Bone remodeling is a highly coordinated process involving bone formation and resorption, and imbalance of this process results in osteoporosis. It has long been recognized that long-term heparin therapy often causes osteoporosis, suggesting that heparan sulfate (HS), the physiological counterpart of heparin, is somehow involved in bone mass regulation. The role of endogenous HS in adult bone, however, remains unclear. To determine the role of HS in bone homeostasis, we conditionally ablated Ext1, which encodes an essential glycosyltransferase for HS biosynthesis, in osteoblasts. Resultant conditional mutant mice developed severe osteopenia. Surprisingly, this phenotype is not due to impairment in bone formation but to enhancement of bone resorption. We show that osteoprotegerin (OPG), which is known as a soluble decoy receptor for RANKL, needs to be associated with the osteoblast surface in order to efficiently inhibit RANKL/RANK signaling and that HS serves as a cell surface binding partner for OPG in this context. We also show that bone mineral density is reduced in patients with multiple hereditary exostoses, a genetic bone disorder caused by heterozygous mutations of Ext1, suggesting that the mechanism revealed in this study may be relevant to low bone mass conditions in humans.
Satoshi Nozawa, Toshihiro Inubushi, Fumitoshi Irie, Iori Takigami, Kazu Matsumoto, Katsuji Shimizu, Haruhiko Akiyama, Yu Yamaguchi
While the prevalence of osteoporosis is growing rapidly with population aging, therapeutic options remain limited. Here, we identify potentially novel roles for CaV1.2 L-type voltage–gated Ca2+ channels in osteogenesis and exploit a transgenic gain-of-function mutant CaV1.2 to stem bone loss in ovariectomized female mice. We show that endogenous CaV1.2 is expressed in developing bone within proliferating chondrocytes and osteoblasts. Using primary BM stromal cell (BMSC) cultures, we found that Ca2+ influx through CaV1.2 activates osteogenic transcriptional programs and promotes mineralization. We used Prx1-, Col2a1-, or Col1a1-Cre drivers to express an inactivation-deficient CaV1.2 mutant in chondrogenic and/or osteogenic precursors in vivo and found that the resulting increased Ca2+ influx markedly thickened bone not only by promoting osteogenesis, but also by inhibiting osteoclast activity through increased osteoprotegerin secretion from osteoblasts. Activating the CaV1.2 mutant in osteoblasts at the time of ovariectomy stemmed bone loss. Together, these data highlight roles for CaV1.2 in bone and demonstrate the potential dual anabolic and anticatabolic therapeutic actions of tissue-specific CaV1.2 activation in osteoblasts.
Chike Cao, Yinshi Ren, Adam S. Barnett, Anthony J. Mirando, Douglas Rouse, Se Hwan Mun, Kyung-Hyun Park-Min, Amy L. McNulty, Farshid Guilak, Courtney M. Karner, Matthew J. Hilton, Geoffrey S. Pitt
Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury–induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.
Frédéric Torossian, Bernadette Guerton, Adrienne Anginot, Kylie A. Alexander, Christophe Desterke, Sabrina Soave, Hsu-Wen Tseng, Nassim Arouche, Laetitia Boutin, Irina Kulina, Marjorie Salga, Beulah Jose, Allison R. Pettit, Denis Clay, Nathalie Rochet, Erica Vlachos, Guillaume Genet, Charlotte Debaud, Philippe Denormandie, François Genet, Natalie A. Sims, Sébastien Banzet, Jean-Pierre Levesque, Jean-Jacques Lataillade, Marie-Caroline Le Bousse-Kerdilès
Advanced breast cancer is frequently associated with skeletal metastases and accelerated bone loss. Recombinant parathyroid hormone [teriparatide, PTH(1-34)] is the first anabolic agent approved in the US for treatment of osteoporosis. While signaling through the PTH receptor in the osteoblast lineage regulates bone marrow hematopoietic niches, the effects of anabolic PTH on the skeletal metastatic niche are unknown. Here, we demonstrate, using orthotopic and intratibial models of 4T1 murine and MDA-MB-231 human breast cancer tumors, that anabolic PTH decreases both tumor engraftment and the incidence of spontaneous skeletal metastasis in mice. Microcomputed tomography and histomorphometric analyses revealed that PTH increases bone volume and reduces tumor engraftment and volume. Transwell migration assays with murine and human breast cancer cells revealed that PTH alters the gene expression profile of the metastatic niche, in particular VCAM-1, to inhibit recruitment of cancer cells. While PTH did not affect growth or migration of the primary tumor, it elicited several changes in the tumor gene expression profile resulting in a less metastatic phenotype. In conclusion, PTH treatment in mice alters the bone microenvironment, resulting in decreased cancer cell engraftment, reduced incidence of metastases, preservation of bone microarchitecture and prolonged survival.
Srilatha Swami, Joshua Johnson, Lance A. Bettinson, Takaharu Kimura, Hui Zhu, Megan A. Albertelli, Rachelle W. Johnson, Joy Y. Wu
Decreased cortical thickness and increased cortical porosity are the key anatomic changes responsible for osteoporotic fractures in elderly women and men. The cellular basis of these changes is unbalanced endosteal and intracortical osteonal remodeling by the osteoclasts and osteoblasts that comprise the basic multicellular units (BMUs). Like humans, mice lose cortical bone with age, but unlike humans, this loss occurs in the face of sex steroid sufficiency. Mice are therefore an ideal model to dissect age-specific osteoporotic mechanisms. Nevertheless, lack of evidence for endosteal or intracortical remodeling in mice has raised questions about their translational relevance. We show herein that administration of the antiosteoclastogenic cytokine osteoprotegerin to Swiss Webster mice ablated not only osteoclasts, but also endosteal bone formation, demonstrating the occurrence of BMU-based endosteal remodeling. Femoral cortical thickness decreased in aged male and female C57BL/6J mice, as well as F1 hybrids of C57BL/6J and BALB/cBy mice. This decrease was greater in C57BL/6J mice, indicating a genetic influence. Moreover, endosteal remodeling became unbalanced because of increased osteoclast and decreased osteoblast numbers. The porosity of the femoral cortex increased with age but was much higher in females of both strains. Notably, the increased cortical porosity resulted from de novo intracortical remodeling by osteon-like structures. Age-dependent cortical bone loss was associated with increased osteocyte DNA damage, cellular senescence, the senescence-associated secretory phenotype, and increased levels of RANKL. The demonstration of unbalanced endosteal and intracortical remodeling in old mice validates the relevance of this animal model to involutional osteoporosis in humans.
Marilina Piemontese, Maria Almeida, Alexander G. Robling, Ha-Neui Kim, Jinhu Xiong, Jeff D. Thostenson, Robert S. Weinstein, Stavros C. Manolagas, Charles A. O’Brien, Robert L. Jilka
Postnatal bone formation is influenced by nutritional status and compromised by disturbances in metabolism. The oxidation of dietary lipids represents a critical source of ATP for many cells but has been poorly studied in the skeleton, where the prevailing view is that glucose is the primary energy source. Here, we examined fatty acid uptake by bone and probed the requirement for fatty acid catabolism during bone formation by specifically disrupting the expression of carnitine palmitoyltransferase 2 (Cpt2), an obligate enzyme in fatty acid oxidation, in osteoblasts and osteocytes. Radiotracer studies demonstrated that the skeleton accumulates a significant fraction of postprandial fatty acids, which was equal to or in excess of that acquired by skeletal muscle or adipose tissue. Female, but not male, Cpt2 mutant mice exhibited significant impairments in postnatal bone acquisition, potentially due to an inability of osteoblasts to modify fuel selection. Intriguingly, suppression of fatty acid utilization by osteoblasts and osteocytes also resulted in the development of dyslipidemia and diet-dependent modifications in body composition. Taken together, these studies demonstrate a requirement for fatty acid oxidation during bone accrual and suggest a role for the skeleton in lipid homeostasis.
Soohyun P. Kim, Zhu Li, Meredith L. Zoch, Julie L. Frey, Caitlyn E. Bowman, Priyanka Kushwaha, Kathleen A. Ryan, Brian C. Goh, Susanna Scafidi, Julie E. Pickett, Marie-Claude Faugere, Erin E. Kershaw, Daniel L. J. Thorek, Thomas L. Clemens, Michael J. Wolfgang, Ryan C. Riddle
Multiple hereditary exostoses (MHE) is characterized by the development of numerous benign bony tumors (osteochondromas). Although it has been well established that MHE is caused by mutations in EXT1 and EXT2, which encode glycosyltransferase essential for heparan sulfate (HS) biosynthesis, the cellular origin and molecular mechanisms of MHE remain elusive. Here, we show that in Ext1 mutant mice, osteochondromas develop from mesenchymal stem cell–like progenitor cells residing in the perichondrium, and we show that enhanced BMP signaling in these cells is the primary signaling defect that leads to osteochondromagenesis. We demonstrate that progenitor cells in the perichondrium, including those in the groove of Ranvier, highly express HS and that Ext1 ablation targeted to the perichondrium results in the development of osteochondromas. Ext1-deficient perichondrial progenitor cells show enhanced BMP signaling and increased chondrogenic differentiation both in vitro and in vivo. Consistent with the functional role for enhanced BMP signaling in osteochondromagenesis, administration of the small molecule BMP inhibitor LDN-193189 suppresses osteochondroma formation in two MHE mouse models. Together, our results demonstrate a role for enhanced perichondrial BMP signaling in osteochondromagenesis in mice, and they suggest the possibility of pharmacological treatment of MHE with BMP inhibitors.
Toshihiro Inubushi, Satoshi Nozawa, Kazu Matsumoto, Fumitoshi Irie, Yu Yamaguchi
Genotypic and phenotypic alterations in the bone marrow (BM) microenvironment, in particular in osteoprogenitor cells, have been shown to support leukemogenesis. However, it is unclear how leukemia cells alter the BM microenvironment to create a hospitable niche. Here, we report that acute myeloid leukemia (AML) cells, but not normal CD34+ or CD33+ cells, induce osteogenic differentiation in mesenchymal stromal cells (MSCs). In addition, AML cells inhibited adipogenic differentiation of MSCs. Mechanistic studies identified that AML-derived BMPs activate Smad1/5 signaling to induce osteogenic differentiation in MSCs. Gene expression array analysis revealed that AML cells induce connective tissue growth factor (CTGF) expression in BM-MSCs irrespective of AML type. Overexpression of CTGF in a transgenic mouse model greatly enhanced leukemia engraftment in vivo. Together, our data suggest that AML cells induce a preosteoblast-rich niche in the BM that in turn enhances AML expansion.
V. Lokesh Battula, Phuong M. Le, Jeffrey C. Sun, Khoa Nguyen, Bin Yuan, Ximin Zhou, Sonali Sonnylal, Teresa McQueen, Vivian Ruvolo, Keith A. Michel, Xiaoyang Ling, Rodrigo Jacamo, Elizabeth Shpall, Zhiqiang Wang, Arvind Rao, Gheath Al-Atrash, Marina Konopleva, R. Eric Davis, Melvyn A. Harrington, Catherine W. Cahill, Carlos Bueso-Ramos, Michael Andreeff
NELL-1 is a secreted, osteogenic protein first discovered to control ossification of the cranial skeleton. Recently, NELL-1 has been implicated in bone maintenance. However, the cellular determinants of NELL-1’s bone-forming effects are still unknown. Here, recombinant human NELL-1 (rhNELL-1) implantation was examined in a clinically relevant nonhuman primate lumbar spinal fusion model. Prolonged rhNELL-1 protein release was achieved using an apatite-coated β-tricalcium phosphate carrier, resulting in a local influx of stem cell antigen-1–positive (Sca-1+) mesenchymal progenitor cells (MPCs), and complete osseous fusion across all samples (100% spinal fusion rate). Murine studies revealed that
Aaron W. James, Jia Shen, Rebecca Tsuei, Alan Nguyen, Kevork Khadarian, Carolyn A. Meyers, Hsin Chuan Pan, Weiming Li, Jin H. Kwak, Greg Asatrian, Cymbeline T. Culiat, Min Lee, Kang Ting, Xinli Zhang, Chia Soo
Osteoarthritis (OA) is the most common form of arthritis worldwide. It is a complex disease affecting the whole joint but is generally characterized by progressive degradation of articular cartilage. Recent genome-wide association screens have implicated distinct DNA methylation signatures in OA patients. We show that the de novo DNA methyltransferase (Dnmt) 3b, but not Dnmt3a, is present in healthy murine and human articular chondrocytes and its expression decreases in OA mouse models and in chondrocytes from human OA patients. Targeted deletion of Dnmt3b in murine articular chondrocytes results in an early-onset and progressive postnatal OA-like pathology. RNA-Seq and methylC-Seq analyses of Dnmt3b loss-of-function chondrocytes show that cellular metabolic processes are affected. Specifically, TCA metabolites and mitochondrial respiration are elevated. Importantly, a chondroprotective effect was found following Dnmt3b gain of function in murine articular chondrocytes in vitro and in vivo. This study shows that Dnmt3b plays a significant role in regulating postnatal articular cartilage homeostasis. Cellular pathways regulated by Dnmt3b in chondrocytes may provide novel targets for therapeutic approaches to treat OA.
Jie Shen, Cuicui Wang, Daofeng Li, Taotao Xu, Jason Myers, John M. Ashton, Ting Wang, Michael J. Zuscik, Audrey McAlinden, Regis J. O’Keefe
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