Dual function of Bmpr1a signaling in restricting preosteoblast proliferation and stimulating osteoblast activity in mouse

Abstract

Exogenous bone morphogenetic proteins (Bmp) are well known to induce ectopic bone formation, but the physiological effect of Bmp signaling on normal bone is not completely understood. By deleting the receptor Bmpr1a in osteoblast lineage cells with Dmp1-Cre, we observed a dramatic increase in trabecular bone mass in postnatal mice, which was due to a marked increase in osteoblast number that was likely to be driven by hyperproliferation of Sp7(+) preosteoblasts. Similarly, inducible deletion of Bmpr1a in Sp7(+) cells specifically in postnatal mice increased trabecular bone mass. However, deletion of Smad4 by the same approaches had only a minor effect, indicating that Bmpr1a signaling suppresses trabecular bone formation through effectors beyond Smad4. Besides increasing osteoblast number in the trabecular bone, deletion of Bmpr1a by Dmp1-Cre also notably reduced osteoblast activity, resulting in attenuation of periosteal bone growth. The impairment in osteoblast activity correlated with reduced mTORC1 signaling in vivo, whereas inhibition of mTORC1 activity abolished the induction of protein anabolism genes by BMP2 treatment in vitro. Thus, physiological Bmpr1a signaling in bone exerts a dual function in both restricting preosteoblast proliferation and promoting osteoblast activity.

Keywords: Bmp; Bmpr1a; Mouse; Osteoblast; Smad4; mTORC1.

Fig. 1.

Dmp1-Cre targets osteoblast lineage cells in mice. (A-D) Representative fluorescence images of longitudinal sections of the tibia from Dmp1-Cre; mT/mG mice at the indicated ages. (A′-D′) Representative trabecular bone regions at higher magnification. (A″-D″) Representative cortical bone regions at higher magnification. Red, mTomato; green, mGFP; blue, DAPI staining of nuclei. Red and green protein fluorescence was captured directly.

Fig. 2.

Dmp1-Cre; Bmpr1a^f/f mice display higher trabecular bone mass but smaller bone diameters at P33. (A) Body weight measurements. (B-D) X-ray radiography of the hindlimb (B), calvarium (C) and sternum (D). Arrows in B denote trabecular bone region (red) or diaphysis of tibia (yellow). Red line in C denotes thickness of calvaria. Arrow in D denotes sternum. (E) μCT 3D reconstruction of trabecular bone. (F) μCT images of mid-diaphyseal region of the tibia. (G-L) μCT quantification of trabecular bone (G-J) or cortical bone (K,L). (M) Serum CTX-I measurement. WT, Bmpr1a^f/f; MT, Dmp1-Cre; Bmpr1a^f/f. *P<0.05, n=3. BV, bone volume; TV, total volume; Tb.N*, trabeculae number; Tb.Th*, trabeculae thickness; Tb.Sp*, trabeculae spacing; Cort.Th., cortical thickness; T.Ar., total cross-sectional area.

Fig. 3.

Bmpr1a deletion increases trabecular osteoblast number but reduces osteoblast activity. Mice were analyzed at P33 (A-H) or P60 (I-L). (A,A′) H&E staining of longitudinal sections of the femur. Blue or red boxes denote areas of secondary or primary ossification centers, respectively, shown at higher magnification beneath. (B,B′) Secondary ossification centers. (C,C′) Primary ossification centers. Insets show representative osteoblast morphology at higher magnification. (D,D′) H&E staining of longitudinal sections through the cortical bone of the femur. (E-G) Histomorphometric quantification of osteoblast number (Ob.N; E), osteocyte density (Os.Dn) in cortical bone (F) and osteoclast number (Oc.N; G), all in the femur. (H) Dynamic histomorphometry in the tibia. (I,K) Representative images of double labeling in the trabecular bone region (I) or periosteum (K) in the tibia. (J,L) Dynamic histomorphometric parameters in trabecular bone (J) or periosteum (L) in the tibia. WT, Bmpr1a^f/f; MT, Dmp1-Cre; Bmpr1a^f/f. *P<0.05, n=3. MS, mineralizing surface; BS, bone surface; MAR, mineral apposition rate.

Fig. 4.

Bmpr1a deletion increases the proliferation of Sp7⁺ preosteoblasts. (A) Representative images for BrdU staining of longitudinal femur sections at P33. (B) Quantification of BrdU-positive cells (%) at the chondro-osseous junction. (C) Representative images for EdU labeling (green) and Sp7 immunostaining (red). Area in red box is shown at higher magnification to the right. Arrows indicate Sp7⁺ EdU⁺ cells (yellow). GP, growth plate; M, marrow. (D) Quantification of Sp7⁺ EdU⁺ cells among total Sp7⁺ cells. WT, Bmpr1a^f/f; MT, Dmp1-Cre; Bmpr1a^f/f. *P<0.05, n=3.

Fig. 5.

Postnatal deletion of Bmpr1a by Sp7-Cre increases trabecular bone formation. (A) Direct fluorescence images of longitudinal sections of the tibia from Sp7-Cre; mT/mG mice with or without doxycycline (Dox) withdrawal (P21-P33) at low (top row) or high (bottom row) magnification. Red, mTomato; green, mGFP; blue, DAPI staining of nuclei. (B) X-ray radiography of the hindlimb bones from mice of genotype Sp7-Cre; Bmpr1a^f/f subjected to the same Dox regimen as above. (C) μCT 3D reconstruction of trabecular bone of the tibia. (D-F) μCT quantification of trabecular bone in the tibia. (G,H) Histomorphometric analyses of osteoblast and osteoclast numbers in trabecular bone of the femur. (I) Serum CTX-I measurements. +DOX, Sp7-Cre; Bmpr1a^f/f mice were maintained on Dox water from conception until sacrifice at P33. −DOX, Sp7-Cre; Bmpr1a^f/f mice were raised on Dox water from conception until P21, when they were weaned off Dox until sacrifice at P33. *P<0.05, n=3.

Fig. 6.

Deletion of Smad4 mildly affects trabecular bone mass. (A,B) Picrosirius Red staining of longitudinal sections of the tibia. (C) μCT quantification of trabecular bone of the tibia. −DOX, mice raised on Dox from conception until P21, and then weaned off Dox for 9 weeks before harvest. +DOX, mice maintained on Dox from conception until harvest at the same age as the −Dox mice. *P<0.05, n=3.

Fig. 7.

Bmp-mTORC1 signaling induces expression of protein anabolism genes. (A) Western blot of protein extracts from long bones at P33. Examples are shown for two mice with each genotype. (B) qRT-PCR with RNA isolated from calvaria at P33. (C) qRT-PCR with RNA isolated from Bmpr1a^f/f newborn calvarial cells infected with Ad-Cre or Ad-Gfp virus followed by vehicle or BMP2 treatment for 96 h. (D) Western blot in ST2 cells following 1 h treatment with vehicle (Veh) or BMP2. Phosphorylated ribosomal protein S6 (P-S6) is normalized to S6. Quantification (average with s.d.) is shown for three independent experiments. (E) qRT-PCR with RNA from ST2 cells treated with BMP2 or vehicle for 72 h with or without chemical inhibitors. *P<0.05, n=3. WT, Bmpr1a^f/f; MT, Dmp1-Cre; Bmpr1a^f/f.