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

doi:10.1016/j.bone.2014.06.013

. 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 Adapala¹, Danielle Holland², Vanessa Scanlon¹, Mary F Barbe², Wallace Y Langdon³, Alexander Y Tsygankov⁴, Joseph A Lorenzo⁵, Archana Sanjay⁶

Affiliations

¹ Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT 06032, USA.
² Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
³ School of Pathology and Laboratory Medicine, University of Western Australia, Crawley 6009, Australia.
⁴ Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
⁵ Department of Medicine, University of Connecticut Health Center, Farmington, CT 06032, USA.
⁶ Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT 06032, USA. Electronic address: asanjay@uchc.edu.

PMID: 24994594
PMCID: PMC4149851
DOI: 10.1016/j.bone.2014.06.013

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

Naga Suresh Adapala et al. Bone. 2014 Oct.

. 2014 Oct:67:1-9.

doi: 10.1016/j.bone.2014.06.013. Epub 2014 Jul 1.

Authors

Naga Suresh Adapala¹, Danielle Holland², Vanessa Scanlon¹, Mary F Barbe², Wallace Y Langdon³, Alexander Y Tsygankov⁴, Joseph A Lorenzo⁵, Archana Sanjay⁶

Affiliations

¹ Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT 06032, USA.
² Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
³ School of Pathology and Laboratory Medicine, University of Western Australia, Crawley 6009, Australia.
⁴ Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
⁵ Department of Medicine, University of Connecticut Health Center, Farmington, CT 06032, USA.
⁶ Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT 06032, USA. Electronic address: asanjay@uchc.edu.

PMID: 24994594
PMCID: PMC4149851
DOI: 10.1016/j.bone.2014.06.013

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. 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. 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. 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. 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. 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. 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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References

1. Turner RT. Mice, estrogen, and postmenopausal osteoporosis. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1999;14:187–191. - PubMed
1. Swaminathan G, Tsygankov AY. The Cbl family proteins: ring leaders in regulation of cell signaling. Journal of cellular physiology. 2006;209:21–43. - PubMed
1. Thien CB, Langdon WY. c-Cbl and Cbl-b ubiquitin ligases: substrate diversity and the negative regulation of signalling responses. Biochem J. 2005;391:153–166. - PMC - PubMed
1. Thien CBF, Langdon WY. Cbl: many adaptations to regulate protein tyrosine kinases. Nat. Rev. Mol. Cell. Biol. 2001;2:294–305. - PubMed
1. Naramura M, Jang I-K, Kole H, Huang F, Haines D, Gu H. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nature immunology. 2002;3:1192–1199. - PubMed

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[1] Turner RT. Mice, estrogen, and postmenopausal osteoporosis. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1999;14:187–191. - PubMed

[2] Turner RT. Mice, estrogen, and postmenopausal osteoporosis. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 1999;14:187–191. - PubMed

[3] Swaminathan G, Tsygankov AY. The Cbl family proteins: ring leaders in regulation of cell signaling. Journal of cellular physiology. 2006;209:21–43. - PubMed

[4] Swaminathan G, Tsygankov AY. The Cbl family proteins: ring leaders in regulation of cell signaling. Journal of cellular physiology. 2006;209:21–43. - PubMed

[5] Thien CB, Langdon WY. c-Cbl and Cbl-b ubiquitin ligases: substrate diversity and the negative regulation of signalling responses. Biochem J. 2005;391:153–166. - PMC - PubMed

[6] Thien CB, Langdon WY. c-Cbl and Cbl-b ubiquitin ligases: substrate diversity and the negative regulation of signalling responses. Biochem J. 2005;391:153–166. - PMC - PubMed

[7] Thien CBF, Langdon WY. Cbl: many adaptations to regulate protein tyrosine kinases. Nat. Rev. Mol. Cell. Biol. 2001;2:294–305. - PubMed

[8] Thien CBF, Langdon WY. Cbl: many adaptations to regulate protein tyrosine kinases. Nat. Rev. Mol. Cell. Biol. 2001;2:294–305. - PubMed

[9] Naramura M, Jang I-K, Kole H, Huang F, Haines D, Gu H. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nature immunology. 2002;3:1192–1199. - PubMed

[10] Naramura M, Jang I-K, Kole H, Huang F, Haines D, Gu H. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nature immunology. 2002;3:1192–1199. - PubMed

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

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

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