QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 24 July 2007
doi: 10.1242/jcs.003855

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nissen-Meyer, L. S. H. Articles by Gautvik, K. M. Search for Related Content PubMed PubMed Citation Articles by Nissen-Meyer, L. S. H. Articles by Gautvik, K. M.

Osteopenia, decreased bone formation and impaired osteoblast development in Sox4 heterozygous mice

Lise Sofie Haug Nissen-Meyer^1,*, Rune Jemtland², Vigdis T. Gautvik¹, Mona E. Pedersen¹, Rita Paro^1,, Dario Fortunati¹, Dominique D. Pierroz³, Vincent A. Stadelmann⁴, Sjur Reppe¹, Finn P. Reinholt⁵, Andrea Del Fattore⁶, Nadia Rucci⁶, Anna Teti⁶, Serge Ferrari³ and Kaare M. Gautvik^1,7,

¹ Department of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
² Endocrine Section, Department of Medicine, University of Oslo, Rikshospitalet-Radiumhospitalet Medical Centre, N-0027 Oslo, Norway
³ Service of Bone Diseases, WHO Collaborating Center for Osteoporosis Prevention, Geneva University Hospital, 1211 Geneva, Switzerland
⁴ Laboratory of Biomechanical Orthopedics, EPFL-HOSR, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland
⁵ Institute of Pathology, University of Oslo, and The Pathology Clinic, Rikshospitalet-Radiumhospitalet Medical Centre, N-0027 Oslo, Norway
⁶ Department of Experimental Medicine, University of L'Aquila, 67100 L'Aquila, Italy
⁷ Department of Clinical Chemistry, Ullevål University Hospital, N-0407 Oslo, Norway

View larger version (27K): [in this window] [in a new window]   Fig. 1. Bone densitometric, morphological and biomechanical characteristics of Sox4+/– mice compared with WT. (A) BMC-changes with age measured with DXA in WT and Sox4+/– mice (n=11-24). Mean age for each measurement: 48, 70, 104, 134, 168, 182, 222, 260, 300, 335 and 374 days. The growth curves for each genotype in both genders (corrected for body weight and bone area) were significantly different (P<0.001) throughout the observation period. Plots of mean BMC ± s.d. (WT males and females: n=24; Sox4+/– males: n=11, Sox4+/– females: n=14). Error bars on one side were omitted for clarity. (B) Microarchitecture of diaphyseal and metaphyseal bone from WT and Sox4+/– mice analyzed by µCT. Typical example of femurs from 3-month-old males, left: diaphysis (cortical bone), right: methaphysis (trabecular bone). (C) Cortical bone volume (Cort. BV), cortical thickness, trabecular bone volume (Trab. BV/TV, %) and trabecular thickness in 3-month-old males. Bars, mean ± s.d. (WT: n=11, open bars; Sox4+/–: n=8, solid bars). For cortical thickness, P=0.052. (D) Dynamic assessment of mineral acquisition rate (MAR) in WT and Sox4+/– mice. Fluoroscopic microphotograph showing fluorochrome labeling in a 3-month-old female injected with Alizarin (red) and calcein (green) 10 and 3 days before sacrifice, respectively. Distance between arrows: MAR during 7 days (see Table 3 for quantitative analysis). (E) Bone formation rate/bone surface/year (BFR/BS/year) in 3-month-old males treated as described in D. Bars, mean ± s.d. (WT: n=5, open bars; Sox4+/–: n=4, solid bars). (F) Biomechanical properties: moment of inertia (Ix) and stiffness in femurs from 3-month-old males, presented as mean ± s.d. (WT: n=11, open bars; Sox4+/–: n=8, solid bars). ***P<0.001; *P<0.05 compared with WT.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Primary cultures of calvarial osteoblasts derived from Sox4+/– and WT mice. (A) Real-time PCR analyses of osteoblast characteristic mRNAs extracted from Sox4+/– and WT primary osteoblast cultures: Osx, osterix; OCN, osteocalcin; Coll1A2, collagen1A2; ALP, alkaline phosphatase; Runx2; BMP-2, bone morphogenetic protein-2; OPN, osteopontin; PTHR1, PTH/PTHrP-receptor-1; PTHrP, PTH-related peptide. Results (from three independent experiments) were normalized to -actin or GAPDH, and expression in Sox4+/– cells is presented relative to WT (set to 1, broken line). (B) Representative immunoblot of lysates from WT and Sox4+/– osteoblasts, using anti-Sox4 and anti-actin antibodies as described in the Materials and Methods. (C) Representative micrographs of ALP histochemical staining (20x magnification) in WT and Sox4+/– osteoblasts cultured for 7 days (n=4). (D) Biochemical quantification of ALP activity in the osteoblast cultures shown in C. (E) Micrographs of von Kossa-stained osteoblast cultures (20x magnification) demonstrating mineralization (dark areas) of nodules after 3 weeks of culture in medium containing ascorbic acid and -glycerophosphate. (F) Densitometric quantification of von Kossa-stained mineralized nodules expressed as per cent of WT staining. In each of three independent experiments, 4-10 representative microscopic fields were examined. (G) Incorporation of [3H]thymidine in proliferating osteoblasts. Data from two independent experiments were normalized and compared with mean WT values (n=24 for each genotype). Bars, mean ± s.d. ***P<0.001, *P<0.05, unpaired Student's t-test (with Welch correction in F).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Silencing of Sox4 mRNA in WT primary osteoblast cultures with siRNA. (A) Real-time RT-PCR analyses of mRNA levels in siRNA-treated osteoblasts expressed relative to osteoblasts treated with control (scrambled) siRNA (set to 1, broken line), and normalized to GAPDH mRNA. Osx, osterix; OCN, osteocalcin; Coll1A2, collagen1A2; ALP, alkaline phosphatase; Runx2; OPN, osteopontin; PTHR1, PTH/PTHrP-receptor-1; PTHrP, PTH-related peptide. (B) Incorporation of [3H]thymidine in proliferating WT osteoblasts following treatment with control or Sox4 siRNA, respectively. (C) Photomicrographs of WT osteoblast cultures treated with control or Sox4 siRNA, respectively, stained for ALP activity. (D) Quantification of ALP-positive cells per well in C. (E) Biochemical activity of ALP in siRNA-treated osteoblasts. (F) mRNA levels of Runx2, OCN and Sox4 following treatment of WT cells with specific Runx2 siRNA compared with control siRNA, quantified by real-time RT-PCR and related to GAPDH mRNA as control (set to 1, broken line). In A,B,D-F: ***P<0.001, *P<0.05 versus siRNA control (ctrl); bars, mean ± s.d. of triplicates (n=2).

View larger version (6K): [in this window] [in a new window]   Fig. 4. Proposed model of Sox4 regulation of osteoblast proliferation and differentiation. Represented is the hierarchy of the osteoblast lineage and the predicted Sox4 actions. According to the experimental data, Sox4 could affect proliferation of osteoprogenitors as well as differentiation of mature osteoblasts. This latter action could be exerted by downregulation of osterix, with a mechanism apparently independent of Runx2.