Publications by authors named "Alexander G Robling"

108 Publications

The Wnt pathway: An important control mechanism in bone's response to mechanical loading.

Bone 2021 Jul 5;153:116087. Epub 2021 Jul 5.

Department of Anatomy, Cell Biology & Physiology, Indiana University School of Medicine, Indianapolis, IN, USA; Department of Biomedical Engineering, Indiana University-Purdue University at Indianapolis, Indianapolis, IN, USA; Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, IN, USA; Indiana Center for Musculoskeletal Health, Indianapolis, IN, USA. Electronic address:

The conversion of mechanical energy into biochemical changes within living cells is process known as mechanotransduction. Bone is a quintessential tissue for studying the molecular mechanisms of mechanotransduction, as the skeleton's mechanical competence is crucial for vertebrate movement. Bone cell mechanotransduction is facilitated by a number of cell biological pathways, one of the most prominent of which is the Wnt signaling cascade. The Wnt co-receptor Lrp5 has been identified as a crucial protein for mechanical signaling in bone, and modifiers of Lrp5 activity play important roles in mediating signaling efficiency through Lrp5, including sclerostin, Dkk1, and the co-receptor Lrp4. Mechanical regulation of sclerostin is mediated by certain members of the Hdac family. Other mechanisms that influence Wnt signaling-some of which are mechanoresponsive-are coming to light, including R-spondins and their role in organizing the Rnf43/Znrf3 and Lgr4/5/6 complex that liberates Lrp5. While the identity of the key Wnt proteins involved in bone cell mechanical signaling are elusive, the likely pool of key players is narrowing. Identification of Wnt-based molecular targets that can be modulated pharmacologically to make mechanical stimulation (e.g., exercise) more beneficial is an emerging approach to improving skeletal integrity and reducing fracture risk.
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http://dx.doi.org/10.1016/j.bone.2021.116087DOI Listing
July 2021

Notum Deletion From Late-Stage Skeletal Cells Increases Cortical Bone Formation and Potentiates Skeletal Effects of Sclerostin Inhibition.

J Bone Miner Res 2021 Jul 5. Epub 2021 Jul 5.

Department of Anatomy, Cell Biology, and Physiology, Indiana University School of Medicine, Indianapolis, IN, USA.

Wnt signaling plays a vital role in the cell biology of skeletal patterning, differentiation, and maintenance. Notum is a secreted member of the α/β-hydrolase superfamily that hydrolyzes the palmitoleoylate modification on Wnt proteins, thereby disrupting Wnt signaling. As a secreted inhibitor of Wnt, Notum presents an attractive molecular target for improving skeletal health. To determine the cell type of action for Notum's effect on the skeleton, we generated mice with Notum deficiency globally (Notum ) and selectively (Notum ) in limb bud mesenchyme (Prx1-Cre) and late osteoblasts/osteocytes (Dmp1-Cre). Late-stage deletion induced increased cortical bone properties, similar to global mutants. Notum expression was enhanced in response to sclerostin inhibition, so dual inhibition (Notum/sclerostin) was also investigated using a combined genetic and pharmacologic approach. Co-suppression increased cortical properties beyond either factor alone. Notum suppressed Wnt signaling in cell reporter assays, but surprisingly also enhanced Shh signaling independent of effects on Wnt. Notum is an osteocyte-active suppressor of cortical bone formation that is likely involved in multiple signaling pathways important for bone homeostasis © 2021 American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4411DOI Listing
July 2021

Improving Bone Health by Optimizing the Anabolic Action of Wnt Inhibitor Multitargeting.

JBMR Plus 2021 May 6;5(5):e10462. Epub 2021 May 6.

Department of Anatomy, Cell Biology & Physiology Indiana University School of Medicine Indianapolis IN USA.

Sclerostin antibody (romosozumab) was recently approved for clinical use in the United States to treat osteoporosis. We and others have explored Wnt-based combination therapy to disproportionately improve the anabolic effects of sclerostin inhibition, including cotreatment with sclerostin antibody (Scl-mAb) and Dkk1 antibody (Dkk1-mAb). To determine the optimal ratio of Scl-mAb and Dkk1-mAb for producing maximal anabolic action, the proportion of Scl-mAb and Dkk1-mAb were systematically varied while holding the total antibody dose constant. A 3:1 mixture of Scl-mAb to Dkk1-mAb produced two to three times as much cancellous bone mass as an equivalent dose of Scl-mAb alone. Further, a 75% reduction in the dose of the 3:1 mixture was equally efficacious to a full dose of Scl-mAb in the distal femur metaphysis. The Scl-mAb/Dkk1-mAb combination approach was highly efficacious in the cancellous bone mass, but the cortical compartment was much more subtly affected. The osteoanabolic effects of Wnt pathway targeting can be made more efficient if multiple antagonists are simultaneously targeted. © 2021 The Authors. published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101614PMC
May 2021

Skeletal Functions of Voltage Sensitive Calcium Channels.

Curr Osteoporos Rep 2021 Apr 15;19(2):206-221. Epub 2021 Mar 15.

Department of Physical Therapy, School of Health and Rehabilitation Sciences, Indiana University, Indianapolis, IN, 46202, USA.

Voltage-sensitive calcium channels (VSCCs) are ubiquitous multimeric protein complexes that are necessary for the regulation of numerous physiological processes. VSCCs regulate calcium influx and various intracellular processes including muscle contraction, neurotransmission, hormone secretion, and gene transcription, with function specificity defined by the channel's subunits and tissue location. The functions of VSCCs in bone are often overlooked since bone is not considered an electrically excitable tissue. However, skeletal homeostasis and adaptation relies heavily on VSCCs. Inhibition or deletion of VSCCs decreases osteogenesis, impairs skeletal structure, and impedes anabolic responses to mechanical loading. RECENT FINDINGS: While the functions of VSCCs in osteoclasts are less clear, VSCCs have distinct but complementary functions in osteoblasts and osteocytes. PURPOSE OF REVIEW: This review details the structure, function, and nomenclature of VSCCs, followed by a comprehensive description of the known functions of VSCCs in bone cells and their regulation of bone development, bone formation, and mechanotransduction.
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http://dx.doi.org/10.1007/s11914-020-00647-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216424PMC
April 2021

The effect of overexpression of Lrp5 on orthodontic tooth movement.

Orthod Craniofac Res 2021 Aug 26;24(3):430-437. Epub 2020 Dec 26.

Department of Orthodontics and Oral Facial Genetics, Indiana University School of Dentistry, Indianapolis, IN, USA.

Objective: To analyse the effect of gain-of-function mutations in the low-density lipoprotein receptor-related protein 5 (Lrp5) on orthodontic tooth movement (OTM).

Setting And Sample Population: A split-mouth study design was utilized. Thirty-two male Lrp5-high bone mass (HBM) knock-in mice including A214V and G171V mutants (n = 16/group) and sixteen C57BL/6 wild-type (WT) mice were included in the study.

Materials And Methods: A mouse model of OTM was used for mesial movement of the maxillary first molar using a closed-coil nickel titanium (NiTi) spring attached between the molar and the incisors. After 21 days, the dissected maxillae were scanned for micro-computed tomography (micro-CT) analyses and embedded in methyl methacrylate and paraffin for histological staining and imaging. Histological analyses included immunohistochemistry for sclerostin (Sost), tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts and fluorescent imaging.

Results: OTM in the A214V and G171V groups was significantly less than the WT group. Bone volume (BV), per cent bone volume (BV/TV) and trabecular thickness (Tb.Th) were significantly increased in both A241V and G171V animals compared to the WT animals. On the compression side, decreased osteoclast activity was seen in both A214V and G171V groups compared to the WT group. Fluorescent labelling demonstrated that the pattern of bone deposition in the A214V animals was periosteal whereas the G171V animals added bone endocortically.

Conclusion: Gain-of-function mutations of Lrp5 decrease orthodontic tooth movement by increasing alveolar bone mass and reducing osteoclast-mediated bone resorption.
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http://dx.doi.org/10.1111/ocr.12459DOI Listing
August 2021

Co-deletion of Lrp5 and Lrp6 in the skeleton severely diminishes bone gain from sclerostin antibody administration.

Bone 2021 02 24;143:115708. Epub 2020 Oct 24.

Department of Anatomy, Cell Biology & Physiology, Indiana University School of Medicine, Indianapolis, IN, USA; Department of Biomedical Engineering, Indiana University-Purdue University at Indianapolis, Indianapolis, IN, USA; Richard L. Roudebush VA Medical Center, Indianapolis, IN, USA; Indiana Center for Musculoskeletal Health, Indianapolis, IN, USA. Electronic address:

The cysteine knot protein sclerostin is an osteocyte-derived secreted inhibitor of the Wnt co-receptors LRP5 and LRP6. LRP5 plays a dominant role in bone homeostasis, but we previously reported that Sost/sclerostin suppression significantly increased osteogenesis regardless of Lrp5 presence or absence. Those observations suggested that the bone forming effects of sclerostin inhibition can occur through Lrp6 (when Lrp5 is suppressed), or through other yet undiscovered mechanisms independent of Lrp5/6. To distinguish between these two possibilities, we generated mice with compound deletion of Lrp5 and Lrp6 selectively in bone, and treated them with sclerostin monoclonal antibody (Scl-mAb). All mice were homozygous flox for both Lrp5 and Lrp6 (Lrp5; Lrp6), and varied only in whether or not they carried the Dmp1-Cre transgene. Positive (Cre+) and negative (Cre-) mice were injected with Scl-mAb or vehicle from 4.5 to 14 weeks of age. Vehicle-treated Cre+ mice exhibited significantly reduced skeletal properties compared to vehicle-treated Cre- mice, as assessed by DXA, μCT, pQCT, and histology, indicating that Lrp5/6 deletions were effective and efficient. Scl-mAb treatment improved nearly every bone-related parameter among Cre- mice, but the same treatment in Cre+ mice resulted in little to no improvement in skeletal properties. For the few endpoints where Cre+ mice responded to Scl-mAb, it is likely that antibody-induced promotion of Wnt signaling occurred in cell types earlier in the mesenchymal/osteoblast differentiation pathway than the Dmp1-expressing stage. This latter conclusion was supported by changes in some histomorphometric parameters. In conclusion, unlike with the deletion of Lrp5 alone, the bone-selective late-stage co-deletion of Lrp5 and Lrp6 significantly impairs or completely nullifies the osteogenic action of Scl-mAb, and highlights a major role for both Lrp5 and Lrp6 in the mechanism of action for the bone-building effects of sclerostin antibody.
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http://dx.doi.org/10.1016/j.bone.2020.115708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770084PMC
February 2021

Sensitive detection of Cre-mediated recombination using droplet digital PCR reveals Tg(BGLAP-Cre) and Tg(DMP1-Cre) are active in multiple non-skeletal tissues.

Bone 2021 01 6;142:115674. Epub 2020 Oct 6.

Orthopedic Research Laboratories, Department of Orthopedic Surgery, Boston Children's Hospital, Boston, MA, United States of America; Department of Genetics, Harvard Medical School, Boston, MA, United States of America. Electronic address:

In humans, somatic activating mutations in PIK3CA are associated with skeletal overgrowth. In order to determine if activated PI3K signaling in bone cells causes overgrowth, we used Tg(BGLAP-Cre) and Tg(DMP1-Cre) mouse strains to somatically activate a disease-causing conditional Pik3ca allele (Pik3ca) in osteoblasts and osteocytes. We observed Tg(BGLAP-Cre);Pik3ca offspring were born at the expected Mendelian frequency. However, these mice developed cutaneous lymphatic malformations and died before 7 weeks of age. In contrast, Tg(DMP1-Cre);Pik3ca offspring survived and had no cutaneous lymphatic malformations. Assuming that Cre-activity outside of the skeletal system accounted for the difference in phenotype between Tg(BGLAP-Cre);Pik3ca and Tg(DMP1-Cre);Pik3ca mice, we developed sensitive and specific droplet digital PCR (ddPCR) assays to search for and quantify rates of Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated recombination in non-skeletal tissues. We observed Tg(BGLAP-Cre)-mediated recombination in several tissues including skin, muscle, artery, and brain; two CNS locations, hippocampus and cerebellum, exhibited Cre-mediated recombination in >5% of cells. Tg(DMP1-Cre)-mediated recombination was also observed in muscle, artery, and brain. Although we cannot preclude that differences in phenotype between mice with Tg(BGLAP-Cre)- and Tg(DMP1-Cre)-mediated PIK3CA activation are due to Cre-recombination being induced at different stages of osteoblast differentiation, differences in recombination at non-skeletal sites are the more likely explanation. Since unanticipated sites of recombination can affect the interpretation of data from experiments involving conditional alleles, we recommend ddPCR as a good first step for assessing efficiency, leakiness, and off-targeting in experiments that employ Cre-mediated or Flp-mediated recombination.
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http://dx.doi.org/10.1016/j.bone.2020.115674DOI Listing
January 2021

Generation and Characterization of Mouse Models for Skeletal Disease.

Methods Mol Biol 2021 ;2221:165-191

Program for Skeletal Disease and Tumor Microenvironment, Center for Cancer and Cell Biology, Van Andel Institute, Grand Rapids, MI, USA.

Our laboratories have used genetically engineered mouse models (GEMMs) to assess genetic contributions to skeletal diseases such as osteoporosis and osteoarthritis. Studies on the genetic contributions to OA are often done by assessing how GEMMs respond to surgical methods that induce symptoms modeling OA. Here, we will describe protocols outlining the induction of experimental OA in mice as well as detailed descriptions of methods for analyzing skeletal phenotypes using micro-computerized tomography and skeletal histomorphometry.
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http://dx.doi.org/10.1007/978-1-0716-0989-7_11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934896PMC
March 2021

The mTORC2 Component Rictor Is Required for Load-Induced Bone Formation in Late-Stage Skeletal Cells.

JBMR Plus 2020 Jul 18;4(7):e10366. Epub 2020 Jun 18.

Department of Anatomy & Cell Biology Indiana University School of Medicine Indianapolis IN USA.

Bone relies on mechanical cues to build and maintain tissue composition and architecture. Our understanding of bone cell mechanotransduction continues to evolve, with a few key signaling pathways emerging as vital. Wnt/β-catenin, for example, is essential for proper anabolic response to mechanical stimulation. One key complex that regulates β-catenin activity is the mammalian target of rapamycin complex 2 (mTORc2). mTORc2 is critical for actin cytoskeletal reorganization, an indispensable component in mechanotransduction in certain cell types. In this study, we probed the impact of the mTORc2 signaling pathway in osteocyte mechanotransduction by conditionally deleting the mTORc2 subunit Rictor in Dmp1-expressing cells of C57BL/6 mice. Conditional deletion of the Rictor was achieved using the Dmp1-Cre driver to recombine Rictor floxed alleles. Rictor mutants exhibited a decrease in skeletal properties, as measured by DXA, μCT, and mechanical testing, compared with Cre-negative floxed littermate controls. in vivo axial tibia loading conducted in adult mice revealed a deficiency in the osteogenic response to loading among Rictor mutants. Histological measurements of osteocyte morphology indicated fewer, shorter cell processes in Rictor mutants, which might explain the compromised response to mechanical stimulation. In summary, inhibition of the mTORc2 pathway in late osteoblasts/osteocytes leads to decreased bone mass and mechanically induced bone formation. © 2020 The Authors. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340445PMC
July 2020

Independent validation of experimental results requires timely and unrestricted access to animal models and reagents.

PLoS Genet 2020 06 26;16(6):e1008940. Epub 2020 Jun 26.

Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Institute, Grand Rapids, Michigan, United States of America.

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http://dx.doi.org/10.1371/journal.pgen.1008940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319330PMC
June 2020

Pten deletion in Dmp1-expressing cells does not rescue the osteopenic effects of Wnt/β-catenin suppression.

J Cell Physiol 2020 12 11;235(12):9785-9794. Epub 2020 Jun 11.

Department of Anatomy, Cell Biology & Physiology, Indiana University School of Medicine, Indianapolis, Indiana.

Skeletal homeostasis is sensitive to perturbations in Wnt signaling. Beyond its role in the bone, Wnt is a major target for pharmaceutical inhibition in a wide range of diseases, most notably cancers. Numerous clinical trials for Wnt-based candidates are currently underway, and Wnt inhibitors will likely soon be approved for clinical use. Given the bone-suppressive effects accompanying Wnt inhibition, there is a need to expose alternate pathways/molecules that can be targeted to counter the deleterious effects of Wnt inhibition on bone properties. Activation of the Pi3k/Akt pathway via Pten deletion is one possible osteoanabolic pathway to exploit. We investigated whether the osteopenic effects of β-catenin deletion from bone cells could be rescued by Pten deletion in the same cells. Mice carrying floxed alleles for Pten and β-catenin were bred to Dmp1-Cre mice to delete Pten alone, β-catenin alone, or both genes from the late-stage osteoblast/osteocyte population. The mice were assessed for bone mass, density, strength, and formation parameters to evaluate the potential rescue effect of Pten deletion in Wnt-impaired mice. Pten deletion resulted in high bone mass and β-catenin deletion resulted in low bone mass. Compound mutants had bone properties similar to β-catenin mutant mice, or surprisingly in some assays, were further compromised beyond β-catenin mutants. Pten inhibition, or one of its downstream nodes, is unlikely to protect against the bone-wasting effects of Wnt/βcat inhibition. Other avenues for preserving bone mass in the presence of Wnt inhibition should be explored to alleviate the skeletal side effects of Wnt inhibitor-based therapies.
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http://dx.doi.org/10.1002/jcp.29792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529875PMC
December 2020

An osteocalcin-deficient mouse strain without endocrine abnormalities.

PLoS Genet 2020 05 28;16(5):e1008361. Epub 2020 May 28.

Program in Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Institute, Grand Rapids, Michigan, United States of America.

Osteocalcin (OCN), the most abundant noncollagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc-) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double-knockout (dko) allele (Bglap/2p.Pro25fs17Ter) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full-length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their serum. FTIR imaging of cortical bone in these homozygous knockout animals finds alterations in the collagen maturity and carbonate to phosphate ratio in the cortical bone, compared with wild-type littermates. However, μCT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc-allele, serum glucose levels and male fertility in the OCN-deficient mice with the Bglap/2pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Possible explanations include the effects of each mutated allele on the transcription of neighboring genes, or differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double-knockout strain to the Jackson Laboratories for academic distribution.
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http://dx.doi.org/10.1371/journal.pgen.1008361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255615PMC
May 2020

Skeletal loading regulates breast cancer-associated osteolysis in a loading intensity-dependent fashion.

Bone Res 2020 14;8. Epub 2020 Feb 14.

1Department of Pharmacology, School of Pharmacy, Harbin Medical University, Harbin, 150081 China.

Osteocytes are mechanosensitive bone cells, but little is known about their effects on tumor cells in response to mechanical stimulation. We treated breast cancer cells with osteocyte-derived conditioned medium (CM) and fluid flow-treated conditioned medium (FFCM) with 0.25 Pa and 1 Pa shear stress. Notably, CM and FFCM at 0.25 Pa induced the mesenchymal-to-epithelial transition (MET), but FFCM at 1 Pa induced the epithelial-to-mesenchymal transition (EMT). This suggested that the effects of fluid flow on conditioned media depend on flow intensity. Fluorescence resonance energy transfer (FRET)-based evaluation of Src activity and vinculin molecular force showed that osteopontin was involved in EMT and MET switching. A mouse model of tumor-induced osteolysis was tested using dynamic tibia loadings of 1, 2, and 5 N. The low 1 N loading suppressed tumor-induced osteolysis, but this beneficial effect was lost and reversed with loads at 2 and 5 N, respectively. Changing the loading intensities in vivo also led to changes in serum TGFβ levels and the composition of tumor-associated volatile organic compounds in the urine. Collectively, this study demonstrated the critical role of intensity-dependent mechanotransduction and osteopontin in tumor-osteocyte communication, indicating that a biophysical factor can tangibly alter the behaviors of tumor cells in the bone microenvironment.
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http://dx.doi.org/10.1038/s41413-020-0083-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021802PMC
February 2020

The Osteocyte: New Insights.

Annu Rev Physiol 2020 02;82:485-506

Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA; email:

Osteocytes are an ancient cell, appearing in fossilized skeletal remains of early fish and dinosaurs. Despite its relative high abundance, even in the context of nonskeletal cells, the osteocyte is perhaps among the least studied cells in all of vertebrate biology. Osteocytes are cells embedded in bone, able to modify their surrounding extracellular matrix via specialized molecular remodeling mechanisms that are independent of the bone forming osteoblasts and bone-resorbing osteoclasts. Osteocytes communicate with osteoclasts and osteoblasts via distinct signaling molecules that include the RankL/OPG axis and the Sost/Dkk1/Wnt axis, among others. Osteocytes also extend their influence beyond the local bone environment by functioning as an endocrine cell that controls phosphate reabsorption in the kidney, insulin secretion in the pancreas, and skeletal muscle function. These cells are also finely tuned sensors of mechanical stimulation to coordinate with effector cells to adjust bone mass, size, and shape to conform to mechanical demands.
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http://dx.doi.org/10.1146/annurev-physiol-021119-034332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8274561PMC
February 2020

Combination therapy in the Col1a2 mouse model of Osteogenesis Imperfecta reveals an additive effect of enhancing LRP5 signaling and inhibiting TGFβ signaling on trabecular bone but not on cortical bone.

Bone 2020 02 21;131:115084. Epub 2019 Oct 21.

Divisions of Endocrinology and Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA. Electronic address:

Enhancing LRP5 signaling and inhibiting TGFβ signaling have each been reported to increase bone mass and improve bone strength in wild-type mice. Monotherapy targeting LRP5 signaling, or TGFβ signaling, also improved bone properties in mouse models of Osteogenesis Imperfecta (OI). We investigated whether additive or synergistic increases in bone properties would be attained if enhanced LRP5 signaling was combined with TGFβ inhibition. We crossed an Lrp5 high bone mass (HBM) allele (Lrp5) into the Col1a2 mouse model of OI. At 6-weeks-of-age we began treating mice with an antibody that inhibits TGFβ1, β2, and β3 (mAb 1D11), or with an isotype-matched control antibody (mAb 13C4). At 12-weeks-old, we observed that combining enhanced LRP5 signaling with inhibited TGFβ signaling produced an additive effect on femoral and vertebral trabecular bone volumes, but not on cortical bone volumes. Although enhanced LRP5 signaling increased femur strength in a 3-point bending assay in Col1a2 mice, femur strength did not improve further with TGFβ inhibition. Neither enhanced LRP5 signaling nor TGFβ inhibition, alone or in combination, improved femur 3-point-bending post-yield displacement in Col1a2 mice. These pre-clinical studies indicate combination therapies that target LRP5 and TGFβ signaling should increase trabecular bone mass in patients with OI more than targeting either signaling pathway alone. Whether additive increases in trabecular bone mass will occur in, and clinically benefit, patients with OI needs to be determined.
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http://dx.doi.org/10.1016/j.bone.2019.115084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232829PMC
February 2020

YAP and TAZ Mediate Osteocyte Perilacunar/Canalicular Remodeling.

J Bone Miner Res 2020 01 14;35(1):196-210. Epub 2019 Oct 14.

Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA, USA.

Bone fragility fractures are caused by low bone mass or impaired bone quality. Osteoblast/osteoclast coordination determines bone mass, but the factors that control bone quality are poorly understood. Osteocytes regulate osteoblast and osteoclast activity on bone surfaces but can also directly reorganize the bone matrix to improve bone quality through perilacunar/canalicular remodeling; however, the molecular mechanisms remain unclear. We previously found that deleting the transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-motif (TAZ) from osteoblast-lineage cells caused lethality in mice due to skeletal fragility. Here, we tested the hypothesis that YAP and TAZ regulate osteocyte-mediated bone remodeling by conditional ablation of both YAP and TAZ from mouse osteocytes using 8 kb-DMP1-Cre. Osteocyte-conditional YAP/TAZ deletion reduced bone mass and dysregulated matrix collagen content and organization, which together decreased bone mechanical properties. Further, YAP/TAZ deletion impaired osteocyte perilacunar/canalicular remodeling by reducing canalicular network density, length, and branching, as well as perilacunar flourochrome-labeled mineral deposition. Consistent with recent studies identifying TGF-β as a key inducer of osteocyte expression of matrix-remodeling enzymes, YAP/TAZ deletion in vivo decreased osteocyte expression of matrix proteases MMP13, MMP14, and CTSK. In vitro, pharmacologic inhibition of YAP/TAZ transcriptional activity in osteocyte-like cells abrogated TGF-β-induced matrix protease gene expression. Together, these data show that YAP and TAZ control bone matrix accrual, organization, and mechanical properties by regulating osteocyte-mediated bone remodeling. Elucidating the signaling pathways that control perilacunar/canalicular remodeling may enable future therapeutic targeting of bone quality to reverse skeletal fragility. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066596PMC
January 2020

Lrp4 Mediates Bone Homeostasis and Mechanotransduction through Interaction with Sclerostin In Vivo.

iScience 2019 Oct 18;20:205-215. Epub 2019 Sep 18.

Department of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Dr., MS 5035, Indianapolis, IN 46202, USA; Indiana Center for Musculoskeletal Health, Indianapolis, IN 46202, USA; Richard L. Roudebush VA Medical Center, Indianapolis, IN 46202, USA. Electronic address:

Wnt signaling plays a key role in regulating bone remodeling. In vitro studies suggest that sclerostin's inhibitory action on Lrp5 is facilitated by the membrane-associated receptor Lrp4. We generated an Lrp4 R1170W knockin mouse model (Lrp4), based on a published mutation in patients with high bone mass (HBM). Lrp4 mice have an HBM phenotype (assessed radiographically), including increased bone strength and formation. Overexpression of a Sost transgene had osteopenic effects in Lrp4-WT but not Lrp4 mice. Conversely, sclerostin inhibition had blunted osteoanabolic effects in Lrp4 mice. In a disuse-induced bone wasting model, Lrp4 mice exhibit significantly less bone loss than wild-type (WT) mice. In summary, mice harboring the Lrp4-R1170W missense mutation recapitulate the human HBM phenotype, are less sensitive to altered sclerostin levels, and are protected from disuse-induced bone loss. Lrp4 is an attractive target for pharmacological targeting aimed at increasing bone mass and preventing bone loss due to disuse.
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http://dx.doi.org/10.1016/j.isci.2019.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817631PMC
October 2019

Skeletal adaptations in young male mice after 4 weeks aboard the International Space Station.

NPJ Microgravity 2019 24;5:21. Epub 2019 Sep 24.

1Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN USA.

Gravity has an important role in both the development and maintenance of bone mass. This is most evident in the rapid and intense bone loss observed in both humans and animals exposed to extended periods of microgravity in spaceflight. Here, cohabitating 9-week-old male C57BL/6 mice resided in spaceflight for ~4 weeks. A skeletal survey of these mice was compared to both habitat matched ground controls to determine the effects of microgravity and baseline samples in order to determine the effects of skeletal maturation on the resulting phenotype. We hypothesized that weight-bearing bones would experience an accelerated loss of bone mass compared to non-weight-bearing bones, and that spaceflight would also inhibit skeletal maturation in male mice. As expected, spaceflight had major negative effects on trabecular bone mass of the following weight-bearing bones: femur, tibia, and vertebrae. Interestingly, as opposed to the bone loss traditionally characterized for most weight-bearing skeletal compartments, the effects of spaceflight on the ribs and sternum resembled a failure to accumulate bone mass. Our study further adds to the insight that gravity has site-specific influences on the skeleton.
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http://dx.doi.org/10.1038/s41526-019-0081-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760218PMC
September 2019

Twist1 Inactivation in Dmp1-Expressing Cells Increases Bone Mass but Does Not Affect the Anabolic Response to Sclerostin Neutralization.

Int J Mol Sci 2019 Sep 9;20(18). Epub 2019 Sep 9.

Department of Anatomy & Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

Wnt signaling plays a major role in bone metabolism. Advances in our understanding of secreted regulators of Wnt have yielded several therapeutic targets to stimulate osteoanabolism-the most promising of which is the Wnt inhibitor sclerostin. Sclerostin antibody recently gained approval for clinical use to treat osteoporosis, but the biology surrounding sclerostin antagonism is still incompletely understood. Numerous factors regulate the efficacy of sclerostin inhibition on bone formation, a process known as self-regulation. In previous communications we reported that the basic helix-loop-helix transcription factor Twist1-a gene know to regulate skeletal development-is highly upregulated among the osteocyte cell population in mice treated with sclerostin antibody. In this communication, we tested the hypothesis that preventing Twist1 upregulation by deletion of Twist1 from late-stage osteoblasts and osteocytes would increase the efficacy of sclerostin antibody treatment, since Twist1 is known to restrain osteoblast activity in many models. Twist1-floxed loss-of-function mice were crossed to the Dmp1-Cre driver to delete Twist1 in Dmp1-expressing cells. Conditional Twist1 deletion was associated with a mild but significant increase in bone mass, as assessed by dual energy x-ray absorptiometry (DXA) and microCT (µCT) for many endpoints in both male and female mice. Biomechanical properties of the femur were not affected by conditional mutation of Twist1. Sclerostin antibody improved all bone properties significantly, regardless of Twist1 status, sex, or endpoint examined. No interactions were detected when Twist1 status and antibody treatment were examined together, suggesting that Twist1 upregulation in the osteocyte population is not an endogenous mechanism that restrains the osteoanabolic effect of sclerostin antibody treatment. In summary, Twist1 inhibition in the late-stage osteoblast/osteocyte increases bone mass but does not affect the anabolic response to sclerostin neutralization.
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http://dx.doi.org/10.3390/ijms20184427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769567PMC
September 2019

Expression of a Degradation-Resistant β-Catenin Mutant in Osteocytes Protects the Skeleton From Mechanodeprivation-Induced Bone Wasting.

J Bone Miner Res 2019 10 5;34(10):1964-1975. Epub 2019 Aug 5.

Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA.

Mechanical stimulation is a key regulator of bone mass, maintenance, and turnover. Wnt signaling is a key regulator of mechanotransduction in bone, but the role of β-catenin-an intracellular signaling node in the canonical Wnt pathway-in disuse mechanotransduction is not defined. Using the β-catenin exon 3 flox (constitutively active [CA]) mouse model, in conjunction with a tamoxifen-inducible, osteocyte-selective Cre driver, we evaluated the effects of degradation-resistant β-catenin on bone properties during disuse. We hypothesized that if β-catenin plays an important role in Wnt-mediated osteoprotection, then artificial stabilization of β-catenin in osteocytes would protect the limbs from disuse-induced bone wasting. Two disuse models were tested: tail suspension, which models fluid shift, and botulinum-toxin (botox)-induced muscle paralysis, which models loss of muscle force. Tail suspension was associated with a significant loss of tibial bone mass and density, reduced architectural properties, and decreased bone formation indices in uninduced (control) mice, as assessed by dual-energy X-ray absorptiometry (DXA), micro-computed tomography (µCT), and histomorphometry. Activation of the βcatCA allele in tail-suspended mice resulted in little to no change in those properties; ie, these mice were protected from bone loss. Similar protective effects were observed among botox-treated mice when the βcatCA was activated. RNAseq analysis of altered gene regulation in tail-suspended mice yielded 35 genes, including Wnt11, Gli1, Nell1, Gdf5, and Pgf, which were significantly differentially regulated between tail-suspended β-catenin stabilized mice and tail-suspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of β-catenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813861PMC
October 2019

Osteocytes and mechanical loading: The Wnt connection.

Orthod Craniofac Res 2019 May;22 Suppl 1:175-179

Department of Anatomy & Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana.

Bone adapts to the mechanical forces that it experiences. Orthodontic tooth movement harnesses the cell- and tissue-level properties of mechanotransduction to achieve alignment and reorganization of the dentition. However, the mechanisms of action that permit bone resorption and formation in response to loads placed on the teeth are incompletely elucidated, though several mechanisms have been identified. Wnt/Lrp5 signalling in osteocytes is a key pathway that modulates bone tissue's response to load. Numerous mouse models that harbour knock-in, knockout and transgenic/overexpression alleles targeting genes related to Wnt signalling point to the necessity of Wnt/Lrp5, and its localization to osteocytes, for proper mechanotransduction in bone. Alveolar bone is rich in osteocytes and is a highly mechanoresponsive tissue in which components of the canonical Wnt signalling cascade have been identified. As Wnt-based agents become clinically available in the next several years, the major challenge that lies ahead will be to gain a more complete understanding of Wnt biology in alveolar bone so that improved/expedited tooth movement becomes a possibility.
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http://dx.doi.org/10.1111/ocr.12282DOI Listing
May 2019

Differential changes in bone strength of two inbred mouse strains following administration of a sclerostin-neutralizing antibody during growth.

PLoS One 2019 4;14(4):e0214520. Epub 2019 Apr 4.

Department of Orthopaedic Surgery, University of Michigan, Ann Arbor, Michigan, United States of America.

Administration of sclerostin-neutralizing antibody (Scl-Ab) treatment has been shown to elicit an anabolic bone response in growing and adult mice. Prior work characterized the response of individual mouse strains but did not establish whether the impact of Scl-Ab on whole bone strength would vary across different inbred mouse strains. Herein, we tested the hypothesis that two inbred mouse strains (A/J and C57BL/6J (B6)) will show different whole bone strength outcomes following sclerostin-neutralizing antibody (Scl-Ab) treatment during growth (4.5-8.5 weeks of age). Treated B6 femurs showed a significantly greater stiffness (S) (68.8% vs. 46.0%) and maximum load (ML) (84.7% vs. 44.8%) compared to A/J. Although treated A/J and B6 femurs showed greater cortical area (Ct.Ar) similarly relative to their controls (37.7% in A/J and 41.1% in B6), the location of new bone deposition responsible for the greater mass differed between strains and may explain the greater whole bone strength observed in treated B6 mice. A/J femurs showed periosteal expansion and endocortical infilling, while B6 femurs showed periosteal expansion. Post-yield displacement (PYD) was smaller in treated A/J femurs (-61.2%, p < 0.001) resulting in greater brittleness compared to controls; an effect not present in B6 mice. Inter-strain differences in S, ML, and PYD led to divergent changes in work-to-fracture (Work). Work was 27.2% (p = 0.366) lower in treated A/J mice and 66.2% (p < 0.001) greater in treated B6 mice relative to controls. Our data confirmed the anabolic response to Scl-Ab shown by others, and provided evidence suggesting the mechanical benefits of Scl-Ab administration may be modulated by genetic background, with intrinsic growth patterns of these mice guiding the location of new bone deposition. Whether these differential outcomes will persist in adult and elderly mice remains to be determined.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214520PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448823PMC
December 2019

Loss of optimizes osteogenic metabolism and secretion to enhance bone quality.

Am J Physiol Endocrinol Metab 2019 05 15;316(5):E749-E772. Epub 2019 Jan 15.

Department of Medical and Molecular Genetics, Indiana University School of Medicine , Indianapolis, Indiana.

A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor nuclear matrix protein 4 (, , , ) respond to several classes of osteoporosis drugs with enhanced bone formation compared with wild-type (WT) animals. mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyperanabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that MSPCs exhibited an enhanced capacity for glycolytic conversion: a key step in bone anabolism. cells showed elevated collagen translation and secretion. The expression of matrix genes that contribute to bone material-level mechanical properties was elevated in cells, an observation that was supported by biomechanical testing of bone samples from and WT mice. We conclude that loss of increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality.
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http://dx.doi.org/10.1152/ajpendo.00343.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580174PMC
May 2019

Finite Element Analysis of the Mouse Distal Femur with Tumor Burden in Response to Knee Loading.

Int J Orthop (Hong Kong) 2018 28;5(1):863-871. Epub 2018 Feb 28.

Department of Mechanical Engineering, Indiana University Purdue University Indianapolis, Indianapolis, IN 46202, USA.

Breast cancer-associated bone metastasis induces bone loss, followed by an increased risk of bone fracture. To develop a strategy for preventing tumor growth and protecting bone, an understanding of the mechanical properties of bone under tumor burden is indispensable. Using a mouse model of mammary tumor, we conducted finite element analysis (FEA) of two bone samples from the distal femur. One sample was from a placebo-treated mouse, and the other was from a mouse treated with the investigational drug candidate, PD407824, an inhibitor of checkpoint kinases. Mechanical testing and microCT images revealed that bone strength is improved by administration of PD407824. In response to loading to the knee, FEA predicted that the peaks of von Mises stress, an indicator of fracture yielding, as well as the third principal compressive stress, were higher in the placebo-treated femur than the drug-treated femur. Higher peak stresses in trabecular segments were observed in the lateral condyle, a critical region for integrity of the knee joint. Collectively, this FE study supports the notion that mechanical weakening of the femur was observed in the tumor-invaded trabecular bone, and chemical agents such as PD407824 may potentially assist in preventing bone loss and bone fracture.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261479PMC
February 2018

Induction of Lrp5 HBM-causing mutations in Cathepsin-K expressing cells alters bone metabolism.

Bone 2019 03 25;120:166-175. Epub 2018 Oct 25.

Department of Anatomy & Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA; Richard L. Roudebush VA Medical Center, Indianapolis, IN, USA; Department of Biomedical Engineering, Indiana University-Purdue University at Indianapolis, Indianapolis, IN, USA. Electronic address:

High-bone-mass (HBM)-causing missense mutations in the low density lipoprotein receptor-related protein-5 (Lrp5) are associated with increased osteoanabolic action and protection from disuse- and ovariectomy-induced osteopenia. These mutations (e.g., A214V and G171V) confer resistance to endogenous secreted Lrp5/6 inhibitors, such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). Cells in the osteoblast lineage are responsive to canonical Wnt stimulation, but recent work has indicated that osteoclasts exhibit both indirect and direct responsiveness to canonical Wnt. Whether Lrp5-HBM receptors, expressed in osteoclasts, might alter osteoclast differentiation, activity, and consequent net bone balance in the skeleton, is not known. To address this, we bred mice harboring heterozygous Lrp5 HBM-causing conditional knock-in alleles to Ctsk-Cre transgenic mice and studied the phenotype using DXA, μCT, histomorphometry, serum assays, and primary cell culture. Mice with HBM alleles induced in Ctsk-expressing cells (TG) exhibited higher bone mass and architectural properties compared to non-transgenic (NTG) counterparts. In vivo and in vitro measurements of osteoclast activity, population density, and differentiation yielded significant reductions in osteoclast-related parameters in female but not male TG mice. Droplet digital PCR performed on osteocyte enriched cortical bone tubes from TG and NTG mice revealed that ~8-17% of the osteocyte population (depending on sex) underwent recombination of the conditional Lrp5 allele in the presence of Ctsk-Cre. Further, bone formation parameters in the midshaft femur cortex show a small but significant increase in anabolic action on the endocortical but not periosteal surface. These findings suggest that Wnt/Lrp5 signaling in osteoclasts affects osteoclastogenesis and activity in female mice, but also that some of the changes in bone mass in TG mice might be due to Cre expression in the osteocyte population.
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http://dx.doi.org/10.1016/j.bone.2018.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360125PMC
March 2019

Vhl deficiency in osteocytes produces high bone mass and hematopoietic defects.

Bone 2018 11 30;116:307-314. Epub 2018 Aug 30.

Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California Davis, Davis, CA, USA. Electronic address:

Tissue oxygen (O) levels vary during development and disease; adaptations to decreased O (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are active in the skeleton, and stabilizing HIF-α isoforms cause high bone mass (HBM) phenotypes. A fundamental limitation of previous studies examining the obligate role for HIF-α isoforms in the skeleton involves the persistence of gene deletion as osteolineage cells differentiate into osteocytes. Because osteocytes orchestrate skeletal development and homeostasis, we evaluated the influence of Vhl or Hif1a disruption in osteocytes. Osteocytic Vhl deletion caused HBM phenotype, but Hif1a was dispensable in osteocytes. Vhl cKO mice revealed enhanced canonical Wnt signaling. B cell development was reduced while myelopoiesis increased in osteocytic Vhl cKO, revealing a novel influence of Vhl/HIF-α function in osteocytes on maintenance of bone microarchitecture via canonical Wnt signaling and effects on hematopoiesis.
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http://dx.doi.org/10.1016/j.bone.2018.08.022DOI Listing
November 2018

Finite-element analysis of the mouse proximal ulna in response to elbow loading.

J Bone Miner Metab 2019 May 30;37(3):419-429. Epub 2018 Jul 30.

Department of Mechanical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202, USA.

Bone is a mechano-sensitive tissue that alters its structure and properties in response to mechanical loading. We have previously shown that application of lateral dynamic loads to a synovial joint, such as the knee and elbow, suppresses degradation of cartilage and prevents bone loss in arthritis and postmenopausal mouse models, respectively. While loading effects on pathophysiology have been reported, mechanical effects on the loaded joint are not fully understood. Because the direction of joint loading is non-axial, not commonly observed in daily activities, strain distributions in the laterally loaded joint are of great interest. Using elbow loading, we herein characterized mechanical responses in the loaded ulna focusing on the distribution of compressive strain. In response to 1-N peak-to-peak loads, which elevate bone mineral density and bone volume in the proximal ulna in vivo, we conducted finite-element analysis and evaluated strain magnitude in three loading conditions. The results revealed that strain of ~ 1000 μstrain (equivalent to 0.1% compression) or above was observed in the limited region near the loading site, indicating that the minimum effective strain for bone formation is smaller with elbow loading than axial loading. Calcein staining indicated that elbow loading increased bone formation in the regions predicted to undergo higher strain.
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http://dx.doi.org/10.1007/s00774-018-0943-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353704PMC
May 2019

Sclerostin neutralization unleashes the osteoanabolic effects of Dkk1 inhibition.

JCI Insight 2018 06 7;3(11). Epub 2018 Jun 7.

Department of Anatomy & Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

The WNT pathway has become an attractive target for skeletal therapies. High-bone-mass phenotypes in patients with loss-of-function mutations in the LRP5/6 inhibitor Sost (sclerosteosis), or in its downstream enhancer region (van Buchem disease), highlight the utility of targeting Sost/sclerostin to improve bone properties. Sclerostin-neutralizing antibody is highly osteoanabolic in animal models and in human clinical trials, but antibody-based inhibition of another potent LRP5/6 antagonist, Dkk1, is largely inefficacious for building bone in the unperturbed adult skeleton. Here, we show that conditional deletion of Dkk1 from bone also has negligible effects on bone mass. Dkk1 inhibition increases Sost expression, suggesting a potential compensatory mechanism that might explain why Dkk1 suppression lacks anabolic action. To test this concept, we deleted Sost from osteocytes in, or administered sclerostin neutralizing antibody to, mice with a Dkk1-deficient skeleton. A robust anabolic response to Dkk1 deletion was manifest only when Sost/sclerostin was impaired. Whole-body DXA scans, μCT measurements of the femur and spine, histomorphometric measures of femoral bone formation rates, and biomechanical properties of whole bones confirmed the anabolic potential of Dkk1 inhibition in the absence of sclerostin. Further, combined administration of sclerostin and Dkk1 antibody in WT mice produced a synergistic effect on bone gain that greatly exceeded individual or additive effects of the therapies, confirming the therapeutic potential of inhibiting multiple WNT antagonists for skeletal health. In conclusion, the osteoanabolic effects of Dkk1 inhibition can be realized if sclerostin upregulation is prevented. Anabolic therapies for patients with low bone mass might benefit from a strategy that accounts for the compensatory milieu of WNT inhibitors in bone tissue.
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http://dx.doi.org/10.1172/jci.insight.98673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124404PMC
June 2018

Conditional Deletion of Sost in MSC-Derived Lineages Identifies Specific Cell-Type Contributions to Bone Mass and B-Cell Development.

J Bone Miner Res 2018 10 29;33(10):1748-1759. Epub 2018 Jun 29.

Physical and Life Sciences Directorate, Lawrence Livermore National Laboratories, Livermore, CA, USA.

Sclerostin (Sost) is a negative regulator of bone formation and blocking its function via antibodies has shown great therapeutic promise by increasing both bone mass in humans and animal models. Sclerostin deletion in Sost KO mice (Sost ) causes high bone mass (HBM) similar to sclerosteosis patients. Sost mice have been shown to display an up to 300% increase in bone volume/total volume (BV/TV), relative to age-matched controls. It has been postulated that the main source of skeletal sclerostin is the osteocyte. To understand the cell-type specific contributions to the HBM phenotype described in Sost mice, as well as to address the endocrine and paracrine mode of action of sclerostin, we examined the skeletal phenotypes of conditional Sost loss-of-function (Sost ) mice with specific deletions in (1) the limb mesenchyme (Prx1-Cre; targets osteoprogenitors and their progeny); (2) midstage osteoblasts and their progenitors (Col1-Cre); (3) mature osteocytes (Dmp1-Cre); and (4) hypertrophic chondrocytes and their progenitors (ColX-Cre). All conditional alleles resulted in significant increases in bone mass in trabecular bone in both the femur and lumbar vertebrae, but only Prx1-Cre deletion fully recapitulated the amplitude of the HBM phenotype in the appendicular skeleton and the B-cell defect described in the global KO. Despite WT expression of Sost in the axial skeleton of Prx1-Cre deleted mice, these mice also had a significant increase in bone mass in the vertebrae, but the sclerostin released in circulation by the axial skeleton did not affect bone parameters in the appendicular skeleton. Also, both Col1 and Dmp1 deletion resulted in a similar 80% significant increase in trabecular bone mass, but only Col1 and Prx1 deletion resulted in a significant increase in cortical thickness. We conclude that several cell types within the Prx1-osteoprogenitor-derived lineages contribute significant amounts of sclerostin protein to the paracrine pool of Sost in bone. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jbmr.3467DOI Listing
October 2018

Skeletal cell YAP and TAZ combinatorially promote bone development.

FASEB J 2018 05 10;32(5):2706-2721. Epub 2018 Jan 10.

Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

The functions of the paralogous transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in bone are controversial. Each has been observed to promote or inhibit osteogenesis in vitro, with reports of both equivalent and divergent functions. Their combinatorial roles in bone physiology are unknown. We report that combinatorial YAP/TAZ deletion from skeletal lineage cells, using Osterix-Cre, caused an osteogenesis imperfecta-like phenotype with severity dependent on allele dose and greater phenotypic expressivity with homozygous TAZ vs. YAP ablation. YAP/TAZ deletion decreased bone accrual and reduced intrinsic bone material properties through impaired collagen content and organization. These structural and material defects produced spontaneous fractures, particularly in mice with homozygous TAZ deletion and caused neonatal lethality in dual homozygous knockouts. At the cellular level in vivo, YAP/TAZ ablation reduced osteoblast activity and increased osteoclast activity, in an allele dose-dependent manner, impairing bone accrual and remodeling. Transcriptionally, YAP/TAZ deletion and small-molecule inhibition of YAP/TAZ interaction with the transcriptional coeffector TEAD reduced osteogenic and collagen-related gene expression, both in vivo and in vitro. These data demonstrate that YAP and TAZ combinatorially promote bone development through regulation of osteoblast activity, matrix quality, and osteoclastic remodeling.-Kegelman, C. D., Mason, D. E., Dawahare, J. H., Horan, D. J., Vigil, G. D., Howard, S. S., Robling, A. G., Bellido, T. M., Boerckel, J. D. Skeletal cell YAP and TAZ combinatorially promote bone development.
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http://dx.doi.org/10.1096/fj.201700872RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5901392PMC
May 2018
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