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First published online 5 June 2007
doi: 10.1242/jcs.03466

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Smad4 is required for maintaining normal murine postnatal bone homeostasis

Xiaohong Tan^1,*, Tujun Weng^1,*, Jishuai Zhang¹, Jian Wang¹, Wenlong Li¹, Haifeng Wan¹, Yu Lan¹, Xuan Cheng¹, Ning Hou¹, Haihong Liu², Jun Ding³, Fuyu Lin¹, Ruifu Yang², Xiang Gao³, Di Chen⁴ and Xiao Yang^1,

¹ Genetic Laboratory of Development and Disease, Institute of Biotechnology, Beijing 100071, P.R. China
² Laboratory of Analytical Microbiology, Institute of Microbiology and Epidemiology, Beijing 100071, P.R. China
³ Model Animal Research Center, Nanjing University, P.R. China
⁴ Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY, USA

View larger version (53K): [in this window] [in a new window]   Fig. 1. Targeted disruption of Smad4 in mouse osteoblasts. (A-I) Characterization of Cre activity in 6-week-old OC-Cre;ROSA26 double transgenic mice. LacZ staining was detected in osteoblasts on trabecular bone (A), under the periosteum (B) and in osteocytes embedded in cortical bone (C). (D-I) Primary bone marrow cells of OC-Cre/ROSA26 double heterozygous (D-F) and ROSA26 (G-I) mice cultured for 12 days were subjected to LacZ staining (D,E,G,H) or Alizarin Red staining (F,I). E and H were higher magnification of areas in D and G, respectively. (J) Southern blot showing Cre-mediated recombination in bone tissues. The 4.3 kb fragment stems from the Smad4 conditional allele, the 7.2 kb fragment stems from the Smad4 allele after Cre-mediated recombination in osteoblasts. (K) Southern, using the probe previously described (Zhang et al., 2005), blot of Smad4 mutant calvarial genomic DNA at different ages. (L-M) Blockage of BMP and TGF signaling. Primary Smad4Co/Co osteoblasts were transfected by pCMV2 and pCMV2-Cre vector together with BMP reporter construct, 12xSBE-OC-Luc (L) or TGF reporter construct, 3TP-Lux (M), and then treated with 50 ng/ml BMP-2 (L) or 5 ng/ml TGF1 (M). Inactivation of Smad4 blocked luciferase activity stimulated by BMP-2 and TGF1. *P<0.01. Bar, 25 µm (A-C).

View larger version (54K): [in this window] [in a new window]   Fig. 2. Inactivation of Smad4 in differentiated osteoblasts caused growth retardation and reduced bone density. (A) Smad4 mutant (Cre/Co/+ and Cre/Co/Co) and control (Co/Co) mice at 6 weeks of age showing impairment of bone growth in Smad4 mutants. (B) Soft X-ray images of femurs and tibias from 6-week-old female mice. (C) Significant loss of total bone density in Smad4 mutant mice. (D-G) Histological analysis of tibias and calvarial bone in Smad4 mutant and control mice. Sections of tibia (D,E) and lambda sagittal sutures (F,G) from 7-week-old Smad4 mutant (E,G) and control mice (D,F). *P<0.01. Bar, 350 µm (D,E); 180 µm (F,G).

View larger version (73K): [in this window] [in a new window]   Fig. 3. Decreased bone formation in Smad4 mutant mice. (A,B) Von Kossa staining of the proximal tibia sections of Smad4 mutant (B) and control mice (A). (C,D) Representative images of the femora of 4-week-old male Smad4 mutant and control mice labeled with sequential doses of calcein. (E,F) Formation of mineralized ECM in neonatal calvarial osteoblast cultures from Smad4 mutant and control mice. Mineralized nodules were stained with Alizarin Red. (G-L) Quantitative histomorphometric measurements were performed on the secondary spongiosa at the distal femur of 8-week-old male mice. (G) BV/TV, bone volume/tissue volume; (H) TbN, trabecular number; (I) TbSp, trabecular separation; (J) ObS/BS, osteoblast surface/bone surface; (K) MAR, mineral apposition rate; (L) BFR/BS, bone formation rate/bone surface. All values are mean ± s.d. from six control (white bars) or Smad4 mutant mice (grey bars). *P<0.05; **P<0.01. Bar, 100 µm (A,B); 6.25 µm (C,D).

View larger version (37K): [in this window] [in a new window]   Fig. 4. Reduced production of bone extracellular matrix and decreased osteoblast proliferation in Smad4 mutant mice. (A-D) In situ hybridization of proximal tibias from 4-week-old control (A,C) or mutant mice (B,D). The expression of (B) osteopontin (OPN) and (D) osteocalcin (OC) was decreased in Smad4 mutant mice compared with controls (A,C). (E-H) Real-time PCR of Akp1 (ALP), osteocalcin, Col1a2 and Runx2 from calvarial bone extracts of 7-week-old Smad4 mutant (grey bars) and control mice (white bars). Values are presented as relative expression. (I) Sections of calvaria from 3-week-old mice showing a reduced number of BrdU-positive cells (arrowhead) in Smad4 mutant mice compared with control littermates. (J) Osteoblast mitotic index (percentage of BrdU-positive cells per total cell number) in control (white bars) and Smad4 mutant mice (grey bars). *P<0.01. Bar, 400 µm (A-D); 26.5 µm (I).

View larger version (46K): [in this window] [in a new window]   Fig. 5. Increased trabecular bone volume in old Smad4 mutant mice. (A-D) Sections of tibias (A,B) and sagittal sutures (C,D) of 1-year-old Smad4 mutant (B,D) and control mice (A,C). (E-H) Quantitative histomorphometric measurements were performed on the spongiosa at the proximal tibias of 11-month-old female mice. All values were mean ± s.d. of five control (white bars) or Smad4 mutant mice (grey bars). *P<0.05; **P<0.01. Bar, 350 µm (A,B); 100 µm (C,D).

View larger version (57K): [in this window] [in a new window]   Fig. 6. Decreased bone resorption in Smad4 mutant mice. (A,B) Representative images of TRAP-stained distal femur from 8-week-old control (A) and Smad4 mutant mice (B). (C,D) Quantitative assessment by histomorphometric analysis of percentage of bone surface covered by mature osteoclasts (OcS/BS) and number of mature osteoclasts in bone perimeter (NOc/BPm) on the secondary spongiosa of distal femur in control (white bars) and Smad4 mutants (grey bars). All values are mean ± s.d. of eight mice. (E,F) Real-time PCR of TRAP and cathepsin K (CathK) from calvarial bone extracts of 7-week-old Smad4 mutant (grey bars) and control mice (white bars). (G-I) TRAP staining was performed following co-culture of osteoclast progenitor cells derived from the wild-type spleen with control (upper panel in G,H) or Smad4 mutant (lower panel in G,I) osteoblast-like cells generated from bone marrow mesenchymal stem cells. (J) RT-PCR analysis to detect transcripts of OPG, RANKL, Tgfb1, Bmp2 and Bmp4 in calvarial bones of 7-week-old Smad4 mutant and control mice. (K-N) Real-time PCR of OPG, RANKL, Tgfb1 and Bmp4 from calvarial bone extracts of 7-week-old Smad4 mutant (grey bars) and control mice (white bars) *P<0.05; **P<0.01. Bar, 100 µm (A,B).