mice after OVX or sham operation. (g–i) ELISA of serum concentrations of Ocn (g), Trap5b (h), Il-6 and Tnf (i) in WT vs Wnt4 mice after OVX or sham operation. Scale bars, 30µm. (j) Immunostaining and quantification of active p65 in trabecular bone cells and surrounding bone marrow cells in WT and Wnt4 mice after OVX or sham operation. Scale bars, 30 µm. IOD, integral optical density. For b–e, and g–j, n = 8 for sham groups; n = 12 for OVX groups. *P < 0.05, ** P < 0.01, unpaired two-tailed t-test.

Figure 3

Wnt4 inhibits TNF-induced bone loss and NF-κB activation. (a,b) µCT reconstruction (a), BMD and BV/TV (b) of distal femoral metaphysis regions from WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. Scale bars, 200 µm. (c) Comparisons of MAR and BFR in TNFtg mice and TNFtg/Wnt4 mice. (d,e) Morphometric analysis of osteoblast counts (d) and osteoclast counts (e) in TNFtg mice and TNFtg/Wnt4 mice. (f) TRAP staining of osteoclasts surrounding trabecular bones in WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. Scale bars, 40 µm. (g–i) ELISA of Ocn (g), Trap5b (h) and Il-6 (i) concentrations in serum collected from WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. (j) Immunostaining with anti-active p65 and quantification of NF-κB activity surrounding the trabecular bone in WT, Wnt4, TNFtg and TNFtg/Wnt4 mice. Scale bars, 40µm. TNF, TNFtg mice; T/W4, TNFtg/Wnt4 mice. For b–e, and g–j, n = 6 per group for WT and WNT4 mice; n = 8 per group for TNFtg and TNFtg/Wnt mice. *P < 0.05, ** P < 0.01, unpaired two-tailed t-test.

Figure 4

Wnt4 attenuates skeletal aging by inhibiting NF-κB. (a–c) µCT reconstruction (a), BMD and BV/TV (b), as well as H&E staining (c) of distal femoral metaphysis regions from 6-, 18- and 24-months-old WT and Wnt4 mice. Scale bars, 200 µm (a); 300 µm (c). (d) Morphometric analysis of osteoblast counts in distal femoral metaphysis from 3-, 18- and 24-months-old WT and Wnt4 mice. (e) ELISA of Ocn concentrations in serum from 3-, 18- and 24-months-old WT and Wnt4 mice. (f) Morphometric analysis of osteoclast counts in distal femoral metaphysis from 3-, 18- and 24-months-old WT and Wnt4 mice. (g,h) ELISA of Trap5b (g) and Il-6 (h) concentrations in serum from 3-, 18- and 24-months-old WT and Wnt4 mice. (i) Immunostaining with anti-active p65 and quantification of NF-κB activity surrounding the trabecular bones from 24-months-old WT and Wnt4 mice. Scale bars, 25 µm. For b, and d–i, n = 12 mice per group. *P < 0.05, ** P < 0.01, unpaired two-tailed t-test.

Figure 5

Wnt4 inhibits NF-κB by interfering with TAK1-TRAF6 binding. (a) Immunoblots showing the phosphorylation of Tak1, p65 and Iκbα in bone marrow macrophages after treatment of Rankl, rWnt4 and rWnt4 with Rankl. (b) Immunoblots showing p65 and Tata-binding protein (Tbp) in nuclear extracts of bone marrow macrophages treated with Rankl, rWnt4 and rWnt4 with Rankl. (c) Relative NF-κB-dependent luciferase reporter activities in bone marrow macrophages after treatment of Rankl, rWnt4 and rWnt4 with Rankl. (d) Immunoblots showing the Traf6-Tak1-Tab2 complex formation induced by Rankl in bone marrow macrophages. (e) Immunoblots showing the induction of Nfatc1 in bone marrow macrophages after treatment of Rankl, and rWnt4 with Rankl. (f) ChIP assays of the recruitment of p65 to the Nfatc1 promoter induced by Rankl. Anti-IgG and primers designed at 9 kb downstream of transcription start site (TSS) were used as negative control. (g) ChIP assays of Nfatc1 binding to the Nfatc1 promoter. (h) Immunoblots of β-catenin in cytosolic extract (CE) and nuclear extract (NE) of bone marrow macrophages treated with Wnt3a and Wnt4. (i) Relative Topflash luciferase activities in bone marrow macrophages treated with Wnt3a or Wnt4. (j) Real-time RT-PCR of Axin2 and Dkk1 in bone marrow macrophages treated with Wnt3a or Wnt4. n = 3; * P < 0.05; ** P < 0.01; unpaired two-tailed t-test.

Figure 6

rWnt4 proteins attenuates established bone loss by inhibiting NF-κB. (a–c) µCT reconstruction (a), BMD and BV/TV (b), as well as H&E staining (c) of distal femoral metaphysis regions from mice after sham operation, OVX and OVX with rWnt4 injection. Scale bars, 200 µm (a); 300 µm (c). (d,e) Morphometric analysis of osteoblast (d) and osteoclast (e) counts in distal femoral metaphysis from mice after sham operation, OVX and OVX with rWnt4 injection. (f) TRAP staining showing osteoclasts surrounding trabecular bones in mice after sham operation, OVX and OVX with rWnt4 injection. Scale bars, 30µm. (g,h) ELISA of Trap5b (g) and Ocn (h) concentrations in serum from mice after sham operation, OVX and OVX with rWnt4 injection. (i) Immunostaining with anti-active p65 and quantification of NF-κB activity surrounding the trabecular bones from mice after sham operation, OVX and OVX with rWnt4 injection. Scale bars, 30 µm. (j) ELISA of Il-6 and Tnf concentrations in serum from mice after sham operation, OVX and OVX with rWnt4 injection. n = 8 mice for sham group; n = 12 mice per group for mice receiving OVX and OVX with rWnt4 injection. *P < 0.05, ** P < 0.01, unpaired two-tailed t-test.