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

Authors

Naga Suresh Adapala, Danielle Holland, Vanessa Scanlon, Mary F. Barbe, Wallace Y. Langdon, Alexander Y. Tsygankov, Joseph A. Lorenzo, Archana Sanjay

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.

Link To Article

http://dx.doi.org/10.1016/j.bone.2014.06.013

The mineral dissolution function of osteoclasts is dispensable for hypertrophic cartilage degradation during long bone development and growth

Authors

Heiani Touaitahuata, Gaelle Cres, Sylvain de Rossi, Virginie Vives, Anne Blangy

Abstract

During long bone development and post-natal growth, the cartilaginous model of the skeleton is progressively replaced by bone, a process known as endochondral ossification. In the primary spongiosa, osteoclasts degrade the mineralized cartilage produced by hypertrophic chondrocytes to generate cartilage trabeculae that osteoblasts embed in bone matrix. This leads to the formation of the trabecular bone network of the secondary spongiosa that will undergo continuous remodeling. Osteoclasts are specialized in mineralized tissue degradation, with the combined ability to solubilize hydroxyapatite and to degrade extracellular matrix proteins. We reported previously that osteoclasts lacking Dock5 could not degrade bone due to abnormal podosome organization and absence of sealing zone formation. Consequently, adult Dock5−/− mice have increased trabecular bone mass. We used Dock5−/− mice to further investigate the different functions of osteoclast during endochondral bone formation. We show that long bones are overall morphologically normal in developing and growing Dock5−/− mice. We demonstrate that Dock5−/− mice also have normal hypertrophic cartilage and cartilage trabecular network. Conversely, trabecular bone volume increased progressively in the secondary spongiosa of Dock5−/− growing mice as compared to Dock5+/+ animals, even though their osteoclast numbers were the same. In vitro, we show that Dock5−/− osteoclasts do present acidic compartments at the ventral plasma membrane and produce normal amounts of active MMP9, TRAP and CtsK for matrix protein degradation but they are unable to solubilize minerals. These observations reveal that contrarily to bone resorption, the ability of osteoclasts to dissolve minerals is dispensable for the degradation of mineralized hypertrophic cartilage during endochondral bone formation.

Link To Article

http://dx.doi.org/10.1016/j.ydbio.2014.06.020

Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1

Authors

Jean de la Croix Ndong, Alexander J Makowski, Sasidhar Uppuganti, Guillaume Vignaux, Koichiro Ono, Daniel S Perrien, Simon Joubert, Serena R Baglio, Donatella Granchi, David A Stevenson, Jonathan J Rios, Jeffry S Nyman & Florent Elefteriou

Abstract

Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase–activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal–regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2–induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.

Link To Article

http://dx.doi.org/10.1038/nm.3583

Effect of sequential treatments with alendronate, parathyroid hormone (1–34) and raloxifene on cortical bone mass and strength in ovariectomized rats

Authors

Sarah K. Amugongo, Wei Yao, Junjing Jia, Weiwei Dai, Yu-An E. Lay, Li Jiang, Danielle Harvey, Elizabeth A. Zimmermann, Eric Schaible, Neil Dave, Robert O. Ritchie, Donald B. Kimmel, Nancy E. Lane

Abstract

Anti-resorptive and anabolic agents are often prescribed for the treatment of osteoporosis continuously or sequentially for many years. However their impact on cortical bone quality and bone strength is not clear.

Methods Six-month old female rats were either sham operated or ovariectomized (OVX). OVX rats were left untreated for two months and then were treated with vehicle (Veh), hPTH (1–34) (PTH), alendronate (Aln), or raloxifene (Ral) sequentially for three month intervals, for a total of three periods. Mid-tibial cortical bone architecture, mass, mineralization, and strength were measured on necropsy samples obtained after each period. Bone indentation properties were measured on proximal femur necropsy samples.

Results Eight or more months of estrogen deficiency in rats resulted in decreased cortical bone area and thickness. Treatment with PTH for 3 months caused the deposition of endocortical lamellar bone that increased cortical bone area, thickness, and strength. These improvements were lost when PTH was withdrawn without followup treatment, but were maintained for the maximum times tested, six months with Ral and three months with Aln. Pre-treatment with anti-resorptives was also somewhat successful in ultimately preserving the additional endocortical lamellar bone formed under PTH treatment. These treatments did not affect bone indentation properties.

Summary Sequential therapy that involved both PTH and anti-resorptive agents was required to achieve lasting improvements in cortical area, thickness, and strength in OVX rats. Anti-resorptive therapy, either prior to or following PTH, was required to preserve gains attributable to an anabolic agent.

Link To Article

http://dx.doi.org/10.1016/j.bone.2014.04.033

Prostate Cancer Cells Preferentially Home To Osteoblast-Rich Areas In The Early Stages Of Bone Metastasis – Evidence From In Vivo Models

Authors

Ning Wang Ph.D, Freyja E Docherty PhD, Hannah K Brown PhD, Kimberley J Reeves PhD, Anne CM Fowles MSc, Penelope D Ottewell PhD, T. Neil Dear PhD, Ingunn Holen PhD, Peter I Croucher PhD andColby L Eaton PhD

Abstract

It has been suggested that metastasis-initiating cells gain a foothold in bone by homing to a metastastatic microenvironment (or ‘niche’). Whereas the precise nature of this niche remains to be established, it is likely to contain bone cell populations including osteoblasts and osteoclasts. In the mouse tibia, the distribution of osteoblasts on endocortical bone surfaces is non-uniform and we hypothesize that studying co-localization of individual tumour cells with resident cell populations will reveal the identity of critical cellular components of the niche. In this study, we have mapped the distribution of three human prostate cancer cell lines (PC3-NW1, LN-CaP, and C4 2B4) colonising the tibias of athymic mice following intracardiac injection and evaluated their interaction with potential metastatic niches. Prostate cancer cells labelled with the fluorescent cell membrane dye (Vybrant DiD) were found by two-photon microscopy to be engrafted in the tibiae in close proximity (∼40 µm) to bone surfaces and 70% more cancer cells were detected in the lateral compared to the medial endocortical bone regions. This was associated with a 5-fold higher number of osteoblasts and 7-fold higher bone formation rate on the lateral endocortical bone surface compared to the medial side. By disrupting cellular interactions mediated by the chemokine (C-X-C motif) receptor 4 (CXCR4)/chemokine ligand 12 (CXCL12) axis with the CXCR4 inhibitor AMD3100, the preferential homing pattern of prostate cancer cells to osteoblast-rich bone surfaces was disrupted. In this study, we map the location of prostate cancer cells that home to endocortical regions in bone and our data demonstrate that homing of prostate cancer cells is associated with the presence and activity of osteoblast lineage cells, and suggest that therapies targeting osteoblast niches should be considered to prevent development of incurable prostate cancer bone metastases.

Link To Article

http://dx.doi.org/10.1002/jbmr.2300

Runx2 is required for early stages of endochondral bone formation but delays final stages of bone repair in Axin2-deficient mice

Authors

Meghan E. McGee-Lawrence, Lomeli R. Carpio, Elizabeth W. Bradley, Amel Dudakovic, Jane B. Lian, Andre J. van Wijnen, Sanjeev Kakar, Wei Hsu, Jennifer J. Westendorf

Abstract

Runx2 and Axin2 regulate skeletal development. We recently determined that Axin2 and Runx2 molecularly interact in differentiating osteoblasts to regulate intramembranous bone formation, but the relationship between these factors in endochondral bone formation was unresolved. To address this, we examined the effects of Axin2 deficiency on the cleidocranial dysplasia (CCD) phenotype of Runx2+/− mice, focusing on skeletal defects attributed to improper endochondral bone formation. Axin2 deficiency unexpectedly exacerbated calvarial components of the CCD phenotype in the Runx2+/− mice; the endocranial layer of the frontal suture, which develops by endochondral bone formation, failed to mineralize in Axin2−/−:Runx2+/− mice, resulting in a cartilaginous, fibrotic and larger fontanel than observed in Runx2+/− mice. Transcripts associated with cartilage development (e.g., Acan, miR140) were expressed at higher levels, whereas blood vessel morphogenesis transcripts (e.g., Slit2) were suppressed in Axin2−/−:Runx2+/− calvaria. Cartilage maturation was impaired, as primary chondrocytes from double mutant mice demonstrated delayed differentiation and produced less calcified matrix in vitro. The genetic dominance of Runx2 was also reflected during endochondral fracture repair, as both Runx2+/− and double mutant Axin2−/−:Runx2+/− mice had enlarged fracture calluses at early stages of healing. However, by the end stages of fracture healing, double mutant animals diverged from the Runx2+/− mice, showing smaller calluses and increased torsional strength indicative of more rapid end stage bone formation as seen in the Axin2−/− mice. Taken together, our data demonstrate a dominant role for Runx2 in chondrocyte maturation, but implicate Axin2 as an important modulator of the terminal stages of endochondral bone formation.

Link To Article

http://dx.doi.org/10.1016/j.bone.2014.06.022