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. 2014 Oct:67:1-9.
doi: 10.1016/j.bone.2014.06.013. Epub 2014 Jul 1.

Loss of Cbl-PI3K interaction in mice prevents significant bone loss following ovariectomy

Naga Suresh Adapala  1 , Danielle Holland  2 , Vanessa Scanlon  1 , Mary F Barbe  2 , Wallace Y Langdon  3 , Alexander Y Tsygankov  4 , Joseph A Lorenzo  5 , Archana Sanjay  6
Affiliations

Loss of Cbl-PI3K interaction in mice prevents significant bone loss following ovariectomy

Naga Suresh Adapala et al. Bone. 2014 Oct.

Abstract

Cbl and Cbl-b are E3 ubiquitin ligases and adaptor proteins, which perform regulatory roles in bone remodeling. Cbl-/- mice have delayed bone development due to decreased osteoclast migration. Cbl-b-/- mice are osteopenic due to increased bone resorbing activity of osteoclasts. Unique to Cbl, but not present in Cbl-b, is tyrosine 737 in the YEAM motif, which upon phosphorylation provides a binding site for the regulatory p85 subunit of PI3K. Substitution of tyrosine 737 with phenylalanine (Y737F, CblYF/YF mice) prevents Y737 phosphorylation and abrogates the Cbl-PI3K interaction. We have previously reported that CblYF/YF mice had increased bone volume due to defective bone resorption and increased bone formation. Here we show that the lumbar vertebra from CblYF/YF mice did not have significant bone loss following ovariectomy. Our data also suggests that abrogation of Cbl-PI3K interaction in mice results in the loss of coupling between bone resorption and formation, since ovariectomized CblYF/YF mice did not show significant changes in serum levels of c-terminal telopeptide (CTX), whereas the serum levels of pro-collagen type-1 amino-terminal pro-peptide (P1NP) were decreased. In contrast, following ovariectomy, Cbl-/- and Cbl-b-/- mice showed significant bone loss in the tibiae and L2 vertebrae, concomitant with increased serum CTX and P1NP levels. These data indicate that while lack of Cbl or Cbl-b distinctly affects bone remodeling, only the loss of Cbl-PI3K interaction protects mice from significant bone loss following ovariectomy.

Keywords: Bone resorption; Cbl; Cbl-b; Osteoclast; Ovariectomy; PI3K.

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Figures

Figure 1
Figure 1. Trabecular bone parameters in the second lumbar vertebra of mice lacking Cbl-PI3K interaction following ovariectomy
A. Three dimensional (3D) μCT images of second lumbar vertebra (L2) showing bone volume in CblWT/WT and CblYF/YF mice. L2 Vertebra of SHAM operated mice (n=6) are shown in the top panel and ovariectomized (OVX) mice (n=6) in the bottom panel. B. BV/TV% (percentage of Bone volume/Trabecular volume) is in CblWT/WT (white bars) and CblYF/YF (black bars) mice. C. Tb. Th (Trabecular thickness in mm) D. Tb.N (Trabecular Number) E. Tb. Sp (Trabecular Separation). Data are presented as mean ± SD from indicated number of mice and compared with SHAM. p<0.05 was considered statistically significant as compared to respective controls using two way ANOVA with Bonferroni post-test. a, CblWT/WT SHAM vs OVX; b, CblWT/WT SHAM vs CblYF/YF SHAM. ns, not significant.
Figure 2
Figure 2. Trabecular bone parameters in the tibia of mice lacking Cbl-PI3K interaction following ovariectomy
A. Three dimensional (3D) μCT images of tibial trabecular region showing bone volume in CblWT/WT and CblYF/YF mice. Tibiae of SHAM operated mice (n=6) are shown in the top panel and ovariectomized (OVX) mice (n=6) in the bottom panel. B. BV/TV% (Percentage of Bone volume/Trabecular volume) in CblWT/WT (white bars) and CblYF/YF (black bars) mice. C. Tb. Th (Trabecular thickness in mm) D. Tb. N (Trabecular Number) E. Tb. Sp (Trabecular Separation). Data are presented as mean ± SD from indicated number of mice and compared with SHAM. p<0.05 was considered statistically significant as compared to respective controls using two way ANOVA with Bonferroni post-test a, CblWT/WT SHAM vs OVX; b, CblWT/WT SHAM vs CblYF/YF SHAM. ns, not significant.
Figure 3
Figure 3. Effect of ovariectomy on the osteoclast and osteoblast functions in mice lacking Cbl-PI3K interaction
Serum levels of c-terminal telopeptide (CTX) reflective of in vivo osteoclast activity and pro-collagen type 1 amino-terminal pro-peptide (P1NP) were determined. Serum was collected from SHAM operated mice (n=6) and ovariectomized (OVX) mice (n=6) six weeks following surgery, and the levels CTX and P1NP were determined using ELISA. A. Serum levels of CTX in CblWT/WT (white bars) and CblYF/YF (black bars) are presented. B. Serum levels of P1NP are shown. Data are presented as mean ± SD from indicated number of mice and compared with SHAM. p<0.05 was considered statistically significant as compared to respective controls using two way ANOVA with Bonferroni post-test. a, CblWT/WT SHAM vs OVX; b, CblWT/WT SHAM vs CblYF/YF SHAM; c, CblYF/YF SHAM vs OVX. ns, not significant.
Figure 4
Figure 4. Trabecular bone parameters in the second lumbar vertebra of mice lacking Cbl or Cbl-b following ovariectomy
A. Three dimensional (3D) μCT images of second lumbar vertebra (L2) showing decreased bone volume due to ovariectomy (OVX). Vertebrae of SHAM operated mice (n=6) are shown in the top panel and ovariectomized mice (n=6) in the bottom panel. B. BV/TV% (percentage of Bone volume/Trabecular volume) in CblWT/WT (white bars), Cbl−/− (black bars) and Cbl-b−/− (grey bars). C. Tb. Th (Trabecular thickness in mm). D. Tb. N (Trabecular Number). E. Tb. Sp (Trabecular Separation). Data are presented as mean ± SD from indicated number of mice and compared with SHAM. p<0.05 was considered statistically significant as compared to respective controls using two way ANOVA with Bonferroni post-test. Panels B, C: a, CblWT/WT SHAM vs OVX; b, Cbl−/− SHAM vs OVX; c, CblWT/WT SHAM vs Cbl-b−/− SHAM; d, Cbl-b−/− SHAM vs OVX. Panels D, E: c, Cbl-b−/− SHAM vs OVX.
Figure 5
Figure 5. Trabecular bone parameters in the tibia of mice lacking Cbl or Cbl-b following ovariectomy
A. Three dimensional (3D) μCT images of proximal tibial trabecular region showing bone loss due to ovariectomy (OVX). Tibiae of SHAM operated mice (n=6) are shown in the top panel and ovariectomized mice (n=6) in the bottom panel. B. BV/TV% (percentage of Bone volume/Trabecular volume) in CblWT/WT (white bars), Cbl−/− (black bars) and Cbl-b−/− (grey bars). C. Tb. Th (Trabecular thickness in mm). D. Tb. N (Trabecular Number). E. Tb. Sp (Trabecular Separation). Data are presented as mean ± SD from indicated number of mice and compared with SHAM. p<0.05 was considered statistically significant as compared to respective controls using two way ANOVA with Bonferroni post-test. Panels B, E: a, CblWT/WT SHAM vs OVX; b, Cbl−/− SHAM vs OVX; c, CblWT/WT SHAM vs Cbl-b−/− SHAM; d, Cbl-b−/− SHAM vs OVX. Panels C, D: c, Cbl-b−/− SHAM vs OVX.
Figure 6
Figure 6. Effect of ovariectomy on the osteoclast and osteoblast functions in mice lacking Cbl or Cbl-b
Serum levels of c-terminal telopeptide (CTX) reflective of in vivo osteoclast activity and pro-collagen type 1 amino-terminal propeptide (P1NP) were determined. Serum was collected from SHAM operated mice (n=6) and ovariectomized (OVX) mice (n=6) six weeks following surgery, and the levels CTX and P1NP were determined using ELISA. A. Serum levels of CTX in CblWT/WT (white bars), Cbl−/− (black bars) and Cbl-b−/− (grey bars) are presented. B. Serum levels of P1NP are shown. Data are presented as mean ± SD from indicated number of mice and compared with SHAM. p<0.05 was considered statistically significant as compared to respective controls using two way ANOVA with Bonferroni post-test Panel A: a, CblWT/WT SHAM vs OVX; b, Cbl−/− SHAM vs OVX; c, CblWT/WT SHAM vs Cbl-b−/− SHAM; d, Cbl-b−/− SHAM vs OVX. Panel B: c, Cbl-b−/− SHAM vs OVX.
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