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Cited by 148Bone Metabolism and DiseasesConnexins and lens biologyBone health and osteoporosis research

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Enhanced Osteoclastic Resorption and Responsiveness to Mechanical Load in Gap Junction Deficient Bone

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Zhang, Yue, Paul, Emmanuel M., Sathyendra, Vikram, Davison, Andrew, Sharkey, Neil, Bronson, Sarah, Srinivasan, Sundar, Gross, Ted S., Donahue, Henry J.·2011·PLoS ONE
DOI 10.1371/journal.pone.0023516PMC PMC3163577
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Abstract

Emerging evidence suggests that connexin mediated gap junctional intercellular communication contributes to many aspects of bone biology including bone development, maintenance of bone homeostasis and responsiveness of bone cells to diverse extracellular signals. Deletion of connexin 43, the predominant gap junction protein in bone, is embryonic lethal making it challenging to examine the role of connexin 43 in bone in vivo. However, transgenic murine models in which only osteocytes and osteoblasts are deficient in connexin 43, and which are fully viable, have recently been developed. Unfortunately, the bone phenotype of different connexin 43 deficient models has been variable. To address this issue, we used an osteocalcin driven Cre-lox system to create osteoblast and osteocyte specific connexin 43 deficient mice. These mice displayed bone loss as a result of increased bone resorption and osteoclastogenesis. The mechanism underlying this increased osteoclastogenesis included increases in the osteocytic, but not osteoblastic, RANKL/OPG ratio. Previous in vitro studies suggest that connexin 43 deficient bone cells are less responsive to biomechanical signals. Interestingly, and in contrast to in vitro studies, we found that connexin 43 deficient mice displayed an enhanced anabolic response to mechanical load. Our results suggest that transient inhibition of connexin 43 expression and gap junctional intercellular communication may prove a potentially powerful means of enhancing the anabolic response of bone to mechanical loading.

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Figures

Figure 1 Breeding of Cx43 deficient mice. A: Breeding strategy for developing osteoblast and osteocyte Cx43 deficient mice and assessment of their genotype. B: Using the primers described in Table 1, genomic DNA extracted from ears was amplified for genotyping.
Figure 1 Breeding of Cx43 deficient mice. A: Breeding strategy for developing osteoblast and osteocyte Cx43 deficient mice and assessment of their genotype. B: Using the primers described in Table 1, genomic DNA extracted from ears was amplified for genotyping.CC BY · PubMed Central
Figure 2 Confirmation of Cx43 deletion by X-gal staining. The figure shows X-gal staining of osteoblasts isolated from femurs and tibia of a wild type (Cx43flx/flx, WT) and a Cx43 deficient (OC-Cre;Cx43flx/flx, KO) mouse. Cells from wild type mice had no blue staining indicating that floxed Cx43 was not deleted in the absence of OC-Cre. Cells from a Cx43 deficient mouse were positive for β-galactodase indicating Cx43 deleted by Cre.
Figure 2 Confirmation of Cx43 deletion by X-gal staining. The figure shows X-gal staining of osteoblasts isolated from femurs and tibia of a wild type (Cx43flx/flx, WT) and a Cx43 deficient (OC-Cre;Cx43flx/flx, KO) mouse. Cells from wild type mice had no blue staining indicating that floxed Cx43 was not deleted in the absence of OC-Cre. Cells from a Cx43 deficient mouse were positive for β-galactodase indicating Cx43 deleted by Cre.CC BY · PubMed Central
Figure 3 Confirmation of Cx43 deletion by PCR. The figure shows PCR of genomic DNA extracted from bladder (bld), brain (brn), calvaria (cal), heart (ht), humerus (hm), intestine (int), liver (lv), muscle (msl), skin (sk), and spleen (spl) from a OC-Cre; Cx43flx/+ mouse. DNA was amplified using primers (Cx43delForw and Cx43delRev) flanking the junction between the intron of Cx43 and LacZ region. If Oc-Cre is activated the floxed Cx43 is deleted allowing amplification of a 670 bp product. If Oc-Cre is not activated and floxed Cx43 is not deleted, the template is too large to be amplified. We observed a 670 bp PCR product in calvaria and humerus indicating Cx43 is deleted. The same DNA was amplified using primers for Cre and Cx43 were used as positive control.
Figure 3 Confirmation of Cx43 deletion by PCR. The figure shows PCR of genomic DNA extracted from bladder (bld), brain (brn), calvaria (cal), heart (ht), humerus (hm), intestine (int), liver (lv), muscle (msl), skin (sk), and spleen (spl) from a OC-Cre; Cx43flx/+ mouse. DNA was amplified using primers (Cx43delForw and Cx43delRev) flanking the junction between the intron of Cx43 and LacZ region. If Oc-Cre is activated the floxed Cx43 is deleted allowing amplification of a 670 bp product. If Oc-Cre is not activated and floxed Cx43 is not deleted, the template is too large to be amplified. We observed a 670 bp PCR product in calvaria and humerus indicating Cx43 is deleted. The same DNA was amplified using primers for Cre and Cx43 were used as positive control.CC BY · PubMed Central
Figure 4 Quantification of GJIC in osteoblasts isolated from wild type (WT) and Cx43 deficient (KO) mice. Donor hFOB cells were placed in contact with a monolayer of acceptor cells isolated from femurs from wild type or Cx43 deficient mice. Bars represent the mean±SD number of acceptor cells to which dye transferred from each donor hFOB cells. n = 4 **p<0.01 vs wild type.
Figure 4 Quantification of GJIC in osteoblasts isolated from wild type (WT) and Cx43 deficient (KO) mice. Donor hFOB cells were placed in contact with a monolayer of acceptor cells isolated from femurs from wild type or Cx43 deficient mice. Bars represent the mean±SD number of acceptor cells to which dye transferred from each donor hFOB cells. n = 4 **p<0.01 vs wild type.CC BY · PubMed Central
Figure 5 Microstructure of the femur midshaft of wild type (WT) and Cx43 deficient mice (KO). μCT image of femoral midshaft of 8 week old wild type (A left) and Cx43 deficient mice (A right) mice. Periosteal and endocortical expansion is evident in Cx43 deficient mice relative to wild type. B–F: Quantification of μCT in 8 week old Cx43 deficient mice and wild type mice. Cx43 deficient mice displayed a significant decrease in BMD (B) and increase in periosteal (C) and endocortical volumes (D). Cx43 deficient mice displayed no change in cortical bone thickness (E, p = 0.054) or bone porosity (F, p = 0.55). n = 9 mice per group, ** p<0.01, Bar = 1 mm.
Figure 5 Microstructure of the femur midshaft of wild type (WT) and Cx43 deficient mice (KO). μCT image of femoral midshaft of 8 week old wild type (A left) and Cx43 deficient mice (A right) mice. Periosteal and endocortical expansion is evident in Cx43 deficient mice relative to wild type. B–F: Quantification of μCT in 8 week old Cx43 deficient mice and wild type mice. Cx43 deficient mice displayed a significant decrease in BMD (B) and increase in periosteal (C) and endocortical volumes (D). Cx43 deficient mice displayed no change in cortical bone thickness (E, p = 0.054) or bone porosity (F, p = 0.55). n = 9 mice per group, ** p<0.01, Bar = 1 mm.CC BY · PubMed Central
Figure 6 Strength of femurs from 8 week old mice. Femurs were loaded to failure using 3-point bending and maximum tensile stress at failure was determined form force deformation curves. Both load (left) and stress (right) at failure were significantly lower in Cx43 deficient mice (KO) relative to wild type (WT) mice. n = 6–7 mice per group, *p<0.05 vs wild type.
Figure 6 Strength of femurs from 8 week old mice. Femurs were loaded to failure using 3-point bending and maximum tensile stress at failure was determined form force deformation curves. Both load (left) and stress (right) at failure were significantly lower in Cx43 deficient mice (KO) relative to wild type (WT) mice. n = 6–7 mice per group, *p<0.05 vs wild type.CC BY · PubMed Central
Figure 7 Dynamic histomorphometry in 8 week old mice. Eight week old Cx43 deficient (KO) and wild type (WT) mice displayed similar endocortical or periosteal mineralized surface (MS/BS, A, D) bone mineral apposition rates (MAR, B, E) and bone formation rates (BFR/BS, C, F). n = 6 mice per group.
Figure 7 Dynamic histomorphometry in 8 week old mice. Eight week old Cx43 deficient (KO) and wild type (WT) mice displayed similar endocortical or periosteal mineralized surface (MS/BS, A, D) bone mineral apposition rates (MAR, B, E) and bone formation rates (BFR/BS, C, F). n = 6 mice per group.CC BY · PubMed Central
Figure 8 Dynamic histomorphometry in 6 month old mice. Six month old Cx43 deficient (KO) mice show significantly increased endocortical MS/BS (A), and BFR (C), but no significant change in other parameters (B, D, E and F). n = 4–5 mice per group, ** p<0.01.
Figure 8 Dynamic histomorphometry in 6 month old mice. Six month old Cx43 deficient (KO) mice show significantly increased endocortical MS/BS (A), and BFR (C), but no significant change in other parameters (B, D, E and F). n = 4–5 mice per group, ** p<0.01.CC BY · PubMed Central
Figure 9 Quantification of serum markers of bone formation. Four week old Cx43 deficient (KO) and wild type (WT) control mice have similar PINP concentrations in serum. n = 3–9 mice per group.
Figure 9 Quantification of serum markers of bone formation. Four week old Cx43 deficient (KO) and wild type (WT) control mice have similar PINP concentrations in serum. n = 3–9 mice per group.CC BY · PubMed Central
Figure 10 Cx43 deficient mice display increased bone resorption and osteoclast number. Four week old Cx43 deficient mice show increased osteoclast number in the distal epiphyseal area (A) and increased serum CTX concentration (B). n = 4 (A) or 3–9 (B) mice per group, * p<0.05. C–E: Typical TRAP staining of four-week old wild type (WT) (C and D) and Cx43 deficient (KO) (E and F) mouse femur. There are more osteoclasts in bone from Cx43 deficient mice. Scale bar = 100 µm.
Figure 10 Cx43 deficient mice display increased bone resorption and osteoclast number. Four week old Cx43 deficient mice show increased osteoclast number in the distal epiphyseal area (A) and increased serum CTX concentration (B). n = 4 (A) or 3–9 (B) mice per group, * p<0.05. C–E: Typical TRAP staining of four-week old wild type (WT) (C and D) and Cx43 deficient (KO) (E and F) mouse femur. There are more osteoclasts in bone from Cx43 deficient mice. Scale bar = 100 µm.CC BY · PubMed Central
Figure 11 Ablation of Cx43 affects RANKL/OPG mRNA ratio in osteocytic but not osteoblastic cells. Cx43 protein (A) and mRNA (B) levels in primary osteoblasts from Cx43 deficient (KO) mice were decreased relative to wild type (WT), but the ratio of RANKL/OPG mRNA levels were similar in osteoblasts from Cx43 deficient and wild type mice (C). Osteocytic MLO-Y4 cells were transfected with either scrambled control siRNA or siRNA directed against Cx43 mRNA. MLO-Y4 cells transfected with Cx43 siRNA displayed decreased Cx43 protein (D) and mRNA levels (E) but increased RANKL/OPG mRNA levels (F), relative to cells transfected with control siRNA. n = 3 separate experiments for A and D; 11–12 for B and C; and 10 for E and F), ** p<0.01.
Figure 11 Ablation of Cx43 affects RANKL/OPG mRNA ratio in osteocytic but not osteoblastic cells. Cx43 protein (A) and mRNA (B) levels in primary osteoblasts from Cx43 deficient (KO) mice were decreased relative to wild type (WT), but the ratio of RANKL/OPG mRNA levels were similar in osteoblasts from Cx43 deficient and wild type mice (C). Osteocytic MLO-Y4 cells were transfected with either scrambled control siRNA or siRNA directed against Cx43 mRNA. MLO-Y4 cells transfected with Cx43 siRNA displayed decreased Cx43 protein (D) and mRNA levels (E) but increased RANKL/OPG mRNA levels (F), relative to cells transfected with control siRNA. n = 3 separate experiments for A and D; 11–12 for B and C; and 10 for E and F), ** p<0.01.CC BY · PubMed Central
Figure 12 Mechanical loading of bone in Cx43 deficient and wild type mice. An enhanced periosteal osteoblastic response was observed in 6 month old Cx43 deficient mice (KO) relative to wild type (WT) when the right tibias of each were exposed to mechanical loading that induced equivalent peak normal strains. p.MS, p.MAR and p.BFR are reported for contralateral (C) and loaded tibias (L). p.MS and p.BFR were significantly greater in loaded tibia, relative to contralateral, in Cx43 deficient mice but not wild type mice. p.Mar in loaded tibia was not significantly different than in contralateral tibia in Cx43 deficient or wild type mice. n = 7–9 mice per group, * p<0.05.
Figure 12 Mechanical loading of bone in Cx43 deficient and wild type mice. An enhanced periosteal osteoblastic response was observed in 6 month old Cx43 deficient mice (KO) relative to wild type (WT) when the right tibias of each were exposed to mechanical loading that induced equivalent peak normal strains. p.MS, p.MAR and p.BFR are reported for contralateral (C) and loaded tibias (L). p.MS and p.BFR were significantly greater in loaded tibia, relative to contralateral, in Cx43 deficient mice but not wild type mice. p.Mar in loaded tibia was not significantly different than in contralateral tibia in Cx43 deficient or wild type mice. n = 7–9 mice per group, * p<0.05.CC BY · PubMed Central

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  1. MM Saunders, 2001, Gap junctions and fluid flow response in MC3T3-E1 cells., Am J Physiol Cell Physiol, 281, C1917, 10.1152/ajpcell.2001.281.6.C1917 · DOI

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Metadata

Authors
Zhang, Y., Paul, E. M., Sathyendra, V., Davison, A., Sharkey, N., Bronson, S., Srinivasan, S., Gross, T. S., & Donahue, H. J.
Affiliations
  • Pennsylvania State University (US)
  • University of Washington (US)
Corresponding author
Donahue, Henry J.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-08-29
Volume
6
Issue
8
Pages
e23516
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0023516
PMC
PMC3163577
OpenAlex
W2022309828
Cited by
148
References
63
Citations by year
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FWCI
6.75
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • University of Pennsylvania
  • Pennsylvania State University
  • National Institutes of Health
  • Penn State College of Medicine
  • National Institute on Aging (R01AG013087)
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (R03AR057546)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Connexins and lens biology
  • Health Sciences › Medicine › Orthopedics and Sports Medicine › Bone health and osteoporosis research
Keywords
connexin 43, gap junctional intercellular communication, osteoclastogenesis, bone resorption, osteocytes, osteoblasts, RANKL/OPG ratio, mechanical loading, bone loss
MeSH terms
Animals, Biomechanical Phenomena, Bone and Bones, Bone Resorption, Cell Communication, Cell Line, Humans, Male, Osteoclasts, Osteocytes, Osteogenesis, RNA, Messenger, Bone Density, Gene Deletion, Gap Junctions, Connexin 43, Mice, Osteoprotegerin, RANK Ligand, Mechanical Phenomena

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