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First published online 19 September 2006
doi: 10.1242/jcs.03162

Journal of Cell Science 119, 4187-4198 (2006)
Published by The Company of Biologists 2006
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Low peak bone mass and attenuated anabolic response to parathyroid hormone in mice with an osteoblast-specific deletion of connexin43

Dong Jin Chung1,2,*, Charlles H. M. Castro1,3,*, Marcus Watkins1, Joseph P. Stains1,{ddagger}, Min Young Chung2, Vera Lucia Szejnfeld3, Klaus Willecke4, Martin Theis§ and Roberto Civitelli1,¶

1 Division of Bone and Mineral Diseases, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110, USA
2 Department of Internal Medicine, Chonnam University Research Institute of Medical Sciences, Chonnam National University Medical Gwangju, Republic of Korea
3 Universidade Federal de São Paulo-Escola Paulista de Medicina, São Paulo, Brasil
4 Institut für Genetik, Universität Bonn, Germany


Figure 1
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  		Fig. 1. Cre-mediated Cx43 gene deletion in osteoblasts. (A) PCR of genomic DNA extracted from bone (b) or tail (t) tissue revealed the presence of a 670-kb band corresponding to the Cx43-deleted allele selectively in the bone tissue extract. (B) Western blot of bone tissue extracts from mice of the different genotypes showing barely detectable Cx43 immunoreactive bands in Western blots of ColCre;Cx43–/fl tissue, contrasting with strong expression of Cx43 protein in wild-type equivalent littermates, and lower but detectable expression in heterozygous equivalent mice. (C) Quantitation of mRNA for Cre by real-time PCR, showing the presence of the transgene only in extracts of ColCre;Cx43–/fl bone (one femur). (D) Whole mounts of newborn mice revealing strong X-gal stain in areas corresponding to mineralized skeleton of ColCre;Cx43–/fl mice, whereas only very faint stain was observed in Cx43–/fl mice. (E) X-gal stained sections of the mid-shaft (upper left) and metaphysis (lower left) of the tibia, and of one lumbar vertebra (right) of ColCre;Cx43–/fl mice, showing blue stain in cells lining the bone surface in both skeletal sites, but not in bone marrow cells. Non-stained surfaces correspond to areas where the cell layer was artifactually detached from bone. F, female; M, male; Ob, osteoblasts; Ocy, osteocytes.

 

Figure 2
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  		Fig. 2. Low bone mass phenotype in conditionally Cx43 deleted mice. (A) Whole mount of alizarin red and alcian blue staining of newborn mice did not reveal any major skeletal abnormalities in ColCre;Cx43–/fl mice compared with their control littermates. (B) Lower body weight in ColCre;Cx43–/fl (n=46) relative to Cx43+/fl (n=69) and Cx43–/fl groups (n=42) at 6 months of age. (C) Whole-body BMD measured by DEXA monitored in vivo revealed significantly lower bone mass in ColCre;Cx43–/fl relative to wild-type littermates (P<0.05, for genotype effect; two-way ANOVA for repeated measures).

 

Figure 3
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  		Fig. 3. Histomorphometric characterization of osteopenia in conditionally osteoblast Cx43-deleted mice. (A) Trichrome Masson stain of 4 µm undecalcified sections of proximal tibiae from mice of the three different genotypes at 6 months. (B-G) Quantitative histomorphometry showing significantly lower bone volume/total volume, osteoblast number and trabecular thickness in the ColCre;Cx43–/fl, with non-statistically different trabecular number, mineral apposition rate and osteoclast number among the three genotype groups. *P<0.05 versus Cx43+/fl (one-way ANOVA); n=6 per genotype group.

 

Figure 4
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  		Fig. 4. Delayed differentiation of Cx43-deficient osteoblasts. (A) X-gal staining of post-confluent ColCre;Cx43–/fl calvaria cells grown in mineralizing medium, showing blue staining becoming stronger with time in culture. (B) Western analysis demonstrated barely detectable Cx43-specific bands in lysates of conditionally deleted cells, and reduced abundance of Cx43 in heterozygous equivalent cells. (C) Abundance of Cx43 mRNA, assessed by real-time PCR, was reduced in ColCre;Cx43–/fl calvarial cells relative to wild-type equivalent cells after 3 weeks in culture (n=3). (D) Development of alkaline phosphatase activity was significantly reduced in calvarial cells derived from ColCre;Cx43–/fl mice 2 weeks post-confluence (n=4). (E) The abundance of mRNA transcripts for other osteoblast-specific genes was reduced by more than 50%, as measured by real-time PCR, relative to wild-type equivalent cells (*P<0.05 versus Cx43+/fl; one-way ANOVA; n=3). (F) Representative results of von Kossa stain of post-confluent calvaria cells and (G) quantitative data on three different preparations showing delayed in vitro matrix mineralization by ColCre;Cx43–/fl calvaria cell cultures (*P<0.05 versus Cx43+/fl; Tukey test; n=3).

 

Figure 5
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  		Fig. 5. Attenuated BMC response to PTH in osteoblast Cx43-deficient mice. Percent change from baseline of whole body BMC after a 4-week treatment with different doses of teriparatide (PTH), showing a dose-related increase of bone mass in wild-type Cx43+/fl mice (A). Effects of lesser magnitude were observed in heterozygous equivalent Cx43–/fl mice at the intermediate doses (B), whereas the effect of PTH treatment was uniformly attenuated at all doses in ColCre;Cx43–/fl mice (C). *P<0.05, **P<0.01 versus vehicle (one-way ANOVA); n=6-8 per each data point. Data are mean ± s.e.m.; n=12 (vehicle) and 6-8 (PTH-treated groups).

 

Figure 6
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  		Fig. 6. Region-specific changes in BMC in osteoblast Cx43-deficient mice. (A) Percent change from baseline of whole body BMC after a 4-week treatment with 40 µg/kg PTH in a group of 7.4-9.6-month-old mice, showing an attenuated response in conditionally deleted ColCre;Cx43–/fl mice (n=10) relative to wild-type animals (n=15), whereas response in Cx43–/fl mice (n=11) was intermediate. (B) Response was absent at the lumbar spine in both ColCre;Cx43–/fl and heterozygous equivalent Cx43–/fl. (C) Significant increases in bone mass were instead detected in all genotypes on the total femoral area. **P<0.01, *P<0.05 versus vehicle (n=9); #P<0.05 versus ColCre;Cx43–/fl; one-way ANOVA.

 

Figure 7
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  		Fig. 7. Static bone histomorphometric analysis after a 4-week treatment with 40 µg/kg PTH. (A-C) Trichrome Masson stain of the proximal tibia showing less abundant trabecular bone mass in the conditional knockout (cKO) ColCre;Cx43–/fl relative to heterozygous equivalent (Het) Cx43–/fl and wild-type (WT) Cx43+/fl mice. (D) Robust anabolic response occurred in the WT group, and an attenuated response was observed in Het mice. By contrast, no significant increases in bone volume/total volume were detected in the conditionally deleted ColCre;Cx43–/fl mice relative to the other genotype groups. (E) Osteoblast number was significantly increased by PTH treatment in all groups. (F) Trabecular number was significantly increased in the wild-type groups only. (G) The same result was observed for trabecular thickness. (H) No significant changes were detected for cortical thickness. (I) Osteoclast number was not different among the different groups. *P<0.05 versus vehicle (ANOVA); n=9-15.

 

Figure 8
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  		Fig. 8. Dynamic bone histomorphometric analysis and osteoblast proliferation after a 4-week treatment with 40 µg/kg PTH. (A) Fluorescent micrographs (200x) of undecalcified sections of lumbar spine trabecular bone showing double calcein labels in both wild-type Cx43+/fl (WT) and heterozygous equivalent Cx43–/fl (Het) mice, but only single labels in conditional knockout ColCre;Cx43–/fl (cKO) mice. (B) Mineral apposition rate (MAR) in the lumbar spine was significantly lower in cKO ColCre;Cx43–/fl mice than in either HT or WT Cx43+/fl group (n=5-6). (C) Calcein labeling and (D) mineral apposition rate in the endosteal surface of the tibia showing attenuated response to PTH in cKO mice (n=5-6). (E) BrdU stain of endosteal tibial surface, showing positively stained cells equally in mice of all genotypes (n=3-4). (F) Osteoblast mitotic index, expressed as percentage of BrdU-labeled cells per total number of cells on the bone surface. *P<0.05 versus Cx43+/fl, one-way ANOVA.

 




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