WNT7B promotes bone formation in part through mTORC1

Abstract

WNT signaling has been implicated in both embryonic and postnatal bone formation. However, the pertinent WNT ligands and their downstream signaling mechanisms are not well understood. To investigate the osteogenic capacity of WNT7B and WNT5A, both normally expressed in the developing bone, we engineered mouse strains to express either protein in a Cre-dependent manner. Targeted induction of WNT7B, but not WNT5A, in the osteoblast lineage dramatically enhanced bone mass due to increased osteoblast number and activity; this phenotype began in the late-stage embryo and intensified postnatally. Similarly, postnatal induction of WNT7B in Runx2-lineage cells greatly stimulated bone formation. WNT7B activated mTORC1 through PI3K-AKT signaling. Genetic disruption of mTORC1 signaling by deleting Raptor in the osteoblast lineage alleviated the WNT7B-induced high-bone-mass phenotype. Thus, WNT7B promotes bone formation in part through mTORC1 activation.

Figure 1. WNT7B, but not WNT5A, increases bone mass in vivo.

(A) A schematic for generating mice with Cre-dependent overexpression of WNT7B or WNT5A. (B–C) X-ray images of hindlimbs of two-month-old ColI-Cre (Ctrl) (B) or ColI-Wnt5a littermate mice (C). (D–G) X-ray images of the axial skeleton (D, E) and hindlimbs (F, G) of two-month-old ColI-Cre (Ctrl) (D, F) versus ColI-Wnt7b littermate mice (E, G). Arrows denote increased mineral density in sterna, ribs and spine. (H–I) µCT 3D reconstruction of skulls from two-month-old ColI-Cre (Ctrl) (H) or ColI-Wnt7b littermate mice (I). H1, H2, I1, I2 show a single-slice µCT scan at positions indicated by the red or green line. (J, K) µCT 3D reconstruction of tibias from two-month-old ColI-Cre (Ctrl) (J) or ColI-Wnt7b littermate mice (K).

Figure 2. WNT7B increases osteoblast number and activity.

(A, B) H&E staining of longitudinal tibia sections from two-month-old control (A) or ColI-Wnt7b littermates (B). 1°, 2°: primary and secondary ossification center. Shown to the right are higher magnification images of secondary ossification center (A1, B1), growth plate (A2, B2), primary spongiosa (A3, B3), and marrow region (A4, B4). Scale bar: 0.5 mm in panels A, B; 0.1 mm in panels A1–A4, B1–B4. (C) Serum osteocalcin levels of control (C) and ColI-Wnt7b littermates (7b) at one and two months of age. (D) Number of osteoblasts normalized to trabecular bone perimeter on longitudinal tibia sections. (E–F) Representative images of calcein double labeling in the humerus of two-month-old control (E) and ColI-Wnt7b (F) littermates. (G) Dynamic histomorphometry parameters from secondary ossification center of the humerus. MAR: mineral apposite rate; MS/BS: mineralizing surface over bone surface; BFR/BS: bone formation rate. (H) Serum CTX-I levels. (I) Osteoclast parameters from histomorphometry. #Oc/mm: osteoclast number normalized to trabecular bone perimeter, µm/Oc (average osteoclast surface), Oc S/BS (osteoclast surface normalized to bone surface). All bar graphs show mean ± STDEV, *: P<0.05, n = 3.

Figure 3. WNT7B stimulates bone acquisition in the embryo.

(A) Whole-mount skeletal staining at E18.5. Arrows denote more bone in skull and limbs of ColI-Wnt7b embryos. (B) H&E staining of longitudinal tibial sections at E18.5. Shown below are images of the diaphyseal region at a higher magnification. (C, D) Analyses of longitudinal sections of the humerus at E14.5 (C) and E16.5 (D) by histology and in situ hybridization. (E) Immunostaining of GFP and CD31 on longitudinal sections of the humerus in E16.5 Osx-Wnt7b embryos. GFP: green; CD31: red; DAPI: blue. (F) Analyses of longitudinal sections of the humerus at E18.5 by histology and in situ hybridization. In situ hybridization signals shown in red.

Figure 4. Generation and characterization of Runx2-rtTA transgenic mice.

(A) A schematic for generating the Runx2-rtTA BAC transgenic mouse. (B–C) GFP imaging by fluorescence microscopy of whole-mount skeletal elements (left to right: skull, forelimb, ribs, hindlimb, vertebrae) from R26-mTmG (B) or Runx2-rtTA;TetO-cre;R26-mTmG (C) neonates treated with 1 mg/ml Dox in drinking water from E1.5 till birth. (D–G) Fluorescence imaging of frozen sections of the tibia from R26-mTmG (D, E) or Runx2-rtTA; TetO-Cre; R26-mTmG (F, G) neonates treated with 1 mg/ml Dox from E1.5 to birth. D, F: GFP single channel; E, G: GFP and RFP merged image. Boxed areas in G are shown at a higher magnification in G1 (growth plate), G2 (primary spongiosa) and G3 (diaphysis). (H–K) GFP detection on longitudinal tibial sections of R26-mTmG (H, I) or Runx2-rtTA;TetO-Cre;R26-mTmG (J,K) mice treated with 1 mg/ml Dox in drinking water for 15 days starting at 1 month of age. H, J: GFP immunofluorescence; I, K: merged images of GFP and RFP signals. RFP from direct fluorescence microscopy. Boxed areas in K are shown in K1 (primary spongiosa), K2 (cortical bones) and K3 (diaphyseal bone marrow). 1°, 2°: primary and secondary ossification center, respectively. Arrow: GFP⁺ bone marrow stromal cell; arrowhead: GFP⁺ perivascular cells.

Figure 5. WNT7B enhances bone accrual in postnatal life.

All data from Runx2-rtTA;TetO-Cre;R26-Wnt7b mice treated with (+Dox) or without (−Dox) 1 mg/ml Dox in drinking water from one month through two months of age. (A) X-ray images of hindlimbs. Arrows point to the places with increased bone mineral density. (B) µCT 3D reconstruction of metaphyseal trabecular bone of the tibia. (C) H&E staining of sections of the proximal tibias. (D) Serum CTX-I levels of two-month-old mice. (E) Histomorphometric parameters of osteoclasts on tibial sections. #Oc/mm: osteoclast number normalized to trabecular bone perimeter; Oc S/BS: osteoclast surface normalized to bone surface; µm/Oc: average osteoclast surface. (F) Number of osteoblasts normalized to trabecular bone perimeter on tibia sections. Bar graphs show mean ± STDEV, *: P<0.05, n = 3. f: femur; t: tibia; m: metatarsal.

Figure 6. WNT7B and WNT3A activate mTORC1 signaling.

(A) Transient transfection assays with luciferase reporter Lef1-luc in ST2 cells. IE: GFP-expressing control retrovirus; 7B: WNT7B-expressing retrovirus; V: vehicle (0.1% CHAPS in PBS); 3A: WNT3A. (B) Western blot with whole-cell lysates from ST2 cells infected with WNT7B or control (IE) retrovirus. Cells were serum-starved for 16 hours and then treated with inhibitor or vehicle for 2 hours before harvest. (C) Representative image (left) and quantification (right) of Western analyses with bone protein extracts from two-month-old Osx-Cre (Ctrl) and Osx-Wnt7b (7B) littermate mice. Levels of P-S6/S6 in control littermates designated 1. *: P<0.05, n = 3. (D) Western blot with whole-cell lysates from ST2 cells infected with WNT7B or control (IE) retrovirus. Cells were serum-starved for 16 hours and then treated with inhibitor or vehicle for 2 hours before harvest. (E) Western blot of total cell lysates from ST2 cells infected with lentivirus expressing shRNA for β-catenin or LacZ, followed by retroviral infection of WNT7B or GFP (IE). (F) Western blot of total cell lysates from serum-starved ST2 cells treated with WNT3A or vehicle (V) for 1 hour with or without inhibitors (with 1-hr pretreatment). Rapa: rapamycin. (G) Western blot of total cell lysates from serum-starved ST2 cells treated with WNT3A or vehicle (V) for 1 hour with or without DKK1 (with 1-hr pretreatment). (H–I) Effects of LRP5 and/or LRP6 knockdown with shRNA. ST2 cells infected with lentiviruses were serum-starved before WNT3A treatment for 1 hour. (H): representative Western blots; (I): quantification of pS6/S6 from three independent experiments, *: p<0.05. (J) Effects of GSK3 inhibition. Serum-starved ST2 cells were treated with WNT3A for 1 hour in the presence of LiCl or NaCl.

Figure 7. Removal of Raptor partially rescues WNT7B-induced bone formation.

All data from mice treated with Dox from E1.5 till one month of age, then weaned off Dox for three weeks immediately before harvest. (A–D) X-ray images of hindlimbs from Osx-Cre;Raptor^f/+ (A), Osx-Cre;Raptor^f/f (B), Osx-Cre;R26-Wnt7b;Raptor^f/+ (C), and Osx-Cre;R26-Wnt7b;Raptor^f/f mice (D). (E–H) µCT 3D reconstruction of tibias from Osx-Cre;Raptor^f/+ (E), Osx-Cre;Raptor^f/f (F), Osx-Cre;R26-Wnt7b;Raptor^f/+ (G), and Osx-Cre;R26-Wnt7b;Raptor^f/f mice (H). Shown below is mean ± STDEV of combined cortical and trabecular bone volume normalized to tissue volume (BV/TV%) from three mice of each genotype. See Experimental Procedures for details. *: p<0.05 between G and H. (I–L) H&E staining of longitudinal tibia sections from Osx-Cre;Raptor^f/+ (I), Osx-Cre;Raptor^f/f (J), Osx-Cre;R26-Wnt7b;Raptor^f/+ (K) and Osx-Cre;R26-Wnt7b;Raptor^f/f mice (L). (M) Western blot analysis of bone extracts from Osx-Cre;Raptor^f/+ (lane 1), Osx-Cre;Raptor^f/f (lane 2), Osx-Cre;R26-Wnt7b;Raptor^f/+ (lane 3), and Osx-Cre;R26-Wnt7b;Raptor^f/f mice (lane 4). (N) P-S6 immunohistochemistry on longitudinal sections of tibias from Osx-Cre;R26-Wnt7b;Raptor^f/+ (left) and Osx-Cre;R26-Wnt7b;Raptor^f/f mice (right). Shown below are images of a higher magnification for boxed areas (junction between growth plate and primary spongiosa). Signal in brown. (O) Representative images of calcein double labeling in tibias of Osx-Cre;Raptor^f/+ (1), Osx-Cre;Raptor^f/f (2), Osx-Cre;R26-Wnt7b;Raptor^f/+ (3) and Osx-Cre;R26-Wnt7b;Raptor^f/f mice (4). (P) Bone formation parameters from the primary ossification center. (Q) Number of osteoblasts normalized to trabecular bone perimeter (#Ob/mm) on tibia sections. (R) Osteoclast parameters. All bar graphs show mean ± STDEV, *: P<0.05, n = 3.